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+{"text":"\n\nACRYLIC  \nINNOVATION\n\nStyles +  \ntechniques  \nfeaturing 64   \nvisionary  \nartists\n\n**NANCYREYNER** \u2022 Author of _Acrylic Revolution_\n\nwww.artistsnetwork.com\n**Acrylic Innovation**. Copyright \u00a9 2010 by Nancy Reyner. Manufactured in China. All rights reserved. No part of this book may be reproduced in any form or by any electronic or mechanical means including information storage and retrieval systems without permission in writing from the publisher, except by a reviewer who may quote brief passages in a review. Published by North Light Books, an imprint of F+W Media, Inc., 4700 East Galbraith Road, Cincinnati, Ohio, 45236. (800) 289-0963. First edition.\n\nOther fine North Light Books are available from your local bookstore, art supply store or online. Also visit our website at www.fwmedia.com.\n\n14 13 12 11 10 5 4 3 2 1\n\nDISTRIBUTED IN CANADA BY FRASER DIRECT  \n100 Armstrong Avenue  \nGeorgetown, ON, Canada L7G 5S4  \nTel: (905) 877-4411\n\nDISTRIBUTED IN THE U.K. AND EUROPE BY DAVID & CHARLES  \nBrunel House, Newton Abbot, Devon, TQ12 4PU, England  \nTel: (+44) 1626 323200,Fax: (+44) 1626 323319  \nEmail: postmaster@davidandcharles.co.uk\n\nDISTRIBUTED IN AUSTRALIA BY CAPRICORN LINK  \nP.O. Box 704, S. Windsor NSW, 2756 Australia  \nTel: (02) 4577-3555\n\n**Library of Congress Cataloging in Publication Data**\n\nReyner, Nancy.\n\nAcrylic innovation : styles and techniques featuring 64 visionary artists\/ Nancy Reyner. -- 1st ed.\n\np. cm.\n\nISBN 978-1-60061-864-2 (alk. paper)\n\n1. Acrylic painting--Technique. I. Title. II. Title: Styles and techniques featuring 64 visionary artists. III. Title: Styles and techniques featuring sixty four visionary artists.\n\nND1535.R48 2010\n\n751.4'26--dc22\n\n2010018580\n\nEdited by Kelly C. Messerly, Designed by Clare Finney, Production coordinated by Mark Griffin\n**About the Author**\n\nAuthor photo by Reynaldo Villalobos.\n\nBorn and raised on the East Coast in the United States, Nancy Reyner received a BFA from the Rhode Island School of Design and an MFA from Columbia University. Creating costumes and sets for theater and film to coordinating public arts programs for the state of New York, Reyner has had an expansive career in the arts, all of which inform her work. She now lives in Santa Fe, New Mexico, has been a painter for more than thirty years and exhibits and teaches both nationally and internationally. Her previous book, _Acrylic Revolution_ , contains over one hundred acrylic painting techniques, and continues to be a best seller. Please visit her Web site, www.NancyReyner.com, for current work, her painting blog, workshops and gallery representation.\n\n**Artwork on Cover and Back Cover:**\n\nGary Denmark, _Jongleur 2_ , pictured again\n\nPat Forbes, (detail) _Neutrino Blossoms_ , pictured in full\n\nCatherine Mackey, (detail) _Harrison Street Scooter_ , pictured in full\n\nBeth Ames Swartz, (detail) _The Fire and the Rose: But Heard, Half-Heard, in the Stillness_ , pictured in full\n\nJim Waid, _Blue Shimmy_ , pictured again\n\nInes Kramer, _Rooftop Eden_ , pictured again\n\nMartha Kennedy, _Avocado on Orange_ , pictured again\n\nRobin Sierra, _Aquamarine_ , pictured again\n\nRen\u00e9e Phillips, _Field of Dreams_ , pictured again\n\n**Art**\n\nNancy Reyner, _Koi and River_ , acrylic on canvas, 60\" \u00d7 46\" (152cm \u00d7 117cm)\n\n**Art**\n\nNancy Reyner, _Ocean Elixer_ , acrylic and gold leaf on panel 20\" \u00d7 20\" (51cm \u00d7 51cm)\n**Metric Conversion Chart**  \n---  \n|  |   \n**To convert** | **to** | **multiply by**  \nInches | Centimeters | 2.54   \nCentimeters | Inches | 0.4   \nFeet | Centimeters | 30.5   \nCentimeters | Feet | 0.03   \nYards | Meters | 0.9   \nMeters | Yards | 1.1\n\n**Acknowledgments**\n\nSpecial thanks to those who helped connect me with the extraordinary artists included in this book: George Alexander, Bruce Cody, Bill Hinsvark, Katherine Chang Liu, Michael Matassa, Keith Morant and Renee Phillips. Many thanks to Reynaldo Villalobos for photography. Thank you to Bonnie Teitelbaum, Tekla Johnson and Eve Munson for inspiration and assistance. Thank you Phillip Cohen and to my wonderful family and friends for support. I thank my art professors Phyllis Bramson, Jane Wilson and the late Art Wood for their wisdom and encouragement. I cherish my dance teacher, Lori Brody, and all my cool dancer friends in Brody's Ballerinas for making life wonderful. Thank you to Mark and Barbara Golden, and to the staff at Golden Artist Colors for all their wonderful inventions, products and support, with special thanks to their technical department. I have enjoyed immensely working with the F+W and North Light Books staff, with extra thanks to Jamie Markle and Kelly Messerly.\n**Dedication**\n\nThis book is dedicated to my son, Jacob Cooper Cohen, who has been and continues to be a great inspiration for me.\nTABLE OF CONTENTS\n\nIntroduction: How to Use this Book\n\nEssential Acrylic Tips\n\nSECTION 1\n\n**The Realistic Window**\n\nEpic Narrative\n\nPhotorealism\n\nPortrait\n\nStill Life\n\nLandscape\n\nSECTION 2\n\n**Abstracting the Window**\n\nFigure as Form\n\nReductive\n\nFlat Space\n\nRomanticized\n\nExaggeration\n\nSECTION 3\n\n**Changing Perspectives**\n\nInside & Outside\n\nMultiple Perspectives\n\nComposite Arrangements\n\nCollage\n\n**Sherry Loehr**\n\n_Tulips and Oranges_\n\nAcrylic on board\n\n24\" \u00d7 36\" (61cm \u00d7 91cm)\n\n**Gary Denmark**\n\n_Jongleur 2_\n\nAcrylic and collage on board\n\n48\" \u00d7 48\" (122cm \u00d7 122cm)\n\nSECTION 4\n\n**Engaging the Picture Plane**\n\nMapping: Planes & Fragments\n\nConfronting the Viewer\n\nIsolated Imagery\n\nEmbellished Form\n\nHard Edge Graphic\n\nPattern Fields\n\nSECTION 5\n\n**Spatial Push-Pull**\n\nMarking Space\n\nFree-Floating Organics\n\nText, Line & Image\n\nGeometry & Space\n\nSECTION 6\n\n**Minimal**\n\nSheens & Texture\n\nIlluminated\n\nColor Field\n\nA Singular Note\n\nVeiling Forms\n\nContributing Artists\n\n**Bruce Cody**\n\n_Capitol City Sunlight_\n\nAcrylic on canvas\n\n16\" \u00d7 30\" (41cm \u00d7 76cm)\n\n**Hamish Allan**\n\n_House and Hills_\n\nAcrylic on canvas\n\n24\" \u00d7 24\" (61cm \u00d7 61cm)\nINTRODUCTION:  \nHOW TO USE TH IS BOOK\n\n**Nancy Reyner**\n\n_Essence_ (detail)\n\nAcrylic and gold leaf on panel\n\n32\" \u00d7 26\" (81cm \u00d7 66cm)\n\nNeed an idea? Want new motivation? Desiring new tips and techniques? Just flip through the twenty-nine styles in this book ranging from photorealism to minimal color field and everything in between. Read how other artists have taken the journey to create unique and personal art, offering advice and gems from their own experience and encouraging artists to invent, not replicate. An artist's process can be viewed both mentally (the idea) and physically (the technique). A visionary artist develops and combines both to create a personal vision and unique art. This book is meant to inspire artists to find their own personal voice.\n\nThroughout history the discovery of new art products spawned the invention of new art styles, ideas and techniques. The creation of oil paint was a catalyst for new styles of blending and realism that were previously unheard of for the egg tempera painters of that time. The invention of storing oil paint in tubes added portability for painters, taking them out of the studio and paving the way for plein air and onsite painting. Photography, digital imagery and computers have also made their mark.\n\nAcrylic paint, first developed in the 1930s, continued to improve in quality and durability, and eventually established itself as one of the most dependable, archival and permanent artist materials offered. Available to artists now for over sixty years, it is currently helping to push the envelope of contemporary vision. Acrylic is often used as a substitute for oil paint or watercolor. But the real gold mine for the artist is in allowing the medium freedom to do what it does best. New paint requires a new way of thinking. Acrylic has a wide range of possibilities, uses and techniques. My previous book, Acrylic Revolution, demonstrates this variety with over one hundred acrylic painting techniques in an easy resource format. More and more artists are choosing to paint in acrylic, and this growing trend is reflected in the new contemporary art styles emerging on the scene. This book contains incredible artwork from sixty-four acrylic artists worldwide, all contemporary, using acrylic in part or completely in their work, who are taking advantage of acrylic's potential to create unique genres or styles. This book addresses methods and techniques for finding your own personal voice in this medium.\n\nTo view a painting is to experience the idea of space. In this book I take a fresh stance by grouping styles in terms of their perceived spatial qualities, even inventing some of the style names. Notably, many artists in this book have work represented simultaneously in several style categories. A style is merely a tool, meant to facilitate the expression of an idea, and therefore most artists' work will not fit neatly into any specific category. A style is a tool, not a label.\n\nTo create an original idea, we can use the wide variety of visual stimuli around us every day, in advertisements and media, our relationships and experiences, objects, nature and architecture. All these can offer artistic references. Instead of replicating, art can be fresh and original when we add our own individuality, rearrange images, work with unusual combinations, and let materials and mediums influence us.\n\nESSENTIAL ACRYLIC TIPS\n\n**\u2022 Acrylic is available in a wide variety of paints and products.** It is helpful to understand the different categories of products to select the most appropriate one for the technique at hand. In general, acrylic paint color has two components: pigment (for color) and acrylic binder (also known as polymer). Usually, the binder is also called a medium, and, when thickeners are added, it's a gel. The fluid binder without thickeners is combined with pigment to make fluid paints. Binder with thickener added to pigment creates the heavy bodied paints. A small amount of whiting agent added to the binder creates matte products such as matte mediums, matte gels and matte varnishes. Larger quantities of whiting or opaque components make pastes. Adding materials such as pumice, glass beads, metal and fiber can make unusual gels. Any of these products can be used as a ground to change the painting surface, when applied as a first layer to customize and change the surface absorbency, texture or color.\n\n**\u2022 Acrylic products are compatible with each other.** Any acrylic product can be combined together while wet to create new mixtures and hybrids. Gels, mediums and pastes can all be mixed together. Keep in mind that the characteristics of one particular paint or product will change when you mix it with another. This can work to your advantage; for example, mixing a gloss medium with a matte medium will produce an excellent semigloss. However, some specialty products, such as Clear Tar Gel, which makes the paint tar-like and stringy, will lose their special abilities when too much of another paint or product is added. You can also layer acrylic products and paints in any order in your painting process. Gels can go on top of mediums and mediums on gels. Diluted paint works on undiluted paint and vice versa. Glossy can go on top of matte, then on satin and on glossy again.\n\n**\u2022 Acrylic can be used in combination with other mediums.** Most other mediums such as oil, gouache, watercolor, pastel or casein can be applied on top of acrylic grounds or acrylic underpaintings. In general, though, avoid using acrylic on top of other mediums, or combining other mediums together with acrylic in wet form. For example, watercolor can be used to wonderful effect on top of acrylic that is already dry, but mixing the two together wet is less effective.\n\n**\u2022 A few acrylic products are best used alone.**\n\n1. Use acrylic gesso as a primer and not as a white paint. In thick applications, acrylic gesso may crack.\n\n2. Use a removable archival varnish as a final protective coat. Even though it can be used in the middle layers of a painting, it is meant to be removable for cleaning purposes and as a final coat.\n\n3. Additives such as retarder, thinners and flow release need to be used in correct proportions. It is always a good idea to read product labels for correct use and application information.\n\n**\u2022 Acrylic is a glue.** Any acrylic paint or product can function very well as a glue. You can add collage elements and mixed media to acrylic while it's wet and they will adhere nicely.\n\n**\u2022 Stir gently and don't shake.** The small amount of soap remaining in the acrylic products from processing may create bubbles with vigorous stirring or handling. To stir a batch of paint, use a palette knife instead of a stiff bristle brush. After stirring mediums for use in pours, let the bubbles settle out overnight before pouring.\n\n**\u2022 In general, mix paint with a knife.** Brushes are meant to hold paint, while knives more easily release the paint. Mix a batch of paint with a knife for a clean, homogenized mixture. Use a brush to mix paint in a hodgepodge manner, for a painting technique called \"dirty brushing.\"\n\n**\u2022 As you paint, keep brushes in water** until you can clean them well with soap. Soap removes paint from the brush more effectively than water alone. If a brush in use is left out of water too long, the acrylic will dry on it, making it difficult to remove later. To make cleaning easier, slightly dampen the brush bristles with water before dipping them into the acrylic paint. Damp bristles reduce the gripping power of acrylic, making the paint easier to remove later.\n\n**\u2022 There are two choices for thinning acrylic paint:** water or acrylic medium. Adding water will break down the acrylic binder in the paint, creating a thinner paint that appears like watercolor, resulting in a matte finish. The more water you add, the more the paint will be affected by the absorbency of the painting surface. On the other hand, adding acrylic mediums, gels or pastes, while minimizing the addition of water, will maintain the acrylic's properties, offering a rich, glossy appearance. Either choice is effective, depending on your desired result.\n\n**\u2022 Acrylic shrinks in volume while drying.** As acrylic dries, the paint layer will shrink by about a third in volume. This is more noticeable when using thick pastes and gels. Generally, apply a paint layer that is a little thicker than what you want at completion.\n\n**\u2022 Acrylic appears lighter in color when wet.** The acrylic binder is naturally white when wet, but dries clear and glossy. Therefore while painting with acrylic, the color will appear lighter when wet but goes to its natural color when dry. The more mediums and gels you add to a paint color, the greater the difference between this lighter version when wet and its natural color when dry. Paint color added to pastes, however, will not change in color very much between wet and dry. That is because pastes are white when wet, remaining white when dry.\n\n**\u2022 Acrylic is naturally glossy.** Matte products such as matte mediums, matte gels and matte varnishes have matting agent (a fine white powder) added to them to create their characteristic appearance. This matting agent means that matte products slightly cloud and lighten colors underneath. The thicker the application of a matte product, the more this cloudy and lightening effect will be apparent. Acrylic products labeled semigloss or satin have matting agent in them as well, just less in proportion to a matte product. Also, colored paints when dry will vary naturally in sheen, according to the pigment.\n\n**\u2022 Acrylic has a two-part drying process.** The first part of the acrylic drying process, known as \"dry to the touch,\" means the top layer of the paint skin has dried due to the evaporation of the water in the paint, enabling layering. The second part of the drying process involves the curing of the polymer or acrylic in the paint, which takes several days to several weeks to \"lock down,\" or fully cure. The actual curing time is dependent on the layer's thickness and environmental factors. During this curing time, it is important to allow air to flow around it. Avoid tightly wrapping the painting, storing the artwork in a closed environment or exposing it to extreme temperatures during the curing phase.\n\n**\u2022 Be aware of the \"tacky phase.\"** While the paint is still wet, it is very malleable. You can scrape it, wipe it off and rework it with ease. As you continue to work into this wet paint, however, it is already beginning to dry. Once it dries to the touch, it has a wonderfully resistant surface on which you can overlay new paints without disturbing what is underneath. It is between the wet stage and this \"dry to the touch\" stage when problems can occur. Between the wet and dry stages, the acrylic gets tacky, and continued working over this tacky area can create unwanted effects such as streaking or pulling as the paint sticks to your brush. Initially, acrylic paint glides smoothly and easily, but as it reaches this tacky phase, it will begin to pull and feel difficult to manipulate. At this point, stop painting in that area, and move on to a drier area of the painting. If you need to keep working in the tacky area, use a blow-dryer for a minute to dry it quickly, then resume painting. Avoid blow-drying very thick layers. Also, be aware that the paint on your brush will dry quickly. Make a habit of rinsing frequently to keep the paint from getting tacky on your brush.\n\n**\u2022 Do not freeze acrylic.** Oil painters sometimes freeze the excess paint on their palettes to prolong the life of the paint. Avoid this with acrylic. Acrylic contains a certain amount of antifreeze, but after a few freeze\/thaw cycles, the paint will no longer be stable. Acrylic paintings, when fully cured, will be fine if exposed to cold. However, fine art should not be exposed to extreme temperatures. To extend the drying time of wet paint, consider using Golden's slow drying OPEN Acrylics.\n**Section 1**\n\nTHE  \nREALISTIC  \nWINDOW\n\n**Jason de Graaf**\n\n_Untitled (Self Portrait)_\n\nAcrylic on canvas\n\n30\" \u00d7 30\" (76cm \u00d7 76cm)\n\nRealism, convincing portrayals of the physical world, presents imagery that has fascinated artists and viewers for centuries, and continues in popularity today. Still life, landscape and portraiture are all just as potent today as they were in the past. This section includes five realistic styles, each using similar principles. These principles, popularized in the Renaissance, include singular or fixed perspective, the use of a horizon line, modeling of light and shadow to create volume, and the illusion of receding space, all of which turn the canvas into a viewing window. Inspired by evolving technologies in photography, digital imagery and new pigment development, realism has expanded to include hyperrealism, along with new techniques and exciting new color palettes. This section investigates both traditional and new realistic styles with an emphasis on keeping them personal and contemporary.\n\nEPIC NARRATIVE\n\nAn epic narrative style is characterized by multiple figures in a landscape\u2014it's the \"big scene\" that has been used historically to convey weighty narratives such as religious stories and myths.\n\nPaul Pletka's _Tears of the Lord_ is a new vision on the epic narrative. This striking piece uses a bold-colored, flat backdrop and a vibrant color palette to set off multiple figures, along with incredible attention to detail.\n\nPletka works in large formats for greater impact and has centered his work on his desire to explore the ritual side of Native American culture, to bring forth the spiritual side in a respectful way, and to consider this an interpretation from his own cultural point of view.\n\nPletka has painted for many years, working through his own personal desire to invent something original. Each painting is heavily researched to use authentic and accurate material. But for Pletka, making something beautiful is the most important aspect; therefore, some elements may be slightly compromised for aesthetics.\n\n**The Artist's Process**\n\nTwo key motivations for Pletka are scale and a new technical challenge. He creates many thumbnail sketches until a cogent image emerges, then he moves directly to the canvas, drawing first with vine charcoal to transfer his energy and excitement into the work. He then overpaints the line work with Raw Sienna. Even with the initial drawing in place, Pletka will rearrange and redesign to accommodate a piece's evolution. He often obliterates a figure he painstakingly worked and repaints it in a new location, sometimes several times. To maintain the integrity of the canvas's surface, he sands or scrubs off an area, instead of overpainting. By working in acrylic, Pletka is able to maintain a sense of immediacy while working with multiple glaze layers on one painting at a time.\n\n**Other Art in This Style**\n\n\u2022 Contemporary artist Mark Tansey, Modern artist George Bellows\n\n\u2022 Renaissance painters Sandro Botticelli, Masaccio, Leonardo da Vinci, Michelangelo Buonarroti, Titian, Tintoretto and Raphael\n\n\u2022 Historic religious and mythic paintings from French Classicism and Baroque periods\n\n\u2022 Fantastic imagery from Hieronymus Bosch\n\n\u2022 Muralist Diego Rivera\n\n **TIPS**  \n **from the Artist**\n\nGreat art contains the artist's originality, which then shows through the work. Be willing to destroy elements in your work to push the piece further. Your work has to be you, it has to come from your core and be honest. It can't come from someone else and be successful. There are so many permutations available in this world that it's always possible to come up with a unique vision.\n\nFor Pletka, inspiration comes from many trips across the Western United States, visiting museums, national parks and wildlife refuges, reading books and attending cultural events.\n\n**Paul Pletka**\n\n_Tears of the Lord_\n\nAcrylic on linen canvas\n\n96\" \u00d7 72\" (244cm \u00d7 183cm)\n\n\u00a9 copyright Paul Pletka, 2005\n\nCourtesy of the Museum of the American\n\nWest, Autry National Center, Los Angeles\n\nVARIATIONS ON EPIC NARRATIVE\n\n**Paul Pletka**\n\n_The Revelation_\n\nAcrylic on linen canvas\n\n44\" \u00d7 66\" (112cm \u00d7 168cm)\n\n\u00a9 copyright Paul Pletka, 2008\n\nPrivate collection\n\n**Variation 1: Action and Narrative**  \nA wide variety of strong colors and distorted proportions adds visual tension and movement to emphasize action and narrative.\n\n**Daniel Barkley**\n\n_Far Shore_\n\nAcrylic on canvas\n\n52\" \u00d7 114\" (132cm \u00d7 290cm)\n\n**Variation 2: Emphasize Mood**  \nA limited palette with muted tones creates mystery and mood. The simplified and elegant background keeps the focus on the figures and narrative.\n\n**More Variations**\n\n\u2022 Use your own personal stories to paint scenes depicting memorable moments. Take liberties and change, rearrange and re-create to paint your ideal version. Paint yourself into new scenes you desire or fear. Using childhood photographs, replace figures or backgrounds with new ones. Change color palettes.\n\n\u2022 Experiment with arrangements of multiple figures. Cut out figures, using a photocopier to reduce and enlarge for a variety of sizes. Place and arrange on various backgrounds from photographs, drawings or other paintings. Backgrounds can be as simple as a color field and as complex in detail as a photographic landscape.\n\n PREPARING A TONAL BASE\n\nEpic narrative paintings combine multiple figures in a landscape, coordinating many forms in one image. These compositions rely heavily on tonal areas (shapes of light and dark) to create movement and focus.\n\nStarting the painting with a tonal drawing gives you a jumpstart to the composition process. This technique transforms a drawing into tonal shapes, then seals and prepares it for subsequent paint layers.\n\n**Materials**\n\n**\u2022 Drawing Materials:** A variety of water-soluble and waterproof drawing materials (i.e., charcoal, Cont\u00e9, graphite, water-soluble pencils), smudge stick, chamois, erasers\n\n**\u2022 Surface:** Any primed surface\n\n**\u2022 Painting Tools:** Painting knife with stepped handle, paintbrush\n\n**\u2022 Other:** Acrylic polymer medium (matte), water\n\n**1. Create a Line Drawing**  \nMake a drawing outlining the general areas to be painted, using a variety of drawing materials, both water-soluble and waterproof. Make sure to have at least one water-soluble item. Test it first on a scrap surface to see if the drawn line will blend when brushed over with water. Experiment by using a variety of line qualities; change your grip, vary the angle onto the surface, vary pressure applied, and smear lines using a smudge stick, eraser or chamois.\n\n**2. Create Tonal Areas Using Water and Medium**  \nDip a brush in water and\/or acrylic medium. Apply the brush over the drawn lines, softly moving tones out along the surface to create softer edges and tonal areas. When applied over waterproof materials, such as graphite pencil, the line will not create tones. By using both waterproof and water-soluble materials in your drawing, you get more variations with this step. If you need to get some white areas back, selectively overpaint with gesso. In addition, experiment to see the differences using medium versus water. This step alone may suffice for sealing. If some lines, however, did not get painted with water or medium and are still water-soluble, continue to step 3 to seal the drawing completely.\n\n**3. Add a Waterproof Coating**  \nLiberally pour an acrylic medium over the entire surface. If working on a large area, do this step in smaller sections. Using a painting knife with a stepped handle, gently spread an even coat of the acrylic medium over the drawing. Avoid scraping the surface by raising the knife about 1\/8-inch (3mm) from the surface with the acrylic rolling out from under it. Keep the knife at a slight angle so it isn't parallel to the surface. If the knife keeps scraping the surface, apply more acrylic medium.\n\nPHOTOREALISM\n\nPhotorealism relies on sharp detail, with super-real images incorporating photographic characteristics. Daniel Smith, photorealist wildlife painter, considers it a compliment when someone comments that his work is as good as a photo. Yet Smith goes even further. Making his paintings believably realistic while still able to stand alone as good works of art takes daring and creativity beyond copying a photograph. Too much emphasis on the story, however, can feel contrived, like an illustration. Even with his intent for portraying reality truthfully, Smith will stretch things a bit to enhance aesthetics when needed. Photographic images contain inherent limitations from the camera's depth of field, including distortion, compressed images and mixed focal ranges\u2014blurring certain areas while leaving others crisp.\n\nIn _Zero Tolerance_ , Smith transforms the work from photographic generality by adding his own compositional design, ideas and imagery, along with simplifying and editing detail. Here the image is enhanced by the addition of foreground dust, which reduces background detail and places the focus on the animals and action. The lionesses were photographed on one trip, while the elephant was from another. In addition to using multiple references, Smith researches animal behavior to get correct wildlife appearances and habitats. Smith has always been intrigued by wildlife, and has been painting this imagery since childhood.\n\n**The Artist's Process**\n\nSmith works on masonite boards, and rolls out his gesso primer to add a pebbly texture (good for rock textures). Drawing and then underpainting gives Smith his jumpstart, whereupon many thin layers of paint are applied, along with drybrushing techniques. Keeping brushstrokes and texture to a minimum, the final surface is smooth and flat, reducing distraction from the exacting detail.\n\n **TIPS  \nfrom the Artist **\n\nThere's a difference between technical skill and turning it into a true work of art. Composition, a strong focal point, editing, balance, aesthetics and the right amount of negative space are all essential. There's a lifetime of experience that goes into it.\n\nSmith lives near Yellowstone National Park where moose, elk, bear and fox are frequently visible. He also travels to places like Alaska, Africa, Glacier National Park and the Canadian Rockies to sketch and photograph. Firsthand experience is key for Smith, adding adventurous stories and personal relationships with the animals in his paintings.\n\n**Other Art in This Style**\n\n\u2022 Wildlife painters Bob Kuhn, Robert Bate-man and Carl Rungius\n\n\u2022 Urban landscape painter Max Ferguson\n\n\u2022 Photorealist Richard Estes\n\n\u2022 The Dutch Masters\n\n**Daniel Smith**\n\n_Zero Tolerance_\n\nAcrylic on Masonite\n\n55\" \u00d7 42\" (140cm \u00d7 107cm)\n\nVARIATIONS ON PHOTOREALISM\n\n**Tom Palmore**\n\n_Mugsy the Jungle Boy_\n\nAcrylic and oil on canvas\n\n46\" \u00d7 72\" (117cm \u00d7 183cm)\n\n**Variation 1: Unexpected Juxtapositions**  \nUnexpected juxtapositions add humor, making this realism more personal. A trademark for Palmore, stylistic plays between background and object creates surprises. Palmore's tip: Develop your own techniques so you don't paint like someone else.\n\n**Jason de Graaf**\n\n_Vesalius Skeleton_\n\nAcrylic on canvas\n\n24\" \u00d7 36\" (61cm \u00d7 91cm)\n\n**Variation 2: The Real and the Surreal**  \nClose-cropping, a hint of narrative, and an intent to make paintings more intriguing than photographs all add dramatic impact and a sense of the surreal. De Graaf consciously tries to paint things that haven't been done before.\n\n**More Variations**\n\n\u2022 Our best work has roots in our everyday lives and past experiences. Spend time photographing your home, personal spaces and favorite places, friends and activities you like. Experiment by cutting and reassembling your photos to create new combinations.\n\n\u2022 In a corner of a room, create your own stage set using fabric and add friends willing to pose with costumes and objects. Photograph this setup using different lighting and use it for a painting.\n\n  TRANSFERRING DIGITAL IMAGERY\n\nPhotorealism demands exact painting techniques using extreme detail, perfect proportions and scale. Many realist painters enjoy the challenge of painting with minimal references and mostly imagination. Other painters take advantage of photographic processes in their paintings. This technique prints a color image directly onto an acrylic skin for use as an underpainting or collage element.\n\n**Tip**\n\nInstead of using gloss acrylic gel for a skin, try other acrylic products such as colored paint, matte gels or pastes to get an opaque or translucent skin. Consider using unusual papers such as aluminium foil.\n\n**Materials**\n\n\u2022 **Acrylic Products:** Any clear glossy acrylic gel, mineral spirits-based acrylic spray fixative\n\n\u2022 **Surface:** A nonstick surface that will release acrylic (freezer paper\u2014not wax paper, HDPE plastic, garbage bags or cheap plastic wrap\u2014test first)\n\n\u2022 **Painting Tools:** A squeegee tool (piece of cardboard or plasterer knife), sponge applicator, masking tape, paper\n\n\u2022 **Other:** Clear digital coating product or ground for inkjet printing (i.e., inkAID or Golden's Digital Ground for Non-Porous Surfaces), ink-jet printer (with a flat feed, i.e., paper should enter in a different place than it exits)\n\n**1. Make an Acrylic Skin**  \nOn a removable surface where acrylic will not stick, squeeze out a generous amount of gloss acrylic gel. Using a squeegee tool, spread the gel in an even layer about \u00bc-inch (6mm) thick. (Acrylic will shrink by 30 percent in volume. A thicker application will keep the dried skin from tearing.) Let this dry until the gel turns clear.\n\n**2. Coat and Prepare the Skin for Printing**  \nWhile the skin is still on its surface, use a sponge applicator to apply a thin layer of digital ground. Let it dry for about an hour, or quick dry for a minute with a blow-dryer. Peel the skin off the surface and cut the skin to a size smaller than a sheet of regular-sized paper. Leaving about \u00bd-inch (12mm) or more as a bottom margin, secure the skin onto a piece of paper with masking tape, along the bottom and on only one side where the printer grips the paper.\n\n**3. Print the Image Onto the Prepared Skin**  \nSelect an image (here, I'm using a photograph taken by Bruce Cody). If using an all-in-one printer, then place the image in the scanner. Otherwise, send the image through a computer, or use an attached scanner. Set the printer preferences for high quality and to print on gloss. Place the paper with taped skin into the printer's paper feed and print.\n\n**4. Secure With Fixative**  \nHere the image is printed onto the clear glossy skin. Fix the image by using a mineral spirits-based acrylic spray fixative. If desired, you can paint on the back of the skin to change transparent areas to opaque, and\/or paint a background. Glue the skin onto a painting surface using gloss acrylic gel.\n\nPORTRAIT\n\nPortraiture has been a popular style for many artists and audiences throughout the ages. For portrait painter Lea Bradovich, continuity between humanity and nature is a key concept, visible in her portrayals using fashion and headgear to create fanciful portraits. Inspired while looking at a photograph of a giant bee head, Bradovich integrated botany with Renaissance style portraiture in the painting shown opposite, adding irony, freshness and an element of surprise. Note the honeycombed collar, bee-head hat, and mouth located where a bee's mandibles would be in _Queen Bee's Regalia II_ . According to Bradovich, \"We humans seem to fashion everything we find into headgear. And headgear is symbolic.\"\n\nPainted in the style of Italian Master Bronzino (Agnolo de Cosimo), _Queen Bee's Regalia II_ follows his traditional, refined and aristocratic flavor. A \"queen bee\" in human society would be cool, aloof and alluring, and here, appropriately paired with Bronzino's painting style, it creates an interesting dialogue between old and new.\n\n**The Artist's Process**\n\nIf an idea amuses Bradovich and makes her smile, she will investigate it further, even if some ideas seem goofy or silly at first. Bradovich starts by collecting reference materials including historic costumes, old masters portraits, fashion magazines and science photographs. These are maintained in boxes, files and on her computer so that she can leaf through for ideas, allowing for cross-fertilization.\n\nInitial drawings give Bradovich a jumpstart, continuing by painting on panel with acrylic gouache, leaving room for accidental things to happen. On occasion she will work from models. Serendipity keeps the work changing, thus avoiding a paint-by-number appearance. Using washes, dry brush, and any technique that works for her at the time, she sometimes builds up areas with small overlapping brushstrokes.\n\n **TIPS  \nfrom the Artist **\n\nWhile mixing paint simultaneously consider the brush strokes you will be painting with that color. This creates a mindfulness and preparatory gestural memory.\n\nDramatic studio lighting plays off multiple portraits in Lea Bradovich's studio.\n\n**Other Art in This Style**\n\n\u2022 Renaissance masters Agnolo Bronzino and his teacher Jacopo da Pontormo\n\n\u2022 Portrait photographers Annie Leibovitz, Dorothea Lange and Cindy Sherman\n\n\u2022 Psychological aspects and technical mastery of contemporary figurative realists Michael Grimaldi, Lucian Freud, Eric Fischl, Philip Pearlstein, Avigdor Arikha, John Currin, Alice Neel, K\u00e4the Kollwitz, Chuck Close, Gerhard Richter and Bernardo Torrens\n\n\u2022 Photographic portraits by Nan Goldin, Cindy Sherman and William Wegman\n\n\u2022 Lifelike sculptures of Duane Hanson.\n\n**Lea Bradovich**\n\n_Queen Bee's Regalia II_\n\nAcrylic gouache on panel\n\n24\" \u00d7 18\" (61cm \u00d7 46cm)\n\nCollection of Renee Goshin\n\nVARIATIONS ON THE PORTRAIT\n\n**More Variations**\n\n\u2022 Compare painting a portrait from life versus photographs by getting a friend or model to pose. Paint them from life in several sittings, then photograph them to work from the image as reference.\n\n\u2022 Use your most available model\u2014yourself\u2014and paint a self-portrait using a mirror, photographs or memory. Paint self-portraits at intervals in your life to create a series.\n\n\u2022 Research and interview to find out about the person you are painting. What do you like about this person? Translate these personality traits into the work through unusual cropping, varying color palettes, placement in unusual backgrounds, and adding costume and prop elements.\n\n\u2022 Stage your own settings using costumes and props like Cindy Sherman's self-portrait series.\n\n**Daniel Smith**\n\n_Colors of Kenya\u2014_\n\n_Warrior_\n\nAcrylic on panel\n\n14\" \u00d7 28\" (36cm \u00d7 71cm)\n\n**Variation 1: Add Personality With Clothing and Objects**  \nCostume and personal objects add a hint of narrative, personality and culture.\n\n**Daniel Barkley**\n\n_Petit nuage a l'horizon_\n\nAcrylic on canvas\n\n54\" \u00d7 76\" (137cm \u00d7 193cm)\n\n**Variation 2: Enhance Mood**  \nA soft, muted limited palette, simplified background and unusual elongated format adds mood and a contemporary feel to this portrait.\n\n FIXING A LINEAR UNDERDRAWING\n\nPreparing a preliminary detailed line drawing offers a jumpstart to any painting, especially when it's drawn directly on the painting surface. Starting with the flexibility of charcoal, where lines can be easily erased and changed, this technique changes the charcoal drawing into waterproof graphite, making it smudgeproof for subsequent overpainting.\n\n**Tip**\n\nIf your drawing surface is too slick and the charcoal will not easily adhere, apply a thin amount of matte medium onto the surface, let dry, then continue drawing.\n\n**1. Create a Charcoal Drawing**  \nA semi-slick surface is best, particularly a surface primed with acrylic gesso. Draw onto the primed surface with soft vine charcoal. Erase and rework as necessary, using your fingers, eraser, chamois, smudge sticks and rags. Keep working the drawing until it is exactly what you want. Here is a finished portrait underdrawing using vine charcoal.\n\n**Materials**\n\n**\u2022 Drawing Materials:** Vine charcoal, soft graphite pencil, chamois or soft rag **\u2022 Surface:** A primed surface\n\n**2. Retrace With Graphite**  \nUsing a graphite pencil, retrace the important lines with medium pressure directly over the vine charcoal lines.\n\n**3. Wipe off the Charcoal**  \nWipe away the vine charcoal using a chamois or a rag to reveal the graphite lines underneath. Graphite will not be affected by any subsequent overpainting with acrylic, producing an underdrawing ready for painting.\n\nSTILL LIFE\n\nStill life depicts subjects such as flowers, shells, inanimate objects, fruit and skulls\u2014just about any object with the exception of people and animals. This style generally involves a grouping of objects, set off by a closely distanced background. Still life was popularized in the seventeenth century, and still holds fascination for us by implying humanity's symbolic presence, adding perspective on culture, lifestyles and attitudes of the times.\n\n**The Artist's Process**\n\nTraditional still life offers Sherry Loehr the opportunity to evoke a quiet and contemplative mood. For her, simplicity is essential. She chooses all of one type of fruit, for example, or clusters several items together to create one large shape. Loehr stages the objects in a classic still life format, then photographs them, leaving background areas open for interpretation. She uses photographs only to get an initial compositional sketch, then lets go of this reference to allow plenty of room for imagination. \"I can't think of anything more boring than copying a photo because you know what it will be like and the best it can be is as good as the photo,\" states Loehr.\n\nLoehr adds paint layers, continually working and reworking areas to expose varying underlayers. She loosely paints background areas using spontaneous patterns and textural areas in quiet tones. Her simplified backgrounds contrast with tightly detailed objects, keeping Loehr's work fresh and contemporary. Loehr was inspired early on by instructor Katherine Chang Liu (featured). Liu, an abstract painter, encouraged Loehr to find her own voice.\n\n **TIPS  \nfrom the Artist **\n\nLook beyond your normal vision for inspiration and explore a wide variety of work and resources that are not necessarily similar to your own style. Go beyond art museums and art books, and look at science museums, fashion, commercials, your house and local environment.\n\n**Loehr At Work in Her Studio**  \nLoehr's \"love-hate\" relationship with acrylic comes from the struggle necessary to achieve controlled edges and blending, while enjoying freedom and playfulness in the loosely painted areas.\n\n**Other Art in This Style**\n\n\u2022 Seventeenth century painters from the Netherlands, especially Dutch flower painting and trompe l'oeil\n\n\u2022 American modernists such as Raphaelle Peale and his group of early American still life artists\n\n\u2022 American trompe l'oeil painters John Haberle, William Michael Harnett and John Frederick Peto\n\n\u2022 Moody works of masters Francisco Goya, Gustave Courbet and Eug\u00e8ne Delacroix\n\n\u2022 Contemporary artist Janet Fish\n\n**Sherry Loehr**\n\n_Tulips and Oranges_\n\nAcrylic on board\n\n24\" \u00d7 36\" (61cm \u00d7 91cm)\n\nVARIATIONS ON STILL LIFE\n\n**Sherry Loehr**\n\n_Koi Chi_\n\nAcrylic on board\n\n36\" \u00d7 24\" (91cm \u00d7 61cm)\n\n**Variation 1: Using Living Subject Matter**  \nInstead of using the traditional inanimate objects for a still life, Loehr paints live swimming fish. Visible here is Loehr's trademark style contrasting realistically detailed forms with looser experimental backgrounds.\n\n**More Variations**\n\n\u2022 Change your painting formula. Make a list of all the habits and processes you use. Then make another list that contains their opposites. Break up your routine by trying one of the opposites. For instance, if you always start with a drawing or undersketch, switch to starting spontaneously with paint. If you always work from photographic references, try working without them. Vary your usual subject matter. Paint on different surfaces. Use a new medium.\n\n\u2022 Change the balance of elements between the background and foreground. Enlarge objects while minimizing the background, or expand the background and minimize the objects.\n\n**Mary L. Parkes**\n\n_Angelic Apples_\n\nAcrylic and oil on linen\n\n30\" \u00d7 40\" (76cm \u00d7 102cm)\n\n**Variation 2: Add Decorative Flourishes**  \nThe addition of decorative flourishes and geometric patterns turns this still life into a delightful fantasy.\n\n MIXING CLEAN BRIGHT REDS\n\nLoehr loves red; it plays a key role in her color palette and composition. Loehr enjoys employing its full range from a cool magenta-purple red to a warm orangey red. Here are two ways to keep reds looking their brightest with acrylic paint.\n\n**Overlay a Glaze**  \nSqueeze a warm bright red such as Cadmium Red and a cool bright red like Quinacridone Magenta onto your palette. Paint out an area of red by using either the Cadmium Red straight out of the tube or mix a small amount of white with Quinacridone Magenta (use about 5 percent white if you're using an opaque white like Titanium White. With a transparent white like Zinc White, use 20 percent). This will bring out the color intensity of the modern color. Do not add white to the mineral color Cadmium Red or it will lose its intensity and get chalky. Overpaint the red with a thin glaze made from a 1:1 mixture of untinted Quinacridone Magenta and gloss acrylic medium. Apply thinly using a soft flat brush or rag.\n\n**Pair the Red With a Complement**  \nTo make the red appear even brighter, paint a contrasting color next to it, such as a muted red, a green (which is red's complement) or a dark color. A muted red can be mixed using red, a touch of black (or green) and some white. Here a muted green is applied in varying values next to red to make it appear brighter.\n\n**Tip**\n\nAvoid buying paint from manufacturers that add white to their modern pigmented colors like Quinacridones and Phthalos. Start with an unadulterated paint (one that comes out dark from the tube) to get good color mixing control.\n\nThe ideas above can be performed with the other two primaries, yellow and blue. For yellow, use Hansa Yellow Medium, Cadmium Yellow Medium or Hansa Yellow Light. For blue, use Ultramarine Blue or Phthalo Blue (Green Shade). Phthalo Blue is the only color in this grouping of yellows and blues that would intensify with white. For the other colors, use them straight out of the tube for the brightest effect.\n\nLANDSCAPE\n\nLandscape painting depicts natural scenery such as mountains, trees and rivers, and still holds a powerful influence and inspiration for artists and viewers.\n\nDennis Culver, living in the southwestern United States, enjoys the local vistas, especially midday, when colors are quiet with no shadows. Not wanting his paintings to merely re-create nature, Culver always starts with a vision from his imagination. By painting directly outdoors and avoiding any photographic references, he distances himself from photographic reality. In the painting shown on Dennis Culver, Culver has added subtle geometry infused with triangles and native symbols, most visible in the foreground rocks, encouraging the viewer to move from scenery toward ideas.\n\n**The Artist's Process**\n\nIn _Arroyo Logic_ , Culver started with several earth tones on a toothbrush, spritzing a variety of dots along the bottom half of the painting. He then drew lines connecting the dots to create irregular geometric patterns which formed the initial inspiration and unusual composition in the foreground. Much of Culver's starting processes come from playing with materials. This play keeps his work spontaneous and varied. He prefers acrylic for its nontoxic, fast drying qualities, and vast potential in attaining varied sheens and surface treatments. Additionally, he enjoys the freedom of layering acrylic with no rules, as opposed to oil with its \"fat over lean\" restrictions.\n\n **TIPS  \nfrom the Artist **\n\nFamiliarize yourself with all the traditional mediums from watercolor to egg tempera. Experiment. Don't leave out acrylic because it's less traditional\u2014you'll be missing a whole lot of fun and surprising results.\n\n**Dennis Culver at Work in His Studio**  \nCulver's work isn't about being realistic, and always contains something surprising.\n\n**Other Art in This Style**\n\n\u2022 Minimized detail and grand panoramas of imaginary landscapes in early Chinese landscape paintings\n\n\u2022 Large scale and vast epic views of Frederic Edwin Church, Albert Bierstadt and other Hudson River School artists\n\n\u2022 Idealized landscapes of the Italian Renaissance painters like Titian\n\n**Dennis Culver**\n\n_Arroyo Logic_\n\nAcrylic on canvas\n\n56\" \u00d7 62\" (142cm \u00d7 157cm)\n\nVARIATIONS ON THE LANDSCAPE\n\n**Bruce Cody**\n\n_Capitol City Sunlight_\n\nAcrylic on canvas\n\n16\" \u00d7 30\" (41cm \u00d7 76cm)\n\n**Variation 1: Urban Landscapes**  \nFor Bruce Cody, art is about people and architecture, and can be seen as a social symbol. Cody uses inanimate elements such as buildings, cars, signage and telephone transmission poles to transform the objects into layers of space, atmosphere and time of day.\n\n**Phil Garrett**\n\n_Jones Gap\/Red Rocks_\n\nAcrylic on linen\n\n42\" \u00d7 60\" (102cm \u00d7 152cm)\n\nCollection of Palmetto Bank,\n\nGreenville, SC\n\n**Variation 2: Close Cropping**  \nHere Garrett's use of close framing strengthens the composition, creating an intimate and unusual view.\n\n**More Variations**\n\n\u2022 Use multiple references to create a new scene. One photograph can be used for its color palette, another for composition, and other photographs that contain natural forms such as trees, clouds, animals, etc. for added elements. Combine a sky from one photograph with ground from another.\n\n\u2022 Paint the same landscape three ways: Outdoors, from a photograph and from memory, then compare the results. Paint the same scene several ways in varying weather, or light during different times of day (see Claude Monet's Haystacks paintings).\n\n\u2022 Paint a landscape using unexpected colors, such as green for the sky and blue for the ground.\n\n  THE INVISIBLE GRID\n\nSmaller images or sketches (often called maquettes) make great starts for large paintings. This is especially helpful for landscapes. When traveling outdoors, it's easy and convenient to create many quick, small, on-the-spot pieces. Gridding provides a quick way to enlarge these later in the studio. Traditional grids using charcoal or graphite lines are difficult to remove later. This gridding technique, however, leaves behind no marks.\n\n**Tip**\n\nIf your painting surface has no sides, place push pins close along the edges. For large-scale works, use an additional color for main gridding lines to help navigate while transferring.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paints\n\n**\u2022 Surface:** Any support (optimally with sides)\n\n**\u2022 Painting Tools:** A brush\n\n**\u2022 Other:** A small image you want to transfer and enlarge, cotton twine, pushpins, hammer, pencil, ruler, two waterproof markers differing in color, mylar or other clear plastic, scissors, tape, a small piece of cardboard, water\n\n**1. Prepare the Maquette**  \nBegin with a small image you want to transfer to a larger scale. Protect it by taping it onto a piece of cardboard and covering it with a taped piece of clear mylar or plastic.\n\n**2. Grid the Image to be Transferred**  \nUsing a waterproof marker, draw onto the plastic by tracing only essential lines and shapes. Keep detail to a minimum, focusing on large compositional lines (pictured in red). Change marker color for the grid lines (here using black), and create a checkerboard grid. Slip white paper between the plastic and the original drawing to isolate the lines and minimize unnecessary detail.\n\n**3. Grid the Painting Surface With String**  \nUsing a painting surface with sides, hammer pushpins at all points along the sides angled slightly upward. Take one end of a ball of string (the same color as the grid lines) and knot it onto one of the pushpins closest to a corner. Wind the string along the pushpins, creating all the vertical and horizontal lines, winding a bit around each pushpin as you go. Secure the string to the last pushpin with a knot. The string will be slightly raised from the surface if pushpins are angled correctly. Add water to a light colored paint and brush apply to re-create the same compositional lines from the maquette onto the painting surface. Focus on one grid square at a time. Allow the brush to go under the string, continuing lines that travel between grid squares. Instead of erasing to correct a wrong line, try changing color.\n\n**4. Remove Pins and String**  \nWhen all the compositional lines are painted, pull out the pushpins along with the string grid. The end result is a transferred image with no visible grid remaining.\n**Section 2**\n\nABSTRACTING  \nTHE WINDOW\n\n**Tom Palmore**\n\n_Phil With His Favorite Chicken_\n\nAcrylic on canvas\n\n72\" \u00d7 60\" (183cm \u00d7 152cm)\n\nAn abstract painting engages the viewer in a different way than realism. Abstraction minimizes detail and recognizable imagery, moving the emphasis toward aesthetic elements such as line, shape, perspective, form, texture and space. This section explores styles that still use some realistic principles such as approaching the canvas like a viewing \"window,\" and maintaining a sense of background, foreground and fixed perspective. Imagery here combines both realistic and abstract elements directing the eye toward more subtle aesthetics, often resulting in an unexpected twist.\n\nFIGURE AS FORM\n\nAs humans, we have a natural and immediate response to body imagery in art, and its use can ground a work in space and create a connection with viewers.\n\nFigures have always been a focus in Jylian Gustlin's work. She creates characters and sets them in moody and unfamiliar landscapes. By reducing detail in the figure and facial features, Gustlin also obscures the figure's gender and race. In this way, she hopes to move the focus of her work away from particular individuals and toward the idea of being human. Most communication, Gustlin muses, comes from body language or gesture, which carry the meaning of her work.\n\n**The Artist's Process**\n\nGustlin's fascination with and study of science and math, especially theories on the Fibonacci sequence, are visible in the way she divides her backgrounds and proportions the body. Metaphysical ideas, including stillness in motion, also play a key role in her work.\n\nGustlin starts with a wood panel, gesso and many layers of plaster and creates an etched relief using chisels. She continues to build multiple layers with a variety of materials: oil, acrylic, charcoal, wax, gold leaf, pastel and graphite. In the painting process, she makes as many mistakes as she can for as long as she can until the painting is finished. She eventually adds a final thin coat of resin to intensify the colors and emphasize texture. A creative block is rare for her, as she leaves her process open-ended, never deciding where she's going\u2014and, states Gust-lin, \"You can't get lost if you have no destination.\" When frustrated, she likes to load a brush with paint and throw it at a surface until something comes up. Inspirational artists for Gustlin include Nathan Oliveira, Richard Diebenkorn and the brooding, haunting qualities of Odd Nerdrum's portraits.\n\n **TIPS  \nfrom the Artist **\n\nMake art as much as possible to find your voice. Play as much as possible; don't be too serious. Combine everything you learn like a soup\u2014play, relax and paint. Mistakes are a way to bring the image into focus and to find your way to the fin-ish. If it doesn't work, it's not finished.\n\nAn expert in computer science, Gustlin finds parallel painting techniques to those found in Photoshop's layering tool. She even created her own computerized software which takes her scanned images of drawings, textures and photos and randomly combines them to make her abundant reference material.\n\n**Other Art in This Style**\n\nInventive combinations of figure and abstraction, including the Bay Area Figurative Movement, Francis Bacon, Aristide Maillol, Willem de Kooning's Woman series, Philip Guston and Jean Dubuffet.\n\n**Jylian Gustlin**\n\n_Bivium 18_\n\nAcrylic and mixed media (plaster, oil stick, pencil, resin) on panel\n\n48\" \u00d7 48\" (122cm \u00d7 122cm)\n\nCorporate collection\n\nVARIATIONS OF THE FIGURE AS FORM\n\n**Joey Fauerso**\n\n_Mouth to Mouth (1)_\n\nWatercolor and acrylic on paper\n\n48\" \u00d7 46\" (122cm \u00d7 117cm)\n\nPrivate collection\n\n**Variation 1: Isolation**  \nEmphasize or isolate parts of the body. Here an openmouthed portrait echoes the open mouth shape silhouetting or framing it.\n\n**More Variations**\n\n\u2022 Draw or photograph figures in various positions. Cut them out and place them on a variety of backgrounds\u2014solid or multicolored, smooth or textural, patterned or simple, abstract or realistic. Keep playing until ideas emerge that you like.\n\n\u2022 Trace outlines of figures. Working only with these generalized lines, change, expand, eliminate and add lines to transform the figure into something else.\n\n**Dennis Culver**\n\n_Politician_\n\nCharcoal and acrylic on paper\n\n44\" \u00d7 30\" (112cm \u00d7 76cm)\n\n**Variation 2: Transformation**  \nAllow figural shapes to transform and transmute. Starting with a charcoal drawing of textures, and combining with acrylic gesso, this playful investigation of materials formed into a central figure while creating a range of grays.\n\n  WORKING WITH MULTIPLE TEXTURES\n\nGustlin's work features seductive surfaces employing a variety of textures. Her technique uses multiple layers of plaster, chiseled to create etched relief lines and contrasting texture. Here is a variation using acrylic molding paste.\n\n**Materials**\n\n**\u2022 Surface:** A rigid and sturdy primed painting surface such as gessoed wood or panel\n\n**\u2022 Painting Tools:** A large plaster knife or other applicator; an array of texture tools such as combs, rakes, sticks and fingers\n\n**\u2022 Other:** Acrylic molding paste\n\n**1. Etch an Outline Into Wet Paste**  \nUsing a primed rigid support, generously apply acrylic molding paste all over the panel using a palette knife or wide plaster knife and smooth it as best as you can. Keep the layer about \u00bc-inch (6mm) thick or more. Using a tool that will easily create a line, such as a stick or the tip of the painting knife, draw the outline of a central form into the wet paste.\n\n**2. Create Varying Levels**  \nThe painting now has two distinct areas created from the etched lines in the previous step: inside the form and its background. Select one of the areas to be raised up higher than the other. Apply another layer of paste in that area in any level you prefer. The varying heights help to emphasize form and background. Continue to the next step while the paste is still wet.\n\n**3. Contrast Textures**  \nSelect one of the areas to add a texture. If the paste has started to dry in that area, reapply a fresh layer of paste. Apply combs and other tools to the wet paste, scrape and draw to get a variety of lines and patterns. Allowing one area untouched to remain smooth contrasts nicely with the textural area just created. Alternatively, in the smooth area, create a different texture by adding more paste, using different tools, or embedding beads or other small objects. Allow the surface to fully dry before applying paint.\n\n**Tip**\n\nTry adding color to the paste. Use different colored layers, sanding back to reveal the various colors. Sanding is also a good way to reduce texture. If sanding, always use waterproof sandpaper and water to eliminate exposure to toxic dust.\n\nREDUCTIVE\n\nOur eyes absorb an infinite amount of detail as we perceive reality. A reductive style reveals what the artist finds essential. Distillating unnecessary detail places the focus on bold shapes and the artist's personal vision.\n\nFrank Webster portrays postindustrial landscapes\u2014 the modern city\u2014combining minimalism and realism and revealing the connection between nature and technology. His editing process, essential to a reductive style, is driven by intuition and taste used for clarity and impact. Webster succeeds in carrying a hint of narrative with a clean elegance and a feel of minimal essentials.\n\n**The Artist's Process**\n\nInspiration comes from reading science fiction books, including the dystopian works of J.G. Ballard, with a great sense \"of the not too distant future.\" Trips to shoot reference photos, architectural research, even finding great compositions in films with epic cinematography, all add to his collective resources. Webster sketches and works digitally to get to the essence of the image. Multiple glazing layers add intensity to his simplified areas. Some of Webster's favorite artists include Tom Morandi, Dan Graham, Robert Smithson and photographer Robert Adams.\n\n**Other Art in This Style**\n\n\u2022 Reductive process: Compare Piet Mondrian's well-known grid abstractions with his early landscapes.\n\n\u2022 Reductive urban and industrial: Charles Sheeler and Ralston Crawford.\n\n\u2022 Contemporary and modern artists: Ed Ruscha, Andy Warhol, Roy Lichtenstein, Philip Guston, Jacob Lawrence, John Marin, Wolf Kahn, David True and Milton Avery\n\n **TIPS  \nfrom the Artist **\n\nAn editing process is idiosyncratic.\n\nWebster noticed postindustrial changes growing up in the midwestern United States. Charles Sheeler and his modernist contemporaries painted romanticized, optimistic versions of modernity and technology. Now, with hindsight and contemporary eyes, Webster adds a new outlook on the subject, with an intention toward subtlety.\n\n**Frank Webster**\n\n_High Rise_\n\nAcrylic on canvas\n\n86\" \u00d7 65\" (218cm \u00d7 165cm)\n\nVARIATIONS ON REDUCTIVE IMAGERY\n\n**More Variations**\n\n\u2022 Take photographs of places, people or events you like. Paint over areas with opaque paint to eliminate detail. See how much you can eliminate and still portray what you think is important.\n\n\u2022 Transform color images to black and white using photography and\/or computers to see them differently.\n\n\u2022 Play with colored cutout shapes, arranging and collaging onto larger colored paper, as an exercise in working with bold areas and no detail.\n\n**James Strom-botne**\n\n_Escalator Girl_\n\nAcrylic on canvas\n\n48\" \u00d7 60\" (122cm \u00d7 152cm)\n\nCourtesy of Handsel\n\nGallery, Santa Fe, NM\n\n**Variation 1: Contrast Flat Space With Perspective**  \nSimplified forms combine with linear perspective to create an interesting visual tension between flat and deep space.\n\n**Frank Webster**\n\n_Plastic Bags_\n\nAcrylic on canvas\n\n60\" \u00d7 80\" (152cm \u00d7 203cm)\n\n**Variation 2: Seek a Different Point of View**  \nSingling out an atypical view creates an immediate attraction in this piece.\n\n  ELEGANT CURVED EDGES\n\nA reductive style often makes use of simplified lines and edges. Dave Yust, whose work is shown, uses this method to get refined hard edges with curved designs.\n\nFor this technique, use masking tape that's \u00bc-inch (6mm) because it curves easily with minimal crimping.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint colors\n\n**\u2022 Surface:** Any primed painting surface\n\n**\u2022 Painting Tools:** Any soft painting brush\n\n**\u2022 Other:** \u00bc-inch (6mm) masking tape, any clear or non-colored acrylic medium or gel (glossy or matte), optional hair blow-dryer to quicken drying time\n\n**1. Tape the Area**  \nUsing a surface that is primed and\/or pre-painted with a background, plan where you wish to have hard edge painted lines or forms. Apply \u00bc-inch (6mm)-thick masking tape in a design or bordering a shape. Pull the tape firmly with one hand while pressing it into place with the other to keep the tape lying as smooth and flat as possible. Press along the edges to secure. Here a pre-painted background using a gradation of green and pink will remain visible in the final outcome only where tape is applied.\n\n**2. Seal the Tape Edge**  \nReduce paint seepage under the taped edges by applying a clear non-colored acrylic medium or gel with a brush along the edge of the tape. Quickly dry with a hair blow-dryer for a minute and continue to the next step.\n\n**3. Apply Paint Color**  \nApply paint color to either or both sides of the tape depending on your preferences, feeling free to paint over the tape when needed. Immediately remove the tape after painting. If the tape is left on the surface while the paint dries for a prolonged period, it may be difficult to remove later without pulling up the paint along with it. To reduce problems with tape removal, roll or peel it back onto itself rather than pulling upward.\n\n**Finished Example**\n\nFLAT SPACE\n\nSince a painting surface is naturally flat, one challenge for artists is to construct the illusion of space. In the twentieth century, a new trend emerged, rejecting the deep illusionistic space from the Renaissance. Instead, the emphasis was on flat space. The challenge changed to maintaining the integrity of a painting's image when flat to avoid looking like wallpaper. Detail and complexity still inform the work, but emphasis is placed on the painted surface and perspective is minimized. A master of this style, James Strombotne's sophisticated works employ deceivingly simplified areas and forms, clearly visible in his painting _Circus Elephant_ shown.\n\n**The Artist's Process**\n\nStrombotne makes it a point to draw every day, and believes this is an essential tool to keep his work fresh, while paint quality and personal experience are also key. Strombotne says, \"The craft of painting what you see is as rich and compelling as the use of the paint.\" Painting is more than just an illustration telling a story. Using a minimal palette helps Strombotne add simplicity with the appearance of effortlessness. He doesn't want his pieces to look like a lot of work, but instead to carry a sense of freedom and naturalness. Too reductive is boring, so the challenge for him is letting it be reductive but still compelling. After three to six layers of paint, he draws over the layers with charcoal, then paints over the drawing, sometimes covering the lines up entirely. An acrylic painter for over thirty years, he favors this medium over oil, because it maintains its luminosity and the feeling of naturalness.\n\nHe finds Georgia O'Keeffe's watercolors fresh, interesting and accomplished. He also likes artists such as Rembrandt, Francisco Goya, Henri Matisse and Pierre Bonnard.\n\n **TIPS  \nfrom the Artist **\n\nEach painting expresses your own personal vision when you express how you see the subject. You don't need to fill in every square inch of canvas. Being selective comes with maturity.\n\nStrombotne notes that artists in general seem to gravitate towards more reductive and simplified work as they get older. They are more apt to \"cut to the chase,\" as they mature.\n\n**James Strombotne**\n\n_Circus Elephant_\n\nAcrylic on canvas\n\n40\" \u00d7 36\" (102cm \u00d7 91cm)\n\nCourtesy of Handsel Gallery,\n\nSanta Fe, NM\n\n**Other Art in This Style**\n\n\u2022 Pop artist Tom Wesselmann, and the comic book style of Roy Lichtenstein\n\n\u2022 Comic book artists\n\n\u2022 Strong patterns and simplified flat space of Henri Matisse, Milton Avery and Joan Mir\u00f3\n\n\u2022 A wide range of flat space imagery from American modernists like Marsden Hartley and Stuart Davis. Also the work of Fernand L\u00e9ger and Anna (Grandma) Moses\n\n\u2022 Great masters from the Florentine School and Byzantine period\n\nVARIATIONS ON FLAT SPACE\n\n**Barbara Moody**\n\n_Chinois_\n\nAcrylic on canvas\n\n30\" \u00d7 40\" (76cm \u00d7 102cm)\n\nPrivate collection\n\n**Variation 1: Contrast Detail with Space**  \nThe contrast between exact detail with the shallow space adds a compelling quality to this still life.\n\n**More Variations**\n\n\u2022 Any techniques that bring attention to the actual surface of the painting, such as texture or sheen, will create an interesting visual tension when combined with the illusion of spatial depth. Experiment with gels and pastes to create texture before or after applying a painted image.\n\n\u2022 Reduce or eliminate perspective techniques. Take out horizon lines or other lines that angle into the picture. Increase the use of drawn lines and repeated geometric patterns. Play with the idea of spatial depth by enlarging the size of forms that appear far back, and reducing the size of objects and forms that are closer (nearer to the bottom of the picture).\n\n\u2022 Paint a realistic scene minimizing the color shifts or gradations that are normally used to create the illusion of light and volume. Instead, paint some areas using flat, evenly applied color.\n\n**James Strombotne** _Summer_\n\nAcrylic on canvas\n\n56\" \u00d7 68\" (142cm \u00d7 173cm)\n\nCourtesy of Handsel Gallery, Santa Fe, NM\n\n**Variation 2: Reduce Detail and Minimize Color**  \nReducing detail and minimizing the color palette places an emphasis on the artist's intent.\n\n  EDGE CONSCIOUSNESS\n\nPaintings using a flat or shallow space still depend on some illusion of depth within the image. Overlapping forms give the illusion of spatial depth, created through the use of edge relationships. A variety of edges from hard to soft, as well as distinct overlapping adds space and a clean look to the work.\n\n**Tip**\n\nRework and refine edges in a painting with a small brush and work with the colors both inside the forms as well as outside or background.\n\n**Undefined Edges Create Ambiguous Space**  \nHere a variety of forms are painted on a peach background. The edges of these forms are all somewhat ambiguous\u2014they are neither hard nor soft and do not clearly indicate whether one form is in front or in back of another. Notice how the space feels and compare this to the next image.\n\n**Define Edges to Enhance Space**  \nA wider variety of edges are created by intentionally making some edges cleaner, crisper and harder while others are softened and blended. Now it's easier to tell which form is in front of which, and the space feels more expansive.\n\n**Eliminate Edge \"Kissing\"**  \nThis image illustrates a common problem of shape \"kissing.\" On the left there are three shapes all sharing common edges. This makes it difficult to tell which form is in front of another. On the right the same set of three shapes overlap one another, thereby increasing the illusion of space.\n\nROMANTICIZED\n\nRomanticism originated toward the end of the eighteenth century in Europe, using images of untamed nature and eliciting emotions such as loneliness and a dreamlike state of mind. The use of soft edges, soft focus, hazy atmospheric qualities and muted palettes add to the surreal beauty often found in these works. Artists can use these techniques in conjunction with contemporary subject matter to evoke a nostalgic or emotional response. This is visible in the work of McCreery Jordan, whose images take on mysterious and haunting tones. In _Companeros_ , a horse and raven are engaged in some form of secret communication, the ring in the bird's beak underscoring the romantic flavor.\n\n**The Artist's Process**\n\nJordan's inspiration is time. Layers and veiling of time and history are echoed in her surface \"patinas,\" which she creates using antiquing, sanding and texturing, then repeating these processes in many layers. Sometimes she'll find and use old doors with their own history. If an object looks too new, she'll sand and stain it, texturizing and antiquing the surface for a few days while allowing the image to emerge. Collage and the use of photographic images are integral to her work and process. Jordan stays connected with her content on a deep emotional level, encouraging this to come through in the finished work.\n\nJordan finds inspiration in the work of Joseph Campbell, the super-realistic still lifes of William Harnett and Jean-Baptiste-Sim\u00e9on Chardin; the loose painting style and philosophy of contemporary artist Richard Schmid, and the textural imagery of Randall Lagro.\n\n **TIPS  \nfrom the Artist **\n\nThe ability to work from your subconscious will allow your vision to come through. This requires many years to develop the necessary skills and tools: including drawing, mistakes, lessons, workshops and miles of canvas.\n\nMcCreery Jordan in her studio\/gallery space with new work. Jordan uses acrylic for its fast drying capabilities, which allows multiple glaze layers, and the ability to work thickly without cracking.\n\n**Other Art in This Style**\n\n\u2022 Emotional dreamlike imagery of Surrealists such as Andr\u00e9 Breton, Marc Chagall, Salvador Dali, Ren\u00e9 Magritte and Dorothea Tanning\n\n\u2022 Moody work and color palettes of Paul Gauguin and Albert Pinkham Ryder\n\n\u2022 Emotional content of Expressionists such as Edvard Munch\n\n\u2022 Masters of Romanticism such as Joseph Mallord William Turner and Caspar David Friedrich\n\n\u2022 John Constable's romantic interpretations of the English landscape\n\n\u2022 Frida Kahlo, Emil Nolde\n\n**McCreery Jordan**\n\n_Companeros_\n\nAcrylic on wood\n\n48\" \u00d7 36\" (122cm \u00d7 91cm)\n\nVARIATIONS ON ROMANTICIZED IMAGERY\n\n**Ren\u00e9e Phillips**\n\n_Field of Dreams_\n\nAcrylic and oil on canvas with powdered pigments\n\n12\" \u00d7 12\" (30cm \u00d7 30cm)\n\n**Variation 1: Soft Colors and Harmonious Flow**  \nRomantic soft colors are used here along with a soothing directional flow moving toward a distant dreamy horizon.\n\n**More Variations**\n\n\u2022 Make a list of things (colors, images, stories, events, adjectives) you find romantic, emotional or beautiful. Select a few of these to assemble together in one image.\n\n\u2022 Mix a palette of colors that feels romantic to you, and use this to create a painting.\n\n\u2022 Make a series of images you find romantic, each using a different context or subject matter including portrait, landscape, still life and abstraction.\n\n**Beth Ames Swartz**\n\n_The Thirteenth Moon: Only the Mindless Waters Remain_\n\nAcrylic and mixed media on canvas\n\n60\" \u00d7 72\" (152cm \u00d7 183cm)\n\n**Variation 2: Limited Palette**  \nUsing a limited color palette with muted tones, Swartz infuses a spiritual and mysterious quality to this moonlit scene.\n\n  GLAZED EDGE ENHANCEMENT\n\nThis technique of vignette glazing can be used to add a romantic or mysterious feeling to a painting. By staining and darkening the outside edges of an image, the space feels more intimate. It can also add a feeling of containment in areas where the image appears to spill out of its frame.\n\nAvoid extending the glaze color too far beyond the edge into the image or it can become distracting.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint (suggested colors: black, and a variety of earth tones in brown, green, rust and ochre)\n\n**\u2022 Surface:** Any painting close to finish\n\n**\u2022 Painting Tools:** Lint-free rag and\/or painting brushes; color mixing palette\n\n**\u2022 Other:** A slow drying clear acrylic medium in gloss or matte\n\n**1. Select a Painting**  \nSelect a painting close to completion that will benefit by adding a romantic mood or more spatial containment. This painting's sky area feels too expansive in comparison to the dark and moodily painted ground. By selectively applying the vignette glazing technique in only the top sky area, the top and bottom halves will be more integrated, the whole image will feel more intimate and contained, and its romantic mood will be enhanced.\n\n**2. Make a Dirty Mix Glaze**  \nPlace several earth tone colors along with slow drying acrylic medium separately on a palette. Use at least four or five distinctly different color choices (pictured here are Sap Green, Burnt Sienna, Payne's Gray, Raw Umber and Carbon Black).\n\nDip a brush or rag into the medium, picking up about \u00bc teaspoon of it, then directly dip into a small amount of paint color. Both medium and paint will be on your rag or brush simultaneously but not mixed together, which is called a dirty mix glaze.\n\n**3. Glaze Along the Edges**  \nApply this dirty mix along a portion of the outside edge using small circular motions. Keep working in a small area while the paint is still wet. Switch to a clean brush or dry rag to remove some of the excess glaze so the color subtly blends into the image. Repeat with more glaze and keep cleaning the brush or use a new dry rag in areas as needed. As you finish one area, move along the same edge using different combinations of color every 1 (25mm) to 2 (51mm) inches. Avoid using the same color for too long or applying it opaquely. Instead, use a variety of color and transparency to subtly enhance the image.\n\n**Finished Example**  \nCompare this to the original image in step 1. Here, the colored edging in the upper half creates a cozy feel to the sky and enhances its mood.\n\nEXAGGERATION\n\nEmphasizing aesthetic elements such as line, shape, proportions, color perspective, texture and space bring individuality and personality to a painting. Martha Kennedy defines her work as \"bold compositions in mouthwatering color,\" and, sure enough, her color is juicy enough to taste. By exaggerating color, Kennedy enables the viewer to better respond with emotion. Kennedy wants her paintings to give people pleasure, to enable them to escape into another world. \"Colors help you feel better, like sharing a smile,\" she says. Kennedy strives for simplicity. When elements are simplified, exaggeration can then come into play to enhance those aesthetic elements the artist prefers, such as color.\n\n**The Artist's Process**\n\nTo get bold areas of exaggerated color, Kennedy works from photographs to assist in simplifying compositional areas. However, her color selections come from her imagination. She chooses colors that play against each other to pop, push, pull and create space. Once she's applied color to an area, she alters it, going lighter to darker, warmer to cooler, or brighter to duller as she creates volume.\n\nOil paint's slow drying properties allow for subtle blending that creates soft shifts in color, making oil Kennedy's primary medium. To replicate this effect, Kennedy has been experimenting with the new slow drying, or extended, acrylics. She has found ways to further extend her working time by using mediums, gels and thinners to resoften dried areas. The reworked surface creates a different type of texture, adding a new quality that she finds desirable and interesting.\n\n**Other Art in This Style**\n\n\u2022 Exaggeration\/simplification of Fernand L\u00e9ger, Milton Avery, Georgia O'Keeffe, Ed Mell, Amedeo Modigliani and Fernando Botero\n\n\u2022 The bold color of Vincent Van Gogh\n\n **TIPS  \nfrom the Artist **\n\nTemporarily switching mediums (i.e., from oil to acrylic) can add a new twist to your work and process. Stick to one medium long enough, though, to go deeper into an idea. Avoid being seduced or distracted by switching mediums too often. For some artists, however, switching to a new medium can add a sense of play, experimentation and new inspiration.\n\nKennedy tapes reference images to the top of her easel while she works. References include an original composition sketch and ink-jet prints of her photographs. Paints and painting tools are located on easy-to-reach dollies. Natural light from windows and skylights are a boost for color decisions.\n\n**Martha Kennedy**\n\n_Road Through Golden Hills_\n\nAcrylic on panel\n\n16\" x 16\" (41cm x 41cm)\n\nCollection of Debra and Chuck Blitzer,\n\nLas Vegas, NV\n\nVARIATIONS OF EXAGGERAT ION\n\n**Martha Kennedy**\n\n_Avocado on Orange_\n\nAcrylic on panel\n\n24\" x 24\" (61cm x 61cm)\n\n**Variation 1: Enlarge and Zoom In**  \nHere Kennedy changes her subject matter from landscape to still life. Simplicity is achieved by selecting and enlarging one object, close cropping and keeping the background minimal. The large, simplified shapes are then painted with exaggerated color.\n\n**More Variations**\n\n\u2022 Try temporarily switching to a medium you haven't used before, such as pastel, watercolor, oil or gouache, and see how this changes your work.\n\n\u2022 Create a few paintings with the same image, but in varying sizes, going from very small to very large to compare the impact.\n\n\u2022 Create a painting from your head that eliminates all recognizable imagery, perhaps using geometric shapes and other nonrealistic forms, and modulate each color shape to give it volume.\n\n**Jakki Kouffman**\n\n_Red Rock, Near Creek_\n\nAcrylic on canvas\n\n40\" x 30\" (102cm x 76cm)\n\nCollection of New Mexico Arts, Art in Public Places,\n\nSan Miguel County Courthouse Annex\n\n**Variation 2: Exaggerate Brushwork**  \nExaggerated textural brushstrokes add a sensual feel to the surface. In addition, the edges between forms are exaggerated, made crisper like stain glass, emphasizing line and pattern.\n\n  REWORKING SLOW DRY ACRYLICS\n\nSlow drying acrylics have a longer open time, which means they stay wet longer than normal acrylic paints. Plus, when the slow-dry paint layers become dry to the touch, you can still rework them for a day or two by using any of the slow drying mediums, gels, thinners and\/or water. These products can be added directly into fresh paint color, or applied straight over a dried layer to rework it. However, once a paint layer has been drying for more than a day or two, it may not be possible to revive it, and then it's best to apply new layers of fresh paint. There are a variety of extended paints. The best results will be obtained using those that stay wet more than twenty-four hours, like Golden's OPEN Acrylics.\n\n**Materials**\n\n**\u2022 Paints:** Slow drying acrylic paint\n\n**\u2022 Surface:** Any painting surface\n\n**\u2022 Painting Tools:** Brush, palette knife\n\n**\u2022 Other:** Mixing palette, slow drying acrylic medium, slow drying acrylic gel, slow drying acrylic thinner, water\n\n**1. Apply slow drying acrylic paint**  \nUsing two slow drying acrylic paint colors, paint two shapes which touch, creating a hard edge. Let dry for several hours.\n\n**2. Resoften dried areas**  \nWith a brush, apply a slow drying acrylic medium, gel, thinner or water over the dried colors near the area where the two colors touch. Let this remain on the dried paint layer for a few minutes to resoften the paint.\n\n**3. Blend**  \nWith a brush, rework the softened color layers to create a smooth transition or softer edge between the two shapes.\n**Section 3**\n\nCHANGING  \nPERSPECTIVES\n\n**Catherine Mackey**\n\n_Pier With Cruise Liner_\n\nAcrylic and mixed media on wood panels\n\n65\" \u00d7 48\" (165cm \u00d7 122cm)\n\nFor centuries a painting was seen as a \"viewing window.\" Most images included a background, foreground, horizon line and fixed perspective. Cubism emerged in the early twentieth century, offering an alternative to the way an image was perceived. Using multiple perspectives in the same image, the fixed viewing point was eliminated, adding a new sense of movement and an interactive approach to viewing art. Cubist masters Pablo Picasso and Georges Braque spearheaded these concepts, while contemporary artists such as David Hockney continue to push them even further. New concepts are continually emerging based on how we really see. In real life, our eyes constantly move, darting around from close-up detail to the distant horizon. We remember an event or scene using a combination of snapshot views taken with our eyes from multiple perspectives. Multiple viewing points in a painting can often feel more real than a conventional photograph taken by a camera. On the other hand, going against our expectations of visual reality can feel surprising, upsetting or jolting to one not familiar with this new way of seeing in contemporary art. The styles in this section reflect the artists' preference to work with these new concepts and to involve the viewer in perceiving more actively.\n\nINSIDE & OUTSIDE\n\nAn image that combines indoor spaces with outdoor environments can convey a sense of spaciousness, freedom and movement. This journey through both natural and human-made environments often employs a broad spectrum of light and shadow effects ranging from natural light sources to incandescent. Hyperrealist painter Gerard Boersma takes us on a magic carpet ride with his paintings through the world's street corners, shops and stores to underground subways. Emphasizing the theme of modern isolation and society, Boersma's work hints at subtle narratives where solitary figures are placed in mundane activities, suggesting the feeling of being alone even in a crowd.\n\n**The Artist's Process**\n\nWorking from photographs and the computer to adjust color and composition, Boersma draws the image onto his painting panel in pencil and continues with paint, making changes when needed. Since he concentrates on one painting at a time, acrylic's fast drying qualities are an advantage, allowing Boersma to layer quickly. Clarity and realistic detail are important to Boersma, who wants his images to engage the viewer in a virtual experience. Artists inspiring to him include Edvard Munch, Johannes Vermeer, Max Ginsburg, John Currin and his great uncle, also a painter, Jopie Huis-man. Super-realist favorites include Ralph Goings and Richard Estes.\n\n **TIPS  \nfrom the Artist **\n\nPractice! Paint what intrigues you. Outliers: The Story of Success by Malcolm Gladwell reports it takes ten thousand hours of time and practice to make something successful.\n\nGerard Boersma deliberately keeps a clean and organized studio, which he notes helps him paint good realism.\n\n**Other Art in This Style**\n\n\u2022 American scenes focusing on loneliness in a modern city: Edward Hopper\n\n\u2022 Scenes using strong lighting contrasts: Masters such as Caravaggio and Rembrandt\n\n**Gerard Boersma**\n\n_The Smoker (self portrait)_\n\nAcrylic on Masonite\n\n37\" \u00d7 28\" (94cm \u00d7 71cm)\n\nPrivate collection\n\nVARIATIONS ON INSIDE & OUTSIDE\n\n**Paul Sarkisian**\n\n_Untitled (El Paso)_\n\nAcrylic on canvas\n\n14' \u00d7 21' (4.3m \u00d7 6.4m)\n\nPhoto by Eric Swanson\n\n**Variation 1: Blur Boundaries**  \nThis trompe l'oeil painting depicts a storefront fa\u00e7ade with incredible lifelike detail, and is even more startling when viewed in person. Here interior and exterior mingle. The artist's use of a black-and-white palette changes the viewing experience from an immediate association with it as a painting to a more conceptual one.\n\n**Hamish Allan**\n\n_House and Hills_\n\nAcrylic on canvas\n\n24\" \u00d7 24\" (61cm \u00d7 61cm)\n\nCollection of the artist\n\n**Variation 2: Simplicity and Repetition**  \nAreas are simplified, creating a bold abstracted landscape. Here the eye travels back and forth between the outdoor and indoor spaces, assisted by the repetition of green used in both places.\n\n**More Variations**\n\n\u2022 Experiment with small collages using interior and exterior images to find unique ways of combining the two.\n\n\u2022 Go out on a photography mission to find shots that naturally combine interior and exterior, like storefront windows that reflect the street while revealing the store's interior.\n\n\u2022 Journal about your emotional response on how interior and exterior spaces differ in feel, and paint that.\n\n  OBTAINING A RANGE OF VALUES\n\nPainting a convincing image that portrays both indoor and outdoor spaces requires a broad range of light and dark values. One surefire way to obtain a variety of these tones in a painting is to pre-mix a full range of colors ahead of time, offering maximum potential. Here is my favorite full palette to achieve this.\n\n**\u2022 Select Optimal Colors for Maximum Color Mixing.** You need a full palette to obtain a full range of colors. Full palettes contain both a warm and a cool for each of the three primary colors, plus white. Adding other colors such as black, green, orange and violet add convenience, but are not necessary.\n\nAlong the outer rim of a large palette, apply paints in an arc, allowing ample space in the middle for mixing color and placing mediums, if desired. Squeeze out a generous amount of each of these suggested colors plus Titanium White:\n\n1. Choose either Hansa Yellow Light or Cadmium Yellow Light for a cool yellow.\n\n2. Use either Hansa Yellow Medium or Cadmium Yellow Medium for a warm yellow.\n\n3. Select from: Cadmium Red Medium or Light, Naphthol Red Medium or Light, Pyrrole Red or Pyrrole Red Light for a warm red.\n\n4. Quinacridone Magenta, a cool red that mixes a clean violet.\n\n5. Ultramarine Blue, a warm blue.\n\n6. Phthalo Blue (Green shade), a cool blue.\n\n**\u2022 Add a Tinted Swatch Near Each Color.** Modern colors look very dark on the palette, while mineral colors appear bright. It can be deceiving to see the colors only in their thick form or mass-tone. Adding white, which turns the colors into a tint, offers different results depending on whether the pigment is modern or mineral.\n\nNext to each color, add a small amount of white in a separate mixture, giving you a reminder of the true potential of each color. In the tinted form, modern colors get brighter, while mineral colors get chalky and muted.\n\n**\u2022 Pre-Mix Lights.** Most paint colors unmixed out of the tube have a middle to mid-dark value range; therefore, a palette using only paint directly from the tubes will produce a dark painting. Pre-mixing a few light values before starting to paint helps solve this problem. Make a 20:1 mixture of Titanium White with red. If the resultant mixture appears too pink, add more white. Make a second light mixture using the same ratio of white mixed with yellow, and a third using white and blue.\n\n**\u2022 Pre-Mix Darks.** There's nothing wrong about using black straight from the tube. Darks are further enhanced, however, by the addition of pre-mixed custom blacks. Make a mixed black combining a modern red and blue (i.e., Quinacridone Magenta with Phthalo Blue) and a small amount of any yellow. Test your mixed black by adding a small amount of white in a separate mixture to see its tinted version of gray. Make one or two more mixtures using the same three colors, but vary the combination ratios to make rich dark browns and cooler or warmer grays.\n\nMULTIPLE PERSPECTIVES\n\nCombining multiple perspectives in the same image allows a variety of viewing points, movement and an invitation for multiple interpretations. This idea has been used by many artists in the twentieth century, most notably with Cubism. Ines Kramer's imaginary cityscapes with a kaleidoscope of planes offers unexpected associations and an overall surprising image. Born in Caracas, Venezuela, Kramer has moved and traveled extensively. Through this rich variety of surroundings Kramer developed a habit of synthesis, absorbing images from all sources and arranging them in new and different ways.\n\n**The Artist's Process**\n\nKramer calls herself a \"hunter-gatherer of images,\" and she goes on several photographing trips each year, later reassembling these images into landscapes of her imagination. Her current interest in urban landscape takes her to large cities, where she captures objects close up such as windows, doors, architecture, rooftop gardens and city trees. Placing close-up shots next to far shots in these reconstructed cityscapes suggests a live city experience with its jostling, overstimulating space.\n\nKramer takes photographs with the intent of capturing a shape or object, not taking a great photograph. This way, she doesn't mind cutting them up or overpainting the photos later. After manipulating them on the computer, Kramer prints out the images, cuts them up and collages them onto a painting surface, which she overpaints with acrylic paint. Some images are completely buried with layers of opaque paint, while others are still visible through layers of transparent glazes. Kramer also adds watercolor, colored pencil, lead pencil and found imagery. Working in layers, Kramer has found the fast drying nature of acrylic works best. Acrylic is a glue as well as a painting medium, making it a good choice for collage, without harming the photographs as oil would.\n\n **TIPS  \nfrom the Artist **\n\nMake sure to have a designated space devoted exclusively to your art\u2014even if it's just a TV cart (my very first \"studio\")\u2014so that you can work on or just look at and think about your work every day. The easiest way to become inspired and stay inspired is to do the work every day. Avoid teachers whose students' work looks just like the teacher's\u2014that teacher will never help you find your own vision.\n\nInes Kramer in her Santa Fe studio. Her large size paintings can contain over twenty-five photographic images. The collaged photographs give her a jumpstart and add inspiration as she continues to change and shift the work.\n\n**Other Art in This Style**\n\nFor multiple perspectives, check out Cubist artists such as Pablo Picasso, Georges Braque and Paul Klee. David Hockney works with a unique contemporary version of Cubism especially visible in his photo collages. See also Wassily Kandinsky, David Salle, James Rosenquist, Jos\u00e9 Clemente Orozco and Richard Diebenkorn.\n\n**Ines Kramer**\n\n_Rooftop Eden_\n\nAcrylic and collage on panel\n\n32\" \u00d7 36\" (81cm \u00d7 91cm)\n\nVARIATIONS OF MULTI PLE PERSPECTIVE S\n\n**Catherine Mackey**\n\n_Whiz Burgers_\n\nAcrylic and mixed\n\nmedia on wood panel\n\n24\" \u00d7 53\" (61cm \u00d7 135cm)\n\n**Variation 1: Combine Separate Panels**  \nJoining separate painted panels contrasts varying perspectives in this assemblage painting.\n\n**Mary Morrison**\n\n_Grasslands_\n\nAssembled acrylic painted fragments on panel\n\n36\" \u00d7 45\" (91cm \u00d7 114cm)\n\nCollection ofThe Herman Memorial Wellness Center, Houston, TX\n\n**Variation 2: Merge Different Images**  \nCombine two completely different images. Here a landscape and abstract designs are spliced into geometric shapes and reassembled into a completely new image.\n\n**More Variations**\n\n\u2022 Pick one particular topic that interests you. Gather all the images of these you can from varying sources: magazines, photocopies from books, your own photos and drawings; and collage these together in unusual patterns and arrangements.\n\n\u2022 Find and cut out pieces of color with no recognizable imagery (i.e., paint swatches from home decorating stores, cut pieces of ads and product labels). Arrange these and collage them to create an abstract or color field, with or without the addition of painted areas.\n\n\u2022 Exaggerate viewpoints: Pick a subject you like: people, landscapes, skies, pets and animals, food, clothes, etc. Photograph it several times from different viewpoints. Collage these by overlapping images.\n\n\u2022 Create a painting that has no horizon line, thus eliminating a finite point of view.\n\n  THE POWER OF HORIZONTALS\n\nHorizontal lines in a painting are easily interpreted as a horizon, and can add a grounded feeling to the work. Many abstract styles add a horizontal line to the image to ease visual tension, while others eliminate it to move the viewer into another reality. Here are some examples to illustrate this idea.\n\n**Tip**\n\nA horizontal line is a grounding tool, not an essential element in a work. It can be used or avoided depending on your intent for the image.\n\n**Evoking a Landscape**  \nThis painting contains very few elements. With only three colors, the composition consists of a gradation of two colors in the top area and a simple horizontal band of color for the bottom. Note how easy it is to interpret this as a landscape, even with nontraditional sky and ground colors. It is our sense of being human, our relationship with the planet and its continual pull of gravity that defines the essence of this viewing experience.\n\n**No Horizon Line Increases Visual Tension**  \nThis painting has no obvious horizontal lines, giving it a floating quality with a visual tension that can offer a new and vital viewing experience.\n\n**Adding a Horizon Line Grounds the Viewer's Perspective**  \nHere the same painting is changed completely with the addition of a simple horizontal line. There is a new feeling of comfort and ease in the viewing.\n\nCOMPOSITE ARRANGEMENTS\n\nThis style, also called assemblage, is a form of artistic construction where multiple individual images and surfaces are combined to create a new, but segmented larger whole. Catherine Mackey relies on her experiences as an interior architect to create an unusual body of work using this process. In a definite reaction to her former career's pristine and controlled nature, her paintings add elements of dirt and grit. Collisions of color and fragments in her urban environment catch her eye as she notes inadvertent beauty, searching for urban decay on walls that carry a visual history of stories about people.\n\n**The Artist's Process**\n\nTen to thirty bits of found wood, reworked and often repainted, can always be found hanging around Mackey's studio. She uses these to play and arrange. Allowing for accidental associations and chance, her process carries an unpredictable timing. Texture is important, and she'll often use building materials like ceramic tiles or Sheetrock compound, pressing other objects into it. She uses acrylic for most of her painting purposes. Her techniques are varied, and include stenciling and thick stippling for adding text in relief. Colored areas are sanded, scratched and washed down. Scrubbing creates an accelerated look of aging. When pieces come together in a way she likes, she connects them by constructing a backing frame, then continues painting them together as one image.\n\nArtists who inspire Mackey include: Robert Rauschenberg and the complete freedom he used to work with anything, repeat imagery, and reuse things in other works; Frank Auerbach's thick layers using lots of subtraction and peeling back; Anselm Kiefer's monolithic early work, using massive scale and architectural composition with miles of field; famed graffiti artist Jean-Michel Basquiat; David Ireland's unusual installations.\n\n **TIPS  \nfrom the Artist **\n\nMackey works mostly from photographs in her studio. Yet even with plenty of photographs on hand, there are times when she still needs to energize her work and\/or attitude, and ventures out with her camera exploring for more. This will inevitably reveal something new. She recommends one of her daily rituals: spending ten minutes every night looking through art books she might not have picked up for a while. Falling asleep while thinking of new ideas keeps her creative energy alive.\n\nCatherine Mackey surrounded by a variety of assemblage panels and materials in her California studio.\n\nHer term \"architectural palimpsests\" aptly describes her interest in graffiti, signs and wall writing. Ripped posters stuck onto each other year after year build up with a patina she can't resist. She peels these treasures off their original surface, soaking them in a tray of water to become unique collage fodder.\n\n**Other Art in This Style**\n\n\u2022 Assemblage artists: Robert Rauschenberg, John Baldessari, Louise Nevelson, Mike Kelly, Jennifer Bartlett\n\n\u2022 Cubist masters: Georges Braque and Pablo Picasso\n\n\u2022 Fluxus Movement artists\n\n\u2022 Marcel Duchamp's readymades, Kurt Schwitters, Man Ray, Joseph Cornell\n\n**Catherine Mackey**\n\n_Tony's Journey_\n\nMixed media on wood panels\n\n24\" \u00d7 238\" (61cm \u00d7 605cm)\n\nVARIATIONS OF COMPOSITE ARRANGEMENTS\n\n**William Dunlap**\n\n_Landscape & Variable: Landscape Askew _\n\nPolymer paint on canvas (with wood, slate, leather, mixed media)\n\n41\" \u00d7 180\" (104cm \u00d7 457cm)\n\nCollection of Washington Convention Center, Washington D.C.\n\n**Variation 1: Use a Variety of Materials**  \nWilliam Dunlap uses materials both found and fashioned, often confusing viewers over what is made or found. The implied narrative is made exciting and contemporary by Dunlap's arrangement, angled placement and use of materials.\n\n**More Variations**\n\n\u2022 Assemble a collection of objects and images that you find or make. Do not permanently adhere the elements together in the beginning to allow for maximum play and flexibility. As you find arrangements you like, photograph them for later use as reference, while continuing to rearrange.\n\n\u2022 Dig out old paintings and drawings that are no longer of interest to you. Cut them into new shapes and pieces. Rearrange them on a new surface or over another painting.\n\n**Don Quade**\n\n_Winter Lotus_\n\nAcrylic, oil stick, graphite and paper collage on wood panel\n\n48\" \u00d7 48\" (122cm \u00d7 122cm)\n\nPrivate collection\n\n**Variation 2: Assemble Non-Related Sections**  \nThree distinct sections offer a playful dialogue about the artist's interest in nature. During a winter visit to a botanical garden, Don Quade noted flowers trapped in ice. The combination of abstraction and strong divisions turned his experience into a metaphor for space and time. The top sections refer to clouds and sky, while the bottom represents rain.\n\n  ANTIQUE EFFECTS\n\nWhen Mackey needs to make something look old, she likes to work with acrylic washes, alternating with subtractive scrubbing. Here is a technique to get similar results, especially helpful with found objects and collage items that look too new.\n\n**Tip**\n\nBefore starting, hold your surface up to the light, and notice the sheen. If some areas are matte and some are glossy, you'll have a greater variety of effects. Optionally, you can prepare the surface by applying matte and semigloss mediums or gels separately in several places on the surface. Let dry before starting. To emphasize the stained effect even more, reapply semigloss acrylic gel in between wash layers, letting it dry before adding any more washes.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint colors in earthy tones such as black, brown, olive green and rust (suggested colors: Burnt Sienna, Quinacridone Nickel Azo Gold, Viridian Green Hue, Transparent Red Iron Oxide, Green Gold, Bone Black)\n\n**\u2022 Surface:** A primed surface with a semigloss or matte sheen\n\n**\u2022 Painting Tools:** Any brush\n\n**\u2022 Other:** Dish scrubbers or sandpaper, water, spray bottle, paper towel or rag\n\n**1. Apply Acrylic Washes**  \nWet the painting surface with enough water to form small puddles. Select a few colors and dilute heavily with water. Apply all the colors randomly over the wet surface. Lightly spray more water over the applied paint and entire surface. Allow the paint to puddle up and move around on its own. Let this dry on a level surface.\n\n**2. Scrub the Paint**  \nIf the dried stains from the previous step are too noticeable, reduce them with this step. Select a dish scrubber or any abrasive tool such as waterproof sandpaper. Apply water to the surface, then remove some of the paint using your scrubber, applying medium pressure in a variety of scrubbing directions. Wipe the excess paint off with a paper towel or rag, and let dry.\n\n**3. Repeat Steps**  \nIf the stains are too subtle, you can repeat steps 1 and 2, layering over the previous one, until you've obtained the desired results.\n\n**A New Use for Old Water**\n\nInstead of diluting a fresh batch of paint for the washes in step 1, use some leftover dirty paint water from your studio.\n\nCOLLAGE\n\nThe term collage comes from the French word _coller_ which means \"to stick.\" A collage incorporates multiple images applied to a surface, usually overlapping one another to partially reveal and partially cover. A whole new image emerges, offering multiple perspectives.\n\nCollage artist Nancy Scheinman takes her paintings to another level by making most of her unusual collage materials. For instance, she washes hand-embossed sheets of copper with acid to create patinas that she nails onto wood panel, which she paints or prints. The unusual formats and ways she combines these materials add a unique quality to her work.\n\nScheinman presents and assembles new stories incorporating themes such as finding balance in life or the myth of a purer past. Her signature motifs include towers, borders, woman protagonists, birds, leaping dogs and red circles\u2014her personal symbol\u2014which she presents in dreamlike landscape. She presents her stories in snippets, allowing the audience to bring their own experience and interpretation to the work. Using the framework of a modernist geometric grid, borders visually contain the multiple stories within each piece, which are slowly revealed to the viewer.\n\n**The Artist's Process**\n\nScheinman freely applies layers of paint and materials, and, just as freely, takes them off. Like fabric in a quilt, she cuts and layers sheets of copper paint and patterns, and collages them together with nails, tacks and antique materials, literally connecting the past with the present. She is inspired by the late contemporary artist Hollis Sigler, whose work contains an admirable strength in dealing with personal narrative; as well as Italian Baroque painter Artemisia Gentileschi, a bold artist who dared to represent historical scenes at a time when it was not considered appropriate for women artists. Gentileschi was the first female painter to become a member of the famed art academy in Florence.\n\n **TIPS  \nfrom the Artist **\n\nA well-known art instructor, Scheinman uses her theme \"Nine Who Dared,\" based on nine artists who dared to face artistic criticism, as inspiration for students to bravely create more personal work.\n\nNancy Scheinman at work in her Maryland studio. A visual storyteller, her use of personal narratives and internalized images from extensive travels are transformed into collective myths and pieced together as part of a universal, spiritual and domestic experience.\n\n**Other Art in This Style**\n\n\u2022 Contemporary artists David Salle, David Hockney, Jim Dine and Gilbert and George\n\n\u2022 Collage work of modernists Pablo Picasso, Georges Braque, Henri Matisse, Joseph Cornell, Man Ray, Robert Motherwell and Peter Blake\n\n\u2022 Merz pictures of collage artist Kurt Schwitters\n\n**Nancy Scheinman**\n\n_Repeated by a Bird\u2014Melody_\n\nAcrylic; canvas; antique tin; brass; etched and\n\ninked vinyl; hand-embossed printed, painted\n\nand patinated copper on wood panel\n\n21\" \u00d7 21\" (53cm \u00d7 53cm)\n\nVARIATIONS OF COLLAGE\n\n**Cate Goedert**\n\n_Repose_\n\nAcrylic and mixed media on canvas panel\n\n16\" \u00d7 8\" (41cm \u00d7 20cm)\n\n**Variation 1: Merge Photography with Paint**  \nThe use of transfer techniques allows an interplay between photography and paint. A photographer with a theater background, Cate Goedert plays with dramatic lighting and atypical formats. A copyright-free photograph in the top portion of the painting is kept transparent and transferred onto a painted background. The bottom portion of the painting contains a photograph that Goedert shot, as well as paint stenciling, crackle paste and textured surfaces. (See for the digital transfer technique.)\n\n**Darlene McElroy**\n\n_Ode to the Masters_\n\nAcrylic on panel with image transfer, collage and gold leaf\n\n39\" \u00d7 39\" (99cm \u00d7 99cm)\n\n**Variation 2: Unusual Formats**  \nOld covered doors are used here, adding an interesting shaped surface resembling a house or shrine. The image is a tribute to Diego Vel\u00e1zquez, painted in the ornate style of his time.\n\n**More Variations**\n\n\u2022 Find printed images to cut out for a collage, but cut them with excess borders to create shapes that differ from the images themselves.\n\n\u2022 Set up a still life using one of your paintings as the backdrop and assembling real objects in front. Photograph the setup to use as a reference for a whole new painting.\n\n\u2022 Collect and save the numerous paper items you receive daily in junk mail, wrappers, product labels, etc. Color, cut, mix and match them to create something different.\n\nSMOOTHING RAISED EDGES\n\nA collage usually combines paint with other materials. Paper, fabric, magazine images and other printed imagery often use inks and dyes that are not lightfast or permanent. Thicker materials like cardboard or heavy fabric can create a raised edge and present another challenge for artists who prefer the edges to transition smoothly into the painting. A smoother surface can be obtained by applying an acrylic gel over the collaged materials. Fading can be reduced by using a gel with UV protection.\n\n**Tip**\n\nThe gel adds a nice handmade texture to the final surface. Generally, a thick gloss gel will suffice. Use a matte gel if you prefer a slightly cloudy, veiled layer that will somewhat obscure the underlying painting. If you desire a glassy smooth surfboard-like finish, you can add a layer of pourable acrylic. See for additional pouring tips.\n\n**Materials**\n\n**\u2022 Surface:** Any collage acrylic painting that is finished or in-process on any surface\n\n**\u2022 Painting Tools:** Palette knife or other wide, flat application tool\n\n**\u2022 Other:** A thick gloss acrylic gel with UV protection (if unavailable use two separate products\u2014a gel and a UV product)\n\n**1. Preparation**  \nSelect a painting with texture or relief edges that needs smoothing. Using a knife with a stepped handle so that your hand will not drag through the gel while applying, load up the back of the knife with a generous amount of the gel.\n\n**2. Spread the Gel**  \nSpread the gel evenly over the entire surface. While applying the gel, keep the knife lifted slightly off the surface, at least by \u00bc inch (6mm), so the knife never actually touches the surface. This will allow the layer to be thick enough to cover all the texture and relief. If your edges are thicker than \u00bc inch (6mm), apply the layer more thickly, or apply a second layer after the first has dried. Additionally, you can add more objects or images into the gel while wet and they will adhere. The gel will turn clear when dry.\n\n**3. Add UV Protection**  \nIf the gel used in the previous step does not have UV protection, then apply another layer using a UV product such as Golden's Archival Varnish. Follow the label's instructions, diluting if required, and brush or spray accordingly. The more coats of a UV product you apply, the longer the colors will remain intact and resist fading.\n**Section 4**\n\nENGAGING  \nTHE  \nPICTURE  \nPLANE\n\n**Jim Waid**\n\n_Blue Shimmy_\n\nAcrylic on canvas\n\n78\" \u00d7 96\" (198cm \u00d7 244cm)\n\nCollection of Kevin Osborn, Tucson, AZ\n\nImages that create the illusion of coming forward confront the viewer, or break the illusion of pictorial depth, and attempt to visually engage us on the front surface of the canvas. This is a significant change from the Renaissance \"window\" where attention to the front picture plane was avoided. The styles in this section contain works which are sometimes bold, blatant and appear to meet us head-on. Shock imagery quickly comes to mind as one way to create this effect. Highly textured surfaces, flatly painted forms and shallow space can also bring our attention to the front picture plane. This section offers a variety of styles and approaches to engage the picture plane, using a range of aesthetic aspects.\n\nMAPPING: PLANES & FRAGMENTS\n\nMapping involves a schematic display that is readily seen in maps, science book diagrams and graphs. A painting that uses mapping can create a sequence in the way it's viewed, as in comic book strips, scrolls and altered books.\n\nDannielle Tegeder makes use of this new approach, inspired primarily by the architectural blueprints and technological sketches she has been exposed to since childhood. Her use of this style, along with mechanical references and forms, creates abstract fields that evoke postmodern visions of the future. The spaces can feel disorienting with no recognizable horizon line, floating architectural fragments and a precarious balance of objects.\n\n**The Artist's Process**\n\nTegeder sands layers of acrylic molding paste on canvas over panel to create a smooth drawing surface, where she then draws with pencil. She then layers paint, transfer skins and gels together to create the final textured surface. For her, adding handmade elements and imperfections offer a humanizing element and balance the hard edges and analytical motifs. Using an intuitive process, Tegeder keeps the end result unplanned, allowing each step to respond to the one before. The sense of space created in Tegeder's paintings shifted when she started working with installations and sculpture. _Inropataciland_ shows her earlier work using a flatter space, compared with _Deconia_ which shows a more expanded space using angles and loosely worked areas.\n\n**Other Art in This Style**\n\n\u2022 Contemporary artists: Squeak Carnwath, Ed Ruscha, Joyce Kozloff, Newton and Helen Mayer Harrison, Julie Mehretu, Vernon Fisher, \u00d6yvind Fahlstr\u00f6m, Jean-Michel Basquiat, Pat Steir's early work, William Wiley\n\n\u2022 Egyptian hieroglyphics\n\n\u2022 The Map as Art by Katharine Harmon, a book on contemporary artists using this style\n\n **TIPS  \nfrom the Artist **\n\nEmerging artists often overdose on art magazines. Look at things not directly related to your work, including unfamiliar art styles, music, books and poetry. Follow things that interest you, even non-art interests, which can still inspire. Don't buy into other people's stuff.\n\nDannielle Tegeder in her New York studio. Tegeder is inspired by cities and charts organizing information about people, like transportation routes, population charts and aviation maps. Organizing color and form is integral to Tegeder, and she allows some type of narrative to come through and read like a map.\n\n**Dannielle Tegeder**\n\n_Inropataciland: White Winter_\n\n_City with Dot lower Tunnel_\n\n_Routes, Love Circle Production Expulsion Center, with five station Route, Twin Station Igloo Housing, with New Central Hospital and Lace Grid Station, multiple Box Square Hotel Housing and 27 circle; Storage from Above Ground and Below Ground; and Sideways color coding Grid, with Triangle Training Center, 2005_\n\nAcrylic, ink, dye, pencil, design marker and gouache on paper\n\n55\" \u00d7 79\" (140cm \u00d7 201cm)\n\nImages courtesy of Priska C.\n\nJuschka Fine Art, New York,\n\nNew York\n\n**Dannielle Tegeder**\n\n_Deconia: Thermal Atomic Rate at Red Midnight 2007_\n\nAcrylic, ink, colored pencil, graphite, gouache and pastel on paper\n\n55\" \u00d7 79\" (140cm \u00d7 201cm)\n\nImages courtesy of Priska C.\n\nJuschka Fine Art, New York,\n\nNew York\n\nVARIATIONS ON MAPPING\n\n**Catherine Mackey**\n\n_Harrison Street Scooter_\n\nAcrylic and mixed media on wood panel\n\n48\" \u00d7 45\" (122cm \u00d7 114cm)\n\n**Variation 1: Strong Divisions**  \nThis composition presents imagery within strong geometric divisions, presenting a flat diagrammatic space.\n\n**Keith Morant**\n\n_Airs & Graces II _\n\nAcrylic and oil pastel on canvas\n\n18\" \u00d7 24\" (46cm \u00d7 61cm)\n\nBryce Gallery\n\n**Variation 2: No Horizon Line**  \nWith no horizon to ground the viewer, Keith Morant creates a dialogue between the free-floating forms by harmonizing, contrasting, and using structural and directional forces.\n\n**More Variations**\n\n\u2022 Secure several maps and diagrams onto a surface. Extend elements from one diagram into another, exploring how to integrate the aspects and create a flow.\n\n\u2022 Collect diagrams and graphs of all kinds. Select your favorite and replicate its format, replacing the information formerly displayed with personal imagery. Find multiple ways of expressing the same concept (pictures, text, lines, arrows, products, shapes, etc). Books on tarot will often show special \"layouts\" for readings that can be used as inspiration for compositional structures.\n\n\u2022 Instead of writing entries in a journal or diary, find a new way of presenting events. For instance, draw or list images from your day by displaying them differently, e.g., chronologically, by priority, like a family tree, circularly, etc. Use text, arrows and pictures to get your point across.\n\n  SEDUCTIVELY SMOOTH SURFACES\n\nTegeder's paintings end up with a multi-textured finish, but she still likes to start with a very smooth surface, facilitating detail and line work. Here is a technique simulating her surface preparation.\n\n**Tip**\n\nTo lend a warmer white color to the paste, add a small amount of Transparent Red Iron Oxide (about two\u2013four drops per eight ounces of paste) before applying the paste to the surface. Other colors can be added in more quantity to the paste to create a colored ground.\n\n**Materials**\n\n**\u2022 Surface:** Any rigid surface\n\n**\u2022 Painting Tools:** A large flat plaster knife or other application tool\n\n**\u2022 Other:** Acrylic molding paste, waterproof sandpaper, paper towels or rags, water\n\n**1. Apply Acrylic Paste**  \nUsing a large flat plaster knife or other application tool, apply acrylic molding paste onto your surface. Use a larger tool for a larger surface. Apply a generous amount of paste and smooth it the best you can. Let it dry at least twelve hours or until it's no longer cool to the touch.\n\n**2. Wet Sand the Dried Paste**  \nUsing waterproof sandpaper (usually black in color, and sold at home improvement stores) wet-sand the paste surface by always keeping water between the sandpaper and the surface. Dip the sandpaper into water or use a brush or spray bottle to apply water separately to the surface. Using moderate pressure, sand in circular motions, continuing to add water at intervals into your sanding area. Wipe excess paint off frequently with a rag. Work the entire surface until smooth.\n\n**3. Repeat as Desired**  \nRepeat with a second layer of paste, letting it dry before sanding. Keep repeating as desired. The surface will become unusually smooth with a dense feel. Drawing and taping are easily accomplished on this surface.\n\nCONFRONTING THE VIEWER\n\nContent in images that confront the viewer head-on can be organized in two different ways: aesthetic or psychological confrontation. Confrontation using aesthetic concepts was spearheaded by German abstract expressionist Hans Hofmann, an influential artist and teacher, who used color relationships to enhance his \"push-pull\" spatial theory. Blocks of color with no recognizable imagery can create the illusion of an assertively forward or recessive backward movement.\n\nPsychological confrontation uses imagery and associations that confront our societal, political, religious or moral values. This latter mode is easily seen in works by Frances Ferdinands, who uses her training in conceptual art to convey ideas on social and environmental concerns of postmodern society. In _Becoming One_, a package of Wonder bread becomes an offering in the lap of an ancient Buddha, addressing issues on global consumption.\n\nFerdinands wants the ideas in her works to take precedence. She uses humor to diffuse confrontation, which she incorporates into her work by mixing contemporary and historic sources with surprising juxtapositions.\n\n**The Artist's Process**\n\nFerdinands' paintings start from personal experiences she transforms to the universal. She allows the process to take whatever time it needs. Ideas often take years to percolate, and sometimes work needs to be put aside for her to return to later. She allows her process full freedom, adding the risk that some pieces can and will get destroyed or overworked. Multiple glaze applications create depth and richness to her color and surfaces.\n\n **TIPS  \nfrom the Artist **\n\nEveryone's life is sufficiently interesting to provide fodder for unique work. You don't have to look \"out there.\" Capture your vision and combine it with technical mastery. Great art has to touch the soul. The harshest critic is yourself.\n\nFor inspiration, Ferdinands researches art history. Artists that inspire her include: Edward Hopper, Jim Dine, Jasper Johns, Robert Rauschenberg, as well as pop artists, conceptual art and surrealist movements.\n\n**Other Art in This Style**\n\n\u2022 Confrontational works of Hans Hofmann, Damien Hirst, Mike Kelly, Paul McCarthy, Otto Dix, Eric Fischl, John Heartfield, Barbara Kruger\n\n\u2022 Realist Impressionist painter \u00c9douard Manet created controversial work for his time in the mid-nineteenth century\n\n\u2022 Pop artists like Andy Warhol\n\n**Frances Ferdinands**\n\n_Becoming One_\n\nAcrylic on canvas\n\n30\" \u00d7 30\" (76cm \u00d7 76cm)\n\nVARIATIONS ON CONFRONTING THE VIEWER\n\n**Daniel Barkley**\n\n_Cage 1_\n\nAcrylic on canvas\n\n42\" \u00d7 42\" (107cm \u00d7 107cm)\n\n**Variation 1: A Direct Gaze**  \nA Japanese fencing mask only slightly obscures the portrait's direct gaze. The mouth is hidden by leather, adding an intimidating quality.\n\n**Grant Wiggins**\n\n_Where Is Gibarian?_\n\nAcrylic on canvas\n\n21\" \u00d7 16\" (53cm \u00d7 41cm)\n\nCollection of Laurence Blake, Palmdale, CA\n\n**Variation 2: Shockingly Bright Color**  \nFluorescent orange paint vibrates, layers collide and colors bounce off each other. Grant Wiggins likes his work to clash and uses advertising and corporate logos for the basis of his inspiration.\n\n**Darlene McElroy**\n\n_Ma\u00eetresse sans Visage_\n\nAcrylic and mixed media on panel\n\n24\" \u00d7 24\" (61cm \u00d7 61cm)\n\n**Variation 3: Add Surprise**  \nAn unexpected image is created by removing something expected, giving this work a disturbing quality. Darlene McElroy appropriates some imagery from historic painting, while adding her own. Paint and image transfers are used over stenciling.\n\n  PAINTING A LUMINOUS BACKGROUND\n\nFerdinands often uses backgrounds that are simple yet luminous, creating a sensual contrast for her realistically painted imagery. This technique takes a simple yellow background and adds luminosity and a halo effect to frame or accent any centrally painted form.\n\n**Materials**\n\n**\u2022 Paints:** Heavy Body or Fluid Acrylic colors: Naples Yellow Hue, Indian Yellow Hue, Quinacridone\/Nickel Azo Gold, Transparent Yellow Iron Oxide, Nickel Azo Yellow\n\n**\u2022 Surface:** Any primed surface\n\n**\u2022 Painting Tools:** Brushes, mixing knife, rag\n\n**\u2022 Other:** Acrylic slow drying or glazing gloss medium\n\n**1. Paint a Colored Background**  \nBrush-apply an opaque color, such as Naples Yellow Hue, over a primed surface, creating a solid, evenly applied undercoat. Apply a second coat if it appears streaky.\n\n**2. Apply Transparent Color**  \nGenerously brush-apply some uncolored slow drying medium or acrylic glazing medium gloss in the center of the painting, covering an area in excess of any central imagery you plan to paint later. Mix a transparent glazing color using medium to color in a 4:1 ratio. Using a brush or rag, apply the glazing color, starting at the corners and outer edges, where the strongest color will be. Continue spreading the glaze, moving towards the center. As you continue to spread the color toward the center, allow the color to dissipate. When the remaining colored glaze meets the wet medium in the center, the color will blend easily. Let the glaze dry fully.\n\nRepeat this step using a variety of glazes. The example here uses Indian Yellow Hue for the first glaze, Quinacridone\/Nickel Azo Gold for the second and Transparent Yellow Iron Oxide for the third.\n\n**3. Integrate With an Overall Glaze**  \nThe background now has a luminous quality from the several glaze layers. The central area, however, appears like a hole since color was withheld there. To integrate the center, a final glazing color is applied with Nickel Azo Yellow. But this time, brush-apply the glaze over the entire image, including the center. For added luminosity, apply another glaze using Interference Gold or Iridescent Gold.\n\n**Finished Example**\n\nISOLATED IMAGERY\n\nWhile complex or overcrowded compositions have their place, a bold use of simplicity can allow a deeper insight into an idea. By isolating a particular form, fragment or area, the composition takes on a graphic quality.\n\nFormer figurative and portrait painter Joey Fauerso offers a great example of isolated imagery in the work pictured opposite. Isolating the figure from its environment allows it to be seen in a different way. When she expanded her medium to include installation and animation, the figure continued to remain her primary focus. Fauerso films people in various activities, and from this creates work that includes her films, stills and paintings from the stills. Fauerso sees animation as \"moving painting\" and her installations offer \"epic paintings in time.\"\n\n**The Artist's Process**\n\nFauerso warms up by working on fifty or sixty sheets of paper all the same size that are soaked then stretched. She experiments on these sheets, trying out new techniques, materials, surfaces and mediums. What she learns becomes part of her new series. She protects the backgrounds by coating the paper with matte medium. Projecting the stills onto the paper, she then traces the forms using thin washes of color. She masks the background around the form with blue tape and seals the edges with matte medium to ensure the background stays clean during painting. Fauerso then applies paint onto the wet paper using brushes, mops and giant assembled brushes to get big pools of color. Working subtractively, she wipes out areas of paint, then continues working to get the visual impact she wants.\n\nInspiration for Fauerso include artists Max Beckmann and Alice Neel, studying Eastern philosophy, reading favorite writers and poets such as Mark Strand, and listening to the music of Bach and Arthur Russell.\n\n **TIPS  \nfrom the Artist **\n\nMore experimenting produces more discoveries. Avoid only working on finished pieces where you are invested in the outcome. Allow mistakes, since these can often lead to surprising techniques.\n\nJoey Fauerso in her Texas studio. Her intent to convey the inside and outside of things, representing our physical and metaphysical boundaries, is aptly expressed in her work. She strives for strong graphic impact, contrasting form against the white page.\n\n**Other Art in This Style**\n\n\u2022 Andy Warhol and other pop artists\n\n\u2022 Photorealist Chuck Close\n\n\u2022 Susan Rothenberg, Jean Dubuffet, Leon Golub\n\n**Joey Fauerso**\n\n_Wide Open Wide_\n\n_(installation detail)_\n\nWatercolor and acrylic on paper\n\n335 paintings, each 8.5\" \u00d7 11\"\n\n(22cm \u00d7 28cm)\n\n**Joey Fauerso**\n\n_Get Naked (Detail)_\n\nOil and acrylic\n\non paper\n\n8.5\" \u00d7 11\"\n\n(22cm \u00d7 28cm)\n\nVARIATIONS ON ISOLATED IMAGERY\n\n**Frances Ferdinands**\n\n_Seven-Day Wonder_\n\nAcrylic on canvas, mounted on panel\n\n24\" \u00d7 36\" (61cm \u00d7 91cm)\n\n**Variation 1: Repetition**  \nA graphic quality is created by the use of bright, bold background colors, and repeating views of the same object.\n\n**Olga Seem**\n\n_Duality (11)_\n\nAcrylic on paper on canvas\n\n18\" \u00d7 18\" (46cm \u00d7 46cm)\n\nCourtesy Couturier Gallery, Los Angeles, CA\n\nand Davis & Cline Gallery, Ashland, OR\n\n**Variation 2: Fragment**  \nThe unconventional divisions in this painting present separate fragmented sections of the same plant. Olga Seem researches unusual or extinct botanical flora. Her paintings incorporate material from reference guides containing various illustrations using dissections and close-up views.\n\n**More Variations**\n\n\u2022 Find photographs or other images you like. Pick out one object or form in each image, and isolate it by painting over the background or cutting it out and placing it on a piece of colored or white paper.\n\n\u2022 Starting with a picture you like, cut out the strongest forms, for example an animal, figure or flower. Recut forms further into smaller fragments or pieces, (i.e., figure is separated into arms, legs, hair, etc.), and experiment by placing the pieces separately on different backgrounds or combine some together on one common background. Take the original picture with its cut-out, and place it over other images to \"fill\" the hole with something else.\n\n\u2022 Place strips of white cardboard over paintings to recrop and reframe images to view them in a new way.\n\n  MASKING A SIMPLIFIED BACKGROUND\n\nThis technique is a great way to simplify a busy background, and isolate a singular form for emphasis or focus.\n\n**1. Select a Painting**  \nFind a painting with a busy background that's in need of an emphasized focus area.\n\n**2. Cut a Mask**  \nApply contact paper or masking paper over your painting in excess of the focus area. (Pictured here is shelf paper found in home improvement stores.) Select a shape or section to single out as a main focus. Using a permanent marker, draw its outline onto the plastic. Transfer the plastic to an appropriate cutting surface, cut the outline with a craft knife, then situate the cut shape over the focus area once again.\n\n**3. Overpaint the Background**  \nSelect your choice of paint application (pictured here is a Preval sprayer found in home improvement stores), your preferred color and preferred transparency (here I am using fluid Iridescent Translucent Pearl mixed with some water to ease spraying). Keep the color fairly transparent at first by adding more medium or water if necessary. More control can be obtained by starting transparent and repeating layers until desired coverage is achieved. Overpaint the entire painting. Remove the remaining contact paper.\n\n**Finished Example With a Simplified Background**  \nSelecting a dark color for use in the background will increase the brightness of the focus area, while a bright or light color will create more of a silhouette.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint\n\n**\u2022 Surface:** A painting in-process with a busy background\n\n**\u2022 Painting Tools:** Your choice of paint applicator such as a brush, sponge or commercial spray bottle (like Preval) or household water sprayer\n\n**\u2022 Other:** Clear or transparent contact paper or masking paper, scissors or cutting knife, permanent marker\n\nEMBELLISHED FORM\n\nEnhancing an image by using decorative elements such as gems, gold leaf and painted detail adds a unique visual flavor. Depending on how the artist handles the embellishments, emotions and qualities such as playful, delicate, disturbing or disruptive can emerge. Jim Barsness combines pattern with pictorial space. Working on canvases covered in rice paper, his elaborate detail brings to mind an enlarged antique illuminated manuscript. Finished painted canvases are even left unstretched to be hung like a tapestry. Ancient meets contemporary with a touch of humor.\n\n**The Artist's Process**\n\nBarsness makes a point of seeking new subject matter that he knows little about. Looking outside himself for the unknown, Barsness puts in hours of research for each project. This process allows him to fall in love with different images and absorb them while actively engaging his own imagination.\n\nBarsness has created a cast of characters that he reuses while continually inventing new ones. He works with acrylic like oil, using traditional glazing techniques, and layering to develop a depth in the character field. Other techniques he frequently uses include distressing the paint by sanding and using bright analogous glazes to enhance color (i.e., red gets an Alizarin glaze then Magenta\u2014see COLOR ENHANCEMENT). Barsness also works with different materials such as glitter, silicone molds and casts from craft jewels, interference paints and flocking. Borders are hand-drawn.\n\nBarsness is inspired by a wide range of art including graffiti; the Rubin Museum of Art's collection of Tibetan thangka paintings with hand-painted fabric borders; Northern Renaissance artists such as Pieter Bruegel the Elder, who painted huge crowd scenes; Early Netherlandish painter Hieronymus Bosch; Persian miniatures; and the stylization of works from Cambodia and Thailand.\n\n **TIPS  \nfrom the Artist **\n\nStart with what moves you visually, not to duplicate but to get you started. Instead of copying work you admire, try to emulate the way that artist thinks visually. Be prepared to make and embrace mistakes and find little moments that work. Find a path from all of the experimentation and mistakes and find your own territory. It takes a long time.\n\nJim Barsness in his studio. Barsness is intrigued by stylization and how it operates in various cultures. For him, pattern is a metaphor for social and cultural contexts, and these designs become a language.\n\n**Other Art in This Style**\n\n\u2022 Gustav Klimt, particularly work from his Golden Phase; other artists from the Symbolist and Art Nouveau movements\n\n\u2022 Dot paintings in Indigenous Australian art\n\n\u2022 Ancient Italian mosaics; illuminated manuscripts from the Middle Ages\n\n**Jim Barsness**\n\n_Hanuman's Rescue_\n\nAcrylic on canvas\n\n90\" \u00d7 66\" (229cm \u00d7 168cm)\n\nVARIATIONS ON THE EMBELLISHED FORM\n\n**Diana Ingalls**\n\n_Ties that Bind_\n\nAcrylic on canvas with pencil and paper\n\n20\" \u00d7 16\" (51cm \u00d7 41cm)\n\n**Variation 1: Combine Symbols With Realism**  \nA pattern overlay infuses this image with symbols, transforming a portrait into archetype. Graphic elements combine with realism to create a complex image and rich surface.\n\n**More Variations**\n\n\u2022 Collect patterns and designs from books, images, advertisements, labels, fabric and wallpaper. Cut out, trace or transfer these to your painting in select areas or overall. Allow pattern designs to be opaque, covering the painting underneath, or transparent, allowing the underpainting to be visible but partially obscured.\n\n\u2022 Make your own patterns by starting with one thing: a symbol, letter, number or abstract shape. Photocopy, transfer or redraw this one item onto a larger surface. Use the same form repeatedly, attached to itself in a chain, turned, flipped and chopped in various ways and combinations with itself until a pattern is formed that goes beyond the initial singular element.\n\n**Darlene McElroy**\n\n_Ingres Redux 2_\n\nAcrylic and mixed media on panel\n\n8\" \u00d7 8\" (20cm \u00d7 20cm)\n\n**Variation 2: Mix Contemporary With Traditional**  \nA formal portrait is applied as an image transfer over a vintage scrapbook from the 1800s, then painted and collaged to create a contemporary tribute to French Neoclassical painter Jean Auguste Dominique Ingres.\n\n  JEWEL-LIKE STIPPLING\n\nThe painting surfaces in Barsness's work contain multiple techniques such as glazing, sanding, layering and casting. One of his techniques creates a jewel-like surface by applying paint color in small dot patterns using bottle applicators.\n\n**Materials**\n\n\u2022 **Paints:** Any acrylic paint color\n\n**\u2022 Surface:** Any primed surface\n\n**\u2022 Painting Tools:** Plastic squeeze bottle with small application nozzle, brush, palette knife, mixing palette\n\n**\u2022 Other:** Acrylic gel or medium\n\n**1. Prepare the Materials**  \nSelect a paint color and brush-apply the paint onto your surface to create a background.\n\nWhile the background dries, on a palette, combine this same paint color with acrylic medium or gel in a 1:5 ratio. If you use a thick gel, the stippling will hold higher peaks and stronger dot shapes, while fluid mediums create a softer texture. Using a knife, scoop this mixture into a plastic squeeze bottle or applicator found in hobby and art stores. Here, I combined Golden Fluid Cobalt Teal with Golden Soft Gel Gloss and placed the mixture into a squeeze bottle.\n\n**2. Apply Dot Patterning**  \nSqueeze the paint from the bottle onto the painting surface, creating small dots. After all the dots are applied, let this dry for several hours or overnight. Make sure the dots are dry to the touch before any subsequent overpainting.\n\n**Finished Example**  \nThe raised dot pattern creates a jewel-like texture. Experiment with different sized dots, varying patterns and colors to embellish painted areas.\n\n**Tip**\n\nPastry bags with icing tips can also be used to create varying dot patterns. For a different effect, try squeezing clear gel with no added color from a squeeze bottle directly onto a plain surface to create an underlying jewel-like ground, then apply thin paint color diluted with water on top.\n\nHARD EDGE GRAPHIC\n\nThe phrase \"hard edge graphic\"  \nimmediately brings to mind geometric abstraction, popular in California in the 1960s, with roots going back to the late nineteenth century and later artists such as Piet Mondrian. Images in this genre use precision and clarity with crisp delineations between colors, readily visible in the work of Kasarian Dane.\n\nDane is concerned with the presence of a painting, its physical flat surface, and the canvas as an object in space. Instead of a spatial depth, Dane goes for a substance depth. He works on 1\/8-inch (3mm) thick aluminum sheeting, braced to extend out about 1 inch (25mm) into the space from the wall when hanging for exhibition, which makes it appear to float (see _Untitled_). The relationship of the exhibited paintings with each other and the space around them are key. The thin aluminum projected outward creates a presence that differs from a canvas with edges sitting flat against the wall.\n\n**The Artist's Process**\n\nTo generate ideas, Dane works with the computer, experiments on paper, and collects bits of color from found ads, and other things he finds. His aluminum surfaces are first sanded, degreased and primed. He then divides his surface into horizontal and vertical areas. From there the process is allowed to flow, not planned too far ahead.\n\nDane has experimented with all types of paint applications, like knives and rollers, to get the surface exactly the way he wants. He prefers brush-applying matte acrylics to give a smooth sensual quality to the paint with a hint of brushstroke. He paints, repaints, widens areas, changes colors, and allows his process to be visible in the final piece by allowing remnants of the changes to remain, carrying the history of his process. Perfection from the hard edged forms contrasts with subtle irregular elements. The irregularity comes from: paint drips and other remnants of Dane's process which are allowed to remain visible along the thin aluminum sides; the light brushstroke texture reveals a \"hand at work;\" and a surface texture is created from underlying layers where forms were moved and repainted.\n\n **TIPS  \nfrom the Artist **\n\nOften art schools add pressure to form your work around trends. Don't try to fig-ure out what's hot but do the work that keeps you coming back. Look at a lot of art galleries and museums and soak in everything you can. Try out a lot of stuff. Anything that comes to mind\u2014just try it. Make work that gets you excited to go into your studio day after day.\n\nKasarian Dane in his New York studio.\n\n**Kasarian Dane**\n\n_Untitled, 2009_\n\nAcrylic and Flashe on aluminum\n\n16\" \u00d7 24\" (41cm \u00d7 61cm)\n\n**Other Art in This Style**\n\n\u2022 Piet Mondrian and other artists from the De Stijl or Neo-Plasticism art movement\n\n\u2022 Color Field painters such as Kenneth Noland, Joseph Albers, Ellsworth Kelly, Kasimir Malevich, and shaped canvases of Frank Stella\n\n\u2022 Futurist painters such as Joseph Stella\n\n\u2022 Frederick Hammersley, Al Held, Abraham Gelbart, Keith Haring\n\n\u2022 Minimalist Brice Marden\n\n\u2022 Stained glass artists\n\n\u2022 Contemporary artist Ed Mell paints realistic landscapes and florals using hard edge.\n\nVARIATIONS OF HARD EDGE GRAPHIC\n\n**Dave Yust**\n\n_Chromaxiologic\u2014Inclusion VI with catenary_\n\n_Ogee curve_\n\nAcrylic on canvas (on 38 piece laminated wood stretcher frame)\n\n54\" \u00d7 84\" (137cm \u00d7 213cm)\n\n**Variation 1: Unusual Canvas Shape**  \nThis elliptically shaped painting conveys a relationship between inside painted forms and the outer shaped and constructed canvas.\n\n**Harry Doolittle**\n\n_Gentle Pandemonium_\n\nAcrylic on board with glass and aluminum leaf\n\n43\" \u00d7 32\" (109cm \u00d7 81cm)\n\n**Variation 2: Cut Away Layers**  \nThis painting employs an unusual construction. Canvas board is cut away in a variety of shapes. The board is then placed over metal and painted glass. White areas in the image are silver metal. The glass is seen in the areas that are gray, gold, red and blue. Other colors are painted acrylic.\n\n**Anne Seidman**\n\n_Untitled_\n\nWatermedia on wood\n\n20\" \u00d7 20\" (51cm \u00d7 51cm)\n\n**Variation 3: Contrast Hard Edge With Organic**  \nA variety of high key and neutral colored forms contrast with a textural organic background. The shapes are packed into a set and unified to form a larger shape.\n\n  SOFTENING HARD EDGES\n\nTaping is a great way to get a clean hard edge between two paint colors. Taping, however, can appear mechanical, which can enhance your painting or work against it, depending on the results you want to achieve. By taping one color and hand-painting its adjacent color, the mechanical effects are softened, adding a handmade look to the edge.\n\n**Tip**\n\nTo reduce seepage under the tape and ensure a very clean edge, you can apply a clear gloss acrylic gel or medium before applying the first paint color, along the tape's painting edge to seal any gaps between the tape and painting surface. This is especially helpful if your paint is thin or watery. Apply your paint color immediately over the gel\/medium seal once it is dry to the touch.\n\n**Materials**\n\n**\u2022 Paints:** Any two acrylic paint colors\n\n**\u2022 Surface:** A primed painting surface\n\n**\u2022 Painting Tools:** Soft flat brush\n\n**\u2022 Other:** Masking tape or clear tape\n\n**1. Tape and Paint the First Color**  \nOn a primed surface, apply tape, lining up its edge where the two colors will meet. Press the tape along the painting edge firmly.\n\nPaint the first color, allowing the color to go over the tape. Immediately remove the tape after painting. If left on too long, the tape may be difficult to remove later. Let the paint dry.\n\n**2. Hand Apply the Second Color**  \nSelect a second acrylic paint color and load it onto a soft flat brush. Place the brush at one end and press gently, gliding alongside the previous line. Make sure you breathe while painting to relax your hand and help it move in a straight path. If your brush goes over the edge, correct it later with the first color. Go back and forth alternating with both colors until the edge looks clean.\n\nPATTERN FIELDS\n\nRepeated forms or patterns in an overall \"field\" can produce varying spatial effects, depending on the compositional design and treatment. A flat, tight pattern can evoke a shallow space, yet the illusion of depth can be enhanced by overlapping, contrasting, movement and the treatment of negative space. Patterns are tricky elements to use in paintings. Symmetrical patterning risks producing a wallpaper effect\u2014timidly hanging in our periphery and easily ignored. However, patterns organized into asymmetrical areas and combined with spatial effects can become vibrant and seductive.\n\nJim Waid's sophisticated use of aesthetics, especially contrast, transforms flat decorative patterns into a painting that presents the experience of three-dimensional space. Textural areas contrast with smoothly painted ones, reflective sheens contrast with matte, transparent colors play off opaque, and patterns mingle with solidly painted areas. Waid feels the point of his art is to create a sense of \"presence\" in and on the canvas. He strives for convincing space so the image becomes real, not illusion. His paintings are not illustrations, but rather enactments of the world around him.\n\n**The Artist's Process**\n\nEarly in his painting career, Waid made a commitment to find something original. Along the way he passed through several distinct phases, invented many processes and techniques, even creating his own tools. Waid calls his process the \"guerrilla school of technique\"\u2014using whatever it takes to get the image he wants. He rarely starts a piece knowing what it will look like and lets it develop during the act of painting. The physicality of the paint plays an important role. He works wet in wet, adding colored paint directly into wet gel already on the surface, mixing it all directly on the canvas. He never uses mixing palettes. Super large kitchen spatulas are a favorite tool, along with sponges, rags, scrapers, sticks, brooms, spray guns and paint rollers.\n\n **TIPS  \nfrom the Artist **\n\nUse inspiration from the masters and others who inspire you, and combine this with ambition to create the best. Let go of academic dogma. Think with the paint, not about the paint.\n\nJim Waid working large in his Arizona studio. Inspired by his desert surroundings, Waid keeps his forms and patterns hovering at the edge of recognition, and notes \"The Sonoran Desert has the craziest landscapes. If the gods can use every kind of twisty thing in the world, why can't I?\"\n\n**Other Art in This Style**\n\n\u2022 Wassily Kandinsky\n\n\u2022 Modern artists Henri Matisse and Joseph Stella\n\n\u2022 Postmodern artist David Hockney\n\n\u2022 Decorative patterning in Gothic and early Renaissance art frescoes with Italian masters such as Giotto de Bondone (1267\u20131337)\n\n**Jim Waid**\n\n_Evening Notes_\n\nAcrylic on canvas\n\n79\" \u00d7 61\" (201cm \u00d7 155cm)\n\nVARIATIONS ON PATTERN FIELDS\n\n**Pat Forbes**\n\n_Neutrino Blossoms_\n\nAcrylic and paper on panel\n\nThree panels, each 16\" \u00d7 16\" (41cm \u00d7 41cm)\n\n**Variation 1: Combine Pattern and Sheen**  \nCombining decorative papers with acrylic in a unique layering process produces elegant and rich imagery with a touch of whimsy. Pat Forbes creates unusual surfaces using reflective paints to add a shimmering magical sheen.\n\n**Thaneeya McArdle**\n\n_A Salutary Encounter_\n\nAcrylic on wood panel\n\n8\" \u00d7 10\" (20cm \u00d7 25cm)\n\nPrivate collection\n\n**Variation 2: Highly Detailed Design**  \nThaneeya McArdle uses meditative states and tribal sensibilities to create highly detailed designs conveying energy and flow of spirit and matter. Complementary pairs and intensely bold colors add an optical vibration.\n\n**More Variations**\n\n\u2022 Add decorative papers and patterned objects such as mosaics or fabric to a painting. Compare the difference between adding patterns along a border surrounding an image and adding patterns directly into the image.\n\n\u2022 Start with patterned papers secured onto a surface as background. Use opaque paint to overpaint and eliminate some patterned areas, and transparent paint to emphasize or change others.\n\n\u2022 Paint a realistic still life using several patterned fabrics in your setup.\n\n  GHOST IMAGERY: REVERSE PAINTING\n\nJim Waid has a continual desire to invent. One of his earliest processes was to paint on the back of canvas. The paint partially seeped through to the front creating a new \"ghost image,\" offering surprising new patterns and inspiration for the painting's continued emergence.\n\n**Tip**\n\nPretest the canvas by dripping some water onto its surface. If the droplets hold their form, the fabric needs special washing to remove any water-repellent coatings. Wash the canvas in a washing machine using warm water, adding one tablespoon each of Synthrapol and soda ash. Once any coatings are removed, place the dry canvas over protective plastic on the floor. Either side of the canvas is fine to use; however, whatever side faces up in this step will be the back of the painting later.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint colors\n\n**\u2022 Surface:** Untreated, unstretched medium or heavy weight raw canvas or linen\n\n**\u2022 Painting Tools:** A variety of painting brushes, water\n\n**\u2022 Other:** Any liquid soap or Golden Acrylic Flow Release\n\n**1. Apply Soap to the Reverse Side of the Canvas**  \nDilute liquid soap (a more refined version is Golden Acrylic Flow Release) with water using a 1:4 ratio, and generously apply onto the canvas using a large brush, or other applicator, to the entire surface. Apply enough to wet through to the other side of the canvas. Lift the canvas and remove any excess puddling from underneath or move the canvas to a dry area.\n\n**2. Apply Paint Color**  \nWhile the canvas is still wet, apply paint diluted with water. Start with a 1:1 ratio of paint to water, then experiment with the amount of water to create a variety of dilutions and color intensities. Add enough water so the paint can soak through the canvas threads. Lift the painted canvas and remove excess paint from underneath or move to a dry area. Allow to dry flat.\n\n**3. Flip the Canvas to the Reverse Side**  \nFlip the canvas over to see the results on the reverse side. The colors seep through in a variety of ways, but more subtle and softer, creating an interesting new appearance. Use this side as your painting's new front and first layer. If the front and back are identical, then try this again on a thicker piece of canvas, or use less water in the paint colors.\n**Section 5**\n\nSPATIAL  \nPUSH-PULL\n\n**Gary Denmark**\n\n_Depth Charge_\n\nAcrylic on canvas\n\n36\" \u00d7 36\" (91cm \u00d7 91cm)\n\nRenowned teacher and artist Hans Hofmann coined the phrase push-pull to describe a visual play of pictorial space\u2014the sensation of the eye being pushed and pulled into the illusory depths of the painting's space. Here lies a signature of contemporary art, containing both directional movements, a push toward the illusory depths and a pull back toward the front picture plane, and a continued tug of war between the two. This section offers examples of nonobjective abstract imagery that make use of this effect.\n\nMARKING SPACE\n\nThis style has roots in the Abstract Expressionist and Surrealist movements, which focused on the subconscious and spontaneous coming through the work. Here marks carry expression, and are also key to creating the spatial qualities. Jackson Pollock's drip paintings are one example of how an artist collaborates with the material to express a gesture, creating a signature in the paint. An ambiguous shape, form or mark can appear as a Rorschach test, prodding us to find something recognizable and yet also existing as just a mark. Leah Dunaway's paintings have an emphasis on mark making and architectural space. She uses linear elements such as straight lines and corners, delineating architectural spaces. These contrast with organic marks such as squiggles and loops to create an evocative pictorial space, a \"vibratory push-pull.\" Her paintings often involve a sheet of Plexiglas, bolted and overlayed onto the work, adding an industrial component and drama by casting shadows and enhancing the sense of space. Her work keeps evolving, each new series emerging from the previous one.\n\n**The Artist's Process**\n\nGrowing up with an architect father, Dunaway learned to read blueprints and to this day is attracted to architectural forms and spaces, which she continually adds to her subconscious \"file.\" Patterns, shadows and the play of light through forms continually interest her. Architectural elements appear naturally in her work, since her subconscious plays a main role in her process. Dunaway works on several canvases at the same time, usually working flat as opposed to on an easel, encourages drips, and creates most of her marks using charcoal and oil stick. Her agenda is to \"engage in serious play\" like a child, with no expectations. During her painting process she continually turns the painting around in different ways, waiting a long time to declare it finished. Artists who inspire Dunaway include Hans Hofmann, Jackson Pollock, Richard Diebenkorn and former art instructor Katherine Chang Liu (featured).\n\n **TIPS  \nfrom the Artist **\n\nContinual growth will keep your work fresh. Continue to modify, change and expand what your work is about. Your whole life will feed into it. Everything you do goes into the same giant soup pot to make a chili.\n\nDunaway often finds inspiration in familiar surroundings such as hardware stores, where she can find small sample bottles of house paint, in decorator colors both opaque and muted, for experimenting cheaply at home.\n\n**Other Art in This Style**\n\n\u2022 German and Abstract Expressionists such as Jackson Pollock (check out his early work in addition to the drip paintings), Mark Tobey, Joan Mir\u00f3, Wassily Kandinsky, Willem de Kooning, Paul Klee, Henri Michaux, L\u00e1szl\u00f3 Moholy-Nagy\n\n\u2022 Contemporary artists: Sam Scott, Agnes Martin, Vernon Fisher's \"blackboard\" paintings, Joan Mitchell\n\n\u2022 The calligraphic-style scribble paintings of Cy Twombly\n\n**Leah Dunaway**\n\n_Fusion 2_\n\nAcrylic on canvas\n\n56\" \u00d7 46\" (142cm \u00d7 117cm)\n\nVARIATIONS ON MARKING SPACE\n\n**Beth Ames Swartz**\n\n_The Fire and the Rose: But Heard, Half-Heard, in the Stillness_\n\nAcrylic on canvas\n\n54\" \u00d7 66\" (137cm \u00d7 167cm)\n\n**Variation 1: Contrast Marks With Background**  \nSmall vertical strokes repeat to create a background field. This emphasizes the larger lines which are curvy, bold and gestural.\n\n**Katherine Chang Liu**\n\n_Drawing #1_\n\nAcrylic and mixed media on paper\n\n11\u00bd\" \u00d7 12\u00bd\" (29cm \u00d7 32cm)\n\nCourtesy Jenkins Johnson Gallery, San Francisco and New York\n\n**Variation 2: Vary Marks**  \nA limited palette of colors contrasts with the wide variety of marks, including text, hand drawn lines, graphs and brushstrokes.\n\n**More Variations**\n\n\u2022 Experiment making a variety of marks using different tools. Vary how you hold the tool, the type and consistency of paint you use, and the surfaces. Keep these experiments as reference for later ideas.\n\n\u2022 Start with one mark on a blank canvas. Take a moment to consider what this one mark feels like. Make another mark that responds to the first, either by being similar or opposite, or keeping some qualities while changing others. Continue in this dialogue making, more marks, each one responding to the last, and noting how each new mark affects the whole image.\n\n  HOMEMADE PAINTING TOOLS\n\nJim Waid, featured in the previous chapter, likes to invent his own painting tools to obtain the marks he prefers. Here are some ideas to expand your application options.\n\n**Tip**\n\nPriming your cardboard before or after you cut the comb from it will make it more durable. Applying a coat of gloss acrylic medium over the background color will give cleaner marks and ease for the subtractive part in step 3.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint color different than the surface color\n\n**\u2022 Surface:** Any primed surface pre-painted with color\n\n**\u2022 Other:** Gloss acrylic gel, painting knife, cardboard, cutting knife\n\n**1. Cut Out a Cardboard Comb**  \nUsing thick cardboard and a sharp cutting knife, cut small triangles or wedge shapes into the cardboard, leaving a margin at the top. Vary each triangle in size and length to obtain a less predictable comb effect with the paint.\n\nHere is a finished hand-cut cardboard comb, along with a surface pre-painted with brush-applied Pyrrole Orange.\n\n**2. Apply a Gel Layer**  \nSelect a color that contrasts with the background color. Using a knife, mix it with thick gloss gel in a 1:1 ratio. Mixing gloss gel into the paint slows the drying, allowing more working time. Generously apply the color mixture over the background. Here the blue color is made with Primary Cyan, Titanium White and Soft Gel Gloss that is then applied over the Pyrrole Orange background.\n\n**3. Create Subtractive Marks by Combing**  \nComb through the wet blue paint layer to \"subtract\" the blue while creating lines in the underlying orange color. Smooth out some areas with a painting knife, then repeat subtractive combing, moving in a different direction. Repeat as much as desired.\n\n**Other Homemade Tool Ideas**  \nJoin commercial brushes together to make an oversized brush for large strokes and marks. Consider using non-art tools for unusual marks such as kitchen spatulas and other household objects, scrapers, squeegees, combs, mops and whisk brooms. Browse through home improvement, decoration and discount stores for objects, imagining what type of mark they would make, and experiment.\n\nFREE-FLOATING ORGANICS\n\nForms that are abstract or nonobjective can appear to float in space when there is no distinguishing horizon line or sense of ground. Barbara Moody's painting pictured opposite gives a feeling of being underwater or suspended in air. It is easy to see from the organic shapes in the painting that nature is an inspiration for Moody, and no surprise that she lives in a house filled with specimen jars from her incredible self-made nature collection.\n\n**The Artist's Process**\n\nStarting flat on the floor, Moody makes marks on her surface, then applies overlapping layers of paint. The marks she makes determine the content from there. Moody does not rely on reference material on hand, working mostly out of her head. She admits that years of traditional figurative painting instruction and many years of painting experience have made this viable. However, because she is accomplished at drawing and painting realistically, it would be too easy for her to copy reference material, so by keeping them out of sight she is able to make her actions more her own. She relies on accidents that can then be more deliberately worked.\n\nMoody pushes herself into unknown places by using asymmetrical composition, or colors she finds unattractive. She mixes colors in big plastic cups in large quantities, helpful for big paintings, and never uses primary colors right out of the tube. She likes to make \"nameless\" colors, such as celery, peach or apricot, ones that are not as easy to identify as red or blue. Each layer has areas that are softened, quieted down by glazing, and edited, while a few select items are enhanced. Every day she sketches, and at night she goes through her journal and makes notes, often writing about conceptual ideas to get to the essence of the mood or feeling\u2014not so much the subject. After completing a series, Moody moves on, fueling her sense of discovery and her desire to create something that has never been seen before. She feels her work has to come from within and not from an external source, representing who she is and containing a type of authenticity.\n\n **TIPS  \nfrom the Artist **\n\nWork flat or on the floor instead of at an easel. Pour, scrape, use odd tools to add spontaneity. Respond to happy accidents. Feel as if you are back in kindergarten but with wise intent. Allow yourself to discriminate, edit, refine, eliminate and enhance.\n\nBarbara Moody in her Massachusetts studio. Moody is inspired by young cutting-edge painters because they push the boundaries of painting today and have fewer preconceived habits from modernist dogma.\n\n**Other Art in This Style**\n\nWorks by Joan Mir\u00f3, Wassily Kandinsky, Marc Chagall, Ross Bleckner, Joan Snyder, Lee Krasner and Roberto Matta\n\n**Barbara Moody**\n\n_Ecosystem_\n\nAcrylic on canvas\n\n60\" \u00d7 48\" (152cm \u00d7 122cm)\n\nVARIATIONS OF FREE-FLOATING ORGANICS\n\n**Lisa Ferguson**\n\n_Escaping the chaos to a peaceful haven_\n\nAcrylic and mixed media on canvas\n\n80\" \u00d7 60\" (203cm \u00d7 152cm)\n\n**Variation 1: Minimal Color Palette**  \nUsing a minimal color palette places the focus on the fluid flowing forms.\n\n**Sam Scott**\n\nBlues for Charlie Parker\n\nAcrylic on canvas\n\n80\" \u00d7 66\" (203cm \u00d7 168cm)\n\n**Variation 2: No Horizon Line**  \nWith no recognizable horizon line to evoke a landscape, organic forms are energized in space.\n\n**Patti Brady**\n\n_Blue Bonkers_\n\nAcrylic on paper (with transfers and digital images)\n\n27\" \u00d7 27\" (69cm \u00d7 69cm)\n\n**Variation 3: Variety of Patterns and Forms**  \nA variety of patterning and forms are created through collage, stencils and ink-jet printed acrylic skins.\n\n  EDGE GHOSTING\n\nGary Denmark, whose work is shown on Gary Denmark and Gary Denmark, uses this printmaking technique in his paintings to soften some of the edges of his painted forms. It's a great way to integrate hard edged geometric forms with soft, organic areas and shapes.\n\n**Tip**\n\nIf you don't clean the plastic from step 2, you can save it and reuse it in a collage by cutting up the plastic and regluing it into another painting. Another recycling idea: Apply clear acrylic gel over the dry shape left on the plastic. Let dry. Peel off as a skin and use in a collage.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint color\n\n**\u2022 Surface:** A primed painting surface\n\n**\u2022 Painting Tools:** A variety of brushes\n\n**\u2022 Other:** A clear plastic sheet\n\n**1. Paint a Shape**  \nOn a primed or pre-painted surface, brush-apply a shape. This background is painted with a light green color made from Titanium White, Turquoise Phthalo and Permanent Green, while the shape uses a pink made of Titanium White and Quinacridone Red.\n\n**2. Blot the Wet Paint**  \nWhile the painted shape is wet, place a sheet of plastic over it and rub gently to pick up some excess color. This will create an imprint or light textural pattern.\n\n**3. Remove the Plastic**  \nCarefully remove the plastic by lifting straight up. Place the plastic down again slightly off register to the original shape to create a double image along the edge. Rub to transfer the excess color onto the surface. The plastic can be cleaned and reused by wiping with a damp cloth.\n\n**Finished Example**  \nOriginally a hard edge shape, it is now softened with the addition of a ghost or double of itself slightly overlapping.\n\nTEXT, LINE & IMAGE\n\nUsing text in an image can create a peculiar visual play, and an enriched viewing experience. Text can be seen as symbol, sound or as a graphic, and can enhance any narrative qualities. Former graphic designer Debi Pendell learned to see areas of text as shape, color and value, and to respond to letters and words abstractly. Pendell views everything as a symbol: letters, words, trees, brushstrokes, line, color and texture, and combines these to create a readable space. At first she paints a believable reality, and then she purposely \"spoils\" it to change the way we view it, like placing a wrong value or odd size somewhere, or peppering her image with letters to bring attention to the front surface.\n\n**The Artist's Process**\n\nPendell starts by building up an initial background using collaged papers, often adding painted elements. Some imagery, like the trees in _Proposed Possibility_ , are her own photos turned into gel skin transfers. Pendell creates a faux encaustic (wax) appearance by applying a variety of acrylic gels ranging from very thick to very thin, both gloss and matte. The multiple layers of gel add a depth and a real physical space, underscoring the illusion of space she creates with her landscape format. The matte gels add a nice encaustic look, but often obscure the text and imagery too much, so instead she works with Golden High Solid Gel Gloss to get the thickness, followed by several final coats of matte medium on top. This adds control over how much veiling the matte acrylic will produce. To ensure that letters are read as abstract marks, Pendell purposely spaces them apart enough to avoid having them come together as words. Pendell finds inspiration in artists such as Henri Matisse, Robert Rauschenberg, Sylvia Plimack Man-gold, Giorgio Morandi, Sean Scully and Louise Bourgeois.\n\n **TIPS  \nfrom the Artist **\n\nIn the process of painting, many emotions can come up, like frustration, anger and fear. If you hold back, the work shows repression and the desire to be polite, creating kitsch. Instead, welcome all these emotions, including joy, allowing things to flow. All you have inside will then pour out and the censoring stops. Don't be afraid of the struggle.\n\nDebi Pendell in her Massachusetts studio..\n\n**Other Art in This Style**\n\n\u2022 Contemporary artists: Ed Rusha, Jenny Holzer, Julian Schnabel, Robert Indiana\n\n\u2022 Asian woodcuts, especially from Buddhist texts, include poetry and writing seamlessly into the imagery\n\n\u2022 Joseph Kosuth, Gino Severini, Barbara Kruger, Larry Rivers\n\n**Debi Pendell**\n\n_Proposed Possibility_\n\nAcrylic, collage and mixed media on canvas\n\n48\" \u00d7 48\" (122cm \u00d7 122cm)\n\nCollection of Caron and Scott Palladino,\n\nSaratoga Springs, NY\n\nVARIATIONS OF TEXT, LINE & IMAGE\n\n**Teresa Stanley**\n\n_Not At All_\n\nAcrylic and mixed media on wood panel\n\n36\" \u00d7 48\" (91cm \u00d7 122cm)\n\n**Variation 1: Enhance Painted Forms With Linear Elements**  \nA physical three-dimensional quality is sustained by Teresa Stanley's addition of collaged paint skins and paint. Lighting grid plans with technical references were silkscreened into this image, creating a dialogue between art and science.\n\n**Bill Dunlap**\n\n_Unholy Trinity_\n\nPolymer paint on canvas\n\n49\" \u00d7 37\" (124cm \u00d7 94cm)\n\nCollection of the artist\n\n**Variation 2: Use Readable Text for Impact**  \nText here is kept readable as word with attention to placement and shape creating an immediate association and impact.\n\n**More Variations**\n\n\u2022 Experiment with balancing lines, text and imagery by changing the amount of each in a piece. Try almost all text with barely any imagery, and the reverse, mostly imagery with scant text. Change your process by starting with text then adding imagery, or the reverse.\n\n\u2022 Explore the spacing between letters to find the amount of space needed to form words, and the distance needed for them to read as design or graphic elements.\n\n\u2022 Use writing you find inspirational, from books, poetry or other sources, whether personal or found, and find ways of incorporating sections of the writing into your work.\n\n  ACCIDENTAL STENCILING\n\nStencils are used here in an unorthodox way to create accidental marks. When used conventionally, stencils can sometimes offer imagery that is too specific to integrate, with hard edged forms and a mechanical look. This technique shows a way to use stencils that avoid the commercial cutout appearance, adding softer abstract elements by using smaller portions of the stencil design.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic paint colors\n\n**\u2022 Surface:** Any primed painting surface\n\n**\u2022 Painting Tools:** Painting knife with stepped handle\n\n**\u2022 Other:** Heavy or very thick acrylic gloss gel, a plastic stencil, paper towel\n\n**1. Prepare the Materials**  \nSelect a paint color and stencil design. Tape the stencil onto a painting support with or without a background color. Make a mixture of color and acrylic gel in a 1:1 ratio. Adding thick gloss gel slows the drying, eases application and reduces leakage underneath the stencil.\n\n**2. Apply Color to the Stencil**  \nLoad a painting knife along the back side, with plenty of the paint\/gel mixture. Begin by depositing the paint onto the plastic part of the stencil, not too close to the open cutout portion of the design. Press down hard with the flat side of the knife, and swipe across the stencil, allowing mistakes, missed areas and skimpy paint in some places. By pressing hard, only small portions of the design get transferred, creating unusual abstract \"hand-applied\" design elements.\n\n**3. Remove the Stencil**  \nCarefully lift the stencil off to reveal fragments of the design. Repeat using different stencils and new colors to get a buildup of interesting abstract marks.\n\nGEOMETRY & SPACE\n\nComposing with geometric shapes offers a contemporary way to create space. Katherine Chang Liu creates a delicate balance between geometric and organic shapes; color contrasts; and combinations of drawing, collage and paint. Liu's paintings result in a balance between chaotic and controlled emotional content. Her work focuses on our daily sensory overload with visual, literary, musical and verbal information. Of her work, Liu states she maps \"this emotional state with autobiographical marks and a reference to the urban environment in which I live and work.\"\n\n**The Artist's Process**\n\nLiu gets her inspiration from words, through poetry, news articles\u2014anything that triggers a visual possibility. She continually creates lists of words, allowing time for ideas to simmer. Around these ideas, Liu makes twenty or more thumbnail sketches for general design, overall shapes and color atmosphere. Liu also collects items, which she readily collages into her work. Working in a series, she still only focuses on one painting at a time. Each painting in the series is a comment on the last one. Surfaces are built up using a variety of materials, including acrylic, charcoal, fabric, wood, direct drawing and fragmented paint areas. Working for that true image, Lui remains unattached and continues adding and subtracting paint and layers. Sometimes this fails, but Liu doesn't try to please others; she just needs to please herself. The work needs to reflect back to her like a self-portrait or mirror. If the work comes too easily to her technically, she doesn't trust it and changes it. Questions arise for her such as: How do you cover something and still reveal it? Is it just sitting there on the surface, or worse, jumping off the page? Would slightly veiling it make it more integrated and add more mystery? Something too obvious might be improved if veiled and allowed to be discovered at a second glance. How to hold the weight of importance? How much is allowed to sit on the surface is the artist's choice.\n\n **TIPS  \nfrom the Artist **\n\nEvery painting we make is a self-portrait.\n\nLiu frequently curates art exhibitions and acts as juror to many competitions, keeping her current on the art scene. Artists that inspire Liu include Robert Irwin, Robert Rauschenberg, Anselm Kiefer and Joseph Cornell.\n\n**Other Art in This Style**\n\n\u2022 Hans Hofmann, Fernand L\u00e9ger, Mark Rothko and other Color Field artists\n\n\u2022 Antonio Tapies, Eva Hesse, Piet Mondrian, Jean H\u00e9lion, Wassily Kandinsky, Jasper Johns, Franz Kline, Robert Mangold\n\n\u2022 Auguste Herbin, who developed a movement emphasizing color through geometry\n\n**Katherine Chang Liu**\n\n_Night Walk_\n\nAcrylic and mixed media on panel\n\n30\" \u00d7 30\" (76cm \u00d7 76cm)\n\nCourtesy LewAllen Contemporary Gallery,\n\nSanta Fe, NM\n\nVARIATIONS OF GEOMETRY & SPACE\n\n**Sandra Duran Wilson**\n\n_The Other Side_\n\nAcrylic, gold leaf and collage on panel\n\n40\" \u00d7 48\" (102cm \u00d7 122cm)\n\nPrivate collection of Naresh and Kavita Nakra\n\n**Variation 1: Experimental Techniques and Surfaces**  \nThe combination of techniques and surfaces with geometry creates a unique space. Painting on old hollow core doors cut down to size, Sandra Duran Wilson first applies gold leaf to the entire surface. Heat and viscosity are used to push the medium around to create effects. Monoprints and wood block prints are cut in strong shapes then collaged, while glazing shifts colors. The circles are recurring symbols for the artist, representing overlapping dimensions of time.\n\n**Gary Denmark**\n\n_Jongleur 2_\n\nAcrylic on canvas\n\n48\" \u00d7 48\" (122cm \u00d7 122cm)\n\n**Variation 2: Contrast Geometric With Organic Shapes**  \nThe bold use of geometric shapes contrasts well with gradations of color, fluid spaces and organic forms.\n\n**More Variations**\n\n\u2022 Collect a variety of dispensable items such as smooth and textured papers and old paintings you no longer care for. Cut out different geometric shapes in a variety of sizes and in a range of colors. Arrange these on different painting surfaces to see how they change the painting. Rearrange the items to see how the spaces between the shapes change the idea of space in the painting.\n\n\u2022 Find a painting you admire. Overlay tracing paper and trace the large shapes created by elements in the painting. Create a new painting using these large shapes. Try rearranging them, overlapping them differently and adding your own imagery.\n\n  AVOIDING ATTACHMENT\n\nLiu does not get too attached to any part of the painting while working. Favored elements frequently present problems by inhibiting continued progress. Liu often uses materials that are precious and has ways of avoiding this attachment \"trap.\" Try this project below to test your unattachment skills.\n\n**Materials**\n\n**\u2022 Paints:** A variety of opaque and transparent acrylic paint colors\n\n**\u2022 Surface:** Any sturdy surface\n\n**\u2022 Painting Tools:** Favorite painting brushes, knives, rags, a mixing palette\n\n**\u2022 Other:** Any collage item you like (paper, photographs, fabric, objects, drawings), acrylic gloss gel\n\n**1. Glue the Collage Item**  \nUsing a surface that is much bigger than your collage item (could be a new surface or a painting already started), find a placement for the collage item with ample space around it for adding paint later. Glue the item securely in place using an acrylic gel. Here lies the big trick to avoiding attachment syndrome\u2014making a commitment by permanently gluing the object to the surface.\n\n**2. Mix Up a Variety of Opaque and Transparent Colors**  \nFor opacity, choose mineral (inorganic) pigments such as Cadmiums and Titanium White. Transparent pigments can be made opaque by using them thick, straight out of the container or by adding white. For transparent colors, choose modern (organic) pigments such as Phthalos and Quinacridones. Opaque pigments can be made transparent by adding large amounts of gloss mediums, or by applying thinly. Pre-mix a few colors that match the background of your painting and also of the collaged item. Match colors 10 percent lighter than desired, since acrylic appears lighter when wet.\n\n**3. Apply Varying Degrees of Coverage**  \nApply pre-mixed colors over the collage item and onto the painting surface. Obtain various degrees of coverage by heavily applying the paint using a knife in some areas, and by applying lighter applications with a brush or rag in others. Scrape off paint using a knife in some places to add even more variety.\n\n**Tip**\n\nIf your collage item is thin, like tissue paper, a thick acrylic (gel) offers a better choice for a glue than a thin acrylic (medium) and will reduce wrinkling. Use a matte gel if the glue will seep through the collage material, otherwise a gloss gel is best.\n**Section 6**\n\nMINIMAL\n\n**Paul Sarkisian**\n\n_Untitled (black mark\/amber\/vertical3)_\n\nResin epoxy on wood\n\n12'\u00d7 12' (366cm \u00d7 366cm)\n\nPhoto by Eric Swanson\n\nMinimalism presents exciting challenges for both the artist and the viewer. By limiting or eliminating representative imagery, the artist works solely with aesthetic aspects to create work that attracts. A painter's aesthetic tool box consists of color, line, shape, plane, space, surface and light. These tools in themselves carry associative and narrative connections. In this section, styles use a minimum of aesthetic aspects to get maximum impact, usually evoking a meditative mood or an altered state or reality, allowing viewers to lose themselves in the purely visual.\n\nSHEENS & TEXTURE\n\nUsing sheens and texture places the focus on the painting's surface and can create a seductive tactile experience. _Canyon and Poem_ combines metal leaf with acrylic paint. This combination presents an interesting challenge for me, as the metal leaf changes qualities like a chameleon. As available light shifts throughout the day, the metallic changes from a dark value to light, and from bright to dull. Realizing that busy imagery and bold color can overpower the reflective qualities of the metal leaf, I researched historic Asian landscapes and their expert use of minimal forms and color. A play with opposites adds a sense of space in the painting, with areas containing both high and low refractive qualities, smooth and textured areas, and warm and cool glazing on the metal leaf.\n\n**The Artist's Process**\n\nExperimentation is my main painting tool. I keep playing around until I discover something new that takes me over. I allow my techniques, processes and materials to change according to the needs of each series, allowing the idea to best come through the work. Inventing the way I paint is just as exciting to me as what I paint. For _Canyon and Poem_ , metal leaf is applied onto smooth panels then overpainted with acrylic. I get inspired by artists such as Anselm Kiefer, Wassily Kandinsky, Damien Hirst, and not surprisingly, many of the artists I interviewed for this book. Other ideas come from a continual searching for things I find beautiful, whether natural or man-made: fabrics, butterfly wings, cityscapes, mountains, skies, oceans and people. I draw from models once a week to keep my drawing skills alive, and continually enjoy visiting artists' studios, galleries and museums.\n\n**Other Art in This Style**\n\n\u2022 Chinese landscape paintings from about AD 900\u20131600\n\n\u2022 Illuminated manuscripts from the Middle Ages into the Renaissance period\n\n\u2022 Gustav Klimt's (1862\u20131918) use of metal leaf as backgrounds for sensuously painted figures\n\n **TIPS  \nfrom the Artist **\n\nDon't take action on every idea that comes to you. Keep a journal to write down ideas as they come, allowing them to percolate until the right one really fuels you. Then take action. Inspired action tells you the next step. Time, experimenting and experiences bring artists to their personal vision. The inspiration for our unique vision is already in us, making it difficult for anyone to copy what we do successfully. Do what comes naturally, but make the time to create and the bravery to make things you might not have seen before. Workshops and classes are helpful, but take long periods or enough time in between to work in the studio and let the new tools and techniques simmer into your own stew.\n\nNancy Reyner in her Santa Fe studio at work on her metal leaf paintings.\n\n**Nancy Reyner**\n\n_Canyon and Poem_\n\nAcrylic and gold leaf on panel\n\n48\" \u00d7 36\" (122cm \u00d7 91cm)\n\nPrivate collection\n\nVARIATIONS OF SHEENS & TEXTURE\n\n**Bette Ridgeway**\n\n_It Was a Rainy Red Autumn_\n\nAcrylic with resin on aluminum\n\n32\" \u00d7 24\" (81cm \u00d7 61cm)\n\n**Variation 1: Create a Glasslike Surface with Resin**  \nClear layers of resin are applied in between colored layers, generating a glasslike surface with embedded textural effects. An aluminum surface is allowed to reflect through multiple transparent layers.\n\n**Reynaldo Villalobos**\n\n_Redemption 1_\n\nAcrylic on panel\n\n60\" \u00d7 48\" (152cm \u00d7 122cm)\n\n**Variation 2: Textured, Monochromatic Palette**  \nA monochromatic palette places focus on textures and sheens. The texture here is actually only visual, with no relief or physical texture. The effect is created by allowing paint to seep under previously taped areas, creating a thin but dramatic variation in darks and lights.\n\n**More Variations**\n\n\u2022 Create a textured ground or surface under metal leaf by first applying thick acrylic pastes or gels and letting it dry before applying the leaf. The leaf will then emphasize this underlying texture.\n\n\u2022 Create a collage using materials with a variety of sheens and textures.\n\n  CONTRASTING METALLICS\n\nMetal leaf is an appealing material to use. There is a noticeable difference in the refraction between metal leaf and metallic paints. By contrasting them together in the same painting an interesting result can be obtained.\n\n**Tip**\n\nUse imitation gold leaf with a waterbased adhesive to avoid tarnishing. When using paint color, avoid reducing the reflective quality of the metal leaf by using modern (organic) pigmented paints like Phthalos and Quinacridones instead of the mineral (inorganic) paints like Cadmiums, which are opaque. Avoid matte or satin acrylic products as they will reduce the leaf's reflective qualities.\n\n**Materials**\n\n**\u2022 Paints:** A variety of metallic acrylic paints (copper, gold, bronze, silver)\n\n**\u2022 Surface:** Any painting support\n\n**\u2022 Painting Tools:** Soft flat or foam brush, painting knife\n\n**\u2022 Other:** Metal leaf, leafing adhesive, mixing palette, gloss acrylic gel, mineral spirits-based acrylic varnish in spray or jar\n\n**1. Seal the Metal Leaf**  \nApply metal leaf on a support using leaf adhesive and following directions on the product's label. Overpaint or spray the leaf with a mineral spirits-based acrylic and let dry. This coating will ensure good adhesion for any subsequent acrylic paint layers.\n\n**2. Create a Variety of Metallic Mixtures**  \nUsing a variety of metallic acrylic paints (i.e., copper, bronze, gold, silver, etc.), mix them together in various combinations to create new metallic colors. As an option, add clear gloss acrylic gels into the paint to add texture.\n\n**3. Overpaint the Metal Leaf**  \nApply the various metallic paint mixtures over selected areas of the metallic leaf surface. Use a brush or knife, and apply thinly for a smooth application or thickly to create texture. Avoid covering the leaf surface completely so the leaf can be seen along with the paint. Metallic paint will result in a satin finish as compared to the leaf's highly reflective sheen.  \nAs an alternative in this step, substitute thinly applied transparent glazes of color to subtly shift the metallic leaf instead of overpainting with the metallic paints.\n\nILLUMINATED\n\nIllumination or the representation of light in a painting can be conveyed both figuratively or literally. Light has always played an essential role for painters. Chiaroscuro, developed in the Renaissance, uses paint to create dramatic lights and dark shadows indicating a strong light source and adding depth and mystery to the image. Contemporary artists such as Dan Flavin create art using actual light as the medium itself. Stained glass, photography and light projections are all art forms that use actual or physical light sources in the final art.\n\nPaul Sarkisian's art career spans an astonishing range. Sarkisian continually invents and reinvents to create works that allow us to question our perception of reality. _Untitled (bronze\/ green42)_ is from a series of three hundred works by Sarkisian and uses contoured surfaces and pearlescent pigment to create a surface that color-shifts from different viewing angles and in different light. Sarkisian sees them as fields of energy, greatly affected by their placement in space.\n\n**The Artist's Process**\n\nSarkisian's favorite tool is his creative thoughts. He thinks of how to make his idea in its purest form, resulting in a wide range of mediums, styles and processes. Works are created using tools such as airbrush, trowel and brush and in modes such as collage, photography, photorealism and minimalist abstraction, with mediums including acrylic, oil, asphalt, tar, gouache, gypsum, resin and foam. What he does with the technology he chooses is very important to him. In general, Sarkisian works on one piece at a time within a series.\n\n **TIPS  \nfrom the Artist **\n\nLots of people are afraid to use their talents, and are intimidated by the art scene and superstars. If you have talent, why not use it?\n\nPaul Sarkisian in his studio, in front of one of his paintings.\n\n**Other Art in This Style**\n\nWorks using real light sources by artists such as Dan Flavin, Olafur Eliasson, James Turrell, Waltraut Cooper, Aleksandra Stratimirovic and Austine Wood Comarow.\n\n**Paul Sarkisian**\n\n_Untitled (bronze\/green42)_\n\nPolymer resin and automotive enamel on wood\n\n6'5\" \u00d7 11'8\" (196cm \u00d7 336cm)\n\nPhoto by Eric Swanson\n\n**Detail**\n\nVARIATIONS ON ILLUMINATION\n\n**Sandy Keller**\n\n_Lapis_\n\nAcrylic on panel\n\n24\" \u00d7 36\" (61cm \u00d7 91cm)\n\n**Variation 1: A Rich Value Range in a Single Color**  \nThe use of a monochromatic palette places emphasis on the rich value range and feeling of illumination from within the painting's depths.\n\n**More Variations**\n\n\u2022 Find different lighting situations and paint, photograph or draw these to capture their mood and feeling.\n\n\u2022 Notice shadows and patterns created from the sun and other light sources and use these for painting inspirations.\n\n\u2022 Create a still life using one light source. Paint the dark cast shadows and highlighted areas to capture the feel of light on the objects.\n\n**Bette Ridgeway**\n\n_Yellow Moon_\n\nAcrylic with resin on aluminum\n\n30\" \u00d7 24\" (76cm \u00d7 61cm)\n\n**Variation 2: Colorful Layers Over an Aluminum Surface**  \nResin layers over an aluminum surface creates refraction and illumination. The image itself, although minimal in forms, parallels this light idea by subtly referring to a moonlit landscape.\n\n  ILLUMINATED POURING\n\nThis technique takes advantage of two categories of reflective paints: Iridescent and Interference. Both can be used singly or in combinations to create new, unusual, contemporary illuminated effects. This technique uses Iridescent in the background and Interference in a transparent overpour. Alternatively, the addition of color to this technique will create a vast array of possibilities. Some helpful tips and definitions before starting:\n\n**\u2022 Rigid surface:** Pouring is easiest when done over a rigid, sturdy support such as panel or wood.\n\n**\u2022 Reflective Mixtures:** Adding any Iridescent or Interference to a clear medium or gel will make a reflective mixture. Adding a small amount of black to interference enhances its color. If adding color, use small amounts to keep the color transparent.\n\n**\u2022 Pourable Mediums:** Any acrylic medium that will dry clear and glossy and readily pours from its container can be used in a pour technique. Th in the medium with up to 20 percent water or use specialty pouring products like Golden's GAC 800 or commercial resins.\n\n**Materials**\n\n**\u2022 Paints:** Several Iridescent and Interference paints (optional addition: any transparent modern paint color)\n\n**\u2022 Surface:** Any rigid or sturdy paint surface\n\n**\u2022 Painting Tools:** Any brush or other painting application tool, plastic mixing container and stirrer, wide flat spreading tool or knife, mixing palette\n\n**\u2022 Other:** A pourable acrylic medium, household spray bottle, isopropyl alcohol, water\n\n**1. Paint the Background with Reflective Paint**  \nUsing a brush or knife, apply any Iridescent and\/or Interference paint in any combination to create a reflective background. Here Iridescent Silver (fine) is knife-applied. Let the background dry fully.\n\n**2. Add Black Washes**  \nWith a brush, add enough clean water to the entire surface to create puddles. On a separate palette, mix black acrylic paint with water in a 1:10 ratio. Apply this black wash to the surface puddles. Let dry naturally, and the black washes will form small irregular shapes and patterns. These dark shapes help enhance the pour you will do in Step 3.\n\n**3. Pour a Transparent Reflective Mixture**  \nCreate a reflective mixture by adding any reflective paint to a pourable medium in a 1:10 ratio to maintain transparency. (Pictured here is 9 parts Clear Tar Gel with 1 part water and 5 drops each of Interference Blue, Interference Violet and Interference Red.) Lightly stir, then pour the mixture over the entire surface. Mist lightly with isopropyl alcohol. Let dry flat. Repeat Steps 2 and 3 as much as desired, letting each layer dry before adding another.\n\n**Finished Example**\n\nCOLOR FIELD\n\nEmerging out of New York City in the 1940s and 1950s, Color Field paintings generally consist of large unbroken areas of pure color, often creating an emotional or expressive field, with minimal attention to brushstroke, texture and forms. Aleta Pippin's desire to experiment and work with color as her primary tool is readily visible in her work, and her choice of working minimally allows an internal viewing.\n\n**The Artist's Process**\n\nPippin does not like to over-plan. She usually starts by pouring a color onto the surface, setting up spontaneous events or color combinations and continuing until she finds something beautiful. She then decides to enhance, add or change areas to bring visual depth and light to the color. Pippin chooses application techniques that minimize the brush mark. She finds the color richer when applied with a knife. Applied thinly, the knife allows the paint to overlay without affecting the bottom layers, easing a building up of paint layers. Pippin continually revisits and reevaluates what she is doing and why she is doing it. She is not afraid to take a different direction and try something new, realizing each new path has a learning curve, requiring a sense of bravery to allow the feeling of being a beginner again. Early in her career Pippin was inspired by Wassily Kandinsky, Willem de Kooning and Georgia O'Keeffe. Now she is more influenced by artists such as Gustav Klimt and Helen Frankenthaler. Additional inspiration comes from listening to music from Pink Floyd to George Gershwin.\n\n **TIPS  \nfrom the Artist **\n\nBelieve in what you're doing. Be focused and don't listen to what others say. Artists are entrepreneurs\u2014don't buy into the starving artist myth. Learn basic business skills to get started.\n\nAleta Pippin at work in her Santa Fe studio.\n\n**Other Art in This Style**\n\n\u2022 Pioneering Color Field painters: Mark Rothko, Helen Frankenthaler, Clyfford Still, Robert Motherwell, Barnett Newman, Hans Hofmann, Ad Reinhardt, Sam Francis, Anselm Kiefer\n\n\u2022 Pat Steir's waterfall paintings\n\n**Aleta Pippin**\n\n_The Fabric of Life #4_\n\nAcrylic on Yupo paper mounted on panel\n\n8\" \u00d7 8\" (20cm \u00d7 20cm)\n\nCollection of Patti and Bill Nash\n\nVARIATIONS ON COLOR FIELD\n\n**Robin Sierra**\n\n_Aquamarine_\n\nAcrylic on canvas\n\n25\" \u00d7 27\" (64cm \u00d7 69cm)\n\nCollection of Anton Lengmueller\n\n**Variation 1: Minimize Forms**  \nThis strong unified color field contains a range of color in softly integrated formless areas. The image verges on becoming recognizable as water or reflections.\n\n**Sandy Keller**\n\n_Time Lapse_\n\nAcrylic on panel\n\n48\" \u00d7 36\" (122cm \u00d7 91cm)\n\n**Variation 2: Flowing Areas**  \nA bold use of flowing forms creates movement, light and pattern within a strong field of color. \"It is always about color and creating that magic moment when two or more colors vibrate against each other. That compelling force draws the viewer in, perhaps evoking a memory of a transient moment once seen,\" says Keller.\n\n**More Variations**\n\n\u2022 Collect pieces of pure color (without recognizable forms to distract attraction) by ripping pieces of magazine pictures and collecting chips from paint stores and swatches of fabric. Remake these colors in paint form and build them up in paint layers to enhance the color's richness.\n\n\u2022 Minimize the forms from an image you like by only painting its colors and shapes, and avoiding any detail. Then remove your original reference from view to continue painting. Merge and blend shapes into each other by softening edges to create an overall field of color.\n\n  COLOR ENHANCEMENT\n\nWhen color is applied in several layers, the pigment takes on a richer quality. With multiple layers, light is able to refract in more complex ways, enhancing color and surface quality. Here is a technique using three layers to enhance color quality without changing hue.\n\n**Tip**\n\nThis technique works best when each step uses very transparent layers. For the pour mixture in step 3 it is important to use very little paint color and\/or keep application thin.\n\n**Materials**\n\n**\u2022 Paints:** Any acrylic color in several variants of its hue\n\n**\u2022 Surface:** Any rigid absorbent surface\n\n**\u2022 Painting Tools:** Soft flat blending brush, mixing palette\n\n**\u2022 Other:** Acrylic Glazing Medium or other slow drying medium, household spray bottle, mixing cup, mixing stick, palette knife, isopropyl alcohol, water\n\n**1. Apply Color as a Wash**  \nStart with an absorbent painting surface, or make it absorbent by applying an absorbent product on first as a ground. A surface merely primed with gesso will not be as effective. Products that make good absorbent grounds will dry matte with a discernible tooth or texture, such as acrylic pastes, matte gels or gels with added materials like pumice. Alternatively, you can adhere watercolor paper to your painting surface.\n\nOnce you've attained an absorbent surface, select a color and heavily dilute the acrylic paint with water in a 1:5 ratio. Apply this color wash using a soft, wide, flat brush over the entire surface. Here Cadmium Red Medium Hue, properly diluted with water, is applied over a panel prepared with Golden's Acrylic Ground for Pastel. Let the surface dry. Repeat this step if more color is desired using less water in the wash mixtures.\n\n**2. Apply the Color as a Glaze**  \nUsing the same color from the prior step, or a variant of the same hue, combine color to gloss medium in a 1:10 ratio without adding any water. Optimally, use a slow-dry medium for this mixture to make glazing easier. Here, Primary Magenta is mixed with Acrylic Glazing Liquid and evenly brush-applied over the dried surface from the previous step. Let dry.\n\n**3. Pour the Color**  \nRaise the painting surface off your work table with jars or other objects for support and place on plastic. Add the same color as before, or a variant of the same hue into a pourable gloss medium in a 1:20 ratio. Optionally, add up to 20 percent water if the medium is thick. Here Pyrrole Red is mixed with GAC 800 (no water is needed with this Golden product). Pour this mixture in a puddle in the center of the surface. Gently spread the mixture outward toward the corners and edges with a wide knife. Immediately spray the surface lightly with isopropyl alcohol in a household sprayer to remove any bubbles. Keep level while drying.\n\nA SINGULAR NOTE\n\nA dramatic visual impact can be obtained by presenting one strong form against a minimal field of color. This is readily seen in Jim Alford's painting _Confluence_. A hard edged circle placed against a soft background allows the illusion of floating. Jim Alford aligns with Minimalist philosophies, in particular with the difference between gazing at a painting and staring at it in a conventional Western way. Alford's intent is to offer a meditative experience for the viewer. Interestingly, Alford has his own mind-altering experience in the painting process. Alford typically paints using an airbrush. As he works on layer after layer, he progressively steps back with the sprayer farther and farther from the work, in a rhythmic movement similar to Tai Chi, often placing Alford in a meditative state as well.\n\n**The Artist's Process**\n\nAlford's work spans an impressive range from super-realistic to minimal abstraction. This diversity mandates an equal range in techniques and processes. Alford often begins his paintings on the computer, experimenting with colors and shapes, sometimes constructing an image with combinations of several photographs he has taken. Once he finds an image he likes, he takes it to his studio, transferring the idea into paint. Acrylic is his choice for spraying, which is less toxic than oil. Alford finds his inspiration in nature and landscape, walking along California beaches looking at the sun reflecting in the ocean, and in horizon lines and figures silhouetted against dark skies. Artists inspiring to him include Larry Rivers, Richard Dieben-korn, Mark Rothko, James Terrell, and contemporary landscape painters such as Robert Workman, Peter Nisbet and Doug West.\n\n **TIPS  \nfrom the Artist **\n\nEvery artist has to find his voice and it takes a long time. Graduate school is one way to assist this process, especially by selecting teachers who allow freedom.\n\nJim Alford's studio view offers a continual reminder of nature and landscape.\n\n**Other Art in This Style**\n\nWorks by Morris Louis, Barnett Newman's zip paintings, Yves Klein, Agnes Martin and Susan Rothenberg\n\n**Jim Alford**\n\n_Confluence_\n\nAcrylic on canvas\n\n60\" x 48\" (152cm x 122cm)\n\nVARIATIONS ON A SINGULAR NOTE\n\n**Steve Stone**\n\n_Nol_\n\nAcrylic on canvas\n\n60\" \u00d7 36\" (152cm \u00d7 91cm)\n\nPrivate collection\n\n**Variation 1: A Subtle, Singular Form**  \nA textural field supports a subtle, singular emerging form, a cross, in the upper right through the use of acrylic pastes and gels and a variety of mark-making tools.\n\n**Grant Wiggins**\n\n_Spaceloop Two_\n\nAcrylic on canvas\n\n30\" \u00d7 30\" (76cm \u00d7 76cm)\n\nCollection of the artist\n\n**Variation 2: A Bold Singular Form**  \nRacing stripes are graphically illuminated with fluorescent paint and emphasized on a solid black background.\n\n**More Variations**\n\n\u2022 Select some favorite forms including geometric, organic, realistic and abstract. Draw or cut them out of colored paper. Arrange each one on different surfaces as backgrounds using paintings, tiles, fabric, wallpaper, etc. Use these as a model for creating a painting from scratch.\n\n\u2022 Experiment with materials and techniques to create an overall background or field of color containing some accidental marks or forms. Select one of these accidental forms and make it more visible with painting techniques such as increasing the color hue or intensity, making it lighter or brighter, sharpening the edges and\/or blurring or softening everything around it.\n\n  TEXTURE WITH MESH TAPE\n\nA subtly textured background can add visual appeal to a minimal abstraction. The grid pattern of dry wall mesh tape creates an interesting effect when used in this two-layer technique using paste and washes.\n\n**Tip**\n\nEliminate step 3 by substituting thick acrylic paint color for the paste in step 2.\n\n**Materials**\n\n**\u2022 Paints:** Several acrylic paint colors\n\n**\u2022 Surface:** A primed or pre-painted surface\n\n**\u2022 Painting Tools:** Any brush, painting knife or other applicator tool\n\n**\u2022 Other:** Drywall self-adhesive mesh tape, absorbent acrylic paste, scissors\n\n**1. Attach Drywall Tape**  \nOn a dry surface, pre-painted with a background color, apply strips of drywall mesh tape. Press firmly to stick the tape to the surface. The background color pictured here is a mixture of Titanium White and Primary Blue.\n\n**2. Apply Paste**  \nGenerously load an acrylic paste that is absorbent or dries matte to the back of a painting knife (Light Molding Paste is shown here). Apply the paste over the tape using medium pressure. For variety, allow some areas of the tape to remain without any paste. Remove the tape and allow paste to dry at least a few hours or overnight.\n\n**3. Overpaint With Washes**  \nDilute each of several acrylic paint colors with water in a 1:5 ratio to create several wash colors. You can also pre-wet the dry paste surface with water in some areas to increase a bleed effect. Brush-apply the diluted colored washes separately over the paste, varying colors in areas. Blot with paper towel in some areas to lighten. Pictured here are washes using Phthalo Blue, Carbon Black, Dioxazine Purple, Anthraquinone Blue, Phthalo Turquoise, Quinacridone Burnt Orange and Nickel Azo Yellow.\n\n**Finished Example**\n\nVEILING FORMS\n\nA veil is something that hides yet reveals. Veiling in painting happens when a transparent or translucent layer is applied over another layer, partially hiding what's underneath. Veiling can soften a focus, shape or edge. It can create a misty atmospheric space or add a surface quality similar to encaustic or wax. Veiling adds a sense of mystery in the act of partially hiding things. Bonnie Teitelbaum's paintings use multiple layers, building one on top of another, veiling forms and color areas to create fluid fields. Teitelbaum feels veiling adds more dimension, simulating nature, while flat applications of color feel more inorganic. Her paintings often evoke distilled ecosystems.\n\n**The Artist's Process**\n\nMeditative processing is a key part of Teitelbaum's painting process. She starts her day painting early in the morning to take advantage of feeling centered. Teitelbaum sees painting as a collaboration between herself and the chemistry of the paint, noting that she needs to put in a certain amount of paint and time to get the image to a certain point. First she experiments to see what happens, often discovering new and unusual forms. These accidental discoveries then get developed further with more paint. She frequently applies gels over the painting in stages between layers. Gels are mixed with color, then poured on top of other layers. While the gels are still wet, she adds more color and manipulates the work.\n\n **TIPS  \nfrom the Artist **\n\nIt's good to discover new tools. Abstraction relies on experimentation and discovery. Education is also important, learning about the characteristics of paint and materials.\n\nTeitelbaum gets inspiration out in nature, offering her peace of mind, and which she intends to convey through her work. Teitelbaum is inspired by James Abbott McNeil Whistler's reduction of elements to distill a landscape. She is also inspired by Vincent Van Gogh for movement, brushwork and color.\n\n**Other Art in This Style**\n\n\u2022 The painted forms of Joseph Mallord William Turner (1775\u20131851) that dissolve into atmosphere\n\n\u2022 The evocative, moody works of James Abbott McNeill Whistler's (1834\u20131903) Nocturnes series\n\n\u2022 Abstract Expressionist painters Morris Louis and Helen Frankenthaler\n\n**Bonnie Teitelbaum**\n\n_Tidal Flow_\n\nAcrylic on panel\n\n24\" \u00d7 24\" (61cm \u00d7 61cm)\n\nVARIATIONS ON VEILING FORMS\n\n**Doug Trump**\n\n_Breezeway_\n\nMixed media on polymesh\n\n21\" \u00d7 33\" (53cm \u00d7 84cm)\n\n**Variations: Neutral Colors and Obscured Forms**  \nThese paintings by Doug Trump represent excellent examples of veiling. Trump uses neutral colors plus obscured forms to create a sense of mystery, allusiveness and space. For Trump a painting is a demonstration of its history. He works it until it breathes on its own.\n\n**Doug Trump**\n\n_Tiptoe_\n\nMixed media on paper\n\n32\" \u00d7 40\" (81cm \u00d7 102cm)\n\n**More Variations**\n\n\u2022 While creating a painting, place pieces of tissue paper over areas without attaching them just to see how veiling will change its appearance. When you find a place where veiling improves the work, apply the tissue permanently using acrylic as glue, or apply layers of matte gel, or transparent applications of colored paint.\n\n\u2022 Dry-brushing or using thin layers of paint can also produce translucent layers of color that can act as veils. Try finding several ways of applying a layer of color on top of another without being so opaque that it completely hides the underlayer.\n\n  EMBEDDING PAINTED FORMS\n\nAn intriguing spatial depth can be created when paint is embedded in transparent layers of acrylic. You can create some interesting options and effects with this technique.\n\n**Tips**\n\n\u2022 Use a matte gel for this technique to create an encaustic or waxy look.\n\n\u2022 Gloss will give the most clarity, while matte or satin will give a frosted or veiled appearance. Try varying the types of gel each time you repeat Step 1 for a new layer.\n\n**Materials**\n\n**\u2022 Paints:** A variety of acrylic paint colors\n\n**\u2022 Surface:** A nonstick surface for acrylic such as freezer paper, HDPE plastic or garbage bags\n\n**\u2022 Painting Tools:** Palette knife or spatula, brush\n\n**\u2022 Other:** Acrylic gel (matte, satin or gloss)\n\n**1. Apply Gel to a NonStick Surface**  \nSelect an acrylic gel. Using a wide spatula, apply gel onto a nonstick surface in a thick layer, at least 1\/8-inch (3mm) thick. Work with the knife to get the surface fairly smooth.\n\nIt's your choice to continue to the next step while the gel is still wet, or to wait until it's dry. Each option will produce different results. Experiment both ways to find your preference.\n\n**2. Apply Paint Color**  \nSelect several acrylic paint colors using a range of hues and transparencies. Add water to each of the paint colors. Start with a 1:1 ratio, then create variety by adding more water to increase the transparency, and less to increase the opacity. Apply paint to the top surface of the wet or dry gel using a brush or knife. If desired, spray water over the paint before it dries to get softer edges and blurred forms. Let dry on a level surface.\n\n**3. See the Reverse Side**  \nThe gel should dry for at least a day or more until the white of the acrylic has turned clear. Peel the gel off the surface. Note its reverse side is smoother and the painted forms appear embedded. This \"skin,\" or unattached piece of painted gel, can now be glued onto a background with either side facing up.\n\n**Alternative for Step 3**  \nBefore securing the skin onto a background, repeat the first two steps to create more skin layers. Combine skins one on top of the other, attaching them by gluing with more gel.\nCONTRIBUTING ARTISTS\n\n**Jim Alford**\n\nwww.jimalford.com\n\nNew Mexico\n\n**Hamish Allan**\n\nwww.hamishallan.co.nz\n\nChristchurch, New Zealand\n\n**Daniel Barkley**\n\nwww.danielbarkley.com\n\nMontreal, Canada\n\n**James Barsness**\n\nwww.georgeadamsgallery.com\n\nwww.cclarkgallery.com\n\nGeorgia\n\n**Gerard Boersma**\n\nwww.gerardboersma.nl\n\nThe Netherlands\n\n**Lea Bradovich**\n\nwww.leabradovich.com\n\nwww.nuartgallery.com\n\nNew Mexico\n\n**Patti Brady**\n\nwww.pattibrady.com\n\nSouth Carolina\n\n**Bruce Cody**\n\nwww.brucecody.com\n\nNew Mexico\n\n**Dennis Culver**\n\nwww.doculver.com\n\nNew Mexico\n\n**Kasarian Dane**\n\nwww.kasariandane.com\n\nNew York\n\n**Jason de Graaf**\n\njasondegraaf.blogspot.com\n\nQuebec, Canada\n\n**Gary Denmark**\n\nwww.garydenmark.com\n\nNew Mexico\n\n**Harry Doolittle**\n\nwww.harrycdoolittle.com\n\nNew York\n\n**Leah Dunaway**\n\nwww.leahdunaway.com\n\nTexas\n\n**William Dunlap**\n\nwww.williamdunlap.com\n\nVirginia\n\n**Joey Fauerso**\n\nwww.joeyfauerso.com\n\nTexas\n\n**Frances Ferdinands**\n\nwww.francesferdinands.com\n\nToronto, Canada\n\n**Lisa Ferguson**\n\nwww.lisaferguson.com\n\nNoosa, Australia\n\n**Pat Forbes**\n\nwww.patriciaforbesart.com\n\nNew Mexico\n\n**Phil Garrett**\n\nwww.philgarrett.com\n\nSouth Carolina\n\n**Cate Goedert**\n\nwww.categoedert.com\n\nNew Mexico\n\n**Jylian Gustlin**\n\nwww.jyliangustlin.com\n\nCalifornia\n\n**Diana Ingalls Leyba**\n\nwww.dianaingallsleyba.com\n\nNew Mexico\n\n**McCreery Jordan**\n\nwww.mccreeryjordan.com\n\nNew Mexico\n\n**Sandy Keller**\n\nwww.sandykellerart.com\n\nNew Mexico\n\n**Martha Kennedy**\n\nwww.marthakennedy.com\n\nNew Mexico\n\n**Jakki Kouffman**\n\nwww.jakkikouffman.com\n\nNew Mexico\n\n**Ines Kramer**\n\nwww.ineskramer.com\n\nNew Mexico\n\n**Katherine Chang Liu**\n\nwww.lewallencontemporary.com\n\nwww.jenkinsjohnsongallery.com\n\nCalifornia\n\n**Sherry Loehr**\n\nwww.sherryloehr.com\n\nCalifornia\n\n**Catherine Mackey**\n\nwww.catherinemackey.com\n\nCalifornia\n\n**Thaneeya McArdle**\n\nwww.thaneeya.com\n\nFlorida\n\n**Darlene McElroy**\n\nwww.darleneoliviamcelroy.com\n\nNew Mexico\n\n**Barbara Moody**\n\nwww.barbaramoody.com\n\nMassachusetts\n\n**Keith Morant**\n\nwww.keithmorant.com\n\nChristchurch, New Zealand\n\n**Mary Morrison**\n\nwww.marymorrison.info\n\nColorado\n\n**Tom Palmore**\n\nwww.flyingpaintbrush.com\n\nOklahoma\n\n**Mary L. Parkes**\n\nwww.marylparkes.com\n\nNew Mexico\n\n**Debi Pendell**\n\nwww.debipendell.com\n\nMassachusetts\n\n**Ren\u00e9e Phillips**\n\nwww.ReneePhillipsArt.com\n\nNew York\n\n**Aleta Pippin**\n\nwww.aletapippin.com\n\nNew Mexico\n\n**Paul Pletka**\n\nwww.rivayaresgallery.com\n\nNew Mexico\n\n**Don Quade**\n\nwww.donquade.com\n\nColorado\n\n**Nancy Reyner**\n\nwww.nancyreyner.com\n\nNew Mexico\n\n**Bette Ridgeway**\n\nwww.ridgewaystudio.com\n\nNew Mexico\n\n**Paul Sarkisian**\n\nNew Mexico\n\n**Nancy Scheinman**\n\nwww.scheinman.com\n\nMaryland\n\n**Sam Scott**\n\nwww.samscottart.com\n\nNew Mexico\n\n**Olga Seem**\n\nolgaseem@earthlink.net\n\nCalifornia\n\n**Anne Seidman**\n\nwww.anneseidman.com\n\nPennsylvania\n\n**Robin Sierra**\n\nwww.rsierra.net\n\nNew Mexico\n\n**Daniel Smith**\n\nwww.danielsmithwildlife.com\n\nMontana\n\n**Teresa Stanley**\n\nwww.teresastanley.com\n\nCalifornia\n\n**Steve Stone**\n\nwww.stevestoneart.com\n\nArizona\n\n**James Strombotne**\n\nwww.strombotnestudio.com\n\nCalifornia\n\n**Beth Ames Swartz**\n\nwww.bethamesswartz.com\n\nArizona\n\n**Dannielle Tegeder**\n\nwww.dannielletegeder.com\n\nNew York\n\n**Bonnie Teitelbaum**\n\nwww.bonnieteitelbaum.com\n\nNew Mexico\n\n**Doug Trump**\n\nwww.dougtrump.com\n\nVermont\n\n**Reynaldo Villalobos**\n\nreynaldovillalobos@earthlink.net\n\nCalifornia\n\n**Jim Waid**\n\nwww.jimwaidart.com\n\nArizona\n\n**Frank Webster**\n\nwww.fwebster.com\n\nNew York\n\n**Grant Wiggins**\n\nwww.wiggz.com\n\nArizona\n\n**Sandra Duran Wilson**\n\nwww.sandraduranwilson.com\n\nNew Mexico\n\n**Dave Yust**\n\ndavyust@lamar.colostate.edu\n\nColorado\n\n","meta":{"redpajama_set_name":"RedPajamaBook"}}
+{"text":" \n# Milk Proteins\n\n## From Expression to Food\n\nSecond edition\n\nHarjinder Singh\n\nMike Boland\n\nAbby Thompson\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\n# Table of Contents\n\nCover\n\nTitle page\n\nFood Science and Technology International Series\n\nCopyright\n\nList of Contributors\n\nPreface to the Second Edition\n\nPreface to the First Edition\n\nChapter 1: The World Supply of Food and the Role of Dairy Protein\n\nAbstract\n\nIntroduction\n\nHunger and the need for food\n\nThe dietary essential amino acids in proteins\n\nIdentifying the countries deficient in dietary essential amino acids\n\nDemographic changes, aging populations, and the need for quality protein and essential amino acids\n\nGlobal trade in proteins, the long-term prospects, with a focus on dairy foods\n\nConclusions\n\nChapter 2: Milk: An Overview\n\nAbstract\n\nIntroduction\n\nEvolution of mammals and lactation\n\nUtilization of milk\n\nComposition of milk\n\nMilk constituents\n\nSummary\n\nChapter 3: The Comparative Genomics of Monotremes, Marsupials, and Pinnipeds: Models to Examine the Functions of Milk Proteins\n\nAbstract\n\nIntroduction\n\nThe echidna (Tachyglossus aculeatus)\n\nThe tammar wallaby (Macropus eugenii)\n\nA role for milk in the control of mammary function\n\nThe fur seal\n\nNew player in milk bioactives; MicroRNA\n\nConclusions\n\nChapter 4: Significance, Origin, and Function of Bovine Milk Proteins: The Biological Implications of Manipulation or Modification\n\nAbstract\n\nIntroduction\n\nOrigins of milk proteins\n\nConstraints and opportunities for evolution or manipulation of bovine milk proteins\n\nConclusion\n\nChapter 5: Post-translational Modifications of Caseins\n\nAbstract\n\nIntroduction\n\nThe caseins\n\nCaseins from other species\n\nConclusions\n\nChapter 6: Casein Micelle Structure and Stability\n\nAbstract\n\nIntroduction\n\nCasein primary structure and interactions\n\nCasein micelle properties\n\nModels of casein micelle structure\n\nConcluding remarks\n\nChapter 7: Structure and Stability of Whey Proteins\n\nAbstract\n\nIntroduction\n\nBovine \u03b2-Lactoglobulin\n\n\u03b1-Lactalbumin\n\nSerum albumin\n\nImmunoglobulins\n\nLactoferrin\n\nConcluding remarks\n\nAcknowledgments\n\nChapter 8: Effects of High-pressure Processing on Structure and Interactions of Milk Proteins\n\nAbstract\n\nIntroduction\n\nHigh-pressure-induced changes in caseins\n\nEffects of high pressure on interactions of milk proteins involving whey proteins\n\nConcluding remarks\n\nAcknowledgment\n\nChapter 9: The Whey Proteins in Milk: Thermal Denaturation, Physical Interactions, and Effects on the Functional Properties of Milk\n\nAbstract\n\nIntroduction\n\nThe casein micelle\n\nThe heat treatment of milk\n\nRelationships between denaturation\/interactions of the whey proteins in heated milk and the functional properties of milk\n\nConclusion\n\nChapter 10: Effects of Drying on Milk Proteins\n\nAbstract\n\nIntroduction\n\nProperties of spray-dried milk products\n\nPrinciples of spray drying\n\nProcess improvement\n\nDrying of proteins\n\nConclusions\n\nChapter 11: Changes in Milk Proteins during Storage of Dry Powders\n\nAbstract\n\nIntroduction\n\nThe formation of maillard and pre-maillard compounds\n\nFormation of isopeptide bonds\n\nAmino acids other than lysine\n\nImplications for nutritional value of milk proteins\n\nProduct-specific storage trials\n\nConclusions\n\nChapter 12: Interactions and Functionality of Milk Proteins in Food Emulsions\n\nAbstract\n\nIntroduction\n\nAdsorption of Milk Proteins During the Formation of Emulsions\n\nStability of Milk Protein-Based Emulsions\n\nHeat-Induced Changes in Milk Protein-Based Emulsions\n\nPressure-Induced Changes in Milk Protein-Based Emulsions\n\nMilk Protein Hydrolysates and Oil-In-Water Emulsions\n\nLactoferrin-Based Oil-In-Water Emulsions\n\nLipid Oxidation in Milk Protein-Based Emulsions\n\nBehavior of Milk Protein-Stabilized Emulsions Under Physiological Conditions\n\nConclusions\n\nChapter 13: Milk Protein\u2013Polysaccharide Interactions\n\nAbstract\n\nIntroduction\n\nMixing behavior of biopolymers\n\nPhase diagram\n\nNature of interactions in protein\u2013polysaccharide systems\n\nMilk protein\u2013polysaccharide interactions in the aqueous phase\n\nMilk protein\u2013polysaccharide interactions at the interface\n\nRheological properties and microstructures of protein\u2013polysaccharide systems\n\nConcluding remarks\n\nChapter 14: Interactions between Milk Proteins and Micronutrients\n\nAbstract\n\nIntroduction\n\nInteractions Between native Milk Proteins and Micronutrients\n\nInteractions between process-modified milk proteins and micronutrients\n\nConclusions\n\nChapter 15: Model Food Systems and Protein Functionality\n\nAbstract\n\nIntroduction\n\nProtein functionality in foods\n\nRole of interactions in determining food characteristics\n\nProcessing effects\n\nUses of model food systems\n\nApplications of model food systems\n\nUse of model food systems for other food components\n\nLimitations\n\nConclusions\n\nChapter 16: Sensory Properties of Dairy Proteins\n\nAbstract\n\nIntroduction\n\nSensory analysis\n\nWhey proteins\n\nMilk proteins\n\nCaseins and hydrolysates\n\nFlavor binding\n\nConclusions\n\nAcknowledgment\n\nChapter 17: Milk Protein Gels\n\nAbstract\n\nIntroduction\n\nRennet-induced gels\n\nAcid-induced milk gels\n\nWhey protein gels\n\nConclusions\n\nAcknowledgment\n\nChapter 18: Milk Proteins\u2014A Cornucopia for Developing Functional Foods\n\nAbstract\n\nIntroduction\n\nFunctional foods\n\nMilk proteins as a source of amino acids\u2014specialized nutritionals\n\nMilk proteins as a source of amino acids\u2014specific physiological roles\n\nMilk proteins as a source of amino acids\u2014role in providing calories and in promoting satiety\n\nMilk proteins as a source of bioactive peptides\n\nConclusions\n\nChapter 19: Milk Proteins and Human Health\n\nAbstract\n\nIntroduction\n\nMilk proteins, metabolic health, and type 2 diabetes\n\nMilk proteins, obesity, and weight control\n\nMilk proteins and bone health\n\nConclusions\n\nChapter 20: Milk Proteins: Digestion and Absorption in the Gastrointestinal Tract\n\nAbstract\n\nIntroduction\n\nDigestion of milk proteins\n\nMilk protein hydrolysis in the intestinal lumen\n\nPeptides released during digestion\n\nImpact of processing on milk protein digestion and absorption\n\nConclusions\n\nChapter 21: Milk Proteins: The Future\n\nAbstract\n\nIntroduction\n\nGlobal issues for food\n\nConsumer demands and trends for food and ingredients\n\nNew technologies and their possible effect on milk protein ingredients and products\n\nConclusions\n\nIndex\n\nFood Science and Technology: International Series\n\n# Food Science and Technology International Series\n\nSeries Editor\n\nSteve L. Taylor\n\nUniversity of Nebraska\u2014Lincoln, USA\n\nAdvisory Board\n\nKen Buckle\n\nThe University of New South Wales, Australia\n\nMary Ellen Camire\n\nUniversity of Maine, USA\n\nRoger Clemens\n\nUniversity of Southern California, USA\n\nHildegarde Heymann\n\nUniversity of California\u2014Davis, USA\n\nRobert Hutkins\n\nUniversity of Nebraska\u2014Lincoln, USA\n\nRon S. Jackson\n\nQuebec, Canada\n\nHuub Lelieveld\n\nBilthoven, The Netherlands\n\nDaryl B. Lund\n\nUniversity of Wisconsin, USA\n\nConnie Weaver\n\nPurdue University, USA\n\nRon Wrolstad\n\nOregon State University, USA\n\nA complete list of books in this series appears at the end of this volume.\n\n# Copyright\n\nAcademic Press is an imprint of Elsevier\n\n32 Jamestown Road, London NW1 7BY, UK\n\n225 Wyman Street, Waltham, MA 02451, USA\n\n525 B Street, Suite 1800, San Diego, CA 92101-4495, USA\n\nCopyright \u00a9 2014, 2009 Elsevier Inc. All rights reserved\n\nNo part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher\n\nPermissions may be sought directly from Elsevier's Science & Technology Rights\n\nDepartment in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier.com. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com\/rights for further information\n\nNotice\n\nNo responsibility is assumed by the publisher for any injury and\/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made\n\nBritish Library Cataloguing-in-Publication Data\n\nA catalogue record for this book is available from the British Library\n\nLibrary of Congress Cataloging-in-Publication Data\n\nA catalog record for this book is available from the Library of Congress\n\nISBN : 978-0-12-405171-3\n\nFor information on all Academic Press publications visit our website at elsevierdirect.com\n\nTypeset by Thomson\n\nPrinted and bound in United States of America\n\n14 15 16 17 10 9 8 7 6 5 4 3 2 1\n\n# List of Contributors\n\nSkelte G. Anema\n\nFonterra Research and Development Centre, Palmerston North, New Zealand\n\nS.D. Berry\n\nSchool of Population Health, University of Auckland, Auckland, New Zealand\n\nSwathi Bisana\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nMike J. Boland\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\nSrikanta Chatterjee\n\nMassey University, Palmerston North, New Zealand\n\nRod Collins\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nTh\u00e9r\u00e8se Considine\n\nFonterra Research and Development Centre, Palmerston North, New Zealand\n\nM. Digby\n\nDepartment of Zoology and CRC for Innovative Dairy Products, University of Melbourne, Victoria, Australia\n\nM.A. Drake\n\nDepartment of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA\n\nDidier Dupont\n\nUMR 1253 INRA, Rennes, France\n\nPatrick J.B. Edwards\n\nCentre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand\n\nJohn Flanagan\n\nRiddet Institute, Massey University, Palmerston North, New Zealand; Naturex S.A., Avignon Cedex, France\n\nP.F. Fox\n\nSchool of Food and Nutritional Sciences, University College, Cork, Ireland\n\nKelvin K.T. Goh\n\nInstitute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand\n\nW. James Harper\n\nDepartment of Food Science and Technology, The Ohio State University, Columbus, Ohio, USA\n\nKerianne Higgs\n\nFonterra Research and Development Centre, Palmerston North, New Zealand\n\nJohn W. Holland\n\nInstitute for Molecular Bioscience, The University of Queensland, Australia\n\nDavid S. Horne\n\nFormerly Hannah Research Institute, Ayr, Scotland, UK\n\nThom Huppertz\n\nNIZO Food Research BV, Ede, The Netherlands\n\nGeoffrey B. Jameson\n\nCentre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand\n\nAmit Kumar\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nJoly Kwek\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nChristophe Lefevre\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nSimon M. Loveday\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\nJohn A. Lucey\n\nDepartment of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA\n\nRobin A. McGregor\n\nHuman Nutrition Unit, Institute for Innovation in Biotechnology, School of Biological Sciences, University of Auckland, Auckland, New Zealand\n\nK. Menzies\n\nDepartment of Zoology and CRC for Innovative Dairy Products, University of Melbourne, Victoria, Australia\n\nR.E. Miracle\n\nDepartment of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA\n\nVengama Modepalli\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nPaul J. Moughan\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\nKevin R. Nicholas\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia; Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia\n\nJ.A. O'Mahony\n\nSchool of Food and Nutritional Sciences, University College, Cork, Ireland\n\nHasmukh A. Patel\n\nDairy Science Department, South Dakota State University, South Dakota, USA\n\nSally D. Poppitt\n\nHuman Nutrition Unit, Institute for Innovation in Biotechnology, School of Biological Sciences, University of Auckland, Auckland, New Zealand; Riddet Institute, Massey University, Palmerston North, New Zealand\n\nAnwesha Sarkar\n\nNestec Ltd., Vevey, Switzerland\n\nArnab Sarkar\n\nSynlait Milk Ltd., Rakaia, New Zealand\n\nPierre Schuck\n\nINRA, UMR 1253, STLO, Rennes, France\n\nJulie A. Sharp\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nP.A. Sheehy\n\nCentre for Advanced Technologies in Animal Genetics and Reproduction (REPROGEN), Faculty of Veterinary Science, The University of Sydney, Camden, New South Wales, Australia\n\nHarjinder Singh\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\nR.G. Snell\n\nSchool of Biological Sciences, University of Auckland, Auckland, New Zealand\n\nDaniel Tome\n\nUMR 914 INRA, Paris, France\n\nStephen Wanyonyi\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nAshalyn Watt\n\nSchool of Medicine, Deakin University, Geelong, Victoria, Australia\n\nP. Williamson\n\nCentre for Advanced Technologies in Animal Genetics and Reproduction (REPROGEN), Faculty of Veterinary Science, The University of Sydney, Camden, New South Wales, Australia\n\nJ.M. Wright\n\nDepartment of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA\n\nP.C. Wynn\n\nCentre for Advanced Technologies in Animal Genetics and Reproduction (REPROGEN), Faculty of Veterinary Science, The University of Sydney, Camden, New South Wales, Australia\n\nAiqian Ye\n\nRiddet Institute, Massey University, Palmerston North, New Zealand\n\n# Preface to the Second Edition\n\nIt is now five years since the first edition of Milk Proteins: From Expression to Food was published. In that time there have been considerable advances in the topics covered in the first edition. There is new awareness both of the importance of proteins in nutrition globally and of a burgeoning demand for animal-derived protein on a global scale, coupled with a recognition of the higher resource demands for producing proteins from animals in comparison with plant proteins. New knowledge is emerging on the roles of milk proteins in the prevention of chronic age-related conditions such as adverse metabolic health and type 2 diabetes, muscle wasting and sarcopenia, atherosclerosis, hypertension, and cardiovascular disease risk, as well as bone health and osteoporosis. Understanding the digestion of proteins and how processing can influence digestion kinetics is another emerging area, supported by the emergent science of peptidomics. Because of these changing research interests, three new chapters have been added to this volume, and several other chapters have been extensively expanded and rewritten. All chapters have been considerably revised and updated. Because not all of the original authors were available, some new authors came aboard to rewrite and update the chapters.\n\nLike the first edition, this book provides a comprehensive overview of the biology and chemistry of milk proteins and is intended for academics, researchers, students, and industry personnel interested in milk proteins. We would like to express our sincere appreciation to all the authors, in particular the new authors, for sharing their knowledge and expertise. Their cooperation and timely delivery of manuscripts made our task as editors a pleasure. Finally, we would like to thank the excellent staff at Elsevier, particularly Nancy Maragioglio, Carrie Bolger, and Caroline Johnson for their efforts in making this volume a reality.\n\nHarjinder Singh\n\nAbby Thompson\n\nMike Boland\n\nFebruary 2014.\n\n# Preface to the First Edition\n\nProteins are vital ingredients for the food industry because they provide all the essential amino acids needed for human health combined with a wide range of dynamic functional properties, such as the capacity to form network structures and stabilize emulsions and foams. The proteins of milk have excellent functional properties and nutritional value, and some have distinctive physiological properties, which are widely exploited in the food industry. Milk proteins have been the subject of intensive research during the last 50 years in an effort to unravel their molecular structures and interactions, relationship between structure and functional attributes, interactions of proteins during processing and, more recently, their physiological functions.\n\nRecent studies on the interactions of milk proteins in complex food systems are leading to a new understanding of the nature of these interactions and their impact on food quality. The knowledge has resulted in the development of several specialized milk protein ingredients tailored to meet specific needs of the food industry. Currently, there is a growing demand by the food industry for milk protein ingredients for specialist high-value applications such as functional foods. In the future, application of novel analytical approaches (genomics, proteomics, nanotechnology) to milk proteins and food materials will provide further understanding of molecular structures and interactions to enable the dairy industry to produce highly functional and healthy protein ingredients for specific applications.\n\nSeveral books have been published about milk and milk proteins\u2014so why another one? Most of the earlier books have addressed different specialist aspects of dairy science and technology. The primary theme of this book is to present a view along the dairy food chain\u2014starting at the cow (and its mammalian relatives) and finishing with nutritional aspects affecting the consumer, dipping into important current research topics along the way. The molecular structures and interactions of milk proteins under various processing environments are covered most prominently. More importantly, the book also contains a considerable amount of material from dairy industry-based or industry-funded research. Thus, it provides fresh perspectives on milk proteins, from an advanced dairy industry point of view.\n\nThe editors particularly thank Fonterra Research and Development Centre for making available time for staff members to contribute their chapters, and for making available hitherto unpublished material. This book is designed to provide an update and call for attention, for industry and academic researchers alike, to important and relevant milk protein science in areas that have the potential to advance the dairy industry.\n\nThe overall theme covered in this book was piloted at a meeting organized by the Riddet Institute and Fonterra Research and Development Centre in February 2006, with invited presentations from a number of experts in the relevant fields from Australasia, the USA and the UK. This meeting was particularly successful, with a large number of international delegates attending from a broad range of disciplines. This confirmed the growing interest of milk protein scientists in looking beyond the boundaries of their immediate topic area to gain an understanding of how the whole food chain fits together. Such an understanding can help elucidate mechanisms and processes, identify novel research opportunities, and provide additional applications for new developments.\n\nThis book includes chapters covering many of the topics addressed at the meeting, as well as some new subjects that we felt were important in order to provide a more complete picture of the journey from expression to food. We would like to thank both the contributors who have been involved from the meeting in 2006 and those who have come on board more recently.\n\nWe have chosen to start the book with a comprehensive overview of the biology and chemistry of milk, to set the stage and give a broad underpinning of the later chapters for readers not familiar with this field. Attention is then turned to the biology, and particularly the molecular biology, of lactation, looking first at some \"extreme\" mammals\u2014the tammar wallaby, which can express two different milk compositions at the same time, and the fur seal, which produces an extremely concentrated milk\u2014to give an idea of the range of biology of milk production. The book thereafter focuses on bovine milk, with mention of the milk from other domestic species as appropriate. This starts with an update on the genomics of bovine milk proteins, and is followed by an overview on post-translational modifications, which completes our view of the biology of milk protein production.\n\nThe structural chemistry of milk proteins, including the latest model of casein micelle and molecular structures of whey proteins, is covered in detail. The behavior of milk proteins under a variety of processing regimes, including ultra-high-pressure, functional systems, drying and storage of powders, is dealt with in a series of chapters. These chapters address our current state of knowledge about existing and emerging processes for the production of milk protein-based food ingredients.\n\nAttention is then turned to the behaviors of milk proteins in real and model food systems, and finally to consumer aspects\u2014the sensory and nutritional\/functional food aspects of milk proteins. A wrap-up chapter gives a view on likely issues of future importance for milk proteins, including the emerging area of nutrigenomics.\n\nAs with any volume written by a large number of contributors, this contains a variety of styles of presentation. We have made no attempt to homogenize the authors' styles, but have provided guidance on chapter content to make for best possible continuity.\n\nA volume of this kind always requires a large amount of work by a large number of people. We would like particularly to thank all the contributing authors for their efforts and their expeditious preparation of manuscripts that allowed for the timely publication of this book. We are pleased to acknowledge Claire Woodhall for assisting with the technical editing, and the staff at Elsevier for producing this book.\n\nAbby Thompson\n\nMike Boland\n\nHarjinder Singh\nChapter 1\n\n# The World Supply of Food and the Role of Dairy Protein\n\nSrikanta Chatterjee*\n\nArnab Sarkar**\n\nMike J. Boland***\n\n* Massey University, Palmerston North, New Zealand  \n** Synlait Milk Ltd., Rakaia, New Zealand  \n*** Riddet Institute, Palmerston North, New Zealand\n\n## Abstract\n\nWorld hunger continues to be a major problem. The main focus of those concerned with world hunger is on the availability of adequate calories for all, but this overshadows another problem: the availability of enough protein and enough dietary essential amino acids. We estimate that about a quarter of the world's population is getting barely enough protein; a particular issue is that of the essential amino acid lysine, which is deficient in cereal proteins, the biggest source of protein nutrition. Milk protein is an important source of dietary protein and is particularly rich in lysine and branched-chain amino acids. It accounts for 10% of all the global protein supply and provides the third highest supply after cereals and meat, but it is more important than the amounts would suggest because of its rich supply of essential amino acids and high digestibility (in contrast to cereal protein) and because of its acceptability to vegetarians. Global trade in dairy is still small (about 8% of dairy production is traded) but growing. Supplementation of cereal protein with milk protein has a potentially important role to play in balancing world protein nutrition.\n\n## Keywords\n\nProtein nutrition, milk protein, dairy trade, dairy production\n\nOutline\n\nIntroduction 2\n\nHunger and the need for food 3\n\nHunger-reduction Targets 3\n\nWorld Hunger and Undernutrition Status 4\n\nGlobal Hunger Index 4\n\nProtein and Its Composition and Bioavailability 6\n\nProtein Composition 6\n\nBioavailability 6\n\nWorld Protein Supply and Its Regional Distribution 7\n\nVegetable and Animal Protein Sources with a Focus on Dairy Foods 8\n\nGrowing Global Demand for Animal Proteins and Implications 8\n\nThe dietary essential amino acids in proteins 9\n\nIdentifying the countries deficient in dietary essential amino acids 9\n\nProtein and Dietary Essential Amino Acid Contents of Food Items 10\n\nDemographic changes, aging populations, and the need for quality protein and essential amino acids 10\n\nProtein Nutritional Needs of the Elderly 12\n\nRole of Essential Amino Acids in Nutrition of the Elderly 13\n\nGlobal trade in proteins, the long-term prospects, with a focus on dairy foods 14\n\nThe Global Dairy Food Scene: An Overview 14\n\nConclusions 16\n\n## Introduction\n\nAs one of the basic necessities of life, the availability, quality, and affordability of food are of concern to individuals and nations alike. The Green Revolution started in the late 1960s and involved the introduction of some new, high-yielding seed varieties, better use of irrigation facilities, and fertilizers. Following these innovations, the world enjoyed several decades of relative stability in the price of basic items of food, including food grains. The period from the early 1970s to 1990 saw world output of food grains and oilseeds rise steadily by an average of 2.2% a year, with periodic fluctuations. With the exception of parts of the African continent, the global rate of growth of food crops exceeded that of the world population, leading to an increase in their per capita availability and to relatively stable prices. Indeed, in 2000 world food prices in real terms were at their lowest for one hundred years (Trostle, 2008).\n\nSince the early 1990s, however, the global rate of growth for grain and oilseed production has declined to 1.3% a year and is projected to decline further to around 1.2% over the next decade. This and a few other adverse factors have contributed to the rapid rise in the world market prices for major food products since early 2006, recording an increase of around 60% in just two years to early 2008. This upward trend has moderated somewhat since then, and the inflation-adjusted food price index stood at 140.3 in June 2013, having risen from its 2002\u20132004 base of 100.0. The most recent figures indicate that the Food and Agriculture Organization (FAO) food price index (FFPI) averaged 210 points in February 2013, remaining virtually stable since November 2012. The index of cereal prices, at 246 points, was 5.4% higher in June 2013 than it was a year earlier. The index of cereal prices was 5 points (2.07%) higher, and the index of dairy prices, after a sharp decline (22%) in the year to June 2012, rose 38% by June 2013 (FAO, 2013a). The general climate of rising retail prices of food items globally has raised the specter of another global food crisis, especially in the poorer countries, where the drive for food security has suffered a major jolt.\n\nSide by side with this worsening situation with respect to the availabilities and prices of the major cereals, there has been another recent development involving food consumption patterns in a number of countries. With growing affluence, tastes change and consumers shift to more varied diets, which usually include larger proportions of noncereal items. Over the last few decades, several poorer countries, among them the two most populous ones, China and India, have experienced rapid growth and transformation in their economies. This economic growth has helped to lift several million people out of extreme poverty and to make many others more affluent, bringing in its trail significant changes in food consumption. One of the more noticeable changes has been a decline in the consumption of cereals and a corresponding increase in the consumption of animal protein. These developments have important policy implications for the global food economy. At the same time, hunger still afflicts a large number of people globally and there is a need for policies to resolve or mitigate this.\n\nIn light of these ongoing changes, this chapter seeks to examine several interrelated issues relating to the evolving world food situation. In particular, it investigates the issue of nutrition and the role proteins and their constituent amino acids play in it. It starts by looking at the issue of hunger, its measurement, global incidence, and mitigation targets. It next discusses the issue of nutrition, its global and regional perspectives, and the role of animal and vegetable proteins. The evolving global demographic trends, with a rapidly increasing elderly population that has special nutritional needs, call for policies to deal with the issue of nutrition for the aged and the role of proteins. Since the production and consumption of protein foods in different countries do not always match, significant international trade in protein products exists. This is briefly discussed to identify the major players in the global protein markets. The chapter concludes with observations on policy implications. In examining the various dimensions of proteins, this chapter focuses on the evolving role of dairy proteins and its implications for future policy.\n\n## Hunger and the need for food\n\nEvery day, millions of people around the globe do not get enough food to eat and remain hungry. Hunger has been referred to as \"the uneasy or painful sensation caused by a lack of food\" and \"the recurrent and involuntary lack of access to food\" (Anderson, 1990). There is no assurance that these hungry people will get the minimum required quantity of food on a daily basis. This unpredictability about where the next meal will come from is called food insecurity. The FAO of the United Nations defines food insecurity as \"a situation that exists when people lack secure access to sufficient amounts of safe and nutritious food for normal growth and development and an active and healthy life\" (FAO, 2000).\n\nAccording to this definition, people are hungry if they do not get enough energy supply from food (fewer than about 1800 kilocalories a day), or if the food they consume is not of sufficiently high quality (i.e., does not contain essential nutrients). Hunger is usually understood to refer to the discomfort associated with lack of food (von Grebmer et al., 2012).\n\n### Hunger-Reduction Targets\n\nHaving thus defined hunger as an operational concept, we need to measure it and to track how it changes over time. This is probably better achieved if a target or targets are set, and the observed incidences of hunger are measured against those targets to ascertain whether the observed trends indicate an improvement or a deterioration.\n\nThe FAO currently monitors two main hunger-reduction targets: the World Food Summit target and Goal 1 of the Millennium Development Goals.\n\n\u2022 During the World Food Summit in Rome (1996), world leaders made the commitment to decrease the number of undernourished people to around 425 million by 2015 (considering 850 million undernourished people as the baseline during the period 1990\u20131992) (FAO, 2011).\n\n\u2022 At the 2000 Millennium Summit in New York, this objective was reiterated when the eight Millennium Development Goals were introduced. The first goal pertains directly to hunger, which is the FAO's fundamental global concern. It aims to reduce the number of people suffering from hunger between 1990 and 2015.\n\nGoal 1 of the Millennium Development Goals calls for a reduction by half of the proportion of people suffering from hunger between 1990 and 2015. Rather than setting a definite number to be reached, this hunger objective therefore depends on the size of the future world population (FAO, 2011; United Nations 2010).\n\n#### World Hunger and Undernutrition Status\n\nAccording to the most recent FAO report, the total number of undernourished people in the world was estimated to be 1023 million in 2009, and it was projected to decrease by 9.6% to 925 million in 2010. The largest numbers of undernourished people live in the developing countries. Two-thirds live in just seven countries (Bangladesh, China, the Democratic Republic of the Congo, Ethiopia, India, Indonesia, and Pakistan), and over 40% live in China and India alone (FAO, WFP, and IFAD, 2012).The territory with the world's largest number of undernourished people continues to be Asia and the Pacific, with an estimated 578 million (Fig. 1.1).\n\nFigure 1.1 Undernourishment in 2012, by region (millions). Data from FAO, WFP, and IFAD (2012).\n\n#### Global Hunger Index\n\nThe International Food Policy Research Institute (IFPRI) has introduced the Global Hunger Index (GHI) tool to measure and track global hunger. The index combines three hunger indicators: (1) the number of undernourished as a proportion of the total population; (2) the proportion of underweight children under the age of 5; and (3) the mortality rate of children under the age of 5. The three indicators are assigned equal weights. On a 100-point scale, the higher the value of the index, the worse is the incidence of hunger, implying that a score of 0 indicates no hunger and a score of 100 indicates the worst possible hunger. Both of these extremes are, obviously, just notional, and are not observed in practice. Different hunger scenarios are defined with the help of the GHI. An index value less than 4.9 indicates \"low hunger\"; values of 5 to 9.9 \"moderate hunger\"; 10 to 19.9 \"serious hunger\"; 20 to 29.9 \"alarming hunger\"; and values in excess of 30 \"extremely alarming hunger.\"\n\nThe 2010 GHI showed some improvement over the 1990 value, falling from 19.8 to 15.1. The index fell by 14% in Sub-Saharan Africa; about 25% in South Asia; 33% in the Near East and North Africa; 40% in Southeast Asia; and 43% in Latin America and the Caribbean compared with the 1990 score (von Grebmer et al., 2012). Figure 1.2 graphs these values for the different regions.\n\nFigure 1.2 Global and regional trends of the Global Hunger Index: Contribution of components in 1990, 1996, 2001, and 2012. Reproduced with permission from the International Food Policy Research Institute www.ifpri.org. The publication from which this figure originates can be found online at <http:\/\/www.ifpri.org\/sites\/default\/files\/publications\/ghi12.pdf>.\n\nTHE IMPORTANCE OF PROTEIN IN WORLD NUTRITION\n\nMost reports on hunger and undernutrition focus primarily on calories, and rightly so. If a person does not get enough calories, his or her well-being will be compromised. However, calories are a necessary, but not sufficient, condition for good nutrition. Many micronutrients, such as vitamins and minerals, are also an important part of the diet, but they are not the focus of this discussion.\n\nA further aspect of undernutrition involves people not getting enough protein, and particularly a sufficient amount of the dietary essential amino acids. This aspect of undernutrition, as well as the role of dairy protein in meeting these needs, is the major concern of the rest of this chapter. Table 1.1 lists the recommended daily intake of protein and of the dietary essential amino acids. Adequate intake of total protein and of all the essential amino acids is essential to maintaining health.\n\nTable 1.1\n\nRecommended Daily Intake for Adults of Protein and Dietary Essential Amino Acids\n\nDietary item | Recommended daily intake (mg\/kg body weight)  \n---|---  \nProtein | 800  \nHistidine | 10  \nIsoleucine | 20  \nLeucine | 39  \nLysine | 30  \nMethionine + cystine | 15  \nPhenylalanine + tyrosine | 25  \nThreonine | 15  \nTryptophan | 4  \nValine | 26\n\nData from WHO (2007).\n\n### Protein and Its Composition and Bioavailability\n\nThe assessment of protein nutrition is more complex than that for calories because proteins vary widely in terms of their composition and bioavailability.\n\n#### Protein Composition\n\nAll proteins are composed of linear chains of amino acids, and each species of protein has its own defined amino acid sequence, which is determined by the genetics of the producing organism. Thus, the amino acid composition, and by implication the amount of each essential amino acid in a given protein, are defined. In practice, most food protein sources contain a complex mixture of proteins. Nevertheless, the overall composition can be determined empirically and is generally quite constant. This has enabled the composition of almost all the major food protein sources in terms of essential amino acids to be determined, and thus intakes of dietary essential amino acids can be estimated from knowledge of the types and amounts of food protein in a diet. In practice, it turns out that intake of most essential amino acids in most diets is adequate, provided total protein intake is adequate. The exception to this rule is the dietary essential amino acid lysine, which is discussed in detail later in this chapter.\n\n#### Bioavailability\n\nAdequate protein supply is one aspect of protein nutrition. A further important aspect is bioavailability: getting the amino acids from the food structures in the gastrointestinal tract to the cells that need them throughout the body. In the adult gastrointestinal tract, proteins must be broken down to very small oligopeptides (at most di- or tripeptides) in order to be taken up, and to single amino acids in order to enter most metabolic pathways. Thus it is necessary for the protein in foods to be both accessible to digestive enzymes and to be broken down by the digestive enzymes in the stomach and small intestine in particular. Furthermore, it is necessary that the broken down protein is able to be taken up into the bloodstream, where it can be redistributed to the tissues that need it. The efficiency of digestion of most of the common food protein sources has been determined, using a range of different methods. Past methodology has largely been based on so-called fecal digestibility. This method is now known to be flawed, particularly with respect to foods with poor digestibility; however it has been widely used and is the only method for which literature values are available for most common foods. For a full discussion of protein quality and nutritional requirements, the reader is referred to a recent FAO report, Protein Quality Evaluation in Human Nutrition (FAO, 2013b).\n\nBy using digestibility values, dietary intake can be converted to an estimated uptake into the body for these proteins, noting that these values are derived from a flawed methodology and so, particularly in the case of plant-derived proteins, represent an upper limit of their true bioavailability. The digestibility of a range of dietary proteins is given in Figure 1.3.\n\nFigure 1.3 True protein digestibility of common food proteins. Data from FAO (1970) and WHO\/FAO (1991).\n\nAnimal-derived proteins generally have good bioavailability and content of dietary essential amino acids, but many plant proteins are deficient in one or more dietary essential amino acids, and many are not efficiently digested and therefore the constituent amino acids are not highly bioavailable. Most Western countries are characterized by a high protein diet with a strong emphasis on animal-derived protein, so protein nutrition is not generally a problem (although there may be some issues with protein nutrition among the elderly). Most developing countries are very dependent on plant protein as the main dietary source, and that protein may be inadequate, due to poor digestibility and poor amino acid balance, particularly in the case of lysine.\n\n### World Protein Supply and Its Regional Distribution\n\nInformation on the amount of protein available per capita and by protein source in each country is available from the FAOSTAT database (<http:\/\/faostat.fao.org\/>). In Figure 1.4, we present the average protein availability for individual countries, along with their total population. These statistics are presented as the total number of people who reside in countries with corresponding average protein availability for each incremental 5 g band. The figure is striking in having two clear peaks, one in the range 55\u201360 g protein per capita per day, and the second in the band 85\u201390 g protein per capita per day, with a considerable tail to the right. The first of these peaks is of some concern: It represents more than a quarter of the world's population, and it is simple to calculate that with a standard body weight of 70 kg; based on the dietary recommendations for protein requirements, a person will need 56 g of protein a day to stay healthy. The band at 55\u201360 includes India, Indonesia, Bangladesh, and the Democratic People's Republic of Korea (North Korea). This band is of concern because, although the average availability figure is just above the minimum requirement, disparities of income and situation in these countries will mean that a large proportion of these people are not getting enough protein. Furthermore, these figures are just for total protein supply, with no correction for bioavailability. When the dietary pattern is corrected for digestibility of the main protein components of the diet (from FAOSTAT), the situation is more serious, with almost 1.8 billion people getting, on average, less than 55 g of protein per day based on FAOSTAT figures for 2009.\n\nFigure 1.4 Protein intake by population on a country average basis. Each bar represents the total population of countries with average per capita daily protein intake in bands of 5 g\/capita\/day. Protein figures for 2007 from FAOSTAT September 2012; population data from United Nations (2009).\n\n### Vegetable and Animal Protein Sources with a Focus on Dairy Foods\n\nThere is considerable debate over the merits of vegetarianism and over eating only vegetable-origin foods in consideration of global sustainability. It is often estimated that production of 1 kg of animal-origin food requires 10 kg of plant-origin food, leading to the simplistic assumption that 10 times as many people could be fed off the same resources if everyone was vegetarian. For a full discussion of the subject, the reader is referred to Fairlie (2010). In the case of dairy products and eggs, the situation is somewhat better than it is for meat, because the animal can continue producing throughout its adult lifetime. This leads to conversion ratios of about 4:1. In fact, the argument is much more complex, partly because of the role of animals in subsistence agriculture, largely eating food waste or processing residue, or grazing and browsing plant species that are not suitable for human consumption, and partly because of the niche many animals occupy in developed agricultural economies, either grazing pastures intensively, or being farmed on land not suitable for arable cultivation (e.g., see Elferink et al., 2008). In this context, we have calculated that in the Canterbury Plains in New Zealand, the main wheat-growing area, the yield per hectare per annum of protein from the wheat crop, processed to the form of white flour and its consequent baked products, is somewhat less than the yield of protein from milk that is produced over the same period in the same area. Thus, the efficiencies of production need to be considered in the context of what is the target of that production (there is no doubt that wheat produces the greater number of calories). Nevertheless, it must be recognized that the changing protein consumption patterns, involving more animal-based products, have significant implications for global land-use patterns, agriculture, agri-food industries, cereal prices, and the environment.\n\n### Growing Global Demand for Animal Proteins and Implications\n\nThe demand for animal protein foods is expected to increase to about double the present consumption by 2050, driven by population growth and by the emerging middle classes in developing countries (FAO, 2006). As people get more money, one of the first priorities is better food, and this usually means animal protein foods. This phenomenon was first described by Bennett (1941), who related comparative studies of the consumption of staple foods leading to what has come to be known as Bennett's Law: the empirical generalization that there is an inverse relationship between the percentage of total calories derived from cereals and other staple foods and per capita income. This principle has since become generalized to mean a move away from carbohydrate-based foods to protein-based foods.\n\nA simple extrapolation from past increases in animal production indicates that we should be able to meet this demand if past rates of increase can be sustained (Boland et al., 2013). However, past increases have been based on bringing in new land for farm production, increases in efficiency through breeding gains, better livestock management and nutrition, and other factors revolving around the Green Revolution. Most of these options are either reaching their limits or entering a phase of diminishing returns. The carbon footprint of livestock production is a further constraint, although the good news is that as animal production has intensified, the carbon footprint has massively decreased. For example, Capper et al. (2009) have calculated that the carbon footprint for milk in the United States in 2007 was just 37% of that for the same milk in 1944. Nonetheless, past increases will not continue ad infinitum, and new ways of sustainably meeting the increasing demand are needed.\n\n## The dietary essential amino acids in proteins\n\nAlthough there are nine dietary essential amino acids, it is rare for a diet with adequate overall protein intake to be deficient in most of them. The exception is lysine.\n\nLysine may be an issue for two reasons: The first is that many staple protein sources, particularly the cereals, are deficient in lysine. The second is that lysine is chemically unstable under heating and undergoes a range of reactions when food is heated. The most important of these reactions is the Maillard reaction, in which the side chains of the lysine residues in the protein cross-react with sugar molecules to produce glycosyl lysine side chains that are indigestible and thus no longer bioavailable. This reaction can occur under mild heating conditions, and under more extreme conditions it is responsible for much of the browning of food that occurs during cooking. Another reaction of importance for dairy products is the reaction with phosphoserine, leading to the formation of lysinoalanine, which is not bioavailable. This problem is specific to casein-containing products (mainly milk powders and caseinate), because of its high phosphoserine content (see Chapter 11 for a detailed discussion of this reaction).\n\n## Identifying the countries deficient in dietary essential amino acids\n\nIn an attempt to obtain an understanding of the dietary availability of the essential amino acids, countries with low intakes of protein were analyzed to determine the dietary essential amino acid content of the mix of protein sources for that country (from FAOSTAT), corrected for digestibility for each protein source. Because literature values were unavailable for some minor protein sources, a sensitivity test was performed, changing the digestibility figure from 1.0 to 0.8 for plant proteins and 1.0 to 0.9 for animal proteins in these cases. Because this change did not make a noticeable difference to overall lysine bioavailability for the countries in question, the method was considered to be robust. The countries found to be lysine deficient are given in Table 1.2, together with information about the main dietary protein sources. Of these countries, only Liberia was found to be deficient for any of the other essential amino acids (leucine and isoleucine in this case).\n\nTable 1.2\n\nLysine-Deficient Countries\n\nCountry | Lysine (% RDI) | Animal protein in diet (%) | Main protein source(s) | Main protein source (%)  \n---|---|---|---|---  \nEritrea | 63.1 | 11 | Other cereals* | 48  \nLiberia | 71.6 | 14 | Rice | 43  \nMozambique | 75.1 | 11 | Maize  \nWheat | 28  \n15  \nGuinea-Bissau | 79.4 | 18 | Rice | 39  \nZambia | 79.9 | 16 | Maize | 57  \nHaiti | 87.5 | 19 | Rice  \nWheat | 19  \n16  \nTogo | 89.0 | 12 | Maize | 30  \nEthiopia | 92.7 | 10 | Other cereals* | 34  \nTajikistan | 98.0 | 21 | Wheat | 61  \nBangladesh | 98.7 | 16 | Rice | 61  \nZimbabwe | 98.8 | 19 | Maize | 46  \nYemen | 99.1 | 22 | wheat | 45\n\n* Other cereals include sorghum, barley, oats, and rye.\n\nThe countries that are lysine deficient show a clear pattern of low levels of consumption of animal protein and strong dependence on cereals for their protein.\n\n### Protein and Dietary Essential Amino Acid Contents of Food Items\n\nThe amino acid composition, particularly the lysine content, of proteins is of particular concern for countries that tend to be protein deficient. Thus maintenance of an adequate intake of lysine, especially in populations with a high dependence on cereals, requires attention. Figure 1.5 indicates the levels of bioavailable lysine in a range of common dietary protein sources. Meat is clearly the best source of lysine but may not be a suitable dietary component for many because of cost and cultural restrictions. Dairy protein is also an excellent source of lysine. Inclusion of supplementary dairy protein in the diet may offer an effective solution that is acceptable to vegetarians, price notwithstanding.\n\nFigure 1.5 Bioavailable lysine content in a range of food protein in g\/100 g protein, corrected for digestibility.\n\n## Demographic changes, aging populations, and the need for quality protein and essential amino acids\n\nRecent global demographic trends indicate a steady increase in the number of people aged 60 years and over. The projection is for this population to more than triple from 600 million in 2000 to over 2 billion in 2050 (United Nations, 2009). As a consequence, in the more developed world, the fastest growing section of the population is that of adults aged 80 years or over. This clearly presents unique challenges for health care, diets, and nutrition, as well as for certain age-specific clinical conditions.\n\nBoth the number and the proportion of older persons are growing in virtually all countries, and these trends are likely to continue worldwide. For example, in 2009, Japan had the highest percentage of the population aged 60 or over, at 29.7%, followed by Italy at 26.4%, whereas in Qatar it was only 1.9%. It is expected that the proportion of the population aged 60 or over will be 22% in 2050 compared to 11% in 2009. Figure 1.6 shows the predicted percentage of the elderly population in 2050 in major continents. By 2050, it is projected that there will be more than 1.4 billion elderly people in Asia alone.\n\nFigure 1.6 Percentage of elderly population in 2050. Sarkar (2012).\n\nAging is a continuous, ongoing, and progressive process of damage accumulation. It is associated with reduction in muscle mass and function, and reduced physical activity. The loss of muscle mass with aging is known as sarcopenia. With the aging of the population globally, the prevalence of sarcopenia is likely to increase. Sarcopenia is accelerated by inadequate diet, mainly due to lack of quality protein in optimal quantity and lack of essential amino acids. The issue of the nutritional needs of the growing aging population in terms of the role of dietary protein and essential amino acids with particular reference to sarcopenia is described in more detail in the following sections.\n\nOverall, a strong case can be made that an aging population will require a substantially increased intake of protein and of essential amino acids (particularly leucine), a demand that milk proteins are particularly well suited to meet.\n\n### Protein Nutritional Needs of the Elderly\n\nThe aging process is characterized by changes in body composition, with a progressive loss of muscle and bone mass, strength, and metabolic function. The loss of muscle with aging is the result of a chronic imbalance between muscle protein synthesis and breakdown. There are many causes of sarcopenia, and an understanding of the complex mechanism is evolving. This degenerative loss of skeletal muscle occurs at a rate of 3 to 8% per decade after the age of 30 and accelerates with advancing age; chronic muscle loss is estimated to affect 30% of people older than 60 years and 50% of those older than 80 years (Katsanos et al., 2006; Paddon-Jones et al. 2008). With the aging of the population, the prevalence of sarcopenia and the resulting burden of disability are likely to increase. Strategies to prevent sarcopenia are, therefore, of considerable importance, and there is a need for public awareness, as simple health strategies can be effective.\n\nResearchers have identified two measures that can play a role in fighting against sarcopenia: diet and exercise. However, in the case of many elderly individuals, the ability to perform exercise is compromised due to disease and disability. In this case, daily high-quality protein intake can be helpful to slow down or prevent muscle protein loss. Different protein sources have been found to stimulate muscle protein synthesis in varying degrees. The most important factor is the amount of essential amino acids in the protein, in particular, leucine. Differences in digestibility and bioavailability of certain protein-rich foods may also influence muscle protein synthesis (Paddon-Jones et al., 2008).\n\nCurrently, there is no agreement on whether dietary protein needs change with advancing age. For adults the recommended dietary allowance for protein is 0.8 g protein per kg body weight per day (WHO, 2007). The report of the FAO\/WHO\/UNU expert consultation, published in 2007, recommends that the essential amino acid requirement for elderly people should be the same as for adults, as the current acceptable methodologies are not appropriate to make a separate set of essential amino acid values for elderly people (WHO, 2007). A more recent FAO-sponsored expert consultation has failed to resolve this issue, with one group maintaining that \"the data based on the currently acceptable methodologies... are inadequate to make a separate recommendation for dietary IAA requirements in elderly people\" (Pillai & Kurpad, 2012), while another group has advised that:\n\ndietary protein intake, and the resulting increased availability of plasma amino acids, stimulates muscle protein synthesis. If all other variables are controlled, increased muscle protein synthesis leads to improved muscle mass, strength, and function over time. Increased muscle mass, strength, and function are related to improved health outcomes in older individuals. Since adverse effects of reasonable increases in protein intake above the recommended dietary allowance (RDA) of 0.8 g protein\/kg\/day have not been reported, it is reasonable to conclude that the optimal protein intake for an older individual is greater than the RDA (Wolfe, 2012).\n\nSome studies suggest that an intake of 1.0\u20131.5 g protein per kg body weight per day or about 15\u201320% of total caloric intake is essential to preserve proper nitrogen balance in the healthy elderly instead of the recommended RDA value (Morais et al., 2006; Wolfe et al., 2008).\n\n### Role of Essential Amino Acids in Nutrition of the Elderly\n\nEssential amino acids are mainly responsible for the stimulation of muscle protein anabolism in the aged (Volpi et al., 2003). It is considered that 15 g of essential amino acids taken as bolus is required for maximum stimulation of muscle protein synthesis (Wolfe, 2002). This indicates that quality of protein is very important in the diet of the elderly.\n\nPreliminary data from a recent randomized controlled trial indicate that it is more important to ingest a sufficient amount of high-quality protein (25\u201330 g) with each meal rather than one large bolus, because more than 30 g in a single meal may not further stimulate muscle protein synthesis (Symons et al., 2009). It is also recognized in recent studies that intake of whey protein brings beneficial effects to muscle protein anabolism in the elderly. Furthermore, ingestion of intact whey protein has been found to provide a greater anabolic benefit than ingestion of the equivalent essential amino acids alone. Thus, whey protein may be more than just a simple source of essential amino acids with respect to providing a stimulus for enhancing muscle protein anabolism in the elderly (Katsanos et al., 2008). For a fuller discussion of the function of whey proteins and other milk proteins in human health, the reader is referred to Chapter 19 of this volume.\n\nThere is a general agreement that the essential amino acid leucine increases protein anabolism and decreases protein breakdown (Paddon-Jones and Rasmussen, 2009). Leucine-rich food sources include legumes such as soybeans and cowpea, and animal products such as beef, fish, and particularly dairy proteins (whey protein). It is reported that amino acid supplements without adequate leucine do not stimulate protein synthesis (Rieu et al., 2007; Hayes and Cribb 2008). Leucine has recently been acknowledged to be especially important as a signaling molecule and a building block for muscle. Rat studies show that leucine can directly stimulate muscle protein synthesis through increasing mRNA translation (Anthony et al., 2000). Insulin and leucine are anabolic stimuli for muscle, and both share a common pathway of action via activation of a kinase known as mTOR. mTOR is the main regulator of cell growth and acts by phosphorylating target proteins involved in mRNA translation. Because insulin sensitivity decreases with age, one possible mechanism by which amino acids (mainly leucine) might improve muscle mass is by providing another anabolic stimulus to activate the mTOR-controlled pathway (Gaffney-Stomberg et al., 2009; Casperson et al. 2012).\n\nNo differences exist in protein balance in the elderly relative to the young following administration of either 30 g of beef protein or 15 g of essential amino acids as a bolus (Paddon-Jones et al., 2004). However, when 6.7 g of a mix of the dietary essential amino acids is given, the overall protein synthetic response is reduced in the elderly relative to the young (Katsanos et al., 2005). This anabolic resistance has been attributed to a decrease in leucine sensitivity and may be overcome by increasing the proportion of this amino acid in the diet. For example, when a 6.7 g bolus of dietary essential amino acids enriched with leucine (46% leucine compared to the 26% normally found in whey protein) was given to the elderly individual, protein synthesis was fully restored (Katsanos et al., 2006).\n\n## Global trade in proteins, the long-term prospects, with a focus on dairy foods\n\nGlobal food consumption patterns have been changing in recent decades in several significant ways. Among them is the noticeable and continuing shift in favor of proteins, especially animal proteins. Global consumption of protein is forecast to grow by 96% over the three decades from 1990 to 2020 (von der Heyde, 2012). The growth is largely due to the rising incomes in the developing world, particularly in some of the more populous countries such as China, Brazil, and, to a lesser extent, India. Over the decade since 2000, however, global protein demand has been driven by increased consumption in other countries and geographic areas too. For example, demand in the African continent has increased by around 70%, in Southeast Asia by 49% and in Central America by 29% (von der Heyde, 2012). Between 1999 and 2011, world protein trade grew by 74% (von der Heyde, 2012). Since only a few countries currently have surplus protein to export, the projected increase in its demand is likely to pose serious challenges to these countries and to the world in general.\n\nIn overall world protein nutrition, milk products, representing about 10% of all protein consumption, are the third most important source of protein after cereals (40%) and meat (18%) (data for 2009 from FAOSTAT). When the low levels of lysine in cereals are taken into account (about one-third of that in dairy products), it is clear that milk protein plays a very important nutritional role in the world today.\n\n### The Global Dairy Food Scene: An Overview\n\nMilk and other dairy products have always been among the major everyday food items in human consumption in many cultures. It is a particularly useful food for the large, and possibly growing, number of vegetarians around the world. Its value for both infants and the elderly is easily recognized. Apart from its consumption in liquid form, there are many other ways in which milk is transformed and consumed. Innovations keep occurring to make new milk-based products available in the market.\n\nWith growing world population and changing food habits, the production and consumption of milk and other milk-based products have also been rising over time. Over the five decades since 1961, world milk production more than doubled from 344 million tons to 703 million tons in 2009 (FAOSTAT) and 749 million tons in 2011 (IDF, 2012).\n\nThe pattern of regional distribution of the production and consumption of milk reveals that, as of 2009, Asia's share is the highest\u2014with 36% of global production and 38% of consumption; Europe comes next, with 31% and 30%, respectively, followed by North America with a balanced 13% of both production and consumption, and South America, again with a balanced 8% of production and consumption. The only region with a significant exportable surplus is Oceania, which produces 3.7% and consumes 1.5% of the global totals.\n\nIt is important to note that much of the milk produced is consumed in the country (or economic bloc, in the case of the EU) where it is produced, and that just over 8% of dairy production is involved in international trade (2011 figures; IDF, 2012). In this context, it is noted that about 80% of whole milk powder, 55% of skim milk powder, and only about 10% of cheese is traded internationally (IDF, 2012). It is of interest to note, too, that trade in cheese within the EU-27, for example, was five times the volume exported, which in turn was only 8% of production. In 2012, the major exporters of cheese were the EU-27, the United States, New Zealand, and Australia, and the main importers were Russia, Japan, the EU-27, Mexico, and Korea (USDA, 2012). There is, evidently, some intraindustry trade in cheese. Given its variety and established regional specialties, this is not difficult to understand.\n\nThe six major exporters account for 80% of world dairy trade in cow's milk (IDF, 2012). They were, in 2011, New Zealand (26%), the EU-27 (26%), the United States (12%), Australia (8%), Argentina, and Belarus (4% each). Of course, all of the exported 'milk' consists of processed products, of which the main ones that contain protein (in order of importance) are whole milk powder, skim milk powder and cheese. Different countries dominate the export markets of the different dairy products (Table 1.3).\n\nTable 1.3\n\nVolume of Major Dairy Exports for the Six Main Exporters, 2011\n\n| Whole milk powder  \n(000 tons) | Skim milk powder  \n(000 tons) | Cheese  \n(000 tons) | Total protein  \n(000 tons)a  \n---|---|---|---|---  \nNew Zealand | 1080.8 | 349.8 | 245.9 | 454.1  \nEU-27 | 390.0 | 517.6 | 680.2 | 448.7  \nUSA | 21.6 | 435.7 | 224.3 | 214.0  \nAustralia | 143.2 | 165.9 | 207.0 | 145.6  \nArgentina | 199.1 | 18.4 | 58.8 | 70.9  \nBelarus | 26.7 | 55.2 | 122.1 | 56.5\n\na Excludes other products such as casein, whey products, and liquid and condensed milks. Values are based on protein of: WMP 25%; SMP 35%; and cheese 25%. The cheese value is based on the bulk of traded cheese being cheddar. Values were obtained from the Canadian Dairy Commission dairy ingredient profiles on www.milkingredients.ca and values near the lower end of each range used.\n\nSource: IDF, 2012.\n\nOne notable feature of our discussion is perhaps the absence of the poorer developing countries among the major exporters and importers of such processed high-value milk products as butter and cheese. The possible explanation for this may be that the consumption of these products is income-sensitive; they are consumed in noticeable quantities only when income has reached a certain level.\n\nThis presumption is further confirmed when one examines the trade patterns in respect of milk powders, which are usually reconstituted for consumption as liquid milk\u2014the demand for which is likely to be less income-sensitive. Among the major importers of whole milk powder are China, Algeria, Brazil, Indonesia, and the Philippines, while the major exporters are New Zealand, Australia, and the EU-27. The situation is very similar in the market for skim milk powder. The major importing countries are Mexico, China, Indonesia, and the Philippines, and the major exporters are New Zealand, Australia, the United States, and the EU-27.\n\nWhile China features as a milk importer, India, perhaps surprisingly, is neither a major importer nor a major exporter of dairy products, although it has the largest bovine herd in the world. With the introduction around the mid-1960s of a system of dairy cooperatives under the umbrella of the National Dairy Development Board (NDDB), India's dairy industry has achieved a remarkable transition. Set up in 1965, the NDDB oversaw the dairy cooperatives collecting the often-small marketable surplus milk from the small herds scattered around the villages and supplying the growing market for milk in the urban areas. This linking of the milk producers with the markets\u2014both of which are scattered in locations and are large in numbers\u2014generated a five-fold growth in India's milk production in three decades from the late 1960s, as domestic consumption of milk also rose steadily (Chatterjee, 1990; Brown 2009). This transformation is all the more remarkable in that India's dairy industry is built almost entirely on crop residues\u2014wheat or rice straw, corn stalks, vegetable residues, and grass gathered from roadside\u2014a rather different protein production model.\n\nAlthough the consumption of dairy products is projected to grow as the standards of living improve in the developing world, some new developments, so far mainly in the more affluent countries, have also been creating additional demand for certain types of foods referred to as 'specialty foods.' These include functional foods, defined as 'food and drink products making a specific health claim,' organic foods, and genetically modified (GM) foods. While international trade in specialty foods is still relatively small and confined to a few countries, evidence suggests that it is growing rapidly (Chatterjee, 2012). Dairy products feature prominently among both functional foods and organic foods currently traded internationally; other animal protein products less so. The market for these products is likely to grow over time as rising affluence spreads globally. Resources including land devoted to mostly export- oriented organic farming, for example, have also been growing, particularly in the developing countries of Asia, Africa, and Latin America.\n\n## Conclusions\n\nWorld hunger continues to be a major problem. Hunger has several dimensions, notably the need first and foremost for adequate intake of calories. A close second is the need for adequate intake of protein and of dietary essential amino acids. Protein nutrition is more complex than calories because all proteins are not equal: Nutritional value depends on the type of protein and on how it has been treated prior to consumption, as much as the amount of protein itself. Milk protein is a very high quality protein, with a good supply of the dietary essential amino acids and high bioavailability. It can therefore be used to supplement poorer plant-derived proteins, such as cereal protein, to greatly improve the nutritional value of the combination. Milk production is growing globally, and the amounts of dairy products (and implicitly milk protein) traded internationally are also growing. Milk protein already accounts for 10% of the global food protein supply and makes a disproportionate contribution to global protein nutrition, based on its bioavailability and desirable composition. The future role of milk proteins in the global food protein economy deserves special attention.\n\n# References\n\nAnderson SA .  Core indicators of nutritional state for difficult-to-sample populations .  _Journal of Nutrition_.  1990 ;120 : 1557 \u2013 1600 .\n\nAnthony JC ,  Yoshizawa F ,  Anthony TG ,  Vary TC ,  Jefferson LS ,  Kimball SR .  Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway .  _Journal of Nutrition_.  2000 ;130 : 2413 \u2013 2419 .\n\nBennett MK .  Wheat in national diets Wheat Studies of the Food Research Institute :  Stanford University, California ;  1941 : 37\u201376 .\n\nBoland MJ ,  Rae AN ,  Vereijken JM ,  Meuwissen MPM ,  Fischer ARH ,  van Boekel MAJS ,  Rutherfurd SM ,  Gruppen H ,  Moughan PJ ,  Hendriks WH .  The future supply of animal-derived protein for human consumption .  _Trends in Food Science and Technology_.  2013 ;29 : 62 \u2013 73 .\n\nBrown LS .  Plan B 4.0: Mobilising to save civilization Washington, DC :  Earth Policy Institute ;  2009 : <http:\/\/www.earth-policy.org\/books\/pb4\/pb4ch9_ss4>. Retrieved February, 14, 2013 .\n\nCapper JL ,  Cady RA ,  Bauman DE .  The environmental impact of dairy production: 1944 compared with 2007 .  _Journal of Animal Science_.  2009 ;87 : 2170\u20132167 .\n\nCasperson SL ,  Sheffield-Moore M ,  Hewlings SJ ,  Paddon-Jones D .  Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein .  _Clinical Nutrition_.  2012 ;31 : 512 \u2013 519 .\n\nChatterjee S .  Changing global food consumption patterns: An economic perspective. Chapter 9 .  In:  Ghosh D ,  Das S ,  Bagchi D ,  Smarta RB ,  eds.  _Innovation in healthy and functional foods_ .  New York :  CRC Press, Taylor and Francis Group ;  2012 : 125 \u2013 140 .\n\nChatterjee S .  Aid, trade and rural development: A review of New Zealand's assistance to Indian dairying. Chapter 15 .  In:  Doornbos M ,  Nair KN ,  eds.  _Resources, institutions and strategies: Operation flood and Indian dairying. Indo-Dutch Studies on Development Alternatives_ .  New Delhi :  Sage Publications ;  1990 : 319 \u2013 338 .\n\nElferink EV ,  Nonhebel S ,  Moll HC .  Feeding livestock food residue and the consequences for the environmental impact of meat .  _Journal of Cleaner Production_.  2008 ;16 : 1227 \u2013 1233 .\n\nFairlie S .  _Meat: A benign extravagance_ .  White River Junction, VT :  Chelsea Green Publishing Co ;  2010 .\n\nFAO., 1970. Amino-acid content of foods and biological data on proteins. Retrieved February 10, 2011, from <http:\/\/www.fao.org\/docrep\/005\/ac854t\/ac854t00.htm>. Food and Agriculture Organization of the United Nations, Rome.\n\nFAO .  _The state of food insecurity in the world_ .  Rome :  Food and Agriculture Organization of the United Nations ;  2000 .\n\nFAO .  _World Agriculture: Towards 2030\/2050, Interim Report_ .  Rome :  Food and Agriculture Organization of the United Nations ;  2006 .\n\nFAO., 2011. Hunger: What are the hunger targets? <http:\/\/www.fao.org\/hunger\/faqs-on-hunger\/en#c41476> Retrieved March 12, 2013. Food and Agriculture Organization of the United Nations, Rome.\n\nFAO., 2013a. World Food Situation: FAO Food Price Index. <http:\/\/www.fao.org\/worldfoodsituation\/wfs-home\/foodpricesindex\/en> Retrieved July 25, 2013. Food and Agriculture Organization of the United Nations, Rome.\n\nFAO .  _Protein quality evaluation in human nutrition. Report of an expert consultation. FAO Food and Nutrition Paper 92_ .  Rome :  Food and Agriculture Organization of the United Nations ;  2013 .\n\nFAO., WFP., IFAD., 2012. The state of food insecurity in the world 2012. Economic growth is necessary but not sufficient to accelerate reduction of hunger and malnutrition. <http:\/\/www.fao.org\/docrep\/016\/i3027e\/i3027e.pdf>. Food and Agriculture Organization of the United Nations, Rome.\n\nGaffney-Stomberg E ,  Insogna KL ,  Rodriguez NR ,  Kerstetter JE .  Increasing dietary protein requirements in elderly people for optimal muscle and bone health .  _Journal of the American Geriatrics Society_.  2009 ;57 : 1073 \u2013 1079 .\n\nHayes A ,  Cribb PJ .  Effect of whey protein isolate on strength, body composition and muscle hypertrophy during resistance training .  _Current Opinion in Clinical Nutrition and Metabolic Care_.  2008 ;11 : 40 \u2013 44 .\n\nIDF .  _World Dairy Situation 2012. Bulletin of the International Dairy Federation 458\/2012_ .  Brussels :  International Dairy Federation ;  2012 .\n\nKatsanos CS ,  Chinkes DL ,  Paddon-Jones D ,  Zhang XJ ,  Aarsland A ,  Wolfe RR .  Whey protein ingestion in elderly persons results in greater muscle protein accrual than ingestion of its constituent essential amino acid content .  _Nutrition Research_.  2008 ;28 : 651 \u2013 658 .\n\nKatsanos CS ,  Kobayashi H ,  Sheffield-Moore M ,  Aarsland A ,  Wolfe RR .  Aging is associated with diminished accretion of muscle proteins after the ingestion of a small bolus of essential amino acids .  _American Journal of Clinical Nutrition_.  2005 ;82 : 1065 \u2013 1073 .\n\nKatsanos CS ,  Kobayashi H ,  Sheffield-Moore M ,  Aarsland A ,  Wolfe RR .  A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly .  _American Journal of Physiology-Endocrinology and Metabolism_.  2006 ;291 : E381 \u2013 E387 .\n\nMorais JA ,  Chevalier S ,  Gougeon R .  Protein turnover and requirements in the healthy and frail elderly .  _Journal of Nutrition Health and Aging_.  2006 ;10 : 272 \u2013 283 .\n\nPaddon-Jones D ,  Rasmussen BB .  Dietary protein recommendations and the prevention of sarcopenia .  _Current Opinion in Clinical Nutrition and Metabolic Care_.  2009 ;12 : 86 \u2013 90 .\n\nPaddon-Jones D ,  Sheffield-Moore M ,  Zhang XJ ,  Volpi E ,  Wolf SE ,  Aarsland A ,  Wolfe RR .  Amino acid ingestion improves muscle protein synthesis in the young and elderly .  _American Journal of Physiology- Endocrinology and Metabolism_.  2004 ;286 : E321 \u2013 E328 .\n\nPaddon-Jones D ,  Short KR ,  Campbell WW ,  Volpi E ,  Wolfe RR .  Role of dietary protein in the sarcopenia of aging .  _American Journal of Clinical Nutrition_.  2008 ;87 : 1562S \u2013 1566S .\n\nPillai RR ,  Kurpad AV .  Amino acid requirements in children and the elderly population .  _British Journal of Nutrition_.  2012 ;108 : S44 \u2013 S49 .\n\nRieu I ,  Balage M ,  Sornet C ,  Debras E ,  Ripes S ,  Rochon-Bonhomme C ,  Pouyet C ,  Grizard J ,  Dardevet D .  Increased availability of leucine with leucine-rich whey proteins improves postprandial muscle protein synthesis in aging rats .  _Nutrition_.  2007 ;23 : 323 \u2013 331 .\n\nSarkar A .  _Global protein nutrition: essential amino acids availability_ .  New Zealand :  Thesis, Massey University ;  2012 .\n\nSymons TB ,  Sheffield-Moore M ,  Wolfe RR ,  Paddon-Jones D .  A moderate serving of high-quality protein maximally stimulates skeletal muscle protein synthesis in young and elderly subjects .  _Journal of the American Dietetic Association_.  2009 ;109 : 1582 \u2013 1586 .\n\nTrostle R .  _Global agricultural supply and demand: Factors contributing to the recent increase in food commodity prices. A Report from the Economic Research Service_ .  United States Department of Agriculture ;  2008 .\n\nUnited Nations, 2009. World population prospects: The 2008 revision. Population Division of the Department of Economic and Social Affairs of the United Nations Secretariat. <http:\/\/esa.un.org\/unpp>, downloaded January 30, 2011.\n\nUnited Nations, 2010. Millennium Development Goals. <http:\/\/www.un.org\/millenniumgoals\/poverty.shtml> Retrieved September 6, 2011.\n\nUSDA .  _Dairy: World markets and trade_ .  United States Department of Agriculture, Foreign Agricultural Service ;  2012, December .\n\nVolpi E ,  Kobayashi H ,  Sheffield-Moore M ,  Mittendorfer B ,  Wolfe RR .  Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults .  _American Journal of Clinical Nutrition_.  2003 ;78 : 250 \u2013 258 .\n\nvon der Heyde, C., 2012. JBS Pilgrims Presentation, 2012 NOPA Industry Forum. <http:\/\/www.nopa.org\/content\/newsroom\/2012\/Charles%20von%20der%20Heyde_JBS%20Pilgrims%20Presentation_2012%20NOPA%20Industry%20Forum.pdf>, retrieved 6 January 2013.\n\nvon Grebmer, K., Ringler, C., Rosegrant, M.W., Olofinbiyi, T., Wiesmann, D., Fritschel, H., Badiane, O., et al., 2012. The challenge of hunger: Ensuring sustainable food security under land, water, and energy stresses. Deutsche Welthungerhilfe; International Food Policy Research Institute; Concern Worldwide. Bonn, Germany; Washington, DC; Dublin, Ireland.\n\nWHO\/FAO .  _Protein quality evaluation: Report of Joint FAO\/WHO Expert Consultation_ .  Rome :  FAO ;  1991 .\n\nWHO .  _Protein and amino acid requirements in human nutrition: Report of a Joint FAO\/WHO\/UNU Expert Consultation_ .  Geneva, Switzerland :  World Health Organization ;  2007 .\n\nWolfe RR .  Regulation of muscle protein by amino acids .  _Journal of Nutrition_.  2002 ;132 : 3219S \u2013 3224S .\n\nWolfe RR .  The role of dietary protein in optimizing muscle mass, function and health outcomes in older individuals .  _British Journal of Nutrition_.  2012 ;108 : S88 \u2013 S93 .\n\nWolfe RR ,  Miller SL ,  Miller KB .  Optimal protein intake in the elderly .  _Clinical Nutrition_.  2008 ;27 : 675 \u2013 684 . \nChapter 2\n\n# Milk: An Overview\n\nJ.A. O'Mahony\n\nP.F. Fox School of Food and Nutritional Sciences, University College, Cork, Ireland\n\n## Abstract\n\nMilk is the characterizing excretion of mammals, of which there are about 4500 species, produced to meet the complete nutritional, and some defensive and other physiological, requirements of the neonate of the species. The milk of all species is basically similar, but there are significant species-specific differences. In addition to supplying all the nutritional requirements of the neonate, many of the minor constituents of milk serve protective roles (e.g., oligosaccharides, immunoglobulins, metal-binding proteins, and enzymes). Milk is an aqueous solution (milk serum) of lactose, inorganic and organic salts, and numerous compounds at trace levels, in which are dispersed colloidal particles of three size ranges: whey proteins dissolved at the molecular level, caseins dispersed as large (50\u2013500 nm) colloidal aggregates (micelles), and lipids emulsified as large (1\u201320 \u03bcm) globules.\n\nThe colloidal stability of milk, especially of the casein micelles, is very important from nutritional and technological viewpoints. The micelles are destabilized and aggregate or gel following limited proteolysis or acidification to \u223cpH 4.6. In vivo, aggregation occurs in the stomach of the neonate, thereby slowing transit and improving digestibility. Technologically, destabilization of the micelles can be undesirable or can be exploited in the production of cheese and fermented milk products.\n\nHumans have used the milk of other species in their diets for about 8000 years, and a major industry has developed around the processing of milk of a few species, especially cattle, buffalo, sheep, and goats, for human foods. Milk processing, which exploits certain physicochemical properties of milk, is practiced worldwide, especially in Europe and North America. Milk is a very flexible raw material, from which a wide range of different products, including about 1000 varieties of cheese, are produced.\n\n## Keywords\n\nMammals, milk, evolution, proteins, lipids, lactose, casein micelle, whey proteins, biological functions, physicochemical properties\n\nOutline\n\nIntroduction 20\n\nEvolution of mammals and lactation 21\n\nClassification of Mammals 22\n\nClassification and Phylogenetic Relationships of the Principal Dairying Species 23\n\nEvolution of the Mammary Gland 24\n\nStructure of the Mammary Gland 24\n\nUtilization of milk 25\n\nComposition of milk 25\n\nMilk constituents 26\n\nCarbohydrates 26\n\nLactose 26\n\nModification of the Concentration of Lactose in Milk through Genetic Engineering 28\n\nNutritional Problems Associated with Lactose 29\n\nProduction and Utilization of Lactose 29\n\nOligosaccharides 30\n\nLipids 32\n\nFatty Acids 32\n\nDistribution of FAs in Triglycerides 34\n\nDegradation of Lipids 34\n\nMilk Lipids as an Emulsion 35\n\nMilk Proteins 37\n\nPreparation of Casein and Whey Proteins 39\n\nComparison of Key Properties of Casein and Whey Proteins 40\n\nHeterogeneity and Fractionation of Casein 41\n\nApplication of Gel Electrophoresis to the Study of Milk Proteins 41\n\nMicroheterogeneity of the Caseins 42\n\nVariability in the Degree of Phosphorylation 42\n\nGenetic Polymorphism 42\n\nDisulfide Bonding 43\n\nVariations in the Degree of glycosylation 43\n\nHydrolysis of the Caseins by Plasmin 43\n\nMolecular Properties of the Milk Proteins 43\n\nNomenclature of Milk Proteins 45\n\nWhey Proteins 45\n\nFractionation of Whey Proteins 46\n\nMajor Characteristics of Whey Proteins 46\n\nBlood Serum Albumin 48\n\nImmunoglobulins 48\n\nWhey Acidic Protein 49\n\nProteose Peptone 3 49\n\nMinor Proteins 50\n\nMetal-binding Proteins 50\n\n\u03b22-Microglobulin 50\n\nOsteopontin 50\n\nVitamin-binding Proteins 51\n\nAngiogenins 51\n\nKininogen 51\n\nGlycoproteins 51\n\nProteins in the Milk Fat Globule Membrane 52\n\nGrowth Factors 52\n\nIndigenous Milk Enzymes 52\n\nBiologically Active Cryptic Peptides 53\n\nNonprotein Nitrogen 53\n\nCasein Micelles 53\n\nStability of Casein Micelles 53\n\nMicelle Structure 54\n\nInterspecies Comparison of Milk Proteins 56\n\nMilk Salts 58\n\nVitamins 60\n\nWater 60\n\nPhysical Properties of Milk 61\n\nMicrobiology of Milk 61\n\nSummary 61\n\n## Introduction\n\nMilk is a fluid secreted by the female of all mammalian species, of which there are about 4500 extant species (about 80% of mammalian species are extinct), and it meets the complete nutritional requirements of the neonate. The principal requirements are for energy (supplied by lipids and lactose and, when in excess, by proteins), essential amino acids and amino groups for the biosynthesis of nonessential amino acids (supplied by proteins), essential fatty acids, vitamins, inorganic elements, and water. The nutritional requirements of the neonate depend on its maturity at birth, its growth rate, and its energy requirements, which in turn depend mainly on environmental temperature. Therefore, the gross composition of milk shows large interspecies differences, which reflect these requirements (see Fox and McSweeney, 1998; Fuquay et al., 2011; McSweeney and Fox 2013). Milk also serves a number of physiological functions, which are performed mainly by proteins and peptides, including immunoglobulins, enzymes, enzyme inhibitors, growth factors, hormones, and antibacterial agents.\n\nOf the 4500 species of mammal, the milk of only about 180 species has been analyzed, and of these, the data for only about 50 species are considered reliable (a sufficient number of properly taken samples, representative sampling, adequate coverage of the lactation period). Milk from the commercially important species\u2014cattle, goat, sheep, buffalo, yak, horse, camel, and pig\u2014are quite well characterized; human milk is also well characterized, as is that of experimental laboratory animals, especially rats and mice. Reviews on nonbovine milks include: general (Evans, 1959; Jenness and Sloan, 1970; Fuquay et al. 2011; and Medhammar et al., 2011), buffalo (Laxminarayana and Dastur, 1968), goat (Parkash and Jenness, 1968; Jenness 1980; Haemlein, 1980), sheep (Bencini and Pulina, 1997), sheep and goats (Jandal, 1996; IDF 1996, Park et al., 2007); camel (Rao et al., 1970; Farah 1993), horse (Doreau and Boulot, 1989; Solaroli et al., 1993; Doreau, 1994; Park et al. 2006), human (Atkinson and Lonnerdal, 1989; Jensen, 1989, ) and sow (Verstegen et al., 1998). The Handbooks edited by Park and Haemlein (2006, 2013) are a particularly useful source of information on the milk of nonbovine mammals; they include chapters on goat, sheep, buffalo, mare, camel, yak, reindeer, sow, llama, minor species (moose, musk ox, caribou, alpaca, ass, elk, seals, sea lion, and polar bear), and human.\n\nThe milk of certain domesticated animals and dairy products derived from them are major components of the human diet in many parts of the world. Domesticated goats, sheep, and cattle have been used for milk production since about 8000 BC (Kindstedt, 2012). Recorded milk production today is greater than 700 \u00d7 106 tonnes per annum, about 84% of which is bovine, 13% is from buffalo, and about 2% each is ovine and caprine, with small amounts produced from horses, donkeys, camels, yaks, and reindeer (IDF, 2010). Milk and dairy products are consumed throughout the world but are particularly important in Europe, the United States, Canada, Argentina, India, Australia, and New Zealand. The contribution of milk and dairy products to dietary intake varies widely for different regions of the world; for example, the kilocalories per day supplied by milk range from 12 in China to 436 in Ireland; and in the UK, milk and dairy products supply \u223c30% of dietary protein consumed by young children, \u223c27% of dietary lipids, and \u223c65% of calcium (Barker, 2003; Patton 2011). Overall, the contribution of milk and dairy products to macro- and micro-nutrient intakes in the human diet is substantial, as reflected by the 2005 Dietary Guidelines for Americans recommendations to consume three servings of low-fat or fat-free milk or equivalent milk products daily as part of a healthy diet and lifestyle (see Cifelli et al., 2011).\n\nThe chemistry and physicochemical properties of milk have been studied for about 200 years and are now understood in considerable detail as well as described in a voluminous literature. The objectives of this chapter are to provide a summary and overview of the evolution of mammals and lactation and of the principal constituents of milk, especially proteins, which are the subject of this book. Where possible, an interspecies comparison of milk and its constituents is made. Numerous textbooks and review articles are cited.\n\n## Evolution of mammals and lactation\n\nThe secretion of milk is one of the characteristic features of mammals that evolved from egg-laying, premammalian reptiles, Synapsids and Cynodonts. Cynodonts are believed to be the ancestors of all mammals, which evolved \u223c200 M years ago (at the end of the Triassic Period). The word 'mammal' is derived from mamma, Latin for breast. Initially, mammals were small shrew-like creatures, but they have evolved and diversified to occupy all niches on land, sea, and air. They range in size from a few grams (pigmy shrew) to 200 tonnes (blue whale). Their dominance occurred especially after the extinction of the dinosaurs, 60\u201370 M years ago, at the interface between the Cretaceous and Tertiary Periods (C\/T interface). Mammals have been successful because the young of most species are born alive (viviparous) and all are supplied with a specially designed food, milk, for the critical period after birth. No other class of animal is so pampered (for an interesting discussion on this point, see Peaker, 2002). Not surprisingly, the evolution of mammals is a popular subject; reviews include Crampton and Jenkins (1973), Kemp (1982), Lillegraven et al. (1987), Lillegraven (2004), Forsyth (2003), Benton (1999), Easteal (1999), Springer et al. (2004), and Oftedal (2013).\n\nMammals are distinguished from other classes of animals by four criteria:\n\n\u2022 They secrete milk to nourish their young.\n\n\u2022 They are endothermic; that is, they can control their body temperature.\n\n\u2022 They grow body hair or wool for insulation; even aquatic mammals have some hair.\n\n\u2022 They have different types of teeth (flat incisors, conical canines, and multicusped molars) which allow them to masticate different types of food.\n\nThe class Mammalia contains two subclasses, Prototheria and Theria (young born alive):\n\nPrototheria: These egg-laying mammals, known as monotremes because they have only one opening for the elimination of waste, mating, and egg laying, were the first mammals. Only five species of these mammals survive: the duck-billed platypus and four species of echidna (also called spiny anteaters), which are found only in Australia and New Guinea. Presumably, there were other species of monotreme that have become extinct.\n\nTheria: About 90 M years ago, the Theria split into two infraclasses, Metatheria and Eutheria. However, the fossil of a eutherian mammal, believed to be \u223c 125 M years old, was discovered recently in northeastern China; it was named Eomonia scansoria, meaning \"earliest eutherian mammal with specialised features for climbing\" (Ji et al., 2002, p. 816).\n\nMetatheria: In this class of mammals, usually called marsupials, there are about 330 species. The young are born alive (viviparous) but are very immature and develop in an abdominal pouch (marse = pouch, purse). Marsupials survive mainly in Australia and the surrounding islands (>200 species), with several species in South America, and one species, the Virginia opossum, in North America; there are none in Europe, Asia, or Africa.\n\nEutheria: The fetus of this mammal develops in utero where it receives nourishment from the maternal blood via a highly-specialized organ, the placenta (these are called placental mammals); \u223c95% of all mammals are eutherians.\n\n### Classification of Mammals\n\nThere are \u223c4500 species of mammal, which are classified into 20 orders (see MacDonald, 2004). It is estimated that about 80% of the species that have evolved over the last 200 M years are extinct. The classification and nomenclature of mammals commenced with the work of Carolus Linnaeus in 1758 and was based initially on morphological characteristics (see MacDonald, 2004). More soundly based classification is now possible based on DNA sequences (Murphy et al., 2001; Madsen et al., 2001; Springer et al. 2004) and on the primary sequence of certain proteins, for example, growth hormone and prolactin (Forsyth and Wallis, 2002). It should be possible to classify mammals based on the structure of milk proteins, especially caseins, which are fast-mutating proteins. Some preliminary work has been reported (Chanat et al., 1999; Goldman, 2002; Rijnkels 2002, Simpson and Nicholas, 2002; Peaker, 2002; Rijnkels, 2002; Kawasaki and Weiss, 2003; Kawasaki et al., 2004, ; Lefevre et al., 2009; Le Parc et al. 2010).\n\nAlthough the sequence of milk proteins from a sufficient number of species has not been determined to make a comprehensive classification scheme possible, considerable progress on the structure of milk proteins is being made (see Martin et al., 2003; 2011; 2013a) and is discussed further later in this chapter.\n\nA minor whey protein, whey acidic protein (WAP), has already been useful for tracing the relationships between mammalian families (Hajjoubi et al., 2006). To date, WAP has been found only in the milk of platypus, echidna, tammar wallaby, possum, mouse, rat, rabbit, camel, and pig. In humans and ruminants, the gene for WAP has been frame-shifted and occurs as a pseudogene that is not transcribed. The distribution of WAP suggests that the loss of a functional WAP gene occurred after the divergence of the pig and ruminantia lines but before the bovidae diverged from the other ruminants. Analysis of the milk from a wider range of species for WAP should be interesting.\n\n### Classification and Phylogenetic Relationships of the Principal Dairying Species\n\nAll of the principal, as well as many of the minor, dairying species belong to the Family Bovidae, a member of the Order Artiodactyla [even-toed ungalates (hoofed) mammals, i.e., cloven-hoofed]. A few minor dairying species (horse and ass) are members of Perissodactyla (odd-toed ungalates). The Bovidae evolved \u223c 18 M years ago; the earliest fossil attributed to the Bovidae is Eotragus, found in 18 M-year-old deposits in Pakistan. The Artiodactyla Order has three suborders: Ruminantia (ruminants, to which all major dairying species belong), Suidae (pigs and related species), and Tylopoda (camels, llama, alpaca, and guanaco).\n\nThe Ruminantia are classified into six families: Tragulidae (chevrotains), Moschidae (musk deer), Antilocapridae (pronghorns), Giraffidae (giraffes and okapi), Cervidae (deer; 43 species in 16 genera), and Bovidae (137\u2013138 species in 46\u201347 genera).\n\nThe Bovidae are divided into six subfamilies, of which the Bovinae is the most important. The Bovinae are divided into three Tribes, of which the Bovini are the most important from our viewpoint. The Bovini are classified into five genera: Bubalus (water buffalo), Bos (cattle), Pseudoryx (Saola), Syncerus (African (Cape) buffalo), and Bison (American and European; the European Bison is also called a wisent).\n\nThere are seven or eight species of Bos: B. primigenus (aurochs, the ancestor of domestic cattle, are extinct, with the last animal killed in Poland in 1627), B. javanicus (banteng), B. gaurus (gaur), B. frontalis (gayol), B. mutus (yak), B. sauvali (kouprey), B. taurus (European cattle), and B. indicus (Indian, humped zebu cattle). (B. taurus and B. indicus may be sub-species rather than species.) The phylogenetic relationships of the Bovini have been studied by molecular biology techniques (see Finlay, 2005).\n\nToday, there are about 1.3 \u00d7 109 cattle worldwide, of which there are two species, B. taurus, of European origin, and B. indicus, which originated in India. B. indicus (zebu) cattle also dominate in Africa, but apparently African zebu cattle have some B. taurus genes, probably as a result of cross-breeding many centuries ago. Zebu are less efficient producers of milk or meat than B. taurus but are more resistant to heat stress and various diseases and therefore dominate in tropical regions.\n\nSince cattle were domesticated \u223c 8000 years ago, they have been bred selectively, especially during the past 200 years (i.e., since Herd Books have been kept). These breeding practices have selected for various characteristics, for example, health, fertility, docility, milk or meat production, or both. Today, there are about 1000 breeds of cattle (Buchanan, 2011), including dairy, beef, or dual-purpose breeds. There are \u223c 200 M dairy cows classified into many (mainly local) breeds; Holstein-Friesian is the principal breed of dairy cow, representing \u223c 35% of the total (\u223c 70 M cows). Other important international dairy breeds are Brown Swiss (\u223c 4 M), Jersey (\u223c2 M), Ayrshire, Guernsey, and Red Dane.\n\nThere are about 170 M buffalo worldwide, of which there are two types, river and swamp, found mainly in Southeast Asia, India, and Egypt, with smaller numbers in Bulgaria, Italy, Brazil, and Australia. Buffalos are usually named after the area from which they come. Depending largely on the region, buffalo are used for milk, meat, work, or combinations of these.\n\n### Evolution of the Mammary Gland\n\nEvolution of the mammary gland is believed to have begun with the synapsids, \u223c 300 M years ago. These reptiles laid membrane-shelled eggs that lost water rapidly through evaporation and were kept moist by an aqueous oily secretion from sebaceous apocrine glands on the breast\/abdomen of the mother. These were somewhat like the brood patches on the breast of birds. The secretions are believed to have contained a range of bactericidal substances, such as oligosaccharides, lysozyme, lactoferrin, transferrin, immunoglobulins, and peroxidases, which protected the eggs against microbial infection. Presumably, the secretions were licked by the neonate from the mother's abdomen and served as a source of nutrients.\n\nConsidering its importance in the evolution of mammals, including humans, the evolution of the mammary gland and the origin of lactation have attracted considerable attention, including Darwin (1872). Reviews on the subject include Pond (1977), Hayssen and Blackburn (1985), Blackburn et al. (1989), Blackburn (1991; 1993), Hayssen (1993), Oftedal (2002a,b; ), and Lemay et al. (2009).\n\n### Structure of the Mammary Gland\n\nThe microscopic structure of the mammary gland of all species, monotremes, marsupials, and eutherians, is basically similar. The structure of the bovine gland from the cellular to the organ level is described by Fox and McSweeney (1998) and Oftedal (2013). The cells (mammocytes), whose structure is basically similar to that of all animal cells, are arranged as a monolayer in a pear-shaped organelle, called an alveolus. The alveoli are connected via a system of ducts to a cistern where the milk is stored until it is expressed from the gland, usually through a teat that is sealed by a sphincter muscle. There is little de novo synthesis in the mammary gland. Rather, the major constituents of milk are synthesized from molecules imported from the blood through the basal cell membrane. Within the mammocyte, mainly in the rough endoplasmic reticulum (RER), these molecules are polymerized to lactose, lipids, or proteins. The mammocytes are provided with a good blood supply through an extensive system of capillaries and are surrounded by contractile myoepithelial cells which, under the control of the hormones, oxytocin and prolactin, contract and express milk from the alveoli through the ducts and eventually from the gland. The hormonal control of mammary growth and function was described by Forsyth (1986).\n\nAlthough at the microscopic level, the mammary gland is essentially similar across species, the number and appearance of the gland are characteristic of the species. Monotremes have many glands on the abdomen; the glands do not end in a teat, and the milk is licked from the abdomen by the young. Marsupials have two or four glands, which end in a teat, within the pouch. On entering the pouch, the young attaches to a teat and remains attached during the period it spends permanently in the pouch. During this period, an older offspring may use another gland during its visits to the pouch. The two glands secrete milk of very different composition, designed for the specific requirements of the neonate; the composition changes markedly when the offspring leaves the pouch intermittently (Sharp et al., 2011). The mammary glands of eutherians are located external to the body cavity and end in a teat; their number varies from 2 (human, goat, sheep, horse, elephant, etc.), 4 (cattle), 12\u201314 (pig) to 24 (some insectivores). The glands are separate anatomically.\n\nThe external location of the mammary gland facilitates study of the biosynthesis of milk constituents by isotope dilution techniques, arteriovenous concentration differences, profusion of the severed gland, tissue slices, and cell homogenates (Larson and Smith, 1974\u20131979; Mepham, 1983).\n\n## Utilization of milk\n\nSince young mammals are born at a different stage of maturity and with different nutritional requirements, the milk of each species is designed to meet the requirements of the neonate of that species, that is, it is species-specific. Milk is intended to be consumed unchanged by the young suckling its mother. However, humans have consumed the milk of other species for at least 8000 years. Several species have been used for milk production, but today, cattle, especially Bos taurus, is the principal dairying species, accounting for \u223c84% of total milk production. The other important dairying species are buffalo (Bubalus bubalus) (13%) and goats and sheep (\u223c2% each); other species are significant in certain regions, or for certain purposes, for example, camel, yak, reindeer, horse, and donkey.\n\nMilk is often described as the most 'nearly perfect' food, and although this is true only for the young of the producing or closely-related species. The milk of all species is a nutrient-rich and well-balanced food (Kon, 1959; du Puis, 2002; Patton 2004; ). Many of the minor constituents of milk have biological properties, which will be described in the appropriate section (see also Korhonen, 2006); these minor constituents have been attracting considerable attention recently. However, milk is very susceptible to the growth of microorganisms that will cause spoilage if the milk is not stored properly. To counteract this problem, humans have developed a range of products that are more stable than milk; some of these date from 4000 BC and have evolved desirable epicurean characteristics, in addition to their nutritional value. Today, several thousand food products are produced from milk; these fall into the following principal groups: liquid\/beverage milk (40%), cheese (35%), milk powders (15%), concentrated milks (2%), fermented milk products (2%), butter (30%; some of which is produced from cream\/fat obtained as a by-product in the manufacture of other products), ice cream, infant formula, creams, protein-rich products, and lactose. Some of these groups are very diverse, for example, 1400 varieties of cheese have been listed.\n\n## Composition of milk\n\nMilk is a very complex fluid containing several hundred molecular species (several thousand if all triglycerides are counted individually). The principal constituents are water, lipids, sugar (lactose), and proteins. In addition, there are numerous minor constituents, mostly at trace levels\u2014for example, minerals, vitamins, hormones, enzymes, and miscellaneous compounds. The chemistry of these compounds is generally similar across species, but in many cases their structure differs in detail, reflecting evolutionary changes. The concentrations of the principal constituents vary widely among species: lipids, 2\u201355%; proteins, 1\u201320%; and lactose, 0\u201310%, reflecting mainly the energy requirements (lipids and lactose) and growth rate (mainly proteins) of the neonate. The concentrations of the minor constituents also vary widely (e.g., lysozyme and lactoferrin in equine, human, and bovine milks).\n\nWithin any species, the composition of milk varies among individual animals, between breeds, with the stage of lactation, feed, and health of the animal, along with many other factors. The fat content of bovine milk shows large inter-breed differences, and within any breed there is a wide range of fat and protein content for individual animals; similar differences occur in the milk of sheep, goat, and buffalo.\n\nReflecting mainly the nutritional and physiological requirements of the neonate, the composition of milk, and even the profile of constituents therein, change markedly during lactation. The changes are most marked during the first few days postpartum, especially in the immunoglobulin fraction of proteins. For marsupials, the milk changes from a high-carbohydrate (mainly oligosaccharides) to a high-fat secretion when the neonate begins to leave the pouch, a time that corresponds roughly to the birth of eutherians. The composition of milk remains relatively constant during mid-lactation but changes considerably in late lactation, reflecting the involution of the mammary gland tissue and the greater influx of blood constituents.\n\n## Milk constituents\n\nIn the following sections, the chemistry of milk carbohydrates, lipids, proteins, salts, and some minor constituents are described; where possible, interspecies comparisons are made.\n\n### Carbohydrates\n\n#### Lactose\n\nThe principal carbohydrate in the milk of most species is the reducing disaccharide, lactose, which is composed of galactose and glucose linked by a \u03b21-4 glycosidic bond. Its concentration varies from 0 to \u223c10% (Fox and McSweeney, 1998; McSweeney and Fox, 2009), and milk is the only known source of lactose. Research on lactose commenced with the work of Carl Scheele in about 1780; its chemistry and its important physicochemical properties have been described very thoroughly. The very extensive literature has been reviewed by Whittier (1925; 1944), Weisberg (1954), Zadow (1984; 1992), Fox (1985; 1997), Fox and McSweeney (1998), McSweeney and Fox (2009), Fox (2011), Paterson (2011), and Schuck (2011).\n\nThe concentration of lactose in milk is inversely related to the concentration of lipids and casein because lactose causes the influx of water into the mammocytes, thereby causing dilution of milk (Jenness and Sloan, 1970; Jenness and Holt 1987). The principal function of lactose and lipids in milk is as a source of energy; since lipids are 2.5 times as energy-dense as lactose, when a highly calorific milk is required, for example, by animals in a cold environment (e.g., marine mammals or hibernating bears, this is achieved by increasing the fat content of the milk. The inverse relationship between the concentrations of lactose and casein reflects the fact that the synthesis of lactose draws water into the Golgi vesicles, thereby diluting the concentration of casein (Jenness and Holt, 1987).\n\nLactose is synthesized in the epithelial mammary cells from two molecules of glucose absorbed from the blood. One molecule of glucose is phosphorylated and converted (epimerized) to galactose-P via the Leloir pathway, which is widespread in animal tissues and bacterial cells. Galactose-P is condensed with a second molecule of glucose through the action of a unique two-component enzyme, lactose synthetase. One component is UDP-galactosyl transferase (EC 2.4.1.22), which transfers galactose from UDP-galactose to any of several acceptor molecules in the biosynthesis of glycoproteins and glycolipids. The specificity of the transferase is controlled and modified by \u03b1-lactalbumin (\u03b1-La), one of the principal milk proteins, which reduces the Michaelis constant (KM) for glucose 1000-fold. In its presence, most of the galactose is transferred to glucose, with the synthesis of lactose. There is a positive correlation between the concentrations of lactose and \u03b1-La in milk; the milk of the California sea lion or the hooded seal lack both \u03b1-La and lactose.\n\nLactose serves two important functions in milk: It is a ready source of energy for the neonate (it provides 30% of the calories in bovine milk); and it is responsible for about 50% of the osmotic pressure of milk, which is isotonic with blood and hence is essentially constant. The synthesis of lactose draws water osmotically into the Golgi vesicles and hence affects the volume of milk and the concentration of casein, which is packaged in the Golgi vesicules.\n\nFor milk with a low level of lactose, the concentration of inorganic salts is high to maintain the osmotic pressure at the desired level; there is an inverse relationship between the concentrations of lactose and salts (ash) in milk (Jenness and Sloan, 1970). During mastitis or in late lactation, the integrity of the mammocyte cell membranes is damaged, and there is an influx of blood constituents into milk, the osmotic pressure increases, and to adjust this imbalance, the concentration of lactose is reduced. This relationship is expressed as the Koesler Number, % chloride \u00d7 100 \u00f7 % lactose, which is normally <2 and a value >3 is considered abnormal. Today, the Koesler Number is rarely used as a diagnostic indicator of mastitis, but the electrical conductivity of milk, which depends mainly on the milk salts and can be measured in-line during milking, is commonly used for this purpose.\n\nWhy milk contains lactose rather than some other sugar(s) is not clear. The presence of a disaccharide rather than a monosaccharide can be explained on the basis that twice as much (mass) disaccharide as monosaccharide can be accommodated for any particular incremental increase in osmotic pressure, which is fixed. Maltose, which consists of two molecules of glucose, would seem to be the obvious choice of disaccharide. Since energy is expended in converting glucose to galactose, some benefit must accrue from this conversion. A possible benefit is that galactose or derivatives thereof occurs in some physiologically important lipids and proteins and a galactose-containing sugar in milk provides the neonate with a ready supply of this important monosaccharide.\n\nThe properties of lactose are generally similar to those of other sugars, but it differs in some technologically important respects. Some important characteristics of lactose are:\n\n\u2022 Lactose is a reducing sugar; that is, it has a free, or potentially free, carbonyl group (an aldehyde group in the case of lactose).\n\n\u2022 Like other reducing sugars, lactose exists partially as an open-chain form with an aldehyde group that can form a hemiacetal and thus a ring structure. Formation of a hemiacetal creates a new chiral center (asymmetric carbon), which may exist as two isomers (enanthiomorphs), \u03b1 or \u03b2. By alternatively opening and forming the ring structure, the molecule can interchange between \u03b1 and \u03b2 isomers, a process referred to as mutarotation.\n\n\u2022 The \u03b1 and \u03b2 isomers of lactose have very different properties, the most important of which are specific rotation, [\u03b1]20 D (+89o and +35o for \u03b1 and \u03b2, respectively) and solubility (70 and 500 g\/L, for \u03b1 and \u03b2, respectively).\n\n\u2022 Like all reducing sugars, lactose can participate in the Maillard (nonenzymatic) browning reaction, resulting in the production of (off-) flavor compounds and brown polymers. The Maillard reaction contributes positively to the flavor and color of many foods (e.g., crust of bread, toast, and deep-fried products), but the effects in dairy products are negative and must be avoided.\n\n\u2022 Redox titration using alkaline CuSO4 (Fehling's solution) or chloramine-T is the principal standard method for the quantitative determination of lactose. It may also be determined by polarimetry, spectrophotometrically after reaction with phenol or anthrone in strongly acidic solution, enzymatically, or by high-performance liquid chromatography.\n\n\u2022 Among sugars, lactose, especially the \u03b1 enanthiomorph, has low solubility in water, but when in solution, it is difficult to crystallize and may cause problems in lactose-rich dairy products (e.g., skim milk powder and especially whey powder), unless precautions are taken to induce and control crystallization.\n\n\u2022 \u03b1 and \u03b2 lactose are soluble in water to the extent of about 70 and 500 g\/L, respectively, at 20 \u00b0C; at equilibrium, the ratio of \u03b1:\u03b2 is about 1:2, giving a total solubility of about 180 g\/L at 20 \u00b0C. The solubility of \u03b1 lactose is more temperature dependent than that of the \u03b2 isomer; the solubility-temperature curves intersect at \u223c94oC, making \u03b1 lactose more soluble than the \u03b2 anomer >94 \u00b0C. Hence, \u03b1-lactose is the form of lactose that crystallizes <94 \u00b0C and is the usual commercial form of lactose; \u03b2 lactose may be prepared by crystallization >94 \u00b0C\n\n\u2022 \u03b1 lactose crystallizes as a monohydrate, while \u03b2 lactose forms anhydrous crystals; thus, the yield of \u03b1 lactose is 5% higher than that of \u03b2 lactose.\n\n\u2022 When milk or whey is spray-dried, any lactose that has not been precrystallized forms an amorphous glass that is stable if the moisture content of the powder is maintained low. If it increases to >6%, however, the lactose crystallizes as \u03b1 hydrate, the crystals of which form interlocking masses and clumps that may render the powder unusable if very extensive; (i.e., inadequately crystallized powder is hygroscopic). The problem can be avoided by adequate crystallization of lactose before drying or by using effective packaging.\n\n\u2022 Interestingly, crystalline lactose has very low hygroscopicity and is used in icing sugar blends.\n\n\u2022 Among sugars, lactose has a low level of sweetness; it is only about 16% as sweet as sucrose at 1% in solution and hence has limited value as a sweetening agent, the principal application of sugars in foods. However, it is a useful bulking agent when excessive sweetness is undesirable.\n\n\u2022 Lactose is important in the manufacture of fermented dairy products where it serves as a carbon source for lactic acid bacteria that produce lactic acid.\n\n#### Modification of the Concentration of Lactose in Milk through Genetic Engineering\n\nBecause lactose is the least valuable constituent in milk, there is considerable interest in modifying the lactose content of milk, as it costs energy on the part of the animal to synthesize it. Therefore, it would be economically advantageous to reduce the lactose content of milk. In addition, lactose effectively controls the water content of milk, and most dairy processes require the removal of water. Hence, it would be advantageous to reduce the amount of water in milk by reducing the level of lactose. Since the concentration of lactose is controlled by the concentration of \u03b1-La in the secretory cells, the approach to changing the concentration of lactose involves altering the level of \u03b1-La by genetic engineering. However, if the level of lactose is reduced too much, the viscosity of the milk will be too high for easy expression from the mammary gland. It has been shown that the viscosity of mouse milk engineered to contain no lactose was so high that the pups were unable to suckle and died (Leaver and Law, 2003). Obviously, this problem could be overcome by reducing the level of lactose rather than eliminating it. Alternatively, it may be possible to modify the milk secretory mechanism to produce a more useful, or at least a less problematic sugar than lactose, for example, glucose, maltose, or lactulose (which is a laxative and a prebiotic). It might be possible to increase the concentration of salts in milk. As discussed below, most adult humans are unable to digest lactose. If the problems arising from high viscosity were resolved, lactose-free or -reduced milk would be nutritionally desirable. The possibility of engineering the mammary cell to secrete \u03b2-galactosidase into milk and to hydrolyze lactose in situ has been suggested.\n\nHowever, in some cases it would be advantageous to increase the lactose content of milk. The economic benefits of increasing the milk output of sows by increasing its lactose content were discussed by Wheeler (2003).\n\n#### Nutritional Problems Associated with Lactose\n\nMammals cannot absorb disaccharides from the small intestine, where they are hydrolyzed to monosaccharides, which are absorbed. Lactose is hydrolyzed by \u03b2-galactosidase (lactase), which is secreted by cells in the brush border of the small intestine. The young of most mammalian species secrete an adequate level of lactase, but as the animal ages, the secretion of lactase declines and eventually becomes inadequate to hydrolyze indigested lactose. The lactose then enters the large intestine where it causes an influx of water, resulting in diarrhea, and is metabolized by bacteria with the production of gas that causes cramps and flatulence. In humans, this condition may occur at 8\u201310 years of age and cause many individuals to exclude milk from the diet. The problems may be avoided by prehydrolyzing the lactose using exogenous \u03b2-galactosidase (see Mahoney, 1997; Shakeel-Ur-Rehman 2009). The frequency and intensity of lactose intolerance\/malabsorption vary widely among populations from \u223c100% in Southeast Asia to \u223c5% in northwest Europe (Mustapha et al., 1997; Ingram and Swallow 2009).\n\n#### Production and Utilization of Lactose\n\nPreviously, whey from cheese or casein production was considered a waste material that was fed to farm animals, irrigated on land, or disposed of in sewers. Environmental and economic considerations now dictate, however, that whey should be used more efficiently. The principal product lines produced from whey are whey powders (various), whey protein products produced by membrane technology, and lactose and its derivatives.\n\nLactose is prepared commercially by crystallization from concentrated whey or ultrafiltrate. The crystals are usually recovered by centrifugation; this process is essentially similar to that used for sucrose or other sugars. About 500,000 tonnes of crystalline lactose are produced annually (Paterson, 2009), compared to \u223c100 M tonnes of sucrose. Owing to its relatively low sweetness and low solubility, the applications of lactose are much more limited than those of sucrose or glucose. Its principal application is in the production of 'humanized' infant formula based on cow's milk (human milk contains \u223c7% lactose in comparison with \u223c4.6% in bovine milk). The lactose used may be in the form of a purified crystalline product or demineralized whey (for physiological reasons, it is necessary to reduce the concentration of inorganic salts in whey).\n\nLactose has a number of low-volume special applications in the food industry, for example, as a free-flowing or agglomerating agent, to accentuate\/enhance the flavor of some foods, to improve the functionality of shortenings, and as a diluent for pigments, flavors, or enzymes. It is widely used in the tabletting of drugs in the pharmaceutical industry where low hygroscopicity is a critical property.\n\nLactose can be converted to several more valuable food-grade derivatives, of which the most significant are glucose-galactose syrups (\u223c3 times as sweet as lactose; produced by hydrolysis by \u03b2-galactosidase), lactulose (galactose-fuctose; a prebiotic and laxative), lactitol (the alcohol of lactose), lactobionic acid (a sweet-tasting acid, which is a very rare property), tagatose, oligosaccharides (prebiotics), and fermentation products (ethanol, lactic, acetic, and propionic acids) (Playne and Crittenden, 2009; Ganzle, 2011a,b).\n\n#### Oligosaccharides\n\nIn addition to lactose, the milk of most, and probably all, species contains other free saccharides, mainly oligosaccharides (OSs), the concentration, proportions and types of which show large interspecies differences. The concentration of OSs is higher in colostrum than in milk. General reviews on the OSs in milk include Newburg and Newbauer (1995), Mehra and Kelly (2006), and Urashima et al. (2001; ; 2011).\n\nAlmost all of the OSs have lactose at the reducing end, they contain three to eight monosaccharides, they may be linear or branched, and contain either or both of two unusual monosaccharides, fucose (a 6-deoxyhexose) and N-acetylneuraminic acid. Fucose occurs quite widely in tissues of mammals and other animals where it serves a wide array of functions (Becker and Lowe, 2003). Its significance in the OSs in milk is not clear; perhaps it is to supply the neonate with preformed fucose.\n\nThe OSs are synthesized in the mammary gland, catalyzed by special transferases that transfer galactosyl, sialyl, N-acetylglucosaminyl, or fucosyl residues from nucleotide sugars to the core structures. These transferases are not affected by \u03b1-La and are probably similar to the transferases that catalyze the glycosylation of lipids and proteins.\n\nThe milk of all species examined contains OSs, but the concentration varies markedly. The highest levels are in the milk of monotremes, marsupials, marine mammals, humans, elephants, and bears. With the exception of humans and elephants, the milk of these species contains little or no lactose, and OSs are the principal carbohydrates.\n\nThe milk of the echidna contains mainly the trisaccharide, fucosyllactose, while that of the platypus contains mainly the tetrasaccharide, difucosyllactose. Among marsupials, the best studied is the Tammar wallaby; presumably, its lactation pattern and milk composition are typical of marsupials. A low level of lactose is produced at the start of lactation, but about 7 days after birth, a second galactosyltransferase appears and tri- to penta-saccharides are produced, which by \u223c180 days are the principal saccharides. During this period the saccharide content is high, \u223c50% of total solids, and the level of lipids is low (\u223c15% of total solids). At about 180 days, the carbohydrate decreases to a very low level and consists mainly of monosaccharides, while the level of lipids increases to \u226560% of total solids (Sharp et al., 2011).\n\nHuman milk contains \u223c130 OSs, at a total concentration of \u223c15 g\/L; these are considered to be important for neonatal brain development. Bear milk contains little lactose but a high level of total sugars (mainly OSs) \u2013 1.7 and 28.6 g\/kg, respectively (Oftedal et al. 1993; ). Elephant milk contains \u223c50 and 12 g\/kg of lactose and OSs, respectively, a few days postpartum, but as lactation progresses, the concentration of lactose decreases while that of OSs increases (e.g., 12 and 18 g\/kg, respectively), at 47 days (Osthoff et al., 2005). The milk of seals contains both lactose and OSs, but milks of the Californian sea lion, Northern fur seal, and Australian fur seal contain neither, probably because they contain no \u03b1-La (Urashima et al., 2001).\n\nBovine, ovine, caprine, and equine milk contain relatively low levels of OSs, which have been characterized (see Urashima et al. 2001; ; ). Caprine milk contains about 10 times as much OSs as bovine and ovine milk, and a process for their isolation by nanofiltration has been reported (Martinez-Ferez et al., 2006). Possible methods for producing OSs similar to those found in human milk, by fermentation or by transgenic animals or by recovering OSs from cow's milk whey or UF permeate were discussed by Mehra and Kelly (2006) and O'Mahony and Tuohy (2013).\n\nAs discussed earlier, OSs with bactericidal properties were probably the saccharides present in the mammary secretions of early mammals; the high level of OSs in the milk of monotremes and marsupials conforms with their secretion early in evolution. It is proposed that the primitive mammary glands of the first common ancestor of mammals produced lysozyme (a predecessor of \u03b1-La), and a number of glycosyltransferases but little or no \u03b1-La. This resulted in the production of a low level of lactose that was utilized in the synthesis of OSs and did not accumulate (Messer and Urashima 2002; Urashima et al. 2009). Initially, the OSs served mainly as bactericidal agents but later became a source of energy for the neonate. Both of these functions persist for monotremes, marsupials, and some eutherians such as bears, elephants, and marine mammals. However, most eutherians evolved to secrete predominantly lactose as an energy source, due to the synthesis of an increased level of \u03b1-La, while OSs continued to play a bactericidal role. Human and elephant milk, both of which contain high levels of lactose and OSs, seem to be anomalous. Work on the OSs of a wider range of species is needed to explain this situation.\n\nThe significance of OSs is not clear, but the following aspects may be significant: For any particular level of energy, they have a smaller impact on osmotic pressure than smaller saccharides, they are not hydrolyzed by \u03b2-galactosidase, and fucosidase or neuraminidase is not secreted in the intestine. Hence the OSs are not hydrolyzed and absorbed in the gastrointestinal tract, and they function as soluble fiber and prebiotics that affect the microflora of the large intestine. It is claimed that they prevent the adhesion of pathogenic bacteria in the intestine; galactose, and especially N-acetylneuraminic acid, are important for the synthesis of glycolipids and glycoproteins, which are vital for brain development. It has therefore been suggested that the OSs are important for brain development (see Kunz and Rudloff, 2006).\n\nIn addition to lactose and free OSs, the milk of all species examined contains small amounts of monosaccharides and some milk proteins, especially \u03ba-casein, are glycosylated, and there are low levels of highly glycosylated glycoproteins, especially mucins, and glycolipids in the milk fat globule membrane.\n\nThere is considerable interest in the development of OS-enriched ingredients from bovine milk (O'Mahony and Tuohy, 2013), primarily for infant formula applications. This interest has been spurred by the demonstrated bioactive functionality of these compounds in humans (Kunz and Rudlof, 2006).\n\n### Lipids\n\nLipids (commonly called oils or fats, which are liquid or solid, respectively, at ambient temperature) are those constituents of tissues, biological fluids, or foods that are soluble in an apolar solvent (e.g., diethyl ether, chloroform, or carbon tetrachloride). Historically, the fat of milk was regarded as its most valuable constituent, and until recently, milk was valued largely or totally on the basis of its fat content. This was due at least partially to S. M. Babcock and N. Gerber's development of rather simple methods for quantifying the fat content of milk in the 1890s, long before comparable fast and simple methods for proteins became available. Milk lipids are very complex chemically and exist as a rather unique emulsion. Milk lipids have been thoroughly studied and characterized (see Fox 1983; ; Fox and McSweeney 1998; ; and the references therein).\n\nThe level of fat in milk shows very large interspecies differences, ranging from \u223c2% to >50% (see Fox and McSweeney, 1998). The fat content of milk reflects the energy requirements of the neonate; the requirement is high in the milk of species that live in a cold environment or need to build up a layer of subcutaneous fat quickly (marine mammals).\n\nLipids are commonly divided into three classes:\n\n\u2022 Neutral lipids. These are esters of glycerol, and one, two, or three fatty acids for mono-, di-, and triglycerides, respectively. Neutral lipids are by far the dominant class of lipids in all foods and tissues, representing 98.5% of total milk lipids.\n\n\u2022 Polar lipids (a complex mixture of fatty acid esters of glycerol or sphingosine). These may contain phosphoric acid, a nitrogen-containing compound (choline, ethanolamine, or serine), or a sugar\/OS. Although present at low levels (\u223c1% of total milk lipids), the polar lipids play critical roles in milk and dairy products. They are very good natural emulsifiers and are concentrated in the milk fat globule membrane that maintains the milk lipids as discrete globules and ensures their physical and biochemical stability.\n\n\u2022 Miscellaneous lipids. This is a heterogeneous group of compounds that are unrelated chemically to each other or to neutral or polar lipids. This group includes cholesterol, carotenoids, and the fat-soluble vitamins, A, D, E, and K.\n\nThe carotenoids are important for two reasons: They are natural pigments (yellow, orange, red), and they are responsible for the color of butter and cheese. Some consumers prefer highly colored cheese, which is obtained by adding a carotenoid-containing extract from annatto beans. Some carotenoids are converted to vitamin A in the liver.\n\n#### Fatty Acids\n\nFatty acids (FAs) are carboxylic acids with the general formula R-COOH, where the alkyl group, R, is a hydrocarbon chain containing 3 to 25 carbons (total number of carbons, 4 to 26), which may be saturated or unsaturated (one to six double bonds), and is usually straight (normal), with small amounts of branched chain, hydroxyl, and keto (oxo) acids. The vast majority of FAs have an even number of carbon atoms because they are synthesized from, and elongated by adding, a 2-C compound, acetyl CoA, on each cycle of the multienzyme fatty acid synthetase (FAS). Although the hydroxy fatty acids are present at low levels, they are important in milk fat because upon heating they are converted to lactones, which give a desirable flavor to milk fat, which is considered the premium cooking fat. Although keto acids are also minor components, they are important flavor precursors since they are converted to highly-flavored methyl ketones.\n\nThe melting point (MP) of FAs increases progressively with molecular weight (MW), while solubility in water decreases. The MP decreases with the introduction of double bonds, and for unsaturated FAs, the MP of the cis isomer is lower than that of the trans isomer.\n\nMilk lipids are chemically similar to all other lipids but contain a wide range of FAs (up to 400 FAs have been reported in milk lipids, although most of these are present at trace levels). The milk lipids of ruminants are unique in that they are the only natural lipids that contain butyric (butanoic) acid (C4:0). They also contain substantial amounts of medium-chain FAs [hexanoic (C6:0), octanoic (C8:0), and decanoic (C10:0)], the only other sources of which are coconut and palm kernel oil. The short- and medium-chain FAs are water-soluble and volatile and have a strong aroma and taste.\n\nThe fatty acids in milk fat are obtained from three sources:\n\n\u2022 Butanoic acid is produced by reducing \u03b2-hydroxybutanoic acid, which is synthesized by bacteria in the rumen.\n\n\u2022 All hexanoic (C6:0) to tetradecanoic (C14:0) acids and 50% of hexadecanoic (C16:0) acid are synthesized in the mammary gland from acetyl CoA (CH3COSCoA). These FAs are released from the FAS by chain-length-specific thioesterases, the relative activities of which are responsible for interspecies differences in the proportions of medium-chain FAs. Decanoic acid (C10:0) and dodecanoic (C12:0) are major FAs in the milk fat of elephant, horse, donkey, zebra, tapir, rhinoceros, rabbit, and hare, but these fats contain very little or no butanoic acid (Glass et al., 1967; Glass and Jenness, 1971; Christie, 1995; Osthoff et al., 2005; MacGibbon and Taylor 2006). These species are nonruminant herbivores with a large secum, a feature that presumably is somehow responsible for the high levels of C10:0 and C12:0; some of the above species also practice coprophagy. All octadecanoic (C18:0) and 50% of hexadecanoic (C16:0) acids are obtained from dietary lipids.\n\nThe unsaturated FAs are synthesized as follows:\n\n\u2022 C18:1 is produced from C18:0 in the liver by \u0394-9 desaturase.\n\n\u2022 C18:2 is obtained from the diet; that is, it is an essential FA.\n\n\u2022 The other unstaturated FAs are produced from C18:2 by further desaturation and\/or elongation.\n\nRuminant milk fats contain low levels of polyunsaturated fatty acids (PUFAs) because PUFAs in the diet are hydrogenated by bacteria in the rumen. Biohydrogenation can be prevented by encapsulating dietary PUFAs or PUFA-rich sources in cross-linked protein or cross-linked crushed oilseeds. PUFA-enriched milk has improved spreadability and perceived improved nutritional qualities (Parodi, 2006; O'Brien and O'Connor 2011).\n\nIncomplete biohydrogenation by the rumen bacterium, Butyrivibrio fibrisolvens, results in the formation of conjugated linoleic acid (CLA; also called rumenic acid), which has potent anticarcinogenic properties. Eight isomers of CLA are possible, but cis-9, trans-11 is the most biologically active. The formation of CLA and its nutritional benefits have been the subject of considerable research during the past 15 years and has been reviewed by Bauman and Lock (2006), Parodi (2006), Bauman et al. (2011), and Mills et al. (2011).\n\n#### Distribution of FAs in Triglycerides\n\nAs well as the constituent FAs, the position of the FAs in triglycerides (TGs) affects their MP and rheological properties. For these reasons and to completely characterize the structure of TGs, the position of FAs in milk TGs has been determined. An index of the length of the FAs can be obtained by determining the acyl carbon number (ACN) of TGs, that is, the sum of the number of carbons in the three-component FAs, which can be done by gas chromatography (GC). Probably the first study on this aspect was done by Breckenridge and Kuksis (1967), who reported the ACN of the milk TGs from seven species. More recent work has been reviewed by Christie (1995), MacGibbon and Taylor (2006), and Taylor and MacGibbon (2011).\n\nThe complete structure of TGs can be determined by stereospecific analysis, the results of which for milk fat are described by Christie (1995) and MacGibbon and Taylor (2006). The most notable feature is the almost exclusive esterification of the short-chain FAs, C4:0 and C6:0, at the Sn3 position. Since many lipases are specific for the Sn3 position, these short-chain FAs (which are highly flavored\/off-flavored) are released rapidly, causing desirable\/undesirable changes in sensory properties.\n\n#### Degradation of Lipids\n\nFood lipids are susceptible to two forms of deterioration: lipid oxidation leading to oxidative rancidity and hydrolysis of lipids by lipases (lipolysis), leading to hydrolytic rancidity. Lipid oxidation involves a very complex set of chemical reactions that have been well characterized; the literature has been comprehensively reviewed by Richardson and Korycka-Dahl (1983) and O'Connor and O'Brien (2006; 2011).\n\nMilk contains an indigenous lipoprotein lipase (LPL) that is normally inactive because it is separated from the TG substrates by the milk fat globule membrane (MFGM), but if the membrane is damaged, lipolysis and hydrolytic rancidity ensue rapidly. When milk lipids are hydrolyzed by milk LPL, the short- and medium-chain FAs, which are esterified mainly at the Sn3 position, are released preferentially, and are major contributors to flavor, which may be desirable or undesirable, depending on the product. Hydrolytic rancidity caused by milk LPL is potentially a very serious problem in raw milk and in some dairy products. Lipolysis in milk has been reviewed comprehensively by Deeth and Fitz-Gerald (1983; ; 2006); and Deeth (2011).\n\nA low level of lipolysis is desirable in all types of cheese, especially in blue cheeses, in which the principal lipases are those secreted by the blue mold, Penicillium roqueforti. The free fatty acids (FFAs) are converted to alk-2-ones, the principal flavor compounds in blue cheeses. The characteristic piquant flavor of some cheeses, such as Pecorino Romano, is due to short- and medium-chain FAs that are released mainly by an added lipase, pregastric esterase. Other important derivatives of FAs are alk-2-ols (secondary alcohols), lactones, esters, and thioesters; these are important flavor compounds in cheese.\n\n#### Milk Lipids as an Emulsion\n\nLipids are insoluble in water or aqueous systems. When mixed, a lipid and water (or aqueous solvent) form distinct layers and a force, interfacial tension (\u03b3), exists between the layers. Lipids can be dispersed in water by vigorous agitation (homogenization), but when agitation ceases, the droplets of lipid coalesce quickly into a single mass (i.e., phase separation), driven by the need to reduce the interfacial area and, consequently, interfacial tension, \u03b3, to a minimum. If \u03b3 is reduced, the droplets of lipid will remain discrete, although they will rise to the surface (i.e., cream) owing to the lower density of lipids compared to water. Interfacial tension can be reduced by using a surface-active agent (emulsifier, detergent). Natural emulsifiers include proteins, phospholipids, mono- and diglycerides; there is a wide range of synthetic emulsifiers.\n\nIn milk, the lipids are dispersed in the milk serum (specific gravity, 1.036) as globules with a diameter in the range <1 to \u223c20 \u03bcm (mean 3\u20134 \u03bcm). The FAs (from the sources described above) and monoglycerides (from blood lipids) are synthesized to TGs in the rough endoplasmic reticulum (RER) at the basal region of the epithelial cells. The TGs form into globules within the RER and are released into the cell cytoplasm. The globules are stabilized by a complex layer of proteins and phospholipids, known as the MFGM. The inner layer of the MFGM is acquired within the epithelial cell as the fat globules, after release from the RER, move toward the apical membrane. The outer layer of the MFGM is the apical membrane of the secretory cell through which the lipid globules are pushed as they are expressed from the cell. Since the stability of the milk emulsion is critical in most dairy products, the structure and stability of the MFGM have been the subject of research for more than 100 years.\n\nSodium dodecyl sulfate (SDS)-PAGE indicates that there are eight main proteins in the MFGM butryophilin (BTN), xanthine oxidoreductase (XOR), adipophilin (ADPH), mucin 1 (MUC 1), mucin 15 (MUC 15), periodic acid Schiff glycoproteins (PAS) 6 and 7 and fatty acid-binding protein (FABP)], which have been isolated and characterized (see [Mather, 2000; Keenan and Mather 2006). However, SDS-PAGE followed by microcapillary HPLC-MS indicates 120 proteins, of which 23% are involved in protein trafficking, 23% in cell signaling, 21% in unknown functions, 11% in fat trafficking\/metabolism, 9% in transport, 7% in protein synthesis\/folding, 4% are immune proteins, and 2% are contaminating skim milk proteins (Reinhardt and Lippolis, 2006). Many of the 70 indigenous enzymes in milk are concentrated in the MFGM. The very extensive literature on the MFGM has been the subject of many reviews, including Keenan and Mather (2006) and Mather (2011), who present an up-to-date model of the MFGM (Fig. 2.1).\n\nFigure 2.1 Topology of the major bovine milk fat globule membrane proteins (from Mather, 2011).\n\nSome of the MFGM is shed during the aging of milk, especially if agitated, and forms vesicles (sometimes called microsomes) in the skim milk. The MFGM may be damaged by agitation, homogenization, whipping, or freezing, which may lead to hydrolytic rancidity and non-globular fat, potentially causing cream plug, oiling-off in coffee and tea, and poor wettability of milk powder (for a review see Evers, 2004). The MFGM is stripped from the fat globules by extensive agitation (usually of cream), a process referred to as 'churning'; the free fat coalesces and is kneaded (worked) to give a water-in-oil emulsion, butter. The MFGM partitions into the aqueous phase, referred to as buttermilk. The phospholipids in buttermilk give it good emulsifying properties, and there is commercial interest in using it as a food ingredient (Singh, 2006). Several approaches, many using membrane filtration technology, have been proposed and evaluated over the last 20 years or so for their suitability to fractionate buttermilk\/butter serum in the enrichment of MFGM material (see Zanabria Eyzaguirre and Corredig, 2011; O'Mahony and Tuohy 2013). Some of the polar lipids in the MFGM are reported to have desirable nutritional properties (see Spitsberg, 2005; Ward et al. 2006), but there are conflicting views (see Moss and Freed, 2003; Riccio 2004).\n\nPresumably, the fat globules in the milk of all species are stabilized by a membrane similar to that in bovine milk, but there is very little information on the MFGM in nonbovine milks. Buchheim et al. (1989) and Welsch et al. (1990) studied the glycoproteins in the MFGM of human, rhesus monkey, chimpanzee, dog, sheep, goat, cow, gray seal, camel, and alpaca by SDS-PAGE with periodic acid Schiff (PAS) staining, Western blotting, and lectin biochemistry. Large intra- and interspecies differences were found; and very highly glycosylated proteins were seen in the MFGM of primates, horse, donkey, camel, and dog. Long (0.5\u20131 \u03bcm) filamentous structures extend from the surface of the fat globules in equine and human milk; the filaments are composed of mucins (highly glycosylated proteins) that dissociate rapidly from the surface of bovine globules into the milk serum on cooling; they are also lost on heating human milk (e.g., at 80oC for 10 min; see Patton, 1999, and references therein). The filaments facilitate the adherence of fat globules to the intestinal epithelium and probably improve the digestion of fat. The mucins prevent bacterial adhesion and may protect mammary tissue against tumors (mammary tumors are very rare in cattle) (Patton, 1999). Why the filaments on bovine milk fat globules are lost much more easily than those in equine and human milk is not known; work in this area is warranted. Proteomic methodology is being applied to study the human MFGM and membranes of the mammary epithelial cells (for references see Reinhardt and Lippolis, 2006; Lopez and Menard 2011).\n\nThe fat globules in bovine milk form a cream layer due to the difference in specific gravity between the fat and aqueous phases, but the cream layer is readily dispersed by gentle agitation. The rate of creaming can be calculated from Stokes's equation:\n\nV = 2 r 2 ( \u03c1 1 \u2212 \u03c1 2 ) g \/ 9 \u03b7\n\nwhere V is the velocity of creaming, r is the radius of the fat globules, \u03c11 and \u03c12 are the specific gravity of the continuous and dispersed phases, respectively, g is acceleration due to gravity, and \u03b7 is the viscosity of the continuous phase. Based on the typical values for r, \u03c11, \u03c12, and \u03b7 for milk, one would expect a cream layer to form in milk in about 60 h, but, in fact, a cream layer forms in about 30 min. The faster than expected rate of creaming is due to the aggregation of fat globules, aided by an immunoglobulin M-type protein, called cryoglobulin, because it precipitates onto the fat globules when the milk is cooled. The clusters of globules behave as a unit with a large radius. Creaming can be prevented by homogenizing the milk, which reduces the size of the globules and denatures the cryoglobulins. The fat globules in buffalo, ovine, caprine, equine, and camel milk do not agglutinate because these milks lack cryoglobulins.\n\nPreviously, the creaming of milk was a very important attribute and was a popular research topic; the extensive literature has been the subject of several reviews, most recently by Huppertz and Kelly (2006). Traditionally, the fat was removed from milk by natural (gravity) creaming. Gravity creaming is still used to standardize the fat content for some cheese varieties (e.g., Parmigiano Reggiano), but the removal of fat from milk is now usually accomplished by centrifugal separation, in which g is replaced by \u03c92R, where \u03c9 is the centrifugal velocity in radians per second and R is the radius of the centrifuge bowl. Centrifugal separation is very efficient, essentially instantaneous, and continuous.\n\n### Milk Proteins\n\nThe properties of milk and most dairy products are affected more by the proteins they contain than by any other constituent. The milk proteins have many unique properties. Because of this and their technological importance, the milk proteins have been studied extensively and are probably the best characterized food protein system.\n\nResearch on milk proteins dates from the early nineteenth century. Pioneering work was reported by J. J. Berzelius in 1814, by H. Schubler in 1818 on the physicochemical status of milk proteins, and by H. Braconnot in 1830 who published the first paper in which the word 'casein' was used. A method for preparing protein from milk by acid precipitation was described in 1938 by J. G. Mulder, who coined the term protein ('primary' or 'of first rank'). The acid-precipitated protein was referred to as casein. Some early authors called acid-precipitated milk protein caseinogen, which was converted by rennet to casein, which coagulated in the presence of Ca2+. This situation is analogous to the conversion of fibrinogen in blood by thrombin to fibrin, which coagulates in the presence of Ca2+. About 70 years ago, the term casein was universally adopted as the English word for the pH 4.6-insoluble protein in milk. The method for acid (isoelectric) precipitation of casein was refined by O. Hammarsten in 1883\u20131885, and, consequently, isoelectric casein is frequently referred to as 'casein nach Hammarsten.' An improved method for isolating casein was published by L. L. van Slyke and J. C. Baker in 1918.\n\nAs early as 1846, J. E. Schlossberger reported the separation of casein into two fractions (see Woodward, 1976) and in 1880, A. Danilewsky and P. Radenhausen suggested that isoelectric casein is heterogeneous (see Hammarsten, 1883; Lindqvist 1963), but Hammarsten (1883) maintained that properly produced casein is homogeneous. Based on differential solubility in ethanol-HCl solutions, evidence began to emerge from the work of T. B. Osborne and A. J. Wakeman in 1918 and of K. Linderstr\u00f8m-Lang and collaborators during the period 1925\u20131929 that isoelectric casein is heterogeneous. Heterogeneity was confirmed by K. O. Pederson in 1936, using analytical ultracentrifugation, and by O. Mellander in 1939, using free-boundary electrophoresis (see McMeekin, 1970, for references to early literature).\n\nThe liquid whey remaining after isoelectric precipitation of casein from skim or whole milk is a dilute solution of proteins (whey or serum proteins; \u223c0.7% in bovine milk), lactose, organic and inorganic salts, vitamins, and several constituents at trace levels. By salting-out with MgSO4, the whey proteins were fractionated by J. Sebelein, in 1885, into soluble (albumin) and insoluble (globulin) fractions. According to McMeekin (1970), A. Wichmann, in 1899, crystallized a protein from the albumin fraction of whey by the addition of (NH4)2SO4 and acidification, a technique used to crystallize blood serum albumin and ovalbumin. Using the techniques available at that time, researchers found the whey proteins to be generally similar to the corresponding fractions of blood proteins and were considered to have passed directly from blood to milk. Consequently, the whey proteins attracted little research effort until the 1930s.\n\nIn addition to the caseins and whey proteins, milk contains two other groups of proteinaceous materials, proteose peptones (PPs) and nonprotein nitrogen (NPN), which S. J. Rowland recognized in 1938. Rowland observed that after heating milk at 95 \u00b0C for 10 min, the whey proteins co-precipitated with the caseins on acidification to pH 4.6. When the pH 4.6-soluble fraction of heated milk was made to 12% trichloroacetic acid (TCA), some nitrogenous compounds precipitated which were designated 'proteose peptone'; nitrogenous compounds which remained soluble in 12% TCA were designated nonprotein nitrogen (NPN). A modified version of Rowland's scheme is now used to quantify the principal nitrogenous groups in milk (Aschaffenburg and Drewry, 1959).\n\nThus, by 1938, the complexity of the milk protein system had been described, that is, caseins, lactalbumin, lactoglobulin, PPs, and NPN, which represent approximately 78, 12, 5, 2, and 3%, respectively, of the nitrogen in bovine milk. However, knowledge of the milk protein system was rudimentary and vague at this stage. Advancement of knowledge on the chemistry of milk proteins during the twentieth century can be followed through the progression of textbooks and reviews on dairy chemistry (see Fox 1982; ; ; Fox and McSweeney, 2003; O'Mahony and Fox 2013).\n\n#### Preparation of Casein and Whey Proteins\n\nThe protein fractions may be prepared from whole or skim milk, but skim milk is usually used since the fat is occluded in isoelectric casein and interferes with further characterization of the proteins. The fat is easily removed from milk by centrifugation (e.g., 3000 \u00d7 g for 30 min), and any remaining fat may be removed by washing the precipitated protein with ether. Isoelectric precipitation is the most widely used method for separating the casein and noncasein fractions of milk protein, but several other techniques are used in certain situations (see Fox, 2003; O'Mahony and Fox, 2013):\n\n\u2022 Isoelectric precipitation at\u223c4.6 at 20oC: The precipitate is recovered by filtration or low-speed centrifugation. Essentially similar methods are used to prepare casein on a laboratory or industrial scale.\n\n\u2022 Ultracentrifugation: In milk, the casein exists as large micelles that may be sedimented by centrifugation at 100,000 \u00d7 g for 1 h; the whey proteins are not sedimentable. The casein pellet can be redispersed in a suitable buffer as micelles with properties similar to those of natural micelles.\n\n\u2022 Salting-out methods: casein can be precipitated by any of several salts, usually by (NH4)2SO4 at 260 gL\u20131 or saturated NaCl. The immunoglobulins co-precipitate with the caseins.\n\n\u2022 Ultrafiltration and microfiltration: All the milk proteins are retained by small-pore, semipermeable membranes and separated from lactose and soluble salts. This process, ultrafiltration, is used widely for the industrial-scale production of whey protein concentrates (WPCs) and to a lesser extent for the production of total milk protein. Intermediate-pore membranes are used to separate casein micelles from whey proteins. In microfiltration (MF), using large-pore membranes (1.4 m), both the caseins and whey proteins are permeable, but >99.9% of bacteria and other large particles are retained; MF is used to produce extended shelf-life beverage milk or cheese milk or to remove lipoprotein particles from whey to improve the functionality of WPC.\n\n\u2022 Gel filtration: It is possible to separate the caseins from the whey proteins by permeation chromatography, but this method is not used industrially and rarely on a laboratory scale.\n\n\u2022 Precipitation by ethanol: The caseins are precipitated from milk by \u223c40% ethanol, while the whey proteins remain soluble. However, precipitation by ethanol is rarely used, either on a laboratory or an industrial scale, for the precipitation of casein.\n\n\u2022 Cryo-precipitation: Caseins, in a micellar form, may be destabilized and precipitated by freezing milk or, preferably, concentrated milk, at about \u201310 \u00b0C. Precipitation is caused by a decrease in pH and an increase in [Ca2+]; the precipitated micelles may be redispersed as micelles by heating to about 55oC. Alternatively, the cryo-precipitated casein may be recovered, washed, and dried; it has many interesting properties for food applications, but it is not produced commercially.\n\n\u2022 Rennet coagulation: The casein micelles are destabilized by specific, limited proteolysis and coagulate in the presence of Ca2+. The properties of rennet-coagulated casein are very different from those of isoelectric casein, and it is very suitable for certain food applications, for example, cheese analogues.\n\n\u2022 Caseinates: Isoelectric casein is insoluble in water, but it may be converted to water-soluble caseinates by dispersion in water and adjusting the pH to \u223c6.7 with alkali, usually NaOH, to yield sodium caseinate. KOH, NH4OH, or Ca(OH)2 give the corresponding caseinates which may be freeze-dried or spray-dried.\n\n#### Comparison of Key Properties of Casein and Whey Proteins\n\n\u2022 Solubility at pH 4.6. The caseins are, by definition, insoluble at pH 4.6, whereas the whey proteins are soluble under the ionic conditions of milk. The isoelectric precipitation of casein is exploited in the production of caseins and caseinates, fermented milk products, and acid-coagulated cheeses.\n\n\u2022 Coagulability following limited proteolysis. The caseins are coagulable following specific, limited proteolysis, whereas the whey proteins are not. This property of the caseins is exploited in the production of rennet-coagulated cheese (\u223c75% of all cheese) and rennet casein.\n\n\u2022 Heat stability. The caseins are very heat-stable. Milk at pH 6.7 may be heated at 100 \u00b0C for 24 h without coagulation and withstands heating at 140 \u00b0C for up to 20\u201325 min; aqueous solutions of sodium caseinate may be heated at 140 \u00b0C for several hours without apparent changes. The heat stability of the whey proteins is typical of globular proteins; they are denatured completely on heating at 90 \u00b0C for 10 min. The remarkably high heat stability of the caseins, which is probably due to their lack of typical stable secondary and tertiary structures, permits the production of heat-sterilized dairy products with relatively small physical changes.\n\n\u2022 Amino acid composition. The caseins contain high levels of proline (17% of all residues in \u03b2-casein), which explains their lack of \u03b1- and \u03b2-structures. The caseins are phosphorylated, while the principal whey proteins are not. Whole isoelectric casein contains approximately 0.8% phosphorus, but the degree of phosphorylation varies among the individual caseins. The phosphate is attached to the polypeptides as phosphomonoesters of serine: the presence of phosphate groups has major significance for the properties of the caseins, for example, molecular charge and related properties, such as hydration, solubility, and heat stability, and metal binding which affects their physicochemical, functional, and nutritional properties. Metal binding by casein is regarded as a biological function since it enables a high concentration of calcium phosphate to be carried in milk in a soluble form (to supply the requirements of the neonate). Otherwise, calcium phosphate would precipitate in and block the ducts of the mammary gland, leading to the death of the gland and perhaps of the animal.\n\n\u2022 Sulfur content. The caseins are low in sulfur (0.8%), while the whey proteins are relatively rich (1.7%). The sulfur in casein is mainly in methionine, with little cystine or cysteine; the principal caseins are devoid of the latter two amino acids. The whey proteins are relatively rich in cysteine and\/or cystine, which have major effects on the physicochemical properties of these proteins and of milk.\n\n\u2022 Site of biosynthesis. The caseins are synthesized in the mammary gland and are unique to this organ. Presumably, they are synthesized to meet the amino acid requirements of the neonate and as carriers of important metals required by the neonate. The principal whey proteins are also synthesized in, and are unique to, the mammary gland, but several minor proteins in milk are derived from blood, either by selective transport or due to leakage. Most of the whey proteins have a biological function.\n\n\u2022 Physical state in milk. The whey proteins exist in milk as monomers or as small quaternary structures, while the caseins exist as large aggregates, known as micelles, with a mass of \u223c108 Da and containing about 5000 molecules. The white color of milk is due largely to the scattering of light by the casein micelles. The structure, properties, and stability of the casein micelles are of major significance for the technological properties of milk and have been the subject of intensive research (see below).\n\n#### Heterogeneity and Fractionation of Casein\n\nHammarsten believed that isoelectric casein was a homogeneous protein, but during the early years of the twentieth century, T. B. Osborne and A. J. Wakeman, and especially K. Linderstr\u00f8m-Lang and collaborators, presented evidence that it was heterogeneous (see McMeekin, 1970). By extraction with ethanol-HCl mixtures, K. Linderstr\u00f8m-Lang and S. Kodoma obtained three major casein fractions, which contained about 1.0, 0.6, or 0.1% P, and several minor fractions. The heterogeneity of casein was confirmed by analytical ultracentrifugation and free boundary electrophoresis by Pedersen and Mellander, respectively (see McMeekin, 1970). Electrophoresis resolved isoelectric casein into three proteins, which were named a-, b- and k- in order of decreasing electrophoretic mobility and represented about 75, 22, and 3% of whole casein, respectively.\n\nFollowing the demonstration of its heterogeneity, several attempts were made to isolate the individual caseins. The first reasonably successful method was developed in 1944 by R. C. Warner, who exploited differences in the solubility of \u03b1- and \u03b2-caseins at pH 4.4 and 2 \u00b0C. A much more satisfactory fractionation method was developed in 1952 by N. J. Hipp and co-workers based on the differential solubility of \u03b1-, \u03b2-, and g-caseins in urea solutions at pH 4.9. This method was widely used for many years until the widespread application of ion-exchange (Visser et al., 1986) and reverse-phase chromatography (Visser et al., 1991; Bobe et al. 1998). Reviews describing the application of high-performance and fast protein-liquid chromatography for the analysis of milk and dairy products include Gonzalez-Llano et al. (1990), Strange et al. (1992), and Dupont et al. (2013).\n\nIn 1956, \u03b1-casein was resolved by D. F. Waugh and P. H. von Hippel into Ca-sensitive and Ca-insensitive proteins that were called \u03b1s\\- and \u03ba-caseins, respectively. \u03ba-Casein, which represents \u223c12% of total casein, is responsible for the formation and stabilization of casein micelles and affects many technologically important properties of the milk protein system. Numerous chemical methods were soon developed for the isolation of \u03ba-casein (see Fox, 2003; O'Mahony and Fox 2013). \u03b1s-Casein prepared by the method of Waugh and von Hippel contains two proteins, now called \u03b1s1\\- and \u03b1s2-caseins (Annan and Manson, 1969).\n\nChemical methods for fractionation of the caseins have now been largely superseded by ion-exchange chromatography, which gives superior results when urea and a reducing agent are used (see Strange et al., 1992; Imafidon et al. 1997).\n\n#### Application of Gel Electrophoresis to the Study of Milk Proteins\n\nZone electrophoresis on a solid medium, paper or cellulose acetate, was introduced in the 1940s. This technique gave good results with many protein systems, but the caseins, owing to a very strong tendency to associate hydrophobically, were resolved poorly on these media. Electrophoresis in starch gels (SGE) using discontinuous buffer systems was introduced to general protein chemistry by M. D. Poulik in 1957 and applied to the study of the caseins by R. G. Wake and R. L. Baldwin in 1961. The resolving power of SGE was far superior to that of any of its predecessors. When urea (7 M) and a reducing agent, usually, 2-mercaptoethanol, were incorporated into the starch gel, isoelectric casein was resolved into about 20 bands, most of which are due to the microheterogeneity of one or more of the caseins.\n\nElectrophoresis on polyacrylamide disc gels (PAGE) was introduced by L. Ornstein in 1964 and applied to the study of the caseins by R. F. Peterson in 1966. PAGE and SGE give similar results, but PAGE is far easier to use and has become the standard electrophoretic method for the analysis of caseins (and most other protein systems). Gel electrophoretic methods for the analysis of milk proteins have been reviewed by Swaisgood (1975), Strange et al. (1992), Tremblay et al. (2003), Chevalier et al. (2011a), and Dupont et al. (2013).\n\nSodium dodecyl sulfate (SDS)-PAGE, which resolves proteins mainly on the basis of molecular mass, is very effective for most proteins, but since the masses of the four caseins are quite similar, SDS-PAGE is not very effective. \u03b2-Casein, which has very high surface hydrophobicity, binds a disproportionately high amount of SDS and, consequently, has a higher electrophoretic mobility than \u03b1s1-casein, although it is a larger molecule. SDS-PAGE is very effective for the resolution of whey proteins and is the method of choice.\n\nThe study of proteins (proteomics) has evolved considerably in recent years to incorporate electrophoresis, chromatography, mass spectrometry, and immunoassays (O'Donnell et al., 2004; Manso et al., 2005; Chevalier 2011b; Roncada et al., 2012; Dupont et al. 2013).\n\n#### Microheterogeneity of the Caseins\n\n\u03b1s1-, \u03b1s2-, b-, and k-caseins represent approximately 38, 10, 35, and 12%, respectively, of whole bovine casein. However, SGE or PAGE indicates much greater heterogeneity due to small differences in one or more of the caseins, referred to as microheterogeneity, which arises from five factors which are described in the following sections.\n\n#### Variability in the Degree of Phosphorylation\n\nAll the caseins are phosphorylated but to a variable degree \u03b1s1-, 8 or 9P; \u03b1s2-, 10, 11, 12, or 13P; \u03b2-, 4 or 5P; \u03ba-, 1 or 2P per molecule). The number of phosphate residues is indicated thus: \u03b1s1-CN 8P, \u03b2-CN 5P, and so on. See Chapter 5 for more details.\n\n#### Genetic Polymorphism\n\nIn 1955, R. Aschaffenburg and J. Drewry discovered that \u03b2-lactoglobulin exists in two forms (variants, polymorphs) now called A and B, which differ by only two amino acids. The variant in the milk is genetically controlled, and the phenomenon is called genetic polymorphism. It was soon shown that all milk proteins exhibit genetic polymorphism, and at least 45 polymorphs have been detected by PAGE, which differentiates on the basis of charge, and therefore only polymorphs that differ in charge have been detected (Ng-Kwai-Hang, 2011). It is very likely that only a small proportion of the genetic polymorphs of milk proteins have been detected. The potential of peptide mapping of enzymatic hydrolysates by HPLC-MS has been assessed (Leonil et al., 1995). The genetic polymorph(s) present is indicated by a Latin letter as follows: \u03b2-CN A 5P, \u03b1s1-CN B 9P, \u03ba-CN A 1P, and so on. Genetic polymorphism also occurs in the milk of sheep, goat, buffalo, pig, and horse, and probably of all species.\n\nTechnologically important properties of milk (e.g., rennetability, heat stability, yield, and proportions of milk proteins) are affected by the genetic polymorphs of the milk proteins present, and work in this area is being expanded and refined (Jakob and Puhan, 1992). The extensive literature on the genetic polymorphism of milk proteins has been the subject of several reviews, including Ng-Kwai-Hang and Grosclaude (2003), Ng-Kwai-Hang (2011), and Martin et al. (2013b).\n\n#### Disulfide Bonding\n\n\u03b1s1\\- and \u03b2-caseins lack cysteine and cystine, but both \u03b1s2\\- and \u03ba-caseins contain two 1\/2 cystine residues, which occur as intermolecular disulfide bonds. \u03b1s2-Casein exists as a disulfide-linked dimer, while up to 10 \u03ba-casein molecules may be linked by disulfide bonds. Inclusion of a reducing agent (usually 2-mercaptoethanol) in SGE or PAGE gels is required for good resolution of \u03ba-casein; in its absence, \u03b1s2-casein appears as a dimer (originally called \u03b1s5-casein).\n\n#### Variations in the Degree of Glycosylation\n\n\u03ba-Casein is the only glycosylated casein; it contains galactose, N-acetylgalactosamine, and N-acetylneuraminic (sialic) acid, which occur as tri- or tetrasaccharides, the number of which varies from 0 to 4 per molecule of protein (i.e., a total of nine variants) attached to threonine residues. See Chapter 5 for more details.\n\n#### Hydrolysis of the Caseins by Plasmin\n\nMilk contains several indigenous proteinases, the principal of which is plasmin, a trypsin-like, serine-type proteinase from blood; it is highly specific for peptide bonds with a lysine or arginine at the P1 position. The preferred casein substrates are \u03b2- and \u03b1s2-; \u03b1s1\\- is also hydrolyzed, but \u03ba-casein is very resistant, as are the whey proteins. All the caseins contain several lysine and arginine residues, but only a few bonds are hydrolyzed rapidly. \u03b2-Casein is hydrolyzed rapidly at the bonds Lys28-Lys29, Lys105-His106, and Lys107-Glu108. The resulting C-terminal peptides are the \u03b3-caseins (\u03b31: \u03b2-CNf29\u2013209; \u03b32: \u03b2-CNf106\u2013209; \u03b33: \u03b2-CNf108\u2013209), while the N-terminal peptides are proteose peptones 5, 8slow and 8fast. The \u03b3-caseins, which represent \u223c3% of total caseins, are evident in PAGE gels. Other plasmin-produced peptides are probably present but are either too small to be readily detectable by PAGE or their concentrations are very low relative to the principal caseins.\n\nAlthough \u03b1s2-casein in solution is also quite susceptible to plasmin, \u03b1s2-derived peptides have not been identified in milk. \u03b1s1-Casein in solution is also hydrolyzed readily by plasmin; members of a minor casein fraction, \u03b3-casein, are N-terminal fragments of \u03b1s1-casein produced by plasmin (O'Flaherty, 1997).\n\n#### Molecular Properties of the Milk Proteins\n\nBoth the principal and many of the minor milk proteins have been very well characterized. The principal properties of the six milk-specific proteins are summarized in Table 2.1. A number of features warrant comment.\n\nTable 2.1\n\nProperties of the Principal Lactoproteins\n\n| Caseins | \u03b2-Lg | \u03b1-La  \n---|---|---|---  \nProperties | \u03b1s1 | \u03b1s2 | \u03b2- | \u03ba- |  |   \nMW | 23612 | 25228 | 23980 | 19005 | 18362 | 14174  \nResidues | 199 | 207 | 209 | 169 | 162 | 123  \nConc in milk (g\/L) | 12-15 | 3-4 | 9-11 | 2-4 | 3.0 | 0.7  \nPhosphate residues | 8-9 | 10-13 | 4-5 | 1-2 | 0 | 0  \n\u00bd Cystine | 0 | 2 | 0 | 2 | 5 | 8  \nSugars | 0 | 0 | 0 | Yes | 0 | 0  \nProlyl residue per molecule | 17 | 10 | 35 | 20 | 8 | 2  \nA280, 1% 1 cm | 10.1 | 11.1 | 4.6 | 9.6 | 9.4 | 20.1  \nSecondary structure | Low | Low | Low | Low | High | High  \nH\u03d5ave | 4.89 | 4.64 | 5.58 | 5.12 | 5.03 | 4.68  \npI | 4.96 | 5.27 | 5.20 | 5.54 | 5.2 | 4.2-4.5  \nPartial specific volume (ml\/g) | 0.728 | 0.720 | 0.741 | 0.734 | 0.751 | 0.735\n\nThe six principal lactoproteins are small molecules, a feature that contributes to their stability. The primary structure of the principal lactoproteins of several species is known, as are the substitutions in the principal genetic variants (Martin et al., 2013b).\n\nThe whey proteins are highly structured, but the four caseins lack stable secondary structures. Classical physical measurements indicate that the caseins are unstructured, but theoretical considerations indicate that rather than being unstructured, the caseins are very flexible molecules and have been referred to as rheomorphic (Holt and Sawyer, 1993; see also Horne, 2002; Farrell et al. 2006a). The inability of the caseins to form stable structures is due mainly to their high content of the structure-breaking amino acid, proline; \u03b2-casein is particularly rich in proline, with 35 of the 209 residues. The open, flexible structure of the caseins renders them very susceptible to proteolysis, which facilitates their natural function as a source of amino acids. In contrast, the native whey proteins, especially \u03b2-Lg, are quite resistant to proteolysis, and at least some are excreted in the feces of infants. This feature is important since most of the whey proteins play a non-nutritional function in the intestine, and, therefore, resistance to proteolysis is important.\n\nThe caseins are generally regarded as very hydrophobic proteins but, with the exception of \u03b2-casein, they are not exceptionally hydrophobic. Owing to their lack of stable secondary and tertiary structures, most of their hydrophobic residues are exposed. Consequently, they have a high surface hydrophobicity. One of the more notable features of the amino acid sequence of the caseins is that the hydrophobic and hydrophilic residues are not distributed uniformly, thereby giving the caseins a distinctly amphiphatic structure (Huppertz, 2013). This feature, coupled with their open flexible structure, gives the caseins good surface activity, as well as good foaming and emulsifying properties, making casein the functional protein of choice for many food applications. Owing to their hydrophobic sequences, the caseins have a propensity to yield bitter hydrolysates. Also, due to their open structure, the caseins have a high specific volume. Consequently, they form highly viscous solutions, which is a disadvantage in the production of caseinates. Owing to its high viscosity, it is not possible to spray-dry sodium caseinate solutions containing >20% protein, thereby increasing the cost of drying and resulting in low-bulk density powders.\n\nThe lack of stable tertiary structures means that the caseins are not denaturable sensu stricto. Therefore, they are extremely heat stable; sodium caseinate, at pH 7, can withstand heating at 140 \u00b0C for several hours without visible change. This very high heat stability makes it possible to produce heat-sterilized dairy products with very little change in physical appearance; other major food systems undergo major physical changes upon severe heating.\n\nThe caseins have a very strong tendency to associate due mainly to hydrophobic bonding. Even in sodium caseinate, the most soluble form of casein, the molecules form aggregates of 250\u2013500 kDa, that is, containing 10\u201320 molecules. This strong tendency to associate makes it difficult to fractionate the caseins, for which a dissociating agent (e.g., urea or SDS) is required. On the other hand, a tendency to associate is important for some functional applications and in the formation and stabilization of casein micelles. In contrast, the whey proteins are molecularly dispersed in solution.\n\nOwing to their high content of phosphate groups, which occur in clusters, \u03b1s1-, \u03b1s2-, and \u03b2-caseins have a strong tendency to bind metal ions, which in the case of milk are mainly Ca2+. This property has many major consequences; the most important from a technological viewpoint is that these three proteins, which represent approximately 85% of total caseins, are insoluble at Ca concentrations >\u223c6 mM at temperatures >20 \u00b0C. Since bovine milk contains \u223c30 mM Ca, one would expect the caseins to precipitate under the conditions prevailing in milk. However, \u03ba-casein, which contains only one organic phosphate group, binds Ca weakly and is soluble at all Ca concentrations found in dairy products. Furthermore, when mixed with the Ca-sensitive caseins, \u03ba-casein can stabilize and protect up to \u223c10 times its mass of the Ca-sensitive caseins by forming large colloidal particles called casein micelles. The micelles act as carriers of inorganic elements, especially Ca and P, but also Mg and Zn, and are, therefore, very important from a nutritional viewpoint. Through the formation of micelles, it is possible to solubilize much higher levels of Ca and PO4 than would otherwise be possible.\n\n#### Nomenclature of Milk Proteins\n\nDuring the period of greatest activity on the fractionation of casein (1950\u20131970), several casein (and whey protein) fractions were prepared that were either similar to proteins already isolated and named, or were artifacts of the isolation procedure. In order to standardize the nomenclature of the milk proteins, the American Dairy Science Association established a Nomenclature Committee in 1955, which has published seven reports, the most recent of which is by Farrell et al. (2004). In addition to standardizing the nomenclature of the milk proteins, the characteristics of the principal milk proteins are summarized in these articles.\n\n#### Whey Proteins\n\nAbout 20% of the total proteins of bovine milk are whey (serum) proteins. The total whey protein fraction is prepared by any of the methods described for the preparation by casein. That is, the proteins that are soluble at pH 4.6 or in saturated NaCl or after rennet-induced coagulation of the caseins, are permeable on microfiltration, or not sedimented by ultracentrifugation.\n\nThe proteins prepared by these methods differ somewhat: Acid whey contains PPs; immunoglobulins are co-precipitated with the caseins by saturated NaCl; rennet whey contains the macropeptides, produced from \u03ba-casein by rennet, plus small amounts of casein; and small casein micelles remain in the ultracentrifugal serum.\n\nOn a commercial scale, whey protein-rich products are prepared by:\n\n\u2022 ultrafiltration\/diafiltration of acid casein or rennet whey to remove varying amounts of lactose and spray-dried to produce whey protein concentrates (30\u201385% protein)\n\n\u2022 ion-exchange chromatography and spray-dried to yield whey protein isolate, containing \u223c95% protein,\n\n\u2022 demineralization by electrodialysis or ion exchange, thermal evaporation of water, and crystallization of lactose, and\n\n\u2022 thermal denaturation, removal of precipitated protein by filtration\/centrifugation and spray-drying, to yield lactalbumin, which has very low solubility and poor functionality.\n\n#### Fractionation of Whey Proteins\n\nIt was recognized early that acid whey contains two well-defined groups of proteins: lactalbumins, which are soluble in 50% saturated (NH4)2SO4 or saturated MgSO4; and lactoglobulins, which are salted-out under these conditions. The lactoglobulin fraction contains mainly immunoglobulins. The lactalbumin fraction contains two principal proteins, \u03b2-lactoglobulin and a-lactalbumin, and several minor proteins, including blood serum albumin and lactoferrin, which have been isolated by various procedures and crystallized (see Imafidon et al., 1997; Fox, 2003; O'Mahony and Fox 2013).\n\nThere is considerable interest in the production of the major and many minor whey proteins on a commercial scale for nutritional, nutraceutical, or functional applications. Several methods have been developed for the industrial-scale production of several whey proteins (see Mulvihill and Ennis, 2003).\n\n#### Major Characteristics of Whey Proteins\n\n##### \u03b2-Lactoglobulin\n\n\u03b2-Lactoglobulin (\u03b2-Lg) represents \u223c50% of the whey proteins, \u223c12% of the total proteins, in bovine milk. It is a typical globular protein that has been characterized very well. The extensive literature has been reviewed, among others, by Sawyer (2003; 2013); and Creamer et al. (2011).\n\n\u03b2-Lg is the principal whey protein in the milk of cattle, buffalo, sheep, and goat, although there are slight interspecies differences. Initially, it was considered that \u03b2-Lg occurs only in the milk of ruminants, but it is now known that a similar protein occurs in the milk of many other species, including the sow, mare, kangaroo, dolphin, and manatee. However, \u03b2-Lg does not occur in the milk of human, rat, mouse, guinea pig, camel, llama, or alpaca, in which \u03b1-La is the principal whey protein.\n\nBovine \u03b2-Lg consists of 162 amino acid residues per monomer, with an MW of \u223c18 kDa; its amino acid sequence and that of several other species have been established. Its isoelectric point is \u223cpH 5.2. It contains two intramolecular disulfide bonds and one mol of cysteine per monomer. The cysteine is especially important since it reacts, following thermal denaturation, with the intermolecular disulfide of \u03ba-casein and significantly affects the rennet coagulation and heat stability of milk. It is also responsible for the cooked flavor of heated milk. Some \u03b2-Lgs (e.g., porcine) lack a sulfydryl group. Twelve genetic variants of bovine \u03b2-Lg have been identified, the most common being A and B. Genetic polymorphism also occurs in \u03b2-Lg of other species.\n\n\u03b2-Lg is a highly structured protein: in the pH range 2\u20136, 10\u201315% of the molecule exists as \u03b1-helices, 43% as \u03b2-sheets, and 47% as unordered structures, including \u03b2-turns; the \u03b2-sheets occur in a \u03b2-barrel-type calyx. The molecule has a very compact globular structure. Each monomer exists almost as a sphere, measures about 3.6 nm in diameter, and exists as a dimer, MW \u223c36 kDa, in the pH range 5.5\u20137.5, as a monomer <pH 3.5 and >pH 7.5, and as a tetramer (MW, \u223c144 kDa) in the pH range 3.5\u20135.5. Porcine and other \u03b2-Lgs that lack a free thiol do not form dimers, a property that is probably not due to the absence of a thiol group.\n\n\u03b2-Lg is very resistant to proteolysis in its native state; this feature suggests that its primary function is not nutritional. It may have either or both of two biological roles:\n\n\u2022 It binds retinol (vitamin A) in a hydrophobic pocket, protects it from oxidation, and transports it through the stomach to the small intestine where the retinol is transferred to a retinol-binding protein, which has a similar structure to \u03b2-Lg. It is not clear how retinol is transferred from the core of the fat globules, where it occurs in milk, to \u03b2-Lg and why some species lack this protein. \u03b2-Lg can bind many hydrophobic molecules, and hence its ability to bind retinol may be incidental. \u03b2-Lg is a member of the lipocalin family, all of which have binding properties (Akerstrom et al., 2000).\n\n\u2022 Through its ability to bind fatty acids, \u03b2-Lg stimulates lipase activity, which may be its most important physiological function.\n\n\u03b2-Lg is the most allergenic protein in bovine milk for human infants, and there is interest in producing whey protein products free of \u03b2-Lg for use in infant formula. \u03b2-Lg has very good thermogelling properties and determines the gelation of whey protein concentrates (WPCs).\n\n##### \u03b1-Lactalbumin\n\nAbout 20% of the protein of bovine whey (3.5% of total milk protein) is \u03b1-lactalbumin (\u03b1-La), which is the principal protein in human milk. It is a small protein containing 123 amino acid residues, with a mass of \u223c14 kDa, which has been well characterized. The literature has been reviewed, among others, by McKenzie and White (1991) and Brew (2003; ; 2013).\n\n\u03b1-La contains four tryptophan residues per mole, giving it a specific absorbance at 280 nm of 20. It contains four intramolecular disulfide bonds per mole but no cysteine, phosphate, or carbohydrate. Its isoionic point is \u223cpH 4.8. The milk of Bos taurus breeds contains only one genetic variant of \u03b1-La, B, but Zebu cattle produce two variants, A and B. \u03b1-La has been isolated from the milk of cattle, sheep, goat, sow, human, buffalo, rat, guinea pig, horse, and many other species; there are minor interspecies differences in its composition and properties.\n\nThe primary structure of \u03b1-La is homologous with lysozyme. Out of a total of 123 amino acid residues in \u03b1-La, 54 are identical to corresponding residues in chicken egg white lysozyme and 23 others are structurally similar. \u03b1-La is a compact, highly structured globular protein. The tertiary structure of \u03b1-La is similar to that of lysozyme (McKenzie and White, 1991) and x-ray crystallography-based analysis of apo- and holo- bovine \u03b1-La have been published by Chrysina et al. (2000). In evolutionary terms, lysozyme is a very ancient protein; it is believed that \u03b1-La evolved from it through gene duplication (see Nitta and Sugai, 1989).\n\nAs discussed earlier, \u03b1-La is a component of lactose synthetase, the enzyme that catalyses the final step in the biosynthesis of lactose. There is a direct correlation between the concentrations of \u03b1-La and lactose in milk. The milk of the California sea lion or the hooded seal contains no \u03b1-La.\n\n\u03b1-La is a metalloprotein containing two Ca2+ per molecule in a pocket containing four Asp residues. The Ca-containing protein is the most heat-stable of the principal whey proteins, or, more correctly, the protein renatures following heat denaturation, which occurs at a relatively low temperature, as indicated by differential scanning calorimetry. When the pH is reduced to <\u223cpH 5, the Asp residues become protonated and lose their ability to bind Ca2+. The calcium-free protein is denatured at quite a low temperature and does not renature on cooling; this characteristic has been exploited to isolate \u03b1-La from whey. \u03b1-La has poor thermogelling properties. Most lysozymes do not bind a Ca2, but equine lysozyme is an exception (Nitta et al., 1987) and seems to be an intermediate in the evolution of lysozyme to \u03b1-La (see Nitta and Sugai, 1989).\n\n\u03b1-La is synthesized in the mammary gland, but a very low level is transferred, probably via leaky mammocyte junctions, into blood serum, in which the concentration of \u03b1-La increases during pregnancy or following administration of steroid hormones to male or female animals (Akers, 2000). The concentration of \u03b1-La in blood serum is a reliable, noninvasive indicator of mammary gland development and of the potential of an animal for milk production.\n\nA high MW form of \u03b1-La, isolated recently from human acid-precipitated casein, has anticarcinogenic activity; it was named HAMLET (human \u03b1-La made lethal to tumor cells). Bovine \u03b1-La can be converted to a form with similar activity, called BAMLET; it is the molten globular state formed from apo-\u03b1-La and cis\u03949-octadecenoic acid (see Svensson et al., 2000; Chatterton et al. 2006; and Pettersson et al., 2006). Both forms are reported to have comparable cytotoxic activity against three different cancer cell lines (Brinkmann et al., 2011).\n\n#### Blood Serum Albumin\n\nNormal bovine milk contains 0.1\u20130.4 gL\u20131 of blood serum albumin (BSA; 0.3\u20131.0% of total nitrogen), presumably as a result of leakage from blood; it has no known biological function in milk. BSA has been studied extensively; for reviews see Fox (2003) and O'Mahony and Fox (2013). Owing to its low concentration in milk, BSA has little effect on the physicochemical properties of WPC and whey protein isolate (WPI).\n\n#### Immunoglobulins\n\nMature bovine milk contains 0.6\u20131 g immunoglobulins (Ig)L\u20131 (\u223c3% of total nitrogen), but colostrum contains \u224810% (w\/v) Ig, the level of which decreases rapidly postpartum. IgG1 is the principal Ig in bovine, buffalo, caprine, or ovine milk, with lesser amounts of IgG2, IgA, and IgM; IgA is the principal Ig in human milk. The cow, sheep, and goat do not transfer Ig to the fetus in utero, and the neonate is born without Ig in its blood. Consequently, it is very susceptible to bacterial infection with a very high risk of mortality. The young of these species can absorb Ig from the intestine for several days after birth and thereby acquire passive immunity until they synthesize their own Ig, within a few weeks of birth. The human mother transfers Ig in utero, and the offspring is born with a broad spectrum of antibodies. Although the human baby cannot absorb Ig from the intestine, the ingestion of colostrum is still very important because its Igs prevent intestinal infection. Some species, such as the horse, transfer Ig both in utero and via colostrum (see Hurley, 2003; Marnila and Korhonen 2011; Hurley and Thiel, 2013).\n\nIt has been suggested that the neonate secretes chymosin rather than pepsin because chymosin is weakly proteolytic and does not inactivate Ig. Colostrum contains \u03b12-macroglobulin, which may inhibit proteinases in the GIT and protect Ig.\n\nThe modern dairy cow produces colostrum far in excess of what its calf requires; surplus colostrum is available for the recovery of Ig and other nutraceuticals (Pakkanen and Aalto, 1997). Some work has been done on hyperimmunizing cows against certain human pathogens, such as rota virus, for the production of antibody-rich milk for human consumption, especially by infants. Interest in this approach dates back to the 1950s, when L.M. Spolverini suggested using bovine colostrum in the diet of infants to confer protection against shared human and bovine diseases (Campbell and Petersen, 1959). Milk powder manufactured from 'immune milk' is commercially available in several markets (e.g., Stolle Milk Biologics Inc., Cincinnati, Ohio).\n\n#### Whey Acidic Protein\n\nWhey acidic protein (WAP) was first identified in the milk of mouse and has since also been found in the milk of rat, rabbit, pig, camel, wallaby, possum, echidna, and platypus. Since the milk of all of these species lacks \u03b2-Lg, it was thought that these proteins were mutually exclusive. However, porcine milk, which contains \u03b2-Lg, was recently found to contain WAP also (see Saidi et al., 2007). The MW of WAP is 14\u201330 kDa (the variation may be due to differences in glycosylation), and it contains two (in eutherians) or three (in monotremes and marsupials) 4-disulfide domains. Since human milk lacks \u03b2-Lg, it might be expected to contain WAP, but there are no reports to this effect. In humans and ruminants, the WAP gene is frame-shifted and is a pseudogene. WAP functions as a proteinase inhibitor, is involved in terminal differentiation in the mammary gland, and has antibacterial activity (for reviews see Simpson and Nicholas, 2002; Hajjoubi et al., 2006; Martin et al., 2011).\n\n#### Proteose Peptone 3\n\nThe proteose-peptone (PP) fraction of milk protein is a very complex mixture of peptides, most of which are produced by the action of indigenous plasmin (see above), but some are indigenous to milk. The fraction has been only partially characterized; the current status has been described by Fox (2003) and O'Mahony and Fox (2013). PP fractions 5, 8slow, and 8fast, have little or no technological significance; proteose peptone 3 (PP3) has several interesting technological functionalities.\n\nBovine PP3 is a heat-stable phosphoglycoprotein that was first identified in the PP (heat-stable, acid-soluble) fraction of milk. Unlike the other peptides in this fraction, PP3 is an indigenous milk protein, synthesized in the mammary gland. Bovine PP3 consists of 135 amino acid residues, with five phosphorylation and three glycosylation sites. When isolated from milk, the PP3 fraction contains at least three components of MW \u224828, 18, and 11 kDa; the largest of these is PP3, while the smaller components are fragments thereof generated by plasmin (see Girardet and Linden, 1996). PP3 is present mainly in acid whey, but some is also found in the MFGM. Girardet and Linden (1996) proposed changing the name to lactophorin or lactoglycophorin; it has also been referred to as the hydophobic fraction of proteose peptone.\n\nOwing to its strong surfactant properties (Campagna et al., 1998), PP3 can prevent contact between milk lipase and its substrates, thus preventing spontaneous lipolysis. Its emulsifying properties have also been evaluated in dairy products such as ice cream and recombined dairy cream (Vanderghem et al., 2007; Innocente et al. 2011). Although its amino acid composition suggests that PP3 is not a hydrophobic protein, it behaves hydrophobically, possibly owing to the formation of an amphiphilic \u03b1-helix, one side of which contains hydrophilic residues while the other side is hydrophobic. The biological role of PP3 is unknown.\n\n#### Minor Proteins\n\nMilk contains several proteins at very low or trace levels, many of which are biologically active (see Schrezenmeir et al., 2000; Wynn and Sheehy 2013). Some are regarded as highly significant and have attracted considerable attention as nutraceuticals. When ways of increasing the value of milk proteins are discussed, the focus is usually on these minor proteins, but they are, in fact, of little economic value to the overall dairy industry. They are found mainly in the whey, but some are also located in the fat globule membrane. Reviews on the minor proteins include Fox and Flynn (1992), Haggarty (2003), Fox and Kelly (2003), Wynn et al. (2011), O'Mahony and Fox (2013), and Wynn and Sheehy (2013).\n\n#### Metal-binding Proteins\n\nMilk contains several metal-binding proteins: the caseins (Ca, Mg, Zn), \u03b1-La (Ca), xanthine oxidase (Mo, Fe), alkaline phosphatase (Zn, Mg), lactoperoxidase (Fe), catalase (Fe), ceruloplasmin (Cu), glutathione peroxidase (Se), lactoferrin (Fe), and transferrin (Fe).\n\nLactoferrin (Lf), a non-hem iron-binding glycoprotein, is a member of a family of iron-binding proteins, which includes transferrin and ovotransferrin (conalbumin) (see Lonnerdal, 2003; Lonnerdal and Suzuki, 2013). It is present in several body fluids, including saliva, tears, sweat, and semen. Lf has several potential biological functions: It improves the bioavailability of Fe, is bacteriostatic (by sequestering Fe and making it unavailable to intestinal bacteria), and has antioxidant, antiviral, anti-inflammatory, immunomodulatory, and anticarcinogenic activity. Human milk contains a very high level of Lf (\u224820% of total N), and therefore there is interest in fortifying bovine milk-based infant formula with Lf. The pI of Lf is \u22489.0; that is, it is cationic at the pH of milk, whereas most milk proteins are anionic, and it can be isolated on an industrial scale by adsorption on a cation exchange resin. Hydrolysis of Lf by pepsin yields peptides called lactoferricins, which are more bacteriostatic than Lf, and their activity is independent of iron status. Milk also contains a low level of serum transferrin.\n\nMilk contains a copper-binding glycoprotein, ceruloplasmin, also known as ferroxidase (EC 1.16.3.1). Ceruloplasmin is an \u03b12-globulin with a MW of \u223c126 kDa; it binds six atoms of copper per molecule and may play a role in delivering essential copper to the neonate.\n\nGlutathione peroxidase (GTPase) is a Se-containing protein. It has been reported that milk contains GTPase and that it binds 30% of the total Se in milk (see Fox and Kelly, 2006b; O'Mahony et al., 2013). GTPase has no known enzymatic function in milk and the activity attributed to GTPase in milk may be due to sulfhydryl oxidase (Stagsted, 2006).\n\n#### \u03b22-Microglobulin\n\n\u03b22-Microglobulin, initially called lactollin, was first isolated from bovine acid-precipitated casein by M. L. Groves in 1963. Lactollin, reported to have an MW of 43 kDa, is a tetramer of \u03b22-microglobulin, which consists of 98 amino acids, with a calculated MW of 11,636 Da. \u03b22-Microglobulin, a component of the immune system, is probably produced by proteolysis of a larger protein, mainly within the mammary gland; it has no known significance in milk.\n\n#### Osteopontin\n\nOsteopontin (OPN) is a highly phosphorylated acidic glycoprotein, consisting of 261 amino acid residues with a calculated MW of 29,283 Da (total MW of the glycoprotein, \u224860,000 Da). OPN has 50 potential calcium-binding sites, about half of which are saturated under normal physiological concentrations of calcium and magnesium.\n\nOPN occurs in bone (it is one of the major non-collagenous proteins in bone), in many other normal and malignant tissues, in milk and urine, and can bind to many cell types. It is believed to have a diverse range of functions (Bayless et al., 1997), but its role in milk is not clear. A rapid method for the isolation of OPN from milk was reported by Azuma et al. (2006) who showed that it binds Lf, lactoperoxidase, and Igs and may serve as a carrier for these proteins. An acidic whey protein fraction, which contains OPN, reduces bone loss in ovariectomized rats (Kruger et al., 2006; Wynn et al., 2011; O'Mahony and Fox, 2013; Wynn and Sheehy 2013).\n\n#### Vitamin-binding Proteins\n\nMilk contains binding proteins for at least the following vitamins: retinol (vitamin A, i.e., \u03b2-lactoglobulin), biotin, folic acid, and cobalamine (vitamin B12). The precise role of these proteins is not clear, but they probably improve the absorption of vitamins from the intestine or act as antibacterial agents by rendering vitamins unavailable to bacteria. The concentration of these proteins varies during lactation, but the influence of other factors such as individuality, breed, and nutritional status is not known. The activity of these proteins is reduced or destroyed on heating at temperatures somewhat higher than high temperature short time (HTST) pasteurization (see Wynn et al., 2011; Wynn and Sheehy 2013).\n\n#### Angiogenins\n\nAngiogenins induce the growth of new blood vessels, that is, angiogenesis. They have high sequence homology with members of the RNase A superfamily of proteins and have RNase activity. Two angiogenins (ANG-1 and ANG-2) have been identified in bovine milk and blood serum; both strongly promote the growth of new blood vessels in a chicken membrane assay. The function(s) of the angiogenins in milk is unknown. They may be part of a repair system to protect either the mammary gland or the intestine of the neonate and\/or part of the host-defense system (see Wynn et al., 2011; Wynn and Sheehy 2013).\n\n#### Kininogen\n\nTwo kininogens have been identified in bovine milk: a high (88\u2013129 kDa, depending on the level of glycosylation) and a low (16\u201317 kDa) MW form. Bradykinin, a biologically active peptide containing nine amino acids which is released from the high MW kininogen by the action of the enzyme, kallikrein, has been detected in the mammary gland, and is secreted into milk, from which it has been isolated. Plasma kininogen is an inhibitor of thiol proteinases and has an important role in blood coagulation. Bradykinin affects smooth muscle contraction and reduces hypertension. The biological significance of bradykinin and kininogen in milk is unknown (see Wynn et al., 2011; Wynn and Sheehy 2013).\n\n#### Glycoproteins\n\nMany of the minor proteins discussed above are glycoproteins; in addition, several other minor glycoproteins have been found in milk and especially in colostrum, the functions of which have not been elucidated. One of the high MW glycoproteins in bovine milk is prosaposin, a neurotrophic factor that plays an important role in the development, repair, and maintenance of nervous tissue. It is a precursor of saposins A, B, C, and D, which have not been detected in milk. The physiological role of prosaposin in milk is not known, although saposin C, released by digestion, could be important for the growth and development of the young (Patton et al., 1997; Patton, 1999; Campana et al. 1999).\n\n#### Proteins in the Milk Fat Globule Membrane\n\nAbout 1% of the total protein in milk is in the milk fat globule membrane (MFGM). Most of the proteins are present at trace levels, including many of the indigenous enzymes in milk. The principal proteins in the MFGM include mucins, adipophilin, butyrophilin, and xanthine oxidoreductase (see section on milk lipids).\n\n#### Growth Factors\n\nMilk contains many peptide hormones, including epidermal growth factor, insulin, insulin-like growth factors 1 and 2, three human growth factors (\u03b11, \u03b12, and \u03b2), two mammary-derived growth factors (I and II), colony-stimulating factor, nerve growth factor, platelet-derived growth factor, and bombasin. It is not clear whether these factors play a role in the development of the neonate or in the development and functioning of the mammary gland, or both (see Fox and Flynn, 1992; Gauthier et al., 2006; Wynn and Sheehy 2013).\n\n#### Indigenous Milk Enzymes\n\nMilk contains about 70 indigenous enzymes, which are minor but very important members of the milk protein system (see ; O'Mahony et al., 2013). The enzymes originate from the secretory cells or the blood; many are concentrated in the MFGM and originate in the Golgi membranes of the cell or the cell cytoplasm, some of which becomes entrapped as crescents inside the encircling membrane during exocytosis. Plasmin and lipoprotein lipase are associated with the casein micelles, and several enzymes are present in the milk serum; many of the enzymes are derived from the MFGM that is shed as the milk ages.\n\nThe indigenous enzymes are significant for several reasons:\n\n\u2022 Deterioration of product quality: plasmin, lipoprotein lipase, acid phosphatase, xanthine oxidoreductase.\n\n\u2022 Bactericidal agents: lactoperoxidase and lysozyme.\n\n\u2022 Indices of the thermal history of milk: alkaline phosphatase, \u03b3-glutamyltransferase, or lactoperoxidase.\n\n\u2022 Indices of mastitic infection: catalase, acid phosphatase, and especially N-acetylglucosaminidase.\n\nThe concentration\/activity of indigenous enzymes in milk shows greater interspecies variability than any other constituent (e.g., 3000-fold greater concentration of lysozyme in equine and human milk than in bovine milk). Lactoperoxidase is a major enzyme in bovine milk but is absent from human milk. Human milk and the milk of a few other species contain bile salt-stimulated lipase, but the milk of most species lacks this enzyme. The principal lipase in milk is lipoprotein lipase, of which there is \u223c500 times as much in guinea pig milk as in rat milk. Bovine milk has a high level of xanthine oxidoreductase activity (XOR), but all other milks that have been studied have low XOR activity because the protein lacks Mo (XOR plays a major role in the excretion of fat globules from the mammocyte, but in this function it does not act as an enzyme). (see milk lipids section). The reasons for these interspecies differences are not known, but some of them may be significant.\n\n#### Biologically Active Cryptic Peptides\n\nOne of the most exciting recent developments in milk proteins is the discovery that all milk proteins contain sequences that have biological\/physiological activities when released by proteolysis. The best studied are phosphopeptides, angiotensin-converting enzyme inhibitory peptides, platelet-modifying peptides, opiate peptides, immunomodulating peptides, and the caseinomacropeptides which have many biological properties (see FitzGerald and Meisel, 2003; Korhonen, 2006; Korhonen and Pihlanto, 2006; Mills et al. 2011).\n\n#### Nonprotein Nitrogen\n\nThe nonprotein nitrogen (NPN) fraction of milk contains those nitrogenous compounds soluble in 12% TCA; it represents \u223c5% of total nitrogen (\u223c300 mgL\u20131). The principal components are urea, creatine, uric acid, and amino acids. Human milk contains a high level of taurine, which can be converted to cysteine and may be nutritionally important for infants. Urea, the concentration of which varies considerably, has a significant effect on the heat stability of milk (Walstra and Jenness, 1984). The amino acids present in the NPN fraction also support the growth of lactic acid bacteria.\n\n#### Casein Micelles\n\nIt has been known since the work of H. Schuler in 1818 that the casein in milk exists as large particles, now called casein micelles. The stability of the micelles is critically important for many of the technologically important properties of milk and consequently has been the focus of much research, especially since the discovery of \u03ba-casein in 1956 by Waugh and von Hipple (1956). Early views and research on casein micelles were reviewed by Fox and Brodkorb (2008), and a recent review is that of McMahon and Oommen (2013). Although views on the detailed structure of the casein micelle are divided, there is widespread, or unanimous, agreement on their general structure and properties.\n\nElectron microscopy shows that casein micelles are spherical with a diameter in the range 50\u2013500 nm (average \u223c120 nm) and a mass ranging from 106 to 3 \u00d7 109 Da (average \u223c108 Da) for bovine micelles. There are numerous small micelles, but these represent only a small proportion of the mass. There are 1014\u20131016 micelles mL\u20131 in milk, and they are roughly two micelle diameters (\u223c250 nm) apart. The dry matter of the micelles is \u223c94% protein and 6% low-molecular-mass species, referred to collectively as colloidal calcium phosphate (CCP), and consisting mainly of calcium phosphate with some magnesium and citrate and trace amounts of other species. The micelles bind \u223c2.0 g H2O g\u20131 protein. They scatter light, and the white color of milk is due largely to light scattering by the casein micelles. The white color is lost if the micelles are disrupted, by dissolving CCP with citrate, EDTA, or oxalate, by increasing pH, or by urea (>5 M) or ethanol (\u223c35% at 70 \u00b0C).\n\n#### Stability of Casein Micelles\n\nThe micelles are quite stable to the principal processes to which milk is normally subjected. They are very stable at high temperatures, and they withstand heating at 140 \u00b0C for 15\u201320 min at pH 6.7. Coagulation is caused by heat-induced changes, for example, a decrease in pH due to the pyrolysis of lactose to acids, dephosphorylation of casein, cleavage of the carbohydrate-rich moiety of \u03ba-casein, denaturation of the whey proteins and their precipitation on the casein micelles, and precipitation of soluble calcium phosphate on the micelles (see O'Connell and Fox, 2003).\n\nThe micelles are stable to compaction (e.g., they can be sedimented by ultracentrifugation and redispersed by mild agitation), to commercial homogenization, and to Ca2+ up to at least 200 mM at temperatures up to 50 \u00b0C. The effects of high pressure (up to 800 MPa) on the casein micelles in bovine, ovine, caprine, and buffalo milk have been studied; the size of the micelles increases up to 2\u2013300 MPa but decreases at higher pressure (see Huppertz et al., 2006; Huppertz and de Kruif 2007).\n\nAs the pH of milk is reduced, CCP dissolves and is fully soluble at \u2264pH 4.9. Acidification of cold (4 \u00b0C) milk to pH 4.6, followed by dialysis against bulk milk, is a convenient technique for altering the CCP content of milk. If acidified cold milk is readjusted to pH 6.7, the micelles re-form, provided that the pH had not been reduced below 5.5 (Lucey et al., 1996). This result seems to suggest that most of the CCP can be dissolved without destroying the structure of the micelles.\n\nSome proteinases, especially chymosin, catalyze a very specific hydrolysis of \u03ba-casein, as a result of which the casein coagulates in the presence of Ca2+ or other divalent ions. This is the key step in the manufacture of most cheese varieties. The proteinase preparations used for cheesemaking are called rennets.\n\nAt room temperature, the casein micelles are destabilized by \u223c40% ethanol at pH 6.7 or by lower concentrations if the pH is reduced (Horne, 2003a). However, if the system is heated to \u226570 \u00b0C, the precipitate redissolves, and the system becomes translucent. When the system is recooled, the white appearance of milk is restored, and a gel is formed if the ethanol\u2013milk mixture is held at 4 \u00b0C, especially if concentrated milk is used. If the ethanol is removed by evaporation, very large aggregates (average diameter, \u223c3000 nm) are formed. The dissociating effect of ethanol is promoted by increasing the pH (35% ethanol causes dissociation at 20 \u00b0C and pH 7.3) or by adding NaCl (Horne, 2003b). Methanol and acetone have an effect similar to ethanol, but propanol causes dissociation \u223c25 \u00b0C. The mechanism by which ethanol dissociates casein micelles has not been established, but it is not due to the solution of CCP, which is unchanged (O'Connell et al., 2003).\n\nThe micelles are also reversibly dissociated by urea (5 M) (McGann and Fox, 1974; Holt, 1998), SDS (Lefebvre-Cases et al., 1998), or by raising the pH to >9. Under these conditions, the CCP is not dissolved.\n\nThe micelles are destabilized by freezing (cryodestabilization) due to a decrease in pH and an increase in the Ca2+ in the unfrozen phase of milk; concentrated milk is very susceptibile to cryodestabilization (Moon et al. 1988; ). Cryodestabilized casein can be dispersed by warming the thawed milk to 55oC to give particles with micelle-like properties.\n\n#### Micelle Structure\n\nSince the beginning of the twentieth century, there has been speculation on how the casein particles (micelles) are stabilized (see Fox and Brodkorb, 2008), but no significant progress was possible until Waugh and von Hippel (1956) achieved the isolation and characterization of \u03ba-casein. The first attempt to describe the structure of the casein micelle was made by Waugh in 1958, and since then numerous models have been made and refined. Progress has been reviewed regularly; recent reviews include de Kruif and Holt (2003), Horne (2002; 2003a; 2003b; 2006), Farrell et al. (2006b), and McMahon and Oommen (2013). The principal features that any micelle model must meet are the following: \u03ba-casein, which represents \u223c12\u201315% of total casein, must be located so as to be able to stabilize the calcium-sensitive \u03b1s1-, \u03b1s2-, and \u03b2-caseins, which represent approximately 85% of total casein; chymosin and other rennets, which are relatively large molecules (MW\u223c 35 kDa), very rapidly and specifically hydrolyze most of the \u03ba-casein; when heated in the presence of whey proteins, \u03ba-casein, and \u03b2-lactoglobulin (MW \u223c36 kDa) interact to form a disulfide-linked complex that modifies the rennet and heat coagulation properties of the micelles. The arrangement that would best explain these features is a surface layer of \u03ba-casein surrounding the Ca-sensitive caseins, somewhat analogous to a lipid emulsion in which the triglycerides are surrounded by a thin layer of emulsifier. Most models of the casein micelle propose a surface location for \u03ba-casein, but some early models envisaged \u03ba-casein serving as nodes in the interior of the micelle.\n\nRemoval of CCP causes disintegration of the micelles into particles of MW \u223c106 Da, suggesting that the casein molecules are held together in the micelles by CCP. The properties of the CCP-free system are very different from those of normal milk (e.g., it is precipitated by relatively low levels of Ca2+, it is more stable to heat-induced coagulation, and it is not coagulable by rennets). Many of these properties can be restored, at least partially, by an increased concentration of calcium. However, CCP is not the only integrating factor, as indicated by the dissociating effect of urea, SDS, ethanol, or alkaline pH. At low temperatures, casein, especially \u03b2-casein, dissociates from the micelles.\n\nThere has been strong support for the view, first proposed by C. V. Morr in 1967, that the micelles are composed of submicelles (\u223c106 Da and 10\u201315 nm in diameter) linked together by CCP, giving a micelle with an open porous structure. On removing CCP (by acidification\/dialysis, EDTA, citrate, or oxalate) the micelles disintegrate. Disintegration may also be achieved by treatment with urea, SDS, 35% ethanol at 70 \u00b0C, or pH >9. These reagents do not solubilize CCP, suggesting that hydrophobic interactions and hydrogen bonds contribute to micelle structure. Much of the evidence for a submicellar structure relies on electron microscopy studies that appear to show variations in electron density, a raspberry-like structure, which was interpreted as indicating submicelles.\n\nViews on the proposed structure of the submicelles have evolved over the years (see McMahon and McManus, 1998; Dalgleish et al., 2004; Dalgleish, 2011; McMahon and Oommen 2008; ). Proposals have included the following: A rosette-type structure similar to that of a classical soap micelle, in which the polar regions of \u03b1s1-, \u03b1s2-, and \u03b2-caseins are oriented toward the outside of the submicelle to reduce electrostatic repulsion between neighboring charged groups; each submicelle was considered to be surrounded by a layer (coat) of \u03ba-casein, thus providing a \u03ba-casein coat for the entire micelle. Several authors have suggested that the submicelles are not covered completely by \u03ba-casein and that there are \u03ba-casein-rich, hydrophilic, and \u03ba-deficient, hydrophobic, regions on the surface of each submicelle. The latter aggregate via the hydrophobic patches such that the entire micelle has a \u03ba-casein-rich surface layer but also with some of the other caseins on the surface. In a popular version of this model, it was proposed that the hydrophilic C-terminal region of \u03ba-casein protrudes from the surface, forming a layer 5\u201310 nm thick and giving the micelles a hairy appearance. This hairy layer, functioning as an ionic brush, is considered to be responsible for micelle stability through major contributions to zeta potential (\u201320 mV) and steric stabilization. If the hairy layer is removed through specific hydrolysis of \u03ba-casein or collapsed (e.g., by ethanol), the colloidal stability of the micelles is destroyed, and they aggregate.\n\nA further variant of the subunit model envisages two main types of subunits\u2014one consisting of \u03b1s1-, \u03b1s2-, and \u03b2-caseins, which are present in the core of the micelle, and the other consisting of \u03b1s1\\- and \u03b1s2\\- and \u03ba-caseins, which forms a surface layer. It has also been proposed that \u03b2-casein associates to form thread-like structures with which \u03b1s1\\- and \u03b1s2-caseins associate hydrophobically to form the core of the micelle or submicelles which are surrounded by a layer of \u03ba-casein; CCP cements neighboring submicelles within the micelle. A recent study by Bouchoux et al. (2010), using SAXS analysis, supports a submicelle structure.\n\nAlthough the submicelle model of the casein micelle explains many of the principal features of, and physicochemical reactions undergone by, the micelles, and has been supported widely, it has never enjoyed unanimous support. Indeed, new electron microscopy techniques have cast doubts on the authenticity of submicelles. Using cryopreparation electron microscopy with stereo-imaging, McMahon and McManus (1998) found no evidence to support the submicellar model and concluded that if the micelles do consist of submicelles, these must be smaller than 2 nm or less densely packed than previously presumed. Like other forms of electron microscopy, field emission scanning electron microscopy showed that casein micelles have an irregular surface, but Dalgleish et al. (2004) concluded that the caseins form tubular structures rather than spherical submicelles. In principle, this model seems basically similar to earlier subunit models. McMahon and Oommen (2008) also found no evidence for a submicellar structure using high-resolution transmission electron microscopy.\n\nAlternative models have been proposed. Visser (1992) proposed that the micelles are spherical conglomerates of randomly aggregated casein molecules held together by amorphous calcium phosphate and hydrophobic bonds, with a surface layer of \u03ba-casein. Holt (1992) considered the casein micelle to be a tangled web of flexible casein molecules forming a gel-like structure in which microgranules of CCP are an integral feature and from the surface of which the C-terminal region of \u03ba-casein extends, forming a hairy layer. In what he referred to as the dual binding model, Horne (1998; ; 2003a; 2003b; 2006); described how casein molecules interact hydrophobically and through calcium phosphate nanoclusters to form micelles. These three models retain the key features of the submicellar model, that is, the cementing role of CCP and the predominantly surface location and micelle-stabilizing role of \u03ba-casein, and differ from it mainly with respect to the internal structure of the micelle.\n\n#### Interspecies Comparison of Milk Proteins\n\nThe protein content of milk varies widely depending on species, ranging from \u223c1% to \u223c20%. The protein content reflects the growth rate of the neonate of the species, that is, its requirements for essential amino acids. The milk of all species for which data are available contain two groups of protein, caseins and whey proteins, but the ratio of these varies widely. Both groups show genus- and even species-specific characteristics that presumably reflect some unique nutritional or physiological requirements of the neonate of the species. Interestingly, and perhaps significantly, of the milks that have been characterized, human and bovine milks are more or less at opposite ends of the spectrum. Among the general interspecies comparisons of milk proteins are Woodward (1976), Jenness (1973; ; 1982), Ginger and Grigor (1999), and Martin et al. (2003); reviews on milk proteins of individual species include buffalo (Addeo et al., 1977), goat (Trujillo et al. 1997; ), sheep (Amigo et al., 2000), camel (Kappeler et al., 1998; Ochirkhuyag et al., 1997), yak (Ochirkhuyag et al., 1997), horse (Ochirkhuyag et al., 2000; Park et al., 2006; Uniacke-Lowe et al., 2010; Uniacke-Lowe and Fox 2011), and sow (Gallagher et al., 1997).\n\nThere is considerably more and better information on the interspecies comparison of individual milk proteins than on overall milk composition, probably because only one sample of milk from one animal is sufficient to yield a particular protein for characterization. The two principal milk-specific whey proteins, \u03b1-La and \u03b2-Lg, from quite a wide range of species have been characterized and, in general, show a high degree of homology (see Brew 2003; ; Sawyer 2003; ). The caseins show much greater interspecies diversity, especially in the \u03b1-casein fraction; most of the species that have been studied contain a protein with an electrophoretic mobility similar to that of bovine \u03b2-casein (see Fig. 2.2), but the \u03b2-caseins that have been sequenced show a low level of homology (Martin et al. 2003; 2013a). Sheep's milk is used mainly for cheese production, with small amounts used for the production of fermented milks; hence the coagulation and gel-forming properties of ovine milk are particularly important. The \u03b1s1-casein of caprine milk is very heterogeneous. Not only do the properties of the variants differ, but the concentration of \u03b1s1-casein varies from 0 to 26% of total casein, and consequently, the total protein content varies considerably. This, in turn, has major effects on the rennet-induced coagulation properties of ovine and caprine milk as well as on the yield and quality of cheese produced therefrom (Amigo et al., 2000; Clark and Sherbon 2000a; 2000b; Martin et al., 2013a; 2013b).\n\nFigure 2.2 Urea polyacrylamide gel electrophoretogram of milk from 15 species. Lanes: 1, Bovine; 2, Camel; 3, Equine; 4, Asinine; 5, Human; 6, Rhinoceros; 7, Caprine; 8, Ovine; 9, Asian elephant; 10, African elephant; 11, Vervet monkey; 12, Macaque monkey; 13, Rat; 14, Canine; 15, Porcine (from Uniacke-Lowe, unpublished data).\n\nHuman \u03b2-casein occurs in multiphosphorylated forms (0\u20135 mol P per mol protein; see Atkinson and Lonnerdal, 1989), as does mare's \u03b2-casein (Ochirkhuyag et al., 2000; Girardet et al., 2006; Uniacke-Lowe et al. 2013). Considering the critical role played by \u03ba-casein, it would be expected that all casein systems contain this protein, but Ochirkhuyag et al. (2000) failed to identify \u03ba-casein in mare's milk and suggested that the micelle-stabilizing role was played by \u03b2-casein with zero or a low level of phosphorylation. More recent work has shown that equine milk does contain a low level of \u03ba-casein (Iametti et al., 2001; Egito et al., 2002; Uniacke-Lowe et al. 2013). Human \u03ba-casein is very highly glycosylated, containing 40\u201360% carbohydrate (compared with \u223c10% in bovine \u03ba-casein). The \u03b1s-casein fraction differs markedly between species; human milk probably lacks an \u03b1s-casein (Darragh and Lonnerdal, 2011), while the \u03b1-casein fractions in horse and donkey milk are very heterogeneous. The urea-PAGE gels of milk from 15 species are shown in Figure 2.2.\n\nThere are considerable interspecies differences in the minor proteins of milk. The milk of those species that have been studied in sufficient depth contain approximately the same profile of minor proteins, but there are very marked quantitative differences. Most of the minor proteins in milk have some biochemical or physiological function, and the quantitative interspecies differences presumably reflect the requirements of the neonate of the species. The greatest interspecies differences, in some cases 4000-fold, seem to occur in the indigenous enzymes (Fox and Kelly, 2006a,b).\n\nIn the milk of all species, the caseins exist as micelles (at least the milks appear white), but the properties of the micelles in the milk of only a few nonbovine species have been studied: caprine (Ono and Creamer, 1986; Ono et al., 1989); ovine (Ono et al., 1989); buffalo (Patel and Mistry, 1997); camel (Attia et al., 2000); mare (Welsch et al., 1988; Ono et al. 1989, Uniacke-Lowe, 2011); and human (Sood et al. 1997; ). The appearance and size of casein micelles in the milk of 19 species, guinea pig, rat, nutria (coypu), dog, cat, gray seal, rabbit, donkey, horse, alpaca, dromedary camel, cow, red deer, sheep, pig, water buffalo, goat, porpoise, and humans, were studied by Buchheim et al. (1989). The structures of all micelles appeared similar on electron microscopy, but there were large interspecies differences in size: Human micelles were smallest (64 nm) while those of the alpaca, goat, camel, and donkey were very large (300\u2013350 nm); the micelles in equine milk were as large as 700 nm (Uniacke-Lowe, 2011).\n\n#### Milk Salts\n\nWhen milk is heated at 500oC for \u223c5 h, an ash derived mainly from the inorganic salts of milk and representing \u223c0.7 %, w\/w, of bovine milk, remains. However, the elements in the ash are changed from their original form to oxides or carbonates, and the ash contains P and S derived from caseins, lipids, sugar phosphates, or high-energy phosphates. The organic salts, the most important of which is citrate, are oxidized and lost during ashing; some volatile metals, such as sodium, are partially lost. Thus, ash does not accurately represent the salts of milk. However, the principal inorganic and organic ions in milk can be determined directly by potentiometric, spectrophotometric, or other methods. The typical concentrations of the principal elements, the macroelements, are shown in Table 2.2; considerable variability occurs, due, in part, to poor analytical methods and\/or to samples from cows in very early or late lactation or suffering from mastitis.\n\nTable 2.2\n\nConcentration and Distribution of the Principal Milk Salts\n\nSpecies | Concentration (mg\/l) | % Soluble | Form | % Colloidal  \n---|---|---|---|---  \nSodium | 500 | 92 | Completely ionized | 8  \nPotassium | 1,450 | 92 | Completely ionized | 8  \nChloride | 1,200 | 100 | Completely ionized | -  \nSulfate | 100 | 100 | Completely ionized | -  \nPhosfate | 750 | 43 | 10% bound to Ca and Mg  \n54% H2PO4\u2212   \n36% HPO42\u2212 | 57  \nCitrate | 1,750 | 94 | 85% bound to Ca and Mg (undissociated)  \n15% Citr3\u2212 | 6  \nCalcium | 1,200 | 34 | 35% Ca2+   \n55% bound to citrate  \n10% bound to phosphate | 66  \nMagnesium | 130 | 67 | Probably similar to calcium | 33\n\nMilk also contains 20\u201325 elements at very low or trace levels. These microelements are very important from a nutritional viewpoint; many, notably Zn, Fe, Mo, Cu, Ca, Se, and Mg, are present in enzymes, many of which are concentrated in the MFGM; some microelements, such as Fe and Cu, are very potent lipid pro-oxidants. Although the salts are relatively minor constituents of milk, they are critically important for many technological and nutritional properties of milk.\n\nSome of the salts in milk are fully soluble, but others, especially calcium phosphate, exceed their solubility under the conditions in milk and occur partly in the colloidal state, associated with the casein micelles. These salts are referred to as colloidal calcium phosphate (CCP), although some magnesium, citrate, and traces of other elements are also present in the micelles. As discussed earlier, CCP plays a critical role in the structure and stability of the casein micelles. The typical distribution of the principal organic and inorganic ions between the soluble and colloidal phases of bovine milk is summarized in Table 2.2. The actual form of the principal species can be determined or calculated after making certain assumptions. Typical values are shown in the table.\n\nThe solubility and ionization status of many of the principal ionic species are interrelated, especially H+, Ca2+, PO43\u2013 and citrate3\u2013. These relationships have major effects on the stability of the caseinate system and, consequently, on the processing properties of milk. The status of various species in milk can be modified by adding certain salts to milk; for example, Ca2+ is reduced by adding PO43\u2013 or citrate3\u2013. Addition of CaCl2 affects the distribution and ionization status of calcium and phosphate and the pH of milk.\n\nThe precise nature and structure of CCP are uncertain (Choi et al., 2011). CCP is associated with the caseins, probably via the casein phosphate residues. It probably exists as nanocrystals, which include PO4 residues of casein. The simplest stoichiometry is Ca3(PO4)2, but spectroscopic data suggest that CaHPO4 is the most likely form.\n\nThe distribution of species between the soluble and colloidal phases is strongly affected by pH and temperature. As the pH is reduced, CCP dissolves and is completely soluble <Ca. pH 4.9; the reverse occurs when the pH is increased. The solubility of calcium phosphate decreases as the temperature is increased, and soluble calcium phosphate is transferred to the colloidal phase, with the release of H+ and a decrease in pH:\n\nCaHP O 4 \/ Ca ( H 2 P O 4 ) 2 \u2194 C a 3 ( P O 4 ) 2 + 3 H +\n\nThese changes are quite substantial but are at least partially reversible upon cooling. Since milk is supersaturated with calcium phosphate, concentration of milk by evaporation of water increases the degree of supersaturation and the transfer of soluble calcium phosphate to the colloidal state, with the concomitant release of H+. Dilution has the opposite effect.\n\nMilk salts equilibria are also shifted on freezing; as pure water freezes, the concentrations of solutes in unfrozen liquid are increased. Soluble calcium phosphate precipitates as Ca3(PO4)2, releasing H+ (the pH of the unfrozen liquid may decrease to 5.8). The crystallization of lactose as a monohydrate increases the degree of supersaturation by reducing the amount of solvent water.\n\nThere are substantial changes in the concentrations of the macroelements in milk during lactation, especially at the beginning and end of lactation and during mastitic infection. Changes in the concentration of some of the salts in milk, especially calcium phosphate and citrate, have major effects on the physicochemical properties of the casein system and on the processability of milk, especially rennet coagulability and related properties and heat stability.\n\nReviews on the chemistry of milk salts include Pyne (1962), Holt (1985; ; 2003), Holt et al. (1996; 1998), Holt and Carver (2012), Lucey and Fox (1993), Lucey and Horne (2009), and Choi et al. (2011). Reviews on the nutritional significance of milk salts include Flynn and Power (1985), Flynn and Cashman (1997), Cashman (2003a,b; ), and Hunt and Nielsen (2009).\n\n### Vitamins\n\nMilk contains all the vitamins in sufficient quantities to enable normal maintenance and growth of the neonate. Bovine milk is a very significant source of vitamins, especially biotin (B7), riboflavin (B2), and cobalamine (B12), in the adult human diet. For general information on the vitamins and for specific aspects in relation to milk and dairy products, including stability during processing and storage, the reader is referred to a set of articles in Roginski et al. (2003) and Fuquay et al. (2011) and those by Morrissey and Hill (2009) and Nohr and Biesalski (2009).\n\nIn addition to their nutritional significance, four vitamins are significant for other reasons: vitamin A (retinol) and carotenoids are responsible for the yellow-orange color of fat-containing products made from cow's milk; vitamin E (tocopherols) is a potent antioxidant; vitamin C (ascorbic acid) is an antioxidant or pro-oxidant, depending on its concentration; and vitamin B2 (riboflavin), which is greenish-yellow, is responsible for the color of whey or UF permeate, it co-crystallizes with lactose and is responsible for its yellowish color, which may be removed by recrystallization or bleached by oxidation, and acts as a photocatalyst in the development of light-oxidized flavor in milk, which is due to the oxidation of methionine.\n\n### Water\n\nWater is the principal constituent in the milk of most species. In addition to meeting the requirement of the neonate for water, the water in milk serves as solvent for milk salts, lactose, and proteins, and affects their properties and stability. It controls the rate of many reactions, for example, Maillard browning, lipid oxidation, enzyme activity, and microbial growth, thus affecting the stability of milk and milk products. The preservation of milk products is effected by reducing the water activity by dehydration or by adding sugar or NaCl. The stability and quality of most dairy products are strongly affected by their water content, and small differences can cause major instability problems.\n\nThe chemistry of water, its measurement, and its significance in foods in general and in dairy products, will not be discussed in this text. For that information, the reader is referred to Duckworth (1975), Rockland and Beuchat (1987), Fennema (1996), Roos (1997; 2011), Franks (2000), Chieh (2006), and Simatos et al. (2009; 2011).\n\n#### Physical Properties of Milk\n\nThe physical properties of milk are important from several points of view, including appearance, quality, analytical, and processing characteristics. A summary of the key physical properties of milk is provided in Table 2.3, and further information can be found in McCarthy and Singh (2009) and McCarthy (2011).\n\nTable 2.3\n\nSummary of the Principal Physical Properties of Milk\n\nPhysical property | Typical value  \n---|---  \npH1 | 6.5\u20136.7  \nTitratable acidity | 0.14\u20130.16%  \nDensity2 | 1030 kg m\u22123  \nSpecific heat capacity | 3880\u20134000 Jkg\u22121K\u22121  \nThermal conductivity2 | 0.548 Wm\u22121K\u22121  \nThermal diffusivity2 | 1.34 times 10-7m2s\u22121  \nSurface tension2 | 52 mNm\u22121  \nElectrical conductivity3 | 0.460 Sm\u22121  \nDielectric permittivity3 | 67.9  \nDielectric loss | 17.6  \nRefractive index2 | 1.3440\u20131.3485  \nFreezing point | \u22120.512 to \u22120.550 \u00b0C  \nOsmolality4 | 0.285 osmolkg\u22121\n\n1 Measured at 25 \u00b0C\n\n2 Measured at 20 \u00b0C\n\n3 Relative to value for free space\n\n4 Assuming freezing point of \u22120.53 \u00b0C\n\n#### Microbiology of Milk\n\nDue to its high water content, almost neutral pH, and gross chemical composition, milk is a good growth medium for many microorganisms. Milk from a healthy cow is secreted free of microorganisms, but it can quickly become contaminated with microorganisms of varied origin (e.g., air, human intervention, soil, bedding, milking equipment) (Hassan and Frank, 2011). The microorganisms typically found in milk can be divided into three broad groups\u2014animal pathogens and toxin producers, spoilage microorganisms, and microorganisms used to produce fermented products. Milk-handling conditions (e.g., temperature and duration of storage) and thermal processing parameters need to be designed to eliminate pathogenic microorganisms and reduce or control the growth of spoilage microorganisms in milk and dairy products made therefrom (Tamime, 2009).\n\n## Summary\n\nMilk is a very complex fluid. It contains several hundred molecular species, mostly at trace levels. Most of the microconstituents are derived from blood or mammary tissue, but most of the macroconstituents are synthesized in the mammary gland and are milk-specific. The constituents of milk are in true aqueous solution (e.g., lactose and most inorganic salts) or as a colloidal solution (proteins, which may be present as individual molecules or as large aggregates of several thousand molecules, called micelles) or as an emulsion (lipids). The macroconstituents can be readily fractionated and are used widely as food ingredients. The natural function of milk is to supply the neonate with its complete nutritional requirements for a period (sometimes several months) after birth and with many physiologically important molecules, including carrier proteins, protective proteins, and hormones.\n\nThe properties of milk lipids and proteins may be readily modified by biological, biochemical, chemical, or physical means and thus may be converted into novel dairy products. In this overview, the chemical and physicochemical properties of milk sugar (lactose), lipids, proteins, and inorganic salts were discussed. There is a vast primary literature and a range of specialist textbooks on the technology used to convert milk into multiple food products, but these aspects are not considered here.\n\n# References\n\nAddeo F ,  Mercier J-C ,  Ribadeau-Dumas B .  The caseins of buffalo milk .  _J. Dairy Res_.  1977 ;44 : 455 \u2013 468 .\n\nAkers RM .  Selection for milk protein production from a lactation biology viewpoint .  _J. Dairy Sci_.  2000 ;83 : 1151 \u2013 1158 .\n\nAkerstrom B ,  Flower DR ,  Salier JP .  Lipocalins 2000 .  _Biochim. Biophys. Acta_.  2000 ;1482 : 1 \u2013 356 .\n\nAmigo L ,  Recio I ,  Ramos M .  Genetic polymorphism of ovine proteins: its influence on technological properties of milk\u2014a review .  _Int. Dairy J_.  2000 ;10 : 135 \u2013 149 .\n\nAnnan WD ,  Manson W .  A fractionation of the \u03b1s-casein complex of bovine milk .  _J. Dairy Res_.  1969 ;36 : 259 \u2013 268 .\n\nAschaffenburg R ,  Drewry J .  A procedure for the routine determination of the various non-casein proteins in milk .  _Proc. 15th Int. Dairy Congr. (London)_.  1959 ;3 : 1631 \u2013 1637 .\n\nAtkinson SA ,  Lonnerdal B .  _Protein and Non-protein Nitrogen in Human Milk_ .  Boca Raton, FL :  CRC Press ;  1989 .\n\nAttia H ,  Kherouatou N ,  Nasri M ,  Khorchani T .  Characterization of the dromedary milk casein micelle and study of its changes during acidification .  _Le Lait_.  2000 ;80 : 503 \u2013 515 .\n\nAzuma N ,  Maeta A ,  Fukuchi K ,  Kanno C .  A rapid method for purifying osteopontin from bovine milk and interaction between osteopontin and other milk proteins .  _Int. Dairy J_.  2006 ;16 : 370 \u2013 378 .\n\nBarker ME .  Contribution of dairy foods to nutrient intake .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 2133 \u2013 2137 .\n\nBauman DE ,  Lock AL .  Conjugated linoleic acid: Biosynthesis and nutritional significance .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 93 \u2013 136 .\n\nBauman DE ,  Tyburczy C ,  O'Connell AM ,  Lock AL ,  Conjugated linoleic acid .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 660 \u2013 664 .\n\nBayless KJ ,  Davis GE ,  Meininger GA .  Isolation and biological properties of osteopontin from bovine milk .  _Prot. Expr Purif_.  1997 ;9 : 309 \u2013 314 .\n\nBecker DJ ,  Lowe JR .  Fucose: Biosynthesis and biological functions in mammals .  _Glycobiology_.  2003 ;13 : 41R \u2013 53R .\n\nBencini R ,  Pulina G .  The quality of sheep milk .  _Aust. J. Exper. Agric_.  1997 ;37 : 485 \u2013 504 .\n\nBenton MJ .  Early origins of modern birds and mammals: Molecules vs morphology .  _BioEssays_.  1999 ;21 : 1043 \u2013 1051 .\n\nBlackburn DG .  Evolutionary origins of the mammary gland .  _Mammal. Rev_.  1991 ;21 : 81 \u2013 96 .\n\nBlackburn DG .  Lactation: Historical patterns and potential for manipulation .  _J. Dairy Sci_.  1993 ;76 : 3195 \u2013 3212 .\n\nBlackburn DG ,  Hayssen V ,  Murphy CJ .  The origin of lactation and the evolution of milk: A review with new hypothesis .  _Mammal. Rev_.  1989 ;19 : 1 \u2013 26 .\n\nBobe G ,  Beitz DC ,  Freeman AE ,  Lindberg GL .  Separation and quantification of bovine milk proteins by reversed phase high performance liquid chromatography .  _J. Agric. Food Chem_.  1998 ;46 : 458 \u2013 463 .\n\nBouchoux A ,  Gesan-Guiziou G ,  Perez J ,  Cabane B .  How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study .  _Biophys. J_.  2010 ;99 : 3754 \u2013 3762 .\n\nBreckenridge WC ,  Kuksis A .  Molecular weight distribution of milk fat triglycerides from seven species .  _Lipid Res_.  1967 ;8 : 473 \u2013 478 .\n\nBrew K .  \u03b1-Lactalbumin .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Part A, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 387 \u2013 419 .\n\nBrew K ,  \u03b1-Lactalbumin .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 780 \u2013 786 .\n\nBrew K .  \u03b1-Lactalbumin .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 261 \u2013 273 .\n\nBrinkmann CR ,  Thiel S ,  Larsen MK ,  Petersen TE ,  Jensenius JC ,  Heegaard CW .  Preparation and comparison of cytotoxic complexes formed between oleic acid and either bovine or human \u03b1-lactalbumin .  _J. Dairy Sci_.  2011 ;94 : 2159 \u2013 2170 .\n\nBuchanan DS ,  Major Bos taurus breeds .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 284 \u2013 292 .\n\nBuchheim W ,  Lund S ,  Scholtissek J .  Vergleichende Untersuchungen zur Struktur und Grosse von Caseinmicellen in der Milch verschiedener Species .  _Kieler Milchwirtschaftliche Forschungsberichte_.  1989 ;41 : 253 \u2013 265 .\n\nCampagna S ,  Vitoux B ,  Humbert G ,  Girardet J-M ,  Linden G ,  Haertle T ,  Gaillard J-L .  Conformational studies of a synthetic peptide from the putative lipid-binding domain of bovine milk component PP3 .  _J. Dairy Sci_.  1998 ;81 : 3139 \u2013 3148 .\n\nCampana WM ,  O'Brien JS ,  Hiraiwa M ,  Patton S .  Secretion of prosaposin, a multifunctional protein, by breast cancer cells .  _Biochim. Biophys. Acta_.  1999 ;1427 : 392 \u2013 400 .\n\nCampbell B ,  Petersen WE .  Antibodies in milk for protection against human disease .  _Milchwissenschaft_.  1959 ;14 : 469 \u2013 473 .\n\nCashman KD .  Minerals in dairy products: Macroelements, nutritional significance; Trace elements, nutritional significance .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 2051 \u2013 2058 .\n\nCashman KD .  Minerals in dairy products: Trace elements, nutritional significance .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 2058 \u2013 2065 .\n\nCashman KD .  Milk minerals (including trace elements) and bone health .  _Int. Dairy J_.  2006 ;16 : 1389 \u2013 1398 .\n\nChanat E ,  Martin P ,  Ollivier-Bousquet M .  \u03b1s1-Casein is required for the efficient transport of \u03b2\\- and \u03ba-caseins from the endoplasmic reticulum to the Golgi apparatus of mammary epithelial cells .  _J. Cell Sci_.  1999 ;112 : 3399 \u2013 3412 .\n\nChatterton DEW ,  Smithers G ,  Roupas P ,  Brodkorb A .  Bioactivity of \u03b2-lactoglobulin and \u03b1-lactalbumin\u2014technological implications and processing: Review .  _Int. Dairy J_.  2006 ;16 : 1229 \u2013 1240 .\n\nChevalier F ,  Analytical methods: Electrophoresis .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 185 \u2013 192 .\n\nChevalier F ,  Milk Proteins: Proteomics .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 843 \u2013 847 .\n\nChieh C .  Water chemistry and biochemistry .  In:  Hui YH ,  ed.  _Food Biochemistry and Food Processing_ .  Oxford :  Blackwell Publishing ;  2006 : 103 \u2013 133 .\n\nChoi J ,  Horne DS ,  Lucey JA .  Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk .  _J. Dairy Sci_.  2011 ;94 : 3250 \u2013 3261 .\n\nChristie WW .  Composition and structure of milk lipids .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  2nd ed. London :  Chapman & Hall ;  1995 : 1 \u2013 36 .\n\nChrysina ED ,  Brew K ,  Acharya KR .  Crystal structures of apo- and holo-bovine \u03b1-lactalbumin at 2. 2 \u00c5 resolution reveal an effect of Ca2+ on inter-lobe interactions .  _J. Biol. Chem_.  2000 ;275 : 37021 \u2013 37029 .\n\nCifelli CJ ,  German JB ,  O'Donnell JA ,  Nutritional and health-promoting properties of dairy products: Contribution of dairy foods to nutrient intake .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 1003 \u2013 1008 .\n\nClark S ,  Sherbon JW .  Alphas1-casein, milk composition and coagulation properties of goat milk .  _Small Ruminant Res_.  2000 ;38 : 123 \u2013 134 .\n\nClark S ,  Sherbon JW .  Genetic variants of alphas1-casein in goat milk: breed distribution and associations with milk composition and coagulation properties .  _Small Ruminant Res_.  2000 ;38 : 135 \u2013 143 .\n\nCrampton AW ,  Jenkins FA .  Mammals from reptiles: A review of mammalian origins .  _Ann. Rev. Earth Planet Sci_.  1973 ;1 : 131 \u2013 155 .\n\nCreamer LK ,  Loveday SM ,  Sawyer L ,  \u03b2-Lactoglobulin .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 787 \u2013 794 .\n\nDalgleish DG .  On the structural models of bovine casein micelles\u2014review and possible improvements .  _Soft Matter_.  2011 ;7 : 2265 \u2013 2272 .\n\nDalgleish DG ,  Spagnuolo PA ,  Goff HD .  A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy .  _Int. Dairy J_.  2004 ;14 : 1025 \u2013 1031 .\n\nDarragh A ,  Lonnerdal B ,  Human milk .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 581 \u2013 590 .\n\nDarwin C .  _On the Origin of Species by Means of Natural Selection_ .  6th ed. New York :  Appleton-Century-Crofts ;  1872 .\n\nde Kruif CG ,  Holt C .  Casein micelle structure, functions and interactions .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic-Plenum Publishers ;  2003 : 233 \u2013 276 .\n\nDeeth HC ,  Lipolysis and hydrolytic rancidity .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford, UK :  Academic Press ;  2011 : 721 \u2013 726 .\n\nDeeth HC ,  Fitz-Gerald CH ,  Lipolytic enzymes and hydrolytic rancidity in milk and milk products .  Fox PF ,  ed.  _Developments in Dairy Chemistry_ ,  Volume 2 .  London :  Applied Science Publishers ;  1983 : 195 \u2013 239 .\n\nDeeth HC ,  Fitz-Gerald CH .  Lipolytic enzymes and hydrolytic rancidity in milk and milk products .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry. Volume 2., Lipids_ .  London :  Chapman and Hall ;  1995 : 247 \u2013 308 .\n\nDeeth HC ,  Fitz-Gerald CH ,  Lipolytic enzymes and hydrolytic rancidity .  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry_ ,  Volume 2 .  New York :  Springer ;  2006 : 481 \u2013 556 .\n\nDoreau M .  Le lait de jument et sa production: particularites et facteurs de variation .  _Lait_.  1994 ;74 : 401 \u2013 418 .\n\nDoreau M ,  Boulot S .  Recent knowledge on mare milk production: A review .  _Livest. Prod. Sci_.  1989 ;22 : 213 \u2013 235 .\n\ndu Puis EM .  _Nature 's Perfect Food: How Milk Became America's Drink_ .  New York :  New York University Press ;  2002 .\n\nDuckworth RB .  _Water Relations in Foods_ .  London :  Academic Press ;  1975 .\n\nDupont D ,  Croguennec T ,  Brodkorb A ,  Kouaouci R .  _Quantitation of proteins in milk and milk products. Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects_ .  In:  McSweeney PLH ,  Fox PF ,  eds.  4th ed. New York :  Springer ;  2013 : 87 \u2013 134 .\n\nEasteal S .  Molecular evidence for the early divergence of placental mammals .  _BioEssays_.  1999 ;21 : 1052 \u2013 1058 .\n\nEgito AS ,  Miclo L ,  Lopez C ,  Adam A ,  Girardet J-M ,  Gillard J-L .  Separation and characterization of mares' milk \u03b1s1-, \u03b2-, \u03ba-caseins, \u03b3-casein-like, and proteose peptone component 5-like peptides .  _J. Dairy Sci_.  2002 ;85 : 697 \u2013 706 .\n\nEvans DE .  Milk composition of mammals whose milk is not normally used for human composition .  _Dairy Sci. Abstr_.  1959 ;21 : 277 \u2013 288 .\n\nEvers J .  The milkfat globule membrane\u2014compositional and structural changes post secretion by the mammary secretory cell .  _Int. Dairy J_.  2004 ;14 : 661 \u2013 674 .\n\nFarah Z .  Composition and characteristics of camel milk .  _J. Dairy Res_.  1993 ;60 : 603 \u2013 626 .\n\nFarrell Jr HM ,  Jimenez-Flores R ,  Bleck GT ,  Brown EM ,  Butler JE ,  Creamer LK ,  Hicks CL ,  Holler CM ,  Ng-Kwai-Huang K ,  Swaisgood HE .  Nomenclature of the proteins of cows' milk\u2014sixth revision .  _J. Dairy Sci_.  2004 ;87 : 1641 \u2013 1647 .\n\nFarrell Jr HM ,  Qi PX ,  Uversky VN .  New views on protein structure: Application to the caseins: Protein structure and functionality .  In:  Fishman FML ,  Qi PX ,  Whisker FL ,  eds.  _Advances in Biopolymers: Molecules, Clusters, Networks, and Interactions_ .  Washington, DC :  ACS Symposium Series 935, American Chemical Society ;  2006 : 52 \u2013 70 .\n\nFarrell Jr HM ,  Malin EL ,  Browne EM ,  Qi PX .  Casein micelle structure: What can be learned from milk synthesis and structural biology? _Curr Opinion Coll. Interface Sci_.  2006 ;11 : 135 \u2013 147 .\n\nFennema OR .  Water and ice .  In:  Fennema O ,  ed.  _Food Chemistry_ .  3rd ed. New York :  Marcel Dekker ;  1996 : 17 \u2013 94 .\n\nFinlay, E., 2005. Bovini Mitrochondrial DNA: Demographic History and Recombination. PhD Thesis, Dublin University.\n\nFitzGerald RJ ,  Meisel H .  Milk protein hydrolysates and bioactive peptides .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry \u2014Volume 1: Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 675 \u2013 698 .\n\nFlynn A ,  Cashman K .  Nutritional aspects of minerals in bovine and human milks .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Vitamins_ .  2nd. ed. London :  Chapman & Hall ;  1997 : 257 \u2013 302 .\n\nFlynn A ,  Power P .  Nutritional aspects of minerals in bovine and human milks .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry, Volume 3, Lactose, and Minor Constituents_ .  London :  Elsevier Applied Science ;  1985 : 183 \u2013 215 .\n\nForsyth IA .  Variation among species in the endocrine control of mammary growth and function: The roles of prolactin, growth hormone and placental lactogen .  _J. Dairy Sci_.  1986 ;69 : 886 \u2013 903 .\n\nForsyth IA .  Mammals .  In:  Rogenski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 1672 \u2013 1680 .\n\nForsyth IA ,  Wallis IA .  Growth hormone and prolactin\u2014molecular and functional evolution .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 291 \u2013 312 .\n\nFox PF .  _Developments in Dairy Chemistry -1- Proteins_ .  London :  Applied Science Publishers ;  1982 .\n\nFox PF .  _Developments in Dairy Chemistry -2- Lipids_ .  London :  Applied Science Publishers ;  1983 .\n\nFox PF .  _Developments in Dairy Chemistry -3 - Lactose and Minor Constituents_ .  London :  Elsevier Applied Science ;  1985 .\n\nFox PF .  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  2nd ed. London :  Elsevier Applied Science Publishers ;  1992 .\n\nFox PF .  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  2nd ed. London :  Chapman & Hall ;  1995 .\n\nFox PF .  _Advanced Dairy Chemistry, Volume 3: Lactose, Water, Salts, and Vitamins_ .  2nd ed. London :  Chapman & Hall ;  1997 .\n\nFox PF .  Milk proteins: general and historical aspects .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1 \u2013 48 .\n\nFox PF ,  Lactose: chemistry, properties .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 173 \u2013 181 .\n\nFox PF ,  Brodkorb A .  The casein micelle: Historical aspects, current concepts, and significance .  _Int. Dairy J_.  2008 ;18 : 677 \u2013 684 .\n\nFox PF ,  Flynn A .  Biological properties of milk proteins .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  2nd ed. London :  Elsevier Applied Science ;  1992 : 255 \u2013 284 .\n\nFox PF ,  Kelly AL .  Indigenous enzymes in milk: Overview and historical aspects\u2014Part 1 .  _Int. Dairy J_.  2006 ;16 : 500 \u2013 516 .\n\nFox PF ,  Kelly AL .  Indigenous enzymes in milk: Overview and historical aspects\u2014Part 2 .  _Int. Dairy J_.  2006 ;16 : 5517 \u2013 5532 .\n\nFox PF ,  Kelly AL .  Developments in the chemistry and technology of milk proteins. 2. Minor milk proteins .  _Food Australia_.  2003 ;55 : 231 \u2013 234 .\n\nFox PF ,  McSweeney PLH .  _Dairy Chemistry and Biochemistry_ .  London :  Chapman and Hall ;  1998 .\n\nFox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic-Plenum Plublishers ;  2003 .\n\nFox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer Verlag ;  2006 .\n\nFranks F .  _Water \u2014A Matrix of Life_ .  2nd ed. Cambridge :  Royal Society of Chemistry ;  2000 .\n\nFuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ .  2nd ed. Oxford :  Academic Press ;  2011 .\n\nGallagher DP ,  Cotter PF ,  Mulvihill DM .  Porcine milk proteins: a review .  _Int. Dairy J_.  1997 ;7 : 99 \u2013 118 .\n\nGanzle MG ,  Lactose: derivatives .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 202 \u2013 208 .\n\nGanzle MG ,  Lactose: Galacto-oligosaccharides .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 209 \u2013 216 .\n\nGauthier SF ,  Pouliot Y ,  Maubois J-L .  Growth factors in bovine milk and colostrum: Composition, extraction, and biological activities .  _Lait_.  2006 ;86 : 99 \u2013 125 .\n\nGinger MR ,  Grigor MR .  Comparative aspects of milk caseins .  _Comp. Biochem. Physiol. B_.  1999 ;124 : 133 \u2013 145 .\n\nGirardet J-M ,  Linden G .  PP 3 component of bovine milk: A phosphorylated glycoprotein .  _J. Dairy Res_.  1996 ;63 : 333 \u2013 350 .\n\nGirardet J-M ,  Miclo L ,  Florent S ,  Molle D ,  Gaillard J-L .  Determination of the phosphorylation level and deamidation susceptibility of equine \u03b2-casein .  _Proteomics_.  2006 ;6 : 3707 \u2013 3717 .\n\nGlass RL ,  Jenness R .  Comparative biochemical studies of milk. VI. Constituent fatty acids of milk fats of additional species .  _Comp. Biochem. Physiol_.  1971 ;38B : 353 \u2013 359 .\n\nGlass RL ,  Troolin HA ,  Jenness R .  Comparative biochemical studies of milk. IV. Constituent fatty acids of milk fats .  _Comp. Biochem. Physiol_.  1967 ;22 : 415 \u2013 425 .\n\nGoldman AS .  Evolution of mammary gland defence system .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 277 \u2013 289 .\n\nGonzalez de Llano D ,  Polo C ,  Ramos M .  Update on HPLC and FPLC analysis of nitrogen compounds in dairy products .  _Lait_.  1990 ;70 : 255 \u2013 277 .\n\nHaenlein GFW .  Status of world literature on dairy goats: Introductory remarks .  _J. Dairy Sci_.  1980 ;63 : 1591 \u2013 1599 .\n\nHaggarty NW .  Milk proteins: minor proteins, bovine serum albumin and vitamin-binding proteins .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 1939 \u2013 1946 .\n\nHajjoubi, S., Rival-Gervier, S., Hayes, H., Floriot, S., Eggen, A., Pivini, F., Chardon, P., Houdebine, L.M. and Thepot, D., 2006. Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene. Gene, 370:104-112.\n\nHassan AN ,  Frank JF ,  Microorganisms associated with milk .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 447 \u2013 457 .\n\nHammarsten O .  Zur Frage, ob das Casein ein einheitleicher Stoff sei .  _Z. Physiol Chemie_.  1883 ;7 : 227 \u2013 273 .\n\nHayssen V .  Emperical and theoretical constraints on the evolution of lactation .  _J. Dairy Sci_.  1993 ;76 : 3213 \u2013 3233 .\n\nHayssen V ,  Blackburn DG .  \u03b1-Lactalbumin and the origins of lactation .  _Evolution_.  1985 ;39 : 1147 \u2013 1149 .\n\nHolt C .  The milk salts: their secretion, concentration and physical chemistry .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry, Volume 3, Lactose and Minor Constituents_ .  London :  Elsevier Applied Science Publishers ;  1985 : 143 \u2013 181 .\n\nHolt C .  Structure and stability of bovine casein micelles .  _Adv. Prot. Chem_.  1992 ;43 : 63 \u2013 151 .\n\nHolt C .  The milk salts and their interaction with casein .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Vitamins_ .  2nd ed. London :  Chapman & Hall ;  1997 : 233 \u2013 256 .\n\nHolt C .  Casein micelle substructure and calcium phosphate interactions studied by Sephacryl column chromatography .  _J. Dairy Sci_.  1998 ;81 : 2994 \u2013 3003 .\n\nHolt C .  The milk salts and their interaction with casein .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 2007 \u2013 2015 .\n\nHolt C ,  Carver JA .  Darwinian transformation of a \"scarcely nutritious fluid\" into milk .  _J. Evolution. Biol_.  2012 ;25 : 1253 \u2013 1263 .\n\nHolt C ,  Sawyer L .  Caseins as rheomorphic proteins: interpretation of primary and secondary structures of \u03b1s1-, \u03b2\\- and \u03ba-caseins .  _J. Chem. Soc. Faraday Trans_.  1993 ;89 : 2683 \u2013 2692 .\n\nHolt C ,  Timmins PA ,  Errington N ,  Leaver J .  A core-shell model of calcium phosphate nanoclusters stabilised by \u03b2-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements .  _Eur. J. Biochem_.  1998 ;252 : 73 \u2013 78 .\n\nHolt C ,  Wahlgren M ,  Drakenberg T .  Ability of a \u03b2-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters .  _Biochem. J_.  1996 ;314 : 1035 \u2013 1039 .\n\nHorne DS .  Casein structure, self-association and gelation .  _Curr. Opinion Coll. Surface Sci_.  2002 ;7 : 456 \u2013 461 .\n\nHorne DS .  Casein interactions: casting light on the black boxes, the structure in dairy products .  _Int. Dairy J_.  1998 ;8 : 171 \u2013 177 .\n\nHorne DS .  Caseins, micellar structure .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 1902 \u2013 1909 .\n\nHorne DS .  Ethanol stability .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry \u2014Volume 1: Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 975 \u2013 999 .\n\nHorne DS .  Casein micelle structure: models and muddles .  _Curr. Opinion Coll Interface Sci_.  2006 ;11 : 148 \u2013 153 .\n\nHunt C ,  Nielsen FH .  Nutritional aspects of minerals in bovine and human milk .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts, and Vitamins_ .  3rd ed. New York :  Springer ;  2009 : 391 \u2013 456 .\n\nHuppertz T .  Chemistry of the caseins .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 135 \u2013 160 .\n\nHuppertz T ,  de Kruif CG .  High pressure-induced solubilisation of micellar calcium phosphate from cross-linked casein micelles .  _Colloid Surface A_.  2007 ;295 : 264 \u2013 268 .\n\nHuppertz T ,  Kelly AL .  Physical chemistry of milk fat globules .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 173 \u2013 212 .\n\nHuppertz T ,  Kelly AL ,  Fox PF .  High pressure-induced changes in ovine milk. 2. Effects on ovine casein micelles and whey proteins .  _Milchwissenschaft_.  2006 ;61 : 394 \u2013 397 .\n\nHurley WL .  Immunoglobulins in mammary secretions .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry Volume 1 Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 421 \u2013 447 .\n\nHurley WL ,  Theil PK .  Immunoglobulins in mammary secretions .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 275 \u2013 294 .\n\nIametti S ,  Tedeschi G ,  Oungre E ,  Bonomi F .  Primary structure of \u03ba-casein isolated from mares' milk .  _J. Dairy Res_.  2001 ;68 : 53 \u2013 61 .\n\nIDF .  _Production and Utilization of Ewe and Goat Milk. Proc. CIRVAL Seminar, Crete, October 19-21, 1995_ .  Brussels :  International Dairy Federation ;  1996 .\n\nIDF .  _The World Dairy Situation. Bulletin 446_ .  Brussels :  International Dairy Federation ;  2010 .\n\nImafidon GF ,  Farkye NY ,  Spanier AM .  Isolation, purification and alteration of some functional groups of major milk proteins: A review .  _CRC Crit Rev. Food Sci. Nutr_.  1997 ;37 : 663 \u2013 689 .\n\nIngram CJE ,  Swallow D .  Lactose malabsorption .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water Salts and Vitamins_ .  3rd ed. New York :  Springer ;  2009 : 203 \u2013 229 .\n\nInnocente N ,  Biasutti M ,  Blecker C .  HPLC profile and dynamic surface properties of the proteose-peptone fraction from bovine milk and from whey protein concentrate .  _Int. Dairy J_.  2011 ;21 : 222 \u2013 228 .\n\nJakob E ,  Puhan Z .  Technological properties of milk as influenced by genetic polymorphism of milk proteins\u2014A review .  _Int. Dairy J_.  1992 ;2 : 157 \u2013 178 .\n\nJandal JM .  Comparative aspects of goat and sheep milk .  _Small Ruminant Res_.  1996 ;22 : 177 \u2013 185 .\n\nJenness R .  Caseins and caseinate micelles of various species .  _Neth. Milk Dairy J_.  1973 ;27 : 251 \u2013 257 .\n\nJenness R .  Comparative aspects of milk proteins .  _J. Dairy Res_.  1979 ;46 : 197 \u2013 210 .\n\nJenness R .  Composition and characteristics of goat milk: Review 1968-1979 .  _J. Dairy Sci_.  1980 ;63 : 1605 \u2013 1630 .\n\nJenness R .  Inter-species comparison of milk proteins .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry-1 Proteins_ .  London :  Applied Science Publishers ;  1982 : 87 \u2013 114 .\n\nJenness R ,  Sloan RE .  The composition of milk of various species: A review .  _Dairy Sci. Abstr_.  1970 ;32 : 599 \u2013 612 .\n\nJenness R ,  Holt C .  Casein and lactose concentrations in milk of 31 species are negatively correlated .  _Experentia_.  1987 ;43 : 1015 \u2013 1018 .\n\nJensen RG .  _The Lipids of Human Milk_ .  Boca Raton, FL :  CRC Press ;  1989 .\n\nJensen RG .  _Handbook of Milk Composition_ .  San Diego, CA :  Academic Press ;  1995 .\n\nJi Q ,  Luo Z-X ,  Wible JR ,  Zhang J-F ,  Georgi JA .  The earliest known eutherian mammal .  _Nature_.  2002 ;416 : 816 \u2013 822 .\n\nKappeler S ,  Farah Z ,  Puhan Z .  Sequence analysis of Camelus dromedarius milk caseins .  _J. Dairy Res_.  1998 ;65 : 209 \u2013 222 .\n\nKawasaki K ,  Weiss KM .  Mineralized tissue and vertebrate evolution: the secretory calcium-binding phosphoprotein gene cluster .  _Proc. Natl. Acad. Sci. USA_.  2003 ;100 : 4060 \u2013 4065 .\n\nKawasaki K ,  Lafont A-G ,  Sire J-Y .  The evolution of milk casein genes from tooth genes before the origin of mammals .  _Mol. Biol. Evol_.  2011 ;28 : 2053 \u2013 2061 .\n\nKawasaki K ,  Suzuki T ,  Weiss KM .  Genetic basis for the evolution of vertebrate mineralized tissue .  _Proc. Natl. Acad. Sci. USA_.  2004 ;101 : 11356 \u2013 11361 .\n\nKeenan TW ,  Mather IH .  Intracellular origin of milk fat globules and the nature of the milk fat globule membrane .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 137 \u2013 171 .\n\nKemp TS .  _Mammal-like Reptiles and the Origin of Mammals_ .  London :  Academic Press ;  1982 .\n\nKindstedt PS .  _Cheese and Culture_ .  Chelsea Green Publishing :  White River Junction, VT ;  2012 .\n\nKon SK .  _Milk and Its Products in Human Nutrition_ .  Rome :  United Nations-FAO ;  1959 .\n\nKorhonen H .  Technological and health aspects of bioactive components of milk .  _Int. Dairy J_.  2006 ;16 : 1227 \u2013 1428 .\n\nKorhonen H ,  Pihlanto A .  Bioactive peptides: Production and functionality .  _Int. Dairy J_.  2006 ;16 : 945 \u2013 969 .\n\nKruger M ,  Poulsen RC ,  Schollum L ,  Haggarty N ,  Ram S ,  Palmano K .  A comparison between acidic and basic protein fractions from whey or milk for reduction of bone loss in the ovariectomised rat .  _Int. Dairy J_.  2006 ;16 : 1149 \u2013 1156 .\n\nKunz C ,  Rudloff S .  Health promoting aspects of milk oligosaccharides: A review .  _Int. Dairy J_.  2006 ;16 : 1341 \u2013 1346 .\n\nLarson BL ,  Smith VR .  Lactation: A Comprehensive Treatise ,  Volumes 1\u20134, New York :  Academic Press ;  1974\u20131979 .\n\nLaxminarayana H ,  Dastur NN .  Buffalo milk and milk products, Parts I & II .  _Dairy Sci. Abstr_.  1968 ;30 : 177 \u2013 186 : 231\u2013241 .\n\nLe Parc A ,  Leonil J ,  Chanat E .  \u03b1s1-Casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly bound membrane-associated form .  _BMC Cell Biol_.  2010 ;11 : 65 .\n\nLeaver J ,  Law AJR .  Genetic engineering of milk proteins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Vol. 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 817 \u2013 837 .\n\nLefebvre-Cases E ,  Gastaldi E ,  Tarodo de la Fuente B .  Influence of chemical agents on interactions in dairy products: effect of SDS on casein micelles .  _Colloid Surface B_.  1998 ;11 : 281 \u2013 285 .\n\nLefevre CM ,  Sharp JA ,  Nicholas KR .  Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals .  _Reprod. Fert. Develop_.  2009 ;21 : 1015 \u2013 1027 .\n\nLemay DG ,  Lynn DJ ,  Martin WF ,  Neville MC ,  Casey TM ,  Rincon G ,  Kriventseva EV ,  Barris WC ,  Hinrichs AS ,  Molenaar AJ ,  Pollard KS ,  Maqbool NJ ,  Singh K ,  Murney R ,  Zdobnov EM ,  Tellam RL ,  Medrano JF ,  German JB ,  Rijnkels M .  The bovine lactation genome: insights into the evolution of mammalian milk .  _Genome Biol_.  2009 ;10 (4) : Article R43 .\n\nLeonil J ,  Molle D ,  Gaucheron F ,  Arpino P ,  Guenot P ,  Maubois JL .  Analysis of major bovine milk proteins by on-line high-pressure liquid chromatography and electrospray ionization-mass spectrometry .  _Lait_.  1995 ;75 : 193 \u2013 210 .\n\nLillegraven JA .  Polarities of mammalian evolution seen through homologs of the inner cell mass .  _J. Mammal. Evol_.  2004 ;11 : 143 \u2013 202 .\n\nLillegraven JA ,  Thompson SD ,  McNab BK ,  Patton JL .  The origin of eutherian mammals .  _Biol. J. Linnean Soc_.  1987 ;32 : 281 \u2013 336 .\n\nLindqvist B .  Casein and the action of rennin, Parts I and II .  _Dairy Sci. Abstr_.  1963 ;25 : 257 \u2013 264 : 299\u2013308 .\n\nLonnerdal B .  Lactoferrin .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 449 \u2013 466 .\n\nLonnerdal B ,  Suzuki YA .  Lactoferrin .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 295 \u2013 315 .\n\nLopez C ,  Menard O .  Human milk fat globules: Polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane .  _Colloid Surface B_.  2011 ;83 : 29 \u2013 41 .\n\nLucey JA ,  Fox PF .  Importance of calcium and phosphate in cheese manufacture: a review .  _J. Dairy Sci_.  1993 ;76 : 1714 \u2013 1724 .\n\nLucey JA ,  Horne DS .  Milk salts: technological significance .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Vitamins_ .  3rd ed. New York :  Springer ;  2009 : 351 \u2013 389 .\n\nLucey JA ,  Gorry C ,  O'Kennedy BT ,  Kalab M ,  Tan-Kinita R ,  Fox PF .  Effect of acidification and neutralization of milk on some physico-chemical properties of casein micelles .  _Int. Dairy J_.  1996 ;6 : 257 \u2013 272 .\n\nMacDonald D .  _The New Encyclopedia of Mammals_ .  Oxford :  Oxford University Press ;  2004 .\n\nMacGibbon AKH ,  Taylor MM .  Composition and structure of bovine milk lipids .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 1 \u2013 42 .\n\nMadsen O ,  Scally M ,  Douady CJ ,  Kao DJ ,  De Bry RW ,  Adkins R ,  Amrine HM ,  Stanhope MJ ,  de Jong WW ,  Springer MS .  Parallel adaptive radiations in two major classes of placental mammals .  _Nature_.  2001 ;409 : 610 \u2013 614 .\n\nMahoney RR .  Lactose: enzymic modification .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water Salts and Vitamins_ .  2nd ed. London :  Chapman & Hall ;  1997 : 77 \u2013 125 .\n\nManso MA ,  Leonil J ,  Jan G ,  Gagnaire V .  Application of proteomics to the characterisation of milk and dairy products .  _Int. Dairy J_.  2005 ;15 : 845 \u2013 855 .\n\nMarnila P ,  Korhonen H ,  Immunoglobulins .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 807 \u2013 815 .\n\nMartin P ,  Cebo C ,  Miranda G ,  Interspecies comparison of milk proteins: quantitative variability and molecular diversity .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 821 \u2013 842 .\n\nMartin P ,  Cebo C ,  Miranda G .  Interspecies comparison of milk proteins: quantitative variability and molecular diversity .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A, Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 387 \u2013 429 .\n\nMartin P ,  Bianchi L ,  Cebo C ,  Miranda G .  Genetic polymorphism of milk proteins .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A, Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 463 \u2013 514 .\n\nMartin P ,  Ferranti P ,  Leroux C ,  Addeo F .  Non-bovine caseins: Quantitative variability and molecular diversity .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 277 \u2013 317 .\n\nMartinez-Ferez A ,  Rudloff S ,  Gaudix A ,  Henkel CA ,  Pohlentz G ,  Boza JJ ,  Gaudix EM ,  Kunz C .  Goats milk as a natural source of lactose-derived oligosaccharides: isolation by membrane technology .  _Int. Dairy J_.  2006 ;16 : 173 \u2013 181 .\n\nMather IH .  A review and proposed nomenclature for major milk proteins of the milk fat globule membrane .  _J. Dairy Sci_.  2000 ;83 : 203 \u2013 247 .\n\nMather IH ,  Milk fat globule membrane .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 680 \u2013 690 .\n\nMcCarthy OJ ,  Physical and physico-chemical properties of milk .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 467 \u2013 477 .\n\nMcCarthy OJ ,  Singh H .  Physico-chemical properties of milk .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  3rd ed New York :  Springer ;  2009 : 691 \u2013 758 .\n\nMcGann TCA ,  Fox PF .  Physico-chemical properties of casein micelles reformed from urea-treated milk .  _J. Dairy Res_.  1974 ;41 : 45 \u2013 53 .\n\nMcKenzie HA ,  White Jr FH .  Lysozyme and \u03b1-lactalbumin: structure, function, and interrelationships .  _Adv. Prot. Chem_.  1991 ;41 : 173 \u2013 315 .\n\nMcMahon DJ ,  McManus WR .  Rethinking casein micelle structure using electron microscopy .  _J. Dairy Sci_.  1998 ;81 : 2985 \u2013 2993 .\n\nMcMahon DJ ,  Oommen BS .  Supramolecular structure of the casein micelle .  _J. Dairy Sci_.  2008 ;91 : 1709 \u2013 1721 .\n\nMcMahon DJ ,  Oommen BS .  Casein micelle structure, functions, and interactions .  In:  McSweeney H ,  Fox PF ,  eds.  _Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 185 \u2013 209 .\n\nMcMeekin TL ,  Milk proteins in retrospect .  McKenzie HA ,  ed.  _Milk Proteins: Chemistry and Molecular Biology_ ,  Volume I .  New York :  Academic Press ;  1970 : 3 \u2013 15 .\n\nMcSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry. Volume 3, Lactose, Water, Salts and Minor Constituents_ .  New York :  Springer ;  2009 .\n\nMcSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry. Vol 1A, Basic Aspects_ .  New York :  Springer ;  2013 .\n\nMedhammar E ,  Wijesinha-Bettoni R ,  Stadlmayr B ,  Nilsson E ,  Charrondiere UR ,  Burlingame B .  Composition of milk from minor dairy animals and buffalo breeds: a biodiversity perspective .  _J. Sci. Food Agric_.  2011 ;92 : 445 \u2013 474 .\n\nMehra R ,  Kelly P .  Milk oligosaccharides: structural and technological aspects .  _Int. Dairy J_.  2006 ;16 : 1334 \u2013 1340 .\n\nMepham TB ,  ed.  _Biochemistry of Lactation_ .  Amsterdam :  Elsevier ;  1983 .\n\nMesser M ,  Urashima T .  Evolution of milk oligosaccharides and lactose .  _Trends in Glycosci. Glycotechnol_.  2002 ;14 : 153 \u2013 176 .\n\nMills S ,  Ross RP ,  Hill C ,  FitzGerald GF ,  Stanton C .  Milk intelligence: mining milk for bioactive substances associated with human health .  _Int. Dairy J_.  2011 ;21 : 377 \u2013 401 . \nMoon TW ,  Peng IC ,  Lonergan DA .  Chemical properties of cryocasein .  _J. Food Sci_.  1988 ;53 : 1687 \u2013 1693 .\n\nMoon TW ,  Peng IC ,  Lonergan DA .  Functional properties of cryocasein .  _J. Dairy Sci_.  1989 ;72 : 815 \u2013 828 .\n\nMorrissey PA ,  Hill TR .  Fat soluble vitamins and vitamin C in milk and milk products .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  3rd ed. New York :  Springer ;  2009 : 527 \u2013 589 .\n\nMoss M ,  Freed D .  The cow and the coronary: Epidemiology, biochemistry and immunology .  _Int. J. Cardiol_.  2003 ;87 : 203 \u2013 216 .\n\nMulvihill DM ,  Ennis MP .  Functional milk proteins: production and utilization .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1175 \u2013 1228 .\n\nMurphy WJ ,  Eziak E ,  Johnson WE ,  Zhang YP ,  Ryder OA ,  O'Brien SJ .  Molecular phylogenetics and the origin of mammals .  _Nature_.  2001 ;409 : 416 \u2013 418 .\n\nMustapha A ,  Hertzler SR ,  Savaiano DA .  Lactose: Nutritional significance .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water Salts and Vitamins_ .  2nd ed. London :  Chapman & Hall ;  1997 : 127 \u2013 154 .\n\nNewburg DS ,  Newbauer SH .  Carbohydrates in milk: Analysis, quantities and significance .  In:  Jensen RG ,  ed.  _Handbook of Milk Composition_ .  San Diego :  Academic Press ;  1995 : 273 \u2013 349 .\n\nNg-Kwai-Hang KF ,  Heterogeneity, fractionation and isolation .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 751 \u2013 764 .\n\nNg-Kwai-Hang KF ,  Grosclaude F .  _Genetic polymorphism of milk proteins_ .  In:  Fox PF ,  McSweeney PLH ,  eds.  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 739 \u2013 816 .\n\nNitta K ,  Sugai S .  The evolution of lysozyme and \u03b1-lactalbumin .  _Eur. J. Biochem_.  1989 ;182 : 111 \u2013 118 .\n\nNitta K ,  Tsuge H ,  Sugai S ,  Shimazaki K .  The calcium-binding properties of equine lysozyme .  _FEBS Lett_.  1987 ;223 : 405 \u2013 408 .\n\nNohr D ,  Biesalski HK .  Vitamins in milk and milk products; B-group vitamins .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  3rd ed. New York :  Springer ;  2009 : 591 \u2013 630 .\n\nO'Brien N ,  O'Connor TP ,  Lipids: Nutritional significance .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 711 \u2013 715 .\n\nO'Connell JE ,  Fox PF .  Heat-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 879 \u2013 945 .\n\nO'Connell JE ,  Steinle S ,  Reiter F ,  Auty MAE ,  Kelly AL ,  Fox PF .  Properties of casein micelles reformed from heated mixtures of milk and ethanol .  _Colloid Surface A_.  2003 ;213 : 265 \u2013 273 .\n\nO'Connor TP ,  O'Brien NM ,  Lipid oxidation .  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry_ ,  Vol. 2 .  New York :  Springer ;  2006 : 557 \u2013 600 .\n\nO'Connor TP ,  O'Brien NM ,  Lipid oxidation .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 716 \u2013 720 .\n\nO'Donnell R ,  Holland JW ,  Deeth HC ,  Alewood P .  Milk proteomics .  _Int. Dairy J_.  2004 ;14 : 1013 \u2013 1023 .\n\nO'Flaherty, F., 1997. Characterization of Some Minor Caseins and Proteose Peptones of Bovine Milk. MSc. Thesis, National University of Ireland, Cork.\n\nO'Mahony JA ,  Fox PF .  Milk proteins: Introduction and historical aspects .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 43 \u2013 85 .\n\nO'Mahony JA ,  Tuohy JJ .  Further applications of membrane filtration in dairy processing .  In:  Tamime AY ,  ed.  _Membrane Processing: Dairy and Beverage Applications_ .  West Sussex, UK :  Blackwell Publishing ;  2013 .\n\nO'Mahony JA ,  Fox PF ,  Kelly AL .  Indigenous enzymes of milk .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 337 \u2013 385 .\n\nOchirkhuyag B ,  Chobert J-M ,  Dalgalarrondo M ,  Choiset Y ,  Haertle T .  Characterization of caseins from Mongolian yak, khainak, and bacterian camel .  _Lait_.  1997 ;77 : 601 \u2013 613 .\n\nOchirkhuyag B ,  Chorbet J-M ,  Dalgalarrondo M ,  Haertle T .  Characterization of mare caseins. Identification of \u03b1s1- and \u03b1s2-caseins .  _Lait_.  2000 ;80 : 223 \u2013 235 .\n\nOftedal OT .  The mammary gland and its origin during synapsid evolution .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 225 \u2013 252 .\n\nOftedal OT .  The origin of lactation as a water source for parchment-shelled eggs .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 253 \u2013 266 .\n\nOftedal OT .  Origin and evolution of the major constituents of milk .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry: Volume 1A Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 1 \u2013 42 .\n\nOftedal OT ,  Alt GL ,  Widdowson EN ,  Jakubasz MR .  Nutrition and growth of suckling black bears (Urus americanus) during their mother's winter fast .  _Br. J. Nutr_.  1993 ;70 : 59 \u2013 79 .\n\nOno T ,  Creamer LK .  Structure of goat casein micelles .  _NZ. J. Dairy Sci. Technol_.  1986 ;21 : 57 \u2013 64 .\n\nOno T ,  Kohno H ,  Odagiri S ,  Takagi T .  Subunit components of casein micelles from bovine, ovine, caprine and equine milks .  _J. Dairy Res_.  1989 ;56 : 61 \u2013 68 .\n\nOsthoff G ,  de Waal HO ,  Hugo A ,  de Wit M ,  Botes P .  Milk composition of a free-ranging African elephant (Loxodonta africana) cow during early lactation .  _Comp. Biochem. Physiol_.  2005 ;141 : 223 \u2013 229 .\n\nPakkanen R ,  Aalto J .  Growth factors and antimicrobial factors in bovine colostrums .  _Int. Dairy J_.  1997 ;7 : 285 \u2013 297 .\n\nPark YW ,  Haenlein GFW .  _Milk and Dairy Products in Human Nutrition: Production, Composition and Health_ .  Hoboken, NJ :  John Wiley & Sons ;  2013 .\n\nPark YW ,  Haenlein GFW ,  eds.  _Handbook of Milk of Non-bovine Mammals_ .  Oxford :  Blackwell Publishing ;  2006 .\n\nPark YW ,  Juarez M ,  Ramos M ,  Haenlein GFW .  Physico-chemical characteristics of goat and sheep milk .  _Small Rumin. Res_.  2007 ;68 : 88 \u2013 113 .\n\nPark YW ,  Zhang H ,  Zhang B ,  Zhang L .  Mare milk .  In:  Park YW ,  Haenlein GFW ,  eds.  _Handbook of Milk of Non-bovine Mammals_ .  Oxford :  Blackwell Publishing ;  2006 : 275 \u2013 296 .\n\nParkash S ,  Jenness R .  The composition and characteristics of goats' milk: A review .  _Dairy Sci. Abstr_.  1968 ;30 : 67 \u2013 87 .\n\nParodi P .  Nutritional significance of milk lipids .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 601 \u2013 639 .\n\nPatel RS ,  Mistry VV .  Physicochemical and structural properties of ultrafiltered buffalo milk and milk powder .  _J. Dairy Sci_.  1997 ;80 : 812 \u2013 817 .\n\nPaterson AHJ .  Production and uses of lactose .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  New York :  Springer ;  2009 : 105 \u2013 120 .\n\nPaterson AHJ ,  Lactose: production, applications .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 196 \u2013 201 .\n\nPatton S .  Some practical implications of the milk mucins .  _J. Dairy Sci_.  1999 ;82 : 1115 \u2013 1117 .\n\nPatton S .  _Milk: Its Remarkable Contribution to Human Health and Well-being_ .  New Brunswick, NJ :  Transaction Publishers ;  2004 .\n\nPatton S ,  Milk in human health and nutrition .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 607 \u2013 612 .\n\nPatton S ,  Carson GS ,  Hiraiwa M ,  O'Brien JS ,  Sano A .  Prosaposin, a neurotrophic factor: presence and properties in milk .  _J. Dairy Sci_.  1997 ;80 : 264 \u2013 272 .\n\nPeaker M .  The mammary gland in mammalian evolution: A brief commentary on some of the concepts .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 347 \u2013 353 .\n\nPettersson J ,  Mossberg AK ,  Svanborg C .  \u03b1-Lactalbumin species variation, HAMLET formation and tumor cell death .  _Biochem. Biophys. Res. Commun_.  2006 ;345 : 260 \u2013 270 .\n\nPlayne MJ ,  Crittenden RG .  Galacto-oligosaccharides and other products derived from lactose .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  New York :  Springer ;  2009 : 121 \u2013 201 .\n\nPond CM .  The significance of lactation in the evolution of mammals .  _Evolution_.  1977 ;31 : 177 \u2013 199 .\n\nPyne GT .  Reviews on the Progress of Dairy Science. Section C. Dairy Chemisrty. Some aspects of the physical chemistry of the salts of milk .  _J. Dairy Res_.  1962 ;29 : 101 \u2013 130 .\n\nRao MB ,  Gupta RC ,  Dastur NN .  Camels' milk and milk products .  _Indian J. Dairy Sci_.  1970 ;23 : 71 \u2013 78 .\n\nReinhardt TA ,  Lippolis JD .  Bovine milk fat globule membrane proteome .  _J. Dairy Res_.  2006 ;73 : 406 \u2013 416 .\n\nRiccio P .  The proteins of the milk fat globule membrane in the balance .  _Trends Food Sci. Technol_.  2004 ;15 : 458 \u2013 461 .\n\nRichardson T ,  Korycka-Dahl M ,  Lipid oxidation .  Fox PF ,  ed.  _Developments in Dairy Chemistry_ ,  Volume 2 .  London :  Applied Science Publishers ;  1983 : 241 \u2013 363 .\n\nRijnkels M .  Multispecies comparison of the casein gene loci and evolution of casein gene family .  _J. Mammary Gland Biol_.  2002 ;7 : 327 \u2013 345 .\n\nRockland LB ,  Beuchat LR .  _Water Activity: Theory and Application to Food_ .  New York :  Marcel Dekker ;  1987 .\n\nRoginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 .\n\nRoncada P ,  Piras C ,  Soggiu A ,  Turk R ,  Urbani A ,  Bonizzi L .  Farm animal milk proteomics .  _J. Proteom_.  2012 ;75 : 4259 \u2013 4274 .\n\nRoos Y ,  Water in milk products .  Fox PF ,  ed.  _Advanced Dairy Chemistry, Volume 3: Lactose, Water, Salts and Vitamins_ 2nd ed. London :  Chapman & Hall ;  1997 : 306 \u2013 346 .\n\nRoos YH ,  Water in dairy products .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 4 .  2nd ed. Oxford :  Academic Press ;  2011 : 707 \u2013 714 .\n\nSaidi S ,  Rival-Gervier S ,  Daniel-Carlier N ,  Thepot D ,  Morgenthaler C ,  Viglietta C ,  Prince S ,  Passet B ,  Houdebine L-M ,  Jolivet G .  Distal control of the pig whey acidic protein (WAP) locus in transgenic mice .  _Gene_.  2007 ;401 : 97 \u2013 107 .\n\nSawyer L .  \u03b2-Lactoglobulin .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Part A, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 319 \u2013 386 .\n\nSawyer L .  \u03b2-Lactoglobulin .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 211 \u2013 259 .\n\nSchrezenmeir J ,  Korhonen H ,  Williams CM ,  Gill HS ,  Shah NP .  Beneficial natural bioactive substances in milk and colostrums .  _Br. J. Nutr_.  2000 ;84 (Suppl 1) : S1 \u2013 S166 .\n\nSchuck P ,  Lactose: crystallisation .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 182 \u2013 195 .\n\nShakeel-Ur-Rehman .  Reduced lactose and lactose-free dairy products .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3, Lactose, Water, Salts and Minor Constituents_ .  New York :  Springer ;  2009 : 98 \u2013 104 .\n\nSharp JA ,  Menzies K ,  Lefevre C ,  Nicholas KR ,  Milk of monotremes and marsupials .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 1 .  2nd ed. Oxford :  Academic Press ;  2011 : 553 \u2013 562 .\n\nSimatos D ,  Roudaut G ,  Champion D ,  Analysis and measurement of water activity .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 4 .  2nd ed. Oxford :  Academic Press ;  2011 : 715 \u2013 726 .\n\nSimatos D ,  Champion D ,  Lorient D ,  Loupiac C ,  Roudaut G .  Water in dairy products .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 3: Lactose, Water, Salts and Vitamins_ .  3rd ed. New York :  Springer Verlag ;  2009 : 457 \u2013 526 .\n\nSimpson KJ ,  Nicholas KR .  Comparative biology of whey proteins .  _J. Mammary Gland Biol. Neoplasia_.  2002 ;7 : 313 \u2013 326 .\n\nSingh H .  The milk fat globule membrane. A biophysical system for food applications .  _Curr. Opinion Coll. Interface Sci_.  2006 ;11 : 154 \u2013 163 .\n\nSolaroli G ,  Pagliarini E ,  Peri C .  Composition and quality of mare's milk .  _Italian J. Food Sci. V_.  1993 ; : 3 \u2013 10 .\n\nSood SM ,  Erickson G ,  Slattery CW .  Formation of reconstituted casein micelles with human \u03b2-casein and bovine \u03ba-casein .  _J. Dairy Sci_.  2002 ;85 : 472 \u2013 477 .\n\nSood SM ,  Herbert PJ ,  Slattery CW .  Structural studies on casein micelles of human milk: Dissociation of \u03b2-casein of different phosphorylation levels induced by cooling and ethylenediaminetetraacetate .  _J. Dairy Sci_.  1997 ;80 : 628 \u2013 633 .\n\nSpitsberg VL .  Invited review: Bovine milk fat globule membrane as a potential nutraceutical .  _J. Dairy Sci_.  2005 ;88 : 2289 \u2013 2294 .\n\nSpringer MS ,  Stanhope SJ ,  Madsen O ,  de Jong WW .  Molecules consolidate the placental mammal tree .  _Trends Ecol. Evol_.  2004 ;19 : 430 \u2013 438 .\n\nStagsted J .  Absence of both glutathione peroxidase activity and glutathione in bovine milk .  _Int. Dairy J_.  2006 ;16 : 662 \u2013 668 .\n\nStrange ED ,  Malin EL ,  van Hekken DL ,  Basch JJ .  Chromatographic and electrophoretic methods for analysis of milk proteins .  _J. Chromatogr_.  1992 ;624 : 81 \u2013 102 .\n\nSvensson M ,  Hakansson A ,  Mossberg AK ,  Linse S ,  Svanborg C .  Conversion of alpha-lactalbumin to a protein inducing apoptosis .  _Proc. National Acad. Sci. USA_.  2000 ;97 : 4221 \u2013 4226 .\n\nSwaisgood HE .  _Methods of Gel Electrophoresis of Milk Proteins_ .  Champaign, IL :  American Dairy Science Association ;  1975 : pp. 1\u201333 .\n\nTamime AY ,  ed.  _Milk Processing and Quality Management_ .  West Sussex, UK :  John Wiley and Sons ;  2009 .\n\nTaylor MW ,  MacGibbon AKH ,  Triacylglycerols .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 665 \u2013 669 .\n\nTremblay L ,  Laporte MF ,  Lenoil J ,  Dupont D ,  Paquin P .  Quantitation of proteins in milk and milk products .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 49 \u2013 139 .\n\nTrujillo AJ ,  Casals I ,  Guamis B .  Analysis of major caprine milk proteins by reverse-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry .  _J. Dairy Sci_.  2000 ;83 : 11 \u2013 19 .\n\nTrujillo AJ ,  Guamis B ,  Carretero C .  Las proteinas mayoritarias de la leche de cabra .  _Alimentaria_.  1997 ;285 : 19 \u2013 28 .\n\nUniacke-Lowe, T., 2011. Studies on Equine Milk and Comparative Studies on Equine and Bovine Milk Systems. PhD Thesis, University College Cork.\n\nUniacke-Lowe T ,  Fox PF ,  Equid milk .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 518 \u2013 529 .\n\nUniacke-Lowe T ,  Huppertz T ,  Fox PF .  Equine milk proteins: Chemistry, structure and nutritional significance .  _Int. Dairy J_.  2010 ;20 : 609 \u2013 629 .\n\nUniacke-Lowe T ,  Chevalier F ,  Hem S ,  Fox PF ,  Mulvihill DM .  Proteomic comparison of equine and bovine milks on renneting .  _J. Agric. Food Chem. 61, 2839-2850_.  2013 ; .\n\nUrashima T ,  Asakuma S ,  Kitaoka M ,  Messer M ,  Indigenous oligosaccharides in milk .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 241 \u2013 273 .\n\nUrashima T ,  Kitaoka M ,  Asakuma S ,  Messer M .  Indigenous oligosaccharides in milk .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advance Dairy Chemistry, Volume 3, Lactose Water Salts and Vitamins_ .  3rd ed. New York :  Springer ;  2009 : 295 \u2013 349 .\n\nUrashima T ,  Saito T ,  Nakarmura T ,  Messer M .  Oligosaccharides of milk and colostrums in non-humam mammals .  _Glycoconjugate J_.  2001 ;18 : 357 \u2013 371 .\n\nVanderghem D ,  Danthine S ,  Blecker C ,  Deroanne C .  Effect of proteose-peptone addition on some physico-chemical characteristics of recombined dairy creams .  _Int. Dairy J_.  2007 ;17 : 889 \u2013 895 .\n\nVerstegen MWA ,  Moughan PJ ,  Schrama JW .  _The Lactating Sow_ .  Wageningen :  Wageningen Pers ;  1998 .\n\nVisser H .  A new casein micelle model and its consequences for pH and temperature effects on the properties of milk .  In:  Visser H ,  ed.  _Protein Interactions_ .  VCH Weinheim. ;  1992 : 135 \u2013 165 .\n\nVisser S ,  Slangen KJ ,  Rollema HS .  High performance liquid chromatography of bovine caseins with the application of various stationary phases .  _Milchwissenschaft_.  1986 ;41 : 559 \u2013 562 .\n\nVisser S ,  Slangen CJ ,  Rollema HS .  Phenotyping of bovine milk proteins by reversed phase high performance liquid chromatography .  _J. Chromatog_.  1991 ;548 : 361 \u2013 370 .\n\nWalstra P ,  Jenness R .  _Dairy Chemistry and Physics_ .  New York :  John Wiley & Sons ;  1984 .\n\nWard RE ,  German JB ,  Corredig M .  Composition, applications, fractionation, technological and nutritional significance of milk fat globule material .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Lipids_ .  3rd ed. New York :  Springer ;  2006 : 213 \u2013 244 .\n\nWaugh DF ,  von Hipple PH .  \u03ba-Casein and the stabilization of casein micelles .  _J. Am. Chem. Soc_.  1956 ;78 : 4576 \u2013 4582 .\n\nWeisberg SM .  Recent progress in the manufacture and use of lactose: A review .  _J. Dairy Sci_.  1954 ;37 : 1106 \u2013 1115 .\n\nWelsch U ,  Buchheim W ,  Schumacher U ,  Schinko I ,  Patton S .  Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles .  _Histochemistry_.  1988 ;88 : 357 \u2013 365 .\n\nWelsch U ,  Schumaker U ,  Buchheim W ,  Schinko I ,  Jenness R ,  Patton S .  Histochemical and biochemical observations on milk-fat globule membranes from several species .  _Acta Histochemica, Suppl. XL_.  1990 ; : 59 \u2013 64 .\n\nWheeler MB .  Production of transgenic livestock: promise fulfilled .  _J. Anim. Sci_.  2003 ;81 (Suppl 3) : 32 \u2013 37 .\n\nWhittier EO .  Lactose: A review .  _Chem. Rev_.  1925 ;2 : 85 \u2013 125 .\n\nWhittier EO .  Lactose and its utilization: A review .  _J. Dairy Sci_.  1944 ;27 : 505 \u2013 537 .\n\nWoodward DR .  The chemistry of mammalian caseins: A review .  _Dairy Sci Abstr_.  1976 ;38 : 137 \u2013 150 .\n\nWynn PC ,  Sheehy PA .  Milk proteins, including growth factors .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry, Volume 1A: Proteins: Basic Aspects_ .  4th ed. New York :  Springer ;  2013 : 317 \u2013 335 .\n\nWynn PC ,  Morgan AJ ,  Sheehy PA ,  Minor proteins, bovine serum albumin, vitamin-binding proteins .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 795 \u2013 800 .\n\nZadow JG .  Lactose: Properties and uses .  _J. Dairy Sci_.  1984 ;67 : 2654 \u2013 2679 .\n\nZadow JG .  _Lactose and Whey Processing_ .  London :  Elsevier Applied Science ;  1992 .\n\nZanabria Eyzaguirre R ,  Corredig M ,  Buttermilk and milk fat globule membrane fractions .  Fuquay JW ,  Fox PF ,  McSweeney PLH ,  eds.  _Encyclopedia of Dairy Sciences_ ,  Volume 3 .  2nd ed. Oxford :  Academic Press ;  2011 : 691 \u2013 697 . \nChapter 3\n\n# The Comparative Genomics of Monotremes, Marsupials, and Pinnipeds: Models to Examine the Functions of Milk Proteins\n\nJulie A. Sharp*\n\nAshalyn Watt*\n\nSwathi Bisana*\n\nVengama Modepalli*\n\nStephen Wanyonyi*\n\nAmit Kumar*\n\nJoly Kwek**\n\nRod Collins*\n\nChristophe Lef\u00e8vre*, ***\n\nKevin R. Nicholas*\n\n* School of Medicine, Deakin University, Geelong, Victoria, Australia  \n** Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia  \n*** Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia\n\n## Abstract\n\nMilk has many functions, ranging from the provision of factors crucial to the operation of the mammary gland and the development of the suckled young through to their protection from infection. Lactation evolved about 200 million years ago with the aplacental, egg-laying monotremes, but since that time there has been extensive adaptation to reproduction, including a large repertoire of lactation strategies. This chapter discusses three animal models with extreme adaptation to lactation, and examines the option of exploiting their comparative biology to identify milk protein bioactives that may have potential in functional foods or pharmaceuticals. The echidna (Tachyglossus aculeatus, a monotreme) has a fascinating combination of reptilian and mammalian characteristics. It retains a primitive component of reptilian reproduction in the form of laying shelled eggs, but it also has a prototherian lactation process. The tammar wallaby (Macropus eugenii, a marsupial) has adopted a reproductive strategy that includes a short gestation (26.5 days), birth of an immature young, and a relatively long lactation (300 days). The composition of milk changes progressively during the lactation cycle, and these changes in milk composition subsequently control development of the young. The tammar can also practice concurrent asynchronous lactation; the mother provides a concentrated milk for an older animal that is out of the pouch, and a dilute milk from an adjacent mammary gland for a newborn pouch young. The third study species, the Cape fur seal (Arctocephalus pusilluspusillus, a eutherian), has a lactation characterized by a repeated cycle of long at-sea foraging trips (up to 23 days) alternating with short suckling periods of 2\u20133 days ashore. Lactation almost ceases while the seal is offshore, but the mammary gland does not progress to involution and apoptosis.Technology platforms using genomics, proteomics, and bioinformatics have been used to exploit these models to identify milk bioactives. In addition, the availability of sequenced marsupial, dog, platypus, and bovine genomes permits rapid transfer of information to the cow to provide outcomes for the dairy industry.\n\n## Keywords\n\nExtreme lactation, milk composition, comparative genomics, bioactive, growth and development\n\nOutline\n\nIntroduction 76\n\nThe echidna (Tachyglossus aculeatus) 78\n\nMonotremes: An Animal Model to Discover Antimicrobials 79\n\nThe tammar wallaby (Macropus eugenii) 82\n\nMilk Protein Gene Expression; Naturally Occurring Gene Knockouts Used to Assess Protein Function 82\n\nChanges in Milk Composition Regulate Growth of the Tammar Pouch Young 84\n\nIdentification of Milk bioactives in Tammar Milk Using a Functional Genomics Platform 85\n\nTemporal Delivery of Domain-specific Bioactive Proteins in Wallaby Milk 85\n\nCathelicidins\u2014Domain-specific Delivery of Bioactivity 85\n\nWhey Four-disulfide Core-domain- containing Proteins WAP and WAP Four-disulfide Core Domain Protein 2 88\n\nWFDC2 88\n\nWAP 90\n\nS100A19\u2014A New Member of the S100 Family of Proteins 93\n\nA role for milk in the control of mammary function 95\n\nThe fur seal 96\n\nTranscriptomic Analysis of the Lactating Cape Fur Seal Mammary Gland during Suckling and Foraging 97\n\nAbsence of Local Apoptotic Milk Factors in Fur Seal Milk 99\n\nNew player in milk bioactives; microRNA 103\n\nConclusions 104\n\n## Introduction\n\nThe composition of milk includes all the factors required to provide appropriate nutrition for growth of the neonate. However, it is now clear that milk has many functions and also consists of bioactive molecules that play a central role in regulating developmental processes in the young and providing a protective function for both the suckled young and the mammary gland during milk production (Trott et al., 2003; Waite et al., 2005; Kwek et al., 2009b). Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to meet the demand for foods that improve wellbeing and options for prevention and treatment of disease.\n\nIn addition, human targets for milk bioactives are focused on specific and changing health needs during the progression from infancy to old age (Sharp et al., 2009). For example, the need to support growth and development in early life differs from the maintenance of muscle, eyes, and brain function in the elderly. Health issues such as obesity, diabetes, hypertension, and cancer increase with an aging population. Therefore, functional foods that improve both general nutrition and well-being, and also target prevention and treatment of health problems are very attractive. New animal models with unique reproductive strategies are now becoming increasingly relevant in the search for these factors. This review focuses on three animal models that provide new insights into how the lactation cycle is regulated, and when combined with technology platforms that include genomics, proteomics and bioinformatics can be exploited to identify milk proteins with bioactivity and subsequent application as nutraceuticals and pharmaceuticals.\n\nThe lactation cycle is common to all mammals, although monotremes, marsupials, and some pinnipeds have evolved a reproductive strategy that is very different from that of most eutherians. The evolution of lactation has led to new opportunities to better understand the functionality of milk. The first split in mammalian phylogeny resulted in the Prototheria approximately 200 million years ago and produced the monotreme lineages. Australia is the only continent on which the monotremes, platypus (Ornithorhynchus anatinus), and echidnas (Tachyglossus and Zaglosus genera) are still found. The Theria split into the Metatheria (Marsupialia) and Eutheria (Placentalia) lineages over 150 million years ago. These animal models have an extreme adaptation to lactation and have increased our understanding of the function of milk proteins by revealing mechanisms that are present but often not readily apparent in many eutherian mammals (Brennan et al., 2007; Sharp et al., 2009). Eutherians have a long gestation relative to their lactation period, and the composition of the milk doesn't change substantially. This contrasts with reproduction and lactation in the other two classes of mammals, the monotremes (such as platypus and echidna) and the marsupials (such as the tammar wallaby, Macropus eugenii).\n\nMonotremes do not have a placenta, and they lay eggs that are incubated outside the mother's body. This is followed by a prototherian lactation process, marking them as representatives of early mammals. Apart from a short weaning period (Morrow and Nicol, 2013), milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically na\u00efve (Griffiths, 1978). Since much of the development of the monotreme young occurs in the nonsterile, ex-utero environment, the role of milk is of importance in the growth, development, and protection of the young from disease (Bisana et al., 2013). Reproduction in marsupials is characterized by a short gestation followed by a long lactation period, and all the major milk constituents change substantially and progressively during lactation (Tyndale-Biscoe and Janssens, 1988; Nicholas, 1988a,b). The tammar neonate is altricial and remains attached to the teat for the first 100 days of lactation, and it may be considered a fetus maintained in the pouch as opposed to the uterus. The conversion of milk to body mass is very similar to the conversion of precursors to body mass observed in the eutherian fetus in utero (Tyndale-Biscoe and Janssens, 1988). Therefore, examination of the milk provides a unique opportunity to identify specific molecules that play a primary role in regulating the development of the young. The lactation cycle of the Cape fur seal (Arctocephalus pusillus pusillus) consists of maternal foraging and infant nursing that are spatially and temporally separate (Bonner, 1984). Lactating mothers suckle offspring over a period of 4\u201312 months, and females alternate between short periods ashore suckling their young with longer periods of up to 3\u20134 weeks foraging at sea (Gentry and Holt, 1986). Fur seals provide new opportunities to examine a potential role for milk in protecting the mammary gland from cell death and infection while the lactating animal is foraging, and to examine a function for milk in the growth and protection of the pup that remains on shore.\n\nTransmitting passive immunity through milk is a key factor for survival of the suckled young. Understanding the relationship between the developmental delay of the immune system in the newborn marsupial and the role of the mammary gland in providing immunity for the young to survive in a pathogenic environment provides new opportunities to identify molecules with a role in innate immunity. The transmission of passive immunity is very important until the active immune system has matured in the neonate (Bailey et al., 2001; Butler, 1998; Sinkora et al., 2003; Brambell 1970). This process is defined by the reproduction strategies of mammals and includes the type of placenta and gestation period. The placental system has huge diversity both structurally and physiologically, and fetal\/maternal exchanges have evolved from a primitive form of yolk sac placenta in marsupials to well-developed chorioallantoic placenta in eutherians (Carter et al., 2004). Marsupials have a placenta, but the fetal and maternal interaction is very limited, with minimal transfer of immunity to the embryo. In contrast, in eutherians the embryo is implanted even before the germ layers are formed in the blastocyst and the interactions between the fetus and the mother are more developed (Tyndale-Biscoe and Janssens, 1988). This has extended to support of the fetus for longer periods in utero. This diversity among the mammals in acquiring passive immunity prior to birth has led to wide adaptations in postnatal life in order to survive. If the newborn is unable to acquire any immune protection prior to birth, the mammary gland plays a substantial role in providing the immune protection either through colostrum or mature milk. Therefore, it is likely that the mammary gland has evolved prior to the placenta as monotremes are aplacental (Goldman, 2002) and developed a mammary gland by modifying the epidermal gland on the ventral part of the abdomen to provide secretions to support the offspring (Daniel, 1993).\n\n## The echidna (Tachyglossus aculeatus)\n\nThe monotremes occupy an important position in the mammalian tree of evolution because of their egg-laying mode of reproduction, which is a transition between that of reptiles and therian mammals (Lef\u00e8vre et al., 2010). The extant monotremes, the platypus and echidna, are confined to Australia and New Guinea and show a mixture of specialized adaptations and plesiomorphic features (Bininda-Emonds et al., 2007; Nicol 2003). The short-beaked echidna (Tachyglossus aculeatus) is more widely distributed, occupying a diverse range of habitats from coastal areas to above the snow lines (Griffiths, 1978). The maternal care provided by the mother echidna from fertilization until weaning can be divided into four phases (Fig. 3.1). The 20\u201324 days of gestation forms phase 1 and ends when the egg is laid into the pouch. The mammary glands of the mother hypertrophy and form the margins of the rudimentary pouch during the last two weeks of gestation and shortly before laying the egg (Griffiths, 1978). The egg is incubated inside the pouch for about 10.5 days (phase 2). Following hatching, the young is carried in the pouch and is able to suckle continuously (phase 3). Unlike other mammals, the platypus does not have a defined nipple, and the young consumes milk from a milk pad (Griffiths et al., 1988). In phase 4, the young is evicted from the pouch and sequestered in a nursery burrow, where the mother returns to feed it at intervals of 5\u201310 days (Griffiths et al., 1988; Rismiller and McKelvey 2003). The duration of lactation is about 200 days and the young leave the burrow after 180 to 205 days. Key differences in maternal care during the first weeks of lactation have been observed between the geographical subspecies of echidnas. Weaning in the Tasmanian populations of echidnas is reported not to be abrupt as there is a period when the young echidnas begin exploratory foraging while their mothers are still lactating (Morrow and Nicol, 2013).\n\nFigure 3.1 Stages of echidna development.\n\nAlthough the composition of monotreme milk has not been extensively studied, some of the main components have been described, such as caseins (Lef\u00e8vre et al., 2009) and whey proteins including alpha-lactalbumin (Messer et al., 1997; Shaw et al. 1993), lysozyme (Guss et al., 1997), and whey acidic protein (WAP) (Sharp et al., 2007b). Platypus milk is reported to contain more solids than most eutherian milk, as well as having a high iron content (Griffiths et al., 1984; Grant 2007). The composition of milk secreted by the echidna mother changes over time. At the time of birth, the milk is dilute and contains 1.25% fat, 7.85% protein, and 2.85% carbohydrates and minerals. Mature milk is more concentrated, with 31.0% fat, 12.4% protein, and 2.8% carbohydrate (Augee et al., 2006). Near weaning, protein levels continue to increase, which is thought to be due to the need for keratin synthesis for hair and spines.\n\nIn addition to providing general nutrition, monotreme milk is a potential source to discover new antimicrobials. The protective properties of milk in a range of mammals have been described, including the antiparasitic activity of human milk lipase (Gillin et al., 1983), antifungal activity of bovine milk lactoferrin (Manzoni et al., 2012), and bacteriostatic activity of murine milk WAP (Iwamori et al., 2010). Egg laying, incubation, and hatching of monotreme eggs, and the subsequent development of the monotreme young occurs in nonsterile ex-utero environments, which requires that milk provide both the source of nutrition and antibacterial protection (Griffiths, 1978). It has often been speculated that during reproduction in monotremes, the protolacteal secretions enhanced survival of eggs and the young by virtue of their antimicrobial properties (Oftedal 2002; ; Blackburn et al., 1989; Hayssen and Blackburn 1985).\n\n### Monotremes: An Animal Model to Discover Antimicrobials\n\nA recent study using genomics and proteomics platforms identified a novel, monotreme-specific antibacterial milk protein (Bisana et al., 2013). Since the monotremes are protected species and access to their tissue samples is limited, a noninvasive approach for analysis of lactation was employed (Lef\u00e8vre et al., 2009). Echidna milk cells were harvested from milk collected during peak lactation, and cDNA sequencing identified several casein and whey protein genes expressed at high levels, indicating that monotreme milk cells harvested during lactation were enriched for mammary epithelial cells (Fig. 3.2). A novel, monotreme-specific transcript named EchAMP was identified during this analysis (Bisana et al., 2013). An exploration of EchAMP expression relative to GAPDH in different tissues of echidna using Reverse transcription polymerase chain reaction (RT-PCR) further confirmed a high level of expression in milk cells and a low level of expression in other tissues.\n\nFigure 3.2 Echidna milk cell transcriptome. Relative abundance of the EchAMP transcript in echidna milk cells. A novel sequence (EchAMP) was one of the most highly expressed transcripts.\n\nIn silico analyses indicated that the cognate protein of EchAMP would be hydrophilic and secretory in nature. This was confirmed through identification of peptides corresponding to EchAMP protein in a sample of echidna milk (Bisana et al., 2013). The presence of three consecutive sequences in the platypus genome showing homology to EchAMP cDNA with high percentage identity provided supporting evidence for the presence of an orthologous EchAMP gene in platypus, designated as PlatAMP. However, the expression of the PlatAMP gene in the mammary\/milk cells of the platypus still requires confirmation.\n\nWhile characterizing the functional attributes of EchAMP, in silico analyses indicated that EchAMP contained an overall significant alpha-helical structure, suggestive of antimicrobial activity (Boman, 2003; Lai and Gallo 2009). This was confirmed by predictions from the Antimicrobial Peptide Database (Wang and Wang, 2004). Similarly, the predicted post-translational modification of EchAMP, especially the presence of a mucin-type GalNAc O-glycosylation site, also suggested that EchAMP protein may carry antimicrobial activity. To confirm this hypothesis, a recombinant EchAMP protein was synthesized by transfecting HEK293T cells with a c-Flag pcDNA3-EchAMP construct. The protein was secreted into conditioned media (Bisana et al., 2013). The media was analyzed for antibacterial activity with Gram positive and Gram negative bacteria (Fig. 3.3). Significant antibacterial activity was observed against the pathogenic bacteria E. coli, S. enterica, P. aeruginosa, and Staphylococcus spp, which have been reported to be present in monotremes (McOrist and Smales, 1986; Munday et al., 1998). However, there was no antibacterial activity against the Gram positive commensal bacterium Enterococcus faecalis, which is reported to be one of the predominant harmless commensals found in the gastrointestinal tract in a diverse range of species, including humans, marsupials, and most other vertebrates (Fanaro et al., 2003; Yadav et al., 1972; Witte et al. 1999). Mastitis is the most common infection in the mammary gland, and Staphylococcus aureus, E. coli, and Streptococcus spp. have been frequently isolated in conditions of human and bovine mastitis (Barkema et al., 2009; Borm et al., 2006; Bradley and Green, 2001; Oeding 1952). Taken together, it appears that EchAMP, a novel monotreme-specific milk protein, is capable of conferring protection to the underdeveloped, immunologically na\u00efve young outside the uterus. The monotreme mammary gland lacks nipples, and the altricial young is therefore more likely  to ingest pathogens while suckling compared with other species. We propose that the evolution of nipples and development of offspring in utero in the placental mammals (Metatherian and Eutherian) could have led to the loss of selective pressure for the preservation of this gene. This could explain its subsequent disappearance in these species. For example, there is a precedence of the selective loss of genes involved in gastric function in the platypus, which diverged from the Therian lineage early during mammalian evolution. Notwithstanding the high conservation in vertebrates for more than 200 million years, genes encoding the gastric proteinases, hormone gastrin, both the subunits of the gastric H+\/K+-ATPase and the neurogenin-3 transcription factor have either been deleted or inactivated in the platypus genome, giving rise to physiological differences in digestion between monotremes and therians (Ordonez et al., 2008).\n\nFigure 3.3 Antibacterial assays illustrating bacteriostatic activity using (A) E. coli, (B) Salmonella enterica 43971, (C) Staphylococcus aureus 29213, (D) Staphylococcus aureus 25923, (E) Staphylococcus epidermidis, (F) Pseudomonas aeruginosa 27853, and (G) Enterococcus faecalis 10100. * indicates a statistically significant result (P<0.05). Each assay was performed in triplicate, and the experiments were repeated at least three times. Standard error bars are indicated.\n\n## The tammar wallaby (Macropus eugenii)\n\n### Milk Protein Gene Expression; Naturally Occurring Gene Knockouts Used to Assess Protein Function\n\nLactation in the tammar wallaby has been divided into phases that are defined by the composition of milk and apparent sucking pattern of the young (Nicholas et al. 1995; ) (Fig. 3.4). Phase 1 comprises a 26.5-day pregnancy followed by parturition, and the subsequent 200 days of phase 2 are characterized by lactogenesis and the secretion of small volumes of dilute milk high in complex carbohydrate and low in fat and protein. The pouch young (PY) remains attached to the teat for approximately the first 100 days (phase 2A), after which it relinquishes permanent attachment to the teat and sucks less frequently while remaining in the pouch (phase 2B). The onset of phase 3 of lactation (200\u2013330 days) is characterized by temporary exit from the pouch by the young, a large increase in milk production and a change in the composition of milk to include elevated levels of protein and lipid and low levels of carbohydrate. There is a progressive increase in protein concentration (Nicholas, 1988a,b) and protein production during lactation, and the composition of the proteins changes considerably (Nicholas, 1988a; Simpson et al., 2000; Trott et al. 2002). Interestingly, macropodids such as the tammar can practice concurrent asynchronous lactation; the mother provides a concentrated milk high in protein and fat for an older animal which is out of the pouch, and a dilute milk low in fat and protein but high in complex carbohydrate from an adjacent mammary gland for a newborn PY (Nicholas, 1988a,b) (Fig. 3.5). This phenomenon suggests that the mammary gland is controlled locally, and more recent studies (Wanyonyi et al., 2013) have shown that mammary extracellular matrix (ECM) together with endocrine stimuli regulates the expression of the major milk protein genes specific to each phase of lactation.\n\nFigure 3.4 The lactation cycle of the tammar wallaby. (a) Development of the pouch young from day 6 to day 220 of age. (b) The lactation cycle in the tammar has been divided into four phases characterized by changes in milk composition and the sucking pattern of the pouch young. (c) Expression of the major milk protein genes during the lactation cycle. The \u03b2-casein, \u03b1-casein, \u03b1-lactalbumin, and \u03b2-lactoglobulin genes are induced at parturition, and expression remains elevated for the entire lactation. The genes encoding ELP (early lactation protein), WAP (whey acidic protein), and the LLPs (late lactation proteins A and B) are expressed only for specific phases of the lactation cycle.\n\nFigure 3.5 Concurrent asynchronous lactation. The pouch has been retracted to expose the four mammary glands. A 6-day-old young is attached to a teat from a mammary gland secreting phase 2A milk. An older animal at approximately 275 days of age has vacated the pouch and sucks from the elongated teat which provides phase 3 milk from the enlarged mammary gland. The remaining two teats are from quiescent mammary glands.\n\nTo provide more detailed information on changes in milk protein during lactation in the tammar, and to confirm earlier studies describing changes in milk protein gene expression (Bird et al., 1994; Simpson et al., 2000; Trott et al. 2002), we developed a custom tammar mammary EST (expressed sequence tag) array printed with 10,000 cDNAs representing approximately 7000 genes expressed across the lactation cycle. This array was used to transcript profile the mammary gland at all the major stages of pregnancy and lactation. There were two temporally different patterns of milk protein gene expression during the lactation  cycle: One group of genes is induced to high levels around parturition and is expressed throughout lactation, and a second group of genes is expressed highly only during specific phases of lactation (Sharp et al., 2009; Simpson and Nicholas 2002). For example, the genes for the whey proteins \u03b2-lactoglobulin, trichosurin, and \u03b1-lactalbumin, and the \u03b2-casein and \u03b1-casein genes were induced coordinately around the time of parturition, and were expressed for the duration of lactation (Sharp et al., 2009). In contrast, the early lactation protein (ELP) gene was expressed at very high levels in phase 2A; the WAP gene was most highly expressed in phase 2B; and a gene coding for an outlier lipocalin protein referred to as late lactation protein A (LLP-A) was highly expressed in phase 3 (Sharp et al., 2009). A second LLP gene referred to as LLP-B was not on the microarray, but was first expressed after 200 days of lactation (Trott et al., 2005). Whereas some milk proteins may be specific to the tammar, in most cases orthologues of the proteins are found in other species (Simpson and Nicholas, 2002). As a result, the tammar is now emerging as a valuable model to provide a 'temporal gene knockout' which allows for a more accurate assessment of the role of the gene product in regulating either a specific stage of development of the young or mammary function during each phase of the lactation cycle.\n\n### Changes in Milk Composition Regulate Growth of the Tammar Pouch Young\n\nA study by Trott et al. (2003) examined the hypothesis that the sucking pattern of the PY controls the pattern of milk secretion in the tammar wallaby. To test this hypothesis, groups of 60-day-old PY were fostered at 2-week intervals onto one group of host mothers so that a constant sucking stimulus on the mammary gland was maintained for 56 days. This allowed the lactation stage to progress ahead of the age of the young. Analysis of the milk in fostered and control groups showed that the timing of changes in the concentration of protein and carbohydrate was unaffected by altering the sucking pattern. However, rate of growth and development of the foster PY was significantly increased relative to control PY. This most likely resulted from the foster PY ingesting more milk of higher energy content and milk with a composition that was approximately 50 days more advanced than normally consumed at their age (Trott et al., 2003; Waite et al., 2005; Kwek et al. 2009b). It was concluded from these studies that the lactating tammar wallaby regulates both milk composition and the rate of milk production, and these determine the rates of PY growth and development regardless of the age of the PY. The tammar therefore allows studies that correlate milk with a specific composition, particularly the milk proteins, with defined developmental stages of the suckled young.\n\nSubsequent experiments (Kwek et al., 2009b; Waite et al., 2005) extended the studies of Trott et al. (2003) and examined a role for milk in specific changes in the development of the gut of the tammar PY. Parietal cells positive for the alpha subunit of the H + \/K + -ATPase pump were present in both the forestomach and hindstomach of PY at 168 days of age. However, at 260 days of age parietal cells were seen in sections from the hindstomach but were no longer detectable in the forestomach (Kwek et al., 2009b). Experiments where a PY was fostered to a mother at a more advanced stage of lactation showed that this loss of parietal cells was accelerated. However, it is likely that colonization of the gut also has an important role in this process (Kwek et al., 2009b).\n\n### Identification of Milk bioactives in Tammar Milk Using a Functional Genomics Platform\n\nSome bioactives are required continuously for the development of the young, whereas others have a role at specific stages of lactation. Therefore, it is likely that their pattern of secretion can be matched to the developmental profile of the suckled young. A genomics platform may be used to identify the differentially expressed genes that code for a secreted protein (Sharp et al., 2009). This approach used the tammar microarray to identify the differentially expressed genes in the tammar mammary gland that code for secreted proteins. These proteins were synthesized in vitro and examined for activity in cell-based assays for immune modulation, inflammatory responses, growth, and differentiative effects (Sharp et al., 2009). At least 30 of these proteins had at least one of these bioactivities. In addition, some of the proteins showed the capacity to alter the development of three-dimensional structures of tammar mammary epithelial cells in a mammosphere culture model. This raises the possibility that these proteins may also contribute to growth and function of the mammary gland during lactation.\n\n### Temporal Delivery of Domain-specific Bioactive Proteins in Wallaby Milk\n\nApart from the major lactation phase markers, tELP, tWAP, tLLPB-A, and tLLP-B, the wallaby appears to deliver domain-specific bioactive peptides selectively throughout lactation so that specific parts of multifunctional genes are expressed only when they are required (Nicholas et al., 2012). As discussed earlier, a genomics approach has been used to identify differentially expressed genes that were later shown to perform an array of functions (Sharp et al., 2009). The genomics data were corroborated by a later study that used a variety of proteomics tools, including matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and de novo peptide sequencing to identify a mix of proteins associated with various functions, including antimicrobial defense, growth, and development in tammar milk (Joss et al., 2009). This study identified lactoferrin, complement B protein, haptocorrin, amphiphysin, and myeloid cathelicidin (among other proteins), all of which were delivered in milk at specific times during lactation. Lactoferrin and cathelicidin are well-known antimicrobial proteins, while haptocorrin has also been shown to have bacteriocidal activity against E. coli (Adkins and Lonnerdal, 2003). All of them were expressed in phase 2A and phase 2B (Joss et al., 2009). Amphiphysin, which plays a role in endocytosis (Butler et al., 1997), was found in milk produced from days 172\u2013250 of lactation, while complement B protein, which plays a role in pathogen opsonization (Peters et al., 1988) was detected in phase 2B. The temporal delivery of these proteins by the tammar mother may therefore be able to be exploited to assist in developing a better understanding of the function and role of the bioactives in milk for the development of the young and protection of the mammary gland.\n\n### Cathelicidins\u2014Domain-specific Delivery of Bioactivity\n\nCathelicidins are a well-studied family of antimicrobial proteins found in a wide variety of vertebrates (Boman, 2003; Hancock and Lehrer, 1998; Lehrer and Ganz, 1999; Zanetti et al., 1995; Rollins-Smith et al., 2002; Zasloff 2002). Together with many other cationic amphiphilic peptides collectively known as host-defense peptides (Hancock et al., 2006; Nijnik and Hancock 2009), cathelicidins represent a significant constituent of innate immunity in mammals. They display a remarkably broad spectrum of antipathogenic activity, with a single cathelicidin often showing activity against bacteria, fungi, and viruses (Ramanathan et al., 2002).\n\nThe generic primary structure of cathelicidins includes four exons (Fig. 3.6A; Gudmundsson and Agerberth, 1999; Gudmundsson et al., 1995; Zhao et al. 1995), the first three of which consist of the 22\u201335 amino acid N-terminal signal peptide and the cathelin-like region that covers amino acids 90\u2013115 (Yang et al., 2004). The fourth exon consists of the variable C-terminal antimicrobial domain preceded at its N-terminal by a species-specific proteinase cleavage site (Gudmundsson et al., 1996; Zanetti et al. 1991). All cathelicidins have a net positive charge of 2\u20139, and a high percentage of hydrophobic residues that confer the amphipathic properties upon which their antimicrobial activity is dependent (Hancock and Lehrer, 1998).\n\nFigure 3.6 Tammar MaeuCath1 structure and function. (A) Schematic diagram of the cathelicidin gene structure, including the signal-peptide, cathelin-like region and the variable C-terminal antimicrobial region (AMP). The molecular sizes of the respective regions of MaeuCath1 are indicated. (B) MaeuCath1 exon structure. MaeuCath1a includes all four exons, while MaeuCath1b includes exons 1 and 2 and the 3\u2032 untranslated region. Shaded boxes represent the translated sequence, while unshaded boxes indicate spliced-out exons. (C) An agarose gel showing differential expression of MaeuCath1a and MaeuCath1b genes during wallaby lactation. NP = not pregnant, P = day 16 pregnant, Early = phase 2A lactation, Mid = phase 2B lactation and Late = phase 3 lactation. Involution 1, 5, and 10 represents the number of days post-weaning. Samples in each phase were loaded onto the gel in order of increasing days of lactation. (D) Antibacterial activity of MaeuCath1-derived peptides, Con73 and Con218 directed to S. aureus. The bactericidal activity of the peptides was determined relative to the untreated control and is expressed as the relative bacterial survival (y-axis). Control cells were treated with BSA. (E) Cell proliferation of wallaby mammary epithelial cells (Wall-MEC) following treatment with MaeuCath1. Vector represents conditioned media collected after transfection with empty plasmid. FL MaeuCath1 and MaeuCath1b represent full length Maeucath1 and MaeuCath1b conditioned media, respectively. Control cells were treated with BSA.\n\nThe sequence heterogeneity of the antimicrobial domain between species has been proposed to be an adaptation to the extremely diverse pathogenic challenges for the various host animals. This point of view gains credence from the tendency in mammals to have more than one cathelicidin gene (Castiglioni et al., 1996; Daly et al., 2007; Wang et al. 2011b; Wu et al., 1999), each presumably targeting different microbes. There is increasing evidence that cathelicidins also play significant physiological roles in mammals (Yang et al., 2004). Cathelicidins have been shown to play chemotactic roles (Agerberth et al., 2000; Huang et al. 1997), neutralize endotoxins (Golec, 2007), enhance re-epithelialization of dermal wounds (Yang et al., 2004), and modulate angiogenesis (Li et al., 2000). The secretion of cathelicidins in milk (Joss et al., 2009) must therefore raise questions as to whether milk performs multiple roles in the young and perhaps the mammary gland of the mother.\n\nAlthough eight cathelicidin genes, MaeuCath1-8, have previously been reported in the wallaby (Daly et al., 2009), only MaeuCath1 has been investigated for its function in neonatal development. MaeuCath1 is expressed in the spleen, liver, skin, bone marrow, thymus, and gut of the PY (Daly et al., 2007; Lef\u00e8vre et al., 2007) and the adult mammary gland (Lef\u00e8vre et al., 2007; Khalil et al. 2008).\n\nA recent study showed that MaeuCath1 and MaeuCath7, both of which are secreted in wallaby milk, have antimicrobial activity in their variable C-terminal portion and the capacity to enhance mammary epithelial cell proliferation in the cathelin domain (Fig. 3.6E; Wanyonyi et al., 2011). Synthetic peptides Con73 and Con218, designed from the heterogeneous C-terminal portion of MaeuCath1 and MaeuCath7, respectively, were shown to have bactericidal activity against S. aureus, Pseudomonas aureginosa, Salmonella enterica and E. faecalis (Fig. 3.6D). This is consistent with the need to provide antimicrobial protection to the young before it develops its own immunity (Daly et al., 2007). However, the ability of MaeuCath peptides to kill S. aureus also points to a possible role in the protection of the lactating mammary gland against mastitis agents (Oikonomou et al., 2012).\n\nInitially, the bactericidal activity of MaeuCath peptides against E. faecalis was somewhat paradoxical, because E. faecalis is a commensal of the gut and its clearance would perturb the gut microfloral balance. However, upon examining the expression profile of MaeuCath1, it became evident that its delivery through milk is temporally regulated through alternate splicing (Fig. 3.6B,C), so that the antimicrobial domain (MaeuCath1a) is only expressed in early lactation when it is required for protection of the immune-compromised neonate (Daly et al., 2007) and at involution when the mammary gland of the mother would be vulnerable to potential mastitic infections (Sordillo et al., 1987; Williams and Patnode 1948). In this way, the mother provides broad-spectrum antimicrobial molecules through milk without disturbing the microfloral balance of the neonate's gut.\n\nDuring involution (when MaeuCath1a is highly expressed) a large number of immune cells converge in the mammary gland, similar to a wound-healing environment (Schedin et al., 2007; Findlay 1982). Accompanying the complex milieu of pro-inflammatory activities is an elevated risk of mastitic infections (Sordillo et al., 1987; Williams and Patnode 1948) and a possible release of endotoxins due to the bactericidal activity of immune cells. Interestingly, cathelicidins have been shown to play a role in the different levels of immune response, including regulation of immune genes (Yang et al., 2004), chemotaxis of immune cells (Ciornei et al., 2005), and neutralization of endotoxins (Bhunia et al., 2009). It is therefore conceivable that MaeuCath1a plays these roles in the involuting mammary gland.\n\nApart from the possible roles in immune response, MaeuCath1a may participate in the process of involution by directly inducing apoptosis of epithelial cells, as has been demonstrated for BMAP-28, a bovine cathelicidin. BMAP-28 induces apoptosis through the initiation of the mitochondrial permeability transition pore (MPT pore) and the consequent release of cytochrome c (Risso et al., 2002). Although the mechanism by which BMAP-28 initiates MPT is not well understood, this role is likely to link it to a number of signaling pathways related to mitochondria-mediated apoptosis. BMAP-28 has been shown to selectively kill transformed and proliferating lymphocytes relative to normal nonproliferating cells through MPT (Zanetti, 2004). If MaeuCath1a has pro-apoptotic properties similar to BMAP-28, it would be interesting to investigate for the possibility of it having the ability to selectively kill transformed cells and having a role in the inhibition of tumor progression.\n\nMaeuCath1b is expressed during peak lactation (Nicholas et al., 1997), and, in addition to playing a proliferative role, may induce a number of physiological processes in the mammary gland, similar to the porcine cathelicidin PR-39. In a wound-healing environment, PR-39 has been thought to induce cell proliferation via the expression of syndecans (Bernfield et al., 1992), a family of heparan sulfate proteoglycans known for regulating cell adhesion and blood coagulation, among other functions. The syndecans effect cell proliferation by sensitizing cells to growth factors, including the transforming growth factor-\u03b2 (TGF-\u03b2), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (Bernfield et al., 1992; Zanetti 2004). The possibility of MaeuCath1b inducing cell proliferation through the syndecans would therefore link Maeucath1b to multiple TGF-\u03b2-regulated physiological processes, including angiogenesis, extracellular matrix neogenesis, and immune suppression (Ijichi et al., 2004). However, although this proposition is feasible, it is also somewhat controversial because in normal epithelial cells TGF-\u03b2 signals apoptosis instead of proliferation (Massague, 2000). Nevertheless, the possible role of Maeucath1b in mammary growth during peak lactation is worth further investigation.\n\n### Whey Four-disulfide Core-domain-containing Proteins WAP and WAP Four-disulfide Core Domain Protein 2\n\nWAP and WAP four-disulfide core domain protein 2 (WFDC2) are members of a larger family of whey four-disulfide core (WFDC) proteins (Fig. 3.7). The WFDC genes are composed of multiple four-disulfide core (4-DSC) domains that consist of a core of eight cysteine residues in a conserved arrangement that form intramolecular disulfide bonds (Campbell et al., 1984; Bingle 2011).\n\nFigure 3.7 The 4-disulfide domain structure of WFDC2, marsupial WAP, eutherian WAP, and monotreme WAP from platypus and echidna. Modified from Sharp et al. (2007b).\n\n### WFDC2\n\nWFDC2 is a secreted protein that consists of two 4-DSC domains. Tammar wallaby WFDC2 comprises two 4-DSC domains, which have previously been annotated domain III on the amino terminal end and domain II at the carboxyl terminal end (Sharp et al., 2007b). WFDC2 also shows a similar structure in monotremes and eutherians, and consists of domains III and II (Watt et al., 2012) (Fig. 3.7). This conservation of the WFDC2 domain structure between all species suggests that these proteins may have similar conserved functions.\n\nThe WFDC2 gene was first identified in epididymis and was later found expressed in the oral cavity (Bingle et al., 2002; Kirchhoff et al., 1991; Idoji et al. 2008). More recently, the WFDC2 gene has been identified in the mammary gland of the tammar wallaby during lactation (Sharp et al., 2007b). There is limited information on the biological activity of the WFDC proteins and their domains, with the exception of the secretory leukocyte peptidase inhibitor (Hiemstra, 2002; Zhu et al. 2002), Elafin (Tomee et al., 1997), and Eppin (Yenugu et al., 2004), all of which have been shown to function as antibacterial, anti-inflammatory, antiviral, and antiproteinase proteins (Moreau et al., 2008; Williams et al. 2006).\n\nThe WFDC2 gene expression profile in the tammar wallaby mammary gland during the lactation cycle showed relatively low WFDC2 gene expression in the nonpregnant wallaby mammary gland, but elevated expression in pregnancy (phase 1) and early lactation (phase 2A). The WFDC2 gene was downregulated in midlactation (phase 2B) and subsequently increased toward the end of late lactation (phase 3) and into involution (Watt et al., 2012) (Fig. 3.8).\n\nFigure 3.8 Expression profiles of WAP and WFDC2. RNA was extracted from tammar wallaby mammary tissue at the indicated stages of pregnancy and lactation, and RT-PCR was used to show the expression profile with GAPDH as a housekeeping gene. WFDC2 and WAP densitometry values were normalized against the GAPDH densitometry values to show relative gene expression. Modified from Watt et al. (2012).\n\nThe WFDC2 protein and domain II of the protein have been found to have antibacterial activity against S. enterica, P. aeruginosa, and S. aureus (Fig. 3.9). However, these proteins demonstrated no activity against E. faecalis (Watt et al., 2012). The WFDC2 domain III protein showed no significant antibacterial activity, clearly demonstrating that antibacterial activity correlated with domain II of WFDC2, and that this domain accounted for the strain-specific activity\n\nFigure 3.9 Antibacterial activity of WFDC2. Cultures of S. aureus and S. enterica with recombinant WFDC2 protein and WFDC2 domain II protein showed significant inhibition of growth of the bacteria. Recombinant WFDC2 DIII protein showed no significant inhibition of growth when compared to the empty vector (control, no protein) treatment, P<0.05. Bacteriostatic assays of Enterococcus faecalis with WFDC2 protein, WFDC2 domain II protein and WFDC2 domain III protein all showed no significant inhibition of growth of the bacteria when compared to the empty vector (control, no protein) (P>0.05). Modified from Watt et al. (2012).\n\nThe expression pattern of WFDC2 at specific times during the tammar reproductive strategy was similar to Maeucath1b, and relates to times when the function of the protein is required. For example, WFDC2 expression during pregnancy, early lactation (phase 2A), and in involution correlated with stages of the lactation cycle when the mammary gland has increased risk of infection (Basden et al., 1997; Daly et al., 2007; Old and Deane 2000). The most common infection in the mammary gland during lactation is mastitis, which is typically caused by bacterial infection from S. aureus, E coli, and Streptococcus spp. in human and bovine mastitis (Barkema et al., 2009; Borm et al., 2006; Bradley and Green 2001). Mastitis in the tammar wallaby is yet to be observed, which may be explained by the bioactive antimicrobial proteins expressed in the milk. Elevated expression of the WFDC2 gene in early lactation also suggests a role of immune protection to the developing PY when it does not have a fully functioning immune system (Basden et al., 1997).\n\nCommensal microbial flora in the gut of the tammar PY, predominately E. faecalis and E. coli (Yadav et al., 1972), was observed at 50 days of age (phase 2A). Interestingly, WFDC2 is expressed when microbial flora is present, and while WFDC2 antibacterial activity is targeted to S. aureus, S. enterica, and P. aeruginosa, it seems that WFDC2 may selectively contribute to the absence of problematic bacterial strains in the gut of the developing young while having no activity against the commensal E. faecalis.\n\n### WAP\n\nWAP is a secreted protein found only in milk. The WAP found in the milk of eutherians has two 4-DSC domains annotated DI-DIIA (Fig. 3.7) (Sharp et al., 2007b; Simpson and Nicholas, 2002). Not all species express WAP; in humans and in ruminants, WAP is a pseudogene due to a frameshift mutation (Clauss et al., 2002; Hajjoubi et al. 2006). In marsupials studied to date, WAP has three domains annotated DIII-DI-DIIB (De Leo et al., 2006; Demmer et al., 2001; Simpson et al. 2000). The monotreme WAP from platypus has three 4-DSC domains like marsupials, but with the annotation of DIII-DIIA-DIIB (Fig. 3.7). However, the monotreme echidna WAP comprises two 4-DSC domains DIII-DIIA (Sharp et al., 2007b). The differences between the number of domains present in different species and the domain annotation could suggest that WAP from different species may differ in its role and function.\n\nThe WAP gene-expression profile in the tammar wallaby mammary gland during the lactation cycle showed relatively low WAP gene expression in the nonpregnant wallaby mammary gland, which continued into pregnancy (phase 1) and early lactation (phase 2A). The WAP gene was upregulated in mid lactation (phase 2B) and then became downregulated in late lactation (phase 3) and into involution (Fig. 3.8).\n\nEarlier studies have shown that mouse WAP added to culture medium with mouse HC-11 cells (a mouse mammary epithelial cell line) is antiproliferative and may act by an autocrine or paracrine mechanism (Nukumi et al., 2004). This is consistent with reports showing that overexpression of WAP in transgenic mice inhibits development of the mammary gland and secretion of milk (Burdon et al., 1991). Studies in our laboratory have used in vitro models to examine the effect of tammar WAP on proliferation of an epithelial-enriched population of tammar mammary cells. In contrast to the inhibitory action of mouse WAP on proliferation of HC-11 cells, results show that tammar WAP or domain III added to culture media stimulates proliferation of primary wallaby mammary epithelial cells (Wall-MEC) and increases expression of the cell-cycle gene cyclin D1. Interestingly, a protein construct comprised of DI-II, which more closely resembles the 2-domain eutherian WAPs, did not have any effect on the proliferation of Wall-MEC (Topcic et al., 2009) (Fig. 3.10). Previous studies have shown that DNA synthesis in mammary tissue is higher in phase 2 than phase 3 (Nicholas, 1988b), which is consistent with a potential role of tammar WAP in the development of the mammary gland. This is further supported by our studies showing that tammar WAP stimulates DNA synthesis in primary cultures of mammary epithelial cells, most likely by upregulating the expression of cyclin genes known to be essential for development of the mammary gland (Fig. 3.10) (Topcic et al., 2009). Our analyses of the expression of G1-phase cyclins demonstrated that expression of tWAP and DIII in HC11 and Wall-MEC cells significantly upregulated the expression of cyclin D1 and CDK-4 genes compared to control HEK293 cells (Topcic et al., 2009). This increase in gene expression supports the argument that tammar WAP is a positive regulator of cell-cycle progression of mammary epithelial cells in culture by the regulation of cyclin D1 and CDK-4 expression. It is likely that evolution has adapted the structure and function of the protein and the expression pattern of the gene, which presumably relates to changes in the reproductive strategies of marsupials and eutherians.\n\nFigure 3.10 Proliferative activity of WAP. Proliferation assay using primary wallaby mammary epithelial cells. tWAP or the DIII significantly increased cell number after 72 and 96 hours in culture P<0.05 when compared to the empty vector control, whereas DI-II showed no significant increase in proliferation P>0.05. Expression of the cyclin D1 gene in Wall-MEC cells grown in the presence and absence of tammar WAP (day 3 post-treatment). Expression of the cyclin D1 and GAPDH genes was determined by RT-PCR analyses. The relative expression levels of the cyclin D1 gene in Wall-MEC was quantified by NIH. Image presented as a proportion of the housekeeping gene GAPDH. Modified from Topcic et al. (2009).\n\nStudies using a WAP gene knockout mouse have shown that mammary development is normal. However, the pups had limited development at the later stages of lactation (Triplett et al., 2005). As tammar WAP is a major milk protein secreted during the middle third of lactation (phase 2B) when the PY's diet comprises of only milk, it has been speculated that this protein plays a specific role in development of both the mammary gland and the suckled young at this time. It is likely that a putative function for WAP in mammary development is predominantly found only in marsupials and monotremes, and the activity has been lost in humans and ruminants due to the reduction in evolutionary pressure related to changes in the reproductive strategy of eutherians.\n\n### S100A19\u2014A New Member of the S100 Family of Proteins\n\nThe S100 proteins are calcium-binding proteins implicated in governing diverse intracellular and extracellular processes such as contraction, motility, cell growth, differentiation, cell-cycle progression, gene transcription, protein secretion, and antimicrobial function (Donato 1999; ; Donato et al., 2013). A new member of the S100 family has been identified, which is expressed in both gut and mammary gland tissue of the tammar wallaby (Kwek et al., 2013). Initially, S100A19 was identified from a subtracted cDNA library in an effort to identify genes that were differentially regulated between the tammar forestomach and hindstomach (Kwek et al., 2009a). This differential expression pattern was later confirmed by Northern blot analysis showing that S100A19 was expressed in the immature forestomach at a time when it displays a gastric phenotype, but expression is downregulated as the stomach matures into a fermentation sac (Fig. 3.11) (Kwek et al., 2013). This change in gut phenotype corresponds to a time when the diet of the young transitions from milk only to a diet that is supplemented with herbage (Kwek et al., 2009a).\n\nFigure 3.11 Expression analysis of S100-A19 gene during the tammar wallaby stomach development.   \n(A) Northern blot analysis of S100A19 gene expression in tissue isolated from the developing forestomach (F) and hindstomach (H) at days 140, 190, 230, 260, and adult tammar wallabies. (B) Northern blot analysis of S100A19 expression in tissue isolated from the developing forestomach at days 65, 142, 168, 180, 205, 260, and adult tammar wallabies. Analysis shows the presence of a single transcript (625 bp) that increases in expression from day 65 to day 180 and then declines at day 205. S100A19 is no longer expressed from day 230 of age onward.\n\nS100A19 expression in the tammar wallaby mammary gland was also found to be differentially regulated (Kwek et al., 2013). S100A19 is upregulated during pregnancy and involution, but not expressed during the milk production phase of lactation (Fig. 3.12). Comparison of the tammar wallaby S100A19 protein sequence with S100 protein sequences from eutherian, monotreme, and other marsupial species suggests that the marsupial S100A19 has two functional EF hand domains and an extended His tail. It also has close similarity to S100A7 and S100A15 proteins, which have been shown to have antibacterial activity (Kwek et al., 2013; Buchau et al., 2007; Glaser et al. 2005). Human S100A8, S100A9, and S100A12 have been shown to be expressed in the inflamed gastric mucosa of children infected with Helicobacter pylori, but absent in the normal gut, suggesting that these proteins may be involved in the host response at this site (Leach et al., 2008). The regulated differential expression patterns of S100A19 in the tammar wallaby suggest that S100A19 may play a role in gut development, a process that differs between metatherians and eutherians, and\/or possesses potential antibacterial properties to establish the correct flora and protect against bacterial infection in the immature forestomach. In the mammary gland, S100A19 may act to protect the tissue from infection at times of vulnerability during the lactation cycle.\n\nFigure 3.12 Expression analysis of S100A19 across the tammar wallaby lactation cycle.   \nQuantitative RT-PCR was used to determine levels of S100A19 gene expression at different phases of lactation. One of the milk proteins, \u03b2-lactoglobulin (BLG) gene expression, was also measured and used as the lactation marker gene. The pregnancy period (P), lactation period, and involution period (I) consist of 26, 350, and approximately 10 days, respectively. Data represent mean \u00b1 S.E.M. from three animals.\n\n## A role for milk in the control of mammary function\n\nThere is increasing evidence to suggest that milk plays an important role in regulating mammary epithelial function and survival, and this is particularly evident during involution (Brennan et al., 2007). Apoptosis was induced preferentially in the sealed teats of lactating mice (Li et al., 1997; Marti et al. 1997), while the litter suckled successfully on the remaining teats, which indicates that cell death is stimulated by an intramammary mechanism sensitive to milk accumulation (Quarrie et al., 1996). A protein known as the feedback inhibitor of lactation (FIL) has been suggested as a candidate and is secreted in the milk of the tammar (Hendry et al., 1998) and other species. It acts specifically through interaction with the apical surface of the mammary epithelial cell to reduce secretion (Wilde et al., 1995).\n\nMore recent studies using the tammar mammary explant culture model (Nicholas and Tyndale-Biscoe, 1985) to examine the process of involution have confirmed the likely role of milk, and particularly putative autocrine factors, for controlling mammary function during involution (Brennan et al., 2007). Mammary explants from pregnant tammars, cows, and mice were cultured for three days with insulin, cortisol, and prolactin to induce milk protein gene expression. To mimic involution, all hormones were subsequently removed from culture medium for 10 days to downregulate expression of the milk protein genes. Surprisingly, the explants retained the same level of response during a subsequent challenge with lactogenic hormones. Previous studies have shown that there is limited secretion of milk proteins from tammar mammary explants, but it is unlikely that milk constituents accumulated to elevated concentrations (Nicholas and Tyndale-Biscoe, 1985; McFadden, 1997). The maintenance of epithelial cell viability and hormone responsiveness in explants cultured in the absence of hormones is consistent with a more active mechanism, such as the accumulation of local factors in the milk, being the primary stimulus for apoptosis of mammary epithelial cells in the tammar wallaby mammary gland. This model permits the uncoupling of hormone- and milk-regulated involution. A primary outcome of these studies is evidence for the extraordinary capacity for survival and maintenance of hormone responsiveness by tammar mammary epithelial cells cultured in a chemically defined medium with no exogenous hormones and growth factors.\n\nIt is likely that milk components will play a major role in the shape of the lactation curve in dairy cattle following peak lactation at approximately 20 weeks of milk production. The lactation cycle in dairy cattle includes a period of increasing milk yield in early lactation followed by a steadily declining yield for the remainder of lactation. The amount of milk produced during lactation is determined by the peak yield and persistency of lactation. Milk production is largely a function of the number and activity of secretory cells in the udder that decline significantly between the time of peak yield and late lactation. Therefore, it follows that approaches to address the decline in milk yield and lactation persistency after peak lactation must involve changes to the frequency of apoptosis in mammary secretory cells. Endocrine treatment of cattle with bovine sommatotrophin increases milk yield, but in many cases persistency is not altered. Furthermore, although moderate heritability of persistency suggests that selection for this trait is possible, it is achieved at the cost of milk yield. Increased frequency of milking has been shown to increase milk yield, suggesting that the mammary gland has a local intrinsic resistance to regression. Identification of the milk components that impact significantly on mammary cell fate may provide new approaches for strategies to improve lactation persistency.\n\n## The fur seal\n\nThe three families of Pinnipeds (also known as fin-footed mammals) evolved from a carnivorous ancestor around 25 million years ago and diverged into the Phocids (true seals), Odobenids (walrus), and Otariids (sea lions, fur seals) during the middle Miocene (10 million years ago) (Fordyce, 2002). Each group adopted a different approach to lactation. Phocid seals evolved large body sizes to increase body reserves and reduce heat loss and risk of predation, enabling them to adopt a 'fasting strategy' during lactation (Oftedal et al., 1987). In contrast, the small ancestral otariid seals retained smaller body sizes and insulating fur, breeding at rockeries to gain proximity to local prey resources and adopting a 'foraging lactation' strategy (Bonner, 1984). The smaller size of otariid seals made it necessary to feed during lactation in order to replenish the body stores required to continue milk production. Reduced prey availability contributed to lengthening the lactation period (4 to >12 months), and otariid seals began exploring resources farther off shore, increasing the duration but reducing the frequency of foraging trips during lactation. The current-day otariid seals have adopted a lactation strategy that is characterized by alternation between periods of several days of copious milk production on shore and extended periods of maternal foraging at sea. Intersuckling intervals have been recorded in otariid seals of up to 23 days and are among the longest ever recorded for a mammal. The need to increase the duration of foraging trips due to distant foraging grounds during lactation has selected for an adapted otariid mammary gland, which remains functional despite sustained interruptions in suckling activity. This type of lactation is unique among mammals due to the extreme duration of intersuckling bouts and the rapid rate of energy transfer while suckling, and results in a slow growth rate of the otariid pups (Oftedal et al., 1987) (Fig. 3.13).\n\nFigure 3.13 The 'foraging' lactation strategy of the fur seal. The pregnant female arrives at shore to give birth and remains with the pup for approximately one week. For the remainder of lactation (10\u201312 months), females alternate trips to the sea with short trips ashore to suckle their pup.\n\nFur seal milk is considered among the most nutritious mammalian milk and is composed of 30\u201360% fat, 5\u201315% protein, and around 40% water (Oftedal and Iverson, 1995). The high-energy content of fur seal milk allows for a short and rapid period of energy transfer from mother to pup. Interestingly, fur seal milk is devoid of lactose (Dosako et al., 1983; Urashima et al. 2001b), an osmole that normally regulates the water content of milk (Oftedal and Iverson, 1995).\n\nStudies on milk production and mammary gland function in fur seals have shown that during a foraging bout, the lactating mammary gland produces less milk compared to that of the onshore lactating female, but does not progress to involution. For example, milk production in Antarctic fur seals (A. gazella) has been shown to continue while the female is foraging at sea, but at only 19% of the rate of production on land (Arnould and Boyd, 1995). During onshore lactation, the mammary alveoli of the Cape fur seal appear engorged with milk containing a large amount of lipid (Fig. 3.14a,b) (Sharp et al., 2006b). In contrast, during the mother's extended foraging trip, the alveoli appear less distended, epithelial cells surrounding the alveoli appear columnar, and the lipid component is decreased within the milk, consistent with a gland that is producing less milk. During natural weaning, in most mammals the alveoli fill with milk due to cessation of suckling, with a resulting decline in milk protein gene expression in the mammary epithelial cells that causes the epithelium to regress and enter involution (Li et al., 1997). This process is characterized by apoptotic cell loss and mammary gland remodeling (Lund et al., 1996; Metcalfe et al., 1999; Strange et al., 1992; Walker et al. 1989). Apoptosis associated with involution in the mammary gland of the foraging seal has been analyzed and found to be barely detectable. Even after extended periods when there is no sucking stimulus, the gland does not regress (Sharp et al, unpublished).\n\nFigure 3.14 Histological sections of the mammary gland from seals (a) lactating while nursing onshore and (b) lactating while foraging at sea. Sections are stained with hematoxylin and eosin. Immature alveoli in the pregnant gland are present in lipid (white) and milk protein (grey) and are indicated in the onshore and offshore mammary glands. Magnification x100. (c), \u03b2-casein expression during Cape fur seal lactation cycle. Analysis of expression using canine Affymetrix chips hybridized to cDNA probes generated from RNA from pregnant (placental gestation and nonlactating, n = 2); lactating on shore (n = 2) and lactating at sea (n = 1) (animals in embryonic diapause) Cape fur seals. (d) Cluster analysis of gene-expression profiles from the Cape fur seal mammary gland during different stages of lactation. A total of 1020 Cape fur seal mammary messenger RNA (mRNA) transcripts were identified with expression levels above an intensity of 250 in any sample type. Hierarchical clustering was conducted using euclidean distance. Pregnant and onshore lactating data represent an average of two animals. Offshore data represent a single sample.\n\nAs mentioned previously, one candidate factor involved in the regulatory mechanism of milk production is a milk protein termed FIL, which acts within hours to downregulate milk production (Peaker et al., 1998; Wilde et al. 1987). FIL is a heat-labile protein secreted by the mammary gland and present in the whey fraction of milk which, when exposed to mammary cells for an extended period, acts to block translation of milk protein transcripts and inhibiting secretion of milk proteins by binding a putative receptor in the apical surface of the epithelial cells (Blatchford et al., 1998). Fractionated fur seal milk has demonstrated a FIL-like activity at a similar level to that reported for other species (Cane et al., 2005). It is predicted that FIL may play a role in the downregulation of milk volume in the fur seal mammary gland during foraging to assist in the prevention of prolonged engorgement.\n\n### Transcriptomic Analysis of the Lactating Cape Fur Seal Mammary Gland during Suckling and Foraging\n\nThe transcript profile of the Cape fur seal (Arctocephalus pusillus pussillus) lactation cycle has been analyzed using Affymetrix canine arrays to examine the pattern of gene expression in mammary tissue during pregnancy, onshore lactation, and offshore lactation (Fig. 3.14c) (Sharp et al., 2006a). High-sequence conservation between the Cape fur seal and dog, 95% similarity at the DNA level (Sharp et al., 2006a), permits a significant detection rate of measurable hybridization signals between seal cDNA and the Affymetrix Canine microarray.\n\nCluster analysis of expression profiles from microarray data has revealed that the overall expression profile of lactating mammary gland of the foraging Cape fur seal is more closely related to the profile of pregnant nonlactating animals than the profile obtained from onshore lactating animals (Fig. 3.14d). This suggests that the interruption of lactation in foraging animals involves a major reprogramming of mammary gland expression. This analysis also shows that the acute immune response is upregulated in the mammary gland of the foraging seal, presumably to protect the mammary gland from infection during the extended foraging trip while milk remains in the gland (Sharp et al., 2007a). Gene-expression profiles associated with survival and preservation of tissue architecture were also either maintained or upregulated, preventing degradation of mammary tissue. These global gene-expression data suggest that the immune and acute phase responses observed in the mouse at involution (Clarkson and Watson, 2003), and mimicked in the mammary gland of the foraging fur seal, are independent of the apoptotic phase of involution.\n\nAnalysis of individual genes showed that the reduced rate of milk production was controlled at a transcriptional level by a reduction in milk protein gene-expression profiles (Sharp et al., 2006a). For example, expression of \u03b2-casein showed a significant downregulation of expression in the mammary gland of a foraging animal and correlated with decreased milk volume (Fig. 3.14c). This pattern of expression has been confirmed by RT-PCR and is also observed for other milk protein genes such as \u03b1S2-casein, \u03ba-casein, and \u03b2-lactoglobulin. The fur seal mammary gland therefore undergoes repeated cycling of high milk protein gene expression which correlates with high milk production while the pup is suckling, and low milk protein gene expression that correlates with low milk production in the absence of suckling. It has been postulated that the absence of lactose and subsequent production of milk with low water content may prevent extensive distension of the mammary gland during the long foraging trip (Reich and Arnould, 2007). However, this seems unlikely as fur seal milk is devoid of lactose during both onshore and offshore milk production and milk is not viscous. Instead, we hypothesize that the prevention of overdistension of alveoli in the fur seal mammary is achieved by preventing milk accumulation via downregulation of milk protein gene expression (Sharp et al., 2006a), which in turn lowers milk volume. This mechanism prevents the physiochemical signaling triggers that normally lead to involution upon milk accumulation.\n\n### Absence of Local Apoptotic Milk Factors in Fur Seal Milk\n\nFor other mammals, the accumulation of milk in the mammary gland due to cessation of sucking by the young allows factors present within milk to initiate involution, leading to mammary gland remodeling (Li et al., 1997). In vivo studies, such as teat sealing experiments in mice and goats, have demonstrated the importance of local factors controlling mammary gland involution at weaning (Li et al., 1997; Theil et al. 2005). The effects of local milk factors have also been observed in lactating goats, showing reduced cell number and increased apoptosis in individual glands with infrequent milk removal for several weeks; within those glands, apoptotic cells were concentrated in smaller regressing alveoli (Li et al., 1999). Effects due to alveoli distention were discounted as mammary epithelium was not duly compromised by stored milk (Wilde et al., 1999). Conversely, frequent milking of cows during early lactation has also shown an increase in milk production, which is proposed to be due to removal of these local factors (Wall and McFadden, 2007). The presence of local milk apoptotic factors in the mammary gland is therefore likely to act as a stimulus to initiate involution and also may cause a decline in lactation persistency during normal lactation.\n\nThe absence of sucking during the period of fur seal maternal foraging results in milk remaining within the gland. Apoptotic factors present in milk of other species would be predicted to cause involution. However, this does not occur, and fur seal mammary glands remain functional and alveoli remain intact, as demonstrated by apoptotic staining of mammary tissue by Apotag (Chemicon) (Sharp, Hornby, Nicholas, unpublished data). The absence of involution at this time suggests that the local trigger of involution that is present in milk of other species may be absent or limited in the milk of the fur seals. In addition, milk secretion in fur seals does not follow the pattern of secretion of most other mammals, where peak milk secretion tends to occur at the start of lactation. Studies on the amount of milk volume produced in fur seals suggests that the mother secretes larger volumes of milk as lactation progresses in order to meet the needs of her growing young. This observation may also suggest an absence of a local trigger of involution in fur seal milk.\n\nThe \u03b1-lactalbumin protein, which is required for lactose synthesis and is secreted in milk, has also been implicated in the process of apoptosis (Baltzer et al., 2004; Hakansson et al. 1999; ). The \u03b1-lactalbumin protein plays a central role in the mammary gland as the regulatory subunit of lactose synthase (Lonnerdal and Lien, 2003) and as a potential apoptotic milk factor (Baltzer et al., 2004). A multimeric form of \u03b1-lactalbumin (MAL) isolated from the casein fraction of human milk has previously been shown to localize to the nucleus and cause apoptosis in selected cell lines (Hakansson et al., 1999). Similarly, HAMLET (human \u03b1-lactalbumin made lethal to tumor cells) (Svensson et al., 2002), a complex of apo-\u03b1-lactalbumin combined with oleic acid, has been shown to localize to the nucleus, where it binds to histones, disrupts chromatin structure, and leads to cell death (Duringer et al., 2003). However, negatively charged \u03b1-lactalbumin can also bind to positively charged histones without the aid of oleic acid, causing aggregation (Permyakov et al., 2004). Native bovine \u03b1-lactalbumin has also been found to have an antiproliferative effect on human colon adenocarcinoma cell lines which were dose and time dependent (Sternhagen and Allen, 2001). Affymetrix analysis has shown that \u03b1-lactalbumin is expressed at extremely low levels in the fur seal mammary gland compared to expression levels in other species during lactation; however, the fur seal \u03b1-lactalbumin transcripts are not translated into secreted protein (Sharp et al., 2008). This finding supports previous observations that were unable to detect \u03b1-lactalbumin or lactose in fur seal milk (Dosako et al., 1983; Johnson et al., 1972; Messer et al., 1988; Schmidt et al., 1971; Urashima et al. 2001a). Analysis of \u03b1-lactalbumin function showed that \u03b1-lactalbumin had apoptotic effects on a variety of cell types. These apoptotic effects were also observed when seal mammary cells were exposed to \u03b1-lactalbumin, suggesting that the \u03b1-lactalbumin-induced apoptotic pathway is still functional in fur seal mammary cells (Sharp et al., 2008). These observations were also seen for mammary cells derived from other species such as the cow and mouse. It is speculated that the absence of \u03b1-lactalbumin in fur seal milk contributes to the lack of apoptosis in the mammary gland of lactating seals during foraging. Therefore, the pseudogenization of the \u03b1-lactalbumin gene has enabled the adoption of the current lactation strategy of the fur seals, facilitating avoidance of involution while undertaking long foraging bouts.\n\nIn addition to regulating the amount of milk produced by the mammary gland during foraging, transcriptome analysis has shown that the Cape fur seal mammary gland also upregulates a number of genes involved in maintaining cell structure during foraging compared to the level of expression during lactation on shore (Sharp et al., 2007a). This response involves a >2-fold upregulation of the Na-dependent transporter of taurine (TAUT), which is involved in maintaining cell volume (Christensen et al., 2005) and may act to control the viscosity of milk, and the ZO-1\u2013associated Y-box factor (ZONAB), which is involved in regulating cell density and cell proliferation (Balda et al., 2003).\n\nGlobal gene-expression analysis during the fur seal lactation cycle has also revealed that the foraging fur seal mammary gland fails to downregulate cell survival genes, a phenomenon that usually occurs during first stage of involution. For example, Ank3, Mcl1, Bcl2, and BclXL have been shown to be downregulated in the involuting mouse mammary gland (Clarkson et al., 2004). However, these genes showed no change in expression between the mammary glands of onshore lactating and at-sea foraging fur seals (Fig. 3.15).\n\nFigure 3.15 Expression of involution-related genes in the mammary gland during the fur seal lactation cycle. Genes were grouped according to ontological category. The mean intensity for each category was plotted. Analysis shows no changes in expression of genes involved in promotion of   apoptosis (P53, p21\/WAF1, Daxx, Bax) and   cell survival (Ank3, Mcl1, Bcl2, and BclXL) in the transition between onshore lactation (shore) and foraging (sea). However, induction of immune-related genes, such as   immunoglobulins (IgA, IgJ and IgC),   complements factors (C1qa, C1qc, C1r, C1s, and C3a) and   acute phase response (SAA3) is observed. Genes involved in maintenance of   cell structure (TAUT and ZONAB) are also shown to be upregulated in the foraging fur seal mammary gland (sea) compared to the onshore lactating gland (shore). There are also a set of genes,   sea-specific genes (unannotated hypothetical genes), which are specifically upregulated in the mammary gland of foraging ('sea') seals. Values shown on the y-axis represent average intensity values from canine Affymetrix analysis for each group. The x-axis shows mammary gland tissue type collected at different stages of Cape fur seal lactation. 'Pregnant' represents pregnant mammary gland (n = 2), 'Shore' are samples collected from the mammary gland of onshore-lactating Cape fur seals (n = 2), and 'Sea' is a single sample collected from the lactating mammary gland of a seal caught at sea more than 100 km from the nearest colony.\n\nPro-apoptotic genes, such as Bax, shown to be upregulated in the second phase of involution in mice (Clarkson et al., 2004; Lund et al. 1996), also showed no change in expression in the foraging fur seal (Fig. 3.15), suggesting apoptosis is also not initiated by this pathway. Another pro-apoptotic gene, Daxx, an activator of apoptosis in mice (Yang et al., 1997), was also examined and showed reduced expression in the mammary gland of seals at sea. This finding suggested that any apoptosis that may be mediated in the mammary gland by Daxx is actively prevented during foraging. The absence of expression of these pro-apoptotic genes indicates that apoptosis by these pathways is not initiated in the fur seal mammary gland during foraging, and is consistent with earlier observations showing a lack of apoptotic activity in intact mammary gland tissue from these animals (Sharp et al., 2006a).\n\nOther genes associated with this second stage of involution include downregulation of the tissue inhibitors of matrix metalloproteinases (TIMPs) TIMP-1 and TIMP-3 (Fata et al., 2001). Tissue remodeling during the second phase is largely dependent on matrix metalloproteinases (MMPs), which act to remodel the extracellular matrix and stroma within the gland. MMPs are maintained in the gland in an inactive state by TIMPs during the first phase of involution. The ratio of MMP to TIMP expression is critical for determining the two phases of involution (Talhouk et al., 1992). In order for the first phase of involution to maintain its reversibility, MMPs are not activated until day 3 of involution. TIMP expression is high in the first phase, but as involution progresses, TIMP levels decline, leading to activation of MMPs which then remodel the extracellular matrix and stroma (Lund et al., 1996). Upregulation of the TIMP-3 gene has been observed in microarray analysis of mice involution at 96 hours (Clarkson and Watson, 2003), and TIMP-3 deficient mice show an accelerated involution process, indicating that TIMP-3 is necessary to control MMP activity during the first stage of involution (Fata et al., 2001). During fur seal lactation on shore, TIMP-3 is expressed at high levels (5200 intensity units) and is maintained at a high level while at sea (3400 intensity units), suggesting no significant change in expression of TIMP-3. TIMP-1 is expressed at a lower level while lactation occurs on shore (164 intensity units), and expression is elevated 2.3 fold (371 intensity units) while out foraging. Expression of the MMP-2 and the serine proteinase urokinase-type plasminogen activator (uPA), which are normally low during lactation in mice, are strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution (Lund et al., 1996). These genes are expressed at very low levels in the mammary gland of fur seals during lactation on shore and at sea (<100 intensity units), and upregulation of gene expression was absent in the mammary gland of the foraging fur seal. Maintenance of high levels of TIMP-3 and elevated expression of TIMP-1 combined with generally low levels of MMP-2 may help to maintain the stoichiometry between MMPs and TIMPs and prevent involution while on a foraging trip. Indeed, the characteristic upregulation of TIMP-3 expression during the typical first phase of involution was not observed in the foraging fur seal, suggesting that the pathway associated with TIMP and MMP expression is not initiated. This is consistent with the preservation of alveoli architecture observed in mammary tissue of foraging fur seals (Sharp et al., 2006a).\n\nWithin 72\u201396 hours post forced weaning in mice, a sharp rise in immunoglobulin gene expression has been observed (Clarkson and Watson, 2003). It is unknown what purpose these immunoglobulins have during involution. It has previously been suggested that the vast amount of apoptosis associated with involution leads to a localized immunoglobulin response. However, the fur seal also shows a very high level of immunoglobulin expression (IgA, IgJ, and IgC) during foraging at sea in the absence of apoptosis, suggesting that the immunoglobulins are not expressed as a result of apoptosis. Complement factor genes (C1qa, C1qc, C1r, C1s, and C3a) are also upregulated in the mammary gland of the foraging fur seal when compared to the mammary gland of the onshore fur seal. These genes have been observed to be upregulated in the mouse involution mammary gland and are proposed to mark monocyte and lymphocyte attraction to the site (Clarkson et al., 2004). It is unknown why these cells would be attracted to the foraging fur seal mammary gland, but it may be the result of an inflammatory response for prevention of mastitis during milk stasis. The acute phase response gene, serum amyloid A (SAA), was also seen to be upregulated in the mammary gland of the foraging fur seal (Fig. 3.15). SAA is induced during clinical mastitis in cows (Eckersall et al., 2006; Weber et al., 2006) and during involution (Clarkson et al., 2004). Expression of SAA in the fur seal at this time may act to protect the mammary gland from infection. The sea environment may provide adequate protection against bacterial infection for the teat, which is the usual path of mastitis infection.\n\nA number of other genes are specifically upregulated in the mammary gland of the foraging fur seal (Fig. 3.15). These include a number of unannotated hypothetical genes that may act as potential mediators for prevention of involution. These genes are yet to be studied; however, the expression of these genes due to other factors such as disease cannot as yet be ruled out.\n\nThis unique capacity to inhibit apoptosis associated with involution, to maintain mammary epithelial cells in a viable and hormone-responsive state, and to stimulate mammary gland growth to increase milk production during the lactation cycle provides new opportunities to further identify the mechanisms controlling these events. A thorough understanding of the genetic and\/or local factors in the residual milk regulating this process may have applications for improving lactation persistence in the dairy cow, either by improved breeding programs or by manipulation of milk factors.\n\n## New player in milk bioactives; MicroRNA\n\nMicroRNAs (miRNAs) are a class of 18\u201326 nucleotide small noncoding RNAs that regulate post-transcriptional inhibitory effects on gene expression. They are thought to provide a new level of regulation directed to fine-tuning expression of the genome in a tissue- and time-specific manner. MiRNAs show stage-specific expression (Pigati et al., 2010) and are established mediators of mammary gland development (Avril-Sassen et al., 2009). Recent studies have found miRNAs in high concentrations in milk (Chen et al., 2010); these miRNAs are most concentrated in milk exosomes (Zhou et al., 2012). The presence of miRNA in bovine milk provides new opportunities for use as biomarkers for milk quality (Chen et al., 2010) and potentially as markers of udder function. In addition to the cow, miRNAs have been found in milk from a variety of animals, including goat, pig, yak (Li et al., 2012; Gu et al., 2012; Bai et al. 2013), and human (Zhou et al., 2012). Milk miRNAs appear to be very stable and withstand harsh environmental conditions, including prolonged storage at room temperature, multiple freeze-thaw, RNAse digestion, and boiling (Chen et al., 2010; Bail et al. 2010). Studies with human breast milk have shown that miRNAs were able to withstand highly acidic conditions (pH 1) for 1 hour (Kosaka et al., 2010). An interesting report has shown that exogenous miRNAs consumed from plant foods may pass across the gut and directly influence gene expression in animals (Zhang et al., 2012). These observations suggest that miRNA consumed in the diet may have a putative role in mammary gland physiology, together with signaling potential for bioactive effects on growth and development of the young. Rapid advances in miRNA sequencing combined with the option of exploiting mammalian lactation diversity, provide a platform to address the role of miRNA during lactation.\n\n## Conclusions\n\nThis review has described three animals with extreme adaptation to lactation, and explored approaches to exploit their unique reproductive strategies to identify new proteins in milk with bioactivity that have the potential to regulate mammary function and growth of the suckled young. In contrast, the lactation cycle in most eutherian mammals is characterized by the initiation of lactation around parturition and the production of milk in which the individual components vary little during the entire period of lactation. For example, in the dairy cow, evolution and selection pressure has led to a lactation cycle that does not include significant changes in the secretory pattern of the milk proteins. Therefore, identification of new protein bioactive molecules in the dairy cow requires a broad screening program and will most likely identify molecules in milk that are secreted at low and unchanged levels during lactation. In contrast, in the tammar, echidna, platypus, and fur seal the temporal pattern of secretion of a milk bioactive that is correlated with either specific developmental phases of the suckled young or altered mammary function is more likely to indicate a cause-and-effect relationship, and these factors are more likely to have increased commercial value. Monotremes and marsupials are particularly interesting as their genomes have evolved under evolutionary pressure to protect immunologically na\u00efve young with broad spectrum antibiotics (Wang et al., 2011a). This presents a potential opportunity to discover new antimicrobial proteins. In addition, it may be possible to exploit the specificity of these naturally occurring antibacterial proteins in the dairy industry to help treat infections such as mastitis as an alternative to synthesizing new antibiotics. The increasing availability of sequenced genomes in public databases has underpinned increased interest in comparative genomics and bioinformatics, and our results using the echidna, tammar, and fur seal models have led to the identification of many bovine and human orthologues of proteins with bioactivity. Conceptually, it is very likely that the relevance and use of species with an extreme adaptation to reproduction or environmental pressure will become increasingly attractive for improved understanding of the genetic regulation of physiological processes.\n\n# References\n\nAdkins Y ,  Lonnerdal B .  Potential host-defense role of a human milk vitamin B-12-binding protein, haptocorrin, in the gastrointestinal tract of breastfed infants, as assessed with porcine haptocorrin in vitro .  _Am J Clin Nutr_.  2003 ;77 : 1234 \u2013 1240 .\n\nAgerberth B ,  Charo J ,  Werr J ,  Olsson B ,  Idali F ,  Lindbom L ,  Kiessling R ,  Jornvall H ,  Wigzell H ,  Gudmundsson GH .  The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations .  _Blood_.  2000 ;96 : 3086 \u2013 3093 .\n\nArnould JPY ,  Boyd IL .  Temporal patterns of milk production in Antarctic fur seals (Arctocephalus gazella) .  _Journal of Zoology_.  1995 ;237 : 1 \u2013 12 .\n\nAugee M ,  Gooden B ,  Musser A .  _Echidna: Extraordinary Egg-Laying Mammal_ .  CSIRO Publishing, Collingwood, Victoria, Australia. ;  2006 .\n\nAvril-Sassen S ,  Goldstein LD ,  Stingl J ,  Blenkiron C ,  Le Quesne J ,  Spiteri I ,  Karagavriilidou K ,  Watson CJ ,  Tavare S ,  Miska EA ,  Caldas C .  Characterisation of microRNA expression in post-natal mouse mammary gland development .  _BMC Genomics_.  2009 ;10 : 548 .\n\nBai WL ,  Yin RH ,  Yang RJ ,  Khan WA ,  Ma ZJ ,  Zhao SJ ,  Jiang WQ ,  Wang ZY ,  Zhu YB ,  Luo GB ,  Zhao ZH .  Technical note: Identification of suitable normalizers for microRNA expression analysis in milk somatic cells of the yak (Bos grunniens) .  _J Dairy Sci_.  2013 ;96 : 4529 \u2013 4534 .\n\nBail S ,  Swerdel M ,  Liu H ,  Jiao X ,  Goff LA ,  Hart RP ,  Kiledjian M .  Differential regulation of microRNA stability .  _RNA_.  2010 ;16 : 1032 \u2013 1039 .\n\nBailey M ,  Plunkett FJ ,  Rothk\u00f6tter H-J ,  Vega-Lopez MA ,  Haverson K ,  Stokes CR .  Regulation of mucosal immune responses in effector sites .  _Proceedings of the Nutrition Society_.  2001 ;60 : 427 \u2013 435 .\n\nBalda MS ,  Garrett MD ,  Matter K .  The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density .  _J Cell Biol_.  2003 ;160 : 423 \u2013 432 . \nBaltzer A ,  Svanborg C ,  Jaggi R .  Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin .  _Cell Mol Life Sci_.  2004 ;61 : 1221 \u2013 1228 .\n\nBarkema HW ,  Green MJ ,  Bradley AJ ,  Zadoks RN .  Invited review: The role of contagious disease in udder health .  _J Dairy Sci_.  2009 ;92 : 4717 \u2013 4729 .\n\nBasden K ,  Cooper DW ,  Deane EM .  Development of the lymphoid tissues of the tammar wallaby Macropus eugenii .  _Reprod Fertil Dev_.  1997 ;9 : 243 \u2013 254 .\n\nBernfield M ,  Kokenyesi R ,  Kato M ,  Hinkes MT ,  Spring J ,  Gallo RL ,  Lose EJ .  Biology of the syndecans: A family of transmembrane heparan sulfate proteoglycans .  _Annu Rev Cell Biol_.  1992 ;8 : 365 \u2013 393 .\n\nBhunia A ,  Mohanram H ,  Bhattacharjya S .  Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy .  _Biopolymers_.  2009 ;92 : 9 \u2013 22 .\n\nBingle CD .  Towards defining the complement of mammalian WFDC-domain-containing proteins .  _Biochem Soc Trans_.  2011 ;39 : 1393 \u2013 1397 .\n\nBingle L ,  Singleton V ,  Bingle CD .  The putative ovarian tumour marker gene HE4 (WFDC2), is expressed in normal tissues and undergoes complex alternative splicing to yield multiple protein isoforms .  _Oncogene_.  2002 ;21 : 2768 \u2013 2773 .\n\nBininda-Emonds ORP ,  Cardillo M ,  Jones KE ,  MacPhee RDE ,  Beck RMD ,  Grenyer R ,  Price SA ,  Vos RA ,  Gittleman JL ,  Purvis A .  The delayed rise of present-day mammals .  _Nature_.  2007 ;446 : 507 \u2013 512 .\n\nBird PH ,  Hendry KA ,  Shaw DC ,  Wilde CJ ,  Nicholas KR .  Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii) .  _J Mol Endocrinol_.  1994 ;13 : 117 \u2013 125 .\n\nBisana S ,  Kumar S ,  Rismiller P ,  Nicol SC ,  Lef\u00e8vre C ,  Nicholas KR ,  Sharp JA .  Identification and functional characterization of a novel monotreme\u2014Specific antibacterial protein expressed during lactation .  _PLoS One_.  2013 ;8 : e53686 .\n\nBlackburn DG ,  Hassen V ,  Murphy CJ .  The origin of lactation and the evolution of milk: A review with new hypotheses .  _Mamma Rev_.  1989 ;19 : 1 \u2013 26 .\n\nBlatchford DR ,  Hendry KA ,  Wilde CJ .  Autocrine regulation of protein secretion in mouse mammary epithelial cells .  _Biochem Biophys Res Commun_.  1998 ;248 : 761 \u2013 766 .\n\nBoman HG .  Antibacterial peptides: basic facts and emerging concepts .  _Journal of Internal Medicine_.  2003 ;254 : 197 \u2013 215 .\n\nBonner WN .  Lactation strategies in pinnipeds: problems for a marine mammalian group .  _Symp Zool Soc London_.  1984 ;51 : 253 \u2013 272 .\n\nBorm AA ,  Fox LK ,  Leslie KE ,  Hogan JS ,  Andrew SM ,  Moyes KM ,  Oliver SP ,  Schukken YH ,  Hancock DD ,  Gaskins CT ,  Owens WE ,  Norman C .  Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers .  _J Dairy Sci_.  2006 ;89 : 2090 \u2013 2098 .\n\nBradley AJ ,  Green MJ .  Aetiology of clinical mastitis in six Somerset dairy herds .  _Vet Rec_.  2001 ;148 : 683 \u2013 686 .\n\nBrambell FWR .  _The transmission of passive immunity from mother to young_ .  Amsterdam, NY :  North-Holland Pub. Co.; American Elsevier ;  1970 .\n\nBrennan AJ ,  Sharp JA ,  Lef\u00e8vre C ,  Topcic D ,  Auguste A ,  Digby M ,  Nicholas KR .  The tammar wallaby and fur seal: Models to examine local control of lactation .  _J Dairy Sci, 90 Suppl_.  2007 ;1 : E66 \u2013 75 .\n\nBuchau AS ,  Hassan M ,  Kukova G ,  Lewerenz V ,  Kellermann S ,  Wurthner JU ,  Wolf R ,  Walz M ,  Gallo RL ,  Ruzicka T .  S100A15, an antimicrobial protein of the skin: regulation by E. coli through toll-like receptor 4 .  _J Invest Dermatol_.  2007 ;127 : 2596 \u2013 2604 .\n\nBurdon T ,  Wall RJ ,  Shamay A ,  Smith GH ,  Hennighausen L .  Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype .  _Mech Dev_.  1991 ;36 : 67 \u2013 74 .\n\nButler JE .  Immunoglobulin diversity, B-cell and antibody repertoire development in large farm animals .  _Revue Scientifique et Technique De L 'Office International Des Epizooties_.  1998 ;17 : 43 \u2013 70 .\n\nButler MH ,  David C ,  Ochoa GC ,  Freyberg Z ,  Daniell L ,  Grabs D ,  Cremona O ,  De Camilli P .  Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin\/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle .  _J Cell Biol_.  1997 ;137 : 1355 \u2013 1367 .\n\nCampbell SM ,  Rosen JM ,  Hennighausen LG ,  Strech-Jurk U ,  Sippel AE .  Comparison of the whey acidic protein genes of the rat and mouse .  _Nucleic Acids Res_.  1984 ;12 : 8685 \u2013 8697 .\n\nCane K ,  Arnould JPY ,  Nicholas KR .  Characterisation of proteins in the milk of fur seals .  _Comparative Biochemistry and Physiology B_.  2005 ;141 : 111 \u2013 120 .\n\nCarter AM ,  Enders AC ,  Kunzle H ,  Oduor-Okelo D ,  Vogel P .  Placentation in species of phylogenetic importance: The Afrotheria .  _Anim Reprod Sci_.  2004 ; : 82 \u2013 83 : 35\u201348 .\n\nCastiglioni B ,  Scocchi M ,  Zanetti M ,  Ferretti L .  Six antimicrobial peptide genes of the cathelicidin family map to bovine chromosome 22q24 by fluorescence in situ hybridization .  _Cytogenet Cell Genet_.  1996 ;75 : 240 \u2013 242 .\n\nChen X ,  Gao C ,  Li H ,  Huang L ,  Sun Q ,  Dong Y ,  Tian C ,  Gao S ,  Dong H ,  Guan D ,  Hu X ,  Zhao S ,  Li L ,  Zhu L ,  Yan Q ,  Zhang J ,  Zen K ,  Zhang CY .  Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products .  _Cell Res_.  2010 ;20 : 1128 \u2013 1137 .\n\nChristensen ST ,  Voss JW ,  Teilmann SC ,  Lambert IH .  High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts .  _Cell Biol Int_.  2005 ;29 : 347 \u2013 351 .\n\nCiornei CD ,  Sigurdardottir T ,  Schmidtchen A ,  Bodelsson M .  Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37 .  _Antimicrob Agents Chemother_.  2005 ;49 : 2845 \u2013 2850 .\n\nClarkson RW ,  Watson CJ .  Microarray analysis of the involution switch .  _J Mammary Gland Biol Neoplasia_.  2003 ;8 : 309 \u2013 319 .\n\nClarkson RW ,  Wayland MT ,  Lee J ,  Freeman T ,  Watson CJ .  Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression .  _Breast Cancer Res_.  2004 ;6 : R92 \u2013 R109 .\n\nClauss A ,  Lilja H ,  Lundwall A .  A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein .  _Biochem J_.  2002 ;368 : 233 \u2013 242 .\n\nDaly KA ,  Digby M ,  Lef\u00e8vre C ,  Mailer S ,  Thomson P ,  Nicholas K ,  Williamson P .  Analysis of the expression of immunoglobulins throughout lactation suggests two periods of immune transfer in the tammar wallaby (Macropus eugenii) .  _Vet Immunol Immunopathol_.  2007 ;120 : 187 \u2013 200 .\n\nDaly KA ,  Mailer SL ,  Digby MR ,  Lef\u00e8vre C ,  Thomson P ,  Deane E ,  Nicholas KR ,  Williamson P .  Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells stimulated with lipopolysaccharide and lipoteichoic acid .  _Vet Immunol Immunopathol_.  2009 ;129 : 36 \u2013 48 .\n\nDaniel GB .  Lactation: Historical Patterns and Potential for Manipulation .  _Journal of Dairy Science_.  1993 ;76 : 3195 \u2013 3212 .\n\nDe Leo A ,  Lef\u00e8vre C ,  Topcic D ,  Pharo E ,  Cheng JF ,  Frappell P ,  Westerman M ,  Graves J ,  Nicholas K .  Isolation and characterization of two whey protein genes in the Australian dasyurid marsupial the stripe faced dunnart .  _(Sminthopsis macroura) Cytogenetic and Genome Res_.  2006 ;115 : 62 \u2013 69 .\n\nDemmer J ,  Stasiuk SJ ,  Grigor MR ,  Simpson KJ ,  Nicholas KR .  Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula) .  _Biochim Biophys Acta_.  2001 ;1522 : 187 \u2013 194 .\n\nDonato R .  Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type .  _Biochim Biophys Acta_.  1999 ;1450 : 191 \u2013 231 .\n\nDonato R .  Intracellular and extracellular roles of S100 proteins .  _Microsc Res Tech_.  2003 ;60 : 540 \u2013 551 .\n\nDonato R ,  Cannon BR ,  Sorci G ,  Riuzzi F ,  Hsu K ,  Weber DJ ,  Geczy CL .  Functions of S100 proteins .  _Curr Mol Med_.  2013 ;13 : 24 \u2013 57 .\n\nDosako S ,  Taneya S ,  Kimura T ,  Ohmori T ,  Daikoku H ,  Suzuki N ,  Sawa J ,  Kano K ,  Katayama S .  Milk of northern fur seal: Composition, especially carbohydrate and protein .  _J Dairy Sci_.  1983 ;66 : 2076 \u2013 2083 .\n\nD\u00fcringer C ,  Hamiche A ,  Gustafsson L ,  Kimura H ,  Svanborg C .  HAMLET interacts with histones and chromatin in tumor cell nuclei .  _J Biol Chem_.  2003 ;278 : 42131 \u2013 42135 .\n\nEckersall PD ,  Young FJ ,  Nolan AM ,  Knight CH ,  McComb C ,  Waterson MM ,  Hogarth CJ ,  Scott EM ,  Fitzpatrick JL .  Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis .  _J Dairy Sci_.  2006 ;89 : 1488 \u2013 1501 .\n\nFanaro S ,  Chierici R ,  Guerrini P ,  Vigi V .  Intestinal microflora in early infancy: composition and development .  _Acta Paediatr Suppl_.  2003 ;91 : 48 \u2013 55 .\n\nFata JE ,  Leco KJ ,  Voura EB ,  Yu HY ,  Waterhouse P ,  Murphy G ,  Moorehead RA ,  Khokha R .  Accelerated apoptosis in the Timp-3-deficient mammary gland .  _J Clin Invest_.  2001 ;108 : 831 \u2013 841 .\n\nFindlay L .  The mammary glands of the tammar wallaby (Macropus eugenii) during pregnancy and lactation .  _J Reprod Fertil_.  1982 ;65 : 59 \u2013 66 .\n\nFordyce RE ,  ed.  _Fossil record_ .  San Diego, CA :  Academic Press ;  2002 .\n\nGentry RL ,  Holt JR .  Attendance behaviour of northern fur seals .  In:  Gentry RL ,  Kooyman RL ,  eds.  _Fur seals: Maternal strategies on land and at sea_ .  Princeton, NJ :  Princeton University Press ;  1986 .\n\nGillin FD ,  Reiner DS ,  Wang CS .  Human milk kills parasitic intestinal protozoa .  _Science_.  1983 ;221 : 1290 \u2013 1292 .\n\nGlaser R ,  Harder J ,  Lange H ,  Bartels J ,  Christophers E ,  Schroder JM .  Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection .  _Nat Immunol_.  2005 ;6 : 57 \u2013 64 .\n\nGoldman AS .  Evolution of the mammary gland defense system and the ontogeny of the immune system .  _Journal of Mammary Gland Biology and Neoplasia_.  2002 ;7 : 277 \u2013 289 .\n\nGolec M .  Cathelicidin LL-37: LPS-neutralizing, pleiotropic peptide .  _Annals of Agricultural and Environmental Medicine_.  2007 ;14 : 1 \u2013 4 .\n\nGrant T .  _Platypus_ .  4th ed CSIRO Publishing Melbourne. ;  2007 .\n\nGriffiths M .  _The biology of Monotremes_ .  New York :  Academic Press ;  1978 .\n\nGriffiths M ,  Green B ,  Leckie RMC ,  Messer M ,  Newgrain KW .  Constituents of platypus and echidna milk, with particular reference to the fatty acid complement of the triglycerides .  _Australian Journal of Biological Science_.  1984 ; : 323 \u2013 329 .\n\nGriffiths M ,  Kristo F ,  Green B ,  Fogerty AC ,  Newgrain K .  Observations on free-living, lactating echidnas, Tachyglossus aculeatus (Monotremata: tachyglossidae) and sucklings .  _Aust. Mammal_.  1988 ;11 : 135 .\n\nGu Y ,  Li M ,  Wang T ,  Liang Y ,  Zhong Z ,  Wang X ,  Zhou Q ,  Chen L ,  Lang Q ,  He Z ,  Chen X ,  Gong J ,  Gao X ,  Li X ,  Lv X .  Lactation-related microRNA expression profiles of porcine breast milk exosomes .  _PLoS One_.  2012 ;7 : e43691 .\n\nGudmundsson GH ,  Agerberth B .  Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system .  _J Immunol Methods_.  1999 ;232 : 45 \u2013 54 .\n\nGudmundsson GH ,  Agerberth B ,  Odeberg J ,  Bergman T ,  Olsson B ,  Salcedo R .  The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes .  _Eur J Biochem_.  1996 ;238 : 325 \u2013 332 .\n\nGudmundsson GH ,  Magnusson KP ,  Chowdhary BP ,  Johansson M ,  Andersson L ,  Boman HG .  Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39 .  _Proc Natl Acad Sci USA_.  1995 ;92 : 7085 \u2013 7089 .\n\nGuss JM ,  Messer M ,  Costello M ,  Hardy K ,  Kumar V .  Structure of the calcium-binding echidna milk lysozyme at 1.9 A resolution .  _Acta Crystallogr D Biol Crystallogr_.  1997 ;53 : 355 \u2013 363 .\n\nHajjoubi S ,  Rival-Gervier S ,  Hayes H ,  Floriot S ,  Eggen A ,  Piumi F ,  Chardon P ,  Houdebine LM ,  Thepot D .  Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene .  _Gene_.  2006 ;370 : 104 \u2013 112 .\n\nHakansson A ,  Andreasson J ,  Zhivotovsky B ,  Karpman D ,  Orrenius S ,  Svanborg C .  Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei .  _Exp Cell Res_.  1999 ;246 : 451 \u2013 460 .\n\nHakansson A ,  Zhivotovsky B ,  Orrenius S ,  Sabharwal H ,  Svanborg C .  Apoptosis induced by a human milk protein .  _Proc Natl Acad Sci U S A_.  1995 ;92 : 8064 \u2013 8068 .\n\nHancock RE ,  Brown KL ,  Mookherjee N .  Host defence peptides from invertebrates-emerging antimicrobial strategies .  _Immunobiology_.  2006 ;211 : 315 \u2013 322 .\n\nHancock RE ,  Lehrer R .  Cationic peptides: A new source of antibiotics .  _Trends Biotechnol_.  1998 ;16 : 82 \u2013 88 .\n\nHayssen V ,  Blackburn DG .  Alpha-lactalbumin and the origins of lactation .  _Evolution_.  1985 ;39 : 1147 \u2013 1149 .\n\nHendry KA ,  Simpson KJ ,  Nicholas KR ,  Wilde CJ .  Autocrine inhibition of milk secretion in the lactating tammar wallaby (Macropus eugenii) .  _J Mol Endocrinol_.  1998 ;21 : 169 \u2013 177 .\n\nHiemstra PS .  Novel roles of protease inhibitors in infection and inflammation .  _Biochem Soc Trans_.  2002 ;30 : 116 \u2013 120 .\n\nHuang HJ ,  Ross CR ,  Blecha F .  Chemoattractant properties of PR-39, a neutrophil antibacterial peptide .  _J Leukoc Biol_.  1997 ;61 : 624 \u2013 629 .\n\nIdoji Y ,  Watanabe Y ,  Yamashita A ,  Yamanishi K ,  Nishiguchi S ,  Shimada K ,  Yasunaga T ,  Yamanishi H .  In silico study of whey-acidic-protein domain containing oral protease inhibitors .  _Int J Mol Med_.  2008 ;21 : 461 \u2013 468 .\n\nIjichi H ,  Otsuka M ,  Tateishi K ,  Ikenoue T ,  Kawakami T ,  Kanai F ,  Arakawa Y ,  Seki N ,  Shimizu K ,  Miyazono K ,  Kawabe T ,  Omata M .  Smad4-independent regulation of p21\/WAF1 by transforming growth factor-beta .  _Oncogene_.  2004 ;23 : 1043 \u2013 1051 .\n\nIwamori T ,  Nukumi N ,  Itoh K ,  Kano K ,  Naito K ,  Kurohmaru M ,  Yamanouchi K ,  Tojo H .  Bacteriostatic activity of whey acidic protein (WAP) .  _J Vet Med Sci_.  2010 ;72 : 621 \u2013 625 .\n\nJohnson JD ,  Christiansen RO ,  Kretchmer N .  Lactose synthetase in mammary gland of the California sea lion .  _Biochem Biophys Res Commun_.  1972 ;47 : 393 \u2013 397 .\n\nJoss JL ,  Molloy MP ,  Hinds L ,  Deane E .  A longitudinal study of the protein components of marsupial milk from birth to weaning in the tammar wallaby (Macropus eugenii) .  _Dev Comp Immunol_.  2009 ;33 : 152 \u2013 161 .\n\nKhalil E ,  Digby MR ,  O'donnell P ,  Nicholas KR .  Changes in milk protein composition during acute involution at different phases of tammar wallaby (Macropus eugenii) lactation .  _Comp Biochem Physiol B Biochem Mol Biol_.  2008 ;151 : 64 \u2013 69 .\n\nKirchhoff C ,  Habben I ,  Ivell R ,  Krull N .  A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors .  _Biol Reprod_.  1991 ;45 : 350 \u2013 357 .\n\nKosaka N ,  Izumi H ,  Sekine K ,  Ochiya T .  microRNA as a new immune-regulatory agent in breast milk .  _Silence_.  2010 ;1 : 7 .\n\nKwek J ,  De Iongh R ,  Nicholas K ,  Familari M .  Molecular insights into evolution of the vertebrate gut: focus on stomach and parietal cells in the marsupial, Macropus eugenii .  _J Exp Zool B Mol Dev Evol_.  2009 ;312 : 613 \u2013 624 .\n\nKwek JH ,  Iongh RD ,  Digby MR ,  Renfree MB ,  Nicholas KR ,  Familari M .  Cross-fostering of the tammar wallaby (Macropus eugenii) pouch young accelerates fore-stomach maturation .  _Mech Dev_.  2009 ;126 : 449 \u2013 463 .\n\nKwek JH ,  Wynne A ,  Lef\u00e8vre C ,  Familari M ,  Nicholas KR ,  Sharp JA .  Molecular evolution of a novel marsupial S100 protein (S100A19) which is expressed at specific stages of mammary gland and gut development .  _Mol Phylogenet Evol_.  2013 ;69 : 4 \u2013 16 .\n\nLai Y ,  Gallo RL .  AMPed up immunity: How antimicrobial peptides have multiple roles in immune defense .  _Trends in Immunology_.  2009 ;30 : 131 \u2013 141 .\n\nLeach ST ,  Mitchell HM ,  Geczy CL ,  Sherman PM ,  Day AS .  S100 calgranulin proteins S100A8, S100A9 and S100A12 are expressed in the inflamed gastric mucosa of Helicobacter pylori-infected children .  _Can J Gastroenterol_.  2008 ;22 : 461 \u2013 464 .\n\nLef\u00e8vre CM ,  Digby MR ,  Whitley JC ,  Strahm Y ,  Nicholas KR .  Lactation transcriptomics in the Australian marsupial, Macropus eugenii: transcript sequencing and quantification .  _BMC Genomics_.  2007 ;8 : 417 \u2013 425 .\n\nLef\u00e8vre CM ,  Sharp JA ,  Nicholas KR .  Evolution of lactation: ancient origin and extreme adaptations of the lactation system .  _Ann Rev Genomics and Human Genetics_.  2010 ;11 : 219 \u2013 238 .\n\nLef\u00e8vre CM ,  Sharp JA ,  Nicholas KR .  Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals .  _Reprod Fertil Dev_.  2009 ;21 : 1015 \u2013 1027 .\n\nLehrer RI ,  Ganz T .  Antimicrobial peptides in mammalian and insect host defence .  _Curr Opin Immunol_.  1999 ;11 : 23 \u2013 27 .\n\nLi J ,  Post M ,  Volk R ,  Gao Y ,  Li M ,  Metais C ,  Sato K ,  Tsai J ,  Aird W ,  Rosenberg RD ,  Hampton TG ,  Sellke F ,  Carmeliet P ,  Simons M .  PR39, a peptide regulator of angiogenesis .  _Nat Med_.  2000 ;6 : 49 \u2013 55 .\n\nLi M ,  Liu X ,  Robinson G ,  Bar-Peled U ,  Wagner KU ,  Young WS ,  Hennighausen L ,  Furth PA .  Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution .  _Proc Natl Acad Sci USA_.  1997 ;94 : 3425 \u2013 3430 .\n\nLi P ,  Rudland PS ,  Fernig DG ,  Finch LM ,  Wilde CJ .  Modulation of mammary development and programmed cell death by the frequency of milk removal in lactating goats .  _J Physiol_.  1999 ;519 (Pt 3) : 885 \u2013 900 .\n\nLi Z ,  Lan X ,  Guo W ,  Sun J ,  Huang Y ,  Wang J ,  Huang T ,  Lei C ,  Fang X ,  Chen H .  Comparative transcriptome profiling of dairy goat microRNAs from dry period and peak lactation mammary gland tissues .  _PLoS One_.  2012 ;7 : e52388 .\n\nLonnerdal B ,  Lien EL .  Nutritional and physiologic significance of alpha-lactalbumin in infants .  _Nutr Rev_.  2003 ;61 : 295 \u2013 305 .\n\nLund LR ,  Romer J ,  Thomasset N ,  Solberg H ,  Pyke C ,  Bissell MJ ,  Dano K ,  Werb Z .  Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways .  _Development_.  1996 ;122 : 181 \u2013 193 .\n\nManzoni P ,  Stolfi I ,  Messner H ,  Cattani S ,  Laforgia N ,  Romeo MG ,  Bollani L ,  Rinaldi M ,  Gallo E ,  Quercia M ,  Maule M ,  Mostert M ,  Decembrino L ,  Magaldi R ,  Mosca F ,  Vagnarelli F ,  Memo L ,  Betta PM ,  Stronati M ,  Farina D .  Bovine lactoferrin prevents invasive fungal infections in very low birth weight infants: A randomized controlled trial .  _Pediatrics_.  2012 ;129 : 116 \u2013 123 .\n\nMarti A ,  Feng Z ,  Altermatt HJ ,  Jaggi R .  Milk accumulation triggers apoptosis of mammary epithelial cells .  _Eur J Cell Biol_.  1997 ;73 : 158 \u2013 165 .\n\nMassague J .  How cells read TGF-beta signals .  _Nat Rev Mol Cell Biol_.  2000 ;1 : 169 \u2013 178 .\n\nMcfadden T .  _Prospects for Improving Lactational Persistency_ .  Wallingford :  CAB INTERNATIONAL ;  1997 .\n\nMcorist S ,  Smales L .  Morbidity and mortality of free-living and captive echidnas, Tachyglossus aculeatus (Shaw), in Australia .  _J Wildl Dis_.  1986 ;22 : 375 \u2013 380 .\n\nMesser M ,  Crisp EA ,  Newgrain K .  Studies on the carbohydrate content of milk of the crabeater seal (Lobodon carcinophagus) .  _Comp Biochem Physiol B_.  1988 ;90 : 367 \u2013 370 .\n\nMesser M ,  Griffiths M ,  Rismiller PD ,  Shaw DC .  Lactose synthesis in a monotreme, the echidna (Tachyglossus aculeatus): Isolation and amino acid sequence of echidna alpha-lactalbumin .  _Comp Biochem Physiol B Biochem Mol Biol_.  1997 ;118 : 403 \u2013 410 .\n\nMetcalfe AD ,  Gilmore A ,  Klinowska T ,  Oliver J ,  Valentijn AJ ,  Brown R ,  Ross A ,  Macgregor G ,  Hickman JA ,  Streuli CH .  Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland .  _J Cell Sci_.  1999 ;112 : 1771 \u2013 1783 .\n\nMoreau T ,  Baranger K ,  Dade S ,  Dallet-Choisy S ,  Guyot N ,  Zani ML .  Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family .  _Biochimie_.  2008 ;90 : 284 \u2013 295 .\n\nMorrow GE ,  Nicol SC .  Maternal care in the Tasmanian echidna (Tachyglossus aculeatus setosus) .  _Australian Journal of Zoology_.  2013 ;60 : 289 \u2013 298 .\n\nMunday BL ,  Whittington RJ ,  Stewart NJ .  Disease conditions and subclinical infections of the platypus (Ornithorhynchus anatinus) .  _Philos Trans R Soc Lond B Biol Sci_.  1998 ;353 : 1093 \u2013 1099 .\n\nNicholas K ,  Sharp J ,  Watt A ,  Wanyonyi S ,  Crowley T ,  Gillespie M ,  Lef\u00e8vre C .  The tammar wallaby: A model system to examine domain-specific delivery of milk protein bioactives .  _Semin Cell Dev Biol_.  2012 ;23 : 547 \u2013 556 .\n\nNicholas K ,  Simpson K ,  Wilson M ,  Trott J ,  Shaw D .  The tammar wallaby: A model to study putative autocrine-induced changes in milk composition .  _J Mammary Gland Biol Neoplasia_.  1997 ;2 : 299 \u2013 310 .\n\nNicholas KR .  Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii) .  _Biochem Biophys Res Commun_.  1988 ;154 : 529 \u2013 536 .\n\nNicholas KR .  Control of milk protein synthesis in the tammar wallaby: A model system to study prolactin-dependent development .  In:  Tyndale-Biscoe CH ,  Janssens PA ,  eds.  _The developing Marsupial:models for biomedical research_ .  Heidelberg :  Springer-Verlag ;  1988 .\n\nNicholas KR ,  Tyndale-Biscoe CH .  Prolactin-dependent accumulation of alpha-lactalbumin in mammary gland explants from the pregnant tammar wallaby (Macropus eugenii) .  _J Endocrinol_.  1985 ;106 : 337 \u2013 342 .\n\nNicholas KR ,  Wilde C ,  Bird K ,  Hendry K ,  Tregenza K ,  Warner B .  Asynchronous concurrent secretion of milk proteins in the tammar wallaby (Macropus eugenii) .  In:  Wilde C ,  Knight C ,  Peaker M ,  eds.  _Intercellular signalling in the mammary gland_ .  London :  Plenum Press ;  1995 .\n\nNicol S .  Monotreme biology. Comparative biochemistry and physiology Part A .  _Molecular & Integrative Physiology_.  2003 ;136 : 795 \u2013 798 .\n\nNijnik A ,  Hancock RE .  The roles of cathelicidin LL-37 in immune defences and novel clinical applications .  _Curr Opin Hematol_.  2009 ;16 : 41 \u2013 47 .\n\nNukumi N ,  Ikeda K ,  Osawa M ,  Iwamori T ,  Naito K ,  Tojo H .  Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro .  _Dev Biol_.  2004 ;274 : 31 \u2013 44 .\n\nOeding P .  Examinations on penicillin resistant, serologically homogeneous staphylococci isolated from human mastitis .  _Acta Pathol Microbiol Scand_.  1952 ;31 : 145 \u2013 163 .\n\nOftedal OT .  The origin of lactation as a water source for parchment-shelled eggs .  _J Mammary Gland Biol Neoplasia_.  2002 ;7 : 253 \u2013 266 .\n\nOftedal OT .  The evolution of milk secretion and its ancient origins .  _Animal_.  2012 ;6 : 355 \u2013 368 .\n\nOftedal OT ,  Boness DJ ,  Tedmam RA .  The behaviour, physiology, and anatomy of lactation in the Pinnipedia .  _Current Mammalogy_.  1987 ;1 : 175 \u2013 245 .\n\nOftedal OT ,  Iverson SJ ,  eds.  _Phylogenetic variation in the gross composition of mammalian milks_ .  San Diego, CA :  Academic Press ;  1995 .\n\nOikonomou G ,  Machado VS ,  Santisteban C ,  Schukken YH ,  Bicalho RC .  Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA .  _PLoS One_.  2012 ;7 : e47671 .\n\nOld JM ,  Deane EM .  Development of the immune system and immunological protection in marsupial pouch young .  _Dev Comp Immunol_.  2000 ;24 : 445 \u2013 454 .\n\nOrdonez GR ,  Hillier LW ,  Warren WC ,  Grutzner F ,  Lopez-Otin C ,  Puente XS .  Loss of genes implicated in gastric function during platypus evolution .  _Genome Biol_.  2008 ;9 : R81 .\n\nPeaker M ,  Wilde CJ ,  Knight CH .  Local control of the mammary gland .  _Biochem Soc Symp_.  1998 ;63 : 71 \u2013 79 .\n\nPermyakov SE ,  Pershikova IV ,  Khokhlova TI ,  Uversky VN ,  Permyakov EA .  No need to be HAMLET or BAMLET to interact with histones: Binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids .  _Biochemistry_.  2004 ;43 : 5575 \u2013 5582 .\n\nPeters MG ,  Ambrus Jr JL ,  Fauci AS ,  Brown EJ .  The Bb fragment of complement factor B acts as a B cell growth factor .  _J Exp Med_.  1988 ;168 : 1225 \u2013 1235 .\n\nPigati L ,  Yaddanapudi SC ,  Iyengar R ,  Kim DJ ,  Hearn SA ,  Danforth D ,  Hastings ML ,  Duelli DM .  Selective release of microRNA species from normal and malignant mammary epithelial cells .  _PLoS One_.  2010 ;5 : e13515 .\n\nQuarrie LH ,  Addey CV ,  Wilde CJ .  Programmed cell death during mammary tissue involution induced by weaning, litter removal, and milk stasis .  _J Cell Physiol_.  1996 ;168 : 559 \u2013 569 .\n\nRamanathan B ,  Davis EG ,  Ross CR ,  Blecha F .  Cathelicidins: Microbicidal activity, mechanisms of action, and roles in innate immunity .  _Microbes Infect_.  2002 ;4 : 361 \u2013 372 .\n\nReich CM ,  Arnould JP .  Evolution of Pinnipedia lactation strategies: A potential role for alpha-lactalbumin? .  _Biol Lett_.  2007 ;3 : 546 \u2013 549 .\n\nRismiller PD ,  McKelvey MW .  Body mass, age and sexual maturity in short-beaked echidnas .  _Tachyglossus aculeatus. Comparative biochemistry and physiology Part A, Molecular & integrative physiology_.  2003 ;136 : 851 \u2013 865 .\n\nRisso A ,  Braidot E ,  Sordano MC ,  Vianello A ,  Macri F ,  Skerlavaj B ,  Zanetti M ,  Gennaro R ,  Bernardi P .  BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore .  _Mol Cell Biol_.  2002 ;22 : 1926 \u2013 1935 .\n\nRollins-Smith LA ,  Doersam JK ,  Longcore JE ,  Taylor SK ,  Shamblin JC ,  Carey C ,  Zasloff MA .  Antimicrobial peptide defenses against pathogens associated with global amphibian declines .  _Dev Comp Immunol_.  2002 ;26 : 63 \u2013 72 .\n\nSchedin P ,  O'brien J ,  Rudolph M ,  Stein T ,  Borges V .  Microenvironment of the involuting mammary gland mediates mammary cancer progression .  _J Mammary Gland Biol Neoplasia_.  2007 ;12 : 71 \u2013 82 .\n\nSchmidt DV ,  Walker LE ,  Ebner KE .  Lactose synthetase activity in northern fur seal milk .  _Biochim Biophys Acta_.  1971 ;252 : 439 \u2013 442 .\n\nSharp JA ,  Cane KN ,  Lef\u00e8vre C ,  Arnould JP ,  Nicholas KR .  Fur seal adaptations to lactation: insights into mammary gland function .  _Curr Top Dev Biol_.  2006 ;72 : 275 \u2013 308 .\n\nSharp JA ,  Cane KN ,  Mailer SL ,  Oosthuizen WH ,  Arnould JPY ,  Nicholas KR .  Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus) .  _Matrix Biology_.  2006 ;25 : 430 \u2013 442 .\n\nSharp JA ,  Digby M ,  Lef\u00e8vre C ,  Mailer S ,  Khalil E ,  Topcic D ,  August A ,  Kwek J ,  Brennan A ,  Familari M ,  Nicholas KR .  The comparative genomics of tammar wallaby and fur seal lactation; models to examine function of milk proteins .  In:  Thompson A ,  Boland M ,  Singh H ,  eds.  _Milk proteins: From expression to Food_ .  Academic Press\/Elsevier, San Diego, pp. 55\u201379 ;  2009 .\n\nSharp JA ,  Lef\u00e8vre C ,  Brennan AJ ,  Nicholas KR .  The fur seal\u2014a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution .  _J Mammary Gland Biol Neoplasia_.  2007 ;12 : 47 \u2013 58 .\n\nSharp JA ,  Lef\u00e8vre C ,  Nicholas KR .  Molecular evolution of monotreme and marsupial whey acidic protein genes .  _Evol Dev_.  2007 ;9 : 378 \u2013 392 .\n\nSharp JA ,  Lef\u00e8vre C ,  Nicholas KR .  Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution .  _BMC Biol_.  2008 ;6 : 48 .\n\nShaw DC ,  Messer M ,  Scrivener AM ,  Nicholas KR ,  Griffiths M .  Isolation, partial characterisation, and amino acid sequence of alpha-lactalbumin from platypus (Ornithorhynchus anatinus) milk .  _Biochim Biophys Acta_.  1993 ;1161 : 177 \u2013 186 .\n\nSimpson KJ ,  Nicholas KR .  The comparative biology of whey proteins .  _J Mammary Gland Biol Neoplasia_.  2002 ;7 : 313 \u2013 326 .\n\nSimpson KJ ,  Ranganathan S ,  Fisher JA ,  Janssens PA ,  Shaw DC ,  Nicholas KR .  The gene for a novel member of the whey acidic protein family encodes three four-disulfide core domains and is asynchronously expressed during lactation .  _J Biol Chem_.  2000 ;275 : 23074 \u2013 23081 .\n\nSinkora M ,  Sun J ,  Sinkorova J ,  Christenson RK ,  Ford SP ,  Butler JE .  Antibody repertoire development in fetal and neonatal piglets. VI. B cell lymphogenesis occurs at multiple sites with differences in the frequency of in-frame rearrangements .  _Journal of Immunology_.  2003 ;170 : 1781 \u2013 1788 .\n\nSordillo LM ,  Nickerson SC ,  Akers RM ,  Oliver SP .  Secretion composition during bovine mammary involution and the relationship with mastitis .  _Int J Biochem_.  1987 ;19 : 1165 \u2013 1172 .\n\nSternhagen LG ,  Allen JC .  Growth rates of a human colon adenocarcinoma cell line are regulated by the milk protein alpha-lactalbumin .  _Adv Exp Med Biol_.  2001 ;501 : 115 \u2013 120 .\n\nStrange R ,  Li F ,  Saurer S ,  Burkhardt A ,  Friis RR .  Apoptotic cell death and tissue remodelling during mouse mammary gland involution .  _Development_.  1992 ;115 : 49 \u2013 58 .\n\nSvensson M ,  Duringer C ,  Hallgren O ,  Mossberg AK ,  Hakansson A ,  Linse S ,  Svanborg C .  Hamlet-a complex from human milk that induces apoptosis in tumor cells but spares healthy cells .  _Adv Exp Med Biol_.  2002 ;503 : 125 \u2013 132 .\n\nTalhouk RS ,  Bissell MJ ,  Werb Z .  Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution .  _J Cell Biol_.  1992 ;118 : 1271 \u2013 1282 .\n\nTheil PK ,  Labouriau R ,  Sejrsen K ,  Thomsen B ,  Sorensen MT .  Expression of genes involved in regulation of cell turnover during milk stasis and lactation rescue in sow mammary glands .  _J Anim Sci_.  2005 ;83 : 2349 \u2013 2356 .\n\nTomee JF ,  Hiemstra PS ,  Heinzel-Wieland R ,  Kauffman HF .  Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi .  _J Infect Dis_.  1997 ;176 : 740 \u2013 747 .\n\nTopcic D ,  Auguste A ,  De Leo AA ,  Lef\u00e8vre C ,  Digby MR ,  Nicholas KR .  Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene: New insights into the function of the protein .  _Evol Dev_.  2009 ;11 : 363 \u2013 375 .\n\nTriplett AA ,  Sakamoto K ,  Matulka LA ,  Shen L ,  Smith GH ,  Wagner KU .  Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells .  _Genesis_.  2005 ;43 : 1 \u2013 11 .\n\nTrott JF ,  Adams TE ,  Wilson M ,  Nicholas KR .  Positive and negative regulatory elements in the late lactation protein\u2014A gene promoter from the tammar wallaby (Macropus eugenii) .  _Biochim Biophys Acta_.  2005 ;1728 : 65 \u2013 76 .\n\nTrott JF ,  Simpson KJ ,  Moyle RL ,  Hearn CM ,  Shaw G ,  Nicholas KR ,  Renfree MB .  Maternal regulation of milk composition, milk production, and pouch young development during lactation in the tammar wallaby (Macropus eugenii) .  _Biol Reprod_.  2003 ;68 : 929 \u2013 936 .\n\nTrott JF ,  Wilson MJ ,  Hovey RC ,  Shaw DC ,  Nicholas KR .  Expression of novel lipocalin-like milk protein gene is developmentally-regulated during lactation in the tammar wallaby .  _Macropus eugenii. Gene_.  2002 ;283 : 287 \u2013 297 .\n\nTyndale-Biscoe CH ,  Janssens PA .  _The developing marsupial: Models for biomedical research_ .  Heidelberg :  Springer-Verlag ;  1988 .\n\nUrashima T ,  Arita M ,  Yoshida M ,  Nakamura T ,  Arai I ,  Saito T ,  Arnould JP ,  Kovacs KM ,  Lydersen C .  Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk .  _Comp Biochem Physiol B Biochem Mol Biol_.  2001 ;128 : 307 \u2013 323 .\n\nUrashima T ,  Saito T ,  Nakamura T ,  Messer M .  Oligosaccharides of milk and colostrum in non-human mammals .  _Glycoconj J_.  2001 ;18 : 357 \u2013 371 .\n\nWaite R ,  Giraud A ,  Old J ,  Howlett M ,  Shaw G ,  Nicholas K ,  Familari M .  Cross-fostering in Macropus eugenii leads to increased weight but not accelerated gastrointestinal maturation .  _J Exp Zoolog A Comp Exp Biol_.  2005 ;303 : 331 \u2013 344 .\n\nWalker NI ,  Bennett RE ,  Kerr JF .  Cell death by apoptosis during involution of the lactating breast in mice and rats .  _Am J Anat_.  1989 ;185 : 19 \u2013 32 .\n\nWall EH ,  Mcfadden TB .  The milk yield response to frequent milking in early lactation of dairy cows is locally regulated .  _J Dairy Sci_.  2007 ;90 : 716 \u2013 720 .\n\nWang J ,  Wong ES ,  Whitley JC ,  Li J ,  Stringer JM ,  Short KR ,  Renfree MB ,  Belov K ,  Cocks BG .  Ancient antimicrobial peptides kill antibiotic-resistant pathogens: Australian mammals provide new options .  _PLoS One_.  2011 ;6 : e24030 .\n\nWang Y ,  Lu Z ,  Feng F ,  Zhu W ,  Guang H ,  Liu J ,  He W ,  Chi L ,  Li Z ,  Yu H .  Molecular cloning and characterization of novel cathelicidin-derived myeloid antimicrobial peptide from Phasianus colchicus .  _Dev Comp Immunol_.  2011 ;35 : 314 \u2013 322 .\n\nWang Z ,  Wang G .  APD: the Antimicrobial Peptide Database .  _Nucleic Acids Res_.  2004 ;32 : D590 \u2013 D592 .\n\nWanyonyi SS ,  Lef\u00e8vre C ,  Sharp JA ,  Nicholas KR .  The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii) .  _Matrix Biology_.  2013 ;32 : 342 \u2013 351 .\n\nWanyonyi SS ,  Sharp JA ,  Khalil E ,  Lef\u00e8vre C ,  Nicholas KR .  Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity .  _Comparative biochemistry and physiology Part A, Molecular & integrative physiology_.  2011 ;160 : 431 \u2013 439 .\n\nWatt AP ,  Sharp JA ,  Lef\u00e8vre C ,  Nicholas KR .  WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young .  _Dev Comp Immunol_.  2012 ;36 : 584 \u2013 590 .\n\nWeber A ,  Weber AT ,  McDonald TL ,  Larson MA .  Staphylococcus aureus lipotechoic acid induces differential expression of bovine serum amyloid A3 (SAA3) by mammary epithelial cells: Implications for early diagnosis of mastitis .  _Vet Immunol Immunopathol_.  2006 ;109 : 79 \u2013 83 .\n\nWilde CJ ,  Addey CV ,  Boddy LM ,  Peaker M .  Autocrine regulation of milk secretion by a protein in milk .  _Biochem J_.  1995 ;305 (Pt 1) : 51 \u2013 58 .\n\nWilde CJ ,  Calvert DT ,  Daly A ,  Peaker M .  The effect of goat milk fractions on synthesis of milk constituents by rabbit mammary explants and on milk yield in vivo .  _Evidence for autocrine control of milk secretion. Biochem J_.  1987 ;242 : 285 \u2013 288 .\n\nWilde CJ ,  Knight CH ,  Flint DJ .  Control of milk secretion and apoptosis during mammary involution .  _J Mammary Gland Biol Neoplasia_.  1999 ;4 : 129 \u2013 136 .\n\nWilliams SE ,  Brown TI ,  Roghanian A ,  Sallenave JM .  SLPI and elafin: One glove, many fingers .  _Clin Sci (Lond)_.  2006 ;110 : 21 \u2013 35 .\n\nWilliams WL ,  Patnode RA .  Mastitis of the mouse as related to postsecretory mammary involution .  _Arch Pathol (Chic)_.  1948 ;45 : 229 \u2013 238 .\n\nWitte W ,  Wirth R ,  Klare I .  Enterococci .  _Chemotherapy_.  1999 ;45 : 135 \u2013 145 .\n\nWu H ,  Zhang G ,  Ross CR ,  Blecha F .  Cathelicidin gene expression in porcine tissues: roles in ontogeny and tissue specificity .  _Infect Immun_.  1999 ;67 : 439 \u2013 442 .\n\nYadav M ,  Stanley NF ,  Waring H .  The microbial flora of the gut of the pouch-young and the pouch of a marsupial, Setonix brachyurus .  _J Gen Microbiol_.  1972 ;70 : 437 \u2013 442 .\n\nYang D ,  Biragyn A ,  Hoover DM ,  Lubkowski J ,  Oppenheim JJ .  Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense .  _Annu Rev Immunol_.  2004 ;22 : 181 \u2013 215 .\n\nYang X ,  Khosravi-Far R ,  Chang HY ,  Baltimore D .  Daxx, a novel Fas-binding protein that activates JNK and apoptosis .  _Cell_.  1997 ;89 : 1067 \u2013 1076 .\n\nYenugu S ,  Richardson RT ,  Sivashanmugam P ,  Wang Z ,  O'rand MG ,  French FS ,  Hall SH .  Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif .  _Biol Reprod_.  2004 ;71 : 1484 \u2013 1490 .\n\nZanetti M .  Cathelicidins, multifunctional peptides of the innate immunity .  _J Leukoc Biol_.  2004 ;75 : 39 \u2013 48 .\n\nZanetti M ,  Gennaro R ,  Romeo D .  Cathelicidins: A novel protein family with a common proregion and a variable C-terminal antimicrobial domain .  _FEBS Lett_.  1995 ;374 : 1 \u2013 5 .\n\nZanetti M ,  Litteri L ,  Griffiths G ,  Gennaro R ,  Romeo D .  Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides .  _J Immunol_.  1991 ;146 : 4295 \u2013 4300 .\n\nZasloff M .  Antimicrobial peptides of multicellular organisms .  _Nature_.  2002 ;415 : 389 \u2013 395 .\n\nZhang L ,  Hou D ,  Chen X ,  Li D ,  Zhu L ,  Zhang Y ,  Li J ,  Bian Z ,  Liang X ,  Cai X ,  Yin Y ,  Wang C ,  Zhang T ,  Zhu D ,  Zhang D ,  Xu J ,  Chen Q ,  Ba Y ,  Liu J ,  Wang Q ,  Chen J ,  Wang J ,  Wang M ,  Zhang Q ,  Zhang J ,  Zen K ,  Zhang CY .  Exogenous plant MIR168a specifically targets mammalian LDLRAP1: Evidence of cross-kingdom regulation by microRNA .  _Cell Res_.  2012 ;22 : 107 \u2013 126 .\n\nZhao C ,  Ganz T ,  Lehrer RI .  Structures of genes for two cathelin-associated antimicrobial peptides: prophenin-2 and PR-39 .  _FEBS Lett_.  1995 ;376 : 130 \u2013 134 .\n\nZhou Q ,  Li M ,  Wang X ,  Li Q ,  Wang T ,  Zhu Q ,  Zhou X ,  Gao X ,  Li X .  Immune-related microRNAs are abundant in breast milk exosomes .  _Int J Biol Sci_.  2012 ;8 : 118 \u2013 123 .\n\nZhu J ,  Nathan C ,  Jin W ,  Sim D ,  Ashcroft GS ,  Wahl SM ,  Lacomis L ,  Erdjument-Bromage H ,  Tempst P ,  Wright CD ,  Ding A .  Conversion of proepithelin to epithelins: Roles of SLPI and elastase in host defense and wound repair .  _Cell_.  2002 ;111 : 867 \u2013 878 . \nChapter 4\n\n# Significance, Origin, and Function of Bovine Milk Proteins: The Biological Implications of Manipulation or Modification\n\nS.D. Berry*\n\nP.A. Sheehy**\n\nP. Williamson**\n\nJ.A. Sharp******\n\nK. Menzies***\n\nC. Lefevre******\n\nM. Digby***\n\nK.R. Nicholas****\n\nP.C. Wynn**\n\nR.G. Snell*****\n\n* School of Population Health, University of Auckland, Auckland, New Zealand  \n** Centre for Advanced Technologies in Animal Genetics and Reproduction (REPROGEN), Faculty of Veterinary Science, The University of Sydney, NSW, Australia  \n*** Department of Zoology and CRC for Innovative Dairy Products, University of Melbourne, Victoria, Australia  \n**** Victorian Bioinformatics Consortium, Monash University, Victoria, Australia  \n***** School of Biological Sciences, University of Auckland, Auckland, New Zealand  \n****** School of Medicine, Deakin University, Geelong, Victoria, Australia\n\n## Abstract\n\nThe rapid advances in technology for both evaluating and understanding the structure of animal genomes and their functional significance have presented opportunities for scientists to more clearly understand the complexity of bovine milk proteins and the control of their expression. Used in conjunction with protein data, we can begin to understand the significance of naturally occurring genetic variation influencing milk protein production and composition, as well as how milk proteins are processed into the biologically active peptides that are resident within colostrum and milk. The challenge then remains to translate this information into useful food and therapeutic products, helping to underpin the commercial viability of the dairy industry. Recent advances in genetic technologies provide the potential for accelerated selection of existing traits. Of course, composition could be manipulated via genetically modified (GM) approaches, although consumer acceptance of GM foods is still a major issue. In this chapter, we present a review of the current status of bovine milk genomics and functional genomics and describe the roles, characteristics, and key bioactivities of the major bovine milk proteins and their encrypted peptides. The application of modern molecular analytical tools to the full spectrum of lactation strategies adopted by eutherians, marsupials, and monotremes to improve our understanding of the milk proteome is also discussed.\n\n## Keywords\n\nBovine milk proteins, milk genomics, bovine genome science, evolution or manipulation of bovine milk proteins\n\nOutline\n\nIntroduction 114\n\nMilk Genomics: A Contemporary Approach to Milk Composition 114\n\nAdvances in Bovine Genome Science 115\n\nDNA Sequence 116\n\nGenome Map\/Structure 116\n\nPolymorphism 117\n\nFunctional Genomics 117\n\nTransgenic Technologies for Milk Manipulation 118\n\nComparative Milk Genomics 119\n\nOrigins of milk proteins 120\n\nConstraints and opportunities for evolution or manipulation of bovine milk proteins 122\n\nThe Detailed Structure of Milk Proteins and Their Genes 124\n\nThe Function of Bovine Milk Proteins 125\n\nBioactive Peptides Sequestered within Milk Proteins 128\n\nExisting Variation in Bovine Milk Proteins and the Impact on Expression Function and Milk Quality: Experimental Modifications of Bovine Milk Proteins 129\n\nExperimental Modifications of Bovine Milk Proteins 130\n\nAdding Value to Milk through the Use of Milk Protein Genomics 132\n\nConclusion 133\n\n## Introduction\n\nIn addition to being the source of nutrition for the neonates of mammals, milk has long been considered a healthy source of dietary proteins and minerals for human consumption, and a global dairy industry has evolved to cater to that need. Global demand for dairy continues to grow, driven by the increasing world population, improving economic conditions, and transition toward westernization of diets. In addition, increased competition from other foods and beverages providing enhanced nutritive properties has driven an impetus for diversification of dairy-based foods, such as the manipulation of milk composition to remove saturated fats and the fortification of milk with specific fatty acids, minerals, and vitamins.\n\nAlthough the protein component of bovine milk is only \u22482\u20134% of the total weight, which typically is marginally less than both total fat and carbohydrate, the population of proteins present in milk is diverse and impacts on the physical properties of milk, the nutrition of a neonate, and the metabolic homeostasis and development of both the dam and the offspring. Approximately 80% of the protein present in milk consists of four structurally and functionally interrelated proteins called the caseins. These include \u03b1S1\\- and \u03b1S2-caseins as well as \u03b2\\- and \u03ba-caseins. Some of these proteins show some conservation of gene and protein sequence and structure, and there is similar evidence of relatedness between proteins observed in milks of different species. The remaining 20% of the milk protein population includes the major whey proteins \u03b2-lactoglobulin (\u03b2-Lg) and \u03b1-lactalbumin (\u03b1-La) as well as other constituent proteins, including immunoglobulins, serum proteins, milk fat globule proteins, transferrin, lactoferrin, \u03b22-microglobulin, an array of enzymes, and numerous proteolytic products.\n\nThere has been considerable effort to increase the supply of milk and the diversity of milk products, as well as to characterize the biological activities of milk components. This work has been accelerated by rapid advances in genetic and genomic methods, which have allowed (1) the utilization of comparative genomics to characterize the bioactivities in bovine milk and (2) the accurate selection of specific traits occurring in the natural population. More controversially, such technologies also allow the precise engineering of valuable traits tailored to potential market segments.\n\n### Milk Genomics: A Contemporary Approach to Milk Composition\n\nTrends in the consumer market are driving an increased focus on the health and nutritional functions of food products, and there is an increasing global demand for milk products. One major focus in dairy research is increasing the quantity and improving the quality of milk proteins, which are rich in nutritive and bioactive components. Production of milks with optimized quantities of specific milk proteins may therefore be particularly valuable to dairy processors and suppliers, enabling the provision of specialist milk to precise specifications. The completion of the bovine genome sequencing project has accelerated the ability to define milk protein composition, abundance, and function. In addition, the availability of the bovine genome sequence has facilitated the ability to discover naturally occurring genetic variation, which may influence milk protein quantity and composition.\n\nMilk has been a subject for nutrition and dairy science research for many years, but especially during the post-Second World War period, when it received significant support from the British government for its role as a major source of public nutrition. Increasingly, milk is the source of animal protein of choice worldwide because of its ease of handling and excellent reputation as a high-quality food source. Industrial processing of milk into dairy products has been based largely on the insights into milk chemistry that arose from seminal studies conducted during this period. These studies were concerned primarily with nutritional and processing properties of the major milk proteins. Recent advances in bovine genomics, including the sequencing of the bovine genome and the development of related bioinformatics tools, now extend the capacity via analysis of sequence data and gene expression to understand the properties of milk proteins at a more detailed level and, along with parallel advances in humans and other animals, to open a window on the evolutionary origins and specific benefits of milk. This new knowledge will also enable the discovery of animals with naturally occurring beneficial genetic variations. The study of milk genomics has been bolstered in recent years through the development of the Milk Genome Consortium in 2004 (http:\/\/milkgenomics.org), which endeavors to understand the biological processes underlying milk genomics, as well as the function of, and health traits conferred by, milk proteins.\n\n### Advances in Bovine Genome Science\n\nGenomics is the study of an organism's gene sequence, gene expression, and gene function. Genomic tools can be used to study population diversity, the understanding of which provides strategies for the genetic selection of desirable traits. Genome science has developed rapidly in the past decade as a result of a vast and rapid expansion of genome sequence data, initially arising from the human genome sequencing project, and the concomitant development of applied information technology (bioinformatics). In December 2003, only months after the completion of the human genome sequencing project, an international consortium formally declared the initiation of the bovine genome sequencing project. The human genome sequencing project had resulted in the establishment of significant infrastructure, expertise, and technological advances. Consequently, sequencing of the bovine genome progressed quickly and was completed in 2005, well ahead of the original schedule. Since that time, the development of high-throughput, 'next-generation' sequencing technologies has made whole genome sequencing accessible to many research groups. Genome analysis of individuals is well underway in humans and is being progressively applied in other species, including dairy cows, with a number of large-scale private and public bovine genome sequencing projects currently in progress. A major focus of these projects is the identification of naturally occurring genetic variation, which will facilitate opportunities for discovery of proteins or peptides with novel or improved function and will provide the ability to use advanced breeding technologies to optimize the composition of milk for specific product requirements.\n\n### DNA Sequence\n\nBovine genetics, with a focus on the identification of genetic variation underlying quantitative trait loci (QTL) or linked to important biological phenotypes, has been an active area of study for many years (Khatkar et al., 2004). Using classical genetic and molecular genetic approaches, researchers had mapped over 1000 bovine genes by the mid-1990s. A phase then followed that was based on the positional mapping of candidate genes and on the creation and sequencing of bovine expressed sequence tag (EST) libraries. Subsequently, the sequencing strategy employed for the bovine genome sequencing project combined the use of bacterial artificial chromosome sequences with a whole genome shotgun sequencing approach, using an individual Hereford cow as the source of DNA. The total sequencing effort provided approximately 7x coverage of the genome, representing about 2.7 billion base pairs (reviewed in Tellam, 2007). The completion of the bovine genome provided a foundation for developing bovine-specific post-genome research tools and applied industrial applications. For example, the Bovine Genome Database provides an online resource for the analysis and interpretation of the bovine genome (Childers, 2011). Further discussion of the tools and their application to the study of milk protein composition is included below.\n\n### Genome Map\/Structure\n\nThe bovine genome comprises 29 autosomes, plus the X and Y chromosomes. An international bovine bacterial artificial chromosome (BAC) map was produced to assist in assembling the genome, complemented by an integrated mapping strategy using radiation hybrid and existing physical mapping data. A virtual assembly was released in 2005, and the first de novo assembly was released in 2006. More complete genome builds followed (Zimin et al., 2009; Elsik et al., 2009). There are two current genome assemblies available: Btau_6.1 and UMD3.1. The UMD3.1 bovine genome was derived from a total of 36.82 million reads and 2.649 billion bp. Of these, 99% (2.64 billion bp) have been mapped to chromosomes. A total of 26,618 genes are predicted, coding for an estimated 42,806 proteins (<http:\/\/www.cbcb.umd.edu\/research\/bos_taurus_assembly.shtml>).\n\nComparative genetic maps have revealed significant similarity (synteny) between the bovine genome and genomes of other species (Elsik et al., 2009). This can be effectively viewed using the OXGRID display at <http:\/\/oxgrid.angis.org.au\/>, and as described by Elsik et al. (2009). The synteny between human and bovine genome organization is greater than that between the human and rodent genomes. Further, the mean length of syntenic segments between bovine and human is approximately twice as long as the average length of conserved syntenic segments between human and mouse. The sequencing of the bovine genome and subsequent comparative analysis allowed the discovery of 124 evolutionary breakpoint regions, the majority of which were cattle- or ruminant-specific. Thus, genome sequencing has greatly facilitated further understanding of the evolutionary biology of mammals and lactation.\n\n### Polymorphism\n\nUnderstanding the naturally occurring bovine genetic polymorphism was a primary goal of the genome sequencing project. An understanding of such variation underlying milk proteins provides an opportunity to capture the diversity in milk protein function. Such functional variation could affect the processing characteristics of the milk or change the nutritive properties of the resultant milk product. The Bovine HapMap Consortium resulted in the discovery of a large number of single-nucleotide polymorphisms (SNPs). This effort has been extended to re-sequence six breeds: Holstein, Angus, Jersey, Brahman, Limousin, and Norwegian Red, at low coverage, with the aim of identifying genome-wide SNPs (Tellam, 2007). In addition, mining of existing ESTs has contributed a putative 17,344 SNPs (Hawken et al., 2004).\n\nInitial analysis of dairy herds using a subset of SNPs revealed that the predominant cattle breed used internationally for milk production, Holstein Friesian, has low genetic diversity and may be considered to be a single population unit (Zenger et al., 2007). A subsequent report (Gibbs et al., 2009) found that Bos taurus had a large ancestral population size with an SNP diversity similar to that of humans. Selection for domestication has reduced the effective population size of the breed to small numbers. This has implications for animal breeding. The length of linkage disequilibrium blocks was found to be smaller than expected, but larger than that of humans (Gibbs et al., 2009).\n\nThe genes and genetic variations underlying many milk composition traits have subsequently been identified using the molecular tools that were developed as part of the genome sequencing projects. One of the most significant was the discovery of a variant in the bovine acylCoA:diacylglycerol acyltransferase (DGAT1) gene that affects the production and composition of both milk protein and milk fat (Grisart et al., 2002). This was the first time the causative mutation for a bovine QTL had been identified. Subsequently, a large number of genes and mutations have been identified for milk composition traits as diverse as fatty acid saturation (Mele et al., 2007; Moioli et al., 2007), milk fat color (Berry et al., 2009), and IgA content (Berry et al., 2013).\n\n### Functional Genomics\n\nThe bovine genome sequencing project has also led to improved gene annotation, facilitating the ability to study bovine gene expression and gene function in more detail than was previously possible.\n\nGene-expression tools allowing the study of mammary gland gene expression, and consequently lactation functional genomics, include (among others) microarray platforms and next-generation sequencing technologies. Microarray platforms have been used for many years to measure global gene expression in tissues of interest (Dalma-Weiszhausz et al., 2006; Hoheisel, 2006); to determine changes in gene expression in response to physiological stimuli (for example, Littlejohn et al., 2010); and to identify predicted milk polypeptides, which through subsequent validation and functional analysis may become targets for the development of new dairy ingredients or products. The use of microarrays is being gradually phased out by the decreasing cost of next-generation sequencing technologies and the concomitant development of the RNA sequencing (RNA-seq, or Whole Transcriptome Shotgun Sequencing) approach (Wang et al., 2009). This approach is essentially the application of next-generation sequencing technologies to RNA and enables the identification of transcript abundance, alternatively spliced isoforms and individual-specific sequence variants. RNA sequencing has been used effectively to conduct transcriptional profiling of milk (Wickramasinghe et al., 2012) and to identify differential expression of genes related to oligosaccharide metabolism within lactating mammary tissue, despite the abundance of transcripts from the major milk proteins (Wickramasinghe et al., 2011). It has also allowed identification of genetic variation associated with citrate content in milk (C\u00e1novas et al., 2013), as well as identification of miRNA molecules associated with lactation (Li et al., 2012). miRNAs are small nonprotein-coding RNAs that control expression via base pairing to specific transcripts, targeting them for degradation. They are part of a mechanism involving a variety of small RNAs for controlling expression following transcription.\n\nA complementary approach to gene-expression studies of the mammary gland is provided by milk proteomics (Smolenski et al., 2007; Rawson et al., 2012). Cow's milk comprises approximately 3.5% protein, 80% of which are caseins; the remaining proteins are predominantly the major whey proteins, but there are also a large number of low-abundant proteins, some of which have significant bioactivity or useful nutritional properties. This area has also benefited markedly from the bovine genome sequencing project, and the sequencing of comparative genomes, through the detailed annotation and cataloguing of genes, genetic variations, and proteins. It is now possible to identify variants of both major and low-abundant proteins in milk based on bovine protein sequences. Together with rapid advances in mass spectrometry, particularly improvements in sensitivity and quantification, and complemented by gene-expression data, the protein composition of milk can now be dissected with great accuracy and the variation can be captured using breeding techniques.\n\n#### Transgenic Technologies for Milk Manipulation\n\nAnimals produced using transgenic approaches are a potential source of increased milk production or specifically altered milk composition. The original transgenesis methodology applied to farm animals involved the random integration of a transgene at any location within the genome and was heralded by the production of cattle that expressed human lactoferrin (Krimpenfort et al., 1991). It was originally thought that the use of yeast and bacterial artificial chromosomes capable of conveying large gene constructs up to a Mb may be useful in supporting the overexpression of the 200 kb casein locus (with all four caseins), including the regulatory elements co-coordinating their mammary-specific expression (Zuelke, 1998). To date, there have been only a limited number of successes (Brophy et al., 2003; Laible et al., 2007) including examples where both \u03b2-casein and \u03ba-casein were over-expressed. Since that time, considerable improvements have been made in the methodology for generating transgenic animals (for reviews, see Garrels et al., 2012; Gaj et al., 2013). These methods utilize enzymatic approaches via RNA-guided DNA endonucleases (Zinc-fingers, transcription activator-like effector nucleases, TALENs) and clustered, regularly interspaced short palindromic repeats of Cas9 nuclease (CRISPR\/Cas9) that cause site-specific double-stranded breaks. These breaks subsequently stimulate errors in repair, which can then inactivate genes or facilitate homologous recombination of a sequence delivered along with the enzyme coding vectors or RNA. Through use of these methods combined with nuclear transfer or 'cloning,' the potential therefore exists to make any milk composition as long as the resulting trait is biologically possible and does not harm the animal in any way. This includes the potential for the complete removal of specific genes or the full 'humanization' of bovine milk composition (Wilmut et al., 1997). The major advantage of these improved approaches is that (depending on the target) they can be applied in such a way that leaves no exogenous sequence, potentially overcoming one of the public's concerns about genetic modification technologies. Another genetic modification approach, recently applied to the knockdown of bovine \u03b2-Lg (Jabed et al., 2012), is the use of small RNA molecules, specifically designed for a particular transcript, to target the endogenous transcripts, consequently inhibiting gene expression. (For a review of this technology, see Filipowicz et al., 2008; Valencia-Sanchez et al., 2006.)\n\n### Comparative Milk Genomics\n\nThe bovine genome sequencing initiative was not the only project to follow the human genome sequencing effort. As this initial project abated, international consortia formed to sequence the genomes of other organisms, including the chicken, dog, opossum, chimpanzee, macaque, and platypus. A comparison of the genome sequences of 29 eutherian species was reported in 2011, as a result of the effort by the 29 Mammals Genome Project (Lindblad-Toh et al., 2011). The diverse range of species included in the project was selected to maximize the total branch length of the evolutionary tree. The 29 Mammals Project has facilitated the ability to understand the origin and evolution of lactation strategies and milk protein composition, which vary considerably across different species. It also complements a growing body of work on whole genome expression studies in mammary tissue of different species, including cattle, humans, and mice. For example, Lemay et al. (2009) compared the genomes of five mammalian species and showed that the more divergent milk proteins were associated with nutritional and immunological functions, whereas the more conserved milk proteins were associated with milk secretory processes. Further insight into the evolution of milk synthesis and lactation has been provided by the completion of the kangaroo genome sequence in 2011 (Renfree et al., 2011).\n\nAligned with the goal to develop a deep understanding of milk proteins, a comparative approach to assess the genomic basis of diversity in milk composition between species has been developed (Menzies et al., 2009). Experimental models have been chosen on the basis of reproductive\/lactation strategy and have been cross-referenced to the dairy cow. The study is based on gene expression profiles and integration with genomic mapping information and dairy QTL analysis. Within this framework, a specific methodology has been applied to identify those genes that may code for milk proteins that are either inherently active in milk or could be developed as functional dairy products.\n\nOne example of comparative genomics to alter milk protein composition is the use of functional genomics to exploit animal models, with extreme adaptations to lactation as an alternative approach to identifying genes that regulate protein synthesis in the mammary gland. For example, milk protein is the only component of milk that is synthesized de novo in the mammary gland of the Cape fur seal (in comparison to other mammals, where both milk fat and milk carbohydrate components are synthesized, at least in part, de novo in the mammary gland). The protein content of this seal milk is more than double that of bovine milk (Cane et al., 2005). Understanding the molecular basis for such a lactation strategy may provide insight into mechanisms to increase the protein content of bovine milk. In another model organism, the Tammar wallaby, both milk protein concentration and milk protein yield are dramatically increased in the latter half of lactation (Nicholas et al., 1997). Changes in global gene expression comparing mammary tissues from the Tammar wallaby at early and late lactation using a cDNA array (see Chapter 3), and comparing pregnant and lactating seals and dairy cows using an Affymetrix microarray, indicate that folate metabolism, particularly the role of the folate receptor, may be a crucial regulatory point of milk protein synthesis in mammary epithelial cells (Menzies et al., 2009).\n\nFolate may influence milk protein production, either by producing a direct effect on the mammary gland or by reducing the competition for precursors between gluconeogenesis and methylneogenesis, thereby increasing the metabolic efficiency of the mammary gland. The importance of the folate receptor 1 (or \u03b1) for the cellular uptake of folate was established by analyzing renal folate handling in mice that had targeted gene knockouts of folate-binding proteins 1 and 2 (folbp1 and folbp2, equivalent to human and cow folr\u03b1 and folr\u03b2) (Birn, 2006). Molecular studies in human, monkey, and mouse cell lines have shown that the folate receptor \u03b1 mediates cellular uptake of folate and that folate receptor abundance is regulated by multiple mechanisms. Furthermore, the folate receptor population may play a crucial role in the capacity of mammary epithelial cells to respond and utilize circulating serum folate.\n\nThe folate requirements of lactating dairy cows have been extensively reviewed by Girard et al. (2005) and by Regalle et al. (2009). Lactation increases the demands both for methylated compounds (synthesis of milk choline, creatine, creatinine, and carnitine) and for methionine to support milk protein synthesis (Xue & Snoswell, 1985; Girard & Matte, 2005). The high demand for folate during lactation is demonstrated by the observation that total serum folates decrease by 40% across the lactation period in dairy cows (Girard et al., 1989).\n\nFolate supplement experiments in dairy cows have shown a positive response to milk production and milk protein yields (Girard & Matte, 1998; Girard et al., 2005; Graulet et al., 2007). This positive response was not consistent among all experimental cows in the first two studies and appeared to be dependent on cow lactation status (primiparous were nonresponsive, multiparous were responsive), stage of lactation, and serum vitamin B12 status. The more recent study by Graulet et al. (2007) including vitamin B12 supplementation suggested that the increases in milk and milk protein yields as a result of folic acid supplements were not dependent on the supply of vitamin B12 and were probably closely related to the role of folic acid in the DNA cycle. However, this does not diminish the importance of the role that folate metabolism plays in milk protein synthesis. This is an example of how the combined approach of comparative genomics and bioinformatics, plus species with extreme adaptation to lactation, may be utilized to identify key genes and cellular processes involved specifically in modifying milk composition and milk protein production.\n\n## Origins of milk proteins\n\nThe study of milk protein genes and encoded milk proteins of domesticated mammals and undomesticated mammals alike has made these data relevant to dairy science. The data highlight adaptive features and the diversity of milk protein genes over evolutionary time. Evolutionary evidence suggests that complex lactation preceded divergence of the extant mammalian lineages. Monotreme lactation appears to be the most ancestral form of lactation, whereas marsupials and eutherians developed divergent strategies. Study of the evolution of milk proteins helps determine the significance of maintaining certain proteins while changing or losing others. These differences may reflect divergence of lactation strategies and may provide clues as to why some proteins have become nonessential and are in the process of being lost by some eutherians.\n\nBioinformatic predictions of genes encoding secreted proteins upregulated during lactation in the mammary gland suggest that as many as 300 different proteins may be found in milk. However, caseins are the major proteins in milk, representing about 80% of total milk protein content. Expression of a number of casein genes has been confirmed in monotremes (platypus and echidna) and marsupials. Three evolutionarily related calcium-sensing casein-like genes and the functionally related \u03ba-casein were identified in monotremes. As in other mammals, a number of casein splice variants could be identified. Interestingly, all of these casein genes cluster tightly within a 100-kb region of the platypus genome, a physical linkage that has been observed in the casein locus of all mammalian genomes examined so far (Rijnkels, 2002). The respective organization of the casein locus is also highly conserved in all mammalian lineages. Casein genes occur in the order \u03b1S1-, \u03b2-, \u03b1S2-, and \u03ba-, where the \u03b2-casein gene is encoded in the opposite direction from the other genes. The close proximity of \u03b1S1\\- and \u03b2-casein genes in an inverted tail\u2212tail orientation and the relative orientation of the more distant \u03ba-casein genes are similar in all mammalian genome sequences available so far. This observation suggests that the synchronized tissue-specific expression of casein genes in the lactating mammary gland may be controlled, in part, by a common mechanism at this locus. However, genome sequence analysis has also revealed variation in the \u03b1S2-casein gene content of mammalian lineages. Marsupials possess only one copy of \u03b1-casein corresponding to \u03b1S1-casein (Lefevre et al., 2007; ). In the monotreme lineage, a recent duplication or a gene-conversion event involving \u03b2-casein occurred to produce an \u03b1-like casein gene at a genomic location similar to that of the eutherian \u03b1S2-casein. A similar but more ancestral duplication of \u03b2-casein occurred in eutherians to produce \u03b1S2-casein, and further duplications have also occurred since, in human and mouse lineages, to produce \u03b1S2a\\- and \u03b1S2b-casein genes.\n\n\u03b2-Lg is the major whey protein in ruminant milk and is present in milk from a variety of species. However, it is absent in rodents, such as the mouse; lagomorphs, such as the rabbit; and humans, although reports to the contrary exist. \u03b2-Lg in human milk probably arises either from spurious antibody cross-reactivity (Brignon et al., 1985) or from the presence of ingested bovine \u03b2-Lg in the mother's milk (Fukushima et al., 1997). Not all primate milks are devoid of \u03b2-Lg, as both the macaque and the baboon have the protein within their milk (Hall et al., 2001).\n\nBecause of the absence of \u03b2-Lg in human milk, the dairy industry has developed ways in which to convert the protein composition of bovine milk to something more similar to the protein composition of human milk or remove it altogether using transgenic approaches. Genetic variants have been observed in essentially all of the species from which the protein has been found. There are several genes encoding \u03b2-Lgs within the different species. Sequence analysis of species genomes suggests that some species such as the dog and the cat have three \u03b2-Lg genes, whereas others such as the horse and the donkey have two \u03b2-Lg genes, bovine, sheep, and goat have one \u03b2-Lg gene, and bovine has a pseudogene. Marsupials, monotremes, and some primates have one \u03b2-Lg gene. Comparison of \u03b2-Lg protein sequences shows high conservation of the protein during evolution. Bioactive peptides derived from \u03b2-Lg are currently being intensively studied. These peptides are active only once they are released from the precursor protein. Once released, it is suggested that these peptides play important roles in human health, including having antimicrobial, antihypertensive, and antioxidant activities (Hernandez-Ledesma et al., 2008; Saito et al., 2008). The variation of gene number and protein content in milk represents a specific evolution of \u03b2-Lg for each lineage. It has not yet been explained how a major protein in cow's milk such as \u03b2-Lg can be completely abolished in human, rat, or rabbit milk with little to no consequence to the offspring.\n\nWhey acidic protein (WAP) is a major whey protein that is found in the milk of numerous species such as the mouse (Hennighausen & Sippel, 1982), rat (Campbell et al., 1984), pig (Simpson et al., 1998), rabbit (Devinoy et al., 1988), camel (Beg et al., 1986), wallaby (Simpson et al., 2000), brushtail possum (Demmer et al., 2001), echidna, platypus (Sharp et al., 2007), and dog (Seki et al., 2012), but it is absent in the milk of cows, sheep, and humans (Hajjoubi et al., 2006). WAP pseudogenes have been identified in the bovine and the human. A nucleotide deletion at the end of the first exon is reported to have caused a truncation of the bovine WAP protein (Hajjoubi et al., 2006). This deletion is also found in ovine and caprine species. In comparison, it is the absence of an ATG initiation codon in human WAP that is suggested to be the cause of the pseudogene in this species. Interestingly, the polyadenylation signal AATAAA is still present in the ruminant sequence but not in the human sequence. WAP proteins from different species show moderate sequence similarity; however, they differ in the number of four-disulfide core (4-DSC) motifs between the species. Each specific 4-DSC domain is recognizable by sequence similarity, and it is possible to trace the origins of each domain during evolution (Fig. 4.1). Although not expressed, both bovine and human pseudogenes have similar gene structure to the eutherian WAPs, and it is suggested that these were once functional. As suggested in Chapter 2, the function of the WAP proteins appears to differ with the presence or absence of each of these domains. It is interesting to speculate why this protein appears to be dispensable in some species.\n\nFigure 4.1 Schematic representation of the origins of WAP gene structure. (a) Boxes represent exons within the WAP genes. Each 4-DSC domain is encoded by different exons as indicated. In eutherian WAP, exons 2 and 3 encode the two 4-DSC domains (DI and DIIA). In marsupial WAP, there is an additional exon, and exons 2, 3, and 4 encode the three 4-DSC domains (DIII, DI, and DIIB). In echidna WAP, exons 2 and 3 encode the two 4-DSC domains (DIII and DIIA). In platypus WAP, exons 2, 3, and 4 encode the three 4-DSC domains (DIII, DIIA, and DIIB). Comparison of sequence similarity suggests that DIIB of marsupial WAP and DIIB of platypus WAP are derived from similar exons and that DIIA of eutherian WAP and DIIA of monotreme WAP are derived from similar exons. (b) Schematic representation of stepwise evolutionary history of 4-DSC domains of WAP within the major lineages. The ancestral progenitor, represented by six exons (numbered) and four 4-DSC domains (DIII, DI, DIIA, DIIB), is shown, followed by events of exon loss leading to the evolution of the present-day WAPs in platypus, echidna, marsupial, and monotremes. Numbers represent the order of exons relative to the ancient WAP. Adapted from Sharp et al. (2007).\n\nThe apparently dispensable role of proteins such as \u03b2-Lg and WAP may be related to the diet of the young, which is being adequately compensated in more domesticated animals or may be linked to improved care and hygiene in domesticated life.\n\n## Constraints and opportunities for evolution or manipulation of bovine milk proteins\n\nThe gene and protein structures and sequences of the six major bovine milk proteins (the four caseins, \u03b2-Lg, and \u03b1-La) have been well characterized, although the precise secondary and tertiary structures of the caseins have been difficult to demonstrate experimentally, as they have proven to be challenging to investigate via x-ray crystallography. The casein phosphoproteins precipitate at pH 4.6, which is a significant characteristic that is utilized in cheese production, but, in milk, these proteins aggregate to form protein aggregates or micelles of \u224850\u2212300 nm in diameter. \u03ba-Casein stabilizes the surface of the protein micelles, with the other caseins forming the core, which is stabilized by the phosphorylation of seryl residues. The organic phosphate moieties in turn enable calcium binding, which also stabilizes the micelle structure and, hence, as well as being a significant source of dietary amino acids, the micelle aggregates also form a valuable source of calcium and phosphorus for neonatal nutrition and development. The casein micelle is considered in detail in Chapter 6.\n\nApart from the neonatal nutritional and industrial (dairy products) significance of bovine milk proteins, a wide range of biological activities is observed in the neonate as well as the dam. These activities are attributed to both the intact proteins and, more importantly, to their products of in vivo digestion (see the review by Meisel, 2005; Ricci-Cabello et al., 2012; Pepe et al., 2013). When considered together, bovine milk proteins, which provide neonatal nutrition, regulate physiological processes, coordinate neonatal development, and form the basis of an internationally significant agricultural commodity market, are globally important proteins. For this reason, their functional characteristics need to be considered when evaluating the potential for enhancement or modification of their structure or biosynthesis.\n\n### The Detailed Structure of Milk Proteins and Their Genes\n\nAn understanding of the detailed structure of milk proteins and their genes is critical in the consideration of the impact of modifying bovine milk proteins to increase productivity, to enhance milk product manufacturing efficiency, or to exploit inherent or proposed bioactivity. The physical and structural characteristics of bovine milk proteins have been extensively reviewed (Farrell et al., 2004) and are covered in Chapter 7 of this volume. The genomics coordinates and gene information given below are from the Btau_6.1 annotation (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov\/).\n\nThe bovine casein genes are present as single-copy genes per chromosome and are clustered together on chromosome 6 in a 200-kb region at position q31\u221233 in the order \u03b1S1-, \u03b2-, \u03b1S2-, and \u03ba\\- (Threadgill & Womack, 1990). The bovine \u03b1S1-casein gene (CSN1S1 casein alpha s1 Gene ID: 282208) is 21,900 bp in length and contains 19 exons. The protein is the most abundant of the bovine caseins, present at 12\u221215 g\/L. There are up to eight reported genetic variants of this protein, with the B variant being the most common in Bos taurus species and comprising 199 amino acids with a molecular weight of 23.6 kDa. The \u03b1S1-casein protein may contain up to eight serine monophosphate residues, which cluster in a hydrophilic domain between amino acids 43 and 80 and, through modeling studies, are thought to be connected to a hydrophobic domain (amino acids 100\u2212199) by helical and sheet secondary structures. The less abundant bovine \u03b1S2-casein gene (CSN1S2 casein alpha-S2 Gene ID: 282209) is 18,479 bp in length and contains 18 exons, with significant exon duplication (Mercier & Vilotte, 1993). This gene codes for 207 amino acids, with a molecular weight of 25.23 kDa. This protein exhibits a number of phosphorylation states as well as four variants that have been observed in common bovine populations.\n\nIn contrast, the bovine \u03b2-casein gene (CSN2 casein beta Gene ID: 281099) is significantly less complex, containing nine exons in 8505 bp. The protein is present at 9\u221211 g\/L in modern dairy cattle, consists of 209 amino acids, and has a molecular weight of approximately 24 kDa. Up to 12 genetic variants have been observed and the protein exhibits a flexible structure that can adopt multiple conformations, although a small amount of secondary structure in the form of \u03b1 helices and \u03b2 sheets has been predicted. There are five phosphoserine residues that reside at the N-terminal domain of the protein. \u03b2-Casein can self-assemble into micelles in neutral and acidic environments at room temperature (Moitzi et al., 2008). The bovine \u03ba-casein gene (CSN3 casein kappa Gene ID: 281728) exhibits fewer structural similarities than the other casein genes, contains five exons, and is approximately 13,700 bp in length and not completely annotated on the Btau 6.1. build. It has diverged from the fibrinogen gene family but is more highly conserved between species than the other casein genes. Two common variants are observed in dairy cattle (although several others have been reported), with the B variant containing 169 amino acids with a molecular weight of 19 kDa. Up to six threonine residues may be glycosylated, and, similar to the other caseins, \u03ba-casein has a relatively high proline content that influences the predicted secondary structure of two sets of antiparallel \u03b2 sheet structures exhibiting hydrophobic side chains that may be important for micelle formation. As with the other caseins, the detailed structure of bovine \u03ba-casein is poorly characterized, although the N-terminal domain of amino acids 1\u221244 has been experimentally shown to contain a helical structure (Bansal et al., 2006).\n\nThe bovine \u03b1-La gene (LALBA lactalbumin, alpha Gene ID: 281894) contains four exons and is 2022 bp in length. It is situated on bovine chromosome 5 and exhibits significant interspecies homology in exon 4 (significant for its interaction with galactosyltransferase) and the 5' untranslated region. Two variants have been characterized for the protein, with the most common B variant exhibiting a molecular weight of 14.1 kDa and being present at 0.6\u22121.7 g\/L in skim milk. The 123 amino acids are organized into four helical domains and a small sheet structure that is dependent on pH and metal ion concentrations (Sawyer & Holt, 1993). Different glycosylation states have been observed, and structurally bovine \u03b1-La has some similarity to lysozyme proteins (Brew et al., 1970). This protein, like the caseins, also has calcium-binding properties, as described by Hiraoka et al. (1980).\n\nBovine \u03b2-Lg (PAEP progestagen-associated endometrial protein\/ \u03b2-lactoglobulin Gene ID: 280838) is located on chromosome 11, contains four exons within 4893 bp, and is well conserved between species (Folch et al., 1994). Structural similarities occur throughout the lipocalin protein family, and \u03b2-Lg has been shown to bind to a number of hydrophobic ligands, including palmitic acid (Kontopidis et al., 2004; Konuma et al., 2007). There are two common variants of this protein, A and B, with the protein itself containing 162 amino acids, with a molecular weight of 18.3 kDa. It exhibits a number of \u03b2 strands, which form a \u03b2-barrel structure that contains the hydrophobic-ligand-binding site, and a single \u03b1 helical domain is observed at the C-terminal end of the protein.\n\nThe secondary structures of the major bovine milk proteins are diverse, yet are clearly critical for the functions of the proteins, including micelle formation, calcium and phosphate binding, and other bioactivities. Although there may be opportunities for manipulation of structure and therefore function of these proteins, even modest changes to nucleotide and amino acid sequence may significantly alter protein and peptide functionality.\n\n#### The Function of Bovine Milk Proteins\n\nThe structural complexity of these milk proteins suggests that they serve many functions in the neonate, in addition to the mere provision of a source of amino acids for protein biosynthesis in the fast-developing neonate. Yet the range of functions is not likely to be as great as that in more altricial species such as the monotremes and marsupials, in which the milk constituents orchestrate almost the entire developmental process from the fetus through to attaining physiological independence (see Chapter 3). The contributions that individual proteins make to this developmental spectrum are poorly understood, although longitudinal studies on gene expression and related changes in milk composition will help unravel this relationship.\n\nThe protracted emphasis on selection of cows for milk volume has probably also influenced the balance of biological activities residing within these proteins, because contemporary dairy herds are larger in stature than their predecessors. Thus, developmental activities encoded within the protein fraction may have been altered to emphasize growth promotion rather than other key functions such as the storage of body energy reserves to support reproductive activity and even to promote maturation of the immune system.\n\nThe complexity increases further once these proteins are digested enzymatically or are hydrolyzed in the developing gastrointestinal tract and then absorbed to be transported to their site of action. The precise balance between the peptide component that is absorbed as functional entities and the peptide component that remains for further processing into short peptides as a source of dietary amino acids is not well understood. However, the need to conserve a constant supply of developmental molecules is probably more important than the need for protein synthetic substrate in sustaining the development of the neonate.\n\nThe observation that over 100 hormones and growth factors have been identified in colostrum or milk (Koldovsky, 1996) would suggest that the milk proteins may play important roles, working in concert with these established endocrine\/paracrine signals. Therefore, the sustained selection of animals for milk volume output will have altered the balance of bioactivities in colostrum and milk to account for this response. Thus, the balance of bioactive peptides may differ from those in species in which this selection pressure has been absent. From a commercial point of view, these milk constituents also play a role in determining the sensory properties of milk and its manufactured products. Of course, these same sensory factors probably also serve as attractants to the newborn as it seeks its nutrient supply from the udder.\n\nThe extraction of intact proteins of high commercial value from milk has been seen as a lucrative commercial opportunity by dairy farmers who have incomes tied to the ebb and flow of the commodity markets. The profitability of such ventures depends on the ease of extraction, the relative concentration of the protein in milk, and the value placed on the bioactivity. A range of these proteins is presented in Table 4.1.\n\nTable 4.1\n\nMajor Milk Proteins with Potential for Extraction on a Commercial Scale to Exploit Bioactivities\n\nProtein | Concentration in colostrum (g\/L) | Concentration in milk  \n(g\/L) | Molecular weight | Exploitable bioactivity  \n---|---|---|---|---  \nCaseins (\u03b1s1-, \u03b1s2-, \u03b2\\- and \u03ba-) | 26 | 28 | 14,000\u221222,000 | Ion carriers, bioactive peptide sources, immunomodulators  \n\u03b2-Lactoglobulin | 8.0 | 3.3 | 18,400 | Antioxidant, vitamin-A-binding protein, bioactive peptide source  \n\u03b1-Lactalbumin | 3.0 | 1.2 | 14,200 | Lactose synthesis and milk volume. Calcium binding, immunomodulator, bioactive peptide source  \nImmunoglobulins | 20\u2212150 | 0.5\u22121.0 | 150,000\u22121,000,000 | Specific immune protection, potential bioactive peptide source  \nGlycomacropeptide | 2.5 | 1.2 | 8,000 | Antimicrobial, bifidogenic, gastric modulator, anticlotting factor  \nLactoferrin | 1.5 | 0.1 | 80,000 | Antimicrobial, antioxidant, anti-inflammatory, anticarcinogenic, bioactive peptide source, immunomodulator  \nLactoperoxidase | 0.02 | 0.03 | 78,000 | Antimicrobial, immunopotentiator  \nLysozyme | 0.0004 | 0.0004 | 14,000 | Antimicrobial, immunopotentiator  \nSerum albumin | 1.3 | 0.3 | 66,300 | Bioactive peptide source\n\nAdapted from Korhonen & Pihlanto, 2007.\n\nBoth \u03b1-La and \u03b2-Lg have been isolated on a large scale using a range of chromatographic, ultra-high-pressure, and membrane separation techniques (Korhonen & Pihlanto, 2007). Both of these proteins perform important activities (Table 4.1) that provide the impetus for large-scale production. One of the most important clinical findings is the potential ability of these whey proteins to decrease the incidence of certain cancers (Pepe et al., 2013).\n\nMany of the highly expressed proteins clearly have important immunological functions. The passive immunity provided through the immunoglobulin complement of colostrum to the neonate is supplemented by a range of other bioactivities designed to countenance pathogen loads to which the newborn is exposed in its new environment. The activity of these proteins extends to the initiation of mucosal immunity in the gut and an optimal environment for beneficial microflora. Such proteins include immunoglobulins, many chemokines, mucin 1, lactoferrin, cathelicidin 1, lactoperoxidase, S100 calcium-binding proteins, complement proteins, and polymeric immunoglobulin receptor.\n\nIt is beyond the scope of this review to detail all of the bioactivities identified in milk. However, some specific proteins that are illustrative of principles found across the milk proteome are described below.\n\nLactoferrin has dual roles of scavenging iron and acting as a bacteriostat. It is found in exocrine secretions, including milk, tears, tubotympanum and nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus, and seminal fluid, all of which require antibiotic activity. It is also found in neutrophils that are attracted to sites of infection where both iron sequestration and antibiotic activity assist in the healing process. In addition to these antibacterial effects, lactoferrin has significant commercial value because of its antiviral, antioxidant, antitoxigenic, antithrombotic, and, importantly, anticarcinogenic activities. It is important to note that lactoferrin is just one component of a mix of protective molecules in milk and other exocrine secretions, as lysozyme, beta defensins, and the surfactant proteins A and D (SP-A, SP-D), among others, also contribute to this important function.\n\nTypically, lactoferrin is found in concentrations in excess of 100 mg\/L in bovine colostrum and is extracted by conventional fractionation and chromatography. However, the expression of milk proteins in cereal grains such as rice, using recombinant DNA technology (Lonnerdal, 2006) is another possible way of providing these unique milk proteins in our diet. Similarly, recombinant human lactoferrin given orally has been suggested as a mechanism for the prevention of certain gastrointestinal infections in preterm infants (Sherman & Petrak, 2005). More recently, bovine lactoferrin has been tested as a therapeutic approach for diarrhea in children (Ochoa et al., 2013) and as a way to prevent fungal infections in low-birth-weight infants (Manzoni et al., 2012) and, using a recombinant form of human lactoferrin (Talactoferrin), sepsis in adults (Guntupalli et al., 2013). Incidence of diarrhea did not diminish, but longitudinal prevalence and severity were decreased. Oral delivery of bovine lactoferrin reduced the incidence of invasive fungal infections in preterm infants. Talactoferrin reduced mortality in patients with sepsis. There is little doubt that demand for the use of lactoferrin as a therapeutic agent will increase. Without the development of the ability to produce sufficient quantities in milk, transgenic plants or even recombinant bacterial production (Garcia-Montoya et al., 2013) may well supersede milk as a source of this and other unique bioactive milk proteins.\n\n#### Bioactive Peptides Sequestered within Milk Proteins\n\nMilk proteins contain latent biofunctional peptide sequences within their primary structures that exert physiological responses in vivo. They remain latent until released through the processes of enzymatic processing in the gastrointestinal tract. A large range of these peptides have been identified, including opioid, antimicrobial, immunomodulatory, mineral-transporting, growth-promoting, anticancer, and proteinase and angiotensin-converting enzyme (ACE) inhibitory peptides (Shah, 2000; Ferranti et al., 2004; Ricci-Cabello et al., 2012). In an evolutionary sense, the cow is targeting peptides to specific effector sites through the provision of such peptides to the neonate. Presumably, if the availability of metabolic substrates to support maximal biological response is inadequate, then the animal has the ability to limit the quantities of peptides released by suppressing the release of rate-limiting enzymes.\n\nThe biofunctional peptides currently most studied in food proteins appear to be those that inhibit ACE. This enzyme plays a central role in the regulation of blood pressure through production of the potent vasoconstrictor angiotensin (Ang) II and the degradation of the vasodilator bradykinin. Therefore food-sourced ACE inhibitory peptides may have the ability to lower blood pressure in vivo by limiting the vasoconstrictory effects of Ang II and by potentiating the vasodilatory effects of bradykinin (Murray & Fitzgerald, 2007; Miguel et al., 2009; Jiang et al., 2010).\n\nThe development of functional foods with antihypertensive properties provides an attractive and potentially commercially lucrative range of products.\n\nIn view of the extensive range of peptides derived from various precursor proteins that are involved in the regulation of blood pressure, it may be too simplistic to expect a functional food containing just one or two of these peptides to decrease suppressor activity in the long term. However, several products are currently being evaluated as beneficial functional foods\/food ingredients.\n\nOne success story in deriving a commercial product from milk protein tryptic digests is the casein phosphopeptides used in the chewing gum 'Recaldent' that is capable of remineralizing carious lesions in dental enamel (Cross et al., 2007). These peptides, containing the sequence \u2212Pse\u2212Pse\u2212Pse\u2212Glu\u2212Glu\u2212, where Pse is a phosphoseryl residue, stabilize calcium and phosphate ions in aqueous solution and make these essential nutrients bioavailable. Interestingly, these peptides are naturally formed during gastric digestion, making dairy products containing them nutritionally useful only in dental applications. Investigations of the chemistry of these intriguing peptides have shown that they can be altered to form casein phosphopeptide\u2212amorphous calcium phosphate, which in turn is capable of forming a calcium fluoride.\n\nRecent studies have indicated that there is benefit in incorporating dairy products into the diet with wide-reaching clinical effects (McGregor and Poppitt, 2013). Increasing consumption of dairy products, particularly dairy protein, can decrease the prevalence of metabolic disorders, including type 2 diabetes. One mechanism that has been proposed is milk protein effects on increasing insulin secretion. (See Chapter 19 for an expansion of this topic.)\n\nThe potential for the discovery of new peptides is high, and it would seem reasonable to suspect that careful screening of digestion from various segments of the gastrointestinal tract may prove to be a valuable approach for this discovery process.\n\n#### Existing Variation in Bovine Milk Proteins and the Impact on Expression Function and Milk Quality: Experimental Modifications of Bovine Milk Proteins\n\nAs described above, a number of genetic variants of the major bovine milk proteins have been observed and characterized. These variations range from minor amino acid substitutions to larger deletions. Many studies suggest that the presence or absence of a particular variant expressed within an individual may be associated with altered production or processing quality of milk (see the reviews of Jakob & Puhan 1992; Martin et al., 2002; Caroli et al., 2009). Many of these studies are based on statistical associations, but others have been well characterized to elucidate cause\u2212effect relationships. These naturally occurring variations in milk protein amino acid sequences serve as examples for the breadth of potential that modification of milk proteins may have on milk production and quality.\n\nThe relationship between genetic variants of bovine milk proteins and production characteristics has been extensively reviewed. In particular, variation in the \u03ba-casein and \u03b2-Lg genes has significant impact on milk composition (Hill, 1993; Hill et al., 1997). The characteristics observed to be associated with milk protein polymorphism include first lactation milk production, protein production and fat percentage in Californian Jersey cows (Hill, 1993; Ojala et al., 1997); milk and protein yield and fat content in Finnish Ayrshire cows (Ikonen et al., 2001); milk, protein, and fat yield in Holstein and Ayrshire cows (Lin et al., 1986); processing and manufacturing properties (Hill et al., 1997; Manderson et al., 1997; Creamer et al., 2004); the effect of nutritional regimens on milk composition (Mackle et al., 1999; Auldist et al., 2000); somatic cell count (Ng-Kwai-Hang et al., 1987); total solids and milk protein profile (McLean et al., 1984); and lifetime performance (Lin et al., 1989).\n\nYet, despite this body of evidence, milk protein genotype is only slowly being introduced into selection systems to enhance genetic gain for milk production characteristics. This is due to the concern over a potential reduction in genetic diversity, through reducing the pool of sires to select from, based on a few loci. This is now changing rapidly with the introduction and widespread use of DNA- or genomic selection-based methods (Kemper and Goddard, 2012). By using this approach, both positive and negative effects across the genome can be evaluated; therefore, at least in theory, enabling the selection of lines with altered protein characteristics without affecting overall performance or longer term genetic diversity.\n\nThe B variant of bovine \u03b1S1-casein is the most abundant variant in Western dairy populations, possibly because of indirect selection as other variants may in some way be associated with decreased \u03b1S1-casein synthesis, such as the A variant which may be subject to a potential decrease in translational efficiency because of observed polymorphism at a polyadenylation signal site (McKnight et al., 1989) or because of promoter polymorphisms that may alter transcriptional efficiency (Lum et al., 1997; Prinzenberg et al., 2003). Similarly, animals that are observed to have a lower bovine \u03b1S1-casein protein concentration in milk (heterozygous for the G allele) exhibit a lower casein content and a slower clot formation time, yet display a faster curd-firming time and a higher curd firmness (Mariani et al., 2001). In contrast to bovine \u03b1S1-casein, although there are four known variants of the bovine \u03b1S2-casein protein, few studies have shown any relationships between the genotype of this protein and milk production or quality traits.\n\nThe A1 and A2 variants of bovine \u03b2-casein are present in modern dairy populations at high frequencies, and the A2 variant has been shown in defined populations of Danish dairy cattle breeds to be associated with higher milk, fat, and protein yields when homozygous (Bech & Kristiansen, 1990). Some controversy surrounds the A2 protein genotype, as it has been suggested that human populations who consume milk containing higher concentrations of the A2 variant exhibit a lower incidence of cardiovascular disease, a lower incidence of type 1 diabetes, and decreased severity of symptoms of neurological diseases because the A2 variant does not liberate the opioid bioactive peptide casomorphin 7 upon proteolytic digestion (Laugesen & Elliott, 2003; Tailford et al., 2003; Bell et al., 2006; Kaminski et al., 2007). In a review of evidence, however, Truswell (2005) and, more recently, Clemens (2011) suggested that there was limited evidence to suggest that milk containing the A2 variant of \u03b2-casein had any significant human health advantage. Indeed, human trials suggested that there was no differential effect between dietary products containing either the A1 variant or the A2 variant on human plasma cholesterol concentrations (Venn et al., 2006) or cardiovascular health (Chin-Dusting et al., 2006).\n\nWith the significant structural role of bovine \u03ba-casein in the casein micelle, it is not surprising that there are only two common variants, A and B, in modern dairy populations, although up to nine other variants have been characterized in bovine species (Farrell et al., 2004). A number of studies have been conducted to investigate associations between \u03ba-casein protein variants and milk production and quality characteristics. Of all the genetic polymorphisms of dairy cattle affecting milk composition, the \u03ba-casein genotype is one of the more significant, with association differences identified between genotype and lifetime production (Lin et al., 1989), concentrations of individual milk proteins (McLean et al., 1984), protein yield and percentage (Tsiaras et al., 2005), as well as cheese production characteristics, including rennet clotting time, curd formation, and coagulation strength (Pagnacco & Caroli, 1987; Bittante et al., 2012).\n\nOf the 12 identified bovine \u03b2-Lg variants, only the A and B variants occur at high frequencies in dairy cow populations. The B variant has been associated with higher casein percentage (Lund\u00e9n et al., 1997; Berry et al., 2010), higher total solids (McLean et al., 1984), and higher milk yield, fat yield, fat percentage, and lactose yield (Tsiaras et al., 2005), whereas the A variant is associated with higher whey protein content and lower casein content (Auldist et al., 2000). This expression difference has been attributed to promoter variation affecting allele-specific transcription factor binding (Lum et al., 1997). The B variant association with the casein composition of milk therefore has implications for milk quality for cheese production (Boland & Hill, 2001). Of the three bovine \u03b1-La variants, the B variant is the most common in modern dairy populations, yet the A and B variants differ by a single amino acid residue. The role of this protein in lactose biosynthesis is such that there seem to be little genetic divergence and little function or associated differences observed between the common variants.\n\n#### Experimental Modifications of Bovine Milk Proteins\n\nThe experimental manipulation of patterns of expression or characteristics of bovine milk proteins has previously been reviewed (Bremel et al., 1989; Yom & Bremel, 1993; Clark, 1996). Both overexpression and impairment or 'knockout' of milk proteins have been conducted predominantly in rodent models, presumably because of the expense and technical challenges of conducting studies in bovine or other ruminant species. Although observations from experiments conducted in model species must be considered in context, valuable insight into the potential for future manipulation of bovine milk proteins in ruminant species has been gained.\n\nAlthough a number of experiments have been conducted to either overexpress or inhibit expression of milk proteins in mice and other rodents, only a few have further characterized the milk to assess the impact of manipulation of expression profiles on the quality of milk for dairy product manufacture. A transgenic mouse model overexpressing bovine \u03ba-casein (Guti\u00e9rrez-Ad\u00e1n et al., 1989) exhibited no changes in milk protein concentration, yet milk from these mice exhibited a smaller micelle size and a tendency toward stronger curd characteristics. Conversely, a mouse model in which the endogenous \u03ba-casein was suppressed exhibited a loss in micelle stability, resulting in casein precipitation in alveolar lumens, which prevented lactation (Shekar et al., 2006). These investigations highlight the significance of the role of \u03ba-casein in the tertiary structure of protein in milk regardless of species. In comparison, the manipulation of \u03b2-casein expression results in less dramatic consequences. The overexpression of bovine \u03b2-casein in a mouse model (Hitchin et al., 1996) had little observed effect on lactation, although the processing quality of milk was not assessed in this study. In another study, the inhibition of endogenous murine \u03b2-casein (Kumar et al., 1994) changed micelle size and reduced pup growth.\n\nSimilarly, in cloned transgenic cattle in which both bovine \u03b2-casein and \u03ba-casein were overexpressed (Brophy et al., 2003), milk levels of both proteins were increased, demonstrating that milk protein composition can be altered in large ruminants using recombinant gene technologies. Subsequent analysis revealed that the expression of additional copies of the casein genes resulted in a color change in the milk (Laible et al., 2007). The higher expression of \u03ba-casein resulted as expected in smaller casein micelles, which may explain the color change. The smaller micelles also had an effect on cheese making predicted to be because of a more rapid separation of the fat globules and therefore a lower rate of incorporation into the cheese. There was a slight change in amino acid composition of the cheese consistent with the change in \u03ba-casein content. The authors stated that the fat, lactose, and mineral contents of the transgenic milk were within normal ranges.\n\nThe functionality of caseins can be altered through enzymatic modification of the proteins, for example, through dephosphorylation. This results in increased pepsin hydrolysis, which in turn alters digestibility (Li-Chan & Nakai, 1989) and potentially the release of bioactive peptides. Importantly, none of the studies discussed identified defects or significant changes to mammary development or structure, suggesting that manipulation of milk proteins may not have undesirable effects on the mammary gland itself, compromising either the potential to lactate or animal health.\n\nThe whey proteins have also been investigated for manipulation to alter the processing characteristics of milk. Because of its role in the synthesis of lactose and the implications for manipulation of osmotic potential of milk, \u03b1-La has been a target for manipulation. Mice transgenic for expression of bovine \u03b1-La exhibited increased lactose concentration in milk and a slight increase in pup growth rate (Boston et al., 2001). Similarly, transgenic pigs overexpressing bovine \u03b1-La produced milk with lower total solids, higher milk yield in early lactation, higher lactose concentration, and increased litter growth rates (Noble et al., 2002). In contrast, a mouse model of \u03b1-La deficiency resulted in milk that was so viscous that pups were not able to suckle effectively (Stinnakre et al., 1994). Bovine \u03b1-La itself has also been modified to alter its characteristics, and various mutations have resulted in changes in affinity for galactosyltransferase, increased glucose binding (Grobler et al., 1994), and changes in molten globule conformation (Uchiyama et al., 1995).\n\nThe \u03b2-Lg protein has also been evaluated experimentally. Work describing the small RNA-mediated knockdown has been referred to earlier in this chapter, in the section entitled Transgenic Technologies for Milk Manipulation (Jabed et al., 2012). Overexpression in transgenic mice resulted in normal mammary physiology and therefore normal milk secretion and pup growth (Hyttinen et al., 1998; Guti\u00e9rrez-Ad\u00e1n et al., 1999). However, a similar study in which ovine \u03b2-Lg was overexpressed reported an increase in total protein content (Simons et al., 1987). Mutants of bovine \u03b2-Lg have also been studied, with variation in thermal stability (Cho et al., 1994), rate of secretion (Katakura et al., 1999), and rate of denaturation and digestion (Jayat et al., 2004) being reported.\n\n#### Adding Value to Milk through the Use of Milk Protein Genomics\n\nThe potential value of milk as a source of animal protein varying in characteristics that promote human health has been demonstrated by the range of bioactivities residing within the mammary protein phenome. The ease with which dairy products are distributed to consumers regardless of their socioeconomic status also makes this an ideal vehicle for the administration of therapeutics. The hyperimmune nature of colostrum, containing up to 40% by weight of immunoglobulin, suggests that immunization of cows against specific pathogens may provide a rich source of therapeutic antibodies. As human colostrum contains antibodies reactive to colonization factors 1 and 2 of enterotoxigenic Escherichia coli (Correa et al., 2006), thereby preventing diarrhea in infants, the production of similar antibodies in cows seemed to be a logical commercial target. Although progress in this area has been relatively slow, two recent studies suggest the potential of the production of antibodies in milk. Kramski et al. (2012) immunized cows with HIV-1 gp140 envelope (Env) oligomers and found production of a very high anti-gp 140 IgG titer in serum and colostrum. This may ultimately lead to a topical HIV-1 microbicide. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 transferred from cattle to humans is an important cause of intestinal disease and hemolytic uremic syndrome, which is fatal in 5\u201310% of cases. Vaccination in cattle decreases bacterial shedding and can also induce antibodies in colostrum, which may protect calves from colonization (Rabinovitz et al., 2012).\n\nHowever, perhaps the greatest potential that milk proteins and peptides have for human health is through the addition of whey protein and casein peptides to foods to increase their functionality. Estimates of world whey protein production exceed 0.5 million tons, which provides a resource that should be used more effectively than by simply disposing of it as a liquid waste or as an animal feed. Considerable progress has been made in developing methods for separation and then purification of specific proteins from the whey fraction. Native immunoglobulins, lactoferrin, lactoperoxidase, \u03b1-La, and \u03b2-Lg have all been recovered in industrial quantities. The use of recombinant technologies may ultimately provide a cost-competitive alternative supply of these proteins.\n\nIdeally, a milk enriched in peptides promoting immune function, controlling blood pressure, acting as a bacteriostat, and minimizing oxidative stress and cancer risk, while at the same time relieving depression and preventing dental caries, would seem to have the makings of a highly valuable functional food. Combining this with an enrichment with n-3 fatty acids thought to increase insulin sensitivity and therefore prevent diabetes, together with certain milk carbohydrates capable of improving cognition, adds greatly to a product that already serves as a rich source of amino acids and energy to promote normal growth processes. Manipulation of these proteins in milk will inevitably occur in the factory and potentially in the cow. The challenge remains to turn this speculation into a commercial reality for the benefit of societies in both the developed and developing world.\n\n## Conclusion\n\nThe bovine milk proteins form the basis for a global industry in dairy products and as such have been intensively studied by both classical deconstructive observation and in dynamic whole animal systems utilizing postgenomic or functional genomics approaches. From these studies we have a greater understanding of the roles of these proteins in milk, their biological significance in the neonate, and the biochemical characteristics of bovine milk proteins that affect the efficiencies of manufacturing processes.\n\nThe use of bovine milk proteins and their peptides as bioactivities in clinical trials is an exciting new development. The potential of milk proteins, other than as a simple source of nutrition, is slowly being realized. This success will likely encourage further work in this area. The development of the milk-based nutraceutical business has been slowed by the requirements of some regulators to provide clinical evidence for claims of efficacy. This is not an unreasonable request, and developing a product that meets these requirements will ensure market share. The increasing world demand for high-quality dairy protein has perhaps delayed some research initiatives on product differentiation. However, research results indicate that considerable value can be gained from the protein components of milk\n\nThe bovine genome project has provided the underpinning knowledge necessary to develop tools for accelerated selection of superior animals in both production and specific milk composition. These tools are still in an early phase of development, but combined with accelerated breeding methods it is relatively straightforward and time efficient to generate specific herds for pharmaceutical, nutraceutical, or product requirements.\n\nThe further development of efficient methods of transgene integration or gene knockout in theory means that any compositional change could be made relatively quickly and cheaply. These approaches to modifying food are not yet accepted but may ultimately be desirable, depending on the value of the end product to human health and nutrition.\n\n# References\n\nAuldist MJ ,  Thomson NA ,  Mackle TR ,  Hill JP ,  Prosser CG .  Effects of pasture allowance on the yield and composition of milk from cows of different \u03b2-lactoglobulin phenotypes .  _Journal of Dairy Science_.  2000 ;83 : 2069 \u2013 2074 .\n\nBansal PS ,  Grieve PA ,  Marschke RJ ,  Daly NL ,  McGhie E ,  Craik DJ ,  Alewood PF .  Chemical synthesis and structure elucidation of bovine kappa-casein (1-44) .  _Biochemical and Biophysical Research Communications_.  2006 ;340 : 1098 \u2013 1103 .\n\nBech AM ,  Kristiansen KR .  Milk protein polymorphism in Danish dairy cattle and the influence of genetic variants on milk yield .  _Journal of Dairy Research_.  1990 ;57 : 53 \u2013 62 .\n\nBeg OU ,  von Bahr-Lindstrom H ,  Zaidi ZH ,  Jornvall H .  A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides .  _European Journal of Biochemistry_.  1986 ;159 : 195 \u2013 201 .\n\nBell SJ ,  Grochoski GT ,  Clarke AJ .  Health implications of milk containing beta-casein with the A2 genetic variant .  _Critical Reviews in Food Science and Nutrition_.  2006 ;46 : 93 \u2013 100 .\n\nBerry S ,  Coppieters W ,  Davis S ,  Burrett A ,  Thomas N ,  Palmer D ,  Kelly V ,  Obolonkin V ,  Sanders K ,  Spelman R ,  Georges M ,  Lehnert K ,  Snell R .  A triad of highly divergent polymeric immunoglobulin receptor (PIGR) haplotypes with major effect on IgA concentration in bovine milk .  _PLoS One_.  2013 ;8 : e57219 .\n\nBerry SD ,  Davis SR ,  Beattie EM ,  Thomas NL ,  Burrett AK ,  Ward HE ,  Stanfield AM ,  Biswas M ,  Ankersmit-Udy AE ,  Oxley PE ,  Barnett JL ,  Pearson JF ,  van der Does Y ,  MacGibbon AH ,  Spelman RJ ,  Lehnert K ,  Snell RG .  Mutation in bovine beta-carotene oxygenase 2 affects milk color .  _Genetics_.  2009 ;182 : 923 \u2013 926 .\n\nBerry SD ,  Lopez-Villalobos N ,  Beattie EM ,  Davis SR ,  Adams LF ,  Thomas NL ,  Ankersmit-Udy AE ,  Stanfield AM ,  Lehnert K ,  Ward HE ,  Arias JA ,  Spelman RJ ,  Snell RG .  Mapping a quantitative trait locus for the concentration of \u03b2-lactoglobulin in milk, and the effect of \u03b2-lactoglobulin genetic variants on the composition of milk from Holstein-Friesian x Jersey crossbred cows .  _N Z Vet J_.  2010 ;58 : 1 \u2013 5 .\n\nBirn H .  The kidney in vitamin B12 and folate homeostasis: Characterization of receptors for tubular uptake of vitamins and carrier proteins .  _Am J Physiol Renal Physiol_.  2006 ;291 (1) : F22 \u2013 36 .\n\nBoland MJ ,  Hill JP .  Genetic selection to increase cheese yield\u2014the Kaikoura experience .  _Australian J Dairy Technol._ 2001 ;56 : 171 \u2013 176 .\n\nBittante G ,  Penasa M ,  Cecchinato A .  Invited review: Genetics and modeling of milk coagulation properties .  _J Dairy Sci_.  2012 ;95 : 6843 \u2013 6870 .\n\nBoston WS ,  Bleck GT ,  Conroy JC ,  Wheeler MB ,  Miller DJ .  Short communication: effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth .  _Journal of Dairy Science_.  2001 ;84 : 620 \u2013 622 .\n\nBounous G ,  Gold P .  The biological activity of undenatured dietary whey proteins: role of glutathione .  _Clinical and Investigative Medicine_.  1991 ;14 : 296 \u2013 309 .\n\nBremel RD ,  Yom HC ,  Bleck GT .  Alteration of milk composition using molecular genetics .  _Journal of Dairy Science_.  1989 ;72 : 2826 \u2013 2833 .\n\nBrew K ,  Castellino FJ ,  Vanaman TC ,  Hill RL .  The complete amino acid sequence of bovine alpha-lactalbumin .  _Journal of Biological Chemistry_.  1970 ;245 : 4570 \u2013 4582 .\n\nBrignon G ,  Chtourou A ,  Ribadeau-Dumas B .  Does \u03b2-lactoglobulin occur in human milk? .  _Journal of Dairy Research_.  1985 ;52 : 249 \u2013 254 .\n\nBrophy B ,  Smolenski G ,  Wheeler T ,  Wells D ,  L'Huillier P ,  Laible G .  Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein .  _Nature Biotechnology_.  2003 ;21 : 157 \u2013 162 .\n\nCampbell SM ,  Rosen JM ,  Hennighausen LG ,  Strech-Jurk U ,  Sippel AE .  Comparison of the whey acidic protein genes of the rat and mouse .  _Nucleic Acids Research_.  1984 ;12 : 8685 \u2013 8697 .\n\nCane KN ,  Arnould JP ,  Nicholas KR .  Characterisation of proteins in the milk of fur seals .  _Comparative Biochemistry and Physiology \u2014Part B: Biochemistry and Molecular Biology_.  2005 ;141 : 111 \u2013 120 .\n\nC\u00e1novas A ,  Rincon G ,  Islas-Trejo A ,  Jimenez-Flores R ,  Laubscher A ,  Medrano JF .  RNA sequencing to study gene expression and single nucleotide polymorphism variation associated with citrate content in cow milk .  _J Dairy Science_.  2013 ;96 : 2637 \u2013 2648 .\n\nCaroli AM ,  Chessa S ,  Erhardt GJ .  Invited review: Milk protein polymorphisms in cattle: effect on animal breeding and human nutrition .  _Journal of Dairy Science_.  2009 ;92 : 5335 \u2013 5352 .\n\nChilders CP ,  Reese JT ,  Sundaram JP ,  Vile DC ,  Dickens CM ,  Childs KL ,  Salih H ,  Bennett AK ,  Hagen DE ,  Adelson DL ,  Elsik CG .  Bovine Genome Database: integrated tools for genome annotation and discovery .  _Nucleic Acids Res_.  2011 ;39 : D830 \u2013 834 .\n\nChin-Dusting J ,  Shennan J ,  Jones E ,  Williams C ,  Kingwell B ,  Dart A .  Effect of dietary supplementation with beta-casein A1 or A2 on markers of disease development in individuals at high risk of cardiovascular disease .  _British Journal of Nutrition_.  2006 ;95 : 136 \u2013 144 .\n\nCho Y ,  Gu W ,  Watkins S ,  Lee SP ,  Kim TR ,  Brady JW ,  Batt CA .  Thermostable variants of bovine \u03b2-lactoglobulin .  _Protein Engineering_.  1994 ;7 : 263 \u2013 270 .\n\nClark AJ .  Genetic modification of milk proteins .  _American Journal of Clinical Nutrition_.  1996 ;63 : 633S \u2013 638S .\n\nClemens RA .  Milk A1 and A2 peptides and diabetes .  _Nestle Nutr Workshop Ser Pediatr Program_.  2011 ;67 : 187 \u2013 195 .\n\nCorrea S ,  Palmeira P ,  Carneiro-Sampaio MMS ,  Nishimura LS ,  Guth BEC .  Human colostrum contains antibodies reactive to colonization factors I and II of enterotoxigenic Escherichia coli .  _FEMS Immunology and Medical Microbiology_.  2006 ;47 : 199 \u2013 216 .\n\nCreamer LK ,  Nilsson HC ,  Paulsson MA ,  Coker CJ ,  Hill JP ,  Jim\u00e9nez-Flores R .  Effect of genetic variation on the tryptic hydrolysis of bovine \u03b2-lactoglobulin A, B, and C .  _Journal of Dairy Science_.  2004 ;87 : 4023 \u2013 4032 .\n\nCross KJ ,  Huq NL ,  Reynolds EC .  Casein phosphopeptides in oral health\u2014chemistry and clinical applications .  _Current Pharmaceutical Design_.  2007 ;13 : 793 \u2013 798 .\n\nDalma-Weiszhausz DD ,  Warrington J ,  Tanimoto EY ,  Miyada CG .  The Affymetrix GeneChip platform: an overview .  _Methods in Enzymology_.  2006 ;410 : 3 \u2013 28 .\n\nDemmer J ,  Stasiuk SJ ,  Grigor MR ,  Simpson KJ ,  Nicholas KR .  Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula) .  _Biochimica et Biophysica Acta_.  2001 ;1522 : 187 \u2013 194 .\n\nDevinoy E ,  Hubert C ,  Jolivet G ,  Thepot D ,  Clergue N ,  Desaleux M ,  Dion M ,  Servely JL ,  Houdebine LM .  Recent data on the structure of rabbit milk protein genes and on the mechanism of the hormonal control of their expression .  _Reproduction Nutrition Development_.  1988 ;28 : 1145 \u2013 1164 .\n\nElsik, C.G., Tellam, R.L., Worley, K.C., The Bovine Genome Sequencing and Analysis Consortium, 2009. Report: The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution. Science 324, 522\u2013528. DOI: 10.1126\/science.1169588.\n\nFarrell Jr HM ,  Jimenez-Flores R ,  Bleck GT ,  Brown EM ,  Butler JE ,  Creamer LK ,  Hicks CL ,  Hollar CM ,  Ng-Kwai-Hang KF ,  Swaisgood HE .  Nomenclature of the proteins of cows' milk\u2014sixth revision .  _Journal of Dairy Science_.  2004 ;87 : 1641 \u2013 1674 .\n\nFerranti P ,  Traisci MV ,  Picariello G ,  Nasi A ,  Boschi V ,  Siervo M ,  Falconi C ,  Chianese L ,  Addeo F .  Casein proteolysis in human milk: Tracing the pattern of casein breakdown and the formation of potential bioactive peptides .  _Journal of Dairy Research_.  2004 ;71 : 74 \u2013 87 .\n\nFilipowicz W ,  Bhattacharyya SN ,  Sonenberg N .  Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? .  _Nat Rev Genet_.  2008 ;9 : 102 \u2013 114 .\n\nFolch JM ,  Coll A ,  Sanchez A .  Complete sequence of the caprine \u03b2-lactoglobulin gene .  _Journal of Dairy Science_.  1994 ;77 : 3493 \u2013 3497 .\n\nFukushima Y ,  Kawata Y ,  Onda T ,  Kitagawa M .  Consumption of cow milk and egg by lactating women and the presence of \u03b2-lactoglobulin and ovalbumin in breast milk .  _American Journal of Clinical Nutrition_.  1997 ;65 : 30 \u2013 35 .\n\nGaj T ,  Gersbach CA ,  Barbas CF .  ZFN, TALEN, and CRISPR\/Cas-based methods for genome engineering .  _Trends Biotechnol_.  2013 ;31 : 397 \u2013 405 .\n\nGarcia-Montoya I ,  Gonzalez-Chavez SA ,  Salazar-Martinez J ,  Arevalo-Gallegos S ,  Sinagawa-Garcia S ,  Rascon-Cruz Q .  Expression and characterization of recombinant bovine lactoferrin in E. coli .  _Biometals_.  2013 ;26 : 113 \u2013 122 .\n\nGarrels W ,  Ivics Z ,  Kues WA .  Precision genetic engineering in large mammals .  _Trends Biotechnol_.  2012 ;30 : 386 \u2013 393 .\n\nGibbs RA ,  Taylor JF ,  Van Tassell CP ,  Barendse W ,  Eversole KA ,  Gill CA ,  Green RD ,  Hamernik DL ,  Kappes SM ,  Lien S ,  Matukumalli LK ,  McEwan JC ,  Nazareth LV ,  Schnabel RD ,  Weinstock GM ,  Wheeler DA ,  Ajmone-Marsan P ,  Boettcher PJ ,  Caetano AR ,  Garcia JF ,  Hanotte O ,  Mariani P ,  Skow LC ,  Sonstegard TS ,  Williams JL ,  Diallo B ,  Hailemariam L ,  Martinez ML ,  Morris CA ,  Silva LO ,  Spelman RJ ,  Mulatu W ,  Zhao K ,  Abbey CA ,  Agaba M ,  Araujo FR ,  Bunch RJ ,  Burton J ,  Gorni C ,  Olivier H ,  Harrison BE ,  Luff B ,  Machado MA ,  Mwakaya J ,  Plastow G ,  Sim W ,  Smith T ,  Thomas MB ,  Valentini A ,  Williams P ,  Womack J ,  Woolliams JA ,  Liu Y ,  Qin X ,  Worley KC ,  Gao C ,  Jiang H ,  Moore SS ,  Ren Y ,  Song XZ ,  Bustamante CD ,  Hernandez RD ,  Muzny DM ,  Patil S ,  San Lucas A ,  Fu Q ,  Kent MP ,  Vega R ,  Matukumalli A ,  McWilliam S ,  Sclep G ,  Bryc K ,  Choi J ,  Gao H ,  Grefenstette JJ ,  Murdoch B ,  Stella A ,  Villa-Angulo R ,  Wright M ,  Aerts J ,  Jann O ,  Negrini R ,  Goddard ME ,  Hayes BJ ,  Bradley DG ,  Barbosa da Silva M ,  Lau LP ,  Liu GE ,  Lynn DJ ,  Panzitta F ,  Dodds KG ,  Bovine HapMap Consortium .  Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds .  _Science_.  2009 ;324 : 528 \u2013 532 : doi: 10.1126\/science.1167936 .\n\nGirard CL ,  Lapierre H ,  Matte JJ ,  Lobley GE .  Effects of dietary supplements of folic acid and rumen-protected methionine on lactational performance and folate metabolism of dairy cows .  _J Dairy Sci._ 2005 ;88 (2) : 660 \u2013 670 .\n\nGirard CL ,  Matte JJ ,  Temblay GF .  Serum folates in gestating and lactating dairy cows .  _J Dairy Sci._ 1989 ;72 (12) : 3240 \u2013 3246 .\n\nGirard CL ,  Matte JJ .  Dietary supplements of folic acid during lactation: Effects on the performance of dairy cows .  _J Dairy Sci_.  1998 ;81 (5) : 1412 \u2013 1419 .\n\nGirard C ,  Matte J .  Folic acid and vitamin B12 requirements of dairy cows: A concept to be revised .  _Livestock Production Science_.  2005 ;98 : 123 \u2013 133 .\n\nGraulet B ,  Matte JJ ,  Desrochers A ,  Doepel L ,  Palin MF ,  Girard CL .  Effects of dietary supplements of folic acid and vitamin B12 on metabolism of dairy cows in early lactation .  _J Dairy Sci._ 2007 ;90 (7) : 3442 \u2013 3455 .\n\nGrisart B ,  Coppieters W ,  Farnir F ,  Karim L ,  Ford C ,  Berzi P ,  Cambisano N ,  Mni M ,  Reid S ,  Simon P ,  Spelman R ,  Georges M ,  Snell R .  Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition .  _Genome Res_.  2002 ;12 : 222 \u2013 231 .\n\nGrobler JA ,  Wang M ,  Pike AC ,  Brew K .  Study by mutagenesis of the roles of two aromatic clusters of alpha-lactalbumin in aspects of its action in the lactose synthase system .  _Journal of Biological Chemistry_.  1994 ;269 : 5106 \u2013 5114 .\n\nGuntupalli K ,  Dean N ,  Morris PE ,  Bandi V ,  Margolis B ,  Rivers E ,  Levy M ,  Lodato RF ,  Ismail PM ,  Reese A ,  Schaumberg JP ,  Malik R ,  Dellinger RP .  A phase 2 randomized, double-blind, placebo-controlled study of the safety and efficacy of talactoferrin in patients with severe sepsis .  _Crit Care Med_.  2013 ;41 : 706 \u2013 716 .\n\nGuti\u00e9rrez-Ad\u00e1n A ,  Maga EA ,  Meade H ,  Shoemaker CF ,  Medrano JF ,  Anderson GB ,  Murray JD .  Alterations of the physical characteristics of milk from transgenic mice producing bovine kappa-casein .  _Journal of Dairy Science_.  1989 ;79 : 791 \u2013 799 .\n\nGuti\u00e9rrez-Ad\u00e1n A ,  Maga EA ,  Behboodi E ,  Conrad-Brink JS ,  MacKinlay AG ,  Anderson GB ,  Murray JD .  Expression of bovine \u03b2-lactoglobulin in the milk of transgenic mice .  _Journal of Dairy Research_.  1999 ;66 : 289 \u2013 294 .\n\nHajjoubim S ,  Rival-Gervier S ,  Hayes H ,  Floriot S ,  Eggen A ,  Piumi F ,  Chardon P ,  Houdebine LM ,  Thepot D .  Ruminants genome no longer contains whey acidic protein gene but only a pseudogene .  _Gene_.  2006 ;370 : 104 \u2013 112 .\n\nHall AJ ,  Masel A ,  Bell K ,  Halliday JA ,  Shaw DC ,  VandeBerg JL .  Characterization of baboon (Papio hamadryas) milk proteins .  _Biochemical Genetics_.  2001 ;39 : 59 \u2013 71 .\n\nHawken RJ ,  Barris WC ,  McWilliam SM ,  Dalrymple BP .  An interactive bovine in silico SNP database (IBISS) .  _Mammalian Genome_.  2004 ;15 : 819 \u2013 827 .\n\nHennighausen LG ,  Sippel AE .  Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins .  _Nucleic Acids Research_.  1982 ;10 : 2677 \u2013 2684 .\n\nHernandez-Ledesma B ,  Recio I ,  Amigo L .  \u03b2-Lactoglobulin as source of bioactive peptides .  _Amino Acids_.  2008 ;35 : 257 \u2013 265 .\n\nHill JP .  The relationship between \u03b2-lactoglobulin phenotypes and milk composition in New Zealand dairy cattle .  _J. Dairy Sci._ 1993 ;76 : 281 \u2013 286 .\n\nHill JP ,  Boland MJ ,  Smith AF .  The effect of \u03b2-lactoglobulin variants on milk powder manufacture and properties .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 372 \u2013 394 : IDF S. 9702 .\n\nHiraoka Y ,  Segawa T ,  Kuwajima K ,  Sugai S ,  Murai N .  \u03b1-Lactalbumin: A calcium metalloprotein .  _Biochemical and Biophysical Research Communications_.  1980 ;95 : 1098 \u2013 1104 .\n\nHitchin E ,  Stevenson EM ,  Clark AJ ,  McClenaghan M ,  Leaver J .  Bovine beta-casein expressed in transgenic mouse milk is phosphorylated and incorporated into micelles .  _Protein Expression and Purification_.  1996 ;7 : 247 \u2013 252 .\n\nHoheisel JD .  Microarray technology: Beyond transcript profiling and genotype analysis .  _Nat Rev Genet_.  2006 ;7 : 200 \u2013 210 .\n\nHyttinen JM ,  Korhonen VP ,  Hiltunen MO ,  My\u00f6h\u00e4nen S ,  J\u00e4nne J .  High-level expression of bovine \u03b2-lactoglobulin gene in transgenic mice .  _Journal of Biotechnology_.  1998 ;61 : 191 \u2013 198 .\n\nIkonen T ,  Bovenhuis H ,  Ojala M ,  Ruottinen O ,  Georges M .  Associations between casein haplotypes and first lactation milk production traits in Finnish Ayrshire cows .  _Journal of Dairy Science_.  2001 ;84 : 507 \u2013 514 .\n\nJabed A ,  Wagner S ,  McCracken J ,  Wells DN ,  Laible G .  Targeted microRNA expression in dairy cattle directs production of \u03b2-lactoglobulin-free, high-casein milk .  _Proc Natl Acad Sci U S A_.  2012 ;109 : 16811 \u2013 16816 .\n\nJakob E ,  Puhan Z .  Technological properties of milk as influenced by genetic polymorphism of milk proteins\u2014a review .  _International Dairy Journal_.  1992 ;2 : 157 \u2013 178 .\n\nJayat D ,  Gaudin JC ,  Chobert JM ,  Burova TV ,  Holt C ,  McNae I ,  Sawyer L ,  Haertl\u00e9 T .  A recombinant C121S mutant of bovine \u03b2-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating .  _Biochemistry_.  2004 ;43 : 6312 \u2013 6321 .\n\nJiang Z ,  Tian B ,  Brodkorb A ,  Huo G .  Production, analysis and in vivo evaluation of novel angiotensin-I-converting enzyme inhibitory peptides from bovine casein .  _Food Chemistry_.  2010 ;123 : 779 \u2013 786 .\n\nKaminski S ,  Cieslinska A ,  Kostyra E .  Polymorphism of bovine beta-casein and its potential effect on human health .  _Journal of Applied Genetics_.  2007 ;48 : 189 \u2013 198 .\n\nKatakura Y ,  Ametani A ,  Totsuka M ,  Nagafuchi S ,  Kaminogawa S .  Accelerated secretion of mutant \u03b2-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution .  _Biochimica et Biophysica Acta_.  1999 ;1432 : 302 \u2013 312 .\n\nKemper KE ,  Goddard ME .  Understanding and predicting complex traits: Knowledge from cattle .  _Hum Mol Genet_.  2012 ;21 : R45 \u2013 51 .\n\nKhatkar MS ,  Thomson PC ,  Tammen I ,  Raadsma HW .  Quantitative trait loci mapping in dairy cattle: Review and meta-analysis .  _Genetics Selection Evolution_.  2004 ;36 : 163 \u2013 190 .\n\nKoldovsky O .  The potential physiological significance of milk-borne hormonally active substances for the neonate .  _Journal of Mammary Gland Biology and Neoplasia_.  1996 ;1 : 317 \u2013 323 .\n\nKontopidis G ,  Holt C ,  Sawyer L .  Invited review: \u03b2-lactoglobulin: binding properties, structure, and function .  _Journal of Dairy Science_.  2004 ;87 : 785 \u2013 796 .\n\nKonuma T ,  Sakurai K ,  Goto Y .  Promiscuous binding of ligands by \u03b2-lactoglobulin involves hydrophobic interactions and plasticity .  _Journal of Molecular Biology_.  2007 ;368 : 209 \u2013 218 .\n\nKorhonen H ,  Pihlanto A .  Technological options for the production of health-promoting proteins and peptides derived from milk and colostrum .  _Current Pharmaceutical Design_.  2007 ;13 : 829 \u2013 843 .\n\nKramski M ,  Center RJ ,  Wheatley AK ,  Jacobson JC ,  Alexander MR ,  Rawlin G ,  Purcell DF .  Hyperimmune bovine colostrum as a low-cost, large-scale source of antibodies with broad neutralizing activity for HIV-1 envelope with potential use in microbicides .  _Antimicrob Agents Chemother_.  2012 ;56 : 4310 \u2013 4319 .\n\nKrimpenfort P ,  Rademakers A ,  Eyestone W ,  van der Schans A ,  van den Broek S ,  Kooiman P ,  Kootwijk E ,  Platenburg G ,  Pieper F ,  Strijker R ,  et al.  Generation of transgenic dairy cattle using 'in vitro' embryo production .  _Biotechnology_.  1991 ;9 : 844 \u2013 847 .\n\nKumar S ,  Clarke AR ,  Hooper ML ,  Horne DS ,  Law AJ ,  Leaver J ,  Springbett A ,  Stevenson E ,  Simons JP .  Milk composition and lactation of beta-casein-deficient mice .  _Proceedings of the National Academy of Sciences of the USA_.  1994 ;91 : 6138 \u2013 6142 .\n\nLaible G ,  Brophy B ,  Knighton D ,  Wells DN .  Compositional analysis of dairy products derived from clones and cloned transgenic cattle .  _Theriogenology_.  2007 ;67 : 166 \u2013 177 .\n\nLaugesen M ,  Elliott R .  Ischaemic heart disease, Type 1 diabetes, and cow milk A1 beta-casein .  _New Zealand Medical Journal_.  2003 ;116 : U295 .\n\nLefevre CM ,  Digby MR ,  Mailer S ,  Whitley JC ,  Strahm SJY ,  Nicholas KR .  Lactation transcriptomics in the Australian marsupial, Macropus eugenii: transcript sequencing and quantification .  _BMC Genomic_.  2007 ; : 417 \u2013 430 .\n\nLefevre CM ,  Sharp JA ,  Nicholas KR .  Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals .  _Reprod Fertil Dev_.  2009 ;21 : 1015 \u2013 1027 .\n\nLemay DG ,  Lynn DJ ,  Martin WF ,  Neville MC ,  Casey TM ,  Rincon G ,  Kriventseva EV ,  Barris WC ,  Hinrichs AS ,  Molenaar AJ ,  Pollard KS ,  Maqbool NJ ,  Singh K ,  Murney R ,  Zdobnov EM ,  Tellam RL ,  Medrano JF ,  German JB ,  Rijnkels M .  The bovine lactation genome: Insights into the evolution of mammalian milk .  _Genome Biology_.  2009 ;10 (4) : R43 .\n\nLi Z ,  Liu H ,  Jin X ,  Lo L ,  Liu J .  Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation .  _BMC Genomics_.  2012 ;13 : 731 .\n\nLi-Chan E ,  Nakai S .  Enzymic dephosphorylation of bovine casein to improve acid clotting properties and digestibility for infant formula .  _Journal of Dairy Research_.  1989 ;56 : 381 \u2013 390 .\n\nLin CY ,  McAllister AJ ,  Ng-Kwai-Hang KF ,  Hayes JF .  Effects of milk protein loci on first lactation production in dairy cattle .  _Journal of Dairy Science_.  1986 ;69 : 704 \u2013 712 .\n\nLin CY ,  McAllister AJ ,  Ng-Kwai-Hang KF ,  Hayes JF ,  Batra TR ,  Lee AJ ,  Roy GL ,  Vesely JA ,  Wauthy JM ,  Winter KA .  Relationships of milk protein types to lifetime performance .  _Journal of Dairy Science_.  1989 ;72 : 3085 \u2013 3090 .\n\nLindblad-Toh K ,  Garber M ,  Zuk O ,  Lin MF ,  Parker BJ ,  Washietl S ,  Kheradpour P ,  Ernst J ,  Jordan G ,  et al.  A high-resolution map of human evolutionary constraint using 29 mammals .  _Nature_.  2011 ;478 : 476 \u2013 482 .\n\nLittlejohn MD ,  Walker CG ,  Ward HE ,  Lehnert KB ,  Snell RG ,  Verkerk GA ,  Spelman RJ ,  Clark DA ,  Davis SR .  Effects of reduced frequency of milk removal on gene expression in the bovine mammary gland .  _Physiol Genomics 41_.  2010 ; : 21 \u2013 32 .\n\nLonnerdal B .  Recombinant human milk proteins. Nestle Nutrition Workshop Series .  _Paediatric Programme_.  2006 ;58 : 207 \u2013 215 .\n\nLum LS ,  Dovc P ,  Medrano JF .  Polymorphisms of bovine \u03b2-lactoglobulin promoter and differences in the binding affinity of activator protein-2 transcription factor .  _Journal of Dairy Science_.  1997 ;80 : 1389 \u2013 1397 .\n\nLund\u00e9n A ,  Nilsson M ,  Janson L .  Marked effect of \u03b2-lactoglobulin polymorphism on the ratio of casein to total protein in milk .  _Journal of Dairy Science_.  1997 ;80 : 2996 \u2013 3005 .\n\nMackle TR ,  Bryant AM ,  Petch SF ,  Hill JP ,  Auldist MJ .  Nutritional influences on the composition of milk from cows of different protein phenotypes in New Zealand .  _Journal of Dairy Science_.  1999 ;82 : 172 \u2013 180 .\n\nManderson GA ,  Hardman MJ ,  Creamer LK .  Spectroscopic examination of the heat-induced changes in \u03b2-lactoglobulin A, B and C .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 204 \u2013 211 : IDF S.I. 9702 .\n\nManzoni P ,  Stolfi I ,  Messner H ,  Cattani S ,  Laforgia N ,  Romeo MG ,  Bollani L ,  Rinaldi M ,  Gallo ,  et al.  Bovine lactoferrin prevents invasive fungal infections in very low birth weight infants: a randomized controlled trial .  _Pediatrics_.  2012 ;129 : 116 \u2013 123 .\n\nMariani BP ,  Summer A ,  Di Gregorio PD ,  Randoe A ,  Fossa E ,  Pecorari M .  Effects of the CSN1A(G) allele on the clotting time of cow milk and on the rheological properties of rennet-curd .  _Journal of Dairy Research_.  2001 ;68 : 63 \u2013 70 .\n\nMartin P ,  Szymanowska M ,  Zwierzchowski L ,  Leroux C .  The impact of genetic polymorphisms on the protein composition of ruminant milks .  _Reproduction Nutrition Development_.  2002 ;4 : 433 \u2013 459 .\n\nMcGregor RA ,  Poppitt SD .  Milk protein for improved metabolic health: A review of the evidence .  _Nutr Metab (Lond)_.  2013 ;10 : 46 .\n\nMcKnight RA ,  Jimenez-Flores R ,  Kang Y ,  Creamer LK ,  Richardson T .  Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine alpha s1-casein A from mammary tissue of a homozygous B variant cow .  _Journal of Dairy Science_.  1989 ;72 : 2464 \u2013 2473 .\n\nMcLean DM ,  Graham ER ,  Ponzoni RW ,  McKenzie HA .  Effects of milk protein genetic variants on milk yield and composition .  _Journal of Dairy Research_.  1984 ;51 : 531 \u2013 546 .\n\nMeisel H .  Biochemical properties of peptides encrypted in bovine milk proteins .  _Current Medicinal Chemistry_.  2005 ;12 : 1905 \u2013 1919 .\n\nMele M ,  Conte G ,  Castiglioni B ,  Chessa S ,  Macciotta NP ,  Serra A ,  Buccioni A ,  Pagnacco G ,  Secchiari P .  Stearoyl-coenzyme A desaturase gene polymorphism and milk fatty acid composition in Italian Holsteins .  _J Dairy Sci_.  2007 ;90 : 4458 \u2013 4465 .\n\nMenzies KK ,  Lefevre C ,  Sharp JA ,  Macmillan KL ,  Sheehy PA ,  Nicholas KR .  A novel approach identified the FOLR1 gene, a putative regulator of milk protein synthesis .  _Mamm Genome_.  2009 ;20 : 498 \u2013 503 .\n\nMercier JC ,  Vilotte JL .  Structure and function of milk protein genes .  _Journal of Dairy Science_.  1993 ;76 : 3079 \u2013 3098 .\n\nMiguel M ,  Contreras MM ,  Recio I ,  Aleixandre A .  ACE-inhibitory and antihypertensive properties of a bovine casein hydrolysate .  _Food Chemistry_.  2009 ;112 : 211 \u2013 214 .\n\nMoioli B ,  Contarini G ,  Avalli A ,  Catillo G ,  Orru L ,  De Matteis G ,  Masoero G ,  Napolitano F .  Short communication: Effect of stearoyl-coenzyme A desaturase polymorphism on fatty acid composition of milk .  _J Dairy Sci_.  2007 ;90 : 3553 \u2013 3558 .\n\nMoitzi C ,  Portnaya I ,  Glatter O ,  Ramon O ,  Danino D .  Effect of temperature on self-assembly of bovine beta-casein above and below isoelectric pH. Structural analysis by cryogenic-transmission electron microscopy and small-angle X-ray scattering .  _Langmuir_.  2008 ;24 : 3020 \u2013 3029 .\n\nMurray BA ,  Fitzgerald RJ .  Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production .  _Current Pharmaceutical Design_.  2007 ;13 : 773 \u2013 791 .\n\nNg-Kwai-Hang KF ,  Hayes JF ,  Moxley JE ,  Monardes HG .  Variation in milk protein concentrations associated with genetic polymorphism and environmental factors .  _Journal of Dairy Science_.  1987 ;70 : 563 \u2013 570 .\n\nNicholas K ,  Simpson K ,  Wilson M ,  Trott J ,  Shaw D .  The Tammar wallaby: A model to study putative autocrine-induced changes in milk composition .  _Journal of Mammary Gland Biology and Neoplasia_.  1997 ;2 : 299 \u2013 310 .\n\nNoble MS ,  Rodriguez-Zas S ,  Cook JB ,  Bleck GT ,  Hurley WL ,  Wheeler MB .  Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk .  _Journal of Animal Science_.  2002 ;80 : 1090 \u2013 1096 .\n\nOchoa TJ ,  Chea-Woo E ,  Baiocchi N ,  Pecho I ,  Campos M ,  Prada A ,  Valdiviezo G ,  Lluque A ,  Lai D ,  Cleary TG .  Randomized double-blind controlled trial of bovine lactoferrin for prevention of diarrhea in children .  _J Pediatr_.  2013 ;162 : 349 \u2013 356 . \nOjala M ,  Famula TR ,  Medrano JF .  Effects of milk protein genotypes on the variation for milk production traits of Holstein and Jersey cows in California .  _Journal of Dairy Science_.  1997 ;80 : 1776 \u2013 1785 .\n\nOndetti MA ,  Cushman DW .  Enzymes of the renin-angiotensin system and their inhibitors .  _Annual Review of Biochemistry_.  1982 ;51 : 283 \u2013 308 .\n\nPagnacco G ,  Caroli A .  Effects of casein and \u03b2-lactoglobulin genotypes on renneting properties of milk .  _Journal of Dairy Research_.  1987 ;54 : 479 \u2013 485 .\n\nPepe G ,  Tenore GC ,  Mastrocinque R ,  Stusio P ,  Campiglia P .  Potential anticarcinogenic peptides from bovine milk .  _J Amino Acids_.  2013 ; : Article ID: 939804 .\n\nPrinzenberg EM ,  Weimann C ,  Brandt H ,  Bennewitz J ,  Kalm E ,  Schwerin M ,  Erhardt G .  Polymorphism of the bovine CSN1S1 promoter: linkage mapping, intragenic haplotypes, and effects on milk production traits .  _Journal of Dairy Science_.  2003 ;86 : 2696 \u2013 2705 .\n\nRabinovitz BC ,  Gerhardt E ,  Tironi Farinati C ,  Abdala A ,  Galarza R ,  Vilte DA ,  Ibarra C ,  Cataldi A ,  Mercado EC .  Vaccination of pregnant cows with EspA, EspB, gamma-intimin, and Shiga toxin 2 proteins from Escherichia coli O157:H7 induces high levels of specific colostral antibodies that are transferred to newborn calves .  _J Dairy Sci_.  2012 ;95 : 3318 \u2013 3326 .\n\nRawson P ,  Stockum C ,  Peng L ,  Manivannan B ,  Lehnert K ,  Ward HE ,  Berry SD ,  Davis SR ,  Snell RG ,  McLauchlan D ,  Jordan TW .  Metabolic proteomics of the liver and mammary gland during lactation .  _J Proteomics_.  2012 ;75 : 4429 \u2013 4435 .\n\nReese JT ,  Childers CP ,  Sundaram JP ,  Dickens CM ,  Childs KL ,  Vile DC ,  Elsik CG .  Bovine Genome Database: supporting community annotation and analysis of the Bos taurus genome .  _BMC Genomics_.  2010 ;11 : 645 .\n\nRenfree MB ,  Papenfuss AT ,  Deakin JE ,  Lindsay J ,  Heider T ,  Belov K ,  Rens W ,  Waters PD ,  Pharo EA ,  et al.  Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development .  _Genome Biol_.  2011 ;12 : R81 .\n\nRicci-Cabello I ,  Herrera MO ,  Artacho R .  Possible role of milk-derived bioactive peptides in the treatment and prevention of metabolic syndrome .  _Nutr Rev_.  2012 ;70 : 241 \u2013 255 .\n\nRijnkels M .  Multispecies comparison of the casein gene loci and evolution of casein gene family .  _Journal of Mammary Gland Biology and Neoplasia_.  2002 ;7 : 327 \u2013 345 .\n\nSaito T .  Antihypertensive peptides derived from bovine casein and whey proteins .  _Adv Exp Med Biol_.  2008 ;606 : 295 \u2013 317 .\n\nSawyer L ,  Holt C .  The secondary structure of milk proteins and their biological function .  _Journal of Dairy Science_.  1993 ;76 : 3062 \u2013 3078 .\n\nSeki M ,  Matsura R ,  Iwamori T ,  Nukumi N ,  Yamanouchi K ,  Kano K ,  Naito K ,  Tojo H .  Identification of whey acidic protein (WAP) in dog milk .  _Exp Anim_.  2012 ;61 : 67 \u2013 70 .\n\nShah NP .  Effects of milk-derived bioactives: An overview .  _British Journal of Nutrition_.  2000 ;84 (Suppl 1) : S3 \u2013 S10 .\n\nSharp JA ,  Lefevre C ,  Nicholas KR .  Molecular evolution of monotreme and marsupial whey acidic protein genes .  _Evolution and Development_.  2007 ;9 : 378 \u2013 392 .\n\nShekar PC ,  Goel S ,  Rani SD ,  Sarathi DP ,  Alex JL ,  Singh S ,  Kumar S .  Kappa-casein-deficient mice fail to lactate .  _Proceedings of the National Academy of Sciences of the USA_.  2006 ;103 : 8000 \u2013 8005 .\n\nSherman MP ,  Petrak K .  Lactoferrin-enhanced anoikis: a defense against neonatal necrotizing enterocolitis .  _Med Hypotheses_.  2005 ;65 (3) : 478 \u2013 482 .\n\nSimons JP ,  McClenaghan M ,  Clark AJ .  Alteration of the quality of milk by expression of sheep \u03b2\\- lactoglobulin in transgenic mice .  _Nature_.  1987 ;328 : 530 \u2013 532 .\n\nSimpson KJ ,  Bird P ,  Shaw D ,  Nicholas K .  Molecular characterisation and hormone-dependent expression of the porcine whey acidic protein gene .  _Journal of Molecular Endocrinology_.  1998 ;20 : 27 \u2013 35 .\n\nSimpson KJ ,  Ranganathan S ,  Fisher JA ,  Janssens PA ,  Shaw DC ,  Nicholas KR .  The gene for a novel member of the whey acidic protein family encodes three four-disulfide core domains and is asynchronously expressed during lactation .  _Journal of Biological Chemistry_.  2000 ;275 : 23074 \u2013 23081 .\n\nSmolenski G ,  Haines S ,  Kwan FY ,  Bond J ,  Farr V ,  Davis SR ,  Stelwagen K ,  Wheeler TT .  Characterisation of host defence proteins in milk using a proteomic approach .  _Journal of Proteome Research_.  2007 ;6 : 207 \u2013 215 .\n\nStinnakre MG ,  Vilotte JL ,  Soulier S ,  Mercier JC .  Creation and phenotypic analysis of alpha-lactalbumin-deficient mice .  _Proceedings of the National Academy of Sciences of the USA_.  1994 ;91 : 6544 \u2013 6548 .\n\nTailford KA ,  Berry CL ,  Thomas AC ,  Campbell JH .  A casein variant in cow's milk is atherogenic .  _Atherosclerosis_.  2003 ;170 : 13 \u2013 19 .\n\nTellam R .  Capturing benefits from the bovine genome sequence .  _Australian Journal of Experimental Agriculture_.  2007 ;47 : 1039 \u2013 1050 .\n\nThreadgill DW ,  Womack JE .  Genomic analysis of the major bovine milk protein genes .  _Nucleic Acids Research_.  1990 ;18 : 6935 \u2013 6942 .\n\nTruswell AS .  The A2 milk case: a critical review .  _European Journal of Clinical Nutrition_.  2005 ;59 : 623 \u2013 631 .\n\nTsiaras AM ,  Bargouli GG ,  Banos G ,  Boscos CM .  Effect of kappa-casein and \u03b2-lactoglobulin loci on milk production traits and reproductive performance of Holstein cows .  _Journal of Dairy Science_.  2005 ;88 : 327 \u2013 334 .\n\nUchiyama H ,  Perez-Prat EM ,  Watanabe K ,  Kumagai I ,  Kuwajima K .  Effects of amino acid substitutions in the hydrophobic core of alpha-lactalbumin on the stability of the molten globule state .  _Protein Engineering_.  1995 ;8 : 1153 \u2013 1161 .\n\nValencia-Sanchez MA ,  Liu J ,  Hannon GJ ,  Parker R .  Control of translation and mRNA degradation by miRNAs and siRNAs .  _Genes Dev_.  2006 ;20 : 515 \u2013 524 .\n\nVenn BJ ,  Skeaff CM ,  Brown R ,  Mann JI ,  Green TJ .  A comparison of the effects of A1 and A2 beta-casein protein variants on blood cholesterol concentrations in New Zealand adults .  _Atherosclerosis_.  2006 ;188 : 175 \u2013 178 .\n\nWang Z ,  Gerstein M ,  Snyder M .  RNA-Seq: A revolutionary tool for transcriptomics .  _Nat Rev Genet_.  2009 ;10 : 57 \u2013 63 .\n\nWickramasinghe S ,  Hua S ,  Rincon G ,  Islas-Trejo A ,  German JB ,  Lebrilla CB ,  Medrano JF .  Transcriptome profiling of bovine milk oligosaccharide metabolism genes using RNA-sequencing .  _PLoS One_.  2011 ;6 : e18895 .\n\nWickramasinghe S ,  Rincon G ,  Islas-Trejo A ,  Medrano JF .  Transcriptional profiling of bovine milk using RNA sequencing .  _BMC Genomics_.  2012 ;13 : 45 .\n\nWilmut I ,  Schnieke AE ,  McWhir J ,  Kind AJ ,  Campbell KH .  Viable offspring derived from fetal and adult mammalian cells .  _Nature_.  1997 ;385 : 810 \u2013 813 .\n\nXue GP ,  Snoswell AM .  Regulation of methyl group metabolism in lactating ewes .  _Biochem Int_.  1985 ;11 (3) : 381 \u2013 385 .\n\nYom HC ,  Bremel RD .  Genetic engineering of milk composition: Modification of milk components in lactating transgenic animals .  _American Journal of Clinical Nutrition_.  1993 ;58 : 299S \u2013 306S .\n\nZenger KR ,  Khatkar MS ,  Cavanagh JA ,  Hawken RJ ,  Raadsma HW .  Genome-wide genetic diversity of Holstein Friesian cattle reveals new insights into Australian and global population variability, including impact of selection .  _Animal Genetics_.  2007 ;38 : 7 \u2013 14 .\n\nZimin AV ,  Delcher AL ,  Florea L ,  Kelley DR ,  Schatz MC ,  Puiu D ,  Hanrahan F ,  Pertea G ,  Van Tassell CP ,  Sonstegard TS ,  Mar\u00e7ais G ,  Roberts M ,  Subramanian P ,  Yorke JA ,  Salzberg SL .  A whole-genome assembly of the domestic cow, Bos taurus .  _Genome Biol_.  2009 ;10 : R42 : doi: 10.1186\/gb-2009-10-4-r42 .\n\nZuelke KA .  Transgenic modification of cow's milk for value-added processing .  _Reproduction, Fertility and Development_.  1998 ;10 : 671 \u2013 676 . \nChapter 5\n\n# Post-translational Modifications of Caseins\n\nJohn W. Holland*\n\nMike J. Boland**\n\n* Institute for Molecular Bioscience, The University of Queensland, Australia  \n** Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nThe caseins exhibit a high degree of heterogeneity as a result of post-translational modifications (PTMs). Phosphorylation of the \u03b1s1-, \u03b1s2, and \u03b2-caseins and glycosylation of \u03ba-casein are the best known modifications and are critical for the formation and stability of casein micelles. \u03ba-Casein, in particular, has long been known to exhibit a high degree of variability in glycosylation, specifically in the caseinomacropeptide region, which is responsible for stabilization of the micelle. It is somewhat surprising to see so much variability in such important structural features. In recent years, the adoption of proteomic techniques has greatly enhanced our ability to unravel heterogeneity in proteins arising from complex and variable patterns of PTMs. In this chapter, a summary of our knowledge of the PTMs of caseins is attempted, with a particular emphasis on \u03ba-casein and the implications that variations in PTMs have for dairy processors.\n\n## Keywords\n\ncaseins, \u03ba-casein, post-translational modifications, phosphorylation, glycosylation, disulfide bonding, proteomic techniques\n\nOutline\n\nIntroduction 141\n\nThe caseins 142\n\n\u03b1s1-Casein 143\n\n\u03b1s2-Casein 144\n\n\u03b2-Casein 145\n\n\u03ba-Casein 145\n\nPhosphorylation 150\n\nGlycosylation 150\n\nDisulfide Bonding 152\n\nSources and Functional Significance of \u03ba-casein Heterogeneity 153\n\nSources of Heterogeneity 153\n\nFunctional Significance 156\n\nBiological Significance 160\n\nCaseins from other species 161\n\nConclusions 162\n\n## Introduction\n\nThe caseins are phosphoproteins and constitute about 80% of the protein in milk (Swaisgood, 2003; Farrell et al., 2004). They are assembled in a colloidal complex with calcium phosphate and small amounts of other minerals. Although obviously important for the provision of amino acids, calcium, and phosphorus for infant nutrition, the casein micelle structure is also critical in determining the physical properties of milk. The stability of the micelle, or its controlled destabilization in the case of cheese and yogurt manufacture, is of primary concern to the dairy industry.\n\nA number of reviews of micelle structure have been published in recent years (Rollema, 1992; Holt & Horne, 1996; Horne, 1998; Walstra, 1999; Horne, 2002). In simple terms, the micelle is a network of protein molecules held together by a combination of hydrophobic interactions between protein molecules and electrostatic interactions between phosphoserine-rich regions of the \u03b1\\- and \u03b2-caseins and micellar calcium phosphate. Whereas the internal structure is still the subject of debate (Horne, 1998; Walstra 1999; and Chapter 6 of this volume), there is general acceptance of the 'hairy micelle' concept, in which the hydrophilic C-terminal portion of \u03ba-casein extends from the surface, providing steric and electrostatic repulsion, which prevents micelle aggregation.\n\nA critical factor in micelle formation and stability is the presence of post-translational modifications (PTMs) such as phosphorylation on the \u03b1s1-, \u03b1s2, and \u03b2-caseins, and glycosylation on \u03ba-casein. PTMs on secreted proteins such as the caseins occur in the endoplasmic reticulum and\/or Golgi complex after synthesis of the polypeptide chain. As such, they are not encoded by the genes per se but may be dependent on protein (and hence gene) sequence motifs that are necessary. Of themselves, however, they are not sufficient for modification to take place. A number of other factors determine whether or not a PTM occurs, including expression of the genes encoding the enzymes necessary for the modification, the availability of their substrates, and the accessibility of the modification site on the protein, especially after folding. Therefore, although it may be possible to predict the theoretical sites of modification on proteins, determination of the actual sites and the degree to which they are modified requires considerable experimental characterization.\n\nAdvances in our understanding of complex systems such as the casein micelle are frequently preceded by advances in technology. In recent years, the development of proteomic technologies has greatly enhanced our ability to analyze milk proteins, particularly with respect to PTMs. Two-dimensional electrophoresis (2-DE), in particular, provides a high-resolution methodology for displaying the heterogeneity of the major milk proteins. As can be seen in Figure 5.1, genetic variants, phospho-variants, and glyco-variants of the caseins can be resolved on a single 2-D gel. Advances in mass spectrometry (MS) have enhanced our ability to analyze the proteins arrayed on 2-D gels. Therefore, it is possible not only to resolve many proteins and their isoforms but to characterize them, particularly with respect to the many PTMs that affect their electrophoretic mobility.\n\nFigure 5.1 2-D gel of bovine milk. Section of a 2-D gel highlighting the major protein forms found in bovine milk. Adapted from Holland et al. (2004).\n\nAs will be seen below, the four caseins are present in many diverse forms as a result of differential PTMs. The biological reasons behind the diversity are not clear. However, what is clear is that the PTMs of the caseins are critical for their function in micelle formation and stability. The first part of this chapter summarizes what is known about the PTMs of the \u03b1\\- and \u03b2-caseins. The second part focuses on \u03ba-casein, which has been the subject of recent proteomic studies, and includes an extended discussion on the functional significance of \u03ba-casein heterogeneity. For the main part of the chapter, the discussion is specific to the caseins of the cow, Bos taurus. A short review on the caseins of other species is included in a later section.\n\n## The caseins\n\nThe caseins are phosphoproteins that are generally well characterized in terms of their PTMs and have been the subject of a number of reviews (Mercier, 1981; Ginger & Grigor, 1999; Farrell et al., 2004). In fact, caseins have been used as model phosphoproteins in the development of proteomic techniques to examine global phosphorylation patterns (e.g., Cox et al., 2005; Kapkova et al., 2006; Sweet et al., 2006; Zhou et al., 2006; Wu et al., 2007). The multitude of techniques developed for the analysis of phosphorylation is beyond the scope of this chapter but has been well reviewed (Bodenmiller et al., 2007; Collins et al., 2007). Indeed, the topic of phosphoproteomics has become a field of study in its own right. The PTMs of the \u03b1s1-, \u03b1s2, and \u03b2-caseins are summarized in the sections below.\n\nAn important note concerns the numbering of the amino acids in the casein sequences described in subsequent sections. The numbering of residues is based on the Swiss-Prot database entry for the relevant protein (see Figs 5.2 and 5.3) and includes the signal peptide, which is normally removed during processing to generate the mature protein. This differs from the numbering in most of the dairy literature, in which the N-terminal amino acid of the mature protein is numbered 1.\n\nFigure 5.2 Amino acid sequences of the bovine \u03b1\\- and \u03b2-caseins. Swiss-Prot accession numbers, entry names, protein names, species, and sequences for the \u03b1\\- and \u03b2-caseins. The database sequences are for the B variant of \u03b1s1-casein, the A variant of \u03b1s2-casein, and the A2 variant of \u03b2-casein. Potential phosphorylation sites are shown in bold type, and those that have been experimentally confirmed are underlined. The signal peptides are shown in italics.\n\nFigure 5.3 Amino acid sequence of \u03ba-casein A (Swiss-Prot accession number P02668). The N-terminal signal sequence (1\u201321) is shown in italics. The arrow indicates the amino-terminus of the mature protein. Recognized sites of potential phosphorylation and glycosylation are indicated in bold, underlined text. Amino acid substitutions distinguishing the B variant are shown above the main sequence.\n\n### \u03b1s1-Casein\n\nThe predominant form of \u03b1s1-casein in bovine milk contains eight phosphate groups. The phosphates are attached to hydroxyamino acids occurring in the sequence motif Ser\/Thr\u2013Xxx\u2013Glu\/Asp\/pSer (Mercier, 1981). However, the vast majority of casein phosphorylation sites, including the eight sites in the major form of \u03b1s1-casein, occur in the more restricted Ser\u2013Xxx\u2013Glu\/pSer motif. Phosphorylation of threonine or of serine in the Ser\u2013Xxx\u2013Asp motif is relatively uncommon. A minor form of \u03b1s1-casein with nine phosphates, originally called \u03b1s0-casein, also occurs in bovine milk. It contains one extra phosphate on Ser56, which occurs in a Ser\u2013Xxx\u2013Asp motif (Manson et al., 1977). The reference protein for \u03b1s1-casein is \u03b1s1-CN B-8P, where B-8P signifies the B genetic variant with eight phosphates (Farrell et al., 2004). A number of genetic variants of \u03b1s1-casein have been described (Farrell et al., 2004). Only the less common forms, D and F, are likely to have altered phosphorylation profiles. Variant D has Ala68 substituted with Thr, which generates a phosphorylation site of the form Thr\u2013Xxx\u2013Glu. Phosphorylation of this residue was detected when the variant was first identified (Grosclaude et al., 1972a). Variant F has Ser81 substituted with Leu, which disrupts the serine cluster, Ser79\u2013Ile\u2013Ser\u2013Ser\u2013Ser\u2013Glu\u2013Glu85, and eliminates the secondary phosphorylation sites at Ser79 and Ser81. The amino acid sequence and the modifications of \u03b1s1-casein are shown in Figure 5.2.\n\n### \u03b1s2-Casein\n\nThe \u03b1s2-casein component of bovine milk is more varied than the \u03b1s1-casein component. It generally presents as a mixture of four phosphoforms with 10\u201313 phosphates. The reference protein for \u03b1s2-casein is \u03b1s2-CN A-11P (Farrell et al., 2004). The A variant has 12 serine residues in Ser\u2013Xxx\u2013Glu\/pSer motifs and four threonine residues in Thr\u2013Xxx\u2013Glu motifs (Fig. 5.2). Consequently, up to 16 phosphates are theoretically possible. Presumably, the 12 serine residues are the first to be phosphorylated. However, it is not known whether specific residues remain unphosphorylated in the different forms but, given the consistent appearance of the forms in milk, it is likely to be the case. Unfortunately, it is not possible to draw any conclusions with regard to phosphorylation site occupation until the individual phosphoforms are analyzed. This should be possible using gel-based proteomic techniques.\n\nOnly four genetic variants of \u03b1s2-casein have been described (Farrell et al., 2004), and, in each case, an altered phosphorylation profile would be expected. In variant B, Ser23 is changed to Phe as a result of a single-nucleotide substitution (Ibeagha-Awemu et al., 2007). This causes loss of a phosphorylation site in the first phosphoserine cluster. Variant C has three amino acid changes: Glu48 is changed to Gly, with loss of the phosphorylation site at Ser46; Ala62 is changed to Thr, creating a potential site with the motif Thr62\u2013Xxx\u2013Glu64; and Thr145 is changed to Ile, with loss of the potential site at Thr145. Variant D has a deletion of nine amino acids as a result of skipping exon VIII (Bouniol et al., 1993). This results in loss of the first three serines from the second phosphoserine cluster. A second PTM on \u03b1s2-casein is the formation of an intramolecular disulfide bond between the two cysteine residues in the protein (Rasmussen et al., 1994). The functional role of disulfide bonding is not clear at this stage, but it may contribute to micelle stability. (This is discussed further in the section on \u03ba-casein.)\n\n### \u03b2-Casein\n\nBovine \u03b2-casein is usually present as a single form with five phosphates, indicating that all five Ser\u2013Xxx\u2013Glu\/pSer sites in the sequence are constitutively phosphorylated. The reference protein is \u03b2-CN A2-5P (Farrell et al., 2004). Some 12 genetic variants of \u03b2-casein have been characterized, but only two variants appear to have altered phosphorylation profiles. Variant C has a Glu to Lys substitution at residue 52, which removes the phosphorylation site at Ser50. Variant D has a Ser to Lys substitution at residue 33, which removes the primary phosphorylation site at Ser33. Variation in \u03b2-casein arises primarily as a result of proteolysis rather than PTMs. The sequence and the phosphorylation sites of \u03b2-casein are summarized in Figure 5.2.\n\n### \u03ba-Casein\n\n\u03ba-Casein does not contain any phosphoserine clusters and probably plays little part in calcium binding. Its major feature is a variable degree of glycosylation. The keen interest in \u03ba-casein arises largely from its key role as a stabilizer of the micelle structure. In mice, the absence of \u03ba-casein causes a failure of lactation, as the lumina of the mammary gland become clogged with aggregated caseins (Shekar et al., 2006). \u03ba-Casein is usually thought of as having two distinct domains that are separated by the very specific Phe-Met (126\u2013127) bond cleaved by chymosin. The N-terminal para-\u03ba-casein is the hydrophobic part that remains in the micelle and contains the disulfide bonds. The C-terminal caseinomacropeptide is the part that extends into the solution, and it is removed to become part of the whey fraction during cheese making. It contains the glycosylation sites, which is why this peptide is often referred to as the glycomacropeptide, although strictly speaking this term pertains only to forms that are actually glycoslated. The PTMs of \u03ba-casein have been the subject of more recent research and are covered here in much greater detail than those of the other caseins.\n\nThe full amino acid sequence of bovine \u03ba-casein was first reported in 1973 (Mercier et al., 1973). The mature protein consists of a single chain of 169 amino acids (Fig. 5.3) and has a theoretical molecular weight of 18,974 Da and a theoretical pI of 5.93 (A variant). The amino terminal glutamine is cyclized to form a pyrrolidone glutamic acid residue. The bovine \u03ba-casein gene sequence was published in 1988 (Alexander et al., 1988). It consists of five exons spread over about 14 kilobases, with most of the protein-coding region located in exon 4. A cleavable amino terminal signal sequence of 21 amino acids directs secretion of the mature protein. A number of polymorphisms in the \u03ba-casein gene have been identified, resulting in one or more amino acid substitutions in the mature protein. The most common variants are the A and B variants, which differ by two amino acids in the caseinomacropeptide region (Asp169\/Thr157 in variant A and Ala169\/Ile157 in variant B). The genetic variants of \u03ba-casein are summarized in Table 5.1. A number of polymorphisms in the noncoding region have also been identified (Schild et al., 1994; Keating et al., 2007). Although these do not affect the amino acid sequence, they have the potential to affect expression levels. The full amino acid sequences of \u03ba-casein from 32 species are currently in the Swiss-Prot database, with another 112 entries covering subspecies and incomplete sequences (Table 5.2). The reference protein for \u03ba-casein is \u03ba-CN A-1P, Uni-prot P02668 (Farrell et al., 2004).\n\nTable 5.1\n\nGenetic Variants of Bovine \u03ba-Casein\n\nVarianta | Amino acid changes  \n(Relative to A variant) | Reference  \n---|---|---  \nA |  | (Grosclaude, Mahe, Mercier, Ribadeau-Dumas, 1972b)  \nB | Thr157 to Ile, Asp169 to Ala | (Mercier, Brignon, Ribadeau-Dumas, 1973)  \nB2 | Thr157 to Ile, Asp169 to Ala, Ile174 to Thr | (Gorodetskii, Kershulite, Korobko, 1983)  \nC | Arg118 to His, Thr157 to Ile, Asp169 to Ala | (Miranda, Anglade, Mahe, Erhardt, 1993)  \nE | Ser176 to Gly | (Schlieben, Erhardt, Senft, 1991)  \nF1 | Asp169 to Val | (Sulimova, Sokolova, Semikozova, Nguet, Berberov, 1992)  \nF2 | Arg31 to His | (Prinzenberg, Hiendleder, Ikonen, Erhardt, 1996)  \nG1 | Arg118 to Cys | (Prinzenberg et al., 1996)  \nG2 | Asp169 to Ala | (Sulimova, Badagueva Iu, Udina, 1996)  \nH | Thr156 to Ile | (Prinzenberg, Krause, Erhardt, 1999)  \nI | Ser125 to Ala | (Prinzenberg et al., 1999)  \nJ | Thr157 to Ile, Asp169 to Ala, Ser176 to Arg | (Mahe et al., 1999)  \nA(1) | Silent (Pro150, CCA to CCG) | (Prinzenberg et al., 1999)\n\na Other variants have been described but have not been confirmed or have been proven to be identical to those given.\n\nTable 5.2\n\n\u03ba-Casein Entries in the Swiss Protein Database (December 2013)\n\nEntry name | | Organism  \n---|---|---  \nQ6YLM6_AEPME | Fragment | Aepyceros melampus (Impala)  \nP79091_AILFU | Fragment | Ailurus fulgens (Lesser panda) (Red panda)  \nQ95MY6_ALCBU | Fragment | Alcelaphus buselaphus (Hartebeest)  \nQ8HXL0_9CETA | Fragment | Alces alces (Eurasian elk)  \nQ5C9H0_AMMLE | Fragment | Ammotragus lervia (Barbary sheep) (Aoudad)  \nQ95MY0_ANTMR | Fragment | Antidorcas marsupialis (Springbok)  \nQ7JFQ1_ANTAM | Fragment | Antilocapra americana (Pronghorn)  \nK7Z0L5_ANTCE | Fragment | Antilope cervicapra (Blackbuck)  \nCASK_BALPH | Fragment | Balaenoptera physalus (Finback whale) (Common rorqual)  \nQ95MY7_BEAHU | Fragment | Beatragus hunteri (Hunter's antelope) (Damaliscus hunteri)  \nP79093_BISBI | Fragment | Bison bison (American bison) (Bos bison)  \nB2Z897_BISBO | Fragment | Bison bonasus (European bison)  \nO18899_BOSGA | Fragment | Bos gaurus (Seladang) (Indian bison)  \nB6CXY4_BOSGF | Fragment | Bos gaurus frontalis (Domestic gayal) (Bos frontalis)  \nL8IIT8_BOSMU |  | Bos grunniens mutus  \nB6CXY5_BOSIN | Fragment | Bos indicus (Zebu)  \nP79097_BOSJA | Fragment | Bos javanicus (Wild banteng)  \nK9ZTJ1_9CETA | Fragment | Bos mutus (wild yak)  \nI6UFY2_BOSMU |  | Bos mutus grunniens (Wild yak) (Bos grunniens)  \nA3FJ56_BOVIN | Fragment | Bos taurus (Bovine)  \nO18903_BOSTR | Fragment | Boselaphus tragocamelus (Nilgai)  \nQ712N6_BUBBU |  | Bubalus bubalis (Domestic water buffalo)  \nK9ZS88_BUBDE | Fragment | Bubalus depressicornis (Lowland anoa) (Anoa depressicornis)  \nQ5C9G9_BUDTA | Fragment | Budorcas taxicolor (Golden takin)  \nL0P304_CAMBA |  | Camelus bactrianus (Bactrian camel)  \nL0P3Z7_CAMDR |  | Camelus dromedarius (Dromedary) (Arabian camel)  \nS9WI70_9CETA |  | Camelus ferus (Wild Bactrian camel)  \nF5CIN1_CAPMR | Fragment | Caperea marginata (Pigmy right whale) (Balaena marginata)  \nQ71EC2_CAPAE | Fragment | Capra aegagrus (Wild goat)  \nQ5C9G8_CAPFA | Fragment | Capra falconeri (Markhor)  \nCASK_CAPHI |  | Capra hircus (Goat)  \nQ7YRV1_CAPII | Fragment | Capra ibex ibex (Alpine ibex)  \nQ5C9G7_CAPNU | Fragment | Capra nubiana (Nubian ibex) (Capra ibex nubiana)  \nQ7YRU9_CAPSI | Fragment | Capra sibirica (Siberian ibex) (Capra ibex sibirica)  \nCASK_CAPCA | Fragment | Capreolus capreolus (Roe deer)  \nCASK_CAPCR |  | Capricornis crispus (Japanese serow) (Naemorhedus crispus)  \nCASK_CAPSU |  | Capricornis sumatrensis (Serow)  \nCASK_CAPSW |  | Capricornis swinhoei (Taiwan serow) (Naemorhedus swinhoei)  \nCASK_CAVPO |  | Cavia porcellus (Guinea pig)  \nQ6YLM4_CEPDO | Fragment | Cephalophus dorsalis (Bay duiker)  \nCASK_CEREL | Fragment | Cervus elaphus (Red deer)  \nCASK_CERNI |  | Cervus nippon (Sika deer)  \nQ95MY5_CONGN | Fragment | Connochaetes gnou (Black wildebeest)  \nQ6YLM2_DAMPY | Fragment | Damaliscus pygargus (Bontebok)  \nQ28354_9CETA | Fragment | Delphinidae gen. sp.  \nK7ZCW9_9CETA | Fragment | Dorcatragus megalotis (beira)  \nCASK_ELADA | Fragment | Elaphurus davidianus (Pere David's deer)  \nB1PLB8_EQUAS | Fragment | Equus asinus (Donkey)  \nF0V6V5_EQUAS |  | Equus asinus africanus  \nF6VKB4_HORSE |  | Equus caballus (Horse)  \nCASK_EQUGR | Fragment | Equus grevyi (Grevy's zebra)  \nB1PLB7_EQUZE | Fragment | Equus zebra (Mountain zebra)  \nF5CIN0_ESCGI | Fragment | Eschrichtius gibbosus (California gray whale) (Eschrichtius robustus)  \nK7YUI7_9CETA | Fragment | Gazella arabica (Arabian gazelle)  \nK7Z353_GAZDO | Fragment | Gazella dorcas (Dorcas gazelle)  \nK7ZHH2_GAZSU | Fragment | Gazella subgutturosa marica (sand gazelle)  \nCASK_GIRCA | Fragment | Giraffa camelopardalis (Giraffe)  \nF5CIM6_GRAGR | Fragment | Grampus griseus (Risso's dolphin) (Delphinus griseus)  \nQ074L9_HEMHY | Fragment | Hemitragus hylocrius (Nilgiri tahr)  \nQ074M0_HEMJA | Fragment | Hemitragus jayakari (Arabian tahr)  \nQ5C9G5_HEMJE | Fragment | Hemitragus jemlahicus (Himalayan tahr)  \nG5AYC4_HETGA |  | Heterocephalus glaber (Naked mole rat)  \nCASK_HIPAM | Fragment | Hippopotamus amphibius (Hippopotamus)  \nQ95MY8_HIPEQ | Fragment | Hippotragus equinus (Roan antelope)  \nQ6YLM3_HIPNI | Fragment | Hippotragus niger (Sable antelope)  \nCASK_HUMAN |  | Homo sapiens (Human)  \nT2MHA8_HYDVU | Fragment | Hydra vulgaris (Hydra) (Hydra attenuata)  \nA8IY17_INIGE | Fragment | Inia geoffrensis (Amazon dolphin)  \nQ95MY2_KOBLE | Fragment | Kobus leche (Lechwe)  \nCASK_LAMGU | Fragment | Lama guanicoe (Guanaco)  \nQ95MX9_LITWA | Fragment | Litocranius walleri (Gerenuk)  \nG7P5P0_MACFA |  | Macaca fascicularis (Crab-eating macaque) (Cynomolgus monkey)  \nG7MT61_MACMU |  | Macaca mulatta (Rhesus macaque)  \nK7Z0L8_MADSL | Fragment | Madoqua saltiana (Salt's dikdik)  \nCASK_MAZAM | Fragment | Mazama americana (Red brocket)  \nF5CIM8_MESPE | Fragment | Mesoplodon peruvianus (Peruvian beaked whale) (Pygmy beaked whale)  \nQ6YLM7_MOSMO | Fragment | Moschus moschiferus (Siberian musk deer) (Moschus sibiricus)  \nA8IY19_9CETA | Fragment | Moschus sp. JEG-2007  \nCASK_MUNRE | Fragment | Muntiacus reevesi (Reeve's muntjac) (Chinese muntjac)  \nCASK_MOUSE |  | Mus musculus (Mouse)  \nS7Q8N5_MYOBR |  | Myotis brandtii (Brandt's bat)  \nG8FPL3_NANDA | Fragment | Nanger dama (Dama gazelle) (Gazella dama)  \nQ5C9H1_NANGR | Fragment | Nanger granti (Grant's gazelle) (Gazella granti)  \nCASK_NEMGO |  | Nemorhaedus goral (Gray goral)  \nQ95MX5_NEOMO | Fragment | Neotragus moschatus (Suni)  \nCASK_ODOHE | Fragment | Odocoileus hemionus (Mule deer) (Black-tailed deer)  \nCASK_ODOVI | Fragment | Odocoileus virginianus virginianus (Virginia white-tailed deer)  \nQ6YLM8_OKAJO | Fragment | Okapia johnstoni (Okapi)  \nCASK_OREAM |  | Oreamnos americanus (Mountain goat)  \nQ95MX4_OREOR | Fragment | Oreotragus oreotragus (Klipspringer)  \nD0QJA9_ORNAN |  | Ornithorhynchus anatinus (Duckbill platypus)  \nCASK_RABIT |  | Oryctolagus cuniculus (Rabbit)  \nQ95MY9_ORYDA | Fragment | Oryx dammah (Scimitar-horned oryx)  \nQ95MZ0_ORYGA | Fragment | Oryx gazella (Gemsbok)  \nQ5C9G2_OVIMO | Fragment | Ovibos moschatus (Muskox)  \nD2SZ86_9CETA | Fragment | Ovis ammon collium  \nD2SZ87_9CETA | Fragment | Ovis ammon sevetzovi  \nCASK_SHEEP |  | Ovis aries (Sheep)  \nD2SZ88_OVICA | Fragment | Ovis canadensis canadensis  \nD2SZ94_OVICA | Fragment | Ovis canadensis nelsoni (desert bighorn sheep)  \nQ5C9G0_OVIDA | Fragment | Ovis dalli (Dall sheep)  \nD2SZA4_9CETA | Fragment | Ovis orientalis anatolica  \nD2SZA5_9CETA | Fragment | Ovis orientalis gmelini  \nD2SZB9_9CETA | Fragment | Ovis orientalis isphahanica  \nD2SZE2_OVIVI | Fragment | Ovis vignei (Urial sheep)  \nD2SZC5_OVIVI | Fragment | Ovis vignei arkal  \nD2SZD4_OVIVI | Fragment | Ovis vignei blanfordi  \nD2SZD7_OVIVI | Fragment | Ovis vignei bochariensis (Bukhara urial)  \nD2SZD9_OVIVI | Fragment | Ovis vignei cycloceros  \nD2SZE4_OVIVI | Fragment | Ovis vignei punjabensis  \nD2SZE7_OVIVI | Fragment | Ovis vignei vignei  \nQ5C9F9_PANHO | Fragment | Pantholops hodgsonii (Chiru)  \nQ95MY1_PELCP | Fragment | Pelea capreolus (Gray rhebok)  \nQ95MX3_PHIMO | Fragment | Philantomba monticola (Blue duiker) (Cephalophus monticola)  \nF5CIM7_PHOPH | Fragment | Phocoenoides phocoena (Harbor porpoise)  \nP79230_PHYCD | Fragment | Physeter catodon (Sperm whale) (Physeter macrocephalus)  \nK7YUI9_PROGT | Fragment | Procapra gutturosa (Mongolian gazelle)  \nQ5C9F8_PSENA | Fragment | Pseudois nayaur (Bharal)  \nQ5C9H3_PSENG | Fragment | Pseudoryx nghetinhensis (Saola)  \nL5K924_PTEAL |  | Pteropus alecto (Black flying fox)  \nCASK_RANTA | Fragment | Rangifer tarandus (Reindeer) (Cervus tarandus)  \nQ95MX8_RAPCA | Fragment | Raphicerus campestris (Steenbok)  \nQ95MX6_RAPME | Fragment | Raphicerus melanotis (Grysbok)  \nQ95MX7_RAPSH | Fragment | Raphicerus sharpei (Sharpe's grysbok)  \nCASK_RAT |  | Rattus norvegicus (Rat)  \nQ6YLM5_REDFU | Fragment | Redunca fulvorufula (Mountain reedbuck)  \nQ95MY3_REDRE | Fragment | Redunca redunca (Bohor reedbuck)  \nCASK_RUCDU | Fragment | Rucervus duvaucelii (Swamp deer) (Cervus duvaucelii)  \nQ074J7_RUPPY | Fragment | Rupicapra pyrenaica (Pyrenean chamois)  \nCASK_RUPRU |  | Rupicapra rupicapra (Chamois)  \nCASK_RUSUN | Fragment | Rusa unicolor (Sambar) (Cervus unicolor)  \nCASK_SAITA |  | Saiga tatarica (Saiga antelope)  \nCASK_PIG |  | Sus scrofa (Pig)  \nQ95MX2_SYLGR | Fragment | Sylvicapra grimmia (Grey duiker)  \nQ95MZ2_SYNCA | Fragment | Syncerus caffer (Cape buffalo)  \nO18901_SYNCA | Fragment | Syncerus caffer nanus (forest buffalo)  \nD0QJA7_9MAMM |  | Tachyglossus aculeatus (Australian echidna)  \nCASK_TAPIN | Fragment | Tapirus indicus (Asiatic tapir) (Malayan tapir)  \nF5CIM9_TASSH | Fragment | Tasmacetus shepherdi (Shepherd's beaked whale)  \nCASK_TAYTA | Fragment | Tayassu tajacu (Collared peccary) (Pecari tajacu)  \nQ5C9H2_TETQU | Fragment | Tetracerus quadricornis (Four-horned antelope) (Chousingha)  \nO18902_TRAIM | Fragment | Tragelaphus imberbis (Lesser kudu)  \nQ95MZ1_TRAOR | Fragment | Tragelaphus oryx (Eland) (Taurotragus oryx)  \nCASK_TRAJA | Fragment | Tragulus javanicus (Lesser Malay chevrotain) (Lesser mouse deer)  \nQ9XSD6_TRIVU |  | Trichosurus vulpecula (Brush-tailed possum)  \nCASK_UNCUN | Fragment | Uncia uncia (Snow leopard) (Panthera uncia)\n\n#### Phosphorylation\n\n\u03ba-Casein appears to be constitutively phosphorylated at Ser170 and only partially phosphorylated at Ser148 (Talbot & Waugh, 1970; Mercier et al., 1973). A minor tri-phosphorylated form has also been detected (Vreeman et al., 1986; Molle & Leonil, 1995). Other studies have managed to detect only mono-phosphorylated forms (Rasmussen et al., 1997; Riggs et al., 2001), although, in one of these (Riggs et al., 2001), the phosphorylation site appears to have been identified incorrectly. Phosphorylation has also been examined by MS of intact \u03ba-casein extracted from 2-D gels. Both mono- and di-phosphorylated forms were observed, and phosphorylation at Ser170 was confirmed by MS\/MS (Claverol et al., 2003). However, the electrophoretic mobility of some phospho-forms was not consistent with the MS analysis and probably reflected artifactual modification (e.g., deamidation) during purification. Using 2-DE with isoelectric focusing as the first dimension, phosphorylation variants in whole milk can be easily resolved because of the pI shifts caused by the acidic phosphate groups (Holland et al., 2004). The two main phosphorylation sites have been confirmed by tandem MS sequencing of peptic peptides released from protein forms separated by 2-DE. Tri-phosphorylated forms of both the A and B variants have been observed, and the third phosphorylation site has been identified as Thr166 (Holland et al., 2006). This site is consistent with the general observation of casein phosphorylation on the Ser\/Thr\u2013Xxx\u2013Glu\/pSer motif, with only relatively low levels of phosphorylation on threonine residues. A recent study has identified two minor phosphorylation sites at Thr166 \u2013 as above \u2013 and also at Ser187 (Hernandez-Hernandez et al., 2011).\n\n#### Glycosylation\n\nWhereas about 40% of \u03ba-casein has been estimated to be nonglycosylated, the remaining 60% has up to six glycans attached (Vreeman et al., 1986). The presence of sugars on \u03ba-casein was recognized as long ago as 1961 (Alais & Jolles, 1961), and during the 1970s and 1980s, a large number of studies were directed at elucidating the sugar composition, sequence, and sites of attachment to the protein. The major glycan is a tetrasaccharide composed of galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic or neuraminic acid (NeuAc) of the form NeuAc\u03b1(2-3)Gal\u03b2(1-3)NeuAc\u03b1(2-6)]GalNAc, but monosaccharide (GalNAc), disaccharide (Gal\u03b2(1-3)GalNAc), and trisaccharide (NeuAc\u03b1(2-3)Gal\u03b2(1-3)GalNAc or Gal\u03b2(1-3)[NeuAc\u03b1(2-6)]GalNAc) are also found ([Fournet et al., 1975; ; van Halbeek et al., 1980; Fiat et al., 1988; Saito & Itoh, 1992). The relative amounts have been determined by high-performance liquid chromatography (HPLC) as 56.0% tetrasaccharide, 36.9% trisaccharide (18.4% linear and 18.5% branched), 6.3% disaccharide, and 0.8% monosaccharide (Saito & Itoh, 1992). It is not known whether the minor forms arise from incomplete synthesis of the tetrasaccharide in mammary epithelial cells or are products of degradation after synthesis and\/or secretion of \u03ba-casein into the lumen of the mammary gland.\n\nEstablishment of the attachment site(s) of the glycans has been more controversial. On the basis of Edman sequencing of short glycopeptides obtained by enzymatic digestion, Jolles et al. (1973) proposed Thr152 or Thr154 as the glycan attachment site. Kanamori et al. (1980) proposed Thr152, Thr154, and Thr156 (or Thr157) after analyzing a glycopeptide that was derived from \u03ba-casein and that contained three GalNAc residues. Work from the same laboratory on bovine \u03ba-casein from colostrum also indicated glycosylation at Thr152, Thr154, and Thr156 (Doi et al., 1980). Subsequently, using a different peptide fraction prepared from \u03ba-casein of normal milk, glycosylation at Thr154 and Ser162 was reported (Kanamori et al., 1981). Meanwhile, further work from Jolles's laboratory identified Thr152 as the glycan attachment site on \u03ba-casein from normal milk and Thr152 and Thr163 as the attachment sites on \u03ba-casein from colostrum (Fiat et al., 1981). Zevaco and Ribadeau-Dumas (1984) suggested that glycans could be attached to any of the previously identified sites (Thr152, Thr154, Thr156, Thr157, Ser162, or Thr163), but their published study contained no conclusive evidence for any site. All these studies were limited by the technology available at the time. In normal Edman sequencing, glycosylated serine or threonine residues are not detected, and their presence is inferred from a blank in the sequencing cycle where serine or threonine is expected (for a more detailed discussion, see Pisano et al., 1994). This is further complicated by the fact that serine and threonine are themselves low-yield amino acids. Thus, in assigning O-glycosylation sites, Edman sequencing data can easily be misinterpreted. Conclusive identification of glycosylation sites in \u03ba-casein was achieved using solid-phase Edman sequencing, which allows the direct detection of glycosylated serine and threonine residues (Pisano et al., 1994). Variable levels of glycosylation at Thr142, Thr152, Thr154, Thr157 (A variant only), Thr163, and Thr186 were detected, and no evidence of glycosylation at any serine residue was obtained. However, even this study did not give the full picture of \u03ba-casein glycosylation, as it could detect only average glycosylation site occupancy in a crude mixture of glycoforms.\n\nWe have shown that \u03ba-casein glycoforms can be separated by 2-DE (Holland et al., 2004). and the resolution obtained is shown in Figure 5.4. When the glycosylation site occupancy of individual glycoforms was investigated using tandem MS sequencing of chemically tagged peptides, an interesting pattern was observed. The mono-glycoform was glycosylated exclusively at Thr152, the di-glycoform was glycosylated at Thr152 and Thr163, and the tri-glycoform was glycosylated at Thr152, Thr154, and Thr163 (Holland et al., 2005). Further studies using enriched fractions of \u03ba-casein separated on 2-D gels showed up to six glycans on \u03ba-casein B where only five sites had been previously identified. Tandem MS analysis provided evidence for glycosylation at Thr166 on the tetra-glycoform (Holland et al., 2006). The remaining two glycosylation sites were not confirmed but were presumably on Thr142 and Thr186, as proposed earlier (Pisano et al., 1994). This pattern is illustrated in Figure 5.5. As Thr166 can be phosphorylated or glycosylated, there is potential for competition at this site. However, as both the tri-phosphate and tetra-glycoforms are very minor forms, it may be of little significance. Overall, the glycosylation of \u03ba-casein in the mammary epithelial cells appears to take place in a highly controlled manner; this suggests that it is a rather important process with considerable functional significance.\n\nFigure 5.4 Heterogeneity of \u03ba-casein in cow's milk. 2-D gel showing multiple forms of \u03ba-casein. The genetic variant, number of phosphate residues, and glycosylation state are indicated. Numbers in brackets indicate the extra negatively charged residues relative to \u03ba-casein B-1P. Adapted from Holland et al. (2004).\n\nFigure 5.5 Attachment of tetrasaccharide units to the four major glycoforms of \u03ba-casein. Schematic representation of \u03ba-casein highlighting the glycomacropeptide portion and the attachment sites of phosphate and sugar residues in the major glycoforms. ( ) GalNAc; ( ) Gal; (\u2666) NeuAc.\n\n#### Disulfide Bonding\n\n\u03ba-Casein purified from bovine milk occurs in both monomeric and oligomeric forms, with up to eight or more monomers linked by disulfide bonds (Swaisgood et al., 1964; Talbot & Waugh, 1970). A more recent study has shown that reduced and carboxymethylated \u03ba-casein can form large fibrillar structures, although these do not occur in milk (Farrell et al., 2003). The nature of the disulfide-linked complexes has been examined by a number of authors (Groves et al., 1992; ; Rasmussen et al., 1992; ; ; Farrell et al., 2003).\n\nThere are only two cysteine residues (Cys32 and Cys109) in bovine \u03ba-casein (Mercier et al., 1973), both occurring in the para-\u03ba-casein part of the molecule, and they appear to be randomly linked in disulfide bonds in oligomeric forms (Rasmussen et al., 1992). In monomeric \u03ba-casein, the two cysteines form an intramolecular disulfide bond (Rasmussen et al., 1994). As can be seen in Figure 5.6, disulfide-bonded monomers, dimers, and trimers can be resolved on 2-D gels of whole milk when reducing agents are omitted (Holland et al., 2008). It is not clear whether these higher-order complexes of \u03ba-casein have any importance in micellar structure, but it would be expected that they would be less likely to dissociate from the micelles. The cysteine residues are not well conserved across species, with human, porcine, and rodent \u03ba-caseins containing only a single cysteine, precluding the formation of disulfide-linked oligomers larger than dimers (Rasmussen et al., 1999; Bouguyon et al., 2006). However, the ability of \u03ba-casein to form disulfide-linked complexes with itself or with other proteins during heat treatment is relevant to dairy processing (see below). The combined PTMs of bovine \u03ba-casein are summarized in Figure 5.7.\n\nFigure 5.6 Distribution of disulfide-linked isoforms of \u03ba-casein on a non-reducing 2-D gel. Dimers, trimers, and tetramers of \u03ba-casein are labeled to show the participating monomeric forms. The dimers and trimers run as doublets, depending on the disulfide linkages. MALDI-TOF mass spectra show the disulfide-linked peptides obtained from tryptic digests of the homodimer and homotrimer of \u03ba-casein B:1P.\n\nFigure 5.7 Potential modifications on \u03ba-casein B. This schematic summarizes the potential PTMs on \u03ba-casein B. (P) Phosphorylation sites; ( ) GalNAc; ( ) Gal; (\u2666) NeuAc. Note that trisaccharides and disaccharides lacking one or both NeuAc residues also occur. The monomer has an intramolecular disulfide bond between Cys32 and Cys109.\n\n### Sources and Functional Significance of \u03ba-casein Heterogeneity\n\nAlthough the heterogeneity of \u03ba-casein has been recognized for many decades and the structural elements are now fairly well defined, the source of the heterogeneity, particularly in glycosylation, and its functional role(s) are not known. Early studies on the influence of the glycosylation of \u03ba-casein on its biological properties have been reviewed previously (Dziuba & Minikiewicz, 1996). Dziuba and Minikiewicz highlighted a number of studies addressing factors that could influence the degree of glycosylation and what influence glycosylation might have on micelle stability. The sections below cover some of those studies again and highlight more recent work related to the sources and functional significance of \u03ba-casein heterogeneity.\n\n#### Sources of Heterogeneity\n\nA large number of studies have examined the influence of milk protein polymorphism on milk composition and yield, and these have been extensively reviewed (e.g., Ng-Kwai-Hang, 1997; Martin et al., 2002; Heck et al., 2009). However, in many cases, the results have been inconsistent, which probably reflects the multifactorial nature of milk production. It is difficult to isolate the effects of a single protein polymorphism from those of the other major milk proteins, especially as there appears to be a substantial degree of co-ordination of their expression. There are also a number of environmental or cow-related factors such as feed type and lactational stage that are frequently interrelated, as they all vary with the seasonal changes in dairy farming. Studies on specific effects of \u03ba-casein variants have largely focused on the common A and B variants, and there appears to be a consensus that milk from B variant cows contains more fat, protein, casein, and \u03ba-casein than milk from A variant cows (Bovenhuis et al., 1992; Ng-Kwai-Hang, 1997; Bobe et al., 1999).\n\nStudies relating to the glycosylation status of \u03ba-casein are more limited. Robitaille et al. (1991a) identified a number of factors that appeared to contribute to variation in the NeuAc content of bovine \u03ba-casein. The NeuAc content was higher in cows with the \u03ba-casein AB phenotype than in cows with the AA phenotype. It decreased with increasing parity and varied over the course of lactation, dropping to a minimum at 2\u20133 months after calving before increasing over the next 9\u201310 months. Robitaille et al. (1991b) also examined the association between \u03ba-casein glycosylation and milk production\/composition. Although there appeared to be a statistically significant association between the NeuAc content of \u03ba-casein and milk yield, the most striking result of these investigations was the variability of NeuAc\/\u03ba-casein measurements (mean, 64 \u00b1 21 \u03bcg\/mg; range, 23\u2013166 \u03bcg\/mg), which suggests that other factors could have had a large impact on glycosylation or that the inherent variability in the assay masked any true associations. Limited 2-D gel analyses suggest that the pattern of glycosylation is far more consistent than these measurements indicate (Holland et al., 2004; ). It would be very informative to examine some of these supposed extremes in NeuAc and hence the glycosylation level of \u03ba-casein on 2-D gels.\n\nSignificant differences in the content of nonglycosylated \u03ba-casein in milk have been reported for cows of different \u03ba-casein genotypes (Lodes et al., 1996). Nonglycosylated \u03ba-casein levels were higher (as a percentage of total protein) in milk from cows with the B variant than in milk from cows with the A variant. The rarer variants, C and E, were generally associated with lower levels. However, as no measurements of glycosylated \u03ba-casein levels were reported, no effect of genetic variant on glycosylation can be inferred from this report.\n\nElectrophoretic and chromatographic techniques have been used to profile the caseinomacropeptide from cows of the AA and BB phenotypes (Coolbear et al., 1996). They found that the B variant macropeptide was more highly glycosylated than the A variant, with an increased content of both hexosamine (i.e., GalNAc) and sialic acid (i.e., NeuAc). After anion-exchange HPLC on a MonoQ column, the elution profile of the B variant contained more peaks, suggesting that an increased number of oligosaccharide chains were attached. These results were consistent with earlier studies, suggesting more extensive glycosylation of the B variant (Vreeman et al., 1986) compared with the A variant (Molle & Leonil, 1995), despite the fact that the A variant contains an extra (potential) glycosylation site (Pisano et al., 1994). From these and other results, Coolbear et al. (1996) suggested that there were generally consistent patterns of glycosylation for the genetic variants but that the overall extent of glycosylation could vary.\n\nVariations in \u03ba-casein glycosylation during lactation have been touched on above. Early studies indicated a higher degree of glycosylation of \u03ba-casein in colostrum than in mature milk as well as the presence of an additional sugar moiety, N-acetylglucosamine (GlcNAc) (Guerin et al., 1974; Fournet et al., 1975). Subsequently, a number of studies addressed the structure of the oligosaccharides attached to colostral \u03ba-casein and how they varied with time after parturition (Saito et al., 1981a; 1981b; ; van Halbeek et al., 1981; Fiat et al., 1988). As well as the structures already identified above in normal milk, the following structures have been reported: the acidic hexasaccharide, NeuAc\u03b1(2-3)Gal\u03b2(1-3)NeuAc\u03b1(2-3)Gal\u03b2(1-4)GlcNAc\u03b2(1-6)]GalNAc; the acidic pentasaccharide, NeuAc\u03b1(2-3)Gal\u03b2(1-3)[Gal\u03b2(1-4)GlcNAc\u03b2 (1-6)]GalNAc; the acidic tetrasaccharide, GlcNAc\u03b2(1-3)Gal\u03b2(1-3)[NeuAc\u03b1(2-6)]GalNAc; the neutral pentasaccharide Gal\u03b2(1-3)[Gal\u03b2(1-4){Fuc\u03b1(1-3)}GlcNAc\u03b2(1-6)]GalNAc; the neutral tetrasaccharide, Gal\u03b2(1-3)[Gal\u03b2(1-4)GlcNAc\u03b2(1-6)]GalNAc; and the neutral trisaccharide, Gal\u03b2(1-3)[GlcNAc\u03b2(1-6)]GalNAc. This extra complexity is already observable 15 min after parturition but decreases to normal over about 66 h ([Fiat et al., 1988). These results suggest changes in the expression profiles of the glycosyltransferases responsible for assembling the O-linked glycans on \u03ba-casein. The initial step of attachment of GalNAc to a threonine residue is catalyzed by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAcT) and, although not much is known about the bovine enzymes, there are at least 12 mammalian GalNAcTs that have been cloned and functionally expressed (Ten Hagen et al., 2003). In mice, the expression of several isoforms changes markedly during pregnancy and lactation (Young et al., 2003). It is likely that expression of the other required glycosyltransferases also varies, presumably under control of lactogenic hormones.\n\nOther seasonal factors related to climate, such as heat stress, drought, and nutrition (e.g., pasture versus fodder), can have an impact on milk production and composition. However, we are not aware of any specific studies on their effect on \u03ba-casein glycosylation.\n\n### Functional Significance\n\n\u03ba-Casein plays a key role in micelle stability by acting as a hairy layer that provides both steric and electrostatic repulsion between micelles, preventing aggregation. Glycosylation of \u03ba-casein increases both the size of the hydrophilic C-terminal 'hairs' and their charge, because of the bulk of the hydrophilic sugar residues with their hydration shells and the negative charge of the neuraminic acid groups, respectively. Theoretically, the higher the degree of glycosylation of \u03ba-casein, the greater its stabilizing ability should be. As such, it might be expected that the degree of glycosylation of \u03ba-casein would have a marked effect on both the size and the stability of the casein micelles. However, whereas the size of the casein micelles has been shown to be inversely related to their \u03ba-casein content (relative to total casein) by a number of authors (O'Connell & Fox, 2000), there is no clear correlation between micelle size and degree of glycosylation. Slattery (1978) found an apparent inverse relationship between the proportion of glycosylated \u03ba-casein and micelle size, but it did not apply to all of the size fractions isolated. In contrast, Dalgleish (1985; 1986) found that the proportions of glycosylated and nonglycosylated \u03ba-casein did not vary with micelle size. More recently, O'Connell and Fox (2000) showed an apparent increase in \u03ba-casein glycosylation with increasing micelle size. Some of these discrepancies are probably the result of the different experimental approaches adopted. Currently, there is no conclusive evidence for a distinct relationship between micelle size and \u03ba-casein glycosylation.\n\nAs stated above, micellar stability, or controlled destabilization in the case of cheese and yogurt manufacture, is of key importance in dairy manufacturing. A number of authors have looked for effects of \u03ba-casein heterogeneity on micellar stability and the processing properties of milk. Takeuchi et al. (1985) used ion-exchange chromatography to prepare nine fractions of \u03ba-casein A-1P that varied in the level of glycosylation. The ability of these subfractions to stabilize \u03b1s1-casein was shown to increase with increasing carbohydrate content. In cheese manufacture, the initial step is the chymosin- (rennet-) catalyzed cleavage of the Phe126\u2013Met127 bond in \u03ba-casein, resulting in release of the hydrophilic caseinomacropeptide from the micelle surface, which leads to micellar aggregation or clotting. Doi et al. (1979) examined the susceptibility to chymosin action of \u03ba-casein preparations with different degrees of glycosylation. They found that more highly glycosylated forms were less susceptible to hydrolysis not only by chymosin but also by other proteinases. Others have also found an inverse relationship between glycosylation and chymosin susceptibility with purified \u03ba-casein fractions (Addeo et al., 1984; Vreeman et al., 1986) and in model systems (Addeo et al., 1984; Leaver & Horne, 1996).\n\nIn milk, the relationship is not so clear. Chaplin and Green (1980) claimed that all \u03ba-casein molecules were hydrolyzed with equal efficiency, whereas van Hooydonk et al. (1984) found that the rate of chymosin-catalyzed hydrolysis decreased with increasing glycosylation. Again, differences in experimental approach were probably responsible for at least some of the apparent discrepancy. Rennet coagulation time (RCT), rate of curd firming, and curd firmness have been measured to assess the effect of \u03ba-casein glycosylation on the coagulation properties of milk (Robitaille et al., 1993). Whereas no effect on RCT was observed, the rate of curd firming decreased and the curd firmness increased at higher glycosylation levels. A more recent study of more than 2000 samples of milk from Simmental cows (Bonfatti, et al., 2013) observed that RCT decreased when total \u03ba-casein (as a proportion of total casein) and glycosylated-\u03ba-casein increased, whereas unglycosylated-\u03ba-casein exhibited a slightly unfavorable effect on the onset of the coagulation process. A slight decrease of RCT was also observed for milks with a high degree of glycosylation of \u03ba-casein, although this effect was less clear than that of glycosylated-\u03ba-casein. A favorable effect of \u03ba-casein, glycosylated \u03ba-casein, and degree of glycosylation on curd firmness was also detected. In another recent study, a detailed characterization of \u03ba-casein isoforms was conducted on milks with extremes of good or poor coagulation properties, selected from 892 samples of milk from Holstein-Friesian and Jersey cows (Jensen et al., 2012). Six \u03ba-casein isoforms varying in phosphorylation and glycosylation levels from each of the genetic variants of \u03ba-casein were separated and identified, along with an unmodified \u03ba-casein form at low abundance. Relative quantification showed that around 95% of total \u03ba-casein was phosphorylated with 1 or 2 phosphates attached, whereas approximately 35% of the identified \u03ba-casein was glycosylated with 1 to 3 tetrasaccharides. In a comparison of isoforms from individual samples, a very consistent \u03ba-casein isoform pattern was found, with only minor differences in relation to breed, \u03ba-casein genetic variant, and milk coagulation ability.\n\nDifferences in rennet coagulation properties have also been observed for genetic variants of \u03ba-casein. Shorter RCTs, higher curd-firming rates, and higher curd firmness have been reported for milk from cows with the BB variant than for milk from cows with the AA variant (Walsh et al., 1998, and references therein). These differences between A and B variant milks were maintained after heat treatments of up to 80 \u00b0C for 2 min, despite an overall deterioration of the coagulation properties at elevated temperatures (Choi & Ng-Kwai-Hang, 2003). Milks containing the rarer \u03ba-casein C variant form rennet gels even more slowly than the A or B variant milks, possibly because of the substitution of histidine for arginine at residue 118, which may affect chymosin binding (Smith et al., 1997). Similar results have been observed for the \u03ba-casein G variant, which has cysteine at residue 118 (Erhardt et al., 1997), and a similar explanation has been proposed (Smith et al., 1997).\n\nCoagulation of milk can also be induced by acid, as is the case in yogurt manufacture. Because this step does not involve \u03ba-casein cleavage, a lesser or different effect might be expected. There are fewer studies on the effect of the glycosylation of \u03ba-casein on acid coagulability. Cases et al. (2003) found that partial deglycosylation with neuraminidase had little effect on micellar surface charge and solvation but caused a decrease in acid gelation time, a higher rate of gel firming, and a higher final firmness.\n\nHeat treatment of milk can also destabilize the casein micelle structure. The heat-induced coagulation of milk is a very complex process that is affected by many parameters (O'Connell & Fox, 2003). A number of studies have examined the influence of genetic variants of \u03ba-casein on heat stability parameters, and it is generally accepted that B variant milks are more stable than A variant milks (FitzGerald & Hill, 1997). The reason may be related more to the effects on \u03ba-casein concentration and micelle size mentioned above than to the structural differences between the variants (Smith et al., 2002). Again, there are fewer studies related to the influence of the glycosylation of \u03ba-casein on heat stability. Using a model system composed of casein micelles in simulated milk ultrafiltrate, Minkiewicz et al. (1993) showed that enzymatic removal of neuraminic acid using neuraminidase caused a decrease in heat stability. However, Robitaille and Ayers (1995), using whole milk, could not find a significant effect of neuraminidase treatment on heat stability. When milk is heated above 65 \u00b0C, \u03b2-lactoglobulin denatures, exposing a previously buried sulfhydryl group that can participate in disulfide exchange reactions with other cysteine-containing proteins including \u03ba-casein. This interaction has been recognized for many decades (Sawyer, 1969) and has been the subject of numerous investigations and reviews over the years; a detailed analysis is beyond the scope of this review (for an extensive review, see Chapter 9 of this volume). Recent studies have addressed both the mechanism of formation (Guyomarc'h et al., 2003) and the impact on product quality (Vasbinder et al., 2003) of disulfide-linked complexes. Despite the vast amount of literature on this topic, there do not appear to be any studies that have addressed the impact of the variable glycosylation of \u03ba-casein on its ability to form disulfide-linked complexes either with itself or with \u03b2-lactoglobulin, although this is perhaps not surprising because the sulfhydryl amino acids are in the para-\u03ba-casein domain of the molecule and the glycosylation sites are in the caseinomacropeptide.\n\nHeat-induced changes in micelle structure are particularly relevant for ultra-high-temperature (UHT) milk production and storage. The extremes of heat treatment (of the order of 140\u2013145oC for 4\u201310 s) produce a number of changes in the milk, not least of which is the formation of \u03ba-casein\u2013 \u03b2-lactoglobulin complexes. On storage, UHT-treated milks show a variable tendency to form gels, and this phenomenon, known as age gelation, affects product shelf life (for a review, see Datta & Deeth, 2001). Again, despite extensive studies over many years on UHT processing and product performance, the influence of \u03ba-casein heterogeneity, particularly heterogeneity with respect to glycosylation, has not been addressed. From a theoretical perspective, higher initial levels of glycosylation may act to temper the deleterious effects of heat treatment through effects on micellar size, micellar stability, and formation of disulfide-linked complexes. The heat treatment itself may affect the glycosylation level at the surface of the micelle either indirectly, through loss of \u03ba-casein in complex formation with \u03b2-lactoglobulin, or directly, through degradation of glycosidic residues (van Hooydonk et al., 1987; as quoted in Dziuba & Minikiewicz, 1996). Subsequent changes in the glycosylation level during storage could be mediated by the action of heat-stable glycosidases originating from psychrotrophic bacteria present in the raw milk (Marin et al., 1984). Release of monosaccharides during the storage of UHT milk has been observed (Recio et al., 1998; Belloque et al., 2001). Thus, both the initial glycosylation level of the \u03ba-casein and the residual amount after UHT treatment may affect the storage properties of UHT-treated milk. As the actions of heat-resistant proteinases can contribute to the age gelation of UHT milk, the inhibitory effects of glycosylation on the activity of proteinases such as plasmin (Doi et al., 1979) may be important for prolonging shelf life. Unraveling specific effects will require the application of modern proteomic technologies for \u03ba-casein analysis (Claverol et al., 2003; Holland et al., 2004; ; ; O'Donnell et al., 2004). Through use of these technologies, it will be possible to elaborate the heterogeneous glycoforms of \u03ba-casein in raw milk, after pretreatment(s), after UHT processing, and during storage leading up to gelation. This will allow a definitive assessment of the functional significance of \u03ba-casein glycosylation.\n\nFunctional performance of the caseinomacropeptide is of importance both for use of the caseinomacropeptide as a food ingredient in its own right and for the properties it can impart to cheese whey products. Emulsifying performance has been found to vary with glycosylation (Kreuss et al., 2009a). Nonglycosylated caseinomacropeptide showed significantly better emulsifying properties than glycosylated caseinomacropeptide. While nonglycosylated caseinomacropeptide showed an emulsifying activity index of 150.7 g\/m2, glycosylated caseinomacropeptide achieved a value of only 98.5 g\/m2. Stability of emulsions was 1.4 times higher for nonglycosylated than for glycosylated caseinomacropeptide. Droplet size measurements and creaming studies showed a strong influence of pH on both fractions, with minimal emulsion stabilities at pH 4.1 (glycosylated caseinomacropeptide) and 4.9 (nonglycosylated). Investigation of the flocculation behavior and variations of the ionic strength indicated that the glycan side chains induce a combination of electrostatic, steric, and hydrophilic effects, preventing an ordered adsorption of glycosylated caseinomacropeptide molecules at the oil\/water interface, while nonglycosylated caseinomacropeptide builds a stable network at the surface.\n\nIn a further study, caseinomacropeptide fractions, both glycosylated and nonglycosylated, were studied in detail for their foaming properties (Kreuss et al., 2009b). The nonglycosylated caseinomacropeptide-stabilized foams showed significantly higher foam rigidity and stability than foams stabilized with glycosylated caseinomacropeptide, whereas both fractions yielded a high foaming ability with overruns of around 600%. The glycosylated caseinomacropeptide-stabilized foams, in particular, were considerably influenced by pH and showed reduced foaming properties above the pI, but superior properties at strong acidic pH, below the pI. This influence was less significant for nonglycosylated caseinomacropeptide. An increase in ionic strength did not appear to influence either fraction. The combination of electrical, steric, and hydrophilic barriers caused by the glycosylation of glycosylated caseinomacropeptide appears not to allow an ordered adsorption at the air\/water interface, whereas nonglycosylated caseinomacropeptide can build a stable network at the surface.\n\n#### Biological Significance\n\nOne aspect of \u03ba-casein heterogeneity that has not been considered is its influence on the biological properties of milk. This aspect has been reviewed extensively (Dziuba & Minikiewicz, 1996). There are three areas to consider. First, the effect of post-translational modification on digestibility and bioavailability has only received attention relatively recently. The effect of glycosylation on hydrolysis by chymosin has been discussed above and has been intensively studied because of its importance for cheese making. The digestion of the resultant glycosylated or nonglycosylated caseinomacropeptide by brush border membrane peptidases has been described by Boutrou et al. (2008). Their key finding was that the digestion of unglycosylated and glycosylated caseinomacropeptide through the action of exopeptidases was similar, but the activity of endopeptidases on glycosylated caseinomacropeptide was limited, certainly due to the attached O-glycosylations. Consequently, many more peptides were identified from the unglycosylated than from the glycosylated caseinomacropeptide. In addition, the glycosylation core, as well as the number of the attached glycosylated chains, modified the kinetics of digestion, the most heavily glycosylated forms being the slowest digested.\n\nSecond, there is the nutritional contribution of the carbohydrate residues in \u03ba-casein, particularly NeuAc. The importance of NeuAc and its roles in numerous biological functions have been reviewed (Schauer, 2000). NeuAc is commonly found as the terminal sugar residue on mammalian glycoproteins. Although mammals can synthesize NeuAc, the high levels in milk and especially colostrum may be related to a high demand for neonatal growth and development. The normal glycans on \u03ba-casein are part of a class known as the Thomsen-Friedenreich-related antigens (Dall'Olio & Chiricolo, 2001). The terminal NeuAc residues may play a key role in preventing colonization of the gut by pathogenic organisms by providing alternative binding sites that minimize binding to the normal gut epithelium.\n\nThe third area relates to the enormous interest in bioactive peptides derived from milk proteins (Clare & Swaisgood, 2000; Kilara & Panyam, 2003). Numerous in vitro activities have been ascribed to \u03ba-casein, its caseinomacropeptide or peptides derived from them (Dziuba & Minikiewicz, 1996; Brody, 2000). Some of these activities appear to be associated with particular forms of \u03ba-casein (Malkoski et al., 2001) and can be glycosylation-dependent (Li & Mine, 2004). Whether or not the same activities occur in vivo is not always clear because it requires both generation and absorption of the active component during digestion and this is not easy to detect. In vivo production of caseinomacropeptide is known to occur after milk ingestion (Ledoux et al., 1999, and references therein) and has been detected in the plasma of infants after the ingestion of milk (Chabance et al., 1995). Any naturally occurring bioactivity of caseinomacropeptide-derived peptides could be strongly influenced by the glycosylation status of \u03ba-casein either directly, by modifying the activity of the peptide, or indirectly, by affecting proteolysis of \u03ba-casein and hence release of the peptide.\n\n## Caseins from other species\n\nAlthough much of the focus has been on bovine milk, other species have not been neglected (Ginger & Grigor, 1999), as in recent years attention has focused on milk from alternative species as a source of nutrition. It is apparent that considerable variations in caseins and their PTMs occur between different species. In this section of the chapter, all positions reported are for the mature protein, that is, without the promoter region.\n\nThe \u03b2-casein of human milk exists as six different forms with 0 to 5 phosphates (Greenberg et al., 1984). In a recent study on human milk, the phosphorylation status of \u03b2-casein was investigated in human milk samples over time (Molinari et al., 2013). The phosphorylation of \u03b2-casein varied significantly between term and preterm milk. A longitudinal trend was observed among the term population, with the phosphorylation state of \u03b2-casein decreasing during lactation in seven of the eight mothers analyzed. In the preterm population, no change in the distribution of phosphorylated isoforms was observed over time in 10 of the 16 mothers, whereas in the other 6 mothers, the level of phosphorylation increased during lactation. A study on glycosylation in human milk revealed changing patterns of glycosylation of many of the whey proteins, but not for \u03ba-casein (Froehlich et al., 2010).\n\nEquine \u03b2-casein also shows variation in phosphorylation, with typically 3 to 7 phosphates on full-length \u03b2-casein (Girardet et al., 2006) and 1 to 7 phosphates on a low-molecular-weight form that arises from an internal deletion (Miclo et al., 2007). The phosphorylation sites have recently been reported for equine milk (Mateos et al., 2010). The isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P\u20137P, respectively. The results indicate that the in vivo phosphorylation of the equine \u03b2-casein follows a sequential path and is not random.\n\nOvine \u03b2-casein has also been reported to be variably phosphorylated, with 0 to 7 phosphates (Ferranti et al., 2001), although it is not clear where the seventh phosphorylation site is. Caprine \u03b2-casein appears to be more like bovine \u03b2-casein with the same five phosphorylation sites and an additional site on Thr27 (Neveu et al., 2002). A more recent study compared goats' milk from the indigenous Greek breed and from the international breeds Saanen and Alpine (Moatsou et al., 2008). This study identified a wide range of protein polymorphisms and phosphorylations. Phosphorylation of \u03b1s1-casein was 7P, 8P, and 9P, with lesser amounts of 6P and, rarely, 10P; phosphorylation of \u03b1s2-casein ranged from 6P to 11P; phosphorylation of \u03b2-casein was 5P, 6P, and 7P, with small amounts of lesser phosphorylation; and \u03ba-casein was phosphorylated with mostly 2P but some lesser amounts. A recent study using nanoscale liquid chromatography coupled with tandem electrospray mass spectrometry has identified the majority of the phosphorylation sites of the goat caseins (Olumee-Shabon & Boehmer, 2013). These are summarized in Table 5.3.\n\nTable 5.3\n\nPhosphorylation Positions for the Caprine Caseins\n\nCasein | No of phosphorylations | Phosphorylated residues  \n---|---|---  \n\u03b1s1 | 9 | S61, S63, S130 (6 others not detected)  \n\u03b1s2 | 10 | S23, S24, S25, S72, S73, S74, S77, S145, S147, S159  \n\u03b2 | 5 | S32, S33, S34, S37, S50\n\nData from Olumee-Shabon, Boehmer, 2013.\n\nA detailed proteomic analysis of water buffalo milk (D'Ambrosio et al., 2008) showed high consistency with the bovine counterpart proteins (residue numbers here based on the mature protein sequence). The study identified phosphopeptides from \u03b1sl-casein where phosphorylation occurred at Ser 41, 46, 48, 64, 66, 67, 68, and 75; \u03b1s2-casein phosphorylation occurred at the same sites of the bovine counterpart (Ser 129, 131, and 143); \u03b2-casein phosphorylation sites were consistent with bovine (Ser 15, 17, 18, 19, and 35); and the main and secondary sites of phosphorylation in buffalo \u03ba-casein were Ser 149 and Ser 127 as observed for the bovine protein. Diglycosylated forms of \u03ba-casein were identified, but the specific residues modified were not reported. However, there is no reason to believe they would be different from the bovine sites.\n\nA recent study of the donkey 'caseome' (Chianese et al., 2010) identified 11 components for \u03ba-casein, six phosphorylated components for \u03b2\\- and \u03b1s1-casein, and three main phosphorylated components for \u03b1s2-casein.\n\n## Conclusions\n\nPTMs such as phosphorylation, glycosylation, and perhaps disulfide bond formation play a critical role in casein micelle formation and stability. It seems somewhat surprising that so much variability occurs in these PTMs on the caseins. Whereas significant functional differences in milk properties have been consistently reported for milks with different genetic variants of the caseins and are well established, the effects reported for variable PTMs have been investigated only more recently, enabled by proteomic methods and supported by a range of highly sensitive mass spectrometry techniques. A full understanding of these effects and their implications for dairy processing and products is still in its infancy.\n\n# References\n\nAddeo F ,  Martin P ,  Ribadeau-Dumas B .  Susceptibility of buffalo and cow kappa-caseins to chymosin action .  _Milchwissenschaft_.  1984 ;39 : 202 \u2013 205 .\n\nAlais C ,  Jolles P .  Comparative study of the caseinoglycopeptides formed by the action of rennin on caseins from the cow, sheep and goat. II. Study of the non-peptide portion .  _Biochimica et Biophysica Acta_.  1961 ;51 : 315 \u2013 322 .\n\nAlexander LJ ,  Stewart AF ,  Mackinlay AG ,  Kapelinskaya TV ,  Tkach TM ,  Gorodetsky SI .  Isolation and characterization of the bovine kappa-casein gene .  _European Journal of Biochemistry_.  1988 ;178 : 395 \u2013 401 .\n\nBelloque J ,  Villamiel M ,  Lopez FR ,  Olano A .  Release of galactose and N-acetylglucosamine during the storage of UHT milk .  _Food Chemistry_.  2001 ;72 : 407 \u2013 412 .\n\nBobe G ,  Beitz DC ,  Freeman AE ,  Lindberg GL .  Effect of milk protein genotypes on milk protein composition and its genetic parameter estimates .  _Journal of Dairy Science_.  1999 ;82 : 2797 \u2013 2804 .\n\nBodenmiller B ,  Mueller LN ,  Mueller M ,  Domon B ,  Aebersold R .  Reproducible isolation of distinct, overlapping segments of the phosphoproteome .  _Nature Methods_.  2007 ;4 : 231 \u2013 237 .\n\nBonfatti V ,  Rostellato R ,  Chiarot G ,  Vicario D ,  Carnier P .  Effects of kappa-CN glycosylation on rennet coagulation properties of milk in Simmental cattle .  _Agriculturae Conspectus Scientificus (Poljoprivredna Znanstvena Smotra)_.  2013 ;78 : 163 \u2013 166 .\n\nBouguyon E ,  Beauvallet C ,  Huet J-C ,  Chanat E .  Disulphide bonds in casein micelle from milk .  _Biochemical and Biophysical Research Communications_.  2006 ;343 : 450 \u2013 458 .\n\nBouniol C ,  Printz C ,  Mercier J-C .  Bovine \u03b1s2-casein D is generated by exon VIII skipping .  _Gene_.  1993 ;128 : 289 \u2013 293 .\n\nBoutrou R ,  Jardin J ,  Blais A ,  Tome D ,  Leonil J .  Glycosylations of kappa-casein-derived caseinomacropeptide reduce its accessibility to endo- but not exointestinal brush border membrane peptidases .  _Journal of Agricultural and Food Chemistry_.  2008 ;56 : 8166 \u2013 8173 .\n\nBovenhuis H ,  Van Arendonk JA ,  Korver S .  Associations between milk protein polymorphisms and milk production traits .  _Journal of Dairy Science_.  1992 ;75 : 2549 \u2013 2559 .\n\nBrody EP .  Biological activities of bovine glycomacropeptide .  _British Journal of Nutrition_.  2000 ;84 (Suppl 1) : S39 \u2013 S46 .\n\nCases E ,  Vidal V ,  Cuq JL .  Effect of kappa-casein deglycosylation on the acid coagulability of milk .  _Journal of Food Science_.  2003 ;68 : 2406 \u2013 2410 .\n\nChabance B ,  Jolles P ,  Izquierdo C ,  Mazoyer E ,  Francoual C ,  Drouet L ,  Fiat AM .  Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion .  _British Journal of Nutrition_.  1995 ;73 : 583 \u2013 590 .\n\nChaplin B ,  Green ML .  Determination of the proportion of kappa-casein hydrolyzed by rennet on coagulation of skim-milk .  _Journal of Dairy Research_.  1980 ;47 : 351 \u2013 358 .\n\nChianese L ,  Calabrese MG ,  Ferranti P ,  Mauriello R ,  Garro G ,  De Simone C ,  Quarto M ,  Addeo F ,  Cosenza G ,  Ramunno L .  Proteomic characterization of donkey milk \"caseome\" .  _Journal of Chromatography A_.  2010 ;1217 : 4834 \u2013 4840 .\n\nChoi JW ,  Ng-Kwai-Hang KF .  Effects of genetic variants of kappa-casein and \u03b2-lactoglobulin and heat treatment on coagulating properties of milk .  _Asian-Australasian Journal of Animal Sciences_.  2003 ;16 : 1212 \u2013 1217 .\n\nClare DA ,  Swaisgood HE .  Bioactive milk peptides: A prospectus .  _Journal of Dairy Science_.  2000 ;83 : 1187 \u2013 1195 .\n\nClaverol S ,  Burlet-Schiltz O ,  Gairin JE ,  Monsarrat B .  Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels .  _Molecular & Cellular Proteomics_.  2003 ;2 : 483 \u2013 493 .\n\nCollins MO ,  Yu L ,  Choudhary JS .  Analysis of protein phosphorylation on a proteome-scale .  _Proteomics_.  2007 ;7 : 2751 \u2013 2768 .\n\nCoolbear KP ,  Elgar DF ,  Ayers JS .  Profiling of genetic variants of bovine \u03ba-casein macropeptide by electrophoretic and chromatographic techniques .  _International Dairy Journal_.  1996 ;6 : 1055 \u2013 1068 .\n\nCox DM ,  Zhong F ,  Du M ,  Duchoslav E ,  Sakuma T ,  McDermott JC .  Multiple reaction monitoring as a method for identifying protein posttranslational modifications .  _Journal of Biomolecular Techniques_.  2005 ;16 : 83 \u2013 90 .\n\nDalgleish DG .  Glycosylated kappa-caseins and the sizes of bovine casein micelles. Analysis of the different forms of kappa-casein .  _Biochimica et Biophysica Acta_.  1985 ;830 : 213 \u2013 215 .\n\nDalgleish DG .  Analysis by fast protein liquid chromatography of variants of kappa-casein and their relevance to micellar structure and renneting .  _Journal of Dairy Research_.  1986 ;53 : 43 \u2013 51 .\n\nDalgleish DG ,  Horne DS ,  Law AJR .  Size-related differences in bovine casein micelles .  _Biochimica et Biophysica Acta_.  1989 ;991 : 383 \u2013 387 .\n\nDall'Olio F ,  Chiricolo M .  Sialyltransferases in cancer .  _Glycoconjugate Journal_.  2001 ;18 : 841 \u2013 850 .\n\nD'Ambrosio C ,  Arena S ,  Salzano AM ,  Renzone G ,  Ledda L ,  Scaloni A .  A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens .  _Proteomics_.  2008 ;8 : 3657 \u2013 3666 .\n\nDatta N ,  Deeth HC .  Age gelation of UHT milk\u2014a review .  _Food and Bioproducts Processing_.  2001 ;79 : 197 \u2013 210 .\n\nDavies DT ,  Law AJR .  Variation in protein composition of bovine casein micelles and serum casein in relation to casein micelle size and milk temperature .  _Journal of Dairy Research_.  1983 ;50 : 67 \u2013 75 .\n\nDoi H ,  Kawaguchi N ,  Ibuki F ,  Kanamori M .  Susceptibility of kappa-casein components to various proteases .  _Journal of Nutritional Science and Vitaminology (Tokyo)_.  1979 ;25 : 33 \u2013 41 .\n\nDoi H ,  Kobatake H ,  Ibuki F ,  Kanamori M .  Attachment sites of carbohydrate portions to peptide chain of kappa-casein from bovine colostrum .  _Agricultural and Biological Chemistry_.  1980 ;44 : 2605 \u2013 2611 .\n\nDonnelly WJ ,  McNeill GP ,  Buchheim W ,  McGann TC .  A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles .  _Biochimica et Biophysica Acta_.  1984 ;789 : 136 \u2013 143 .\n\nDziuba J ,  Minikiewicz P .  Influence of glycosylation on micelle-stabilising ability and biological properties of C-terminal fragments of cow's \u03ba-casein .  _International Dairy Journal_.  1996 ;6 : 1017 \u2013 1044 .\n\nErhardt G ,  Prinzenberg E-M ,  Buchberger J ,  Krick-Salek H ,  Krause I ,  Miller M .  Bovine kappa-casein G: detection, occurrence, molecular genetic characterisation, genotyping and coagulation properties .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 328 \u2013 329. : IDF Special Issue 9702 .\n\nFarrell Jr HM ,  Cooke PH ,  Wickham ED ,  Piotrowski EG ,  Hoagland PD .  Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures .  _Journal of Protein Chemistry_.  2003 ;22 : 259 \u2013 273 .\n\nFarrell Jr HM ,  Jimenez-Flores R ,  Bleck GT ,  Brown EM ,  Butler JE ,  Creamer LK ,  Hicks CL ,  Hollar CM ,  Ng-Kwai-Hang KF ,  Swaisgood HE .  Nomenclature of the proteins of cows' milk\u2014sixth revision .  _Journal of Dairy Science_.  2004 ;87 : 1641 \u2013 1674 .\n\nFerranti P ,  Pizzano R ,  Garro G ,  Caira S ,  Chianese L ,  Addeo F .  Mass spectrometry-based procedure for the identification of ovine casein heterogeneity .  _Journal of Dairy Research_.  2001 ;68 : 35 \u2013 51 .\n\nFiat AM ,  Jolles J ,  Loucheux-Lefebvre MH ,  Alais C ,  Jolles P .  Localisation of the prosthetic sugar groups of bovine colostrum kappa-casein .  _Hoppe Seylers Zeitschrift fur Physiologische Chemie_.  1981 ;362 : 1447 \u2013 1454 .\n\nFiat AM ,  Chevan J ,  Jolles P ,  De Waard P ,  Vliegenthart JF ,  Piller F ,  Cartron JP .  Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity .  _European Journal of Biochemistry_.  1988 ;173 : 253 \u2013 259 .\n\nFitzGerald RJ ,  Hill JP .  The relationship between milk protein polymorphism and the manufacture and functionality of dairy products .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 355 \u2013 371 : IDF Special Issue 9702 .\n\nFournet B ,  Fiat AM ,  Montreuil J ,  Jolles P .  The sugar part of kappa-caseins from cow milk and colostrum and its microheterogeneity .  _Biochimie_.  1975 ;57 : 161 \u2013 165 .\n\nFournet B ,  Fiat AM ,  Alais C ,  Jolles P .  Cow kappa-casein: structure of the carbohydrate portion .  _Biochimica et Biophysica Acta_.  1979 ;576 : 339 \u2013 346 .\n\nFroehlich JW ,  Dodds ED ,  Barboza M ,  McJimpsey EL ,  Seipert RR ,  Francis J ,  An HJ ,  Freeman S ,  German JB ,  Lebrilla CB .  Glycoprotein Expression in Human Milk during Lactation .  _Journal of Agricultural and Food Chemistry_.  2010 ;58 : 6440 \u2013 6448 .\n\nGinger MR ,  Grigor MR .  Comparative aspects of milk caseins .  _Comparative Biochemistry and Physiology B_.  1999 ;124 : 133 \u2013 145 .\n\nGirardet J-M ,  Miclo L ,  Florent S ,  Moll\u00e9 D ,  Gaillard J-L .  Determination of the phosphorylation level and deamidation susceptibility of equine \u03b2-casein .  _Proteomics_.  2006 ;6 : 3707 \u2013 3717 .\n\nGorodetskii SI ,  Kershulite DR ,  Korobko VG .  [Primary structure of cDNA of Bos taurus kappa-casein macropeptide] .  _Bioorganicheskaya Khimiya_.  1983 ;9 : 1693 \u2013 1695 .\n\nGreenberg R ,  Groves ML ,  Dower HJ .  Human beta-casein. Amino acid sequence and identification of phosphorylation sites .  _Journal of Biological Chemistry_.  1984 ;259 : 5132 \u2013 5138 .\n\nGrosclaude F ,  Mahe MF ,  Mercier JC ,  Ribadeau-Dumas B .  Characterization of genetic variants of alpha-S1 and beta bovine caseins .  _European Journal of Biochemistry_.  1972 ;26 : 328 \u2013 337 .\n\nGrosclaude F ,  Mahe M-F ,  Mercier J-C ,  Ribadeau-Dumas B .  Localization of amino-acid substitutions differentiating the A and B variants of kappa-casein in cattle .  _Annales de Genetique et de Selection Animale_.  1972 ;4 : 515 \u2013 521 .\n\nGroves ML ,  Dower HJ ,  Farrell Jr HM .  Reexamination of the polymeric distributions of kappa-casein isolated from bovine milk .  _Journal of Protein Chemistry_.  1992 ;11 : 21 \u2013 28 .\n\nGroves ML ,  Wickham ED ,  Farrell Jr HM .  Environmental effects on disulfide bonding patterns of bovine kappa-casein .  _Journal of Protein Chemistry_.  1998 ;17 : 73 \u2013 84 .\n\nGuerin J ,  Alais C ,  Jolles J ,  Jolles P .  Kappa-casein from bovine colostrum .  _Biochimica et Biophysica Acta_.  1974 ;351 : 325 \u2013 332 .\n\nGuyomarc'h F ,  Law AJ ,  Dalgleish DG .  Formation of soluble and micelle-bound protein aggregates in heated milk .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 4652 \u2013 4660 .\n\nHeck JML ,  Schennink A ,  van Valenberg HJF ,  Bovenhuis H ,  Visker MHPW ,  van Arendonk JAM ,  van Hooijdonk ACM .  Effects of milk protein variants on the protein composition of bovine milk .  _Journal of Dairy Science_.  2009 ;92 : 1192 \u2013 1202 .\n\nHernandez-Hernandez O ,  Lebron-Aguilar R ,  Quintanilla-Lopez JE ,  Luz Sanz M ,  Javier Moreno F .  Detection of two minor phosphorylation sites for bovine \u03ba-casein macropeptide by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry .  _Journal of Agricultural and Food Chemistry_.  2011 ;59 : 10848 \u2013 10853 .\n\nHolland JW ,  Deeth HC ,  Alewood PF .  Analysis of disulphide linkages in bovine kappa-casein oligomers using two-dimensional electrophoresis .  _Electrophoresis_.  2008 ;29 : 2402 \u2013 2410 .\n\nHolland JW ,  Deeth HC ,  Alewood PF .  Proteomic analysis of kappa-casein micro-heterogeneity .  _Proteomics_.  2004 ;4 : 743 \u2013 752 .\n\nHolland JW ,  Deeth HC ,  Alewood PF .  Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis .  _Proteomics_.  2005 ;5 : 990 \u2013 1002 .\n\nHolland JW ,  Deeth HC ,  Alewood PF .  Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy .  _Proteomics_.  2006 ;6 : 3087 \u2013 3095 .\n\nHolt C ,  Horne DS .  The hairy casein micelle: evolution of the concept and its implications for dairy technology .  _Netherlands Milk and Dairy Journal_.  1996 ;50 : 85 \u2013 111 .\n\nHorne DS .  Casein interactions: casting light on the black boxes, the structure in dairy products .  _International Dairy Journal_.  1998 ;8 : 171 \u2013 177 .\n\nHorne DS .  Casein structure, self-assembly and gelation .  _Current Opinion in Colloid & Interface Science_.  2002 ;7 : 456 \u2013 461 .\n\nIbeagha-Awemu EM ,  Prinzenberg EM ,  Jann OC ,  Luhken G ,  Ibeagha AE ,  Zhao X ,  Erhardt G .  Molecular characterization of bovine CSN1S2*B and extensive distribution of zebu-specific milk protein alleles in European cattle .  _Journal of Dairy Science_.  2007 ;90 : 3522 \u2013 3529 .\n\nJensen HB ,  Holland JW ,  Poulsen NA ,  Larsen LB .  Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties .  _Journal of Dairy Science_.  2012 ;95 : 2891 \u2013 2903 .\n\nJolles J ,  Fiat AM ,  Alais C ,  Jolles P .  Comparative study of cow and sheep kappa-caseinoglycopeptides: Determination of the N-terminal sequences with a sequencer and location of the sugars .  _FEBS Letters_.  1973 ;30 : 173 \u2013 176 .\n\nKanamori M ,  Kawaguchi N ,  Ibuki F ,  Doi H .  Attachment sites of carbohydrate moieties to peptide chain of bovine kappa-casein from normal milk .  _Agricultural and Biological Chemistry_.  1980 ;44 : 1855 \u2013 1861 .\n\nKanamori M ,  Doi H ,  Ideno S ,  Ibuki F .  Presence of O-glycosidic linkage through serine residue in kappa-casein component from bovine mature milk .  _Journal of Nutritional Science and Vitaminology (Tokyo)_.  1981 ;27 : 231 \u2013 241 .\n\nKapkova P ,  Lattova E ,  Perreault H .  Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption\/ionization mass spectrometry .  _Analytical Chemistry_.  2006 ;78 : 7027 \u2013 7033 .\n\nKeating AF ,  Davoren P ,  Smith TJ ,  Ross RP ,  Cairns MT .  Bovine \u03ba-casein gene promoter haplotypes with potential implications for milk protein expression .  _Journal of Dairy Science_.  2007 ;90 : 4092 \u2013 4099 .\n\nKilara A ,  Panyam D .  Peptides from milk proteins and their properties .  _Critical Reviews in Food Science and Nutrition_.  2003 ;43 : 607 \u2013 633 .\n\nKreuss M ,  Strixner T ,  Kulozik U .  The effect of glycosylation on the interfacial properties of bovine caseinomacropeptide .  _Food Hydrocolloids_.  2009 ;23 : 1818 \u2013 1826 .\n\nKreuss M ,  Krause I ,  Kulozik U .  Influence of glycosylation on foaming properties of bovine caseinomacropeptide .  _International Dairy Journal_.  2009 ;19 : 715 \u2013 720 .\n\nLeaver J ,  Horne DS .  Chymosin-catalysed hydrolysis of glycosylated and nonglycosylated bovine kappa-casein adsorbed on latex particles .  _Journal of Colloid and Interface Science_.  1996 ;181 : 220 \u2013 224 .\n\nLedoux N ,  Mah\u00e9 S ,  Dubarry M ,  Bourras M ,  Benamouzig R ,  Tom\u00e9 D .  Intraluminal immunoreactive caseinomacropeptide after milk protein ingestion in humans .  _Nahrung_.  1999 ;43 : 196 \u2013 200 .\n\nLi EWY ,  Mine Y .  Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophage-like cells, U937 .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 2704 \u2013 2708 .\n\nLodes A ,  Krause I ,  Buchberger J ,  Aumann J ,  Klostermeyer H .  The influence of genetic variants of milk proteins on the composition and technological properties of milk. 1. Casein micelle size and the content of non-glycosylated \u03ba-casein .  _Milchwissenschaft_.  1996 ;51 : 368 \u2013 373 .\n\nMahe MF ,  Miranda G ,  Queval R ,  Bado A ,  Zafindrajaona PS ,  Grosclaude F .  Genetic polymorphism of milk proteins in African Bos taurus and Bos indicus populations. Characterisation of variants alphaS1-casein H and kappa-casein .  _Genetics Selection Evolution_.  1999 ;31 : 239 \u2013 253 .\n\nMalkoski M ,  Dashper SG ,  O'Brien-Simpson NM ,  Talbo GH ,  Macris M ,  Cross KJ ,  Reynolds EC .  Kappacin, a novel antibacterial peptide from bovine milk .  _Antimicrobial Agents and Chemotherapy_.  2001 ;45 : 2309 \u2013 2315 .\n\nManson W ,  Carolan T ,  Annan WD .  Bovine \u03b1S0 casein; a phosphorylated homologue of \u03b1S1 casein .  _European Journal of Biochemistry_.  1977 ;78 : 411 \u2013 417 .\n\nMarin A ,  Mawhinney TP ,  Marshall RT .  Glycosidic activities of Pseudomonas fluorescens on fat-extracted skim milk, buttermilk, and milk fat globule membranes .  _Journal of Dairy Science_.  1984 ;67 : 52 \u2013 59 .\n\nMartin P ,  Szymanowska M ,  Zwierzchowski L ,  Leroux C .  The impact of genetic polymorphisms on the protein composition of ruminant milks .  _Reproduction Nutrition Development_.  2002 ;42 : 433 \u2013 459 .\n\nMateos A ,  Girardet J-M ,  Molle D ,  Corbier C ,  Gaillard J-L ,  Miclo L .  Identification of phosphorylation sites of equine beta-casein isoforms .  _Rapid Communications in Mass Spectrometry_.  2010 ;24 : 1533 \u2013 1542 .\n\nMcGann TC ,  Donnelly WJ ,  Kearney RD ,  Buchheim W .  Composition and size distribution of bovine casein micelles .  _Biochimica et Biophysica Acta_.  1980 ;630 : 261 \u2013 270 .\n\nMercier JC .  Phosphorylation of caseins, present evidence for an amino acid triplet code post translationally recognized by specific kinases .  _Biochimie_.  1981 ;63 : 1 \u2013 17 .\n\nMercier JC ,  Brignon G ,  Ribadeau-Dumas B .  Primary structure of bovine kappa B casein. Complete sequence .  _European Journal of Biochemistry_.  1973 ;35 : 222 \u2013 235 .\n\nMiclo L ,  Girardet JM ,  Egito AS ,  Molle D ,  Martin P ,  Gaillard JL .  The primary structure of a low-M(r) multiphosphorylated variant of beta-casein in equine milk .  _Proteomics_.  2007 ;7 : 1327 \u2013 1335 .\n\nMinkiewicz P ,  Dziuba J ,  Muzinska B .  The contribution of N-acetylneuraminic acid in the stabilization of micellar casein .  _Polish Journal of Food and Nutrition Sciences_.  1993 ;2 : 39 \u2013 48 .\n\nMiranda G ,  Anglade P ,  Mahe MF ,  Erhardt G .  Biochemical characterization of the bovine genetic kappa-casein C and E variants .  _Animal Genetics_.  1993 ;24 : 27 \u2013 31 .\n\nMoatsou G ,  Moschopoulou E ,  Molle D ,  Gagnaire V ,  Kandarakis I ,  Leonil J .  Comparative study of the protein fraction of goat milk from the Indigenous Greek breed and from international breeds .  _Food Chemistry_.  2008 ;106 : 509 \u2013 520 .\n\nMolinari CE ,  Casadio YS ,  Hartmann BT ,  Arthur PG ,  Hartmann PE .  Longitudinal analysis of protein glycosylation and beta-casein phosphorylation in term and preterm human milk during the first 2 months of lactation .  _British Journal of Nutrition_.  2013 ;110 : 105 \u2013 115 .\n\nMolle D ,  Leonil J .  Heterogeneity of the bovine \u03ba-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry .  _Journal of Chromatography A_.  1995 ;708 : 223 \u2013 230 .\n\nNeveu C ,  Molle D ,  Moreno J ,  Martin P ,  Leonil J .  Heterogeneity of caprine beta-casein elucidated by RP-HPLC\/MS: genetic variants and phosphorylations .  _Journal of Protein Chemistry_.  2002 ;21 : 557 \u2013 567 .\n\nNg-Kwai-Hang KF .  A review of the relationship between milk protein polymorphism and milk composition\/milk production .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 22 \u2013 37 : IDF Special Issue 9702 .\n\nO'Connell JE ,  Fox PF .  The two-stage coagulation of milk proteins in the minimum of the heat coagulation time-pH profile of milk: Effect of casein micelle size .  _Journal of Dairy Science_.  2000 ;83 : 378 \u2013 386 .\n\nO'Connell JE ,  Fox PF .  Heat-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 879 \u2013 945 .\n\nO'Donnell R ,  Holland JW ,  Deeth HC ,  Alewood P .  Milk proteomics .  _International Dairy Journal_.  2004 ;14 : 1013 \u2013 1023 .\n\nOlumee-Shabon Z ,  Boehmer JL .  Detection of casein phosphopeptides in goat milk .  _Journal of Proteome Research_.  2013 ;12 : 3034 \u2013 3041 .\n\nPisano A ,  Packer NH ,  Redmond JW ,  Williams KL ,  Gooley AA .  Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine kappa-casein .  _Glycobiology_.  1994 ;4 : 837 \u2013 844 .\n\nPrinzenberg EM ,  Hiendleder S ,  Ikonen T ,  Erhardt G .  Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP .  _Animal Genetics_.  1996 ;27 : 347 \u2013 349 .\n\nPrinzenberg EM ,  Krause I ,  Erhardt G .  SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1) .  _Animal Biotechnology_.  1999 ;10 : 49 \u2013 62 .\n\nRasmussen LK ,  Hojrup P ,  Petersen TE .  The multimeric structure and disulfide-bonding pattern of bovine kappa-casein .  _European Journal of Biochemistry_.  1992 ;207 : 215 \u2013 222 .\n\nRasmussen LK ,  Hojrup P ,  Petersen TE .  Disulphide arrangement in bovine caseins: Localization of intrachain disulphide bridges in monomers of kappa- and alpha s2-casein from bovine milk .  _Journal of Dairy Research_.  1994 ;61 : 485 \u2013 493 .\n\nRasmussen LK ,  Sorensen ES ,  Petersen TE ,  Nielsen NC ,  Thomsen JK .  Characterization of phosphate sites in native ovine, caprine, and bovine casein micelles and their caseinomacropeptides: a solid-state phosphorus-31 nuclear magnetic resonance and sequence and mass spectrometric study .  _Journal of Dairy Science_.  1997 ;80 : 607 \u2013 614 .\n\nRasmussen LK ,  Johnsen LB ,  Tsiora A ,  Sorensen ES ,  Thomsen JK ,  Nielsen NC ,  Jakobsen HJ ,  Petersen TE .  Disulphide-linked caseins and casein micelles .  _International Dairy Journal_.  1999 ;9 : 215 \u2013 218 .\n\nRecio MI ,  Villamiel M ,  Mart\u00ednez-Castro I ,  Olano A .  Changes in free monosaccharides during storage of some UHT milks: A preliminary study .  _Zeitschrift f \u00fcr Lebensmitteluntersuchung und -Forschung A_.  1998 ;207 : 180 \u2013 181 .\n\nRiggs L ,  Sioma C ,  Regnier FE .  Automated signature peptide approach for proteomics .  _Journal of Chromatography A_.  2001 ;924 : 359 \u2013 368 .\n\nRobitaille G ,  Ayers C .  Effects of \u03ba-casein glycosylation on heat stability of milk .  _Food Research International_.  1995 ;28 : 17 \u2013 21 .\n\nRobitaille G ,  Ng-Kwai-Hang KF ,  Monardes HG .  Variation of the N-acetylneuraminic acid content of bovine kappa-casein .  _Journal of Dairy Research_.  1991 ;58 : 107 \u2013 114 .\n\nRobitaille G ,  Ng-Kwai-Hang KF ,  Monardes HG .  Association of kappa-casein glycosylation with milk production and composition in Holsteins .  _Journal of Dairy Science_.  1991 ;74 : 3314 \u2013 3317 .\n\nRobitaille G ,  Ng-Kwai-Hang KF ,  Monardes HG .  Effect of \u03ba-casein glycosylation on cheese yielding capacity and coagulating properties of milk .  _Food Research International_.  1993 ;26 : 365 \u2013 369 .\n\nRollema HS .  Casein association and micelle formation .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry_ .  London :  Elsevier Applied Science ;  1992 : 111 \u2013 140 .\n\nSaito T ,  Itoh T .  Variations and distributions of O-glycosidically linked sugar chains in bovine kappa-casein .  _Journal of Dairy Science_.  1992 ;75 : 1768 \u2013 1774 .\n\nSaito T ,  Itoh T ,  Adachi S .  The chemical structure of a tetrasaccharide containing N-acetylglucosamine obtained from bovine colostrum kappa-casein .  _Biochimica et Biophysica Acta_.  1981 ;673 : 487 \u2013 494 .\n\nSaito T ,  Itoh T ,  Adachi S ,  Suzuki T ,  Usui T .  The chemical structure of neutral and acidic sugar chains obtained from bovine colostrum kappa-casein .  _Biochimica et Biophysica Acta_.  1981 ;678 : 257 \u2013 267 .\n\nSaito T ,  Itoh T ,  Adachi S ,  Suzuki T ,  Usui T .  A new hexasaccharide chain isolated from bovine colostrum kappa-casein taken at the time of parturition .  _Biochimica et Biophysica Acta_.  1982 ;719 : 309 \u2013 317 .\n\nSawyer WH .  Complex between \u03b2-lactoglobulin and \u03ba-casein. A review .  _Journal of Dairy Science_.  1969 ;52 : 1347 \u2013 1355 .\n\nSchauer R .  Achievements and challenges of sialic acid research .  _Glycoconjugate Journal_.  2000 ;17 : 485 \u2013 499 .\n\nSchild TA ,  Wagner V ,  Geldermann H .  Variants within the 5'-flanking regions of bovine milk-protein-encoding genes: I. Kappa-casein-encoding gene .  _Theoretical and Applied Genetics_.  1994 ;89 : 116 \u2013 120 .\n\nSchlieben S ,  Erhardt G ,  Senft B .  Genotyping of bovine kappa-casein (kappa-CNA, kappa-CNB, kappa-CNC, kappa-CNE) following DNA sequence amplification and direct sequencing of kappa-CNE PCR product .  _Animal Genetics_.  1991 ;22 : 333 \u2013 342 .\n\nShekar PC ,  Goel S ,  Rani SDS ,  Sarathi DP ,  Alex JL ,  Singh S ,  Kumar S .  \u03ba-Casein-deficient mice fail to lactate .  _Proceedings of the National Academy of Sciences of the USA_.  2006 ;103 : 8000 \u2013 8005 .\n\nSlattery CW .  Variation in the glycosylation pattern of bovine kappa-casein with micelle size and its relationship to a micelle model .  _Biochemistry_.  1978 ;17 : 1100 \u2013 1104 .\n\nSmith MH ,  Hill JP ,  Creamer LK ,  Plowman JE .  Towards understanding the variant effect on the rate of cleavage by chymosin on \u03ba-caseins A and C .  _Milk Protein Polymorphism_ .  Brussels :  International Dairy Federation ;  1997 : 185 \u2013 188 : IDF Special Issue 9702 .\n\nSmith MH ,  Edwards PJ ,  Palmano KP ,  Creamer LK .  Structural features of bovine caseinomacropeptide A and B by 1H nuclear magnetic resonance spectroscopy .  _Journal of Dairy Research_.  2002 ;69 : 85 \u2013 94 .\n\nSulimova GE ,  Sokolova SS ,  Semikozova OP ,  Nguet LM ,  Berberov EM .  Analysis of DNA polymorphism of cluster genes in cattle: Casein genes and major histocompatibility complex (MHC) genes .  _Tsitologiia i Genetika_.  1992 ;26 : 18 \u2013 26 .\n\nSulimova GE ,  Badagueva Iu N ,  Udina IG .  Polymorphism of the kappa-casein gene in populations of the subfamily Bovinae .  _Genetika_.  1996 ;32 : 1576 \u2013 1582 .\n\nSwaisgood HE .  Chemistry of the caseins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  3rd ed New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 139 \u2013 202 .\n\nSwaisgood HE ,  Brunner JR ,  Lillevik HA .  Physical parameters of \u03ba-casein from cow's milk .  _Biochemistry_.  1964 ;20 : 1616 \u2013 1623 .\n\nSweet SMM ,  Creese AJ ,  Cooper HJ .  Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation .  _Analytical Chemistry_.  2006 ;78 : 7563 \u2013 7569 .\n\nTakeuchi M ,  Tsuda E ,  Yoshikawa M ,  Sasaki R ,  Chiba H .  Fractionation and characterization of 9 subcomponents of bovine kappa-casein A .  _Agricultural and Biological Chemistry_.  1985 ;49 : 2269 \u2013 2276 .\n\nTalbot B ,  Waugh DF .  Micelle-forming characteristics of monomeric and covalent polymeric kappa caseins .  _Biochemistry_.  1970 ;9 : 2807 \u2013 2813 .\n\nTen Hagen KG ,  Fritz TA ,  Tabak LA .  All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases .  _Glycobiology_.  2003 ;13 : 1R \u2013 16R .\n\nvan Halbeek H ,  Dorland L ,  Vliegenthart JF ,  Fiat AM ,  Jolles P .  A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow kappa-casein .  _Biochimica et Biophysica Acta_.  1980 ;623 : 295 \u2013 300 .\n\nvan Halbeek H ,  Dorland L ,  Vliegenthart JF ,  Fiat AM ,  Jolles P .  Structural characterization of a novel acidic oligosaccharide unit derived from cow colostrum kappa-casein .  _A 500 MHz 1H-NMR study. FEBS Letters_.  1981 ;133 : 45 \u2013 50 .\n\nvan Hooydonk ACM ,  Olieman C ,  Hagedoorn HG .  Kinetics of the chymosin-catalysed proteolysis of kappa-casein in milk .  _Netherlands Milk and Dairy Journal_.  1984 ;38 : 207 \u2013 222 .\n\nvan Hooydonk ACM ,  De Koster PG ,  Boerrigter IJ .  The renneting properties of heated milk .  _Netherlands Milk and Dairy Journal_.  1987 ;41 : 3 \u2013 18 .\n\nVasbinder AJ ,  Alting AC ,  Visschers RW ,  De Kruif CG .  Texture of acid milk gels: formation of disulfide cross-links during acidification .  _International Dairy Journal_.  2003 ;13 : 29 \u2013 38 .\n\nVreeman HJ ,  Visser S ,  Slangen CJ ,  Van Riel JA .  Characterization of bovine kappa-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography .  _Biochemical Journal_.  1986 ;240 : 87 \u2013 97 .\n\nWalsh CD ,  Guinee TP ,  Reville WD ,  Harrington D ,  Murphy JJ ,  O'Kennedy BT ,  FitzGerald RJ .  Influence of \u03ba-casein genetic variant on rennet gel microstructure, Cheddar cheesemaking properties and casein micelle size .  _International Dairy Journal_.  1998 ;8 : 707 \u2013 714 .\n\nWalstra P .  Casein sub-micelles: Do they exist? .  _International Dairy Journal_.  1999 ;9 : 189 \u2013 192 .\n\nWu HY ,  Tseng VS ,  Liao PC .  Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis .  _Journal of Proteome Research_.  2007 ;6 : 1812 \u2013 1821 .\n\nYoung Jr WW ,  Holcomb DR ,  Ten Hagen KG ,  Tabak LA .  Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR: A new view of a large family .  _Glycobiology_.  2003 ;13 : 549 \u2013 557 .\n\nZevaco C ,  Ribadeau-Dumas B .  A study on the carbohydrate binding sites of bovine kappa-casein using high performance liquid chromatography .  _Milchwissenschaft_.  1984 ;39 : 206 \u2013 210 .\n\nZhou H ,  Xu S ,  Ye M ,  Feng S ,  Pan C ,  Jiang X ,  Li X ,  Han G ,  Fu Y ,  Zou H .  Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis .  _Journal of Proteome Research_.  2006 ;5 : 2431 \u2013 2437 . \nChapter 6\n\n# Casein Micelle Structure and Stability\n\nDavid S. Horne Formerly Hannah Research Institute, Ayr, Scotland\n\n## Abstract\n\nThe physicochemical properties of the casein proteins are reviewed, highlighting the factors controlling the strength of those interactions most important to the assembly and structure of the casein micelle, namely, electrostatic repulsion and hydrophobic attraction, with particular emphasis on their magnitude and range. The various strands are drawn together step by step to develop the dual-binding model of casein micelle assembly and structure as a polymerization of a block copolymer system. The model is then used to predict the behavioral properties of the micelle in fluid milk, where, by considering the rheology of high concentration milks, the hard sphere colloidal approach is shown to be a special case limited to milk of normal concentration and pH. The necessity for a dual-binding approach is then forcefully demonstrated in its ability to provide full mechanistic explanations of observed behavior in the renneting, acid gelation, and alcohol-induced destabilization of skim milk. Bond mobility is identified as a crucial factor but also important is bond location and whether bonding can be extended beyond the protective shell of the micelle, its hairy layer. It is emphasized that the dual-binding model is only a tool and that the most important features are the interactions of the caseins with themselves and mineral calcium phosphate.\n\n## Keywords\n\nCasein physicochemical properties, casein micelle structure, micellar calcium phosphate, dual-binding model, casein micelle stability\n\nOutline\n\nIntroduction 169\n\nCasein primary structure and interactions 171\n\nCasein micelle properties 176\n\nModels of casein micelle structure 178\n\nThe Dual-binding Model for Micelle Assembly and Structure 178\n\nCalcium Phosphate Equilibria in the Dual-binding Model 180\n\nPredictions of Casein Micelle Properties in the Context of the Dual-binding Model 182\n\nSize and Appearance 182\n\nEffects of Urea, pH, Sequestrants, and Temperature 183\n\nThe Dual-binding Model and Micellar Interactions 184\n\nConcentrated Micellar Dispersions 186\n\nThe Dual-binding Model and Micellar Destabilization 189\n\nDual-binding Model and Rennet Curd Formation 189\n\nDual-binding Model and Ethanol Stability 191\n\nDual-binding Model and Acid Gel Formation 192\n\nConcluding remarks 195\n\n## Introduction\n\nThe caseins are a family of phosphoproteins found in the milks of all mammals. They exist in these milks generally as complex aggregates or micelles of the proteins and mineral calcium phosphate (Fox and Brodkorb, 2008). Because the caseins utilize the same calcium-sequestering mechanism to regulate the calcium phosphate concentration of their environment, they have recently been identified as members of a wider family of secretory calcium-binding phosphoproteins descended from a common ancestor gene (Kawasaki and Weiss, 2003; 2006). These secretory phosphoproteins include enamel matrix proteins, dentine, salivary proteins, bone extracellular matrix proteins, and the caseins, among others. All are descended from early primordial genes by duplication and divergence to serve their specialized adaptive functions. Their genes retain common functional and sequence features even after this extensive divergence. It is thought that primordial calcium-sensitive casein genes diverged from enamel matrix protein genes before the appearance of monotremes in the Jurassic era (Kawasaki and Weiss, 2003). More controversially, Kawasaki et al. (2011) have recently argued that all caseins, both calcium-sensitive and calcium-insensitive, that is, \u03ba-type caseins, evolved from the odontogenic ameloblast-associated gene (ODAM) via two different pathways; calcium-sensitive genes are postulated to originate directly from secretory calcium-binding phosphoprotein genes (SCPP), whereas the calcium-insensitive genes directly differentiated from follicular dendritic cell-secreted peptide gene (FDCSP), both SCPP and FDCSP having a common ancestor in ODAM. This ancestral line for \u03ba-casein genes is inconsistent with the hypothesis of Jolles et al. (1978), based on high levels of sequence identity, that \u03ba-casein was derived from \u03b3-fibrinogen, a blood coagulation factor.\n\nCasein allows milk to appear supersaturated with calcium phosphate. Essentially, it transports the mineral calcium phosphate safely through the mammary gland, which is essential for development of bones and teeth in the suckling infant, through the medium of the casein micelle. Locking up the calcium phosphate in this package is one aspect of the biological function of the micelle. Ensuring release of this same calcium phosphate in the gastric destination of the milk is a property of the micelle that is not generally given great consideration. More research effort has been put into trying to give mechanistic understanding to the technological behavior of the micelle, understanding necessary to achieve efficient conversion of milk into products such as cheese and yogurt, or the behavior of milk components in emulsions or reconstituted dairy products.\n\nMany of the physical and technological properties of the casein micelle (diffusion, viscosity, and light scattering) can be described by treating the casein micelles as colloidal hard spheres (Alexander et al., 2002). The initial stages, up to the onset of instability, in processes such as renneting, acid-induced gelation, and flocculation in the presence of ethanol can apparently be well described by allowing these micellar hard spheres to become adhesive, essentially, as described later, treating their \u03ba-casein outer layer as a salted polyelectrolyte brush (De Kruif and Zhulina, 1996; De Kruif and Holt, 2003). Beyond the critical point in all three processes, however, changes in micellar integrity and internal structure render the simple colloidal particle approach inadequate (Horne, 2003a,b; Choi et al., 2007; Ozcan-Yilsay et al., 2007).\n\nNeither is the adhesive sphere approach at all helpful in furthering our understanding of the processes of micellar assembly, the pathways to dissociation or the maintenance of micellar integrity. For that, we must turn to a structural model of the casein micelle, bearing in mind that, notwithstanding the inadequacy of the adhesive sphere approach, our micelle model also has to be adaptable enough to explain why that primitive approach has been so successful within its limitations.\n\nVarious models of casein micelle assembly and structure have appeared over the years and have been subjected to regular review and appraisal, most recently by Fox and Brodkorb (2008). The three principal contenders are: (1) the submicelle model of Slattery and Evard (Slattery and Evard, 1973; Slattery, 1977), subsequently elaborated by Schmidt (1980); (2) the nanocluster model of Holt (Holt 1992; 2004; De Kruif and Holt, 2003); and (3) the dual-binding model proposed by Horne (1998). There has been considerable debate in more recent years over the suitability and success of these models (Farrell et al., 2006; Horne, 2006; Dalgleish, 2011). Dalgleish (2011) repeated the arguments of Horne (1998) in dismissing the submicelle model and proposed minor but dubiously founded amendments to the dual-binding model, as did McMahon and Oommen (2008). De Kruif et al. (2012) have also described the latest incarnation of the nanocluster model in terms that are essentially those of a dual-binding model. The differences between these amendments and the original dual-binding model of Horne (1998) are small and subtle and are considered in detail later. It is not our intention to repeat previous arguments; rather we would point out that the dual-binding model has its basis in the interactions and chemistry of the caseins and calcium phosphate. As we demonstrate in the following, the model is only an aid to visualizing how the casein micelle responds in the production of dairy products such as yogurts or rennet curds, or in stability situations with ethanol or heat. The reality is in how the interactions and chemistry dictate the behavior of the caseins in those situations. We first summarize the physicochemical properties and interactions of the caseins and show how these lead naturally to the dual-binding picture.\n\n## Casein primary structure and interactions\n\nJust as casein micelles are aggregates of all of the casein proteins and micellar calcium phosphates, so does the dual-binding model involve the properties and interactions of all of the caseins. Central to this argument are those features of the proteins that are conserved across species and through millennia. The caseins were identified as members of the wider secretory calcium phosphate binding family by their possession of functional and sequence features common to that family (Kawasaki and Weiss, 2003; 2006). Among the conserved motifs is the SXE peptide (Ser\u2013Xaa\u2013Glu) where Xaa may be any amino acid. In the caseins, this peptide provides a recognition template for post-translational phosphorylation of the serine in the mammary gland by a casein kinase (Mercier, 1981). Moreover, in the caseins, the serine residues are often found clustered in groups of two, three, or four. Such clusters in the \u03b1S\\- and \u03b2-caseins are highly conserved (Martin et al., 2003), and their numbers attest to the significance of the calcium phosphate requirement for postnatal growth in mammals\u2014even more so when it is noted by reference to their sequences (Swaisgood, 2003) that the \u03b1S-caseins, for example, the \u03b1S1\\- and \u03b1S2-caseins of bovine milk, themselves possess two or more such clusters. From now on, discussion will be confined largely to the behavior of bovine caseins because it is for these that the largest body of research data is available. However, the extensions to the behavior in the micelles of other milks will be obvious.\n\nThese clusters of phosphoserine residues and the necessary glutamic acid residues templating their existence give rise to massive downward spikes in the hydrophobicity profiles of the bovine \u03b1S1\\- and \u03b2-caseins (Fig. 6.1). Associated with these are significantly high densities of negative charge at normal milk pH. There is a charge density of \u20139e within the span of residues 65\u201372 of \u03b1S1-casein and a further \u20136e along the sequence 48\u201353 of the same protein. A similarly high-charge density of \u20139e is found between residues 16\u201323 of \u03b2-casein, encompassing the phosphoserine cluster there. Similar high densities are found around the phosphoserine clusters of \u03b1S2-casein. All of these estimates of charge density assume a contribution of \u20131.5e from each phosphoserine residue at or near the natural pH of milk, 6.7.\n\nFigure 6.1 Hydrophobicity plots of (a) \u03b1S1-casein, (b) \u03b1S2-casein, and (c) \u03b2-casein calculated as a moving average (window n = 3) of amino acid hydrophobicities taken from the consensus scale used by Horne (1988). Asterisks denote centers of electrostatic repulsion arising from phosphoserine cluster motifs, the size indicating the number of negative charges associated with each, as listed in the text.\n\nAway from the phosphoserine clusters, the casein molecules are distinctly hydrophobic. This segregation of hydrophilic and hydrophobic residues confers on the caseins a definite amphipathic nature, which contributes to their ability to function successfully as stabilizers in oil-in-water emulsions. The topography of \u03b2-casein adsorbed at the oil\u2013water interface was probed by testing the accessibility of the reactive sites to the proteolytic enzyme trypsin (Leaver and Dalgleish, 1990). In aqueous solution, the reactive sites of \u03b2-casein were attacked randomly at no preferential rate. With \u03b2-casein-stabilized emulsions, however, the peptides released showed the lysines at positions 25 and 28 of the sequence to be readily accessible to trypsin, whereas all other possible attack sites were less so (Leaver and Dalgleish, 1990). These residues lie in the center of the highly charged, hydrophilic N-terminal region containing the four-phosphoserine cluster in \u03b2-casein. Measured by dynamic light scattering, a decrease of approximately 13 nm in hydrodynamic radius of the emulsion droplets also accompanied the scission of these peptides, indicating the extent to which they stretched out into the aqueous phase from the emulsion droplet surface (Dalgleish and Leaver, 1991). The remaining hydrophobic portion of the molecule was speculated to lie along the droplet surface, shielded from trypsin attack. Similar changes in hydrodynamic radius were observed when \u03b2-casein was adsorbed from aqueous buffers on to the surface of polystyrene latex particles, indicating a similar adsorption pattern (Dalgleish, 1990; Brooksbank et al., 1993), a pattern that was replicated at the air\u2013water interface, as observed by neutron reflectivity (Dickinson et al., 1993).\n\nThe combined experimental evidence was therefore consistent with the view that much of the hydrophobic end of the adsorbed \u03b2-casein was directly associated with the hydrophobic interface, with the hydrophilic N-terminal tail extending significantly out into the aqueous phase. Self-consistent-field calculations of the conformation of \u03b2-casein adsorbed at a planar hydrophobic interface confirmed this picture of a tail\u2013train structure, and also predicted a train\u2013loop\u2013train structure for adsorbed \u03b1S1-casein with anchor points at both ends of the molecule (Leermakers et al., 1996; Dickinson et al., 1997a,b). Schematic representations of these structures were drawn by Horne (1998) and were used in depicting assembly of the casein micelle via the dual-binding model. Perhaps rather than depicting the hydrophobic regions of the molecules as rectangular bars, it would have been more realistic to depict these regions as puckered, as in Figure 6.2, because not all amino acids therein are equally hydrophobic, as the profile plots of Figure 6.1 demonstrate.\n\nFigure 6.2 Schematic structures of \u03b1S1\\- and \u03b2-casein, based on self-consistent field calculations of the proteins adsorbed onto a hydrophobic interface, illustrating the train\u2013loop\u2013train structure of \u03b1S1-casein and the loop\u2013train structure of \u03b2-casein. The dashed circles give an idea of the range of the interaction potential components, the larger circle around the loops being the electrostatic repulsion arising from the negative charge centers thereon and the smaller circle being the regions of hydrophobic attraction in the train.\n\nSuch representations were also used by Horne (1998) to picture the aggregates produced by self-association of \u03b2-casein or \u03b1S1-casein (Fig. 6.3). Thus \u03b2-casein was envisaged as a hedgehog-like micelle subject to a monomer\/micelle equilibrium, a mechanism proposed by Payens and co-workers (Payens and van Markwijk, 1963; Payens et al., 1969). In this picture, the hydrophobic trains are buried inside, and the charged hydrophilic tails extend from the surface into solution. More recently, the self-association of \u03b2-casein has been revisited by De Kruif and collaborators (De Kruif and Grinberg, 2002; Mikheeva et al., 2003; O'Connell et al., 2003), who applied high-sensitivity differential scanning calorimetry, as well as static and dynamic light scattering techniques to the problem. These experiments again concluded that a micelle-like structure was adopted but, rather than being formed by the highly cooperative monomer\/micelle equilibrium suggested previously, instead the micellization took place as a series of consecutive additions of monomer to a growing micelle, as suggested in the shell model of Kegeles (1979). In a like fashion, with its hydrophobic chains at opposite ends of the molecule and a central section containing the highly charged phosphoserine clusters, \u03b1S1-casein self-associates to produce a worm-like chain polymer (Payens and Schmidt, 1966; Schmidt, 1970a,b).\n\nFigure 6.3 Diagrammatic representations of the polymeric structures generated when the hydrophobic chains of the caseins interact: (a) the worm-like chain of \u03b1S1-casein; and (b) the micelle of \u03b2-casein, where only two molecules have been included to simplify the diagram.\n\nThough driven by hydrophobic interactions, electrostatic repulsive interactions are also very important in the self-association of these caseins. In particular, note how the equilibrium structures adopted by their polymers place the centers of charge as far apart as possible while still permitting the self-association to take place. Compared with hydrophobic interaction, electrostatic repulsion is a long-range force, an important factor now being recognized in studies of protein\u2013protein interactions (Kegel and Van der Schoot, 2004; Piazza, 2004; Stradner et al., 2004), and certainly manifesting itself in these reactions of the caseins. These electrostatic interactions define the degree of polymerization and limit further growth. Thus, increasing the pH, which increases the protein charge, decreases the polymer size in both \u03b1S1-casein and \u03b2-casein solutions, whereas increasing the ionic strength, which decreases the range of the electrostatic repulsion component, allows the formation of larger polymers for both casein species (Payens et al., 1969; Schmidt, 1970a,b).\n\nThe importance of charge in controlling the extent of aggregation of the caseins cannot be stressed too highly. Precipitation of the caseins can be achieved by lowering the pH and titrating away a sufficient amount of the charge of the phosphoseryl and carboxyl groups to reach the isoelectric points of the proteins. \u03b1S2-Casein, \u03b1S1-casein, and \u03b2-casein are termed the calcium-sensitive caseins because they can be precipitated in the presence of ionic calcium, the order of sensitivity being as given (Swaisgood, 2003). The most extensive dataset is available for \u03b1S1-casein. Here the aggregation shows a lag phase with little change in molecular weight with time until a critical time, beyond which rapid aggregation occurs. Horne and Dalgleish (1980) demonstrated that the logarithm of this critical coagulation time was a linear function of Q2, where Q is the net negative charge of the protein. Thus Q is the algebraic sum of the negative and positive charges of the protein, reduced by twice the number of calcium ions bound to the protein, each calcium carrying two positive charges. Furthermore, this relationship held when changes in the net negative charge were produced by chemical modification, whether by conversion of positively charged lysine residues to neutral or negatively charged derivatives, or even by introduction of new negatively charged sites by iodination of tyrosine residues to the di-iodo form (Horne, 1983; Horne and Moir, 1984). Each of these modifications effectively increases the net negative charge of the protein, thereby reducing its propensity for calcium-induced precipitation and slowing down the rate of aggregation. However, once the protein charge is corrected for the measured extent of modification, the logarithm of the rate of precipitation has been shown to remain linear in Q2, all points lying on the same line as those obtained with the unmodified protein, all other reaction conditions being the same (Horne, 1983; Horne and Moir, 1984). The net charge of the protein therefore dominates its precipitation behavior. Farrell et al. (2006) have suggested that positively charged residues in the N-terminal hydrophobic chain of \u03b1S1-casein could participate in binding to the phosphate groups of phosphoseryl residues, but such +\/\u2013 bridging does nothing to reduce the net negative charge of the protein having already been accounted for in the algebraic summation leading to Q, which, as we have demonstrated, controls the level of aggregation in these proteins.\n\nIt is only when the local balance of electrostatic repulsion and hydrophobic attractive interaction is in favor of attraction that hydrophobic bonds are formed. Sequentially, the major centers of electrostatic repulsion, the phosphoserine clusters, are remote from the hydrophobic regions in the trains of Figure 6.2, though, depending on the adopted conformation, they may not be remote spatially. Figure 6.2 is a depiction of a possible conformation of each protein at a hydrophobic interface, the puckering displaying the tendency to form a multitude of weak, short-range bonds by the hydrophobic chain. Lowering the temperature weakens hydrophobic bonds, hence the tendency to find monomeric \u03b2-casein at low temperatures but a micellar aggregate at room temperature and above. The aggregation of \u03b2-casein induced by calcium also shows a marked temperature dependence, with no precipitation observed at 4 \u00b0C (Parker and Dalgleish, 1981). At higher temperature, these hydrophobic bonds are in general stronger, but, because they are relatively weak overall, statistically individual bonds are readily ruptured by the increased thermal energy available, leading to more mobile, labile interactions between molecules.\n\n## Casein micelle properties\n\nAlmost all of the casein proteins present in bovine milk expressed at 37\u00b0C are incorporated into the casein micelles, together with a high proportion of the available calcium and inorganic phosphate. The calcium and phosphate within the micelle form low-molecular-mass species collectively known variously as colloidal calcium phosphate, micellar calcium phosphate, and latterly calcium phosphate nanoclusters. The micelles are very open, highly hydrated structures, with typical hydration values of 2\u20133 g H2O\/g protein, depending on the method of measurement.\n\nElectron microscopy shows that casein micelles are generally spherical in shape, with diameters ranging from 50 to 500 nm (average \u2248 150 nm) and a molecular weight ranging from 106 to >109 Da (average \u2248108 Da) (Fox and Brodkorb, 2008). For a casein content of 2.5 g\/100 mL milk, there are some 1014\u20131016 micelles\/mL milk, which implies a relatively close packing with inter-surface separations less than one micelle diameter.\n\nMilk is white largely because the colloidal dimensions of the casein micelles are such that they scatter significant amounts of light, an effect compounded by their high number density. Scattering of shorter wavelength radiation (neutrons and x-rays) reveals the internal structure to be heterogeneous, with a correlation length for variations in scattering length density within the particle of approximately 18 nm. Stothart and Cebula (1982) have interpreted this scattering behavior as being due to a structure composed of closely packed spherical subunits of this diameter, a picture that mirrors the raspberry-like appearance in early electron micrographs of the casein micelle (Schmidt, 1982).\n\nMore recent electron microscopy studies (McMahon and McManus, 1998; McMahon and Oommen, 2008) have suggested that these well-defined structures are likely to be artifacts of the fixation process, although micrographs recently obtained by field emission scanning electron microscopy (SEM) show a complex surface structure of cylindrical or tubular, but not spherical, protrusions between 10 and 20 nm in diameter, extending from the surface of the micelle (Dalgleish et al., 2004). These samples were not metal coated, although they were of necessity subjected to a fixation and dehydration process, which might have introduced some collapse of more loosely bound protein on to a denser skeleton. In a careful study using cryo-transmission electron microscopy (cryo-TEM) and small angle x-ray scattering (SAXS), Marchin et al. (2007) have extended previous structural studies to elucidate greater detail of micellar fine structure. Their cryo-TEM pictures show small regions of high electron density, approximately 2.5 nm in diameter, uniformly distributed in a homogeneous web of protein, giving the micelles a granular aspect that diminishes when the pH is reduced from 6.7 to 5.2. Paralleling this change in appearance, the SAXS scattering profile loses its characteristic shoulder at high wave vector when the pH is reduced. This clearly demonstrates that the shoulder is directly linked to the presence of micellar calcium phosphate and its dissociation from the micelle on acidification to pH 5.2. The results also demonstrate that this micellar calcium phosphate is uniformly distributed through the micelle in small particles, approximately 2.5 nm in diameter, and that, after their removal, other forces\/interactions must contrive to maintain micellar structural integrity. Their loss on acidification does not result in micellar disruption. Trejo et al. (2011) have also used cryo-TEM to study internal micellar structure. By varying the angle of incidence of the electron beam onto the same micelle, they have constructed tomographic images that demonstrate the presence of water-filled channels and cavities within the micelle and map the size, number, and location of the calcium phosphate nanoclusters, significantly different from the seemingly homogeneous networks apparent in earlier cryo-TEM studies (Marchin et al., 2007; McMahon and Oommen, 2008). While these interpretations chime with observations on the internal accessibility of the micelles to enzymes and ready release of \u03b2-casein, a word of caution should be added. The analysis by Trejo et al. (2011) is essentially a paint-by-numbers exercise. If the electron density is less than x, color it black and call it a channel or cavity; if the density is greater than y, then color it purple and call it a calcium phosphate nanocluster. There is no chemical identification of the scattering species, so both the number and size of the nanoclusters are likely to be an overestimate and the same is true for the channels and cavities. However, any structural model of casein micelle assembly would have to lead to a structure that could reproduce both the cryo-TEM pictures and the field emission SEM pictures, allowing for possible changes in appearance brought about by techniques of sample preparation and the vagaries of any analysis approach.\n\nCasein micelle structure is not fixed, but dynamic. In various ways, it responds to changes in micellar environment, temperature, and pressure. Cooling milk on release from the udder at 37 \u00b0C to storage at refrigeration temperatures brings about significant solubilization of \u03b2-casein, some \u03ba-casein, and much lower amounts of \u03b1S1\\- and \u03b1S2-casein from the micelles (Dalgleish and Law, 1988). Raising the temperature back to 37 \u00b0C reverses the process. None of this movement of \u03b2-casein does anything to disrupt the internal structure of the micelle, as observed by cryo-TEM and SAXS (Marchin et al., 2007). Almost complete disruption of the micelles, manifested by a loss of their scattering power and removal of the white color of milk, can be achieved by addition of a strong calcium sequestrant such as ethylene diamine tetraacetic acid (EDTA) (Griffin et al., 1988), by addition of urea (McGann and Fox, 1974), by dialysis against a phosphate-free buffer (Holt et al., 1986), by increasing the pH, by exposure to high pressure (Huppertz et al., 2006), or by addition of ethanol at \u224870 \u00b0C (O'Connell et al., 2001). Significantly, the colloidal calcium phosphate can also be solubilized by lowering the pH but, as confirmed by Marchin et al. (2007), without substantial disruption of the micelle structure.\n\nFractionation of the casein micelles according to size can be realized by a stepwise centrifugation protocol. The proportions of \u03b1S1\\- and \u03b1S2-caseins remain constant with micelle size, but \u03ba-casein content increases inversely with that size (Donnelly et al., 1984; Dalgleish et al., 1989). For a solid sphere, the surface-to-volume ratio is inversely proportional to the radius of the sphere, and these results imply that \u03ba-casein resides on the micellar surface, where its content controls the micellar total surface area and hence the micelle size. A surface location for the \u03ba-casein component may also be inferred from the requirement that this protein be readily accessible for rapid and specific hydrolysis by chymosin and similar proteinases, a reaction that destabilizes the micelles and leads to clot formation, which is exploited in cheese manufacture. A surface location is also required to enable the \u03ba-casein to interact with \u03b2-lactoglobulin in milk to form a complex on heating, the formation of which modifies the rennet and acid coagulation properties of the micelles. It is evident that a principal requirement, which must be met by any micelle model, is that it should generate a surface location for \u03ba-casein.\n\n## Models of casein micelle structure\n\nCasein micelle structure and casein micelle models have been extensively reviewed (Schmidt, 1982; Walstra 1990; 1998; Holt, 1992; Rollema, 1992; Horne 1992; 1998; 2006; Fox, 2003; Farrell et al., 2006; Fox and Brodkorb, 2008). As mentioned previously, based on the biochemical and physical properties of the micelles and the casein proteins outlined above, three main models have been proposed: the submicelle model (Slattery and Evard, 1973; Schmidt, 1982; Walstra, 1998); the nanocluster model of Holt (Holt, 1992; De Kruif and Holt, 2003); and the dual-binding model (Horne, 1998; 2002).\n\nIn the first model, the casein micelles are composed of smaller proteinaceous subunits, the submicelles, linked together via colloidal calcium phosphate. In the second model, the nanoclusters of colloidal calcium phosphate are randomly distributed, cross-linking a three-dimensional web of casein molecules. Both of these models have been severely criticized (Farrell et al., 2006; Horne, 2006), and the dual-binding model arose first as an attempt to overcome their deficiencies. It is significant that the most recent description of the nanocluster model (De Kruif et al., 2012) adopts tacitly an essentially dual-binding approach, replacing the terminology 'hydrophobic interactions' with 'nonspecific interactions.' Since most groups now appear to be accepting it or subtle variants, we present first a summary of the dual-binding model as providing a rational mechanism for micelle assembly and structure, and demonstrate how this model may be exploited to explain various observations of micellar properties and behavior.\n\n### The Dual-Binding Model for Micelle Assembly and Structure\n\nThe description presented in this section largely follows that found in Horne (2002), with minor refinement highlighted.\n\nIn the dual-binding model, micellar assembly and growth take place by a polymerization process involving, as the name suggests, two distinct forms of bonding, namely, cross-linking through hydrophobic regions of the caseins and bridging across calcium phosphate nanoclusters. Central to the model is the concept that bond formation is facilitated, and hence micellar integrity and stability are maintained, by a local excess of hydrophobic attraction over electrostatic repulsion, bearing in mind the quite different ranges of these interaction components. The individual casein molecules behave and interact as they do in their self-association equilibria, as described previously.\n\nEach casein molecule effectively functions as a block copolymer, as detailed in Figure 6.2, with the hydrophobic region(s) offering the opportunity for a multitude of individual, weak, hydrophobic interactions. The hydrophilic regions of the casein molecules contain the phosphoserine cluster (or clusters), with the exception of \u03ba-casein, which has no such cluster, each offering multiple functionality for cross-linking. Thus, as we have seen, \u03b1S1-casein can polymerize (self-associate) through the hydrophobic blocks, giving the worm-like chain of Figure 6.3. Further growth is limited by the strong electrostatic repulsion of the hydrophilic regions, but, in the casein micelle situation, the negative charges of the phosphoserine clusters are neutralized by intercalating their phosphate groups into a facet of the calcium phosphate nanocluster. This has two very important implications for the micelle. First, by removal of a major electrostatic repulsion component, it increases the propensity for hydrophobic bonding upstream and downstream of the nanocluster link. It effectively permits and strengthens those bonds. Second, it allows for multiple protein binding to each nanocluster, permitting a different network to be built up. \u03b2-Casein, with only two blocks, a hydrophilic region containing its phosphoserine cluster and the hydrophobic C-terminal tail, can form polymer links into the network through both, allowing further chain extension through both. \u03b1S2-Casein is envisaged in this model as having two of each block, two (possibly three; see below) phosphoserine clusters and two hydrophobic regions. It is only a small fraction of the total bovine casein but, by being able to sustain growth through all its blocks, it is likely to be bound tightly into the network. \u03ba-Casein is the most important of the caseins in the dual-binding model of micellar assembly and structure. It can link into the growing chains through its hydrophobic N-terminal block, but its C-terminal block is hydrophilic and cannot sustain growth by linking hydrophobically to another casein molecule. Neither does \u03ba-casein possess a phosphoserine cluster; therefore it cannot extend the polymer cluster through a nanocluster link. Thus, chain and network growth are terminated wherever \u03ba-casein joins the chain. This leaves the network with an outer layer of \u03ba-casein, satisfying the prime requirement recognized earlier. This assembly process is in its essential requirements identical to the model proposed by McMahon and Oommen (2008). Their main contribution is to describe the structure resulting from the assembly processes as an interlocked lattice, which seems an excellent description of a complex structure.\n\nThe nanocluster bridging pathway through the phosphoserine clusters was the only pathway allowed in the original nanocluster micelle model of Holt (Holt, 1992; De Kruif and Holt, 2003), where around 50 casein molecules are considered to bind to each calcium phosphate nanocluster. Horne (2006) and Horne et al. (2007a) have argued on the basis of mineral content and stoichiometry that the functionality of these nanoclusters will be much lower and that they link to four to six phosphoserine clusters, which may not necessarily originate from different casein molecules. Some consideration also has to be given to what constitutes a phosphoserine cluster capable of linking into the calcium phosphate nanocluster. Aoki et al. (1992) suggested a minimum of three phosphoserine residues, but De Kruif and Holt (2003) argued that two might be sufficient. This would allow the phosphoserine pair at positions 46 and 48 of \u03b1S1-casein, or those at positions 129 and 131 of \u03b1S2-casein, to function as nanocluster linkage sites, particularly if the carboxyls of the neighboring glutamate residues acted as pseudo-phosphate groups. This would give \u03b1S1-casein two linkage sites and \u03b1S2-casein three linkage sites. This level of functionality in these caseins is absolutely essential to the Holt model to build the required three-dimensional network, as, without them, an \u03b1S2-casein molecule with only two linkage sites and with such a low percentage of the total casein would probably prove to be insufficient. Although they are not essential to the dual-binding model, these mini-clusters of pairs of phosphoserines may provide for a weaker bridging link to the calcium phosphate nanocluster, allowing a range of nanocluster bond strengths to prevail.\n\n### Calcium Phosphate Equilibria in the Dual-Binding Model\n\nFrom the mineral viewpoint, casein micelle assembly is a frustrated crystallization of calcium phosphate. Milk is supersaturated in calcium and phosphate and, were it not for the presence and intervention of the highly phosphorylated caseins, a precipitation of calcium phosphate and potentially painful calcification of the mammary gland duct system would occur. There is considerable controversy over the possible (crystalline) structure of the nanocluster. The inorganic components of colloidal calcium phosphate have a stoichiometry close to that of the mineral, hydroxyapatite, whereas, if the phosphates of the casein phosphoserines are included in the mix, the stoichiometry moves closer to that of the mineral, brushite. Other groups have argued that the nanoclusters are actually an amorphous calcium phosphate (Cross et al., 2005), a form found in many biological situations that may have so-called Posner's clusters (Ca9(PO4)6) as basic building blocks (Yin and Stott, 2003). Posner's cluster can readily be detected as a unit within the structure of apatites, but it is also recognizable with distortion in other phosphate minerals. Amorphous calcium phosphate may be the first solid phase to appear upon mixing calcium and phosphate-containing aqueous solutions at pH >7 and concentrations sufficiently high to produce an immediate precipitation (Dorozkhin, 2010), but, in the formation of casein micelles, this ignores the observation of Horne (1982) that the inclusion of phosphate in a solution of \u03b1S1-casein and calcium initiates precipitation at ion-product levels, which are orders of magnitude below those observed in the absence of casein. This would appear to preclude the phosphoserines binding to preformed calcium phosphate nanoparticles, as suggested by DeKruif et al. (2012) but would offer the potential for the phosphoserine clusters of the caseins to act as templates to initiate nanocluster growth, to moderate that growth by the cutting of growth at a particular facet and finally to terminate that growth at the final free facet. In this way, the overall total number of phosphoseryl clusters in a milliliter of milk controls the size and number of nanoclusters present in the milk (Horne et al., 2007a). This can happen only if all (or close to all) of the phosphoserine cluster motifs are involved in nanocluster stabilization. In the scenario developed by Horne et al. (2007a), the phosphoserine clusters are incorporated into the planes of phosphate groups forming the faces of a hexagonal bipyramid. Examination of the molecular model of the brushite crystal in the photograph shown in Figure 6.4 reveals how these planes transect the crystal. In the Golgi vesicle, nanocrystal growth and number are unlikely to be limited by the availability of the inorganic components but only by the number of capping phosphoserine motifs on the caseins. In turn, this must mean that the binding of a serine phosphate group into the nanocluster must present a bonding advantage thermodynamically over that of a free phosphate group going into a growing calcium phosphate crystal in an equilibrium situation. It is a more favorable outcome thermodynamically. For these reasons, the presence of unbound, free phosphoserine clusters on \u03b2-casein in freshly drawn milk at 37 \u00b0C, as envisaged by Dalgleish (2011) is unlikely.\n\nFigure 6.4 Molecular model of the crystal structure of the calcium phosphate mineral, brushite, courtesy of Beever's Miniature Models, Department of Chemistry, University of Edinburgh. Lines are drawn to aid the eye in discerning planes through the crystal containing phosphorus atoms. Such solids were used by Horne et al. (2007a) to calculate a molecular weight and size for the nanocluster.\n\nBut just as the calcium phosphate crystal in equilibrium in solution with free calcium and phosphate is subject to environmental constraints shifting that equilibrium, so too is the micellar nanocluster species. The two equilibria may exist in parallel, though, when micelles are present in normal milk conditions, the nanocluster form is the favored option. However, conditions may change where 'solution' crystal growth is favored; we try to explore these speculations in some of the following discussions. The possibility of shifting calcium phosphate equilibria is an aspect of casein micelle structural behavior that has not been considered previously but, as the nanoclusters are involved, may offer alternative avenues to explore in addressing problems of micellar behavior associated with the application of high pressure, or of the addition of ethanol, or indeed in lowering milk temperature, where in the first edition we reproduced the effects later envisaged by Dalgleish (2011).\n\n### Predictions of Casein Micelle Properties in the Context of the Dual-binding Model\n\n#### Size and Appearance\n\nA major failing of the earlier micelle models was their lack of a plausible mechanism for assembly, growth, and, more importantly, termination of growth. All such elements are in place in the dual-binding model. Furthermore, the product of the dual-binding model satisfactorily represents the appearance and scattering behavior of the native casein micelle. Network growth is envisaged as a random process, and its termination along any particular pathway depends on the serendipitous arrival of a \u03ba-casein molecule. Micelle size will therefore depend on the proportion of \u03ba-casein in the mix, but will also present a range of sizes dependent as it is on random events. The model also reproduces the heterogeneity in structure required by the x-ray and neutron scattering data. The dense calcium phosphate nanoclusters will be rather homogeneously distributed through the matrix and will give rise to the structures observed by cryo-TEM and inferred from SAXS (Marchin et al., 2007). Their size, as predicted by the stoichiometric analysis of Horne et al. (2007a), has been confirmed in terms of molecular weight by Choi et al. (2011) and length scale by De Kruif et al. (2012).\n\nUntil this point, we have been emphasizing the interactions involved in casein micelle assembly, but the final structure of the micelle is dictated by the kinetics of the aggregation processes. Simulations of particle aggregation reactions (Meakin, 1999) demonstrated that open, ramified structures result when weak repulsion exists between particles. Essentially every collision between particles creates a bond and chains, and clusters grow from their extremities. When reaction barriers are higher, more collisions between particles are required before a favorable encounter produces a bond. This extensive sampling of phase space produces denser, more closely packed aggregates. These computer simulations were confirmed experimentally (Weitz et al., 1991). There is no doubt that casein micelles are open, ramified, highly hydrated structures with extensive water-logged channels and cavities, most recently pictured in the cryo-TEM study of Trejo et al. (2011). The creation of such structures is dictated by the kinetics of the assembly process, implying that such reactions are rapid, occurring on almost every encounter, and demonstrate low or insignificant energy barriers to bond formation.\n\nThe dual-binding model presents the casein micelle as a dynamic, 'living' entity. The hydrophobic interactions are individually weak and capable of breaking and recombining on an almost continuous basis. To some extent, the molecular movements this allows will be restricted by the potentially stronger nanocluster linkages and the low probability of rupturing simultaneously all hydrophobic bonds involving any particular molecule. Molecular movement has several consequences, however. The micelle may have an outer layer of individual \u03ba-casein molecules when initially constructed, but conditions within the Golgi vesicle are suitable for disulfide bond formation; otherwise \u03b2-lactoglobulin and the other whey proteins would not fold properly. Movements within that outer 'hairy' layer may bring those \u03ba-caseins into proximity and allow their polymerization through disulfide bridging, the size of the polymer depending on when the chain closes into a loop, but giving rise to the polymeric \u03ba-casein entities observed on micellar dissociation.\n\nAnother consequence of the 'living' nature of these hydrophobic bonds is that the dehydration of the micelle required in the preparation of a sample for some forms of electron microscopy would also tend to be accompanied by the collapse of the more mobile, weaker, and less multitudinously bonded regions on to those more strongly cross-linked\u2014hence, perhaps giving rise to the raspberry-like (Schmidt, 1982) or tubular (Dalgleish et al., 2004) structures seen in some electron micrographs. Even the putative caps suggested for those tubules by Dalgleish et al. (2004) can be provided by the dual-binding model, as the disulfide bridging between the \u03ba-casein molecules would enhance the Velcro effect of the weak hydrophobic bonding of an individual molecule to many such molecules in the chain.\n\n#### Effects of Urea, pH, Sequestrants, and Temperature\n\nThe concept of a local excess of hydrophobic attraction over electrostatic repulsion, as well as permitting the visualization of micellar growth, successfully accommodates the response of the micelle to changes in pH, temperature, urea addition, or removal of calcium phosphate by sequestrants, all in accordance with experimental observations.\n\nUrea disrupts hydrophobic bonds, and high concentrations will bring about micellar disintegration. In some regions of the micelle, this may be only partial because the nanocluster cross-links through the phosphoserines remain, unaffected by this reagent. Micellar fragments in a range of sizes may be produced, some even as large as some of the original micelles, though perhaps more open and swollen from their own starting state before urea treatment. Extensive disruption does occur, however, as is observed by the loss of the white appearance of skim milk (McGann and Fox, 1974). The dual-binding model fully accounts for these observations.\n\nRemoval of calcium from the calcium phosphate nanocluster by sequestrant addition, whether EDTA, citrate, or oxalate, restores the negative charge of the hydrophilic region, if the pH is maintained at the native milk pH. This shifts the hydrophobic attraction\/electrostatic repulsion balance in favor of repulsion, and the micelle breaks up. Decreasing the milk pH solubilizes the colloidal calcium phosphate (Dalgleish and Law, 1989), but the negative charges associated with the cross-linking phosphoseryl groups are also titrated away. The strength of the hydrophobic bonds remains unaffected or may be enhanced if other carboxyl charges are also titrated away. The integrity of the micelles is maintained, but their scattering behavior and their appearance in cryo-TEM micrographs reflect the loss of the nanoclusters (Marchin et al., 2007; Moitzi et al., 2011). The loss of the nanocluster linkages also produces a smoothing and spreading of the micelles as seen by atomic force microscopy (AFM) on lowering the pH around an adsorbed micelle, as internal bonding becomes dominated by the multitudinous but ephemeral hydrophobic bonds (Ouanezar et al., 2012).\n\nIncreasing the pH may be expected to be the reverse of the dissolution process and to favor the formation of calcium phosphate species. Fox (2003) noted that raising the pH to >9.0 does not dissolve colloidal calcium phosphate but rather increases its level. However, increasing the milk pH to these levels does lead to dissociation of the micelles and creation of a translucent solution. Dialysing these high pH solutions against excess of the original milk restores the milk pH and produces milks with enhanced levels of colloidal calcium phosphate. It is argued (Ozcan et al., 2011) that this is incorporated into larger nanoclusters rather than increasing the number of them.\n\nDecreasing the temperature is known to decrease the strength of hydrophobic attraction and to shift the monomer\/micelle equilibrium in \u03b2-casein solutions toward the monomer side at temperatures below 15 \u00b0C (De Kruif and Grinberg, 2002; Aschi et al., 2009). Lowering the temperature of milk to refrigeration levels also brings about dissociation of a large fraction of the \u03b2-casein from the casein micelle (Dalgleish and Law, 1988), possibly some of which is not bound into the micellar matrix through its phosphoserine cluster. However, nature has invested considerable energy in creating the phosphoserine clusters and in preserving those clusters through eons of evolutionary development, not to have all of those clusters involved in their designated role. Moreover, Ca binding to caseins is known to decrease when temperature is lowered (Dalgleish and Parker, 1980). Hence some weakening of Ca-PSer bonds is possible when temperature is lowered, with consequent release of \u03b2-casein where the increased negative charge will contribute enormously to shifting the balance in binding energies without the necessity for postulating 'free' \u03b2-caseins at all temperatures as suggested by Dalgleish (2011). Raising the temperature back to its initial value reverses the process and the \u03b2-casein is reincorporated into the micelle.\n\nThere are also shifts in the soluble calcium phosphate equilibria in milk associated with temperature change. Ultrafiltration permeate is a clear, straw-yellow liquid when prepared at 4 \u00b0C, but it becomes turbid when heated to room temperature and above because of the precipitation of calcium phosphate. Even permeate collected at room temperature clouds on heating but reverts to clarity on cooling.\n\nHilgemann and Jenness (1951) noted that calcium phosphate also precipitates in milk. However, the calcium phosphate precipitate was only slowly re-solubilized (Jenness and Patton, 1959). Weakening the calcium phosphate 'solution' equilibrium would favor preservation of the nanoclusters, but anything that pushes that 'solution' equilibrium to the solid side could have an effect on the continuing existence of the nanoclusters. There are indications that heating milks in the temperature range 50\u201390 \u00b0C brings about increasing mobility in the micelle (Rollema and Branches, 1989), which would be in line with partial disruption. Thachepan et al. (2010) have also found that prolonged heating at 60\u00b0C at pH 7 for weeks in the case of micellar casein and days for \u03b2-casein\/CCP constructs, yields mesocrystals of hydroxyapatite and products of dissociated micelles. The behavior of casein micelles in this temperature range merits further scrutiny, particularly as so many processes in the dairy industry are conducted just in this range.\n\n### The Dual-Binding Model and Micellar Interactions\n\nThe ideas outlined earlier in this chapter allow us to schematically describe in Figure 6.5 how the casein micelle might appear as an interacting species at the various pH values indicated.\n\nFigure 6.5 Representations of casein micelle structures at various pH values as indicated. The pale chains indicate protein molecules, where they cross being a hydrophobic interaction junction, the depth of color indicating the intensity of attraction at that pH. The small black circles are the calcium phosphate nanoclusters that are solubilized when the pH is lowered. The outer circle is indicative of the range of steric repulsion generated between micelles and preventing interaction of the surface protein chains.\n\nInternally, at pH 6.7, the micellar matrix is closely interlinked through a combination of nanocluster bridging bonds (the small black circles) and hydrophobic interactions, occurring randomly along any selected polymer chain. The hydrophobic interactions at this pH (indicated as crossover points in the tangled protein network in the diagrams in Fig. 6.5) are many but relatively weak, being counterbalanced by the negative charges present on ionized carboxyl groups, dispersed along the chains and throughout the network. The micellar outer reaches are mainly \u03ba-casein molecules, which have terminated polymer extension and limited micellar growth in the dual-binding model. The negative charges from the ionized carboxyls and sialic acid groups on the \u03ba-casein macropeptides provide the electrostatic repulsion component in the inter-micellar interaction potential, which inhibits micellar aggregation. Its longer range, illustrated by the thickness of the shell around the micelle, prevents close approach of the hydrophobic regions buried beneath the shell and amply fulfills the requirements of a hard sphere model colloid at this pH, 6.7.\n\nAt the lower intermediate pH of 5.6 in this series of illustrations, the same shell continues to prevent close approach of the micelles. The pK values of the acidic groups giving rise to the negative charge are generally lower than 5.5 and have yet to be titrated away. Internally, however, most of the micellar calcium phosphate nanoclusters have been solubilized and the bridges between phosphoserine cluster motifs have been lost, weakening the overall network structure of the micelle. The bond strengths of hydrophobic interactions remain relatively weak, still being counterbalanced by ionized carboxyl groups dispersed through the micelle. The relatively weak bonding, however, allows for rapid interchange and restructuring of the micelle in this range of pH, smoothing out gross structural features apparent in AFM pictures at pH 6.7 (Ouazenar et al., 2012).\n\nBy pH 5.1, the surface charges are being titrated away; the shell depicted in Figure 6.5 is much thinner, and aggregation begins. Internally, the hydrophobic interactions are effectively being strengthened (indicated by a deepening of the color of the chains) because the counterbalancing electrostatic repulsions are also being removed from the equation, leading to reduced mobility within the micellar particles.\n\n### Concentrated Micellar Dispersions\n\nIn milk as produced from the cow at its natural pH of 6.7 and temperatures from ambient to blood heat, casein micelles closely follow the behavior of hard sphere colloids (De Kruif, 1998; Alexander et al., 2002). Justification for this assertion comes from studies utilizing light and neutron scattering to measure micelle size and polydispersity (Hansen et al., 1996), from sedimentation behavior (De Kruif, 1998), and from measurements of micellar voluminosity (De Kruif, 1998), diffusivity (De Kruif, 1992), and viscosity of micellar suspensions (Griffin et al., 1989).\n\nParalleling colloidal hard sphere behavior holds only for a limited range of concentrations, and, above a critical concentration, micellar suspensions show strong deviations from expected hard sphere behavior (Mezzenga et al., 2005). The viscosity continues to increase but at a slower rate than that expected for hard spheres. This is accompanied by a transition from Newtonian viscosity behavior at natural milk concentration to non-Newtonian viscoelastic behavior in the high concentration regime. More enlightening demonstrations of the departure from hard sphere behavior come from studies of the rheology of high concentration micellar suspensions produced by ultrafiltration (Karlsson et al., 2005), by evaporation to 45% total solids (Bienvenue et al., 2003), by centrifugal sedimentation and pelleting (Horne, 1998), and by osmotic compression (Bouchoux et al., 2010). In these instances, concentrated micellar suspensions are close packed and show a gel-like behavior, which can be interpreted with the assistance of the dual-binding model.\n\nKarlsson et al. (2005) concentrated skim milk by ultrafiltration to produce a micellar suspension with 19.5% casein and studied the effects of pH and ionic strength on its viscoelastic properties. Their suspensions exhibited Newtonian viscosities at very low (Brownian) and very high (hydrodynamic) shear rates, with shear thinning at intermediate shear rates and stresses. The concentration of the micelles by ultrafiltration forced the micelles to interact, jamming them together at this high-volume fraction and producing a honeycomb-like structure in freeze\u2013fracture electron micrographs. The elastic modulus of these gels decreased as the pH was lowered from the value achieved in the ultrafiltration retentate. Addition of NaCl at levels of 0.33 and 0.66 mol\/kg prior to ultrafiltration increased the elasticity of the gels but shifted their pHs to more acidic values. Thereafter in the salt-added systems, lowering the pH produced a decrease in elasticity that paralleled the untreated suspension behavior, the higher salt level giving the greater elasticity throughout. Karlsson et al. (2005) also measured the phase angle, the partitioning between viscous and elastic components in these gels, as the pH was reduced. In the no-added-salt system, they found the phase angle to increase through a maximum close to 45\u00b0 and thereafter decrease with decreasing pH. In the presence of added salt, the maximum in phase angle was again observed but shifted to much lower pH values: in the case of the higher salt level, to a pH value lower than that for acid gel formation in milk of normal concentration, and, in both cases, where elasticity had been observed to increase again in these salted concentrated suspensions.\n\nThe dual-binding model explains this behavior with reference to the schematic of the micellar interaction potential depicted in Figure 6.6. The increase in micellar concentration in the no-added-salt case forces the micelles together and into the secondary minimum generated by hydrophobic interactions. This is the source of the attractive interaction giving rise to the viscoelasticity observed. The micelles are also in a jammed structure, and their internal bonding contributes to the measured elasticity. On lowering the pH, the loss of the calcium phosphate nanocluster bridges weakens this structure, and the elasticity decreases, as observed. The bonding due to hydrophobic interactions is relatively weak, and the loss of the nanocluster bridges further contributes to the mobility in the gel, as evidenced by the observed increase in phase angle. Dropping the pH further titrates away carboxyl groups. However, it reduces the counterbalancing electrostatic component and thereby strengthens hydrophobic bonds in the matrix, reducing mobility and producing the subsequent drop in phase angle.\n\nFigure 6.6 Repulsive inter-micellar interaction potential with inner hydrophobic interaction minimum. The dashed line shows the effect of salt addition on the range of the electrostatic repulsion component.\n\nThe major effect of the addition of salt is to reduce the Debye\u2013Huckel parameter and shorten the range of the electrostatic repulsion between micelles. This makes it easier to enter the secondary minimum in the interaction potential and increases the gel elasticity, as observed, with more salt producing the stronger gel. Again, however, the calcium phosphate nanocluster bridges contribute stress-carrying bonds and their removal by lowering the pH leads to the observed decrease in the elasticity of the gel. Throughout this titration, the bonds in the system are relatively stronger than in the no-salt case\u2014the salt also contributes to decreasing the effectiveness of intra-micellar electrostatic repulsion\u2014and the phase angles are lower in comparison.\n\nKarlsson et al. (2005) suggested that a significant effect of the salt addition is to exchange bound calcium within the micelle for monovalent ions, which would imply no nanoclusters in the system to be solubilized on decreasing the pH and thereby voiding the above explanation for the decrease in elasticity with pH. Huppertz and Fox (2006) did indeed find increased levels of calcium in serum when 600 mM NaCl was added to a two-times-concentrated milk, but they found no increase in serum inorganic phosphate, suggesting that the increase in calcium came from displacement of casein-bound calcium rather than a salt-induced dissociation of the calcium phosphate nanoclusters, leaving these to be solubilized on acidification.\n\nThe evaporated milks produced by Bienvenue et al. (2003) had 45% total solids or were approximately concentrated from normal by a factor of four, rather than the eight times concentration of the micelles in the ultrafiltration retentates of Karlsson et al. (2005). The milks of Bienvenue et al. (2003) increased in viscosity on storage at 50 \u00b0C, with salt addition accelerating the increase. Such behavior is in line with the predictions of the dual-binding model outlined above. The collision rate increased by concentration will be further increased by raising the temperature, bringing about a higher frequency of micelles attempting to enter the secondary minimum. A higher success rate and flocculation due to more thermal energy will give rise to the observed increase in viscosity. The weak flocs can be disrupted by higher shear stresses, giving the observed shear-thinning behavior. The effect of salt, as above, would be to render it easier to enter the secondary minimum and promote the flocculation reaction.\n\nIn another study of the rheological behavior of concentrated micellar sytems, casein micelle pellets were produced by the centrifugation of skim milk at 19,000 g for 60 min, giving protein concentrations of approximately 20% (Horne, 1998). At high temperatures (40oC), this pellet flowed freely. Its viscosity was Newtonian, independent of shear rate or frequency. At low temperature (5 \u00b0C), however, this micellar suspension exhibited all the properties of a classical viscoelastic gel, with elastic moduli independent of frequency and phase angles less than 45\u00b0. At intermediate temperatures, there was a crossover between viscous and elastic behavior. The behavior here is dominated by that of the hydrophobic interactions. At low temperatures, the strength of these interactions is low. Both \u03b2-casein and \u03ba-casein are known to depart from the micelle under such conditions (Dalgleish and Law, 1988), but, in the close-packed conditions prevailing in the pellet, they are liable to migrate or link to neighboring micelles or to become entangled with proteins loosened from those micelles, leading to the gel-like behavior. As the temperature is increased, the strength of the hydrophobic interaction increases, but the ability to break bonds is also enhanced and more mobility is allowed. The strengthening of the bonding may also lead to a tightening up of the micelles, and their becoming more compact may allow the suspension to flow more freely.\n\nFinally, Bouchoux et al. (2010) combined the osmotic stress technique with SAXS to study the structural response of the casein micelle to an increase in concentration. Their SAXS results indicate that as the micelles are compressed, they lose water and shrink to a smaller volume, but this compression is nonaffine; that is, some soft parts of the micelle lose water and readily collapse, whereas other hard parts resist deformation and are pushed closer together. Bouchoux et al. (2010) argue that existing models of the casein micelle fail to reproduce this behavior and suggest a physical model based on hard regions that assume the structure of Voronoi tessellations, but without providing any mechanism for the creation of this structure. Moreover, the structure they believe corresponds to the behavior deduced from their SAXS spectra, with hard regions containing the nanoclusters and major water-filled channels and cavities, is nothing more than the structure deduced by Trejo et al. (2011) from their tomographic analysis of their cryo-TEM pictures. As we saw earlier in this chapter, such a structure can be developed within the dual-binding model, provided due consideration is given to the kinetics of protein aggregation and micellar assembly.\n\n### The Dual-Binding Model and Micellar Destabilization\n\nThe concept of the casein micelle electrosterically stabilized by a 'hairy layer' coat of \u03ba- casein appears to enjoy universal acceptance (Holt, 1975; Walstra, 1979; Holt and Horne, 1996).\n\nBecause the dual-binding model of the casein micelle naturally provides a surface location for \u03ba-casein in a growth-limiting role, it readily explains the destabilization of the casein micelle system on the proteolysis of \u03ba-casein by chymosin and the loss of the steric-stabilizing hairs. Such proteolysis also leads to a significant drop in the micellar zeta potential (Dalgleish, 1984), and consequent reduction in the electrostatic repulsion between micelles. Further confirmation of the importance of electrostatic repulsion in inter-micellar interactions is evinced by the necessary presence of ionic calcium to bring about\/promote the aggregation of the chymosin-treated micelles. Notwithstanding the importance of electrostatics, hydrophobic interactions also play an important part, as evidenced by the fact that fully renneted micelles show no signs of aggregation at low temperatures (<10 \u00b0C) (Dalgleish, 1983) or that rennet gels increase in elasticity as the incubation temperature is raised (Horne, 1998). Unraveling and separating the contributions of electrostatics and hydrophobic attraction still remain a major gap in our understanding of rennet coagulation.\n\nSimilarly, the action of ethanol in collapsing the hairs and inducing micellar aggregation is a major coup for the 'hairy micelle' model, translated into the adhesive sphere picture of De Kruif and Holt (2003). As the \u03ba-casein hairs are also negatively charged, their neutralization on acidifying milk would also remove a component of the stabilizing barrier and induce aggregation (De Kruif and Holt, 2003). Attractive as these scenarios are for explaining these three routes to micellar destabilization, in none of them does the adhesive sphere\/hairy micelle approach tell the whole story. To achieve this, the influence of reaction conditions on micellar integrity has to be considered, and it is here that the full power of the dual-binding model comes into play.\n\n### Dual-Binding Model and Rennet Curd Formation\n\nThe aggregation and gelation of casein micelles induced by the chymosin proteolysis of \u03ba-casein is the reaction that comes closest to the colloidal aggregation model, especially in strictly controlled laboratory studies, many of which maintained the pH of the milk at its natural value of 6.7. It is under such conditions that the casein micelle exhibits most closely the properties of a colloidal hard sphere and, importantly for the aggregation observed, where the internal integrity of the micelle is undisturbed. It seems that the internal binding through calcium phosphate nanocluster bridges limits extensive rearrangements and constrains excursions of hydrophobic regions so that only those close to the surface behind the barrier of the charged macropeptide can take part in micellar aggregation once that barrier is removed. This explains the success of the reaction schemes for the initial stages of aggregate growth based on the particle model (reviewed by Hyslop, 2003, and previously by Dalgleish, 1992). It also explains the success of the gel strength model described by Horne (1995; 1996), with the particles remaining largely unchanged through the gel formation process.\n\nHowever, cheeses are seldom manufactured at the natural pH of milk, because this is not the optimum pH for enzyme action (Dalgleish, 1992). Generally, some acidification of the milk is applied, and this modifies the internal integrity of the micelles, with consequent effects on the rennet coagulation and curd properties. Some of these properties, and their influence on the cheeses produced from such curds, have been studied by Choi et al. (2007; 2008), by varying the milk pH or by adding EDTA at a fixed milk pH of 6.0, the objective being to examine in detail the impact of removing micellar calcium phosphate. In all samples, the elastic modulus of the rennet gel passed through a maximum as the gel was formed, declining during longer reaction times. Rennet gels produced at pH 6.4 had the highest maximum, probably due to a lower electrostatic repulsion because only low levels of micellar calcium phosphate were solubilized at this pH. The maximum elasticity in the gelation profile thereafter decreased with the decreasing pH of the preparation from 6.4 to 5.4. This is explained in the dual-binding model by the decline in the number of nanocluster bridges within the micelle that contribute to the overall strength of the gel matrix. There was also a decrease in the maximum gel elasticity with an increase in the added concentration of EDTA, which removed nanocluster links by sequestration of calcium. In both cases, pH adjustment and EDTA addition, the decrease in the maximum elasticity in the gel curds was accompanied by an increase in the rheological loss tangent. The removal of the nanocluster bridges was permitting greater mobility in these gels; a weaker, more flexible network was being produced.\n\nThe microstructure of these rennet-induced gels was also examined, near the point of their maximum elasticity and again some 2\u201310 h later using fluorescence microscopy (Choi et al., 2007). When elasticity was at its maximum, the gels obtained at pH 6.4 manifested more branched, interconnected networks than those obtained at pH 5.4 where the strands\/clusters were larger with more obvious open regions between. In all cases, there was a decrease in apparent interconnectivity between strands in the gel microstructure during aging, which agreed with the decrease in elasticity beyond the maximum. It is apparent that gel strength is a function not only of the number and strength of potential bonds in a system but also of their spatial distribution. Here the loss of the nanocluster bridges weakens the network but also introduces into it greater mobility. Rearrangements occur at a rate governed by that mobility and apparently toward a more compact clustering of the casein proteins, which weakens the matrix structure. Predicting the relationship between matrix morphology, bond strength, and bond number is one of the challenges yet to be addressed in food materials science.\n\nThese changes in rennet gel matrix structure have a direct impact on the functional properties of the cheeses made from the gels. Choi et al. (2008) demonstrated that removal of micellar calcium phosphate contributed to a greater softening and ease of flow of these cheeses at higher temperature. However, they found that there was an optimum pH of preparation, below which the increasing influence of attractive hydrophobic interactions in the balance of forces reduced bond lability and inhibited curd stretching.\n\n### Dual-Binding Model and Ethanol Stability\n\nAlthough studies of the response of dilute suspensions of casein micelles to the addition of ethanol constituted one of the greater successes of the hairy micelle model, they also provided pointers to the failings of the adhesive sphere concept developed from that model (Horne, 2003a).\n\nDynamic light-scattering studies (Horne 1984; 1986; Horne and Davidson, 1986) demonstrated the collapse of the hairy layer and the consequent loss of the steric-stabilizing component with subcritical concentrations of ethanol, but attempts to measure layer thickness as a function of buffer pH or ionic calcium concentration were confounded by the initial observation that initial micelle size was a function of these parameters. Raising the pH or decreasing the ionic strength produced an increase in the hydrodynamic size of the micelle, presumably due to a loosening up of the micelle as either treatment increased the effective electrostatic repulsion between the hairs. More importantly for the ethanol-induced collapse of the hairs, this loosening of the structure extended deeper into the micelle, and greater apparent layer thickness was shown as the micelle structure was caused to expand. This behavior is accommodated within the dual-binding model as a manifestation of the control of internal binding by the balance of hydrophobic attraction and electrostatic repulsion and also by the presence of the calcium phosphate nanocluster bridges, which can be lost on acidification. The model allows for changes in the rigidity of the micelle structure, particularly in the surface layers, which would then control the apparent size of the micelle when the steric-stabilizing hairs are collapsed by the nonsolvent, ethanol.\n\nThe experiments described above confirm yet again the contribution of steric stabilization to micelle stability but relate to the behavior of the casein micelle in a highly dilute suspension in an artificial environment, devoid of inorganic phosphate. In milk, the results of the alcohol stability test, and particularly the behavior of the alcohol stability\/pH profile as a function of mineral content, demand consideration of a different mechanism of destabilization (Horne, 1987), but a mechanism wholly consistent with the dual-binding model, although initially proposed well in advance of that model.\n\nThe mechanism proposed by Horne (1987) suggests that ethanol has two competing effects on the micellar system: destabilization through loss of the hairy layer, and shifts in the calcium phosphate equilibria, first noted by Pierre (1985). The behavior of calcium phosphate and its colloidal form has been at the center of much discussion in this chapter. If ethanol promotes the precipitation of calcium phosphate external to the micelle, it would first of all reduce the concentration of free calcium, reduce the level of caseinate-bound calcium, and disrupt the binding through calcium phosphate nanoclusters. Moderate losses would increase the negative charge of the caseins and increase the thickness of the steric-stabilizing layer. The higher the alcohol level, the faster and more extensive would be the precipitation of calcium phosphate. The ensuing adjustment in protein charge and conformation, though relatively rapid, still requires a finite response time. Countering these changes are the effects of ethanol as a nonsolvent for the proteins, promoting cross-linking and collapse of the hairy layer. When the coagulation reaction occurs faster than the adjustment of charge and conformation resulting from shifts in the calcium phosphate equilibria or the extent of the latter is limited by insufficient ethanol, the aggregation reaction dominates and precipitation of micelles follows.\n\nThe origin of the sigmoidal ethanol stability\/pH profile can also be explained through the effect of pH on calcium phosphate precipitation. Increasing the pH brings about increased calcium phosphate precipitation, possibly further enhanced by the ethanol, which means that more ethanol is required to precipitate the protein, that is, to overcome the increased energy barrier being erected following the transfer of calcium phosphate from the nanocluster state. Conversely, decreasing the pH acts to diminish the influence of ethanol-induced precipitation of calcium phosphate by titrating away negative charge and reducing electrostatic repulsion between protein species. Other effects of milk serum composition, of forewarming the milk, and of modifying the milk concentration and ionic strength can all be explained in a similar fashion (Horne, 2002).\n\nFinally, what is effectively a competition between mineral precipitation and protein aggregation explains the anomalous destabilization of milk by trifluoroethanol (TFE) (Horne and Davidson, 1987) and the behavior of ethanol in milks at high temperature (\u224870 \u00b0C) (O'Connell et al., 2001). With TFE, it was found that, after passing through a critical range of TFE concentrations, which caused protein precipitation, higher levels gave rise to micellar dissociation and produced translucent suspensions. When ethanol\/milk mixtures were heated by O'Connell et al. (2001), these, too, became translucent as the micelles dissociated. In both instances in the mechanism proposed here, this behavior would be seen as the result of the calcium phosphate precipitation proceeding so fast or to such an extent that the micelle would disintegrate before the micellar aggregation reaction could occur, any protein aggregates remaining small and giving rise to insignificant turbidity.\n\n### Dual-Binding Model and Acid Gel Formation\n\nIt is in trying to describe milk acid gel formation in terms of molecular events that the dual-binding model is most useful. Superficially, the initial stages of acid-induced aggregation of casein micelles can be accommodated by the adhesive sphere model, where titration of micellar charge collapses the 'hairy layer' and allows aggregation to proceed (De Kruif and Holt, 2003), but closer study, particularly of the kinetics of gel formation using glucono-\u03b4-lactone (GDL) as acidulant, reveals anomalies (Horne, 2003b).\n\nFirst, at any given temperature, increasing the quantity of GDL used, and hence the rate of acidification of the milk, leads to stiffer gels. There is no mechanism for this in the adhesive sphere model, which has pH as the only variable, but, in the dual-binding model, we postulate that titration of the negative charges of the caseins will also affect internal bonds in the micelle and that reduction of electrostatic repulsion will deepen the attraction between the molecules. If the acidification is proceeding slowly, then this may allow equilibration and rearrangement into localized denser structures with few linkages between, giving rise to weaker gels. More rapid drops in pH may lock the protein into a more dispersed structure with greater density of possibly stronger strands, as was observed microscopically when similar trends in elasticity were detected in rennet gels (Choi et al., 2007).\n\nSecond, if the skim milk is heat-treated at 90 \u00b0C for 10 min, a process practiced by the dairy industry and known as forewarming, prior to acid-induced gelation by GDL at 40 \u00b0C, not only is the critical coagulation pH shifted to a higher pH value and a much stiffer gel produced but also a distinct step is introduced on to the gelation profile. This is not an artifact introduced by slippage in the rheometer. It is fully reproducible, and its presence is subject to the reaction conditions applied. It can be made to disappear with forewarmed milk, for example, by lowering the incubation temperature to 30 \u00b0C and below (D. S. Horne, unpublished observations). A similar step can be introduced into the profile of a non\u2013heat-treated milk by raising the incubation temperature to 40 \u00b0C or above and employing a high concentration of GDL. Such steps in the profiles were also seen following the acidification of milks in the presence of xanthan (Aichinger, 2005), where their magnitude and definition were dependent on the level of xanthan introduced. With heat-treated milks, the presence, magnitude, and definition of the stepped gelation profile change in response to the temperature and duration of the preheating (Fig. 6.7). The critical pH for gelation and the maximum in complex modulus are monotonic functions of the level of denaturation of the whey proteins brought about by the prior heat treatment (Fig. 6.8). Stepped gelation profiles like this are also seen during the formation of fermented milk gels, again with forewarmed milks, where the step can be removed by suitable modest additions of trisodium citrate (TSC) (Ozcan-Yilsay et al., 2007). The critical pH and the loss tangent remained unaffected by additions of TSC up to 10 mM, although the gel elasticity recorded at pH 4.6 was enhanced and the gelation profile lost its step and assumed a monotonic increase in G' with decreasing pH.\n\nFigure 6.7 Exemplary gelation profiles showing the complex modulus of the developing gel as a function of the measured pH for skim milks preheated at 90 \u00b0C for the durations indicated on each curve. All milks were gelled at 40 \u00b0C using 4% GDL as acidulant.\n\nFigure 6.8 Gelation pH and maximum value of the complex modulus as a function of the level of denaturation of \u03b2-lactoglobulin in milks preheated at temperatures ranging from 60 to 90 \u00b0C. Different symbols indicate different durations of heating. All milks were gelled at 40 \u00b0C using 4% GDL as acidulant.\n\nAll of the above observations can be reconciled within the following picture, which relies heavily on the precepts of the dual-binding model. Aggregation, and thereafter gelation, of the casein micelles on acidification begin only when the electrostatic repulsion component is reduced below a critical level, that is, when a critical pH is reached. The rate of aggregation is a function of a number of factors: the nature of the surface, the energy (or temperature) involved, and the collision rate. Modifying the surface by partially or wholly coating it with denatured whey proteins through formation of a \u03b2-lactoglobulin\/\u03ba-casein complex, raising the incubation temperature, and increasing the micellar concentration by imposing a phase separation through addition of incompatible polysaccharide will all increase the rate of aggregation and shift the critical pH. These are all operational in the above examples, and in all cases we are forcing the micelles to interact, probing further into the inner reaches of the interaction potential with greater frequency and accessing the hydrophobic minimum there. So far, this is no more than another adhesive hard sphere description, but, during acidification, the internal integrity of the casein micelles is also being compromised through the solubilization of the micellar calcium phosphate nanoclusters. Pushing the critical gelation pH to higher values means that more nanoclusters are still present in the aggregating micelles, which are in consequence more rigid than at lower pH. Because the hydrophobic interactions are counterbalanced by any remaining electrostatic repulsion, these hydrophobic bonds are weaker the higher the coagulation pH. Rearrangements and bond breakage are relatively rapid, as evidenced by the higher value of the phase angle at this point. As the pH continues to drop, the loss of the remaining nanoclusters allows more mobility in the gel, giving the increasing phase angle, but also permitting stronger strands to be formed in the rearrangements, and hence the greater elasticity in the gel. However, the elasticity is not increasing so rapidly that it cannot be overtaken by its diminution on the loss of the nanocluster bridges\u2014hence the maximum or inflexion point in G' with system pH. Eventually, the titration of the carboxyl groups wins out, and the increasing contribution from attractive hydrophobic interactions produces increasing elasticity in the gels. Compared with the gel produced in a reaction regime with a low critical gelation pH, this 'hi-pH' gel is anticipated to have a more uniform distribution of contributing strands, as has been observed in yogurt studies (Ozcan-Yilsay et al., 2007). When the critical pH is low, as for an unheated milk for example, the reactions described above relating to the behavior on the loss of the remaining calcium phosphate nanoclusters still occur but are now confined within the micellar particle, which remains unaggregated because the inter-micellar potential is still repulsive. Mobility is introduced and bonds are strengthened, but only within the micelle so that, when eventually aggregation and gelation do take place, fewer inter-cluster bonds are formed and a more open gel structure is obtained. Location and confinement are major factors in defining the final architecture and strength of the gels formed, and the dual-binding model helps us to understand what is going on.\n\n## Concluding remarks\n\nThis chapter has reviewed the physicochemical properties and interactions of the caseins and blended them into the construction of a mechanistic framework for a working model of the casein micelle, the dual-binding model. The various properties of the casein micelle have been rationalized in terms predicted by the model. It is stressed that the dual-binding model is only a framework and that the real emphasis, in explaining, for example, the behavior of the micelle in processing, should be placed on the effects of those conditions on the ongoing interactions in their totality. This is well demonstrated by the mechanism proposed for ethanol-induced destabilization, where the behavior of the calcium phosphate equilibria and consequent reappearance of electrostatic repulsion have to be set against the poor solvent properties of the proteins in ethanol.\n\nAs well as the interactions, however, due consideration must be given to the location of the effects, and time should also be allowed for these to occur. Examples of these effects are seen in the rapid kinetics of micellar assembly leading to open, ramified structures of varying textures and densities, and in the relaxation behavior of the labile hydrophobic bonds in the growing clusters in acidified milk just below the critical gelation pH leading to stronger gels. Just as when critical pH is lower, the same hydrophobic interchange and readjustment occur only within the confines of the micelle and are locked in as the pH continues to drop, eventually reaching the critical value that leads to aggregation but a weaker final gel.\n\nConstraints of space have limited the number of cases we have been able to consider. A major omission has been explaining the dissociation and reassociation behavior of the casein micelle under high pressure, but this aspect has recently been covered by Huppertz et al. (2006) in terms that can be easily aligned with the dual-binding model. Neither have we extensively mentioned the application of this model to explaining the behavior of nonbovine milks, although a recent study by Horne et al. (2007b) has demonstrated its usefulness in this context for marsupial milks. Our objective has not been to be exhaustive but rather to provide examples of how the model can be made to work in various situations. The reality is that we have moved beyond a model and that everything can be explained by giving due consideration to the properties of the caseins, the calcium phosphate, and the interactions of the components.\n\n# References\n\nAichinger P-A .  _Kinetic trapping of microstructures\u2014control of gelation in dairy products_ .  Glasgow, UK :  Thesis. University of Strathclyde ;  2005 .\n\nAlexander M ,  Rojas-Ochoa LF ,  Leser M ,  Schurtenberger P .  Structure, dynamics and optical properties of concentrated milk suspensions: an analogy to hard-sphere liquids .  _Journal of Colloid and Interface Science_.  2002 ;253 : 34 \u2013 46 .\n\nAoki T ,  Umeda T ,  Kako Y .  The least number of phosphate groups for cross-linking of casein by colloidal calcium phosphate .  _Journal of Dairy Science_.  1992 ;75 : 971 \u2013 975 .\n\nAschi A ,  Calmettes P ,  Daoud M ,  Douillard R ,  Gharbi A .  Micelle formation in beta-casein solutions .  _Polymer_.  2009 ;50 : 6024 \u2013 6031 .\n\nBienvenue A ,  Jimenez-Flores R ,  Singh H .  Rheological properties of concentrated skim milk: Importance of soluble minerals in the changes in viscosity during storage .  _Journal of Dairy Science_.  2003 ;88 : 3784 \u2013 3797 .\n\nBouchoux A ,  Gesan-Guiziou G ,  Perez J ,  Cabane B .  How to squeeze a sponge: Casein micelles under osmotic stress, a SAXS study .  _Biophysical Journal_.  2010 ;99 : 3754 \u2013 3762 .\n\nBrooksbank DV ,  Davidson CM ,  Horne DS ,  Leaver J .  Influence of electrostatic interactions on \u03b2-casein layers adsorbed on polystyrene lattices .  _Journal of the Chemical Society, Faraday Transactions_.  1993 ;89 : 3419 \u2013 3425 .\n\nChoi J ,  Horne DS ,  Lucey JA .  Effect of insoluble calcium concentration on rennet coagulation properties of milk .  _Journal of Dairy Science_.  2007 ;90 : 2612 \u2013 2623 .\n\nChoi J ,  Horne DS ,  Johnson ME ,  Lucey JA .  Effect of the concentration of insoluble calcium phosphate associated with casein micelles on the functionality of directly acidified cheese .  _Journal of Dairy Science_.  2008 ;91 : 513 \u2013 522 .\n\nChoi J ,  Horne DS ,  Lucey JA .  Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk .  _Journal of Dairy Science_.  2011 ;94 : 3250 \u2013 3261 .\n\nCross KJ ,  Huq NL ,  Palamara JE ,  Perich JW ,  Reynolds EC .  Physicochemical characterization of casein phosphopeptide- amorphous calcium phosphate nanocomplexes .  _Journal of Biological Chemistry_.  2005 ;280 : 15362 \u2013 15369 .\n\nDalgleish DG .  Coagulation of renneted casein micelles: Dependence on temperature, calcium ion concentration and ionic strength .  _Journal of Dairy Research_.  1983 ;50 : 331 \u2013 340 .\n\nDalgleish DG .  Measurement of electrophoretic mobilities and zeta potentials of particles from milk using laser Doppler electrophoresis .  _Journal of Dairy Research_.  1984 ;54 : 425 \u2013 438 .\n\nDalgleish DG .  The conformations of proteins on solid\/water interfaces: caseins and phosvitin on polystyrene lattices .  _Colloids and Surfaces_.  1990 ;46 : 141 \u2013 145 .\n\nDalgleish DG .  The enzymatic coagulation of milk .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry\u20141. Proteins_ .  Essex :  Elsevier Applied Science, Barking ;  1992 : 579 \u2013 619 .\n\nDalgleish DG .  On the structural models of casein micelles: Review and possible improvements .  _Soft Matter_.  2011 ;7 : 2265 \u2013 2272 .\n\nDalgleish DG ,  Law AJR .  pH-induced dissociation of bovine casein micelles: Analysis of liberated caseins .  _Journal of Dairy Research_.  1988 ;55 : 529 \u2013 538 .\n\nDalgleish DG ,  Law AJR .  pH-induced dissociation of casein micelles: Mineral solubilization and its relation to casein release .  _Journal of Dairy Research_.  1989 ;56 : 727 \u2013 735 .\n\nDalgleish DG ,  Leaver J .  The possible conformations of milk proteins adsorbed at oil\/water interfaces .  _Journal of Colloid and Interface Science_.  1991 ;141 : 288 \u2013 294 .\n\nDalgleish DG ,  Parker TG .  Binding of calcium ions to bovine \u03b1S1-casein and precipitability of the protein-calcium ion complexes .  _Journal of Dairy Research_.  1980 ;47 : 113 \u2013 122 .\n\nDalgleish DG ,  Horne DS ,  Law AJR .  Size-related differences in bovine casein micelles .  _Biochimica et Biophysica Acta_.  1989 ;991 : 383 \u2013 387 .\n\nDalgleish DG ,  Spagnuolo PA ,  Goff HD .  A possible structure of the casein micelle based on high resolution field-emission scanning electron microscopy .  _International Dairy Journal_.  2004 ;14 : 1025 \u2013 1031 .\n\nDe Kruif CG .  Casein micelles: Diffusivity as a function of renneting time .  _Langmuir_.  1992 ;8 : 2932 \u2013 2937 .\n\nDe Kruif CG .  Supra-aggregates of casein micelles as a prelude to coagulation .  _Journal of Dairy Science_.  1998 ;81 : 3019 \u2013 3029 .\n\nDe Kruif CG ,  Grinberg VY .  Micellization of \u03b2-casein .  _Colloids and Surfaces A. Physicochemical and Engineering Aspects_.  2002 ;210 : 183 \u2013 190 .\n\nDe Kruif CG ,  Holt C .  Casein micelle structure, functions and interactions .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 213 \u2013 276 .\n\nDe Kruif CG ,  Zhulina EB .  \u03ba-Casein as a polyelectrolyte brush on the surface of casein micelles .  _Colloids and Surfaces A. Physicochemical and Engineering Aspects_.  1996 ;117 : 151 \u2013 159 .\n\nDe Kruif CG ,  Huppertz T ,  Urban VS ,  Petukhov AV .  Casein micelles and their internal structure .  _Advances in Colloid and Interface Science_.  2012 ; : 171 \u2013 172 : 36-52 .\n\nDickinson E ,  Horne DS ,  Phipps JS ,  Richardson RM .  A neutron reflectivity study of the adsorption of \u03b2-casein at fluid interfaces .  _Langmuir_.  1993 ;9 : 242 \u2013 248 .\n\nDickinson E ,  Horne DS ,  Pinfield VJ ,  Leermakers FAM .  Self-consistent-field modelling of casein adsorption .  _Comparison of results for \u03b1 S1-casein and \u03b2-casein. Journal of the Chemical Society, Faraday Transactions_.  1997 ;93 : 425 \u2013 432 .\n\nDickinson E ,  Pinfield VJ ,  Horne DS ,  Leermakers FAM .  Self-consistent-field modelling of adsorbed casein. Interaction between two protein-coated surfaces .  _Journal of the Chemical Society, Faraday Transactions_.  1997 ;93 : 1785 \u2013 1790 .\n\nDonnelly WJ ,  McNeill GP ,  Buchheim W ,  McGann TCA .  A comprehensive study of the relationship between size and protein composition in natural casein micelles .  _Biochimica et Biophysica Acta_.  1984 ;789 : 136 \u2013 143 .\n\nDorozkhin SV .  Amorphous calcium (ortho)phosphates .  _Acta Biomaterials_.  2010 ;6 : 4457 \u2013 4475 .\n\nFarrell Jr HM ,  Malin EL ,  Brown EM ,  Qi PX .  Casein micelle structure: What can be learned from milk synthesis and structural biology? .  _Current Opinion in Colloid and Interface Science_.  2006 ;11 : 135 \u2013 147 .\n\nFox PF .  Milk proteins: general and historical review .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1 \u2013 48 .\n\nFox PF ,  Brodkorb A .  The casein micelle: Historical aspects, current concepts and significance .  _Int. Dairy J._ 2008 ;18 : 677 \u2013 684 .\n\nGriffin MCA ,  Lyster RLJ ,  Price JC .  The disaggregation of calcium-depleted micelles .  _European Journal of Biochemistry_.  1988 ;174 : 339 \u2013 343 .\n\nGriffin MCA ,  Price JC ,  Griffin WG .  Variation of the viscosity of a concentrated, sterically stabilized colloid: Effect of ethanol on casein micelles of bovine milk .  _Journal of Colloid and Interface Science_.  1989 ;128 : 223 \u2013 229 .\n\nHansen S ,  Bauer R ,  Lomholt SB ,  Qvist KB ,  Pedersen JS ,  Mortensen K .  Structure of casein micelles studied by small-angle neutron scattering .  _European Biophysics Journal_.  1996 ;24 : 143 \u2013 147 .\n\nHilgemann M ,  Jenness R .  Observations on the effect of heat treatment upon the dissolved calcium and phosphorus in milk .  _Journal of Dairy Science_.  1951 ;34 : 483 \u2013 484 .\n\nHolt C ,  The stability of casein micelles .  Wolfram E ,  ed.  _Proceedings of an International Conference on Colloid and Interface Science, Budapest_ ,  Volume 1 .  Budapest :  Akademia Kiado ;  1975 : 641 \u2013 644 .\n\nHolt C .  Structure and stability of casein micelles .  _Advances in Protein Chemistry_.  1992 ;43 : 63 \u2013 151 .\n\nHolt C .  An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of milk salts in milk .  _European Biophysics Journal_.  2004 ;33 : 421 \u2013 434 .\n\nHolt C ,  Horne DS .  The hairy casein micelle: Evolution of the concept and its implications for dairy technology .  _Netherlands Milk and Dairy Journal_.  1996 ;50 : 85 \u2013 111 .\n\nHolt C ,  Davies DT ,  Law AJR .  Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles .  _Journal of Dairy Research_.  1986 ;53 : 557 \u2013 572 .\n\nHorne DS .  Calcium-induced precipitation of \u03b1S1-casein: Effect of inclusion of citrate or phosphate .  _Journal of Dairy Research_.  1982 ;49 : 107 \u2013 118 .\n\nHorne DS .  The calcium-induced precipitation of \u03b1S1-casein: Effect of modification of lysine residues .  _International Journal of Biological Macromolecules_.  1983 ;5 : 296 \u2013 300 .\n\nHorne DS .  Steric effects in the coagulation of casein micelles by ethanol .  _Biopolymers_.  1984 ;23 : 989 \u2013 993 .\n\nHorne DS .  Steric stabilization and casein micelle stability .  _Journal of Colloid and Interface Science_.  1986 ;111 : 250 \u2013 260 .\n\nHorne DS .  Ethanol stability of casein micelles\u2014a hypothesis concerning the role of calcium phosphate .  _Journal of Dairy Research_.  1987 ;54 : 389 \u2013 395 .\n\nHorne DS .  Predictions of protein helix content from an autocorrelation analysis of sequence hydrophobicities .  _Biopolymers_.  1988 ;27 : 451 \u2013 477 .\n\nHorne DS .  Ethanol stability .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry\u20141. Proteins_ .  Essex :  Elsevier Applied Science, Barking ;  1992 : 657 \u2013 689 .\n\nHorne DS .  Scaling behaviour of shear moduli during the formation of rennet milk gels .  In:  Dickinson E ,  Lorient D ,  eds.  _Food Macromolecules and Colloids_ .  Cambridge :  Royal Society of Chemistry ;  1995 : 456 \u2013 461 .\n\nHorne DS .  Aspects of scaling behaviour in the kinetics of particle gel formation .  _Journal de Chimie Physique et de Physico-Chimie Biologique_.  1996 ;96 : 977 \u2013 986 .\n\nHorne DS .  Casein interactions: casting light on the black boxes, the structure in dairy products .  _International Dairy Journal_.  1998 ;8 : 171 \u2013 177 .\n\nHorne DS .  Caseins\u2014molecular properties, casein micelle formation and structure .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  New York :  Elsevier ;  2002 : 1902 \u2013 1909 .\n\nHorne DS .  Ethanol stability .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 975 \u2013 999 .\n\nHorne DS .  Casein micelles as hard spheres: Limitations of the model in acidified gel formation .  _Colloids and Surfaces A. Physicochemical and Engineering Aspects_.  2003 ;213 : 255 \u2013 263 .\n\nHorne DS .  Casein micelle structure: models and muddles .  _Current Opinion in Colloid and Interface Science_.  2006 ;11 : 148 \u2013 153 .\n\nHorne DS ,  Dalgleish DG .  Electrostatic interactions and the kinetics of protein aggregation: \u03b1S1-casein .  _International Journal of Biological Macromolecules_.  1980 ;2 : 154 \u2013 160 .\n\nHorne DS ,  Davidson CM .  The effect of environmental conditions on the steric stabilization of casein micelles .  _Colloid and Polymer Science_.  1986 ;264 : 727 \u2013 734 .\n\nHorne DS ,  Davidson CM .  Alcohol stability of bovine milk: Anomalous effects with trifluoroethanol .  _Milchwissenschaft_.  1987 ;42 : 509 \u2013 512 .\n\nHorne DS ,  Moir PD .  The iodination of \u03b1S1-casein and its effect on the calcium-induced aggregation reaction of the modified protein .  _International Journal of Biological Macromolecules_.  1984 ;6 : 316 \u2013 320 .\n\nHorne DS ,  Lucey JA ,  Choi J .  Casein interactions: Does the chemistry really matter? .  In:  Dickinson E ,  Leser M ,  eds.  _Food Colloids: Self-Assembly and Materials Science_ .  Cambridge :  Royal Society of Chemistry ;  2007 : 155 \u2013 166 .\n\nHorne DS ,  Anema S ,  Zhu X ,  Nicholas KR ,  Singh H .  A lactational study of the composition and integrity of casein micelles from the milk of the Tammar wallaby (Macropus eugenii) .  _Archives of Biochemistry and Biophysics_.  2007 ;467 : 107 \u2013 118 .\n\nHuppertz T ,  Fox PF .  Effect of NaCl on some physicochemical properties of concentrated bovine milk .  _International Dairy Journal_.  2006 ;16 : 1142 \u2013 1148 .\n\nHuppertz T ,  Kelly AL ,  De Kruif CG .  Disruption and reassociation of casein micelles under high pressure .  _Journal of Dairy Research_.  2006 ;73 : 294 \u2013 298 .\n\nHyslop DB .  Enzymatic coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 839 \u2013 878 .\n\nJenness R ,  Patton S .  _Principles of Dairy Chemistry_ .  New York :  Wiley ;  1959 .\n\nJolles P ,  Loucheux-Lefebvre MH ,  Henschen A .  Structural relatedness of \u03ba-casein and fibrinogen \u03b3-chain .  _J. Mol. Evol._ 1978 ;11 : 271 \u2013 277 .\n\nKarlsson AO ,  Ipsen R ,  Schrader K ,  Ardo Y .  Relationship between physical properties of casein micelles and rheology of skim milk concentrate .  _Journal of Dairy Science_.  2005 ;88 : 3784 \u2013 3797 .\n\nKawasaki K ,  Weiss KM .  Mineralized tissue and vertebrate evolution: The secretory calcium-binding phosphoprotein gene cluster .  _Proceedings of the National Academy of Sciences of the USA_.  2003 ;100 : 4060 \u2013 4065 .\n\nKawasaki K ,  Weiss KM .  Evolutionary genetics of vertebrate tissue mineralization: The origin and evolution of the secretory calcium-binding phosphoprotein family .  _Journal of Experimental Zoology (Molecular Development and Evolution)_.  2006 ;306B : 295 \u2013 316 .\n\nKawasaki K ,  Lafont A-G ,  Sire J-Y .  The evolution of milk casein genes from tooth genes before the origin of mammals .  _Molecular Biology and Evolution_.  2011 ;28 : 2053 \u2013 2061 .\n\nKegel WK ,  Van der Schoot P .  Competing hydrophobic and screened Coulomb interactions in hepatitis B virus capsid assembly .  _Biophysical Journal_.  2004 ;86 : 3905 \u2013 3913 .\n\nKegeles G .  A shell model for size distribution in micelles .  _Journal of Physical Chemistry_.  1979 ;83 : 1728 \u2013 1732 .\n\nLeaver J ,  Dalgleish DG .  The topography of bovine (-casein at an oil\/water interface as determined from the kinetics of trypsin-catalyzed hydrolysis .  _Biochimica et Biophysica Acta_.  1990 ;1041 : 217 \u2013 222 .\n\nLeermakers FAM ,  Atkinson PJ ,  Dickinson E ,  Horne DS .  Self-consistent-field modelling of adsorbed \u03b2-casein: Effect of pH and ionic strength .  _Journal of Colloid and Interface Science_.  1996 ;178 : 681 \u2013 693 .\n\nMarchin S ,  Putaux J-L ,  Pignon F ,  Leonil J .  Effects of the environmental factors on the casein micelle structure studied by cryo-transmission electron microscopy and small-angle X-ray scattering\/ultrasmall-angle X-ray scattering .  _Journal of Chemical Physics_.  2007 ;126 : 045101 .\n\nMartin P ,  Ferranti P ,  Leroux C ,  Addeo F .  Non-bovine caseins: Quantitative variability and molecular diversity .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 277 \u2013 317 .\n\nMcGann TCA ,  Fox PF .  Physico-chemical properties of casein micelles reformed from urea-treated milk .  _Journal of Dairy Research_.  1974 ;41 : 45 \u2013 53 .\n\nMcMahon DJ ,  McManus WR .  Rethinking casein micelle structure using electron microscopy .  _Journal of Dairy Science_.  1998 ;81 : 2985 \u2013 2993 .\n\nMcMahon DJ ,  Oommen BS .  Supramolecular structure of casein micelles .  _Journal of Dairy Science_.  2008 ;91 : 1709 \u2013 1721 .\n\nMeakin P .  A historical introduction to computer models for fractal aggregates .  _Journal of Sol-Gel Science and Technology_.  1999 ;15 : 97 \u2013 117 .\n\nMercier J-C .  Phosphorylation of casein. Present evidence for an amino acid triplet code post-translationally recognized by specific kinases .  _Biochimie_.  1981 ;68 : 1 \u2013 17 .\n\nMezzenga R ,  Schurtenberger P ,  Burbridge A ,  Michel M .  Understanding foods as soft materials .  _Nature Materials_.  2005 ;4 : 729 \u2013 740 .\n\nMikheeva LM ,  Grinberg NV ,  Grinberg VY ,  Khokhlov AR ,  De Kruif CG .  Thermodynamics of micellization of bovine \u03b2-casein studied by high sensitivity differential scanning calorimetry .  _Langmuir_.  2003 ;19 : 2913 \u2013 2921 .\n\nMoitzi C ,  Menzel A ,  Schurtenberger P ,  Stradner A .  The pH induced sol-gel transition in skim milk revisited. A detailed study using time-resolved light and X-ray scattering experiments .  _Langmuir_.  2011 ;27 : 2195 \u2013 2203 .\n\nO'Connell JE ,  Kelly AL ,  Fox PF ,  De Kruif CG .  Mechanism for the ethanol-dependent, heat-induced dissociation of casein micelles .  _Journal of Agricultural and Food Chemistry_.  2001 ;49 : 4424 \u2013 4428 .\n\nO'Connell JE ,  Grinberg VY ,  De Kruif CG .  Association behaviour of \u03b2-casein .  _Journal of Colloid and Interface Science_.  2003 ;258 : 33 \u2013 39 .\n\nOuanezar M ,  Guyomarc'h F ,  Bouchoux A .  AFM imaging of casein micelles: Evidence for structural rearrangement upon acidification .  _Langmuir_.  2012 ;28 : 4915 \u2013 4919 .\n\nOzcan-Yilsay T ,  Lee W-J ,  Horne D ,  Lucey JA .  Effect of tri-sodium citrate on the rheological and physical properties and microstructure of yogurt .  _Journal of Dairy Science_.  2007 ;90 : 1644 \u2013 1652 .\n\nOzcan T ,  Horne D ,  Lucey JA .  Effect of increasing the colloidal calcium phosphate of milk on the texture and microstructure of yogurt .  _Journal of Dairy Science_.  2011 ;94 : 5278 \u2013 5288 .\n\nParker TG ,  Dalgleish DG .  Binding of calcium ions to bovine \u03b2-casein .  _Journal of Dairy Research_.  1981 ;48 : 71 \u2013 76 .\n\nPayens TAJ ,  Schmidt DG .  Boundary spreading of rapidly polymerizing \u03b1S1-casein B and C during sedimentation. Numerical solutions of the Lamm-Gilbert-Fujita equation .  _Archives of Biochemistry and Biophysics_.  1966 ;115 : 136 \u2013 145 .\n\nPayens TAJ ,  Van Markwijk BW .  Some features of the self-association of \u03b2-casein .  _Biochimica et Biophysica Acta_.  1963 ;71 : 517 \u2013 530 .\n\nPayens TAJ ,  Brinkhuis JA ,  Van Markwijk BW .  Self-association in non-ideal systems. Combined light scattering and sedimentation measurements in \u03b2-casein solutions .  _Biochimica et Biophysica Acta_.  1969 ;175 : 434 \u2013 437 .\n\nPiazza R .  Protein interactions and association: An open challenge for colloid science .  _Current Opinion in Colloid and Interface Science_.  2004 ;8 : 515 \u2013 522 .\n\nPierre A .  Milk coagulation by alcohol. Studies on the solubility of the milk calcium and phosphate in alcoholic solutions .  _Lait_.  1985 ;65 : 201 \u2013 212 .\n\nRollema HS .  Casein association and micelle formation .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry\u20141. Proteins_ .  Essex :  Elsevier Applied Science, Barking ;  1992 : 111 \u2013 140 .\n\nRollema HS ,  Branches JA .  A 1H-NMR study of bovine casein micelles: Influence of pH, temperature and calcium ions on micellar structure .  _Journal of Dairy Research_.  1989 ;46 : 417 \u2013 425 .\n\nSchmidt DG .  The association of \u03b1S1-casein at pH 6 .  _6. Biochimica et Biophysica Acta_.  1970 ;207 : 130 \u2013 138 .\n\nSchmidt DG .  Differences between the association of genetic variants B, C and D of \u03b1S1-casein .  _Biochimica et Biophysica Acta_.  1970 ;221 : 140 \u2013 142 .\n\nSchmidt DG .  Colloidal aspects of the caseins .  _Netherlands Milk and Dairy Journal_.  1980 ;34 : 42 \u2013 64 .\n\nSchmidt DG .  Association of caseins and casein micelle structure .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry\u20141_ .  London :  Applied Science Publishers ;  1982 : 61 \u2013 86 .\n\nSlattery CW .  Model calculations of casein micelle size distribution .  _Biophysical Chemistry_.  1977 ;6 : 59 \u2013 64 .\n\nSlattery CW ,  Evard R .  A model for the formation and structure of casein micelles from subunits of variable composition .  _Biochimica et Biophysica Acta_.  1973 ;317 : 529 \u2013 538 .\n\nStradner A ,  Sedgwick H ,  Cardinaux F ,  Poon WCK ,  Egelhaaf SU ,  Schurtenberger P .  Equilibrium cluster formation in concentrated protein solutions and colloids .  _Nature_.  2004 ;432 : 492 \u2013 495 .\n\nStothart PH ,  Cebula DJ .  Small angle neutron scattering study of bovine casein micelles and sub-micelles .  _Journal of Molecular Biology_.  1982 ;160 : 391 \u2013 395 .\n\nSwaisgood HE .  Chemistry of the caseins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry\u20141. Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 139 \u2013 201 .\n\nThachepan S ,  Li M ,  Mann S .  Mesoscale crystallization of calcium phosphate nanostructures in protein (casein) micelles .  _Nanoscale_.  2010 ;2 : 2400 \u2013 2405 .\n\nTrejo R ,  Dokland T ,  Jurat-Fuentes J ,  Harte F .  Cryo-transmission electron tomography of native casein micelles from bovine milk .  _Journal of Dairy Science_.  2011 ;94 : 5770 \u2013 5775 .\n\nWalstra P .  The voluminosity of bovine casein micelles and some of its implications .  _Journal of Dairy Research_.  1979 ;46 : 317 \u2013 323 .\n\nWalstra P .  On the stability of casein micelles .  _Journal of Dairy Science_.  1990 ;73 : 1965 \u2013 1979 .\n\nWalstra P .  Casein sub-micelles: do they exist? .  _International Dairy Journal_.  1998 ;9 : 189 \u2013 192 .\n\nWeitz DA ,  Lin MY ,  Lindsay HM .  Universality laws in coagulation .  _Chemometrics and Intelligent Laboratory Systems_.  1991 ;10 : 133 \u2013 140 .\n\nYin X ,  Stott MJ .  Biological calcium phosphates and Posner's cluster .  _Journal of Chemical Physics_.  2003 ;118 : 3717 \u2013 3723 . \nChapter 7\n\n# Structure and Stability of Whey Proteins\n\nPatrick J.B. Edwards\n\nGeoffrey B. Jameson Centre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nThe chemical and physical stability of the more common proteins of bovine and, where available, ovine, caprine, and equine whey (\u03b2-lactoglobulin, \u03b1-lactalbumin, serum albumin, immunoglobulins, and lactoferrin) is reviewed with regard to their molecular structures and dynamics. The behavior of the proteins separately and in combination with temperature, pressure, pH, denaturants (such as guanidinium chloride and urea), and stabilizers (such as fatty acids and metal ions) has been considered. The combination of high temperature and low pH in the hydrolysis and subsequent formation of fibrils constitutes a new body of study and knowledge since the first edition of this chapter in 2009. Particular emphasis has been placed on studies that have utilized x-ray, NMR, fluorescence, and circular dichroism techniques. Attention is directed to the role of cysteines and disulfide bridges with regard to chemical stability. Whereas there is considerable knowledge of structure\u2013function relationships of individual proteins, there is a dearth of three-dimensional structural knowledge of combinations of proteins, despite the clear importance of such knowledge to functionality, especially with regard to food processes.\n\n## Keywords\n\nWhey protein structure\n\nwhey protein stability\n\nbovine whey\n\nbovine \u03b2-lactoglobulin\n\nx-ray\n\nNMR\n\nfluorescence\n\ncircular dichroism techniques\n\nOutline\n\nIntroduction 202\n\nBovine \u03b2-lactoglobulin 203\n\nMolecular Structure of Bovine \u03b2-Lactoglobulin 203\n\nStructure of Bovine \u03b2-Lactoglobulin in Aqueous Solution 205\n\nThe Monomer\u2013Dimer Equilibrium of Bovine \u03b2-Lactoglobulin in Aqueous Solution 206\n\nStudies of Bovine \u03b2-Lactoglobulin by NMR at Neutral pH 207\n\nBovine \u03b2-Lactoglobulin Dynamics 208\n\nStructures of \u03b2-Lactoglobulins from other Species 209\n\nLigand Binding to \u03b2-Lactoglobulin 210\n\nEffect of Temperature on Bovine \u03b2-Lactoglobulin 213\n\nEffect of Pressure on Bovine \u03b2-Lactoglobulin 216\n\nEffect of Chemical Denaturants on Bovine \u03b2-Lactoglobulin 218\n\nFibrillar Formation from Bovine \u03b2-Lactoglobulin 219\n\n\u03b1-Lactalbumin 220\n\nMolecular Structure of Bovine \u03b1-Lactalbumin 221\n\nEffect of Temperature on Bovine \u03b1-Lactalbumin 222\n\nEffect of Pressure on Bovine \u03b1-Lactalbumin 222\n\nEffect of Denaturants on Bovine \u03b1-Lactalbumin 223\n\nSerum albumin 223\n\nStructure of Serum Albumins 225\n\nEffect of Temperature on Serum Albumins 225\n\nEffect of Pressure on Serum Albumins 226\n\nEffect of Chemical Denaturants on Serum Albumins 226\n\nImmunoglobulins 227\n\nStructure of Immunoglobulin G 227\n\nEffects of Temperature, Pressure, and Chemical Denaturants on Ig Structure and Stability 227\n\nLactoferrin 229\n\nBovine Lactoferrin Structure 229\n\nEffects of Temperature, Pressure, and Chemical Denaturants on Lactoferrin Structure and Stability 231\n\nConcluding remarks 231\n\nAcknowledgments 232\n\n## Introduction\n\nInformation regarding whey protein structure and stability has great potential to facilitate knowledge-based product design. Reviews that highlight the importance of knowledge of structure and stability, including the effects of pressure, temperature, and chemical denaturants, have been made by L\u00f3pez-Fandi\u00f1o (2006). In this chapter, we discuss the structures of the whey proteins shown in Table 7.1 under quiescent and destabilizing conditions (change in pH, temperature, and pressure and addition of chaotropes) and in the presence or absence of small-molecule ligands. A host of other proteins are found in the whey of milk, but only in trace amounts, including the hyperphosphorylated osteopontin, trefoil factors, and growth factors (TGF-\u03b2, IGF-I and IGF-II, EGF, HB-EGF), and the enzymes lactoperoxidase, superoxide dismutase, platelet-activating factor acetylhydrolase, and alkalinephosphatase (Chatterton et al., 2006; Chatterton et al., 2013). Particular emphasis is placed on information that has been obtained via high-resolution x-ray crystallographic and high-field nuclear magnetic resonance (NMR) studies. The results of these studies have been applied to many projects reported in the present volume (e.g., Chapters 8, , , and ). The focus of this chapter is directed toward properties of proteins in milk whey that impinge on functionality, rather than the intrinsic function and effects on organisms imbibing milk.\n\nTable 7.1\n\nTypical Protein Composition of Whey\n\nProtein | Proportion by mass | No. amino acids | Mol mass (\/ Da) | Iso-ionic point | Disulfide bond\/thiols | Comments  \n---|---|---|---|---|---|---  \n\u03b2-Lactoglobulin  \n(\u03b2-Lg) | 60% | 162 | 18 363a | 5.35 | 2\/1 | Two common variants, A and B  \n\u03b1-Lactalbumin  \n(\u03b1-La) | 20% | 123 | 14 178 | 4.80 | 3 | About 10% of molecules are glycosylated  \nBovine serum albumin (BSA) | 3% | 583 | 66 399 |  | 17\/1 | Also present in blood serum  \nImmunoglobulin G (IgG) | 10% | >500 | 161 000 (G1)b | Many isoforms |  | Passive transfer of immunities  \nLactoferrin (Lf) | <0.1% | 689 | 76 110 | 8.95 | 17 | Bacteriostatic role; glycoprotein\n\na Molar mass for the A variant.\n\nb G1 is the major immunoglobulin; two other classes, IgM and IgA, are present in much lower abundance.\n\nBased on Farrell et al., 2004.\n\n## Bovine \u03b2-Lactoglobulin\n\n\u03b2-Lactoglobulin (\u03b2-Lg) contains 162 amino acids and has a molecular weight of 18.3 kDa (Hambling et al., 1992). It is a member of the lipocalin (a contraction of the Greek lipos, meaning 'fat, grease' and calyx, meaning 'cup') family of proteins (Banaszak et al., 1994; Flower, 1996), so called because of their ability to bind small hydrophobic molecules into a hydrophobic cavity. This led to the proposal that \u03b2-Lg functions as a transport protein for retinoid species, such as vitamin A (Papiz et al., 1986).\n\n\u03b2-Lg is the most abundant whey protein in the milk of most mammals (\u223c10% of total protein or \u223c50% of whey protein), but has not been detected in the milk of humans, rodents, or lagomorphs. In the case of human milk, \u03b1-lactalbumin \u03b1-la (see below) is the dominant whey protein. Bovine \u03b2-Lg is by far the most commonly studied milk protein.\n\nThere are ten known genetic variants of bovine \u03b2-Lg. The most abundant variants are labeled \u03b2-Lg A and \u03b2-Lg B (Farrell et al., 2004) and differ by two amino acid substitutions, Asp64Gly and Val118Ala, respectively. The quaternary structure of the protein varies among monomers, dimers, or oligomers depending on the pH, temperature, and ionic strength, with the dimer being the prevalent form under physiological conditions (Kumosinski and Timasheff, 1966; McKenzie and Sawyer, 1967; Gottschalk et al., 2003). This variable state of association is likely to be the result of a delicate balance among hydrophobic, electrostatic, and hydrogen-bond interactions (Sakurai et al., 2001; Sakurai and Goto, 2002).\n\n### Molecular Structure of Bovine \u03b2-Lactoglobulin\n\n\u03b2-Lg was an early target of x-ray diffraction as newly applied at the Royal Institution to protein crystals. This was due to its high abundance and relatively easy purification from milk and to its propensity to form suitable crystals. In retrospect, this was a very ambitious project because \u03b2-Lg was not the easiest protein to analyze (Green et al., 1979) partly because of the multiple crystal forms. Nevertheless, this study established that the protein monomer was near spherical with a block of electron density with a rod-like structure across one face.\n\nThe next attempt (Creamer et al., 1983) to determine the structure was by calculation using sequence data and structural probabilities to estimate which portions of the amino acid sequence might form into the helices, strands, and sheets. The secondary structure of \u03b2-Lg was predicted to comprise \u223c15% \u03b1-helix, \u223c50% \u03b2-sheet, and 15\u201320% reverse turn (Creamer et al., 1983). It is interesting to note that many of the residues that are present in the extended structures of the native protein have been shown to have a nascent propensity to form \u03b1-helical structures in the presence of trifluoroethanol or amphiphiles (Hamada et al., 1995; Kuroda et al., 1996; Chamani et al., 2006).\n\nIn 1986, the first medium resolution structure of \u03b2-Lg was published (Papiz et al., 1986). Structural similarity to a seemingly different type of protein, plasma retinol-binding protein, has given rise to much speculation as to the role of \u03b2-Lg in bovine milk. Higher resolution structures subsequently revealed the now familiar eight-stranded \u03b2-barrel (calyx), flanked by a three-turn \u03b1-helix. A final ninth strand forms the greater part of the dimer interface at neutral pH (Papiz et al., 1986; Bewley et al., 1997; Brownlow et al., 1997). The \u03b2-barrel is formed by two \u03b2-sheets, where strands A to D form one sheet and E to H the other (with some participation from the A strand, facilitated by a 90o bend at Ser21). Two disulfide bonds link Cys66 on loop CD (which, as its name suggests, connects strands C and D) with Cys160 near the C terminus, and Cys106 on strand G with Cys119 on strand H, leaving Cys121 as a free, but unexposed thiol. The loops connecting the strands BC, DE, and FG are relatively short, whereas those at the open end of the barrel, AB, CD, EF, and GH, are longer and more flexible. These features are illustrated in Figure 7.1.\n\nFigure 7.1 Diagram of the dimeric structure of bovine \u03b2-Lg A looking down the two-fold axis. The coordinates are taken from the structure of \u03b2-Lg A in the trigonal Z lattice with 12-bromododecanoic acid bound (PDB code: 1bso). The strands that form the \u03b2 barrel are labeled A to H. The I strand, together with part of the AB loop, forms the dimer interface at neutral pH. The locations of the sites of difference between the A and B variants are also shown. The structure is rainbow colored, beginning with blue at the N-terminus and ending with red at the C-terminus. Ser21, which shows conformational flexibility, and the 12-bromododecanoate anion, are shown as spheres. Figure drawn with PyMOL (Delano, 2002).\n\nThe structures of the A and B variants are very similar. However, the Asp64Gly substitution results in the CD loop adopting differing conformations (Qin et al., 1999). The Val118Ala causes no detectable change to the structures, but the void created by substituting the bulky isopropyl substituent, with the smaller methyl group results in the hydrophobic core of the B variant being less well packed, and may account for the lower thermal stability of the B variant under some measurement conditions (Qin et al., 1999).\n\nVery careful titrimetric and thermodynamic measurements in the late 1950s (Tanford et al., 1959; Tanford and Nozaki, 1959) established the presence of a carboxylic acid residue with an anomalously high pK a value of 7.3. This was attributed to a pH-dependent conformational change, a conclusion that rationalized earlier measurements of pH-dependent sedimentation coefficients (Pedersen, 1936) and specific optical rotation data (Groves et al., 1951). Much later, x-ray structure analyses (Qin et al., 1998a) at pH values above and below this so-named Tanford transition established that, at pH 6.2, the EF loop is closed over the top of the barrel, burying Glu89 (the carboxylic acid with the anomalous pK a) inside the calyx. At pH 8.1 this loop is articulated away from the barrel such that the formerly buried glutamic acid becomes exposed in the carboxylate form (Qin et al., 1998a).\n\nAn early structure of bovine \u03b2-Lg crystallized in the presence of retinol appeared to show retinol bound externally to the protein (Monaco et al., 1987), apparently later confirmed by a body of fluorescence data (Dufour et al., 1994; Lange et al., 1998; Narayan and Berliner, 1998). Subsequent structural analyses have shown, however, that fatty acids, retinoid species (including vitamin A), and cholesterol (including vitamin D) all bind inside the calyx (Kontopidis et al., 2004). Induced circular dichroism (CD) measurements and NMR measurements confirm the x-ray crystallographic observations. Ligand binding will be discussed in more detail below, since it relates to the probable physiological function of \u03b2-Lg as well as to the stability of this molecule and current technological interest in the role of protein-ligand interactions in flavor perception (see Chapter 14). At this stage, there is no evidence for binding of fatty acids or retinoid species outside the calyx. Except for very bulky ligands (see below), ligands bind inside the calyx of \u03b2-Lg at pH \u223c7.\n\nAt about the same time as the Tanford transition and ligand-binding modes were elucidated by high-resolution x-ray crystallography (Qin et al., 1998a), NMR studies of \u03b2-Lg structure in low-pH (\u223c2.5), very low ionic strength (<10 mM) solutions, where the protein is monomeric, were initiated (Ragona et al., 1997; Fogolari et al., 1998; Kuwata et al., 1998; Uhr\u00ednov\u00e1 et al., 1998). These NMR studies, described below, have provided proof of persistence of tertiary structure down to pH 2 and have yielded a depth of insight into structural stability and protein dynamics that is not possible by standard x-ray crystallographic techniques.\n\n### Structure of Bovine \u03b2-Lactoglobulin in Aqueous Solution\n\nNMR spectroscopy is used to obtain protein structures in solution (Cavanagh et al., 1995). The technique is best suited to monomeric proteins with molecular weights <\u223c25 kDa and usually requires recombinant singly (15N) or doubly (15N \/13C) labeled material for molecules with molecular weights >\u223c8 kDa. Therefore, most NMR studies of bovine \u03b2-Lg have been at a pH of between 2 and 3, and very importantly at very low ionic strength, where the molecule is monomeric. Early studies by Molinari's group using wild-type B-variant protein (Fogolari et al., 1998) confirmed the presence of the eight-stranded \u03b2-barrel. However, the full structure (of the A variant) (Kuwata et al., 1999; Uhr\u00ednov\u00e1 et al., 2000) required the use of isotopically labeled recombinant material (Kim et al., 1997; Denton et al., 1998). As the full structure was determined by NMR techniques independently and near-simultaneously by two groups from Tokyo and from Edinburgh and Palmerston North, this has provided objective and very useful comparisons (Jameson et al., 2002a).\n\nThe solution structure was shown by both Kuwata et al. (1999) and Uhr\u00ednov\u00e1 et al. (2000) to have an overall similarity to that established earlier by x-ray crystallography at pH 6.2, despite the considerably lower pH (and concomitant increase in the protein's surface charge) necessary to obtain usable NMR spectra from monomeric bovine \u03b2-Lg. The EF loop is closed over the open end of the \u03b2-barrel at this pH, and the side chain of the Glu89 'latch' is buried, as in the x-ray structures. The biggest difference, when compared with the Z lattice x-ray structure at pH 6.2 (Qin et al., 1998a), is that the three-turn \u03b1-helix adopts a different position with respect to the \u03b2-barrel, possibly because of the pH-induced increase in positive charge on this part of the protein's surface (Uhr\u00ednov\u00e1 et al., 2000). In addition, the loss of the dimer interface at low pH removes restraints on the position of the helices, which in the dimer are in proximity but not direct contact (see Fig. 7.1), and on the conformation of the AB loop, which moves by up to 3.5 \u00c5.\n\nFurther differences were found at the N- and C-termini, but these can be ascribed to limitations imposed by the use of recombinant protein with a non-native N terminus for the NMR structure and possible crystal packing effects at the C terminus for the x-ray structure. In some crystal forms, much of the C-terminus from residue \u223c152 to 162 is not observed or is very poorly defined in electron density maps.\n\n#### The Monomer\u2013Dimer Equilibrium of Bovine \u03b2-Lactoglobulin in Aqueous Solution\n\nThe monomer-dimer equilibrium of bovine \u03b2-Lg has been widely studied over a long period of time by a host of techniques, including analytical ultracentrifugation (AUC), light scattering, small-angle x-ray scattering (SAXS), and isothermal dilution calorimetry (for a comprehensive and critical tabulation, see Table S1 in Supporting Information in Mercadante et al., 2012). What had not been measured until recently were the rate constants associated with the monomer-dimer equilibrium, despite the importance of the dimer dissociation step as the first step in the denaturation of bovine \u03b2-Lg and in subsequent chemical processes. Analytical centrifugation measurements confirmed by earlier values for the dimer dissociation equilibrium were constant over the pH range 2.5 to 7.5 at an essentially constant ionic strength provided by 100 mM NaCl. The equilibrium constants and rate constants, where obtainable by AUC, are summarized in Table 7.2. At pH 2.5\u20133.5 and 6.5\u20137.5, the equilibrium dissociation constants K D are \u223c10 \u03bcM. At pH around the isoelectric point, K D decreases markedly and falls outside the range of standard AUC detection. The dissociation rate constant is quite slow (\u223c0.008 s\u22121), and the association rate constant is well removed from the diffusion-controlled limit. This was attributed (Mercadante et al., 2012) to the considerable restructuring of the cloud of counter-ions on dimerization: At low pH, where \u03b2-Lg is strongly positively charged, there is substantial restructuring of counter-anions, and at pH >7, where it is mildly negatively charged, there is a restructuring of counter-cations as well as a sharp distance dependence for optimization of hydrophobic contacts.\n\nTable 7.2\n\nEquilibriuma and Rateb Constants Calculated for \u03b2Lg A and \u03b2Lg B Dimer Dissociation over the pH Range 2.5 to 7.5 in 100 mM NaCl from Global Fitsc of SE and SV Data\n\n| K D2-1 (\/\u03bcM) | k off (\/s\u22121) | k on (calc.) (\/M\u22121 s\u22121)  \n---|---|---|---  \n| \u03b2Lg A | \u03b2Lg B | \u03b2Lg A | \u03b2Lg B | \u03b2Lg A | \u03b2Lg B  \npH 2.5d | 15 \u00b1 3 | 8 \u00b1 3 | 0.008 \u00b1 0.005 | 0.009 \u00b1 0.005 | 540 | 1,100  \npH 3.5d | 4.0 \u00b1 1.5 | 1.4 \u00b1 1.5 | [0.007]f | [0.041]f | \\---f | \\---f  \npH 6.5e | 4.0 \u00b1 1.5 | 2.5 \u00b1 1.5 | Fast (> 0.1) | Fast (> 0.1) | >25,000 | >40,000  \npH 7.5e | 11 \u00b1 3 | 9 \u00b1 2 | Fast (> 0.1) | Fast (> 0.1) | >9,200 | >12,000\n\na Calculated from global fitting of sedimentation velocity (SV) and sedimentation equilibrium (SE) data.\n\nb Calculated from global fitting of SV data (SE data contain no kinetic signal).\n\nc Calculated error ranges represent the sensitivity of the values to changes in other fitting parameters.\n\nd Citrate buffer.\n\ne 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.\n\nf Indicative value, as no error range could be determined; kon not calculated.\n\nReproduced from Mercadante, Melton et al., 2012.\n\nBello et al. (2011) also observed by isothermal dilution calorimetry that the dimer dissociation constant of variant B of \u03b2-Lg is smaller than that of variant A at temperatures up to 35 \u00b0C, consistent with analytical ultracentrifugation data (Mercadante et al., 2012). Indeed, the dissociation constant of 14.5(1) \u03bcM measured at pH 7.0 in 50 mM phosphate buffer and 100 mM NaCl is very similar to that determined by analytical ultracentrifugation measurements (Table 7.2).\n\n### Studies of Bovine \u03b2-Lactoglobulin by NMR at Neutral pH\n\nThe large size of the bovine \u03b2-Lg dimer at pH 7 is expected to cause some broadening of the peaks in its1H NMR spectrum due to slower molecular reorientation. However, this problem is exacerbated by chemical exchange broadening of peaks in the vicinity of the dimer interface by the dynamic equilibrium of molecules between the associated (dimeric) and unassociated (monomeric) states. These factors render the resulting spectra unsuitable for structure determination. Several methods have been employed to allow NMR studies at neutral pH. The most straightforward of these methods has been to use a nonruminant \u03b2-Lg that is intrinsically monomeric, yet with the same overall tertiary structure as the bovine protein, in this case equine \u03b2-Lg. (Kobayashi et al., 2000). Alternatively, the dimer interface may be disrupted by producing bovine \u03b2-Lg mutants with amino acid substitutions carefully chosen to disrupt the intermolecular interactions between either the I strands or the AB loops (Sakurai and Goto, 2002) (see Fig. 7.1).\n\nAn attempt to form dimeric equine \u03b2-Lg by producing a mutant with amino acid substitutions chosen to mimic those of the bovine protein at the interface was not successful (Kobayashi et al., 2002). This failure indicates that subtle features in \u03b2-Lg conformation remote from the interface have an impact on successful dimer formation. Indeed, reaction of the free thiol of Cys121 (located away from the interface in the H strand and covered by the main \u03b1-helix, Asp129\u2014Lys141; see Fig. 7.2) with 2-nitro-5-thiobenzoic acid produces a monomeric species with native structure at pH 2 and a monomeric but unfolded structure at pH 7 (Sakai et al., 2000). The configuration of the \u03b1-helix is known to change with pH (Uhr\u00ednov\u00e1 et al., 2000), and this may therefore also have an important influence on both the protein's stability and quaternary state.\n\nFigure 7.2 Ligand-binding sites on \u03b2-Lg as inferred from NMR measurements of binding of small (<12 atoms) ligands to \u03b2-Lg at acidic pH (Luebke et al., 2002). The binding site of 12-bromododecanoic acid is shown for reference (Qin et al., 1998a, b). (a) View into the calyx showing primary binding site of fatty acids at pH \u223c7 and of flavor components at \u223cpH 2, highlighted in yellow. (b) Secondary binding site for flavor components at pH 2 at N-terminal ends of strands A, B, C, and D, and the C-terminal strand, highlighted in pink. (c) Secondary binding site for flavor components at pH 2 adjacent to the three-turn helix and strand G, highlighted in cyan. To show more clearly the attachment of side chains to the main chain, loops and strands have not been smoothed. The pH-sensitive EF loop is colored in magenta. Figure drawn with PyMOL (Delano, 2002) using coordinates with PDB code 1bso.\n\nThe third approach to overcome the problems of the rate constants for the dissociation\/ reassociation equilibrium of the bovine \u03b2-Lg dimer being in the intermediate exchange regime has been to covalently bond two monomers via an Ala34Cys mutant (Sakurai and Goto, 2006). This variant was used to study the dynamics of the EF loop across the Tanford transition (see the following section) and to examine the nature of ligand binding to \u03b2-Lg (Konuma et al., 2007). Although no full structure determination was reported, the amide chemical shifts of the mutant were within 0.1 ppm of those from monomeric \u03b2-Lg (except for seven residues that encompassed the substitution site). This fact, combined with the similarity of the mutant and wild-type \u03b2-Lg CD spectra, indicated that the tertiary structures of the mutant and wild-type protein were similar.\n\n### Bovine \u03b2-Lactoglobulin Dynamics\n\nCrystallographic atomic displacement parameters, often loosely referred to as temperature factors or just B factors, describe the spread of an atom's electron density in space and can therefore be used to infer residue-specific mobility. However, for surface residues, the B factors of both main-chain and side-chain atoms are highly sensitive to intermolecular crystal-packing contacts. Moreover, except where data are available to ultra-high resolution (better than 1.0 \u00c5, which is not yet the case for any \u03b2-Lg), similarity restraints are imposed on B values of adjacent atoms and residues along the polypeptide chain to ensure stable refinement.\n\nNonetheless, in the case of isomorphous structures at similar resolution (where structures share the same average B value, the same space group, very similar unit cell parameters, and, hence, intermolecular contacts), or in regions where non-isomorphous structures lack intermolecular contacts, some meaning can be placed on differences observed in B values. These differences can be both within a particular structure or between structures determined, for example, at different pH or in the presence\/absence of added ligands. High B values, indicating apparent high mobility, can also arise from a distribution of slightly different yet immobile conformations or from errors in model building. For these reasons, it is advantageous to study dynamics of the protein (particularly those of the backbone) by NMR techniques, using uniformly15N-labeled protein.\n\nFlexibility on the nanosecond timescale can be inferred from low15N steady-state nuclear Overhauser effect values. As might be expected, mobile residues for \u03b2-Lg tend to have highly accessible surface areas (and such residues identified in NMR studies correlate in general with those that have relatively high B values; see Kuwata et al., 1999). Slower conformational exchange processes can be indicated by large values for the ratio of the T1 (spin-lattice) and T2 (spin-spin) relaxation times of15N nuclei. Such residues include Ser21 at the midpoint (kink) of the A strand, possibly caused by fluctuations of the barrel, and residues 61 and 66 at either end of the CD loop, consistent with a slow segmental or a hinging motion of this loop (Uhr\u00ednov\u00e1 et al., 2000).\n\nNMR measurements of the dynamics of the covalently bonded Ala34Cys dimer mutant have recently given complementary information regarding the structural changes associated with the Tanford transition established previously using x-ray crystallography (Qin et al., 1998a). The15N dynamics of the EF loop measuring either side of the transition indicates a three-step process. With increasing pH, the first event is a conformational change to the GH loop. This is followed first by the breaking of hydrogen bonds at the hinges of the EF loop and then by the articulation of the EF loop away from the calyx (Sakurai and Goto, 2006; Sakurai et al., 2009). The dynamic flexibility of the EF loop at pH \u223c2 is important because it means that at low pH neither ingress into nor egress from the hydrophobic pocket is kinetically prevented.\n\nRecent molecular dynamics simulations confirm what has been suspected from x-ray crystallographic studies that dimerization of bovine \u03b2-Lg is accompanied by an increase in molecular flexibility, an entropically favorable contribution to the free energy of dimer formation (Bello et al., 2011).\n\n### Structures of \u03b2-Lactoglobulins from Other Species\n\nEquine (horse) \u03b2-Lg, which shares 58% identity with bovine \u03b2-Lg, has been shown to be monomeric over a wide pH range (Kobayashi et al., 2000), whereas porcine (pig), which shares 63% identity with bovine \u03b2-Lg, is dimeric below pH 5 and monomeric at pH 5 and above (in contrast to bovine \u03b2-Lg) (Ugolini et al., 2001). At pH 7 both equine and porcine \u03b2-Lg are monomeric and therefore amenable to NMR study. Equine \u03b2-Lg has been extensively studied by NMR with regard to denaturation processes, especially at low pH (<4) where it assumes a molten globule conformation, (Yamamoto et al., 2011), but full structural characterization by either NMR or x-ray methods has yet to be published. However, the x-ray crystal structure at 2.0-\u00c5 resolution and at near-neutral pH of Gyuba \u03b2-Lg, a chimeric protein formed from bovine \u03b2-Lg secondary structure elements and equine \u03b2-Lg loops, revealed a bovine-like dimeric structure (Ohtomo et al., 2011). This is in contrast to an earlier chimera where replacing just the I strand and AB loop of equine \u03b2-Lg by the dimer-forming I-strand and AB loop of bovine \u03b2-Lg failed to elicit dimerization of the chimera (Kobayashi et al., 2002).\n\nThe x-ray crystal structure of porcine \u03b2-Lg at pH 3.2 clearly revealed a dimeric structure formed by domain swapping of N-terminal regions, a quaternary structure quite different from that observed for bovine \u03b2-Lg (Hoedemaeker et al., 2002). The EF loop adopts the closed conformation over the calyx, as found also for bovine \u03b2-Lg at acidic pH, consistent with the notion that this loop acts as a lid to the calyx. However, the porcine protein is much less conformationally stable at acidic pH than its bovine counterpart (Burova et al., 2002; Invernizzi et al., 2006), which has led to questioning of the role of \u03b2-Lg as a transporter of hydrophobic molecules through the acidic environment of the stomach (Burova et al., 2002). Despite 63% identity between bovine and porcine \u03b2-Lg, the RMS difference in C\u03b1 positions between these two structures is remarkably high at 2.8 \u00c5, although inspection of the two structures shows that the core \u03b2-barrel structure superimposes closely and that these differences are concentrated in the flexible loop regions, which comprise nearly a third of the structure.\n\nThe structure of rangiferine (reindeer) \u03b2-Lg at pH \u223c6.5 has been determined to 2.1 \u00c5 resolution (Oksanen et al., 2006). Both the monomeric tertiary structure and the dimeric quaternary structure are very similar to those of bovine \u03b2-Lg, which is not unexpected as polypeptide lengths are identical and sequence identity is greater than 94%. At pH \u223c6.5, the EF loop was observed to be in the closed position. There are few structural data on ovine (sheep) and caprine (goat) \u03b2-Lg, despite the commercial importance of their milk.\n\nAlthough \u03b2-Lg is absent from human milk, humans secrete two other lipocalins that share limited sequence identity (but close structural similarity) with bovine \u03b2-Lg. Tear lipocalin is the major protein in tears, and structural and functional studies indicate that it, like \u03b2-Lg, binds a broad range of hydrophobic molecules (Glasgow et al., 1995). Glycodelin, a heavily glycosylated lipocalin, is found in the human endometrium in early pregnancy where its function remains unclear. Kontopidis et al. (2004) have suggested that \u03b2-Lg arose by gene duplication of glycodelin and exists solely for nutritive value in milk. However, there appears to have been considerable selection pressure to retain not only the Glu89 in all sequences of \u03b2-Lgs found to date but also the pH-sensitive conformational switch for the EF loop of all \u03b2-Lgs (and indeed for tear lipocalin) that have been functionally characterized. Such preservation is not consistent with a solely nutritive role for \u03b2-Lg, although the origin of the \u03b2-Lg gene from glycodelin remains an intriguing possibility.\n\n### Ligand Binding to \u03b2-Lactoglobulin\n\n\u03b2-Lg has the ability to bind a large number of small molecules (for a review see Sawyer et al., 1998), although the location of the bound ligand continues to generate controversy (Dufour et al., 1994; Lange et al., 1998; Narayan and Berliner, 1998; Yang et al., 2008). However, in recent years, NMR techniques have been used to map possible ligand-binding sites on the protein through changes in chemical shift and relaxation times of protein residues.\n\nAnother technique that provides reliable evidence of binding to the protein is induced circular dichroism, where an achiral chromophore 'lights up' in the CD spectrum on being placed into a chiral environment on or in the protein. Creamer et al. (2000) applied this technique to bovine \u03b2-Lg to show unequivocally that retinol and fatty-acid binding (e.g., palmitic and cis-parinaric acids) is competitive. The binding of these and other chirally active ligands, including trans-parinaric acid and retinoic acid, were explored over a range of pH and with nonchromophoric fatty acid ligands to gain an understanding of the parameters surrounding the Tanford transition. More recently Zsila et al. have used induced circular dichroism to study binding of these (Zsila et al., 2002) and other ligands (Zsila, 2003; Zsila et al., 2005), including piperidine, to \u03b2-Lg and other lipocalins.\n\nLigand binding has also been extensively studied by fluorescence spectroscopy, in which, typically, the fluorescence from tryptophan residues is monitored for changes, which may be positive or negative. For bovine \u03b2-Lg, one tryptophan (Trp19) is buried and is part of the highly conserved lipocalin Gly-X-Trp motif at the beginning of strand A (see Fig. 7.2b), whereas the other (Trp61) is largely exposed and is part of the mobile CD loop. Fluorescence by both tryptophans is sensitive to small changes in positions of nearby quenchers, respectively, a nearby charged side-chain of Arg124 and the Cys66-Cys160 disulfide bond. NMR spectroscopy, induced CD, and x-ray results clearly indicate that interpretation of fluorescence measurements for evidence of ligand binding poses hazards.\n\nRagona et al. (2003) used a combination of electrostatic calculations, docking simulations, and NMR measurements to suggest that the pH-dependent conformational change of the EF loop triggered by the protonation of Glu89 is common to all \u03b2-Lgs and that ligand binding (of palmitic acid) is determined by the opening of this loop. In earlier work using13C-labeled palmitic acid, this group had shown that the ligand also undergoes conformational change with increasing pH (Ragona et al., 2000).\n\nRecent NMR studies of the binding of palmitic acid to the 'NMR friendly' Ala34Cys mutant dimer of bovine \u03b2-Lg have indicated that, although a rigid connection is made by the protein with the ligand at the bottom of the calyx, the interaction at the open end of the calyx is more dynamic (Konuma et al., 2007). These observations complement those of Ragona et al. (2003) and also the x-ray studies on binding of fatty acids (Qin et al., 1998b; Wu et al., 1999), which showed the carboxylate head group to be substantially less well ordered than the hydrophobic tail. The results of the study with the Ala34Cys mutant suggest that it is the plasticity of the D strand and the EF and GH loops that allows \u03b2-Lg to accommodate such a wide range of ligands (Konuma et al., 2007). With the exception of changes in conformations of the side chains of Phe105 and Met107, NMR and x-ray studies show that the core lipocalin structure remains invariant upon ligand binding.\n\nCrystallographic data clearly show that both fatty acids and retinoids bind in the calyx of bovine \u03b2-Lg (Qin et al., 1998b; Wu et al., 1999; Sawyer et al., 1998; Kontopidis et al., 2002). Recently, complexes of bovine \u03b2-Lg with a host of different ligands have been crystallographically characterized: dodecyl sulfate (separately bovine variants A and B) (Guti\u00e9rrez-Magdaleno et al., 2013; Loch et al., 2013), unsaturated C18 fatty acids, oleic acid, and linoleic acid (Loch et al., 2013a), saturated fatty acids, lauric (separately bovine variants A and B), myristic, stearic, palmitic, capric, caprylic and acid (Loch et al., 2011; ), trialkyl ammonium species, dodecylammonium (separately bovine variants A and B) (Loch et al., 2013b), and an alkyl-tailed rhodium(III) organometallic complex (Cherrier et al., 2013).\n\nAs a crystallization artifact, n-dodecyl-\u03b2-D-maltoside was found in the canonical binding pocket of a complex of bovine \u03b2-Lg with the D1\/Fab fragment of human IgE (immunoglobulin type E) (Niemi et al., 2008).\n\nAlthough all crystallographic studies of ligand binding have been under conditions of high ionic strength, congruence of these data with NMR and induced CD data collected under conditions of low ionic strength indicate that the x-ray results are not an artifact of ionic strength. The preservation of structure of \u03b2-Lg, in particular the hydrophobic cavity at conditions of near-zero ionic strength at the pI of the protein (\u223c5.3) (Adams et al., 2006) further demonstrates that the primary, and possibly only, ligand-binding site at \u223cpH 7 is inside the calyx.\n\nThe structural studies of Loch et al. (2011; 2012), and Guti\u00e9rrez-Magdaleno et al. (2013) have been complemented by measurements of the thermodynamics of ligand binding by isothermal titration calorimetry (see Table 7.3), with several key provisos: The structural studies were on crystals grown at very high ionic strength (1.34 M sodium citrate at pH 7.5), while ligand-binding thermodynamics were conducted at much lower ionic strength (\u223c50 mM Tris-HCl\/5 mM KOH) and where \u223c0.2 mM protein solution is typically titrated into \u223c20 \u03bcM ligand solutions at initial protein concentrations where the \u03b2-Lg will dissociate from dimer into monomer (see Table 7.2).\n\nTable 7.3\n\nBinding of C8 to C18 Species Bovine \u03b2-Lg\n\n| K(assoc)  \n\/M\u22121 x 105 | \u0394 G   \n\/kJ mol\u22121 | \u0394 H   \n\/kJ mol\u22121 | T \u0394 S   \n\/kJ mol\u22121  \n---|---|---|---|---  \nCaprylate (C8:0)a | 0.107(17) |  |  |   \nCaprate (C10:0)a | 0.060(5) |  |  |   \nLaurate (C12:0)b |  |  |  |   \n\u03b2-Lg A | 1.88 \u00b1 0.08 | \u221231.2 \u00b1 0.8 | 19.2 \u00b1 0.3 | 50.5 \u00b1 0.8  \n\u03b2-Lg B | 3.70 \u00b1 0.02 | \u221232.8 \u00b1 0.7 | 6.5 \u00b1 0.2 | 39. \u00b1 1  \nLauryl sulfateb |  |  |  |   \n\u03b2-Lg A | 1.07 \u00b1 0.02 | \u221229.2 \u00b1 0.5 | \u221210.54 \u00b10.08 | 18.7 \u00b1 0.4  \n\u03b2-Lg B | 5.79 \u00b1 0.06 | \u221266.5 \u00b1 0.7 | \u221217.54 \u00b10.04 | 15.94 \u00b1 0.04  \nLaurate (C12:0)c | 1.72 \u00b1 0.15 | \u221229.9 | \u221223.2 \u00b11.2 | 6.7  \nMyristate (C14:0)c | 7.8 \u00b1 1.6 | \u221233.5 | \u221218.7 \u00b1 0.9 | 14.9  \nPalmitate (C16:0)c | 20. \u00b1 4 | \u221236. | \u221211.6 \u00b1 0.3 | 82.1  \nOleate (C18:1) | 10 \u00b1 3 | \u221234.0 \u00b1 0.1 | \u221215 \u00b1 4 | 19 \u00b1 4  \nLinoleate (C18:2) | 9.0 \u00b1 0.8 | \u221233.2 \u00b1 0.1 | \u22128.8 \u00b1 1.9 | 25 \u00b1 2\n\na Loch et al. (2011). Fluorescence measurements on 20 \u03bcM \u03b2-Lg variant B in 50 mM Tris, pH 7.5 at room temperature.\n\nb Bello et al. (2011). Measurements performed at pH 7.0 in a 50 mM phosphate buffer, 100 mM NaCl, at 35 and 30 \u00b0C for laurate and dodecyl sulfate, respectively. Ligand (30 mM) titrated into \u03b2-Lg (\u223c13 \u03bcM as monomer). Values converted from reported values in kcal mol\u22121.\n\nc Loch et al. (2012). Measurements performed at pH 7.5 in a 50 mM Tris-HCl\/5 mM KOH buffer at 25 \u00b0C. Laurate (2 mM) and myristate (0.6 mM) were titrated into 50\u2013100 \u03bcM \u03b2-Lg (variant B). Palmitate (20 \u03bcM) was titrated into 0.2 mM \u03b2-Lg (variant B). Stearate (C18:0) gave a very small enthalpy of binding, and so its binding is not accessible by calorimetric methods. Heats of dilution were measured by titration beyond saturation of \u03b2-Lg by ligand, and so may be affected by enthalpy of dissociation of the \u03b2-Lg dimer.\n\nd Loch et al. (2013a). Measurements performed at pH 7.5 in a 50 mM Tris-HCl buffer at 25 \u00b0C. Bovine \u03b2-Lg (variant B) (0.25\u20130.40 nM) titrated into oleic acid (20 \u03bcM) or linoleic acid (25\u201330 \u03bcM). Heats of dilution were measured by titration beyond saturation of \u03b2-Lg by ligand.\n\nIsothermal titration calorimetric methods, when properly conducted, give not only binding equilibrium constants and stoichiometry of binding, but also the enthalpy of binding and, after conversion of equilibrium constant to Gibbs free energy, the entropy of binding.\n\nThat ligand binding is a complex process is revealed by the observation that binding of laurate (dodecanoate) and lauryl sulfate (dodecyl) to bovine \u03b2-Lg (variant A) at pH 7.5 is endothermic in phosphate buffer (proton ionization enthalpy of 3.6 kJ mol\u22121) and exothermic in Tris buffer (proton ionization enthalpy of 47.5 kJ mol\u22121) (Loch et al., 2013b). Furthermore, ligand binding may be accompanied by dissociation of the dimer (Bello et al., 2011; Guti\u00e9rrez-Magdaleno et al., 2013). The ligand-binding enthalpy with proton transfer effects removed (\u0394 H dissoc,b) is given by Baker and Murphy (1996)\n\n\u0394  H dissoc,obs = \u0394  n H * \u0394  H ion + \u0394  H dissoc,b\n\nwhere \u0394 H ion have been measured for a number of common buffers (Goldberg et al., 2002) and \u0394 n H is the number of protons transferred in ligand binding (for \u03b2-Lg and fatty acid ligands \u0394 n H \u223c0.25\u22120.6; see Loch et al., 2013a,b).\n\nLoch et al. (2011; ; 2013a,b) systematically determined ligand:\u03b2-Lg binding stoichiometries to be considerably less than the expected value of unity. Errors in measurement of \u03b2-Lg concentrations are probably the cause, since Bello et al. (2011), in work remarkably paralleled by that of on the binding of dodecyl sulfate and laurate (dodecanoate) to \u03b2-Lg variants A and B, report stoichiometries that are not significantly different from 1:1. Affinities for fatty acids and other ligands are consistently smaller (by more than an order of magnitude) when measured by isothermal titration calorimetry than by fluorescence methods.\n\nRemarkably, enthalpic and entropic contributions to the free energy of binding dodecanoate, dodecyl sulfate, and dodecyltrimethyl ammonium differ between variant A and B. Moreover, whereas binding of dodecanoate is primarily entropically driven, that of dodecyl sulfate has both enthalpic and entropic contributions to binding to \u03b2-Lg (Bello et al., 2011). Bello et al. (2012) report significantly stronger binding of dodecanoate and dodecyl sulfate to variant B than to variant A, in contrast to who report the opposite, but in the latter case the differences in affinity are not statistically significant.\n\nMeasurements of ligand-binding affinity, especially of amphipathic ligands such as fatty acids, by spectroscopic means are often compromised by light scattering from ligand micelles unless care is taken to reduce the ligand concentration below the critical micelle limit. The fatty acid ligand is typically dissolved first in ethanol before diluting into buffer, which leaves a residual \u223c0.1% trace in the ligand-protein mixture.\n\nVitamin D3 was reported to bind in half occupancy not only at the canonical site but also at an external site in the groove between the subunits comprising the canonical dimer of bovine \u03b2-Lg (Yang et al., 2008). Quite apart from unconvincing discontinuous electron density for the vitamin D3 in this external site (reconstructed from coordinates and structure factors deposited in the protein data bank with PDB Code 2gj5) (Berman et al., 2000), there are a host of uncomfortably close contacts involving the hydrophobic parts of vitamin D3 with polar (Asp137_O, Ser150_OG) or charged (Arg148) groups and the hydroxyl moiety eschews close contacts with polar residues. Although a number of hydrophobic contacts are reported, most of the residues proposed as being involved in hydrophobic contacts, Phe136, Ala139, Ile147, Ala142, Ile143, and Pro144, have their side chains more than 5.4 \u00c5 distant from the putative vitamin D3. Arg148 is noted as buried, but is only buried by the putative vitamin D3; otherwise it is well exposed to solvent. In brief, there remains no unequivocal crystallographic evidence for the binding of (largely) hydrophobic molecules to an external site on bovine \u03b2-Lg.\n\nAlthough ligands such as palmitic acid appear to be released at acid pH (Ragona et al., 2000), NMR evidence (based on perturbations of backbone chemical shifts) for the binding of the flavor compounds \u03b3-decalactone and \u03b2-ionone at pH 2 has been reported (Luebke et al., 2002; Tromelin and Guichard, 2006). There is, then, evidence for three binding sites to \u03b2-Lg: the canonical site inside the calyx, a second site involving perturbation of residues Trp19, Tyr20, Tyr42, Glu44, Gln59, Gln68, Leu156, Glu157, Glu158, and His161, and a third site involving perturbation of residues Tyr102, Leu104, and Asp129. These regions are illustrated in Figure 7.2.\n\nInitially it was thought that porcine \u03b2-Lg did not bind fatty acids (Frapin et al., 1993). However, NMR studies have shown that the pH for 50% uptake of ligand has shifted by nearly 4 pH units from \u223c5.8 for bovine \u03b2-Lg to 9.7 for porcine \u03b2-Lg, whereupon the EF loop undergoes a structural change analogous to that of its bovine counterpart (Ragona et al., 2003). Indeed, this loop with its absolutely key Glu residue (Glu89 in bovine \u03b2-Lg) is predicted to be pH-gated for all \u03b2-Lgs (Ragona et al., 2003).\n\n### Effect of Temperature on Bovine \u03b2-Lactoglobulin\n\nThe thermal properties of \u03b2-Lg variants are of considerable commercial relevance due to their role in the fouling of processing equipment as well as the functional qualities that can be imparted to dairy products by thermally induced \u03b2-Lg aggregation. Consequently, this aspect of the protein's behavior has been the focus of extensive experimental work.\n\nAt neutral pH the midpoint of the thermal unfolding transitions, as determined by differential scanning calorimetry (DSC), is \u223c70 \u00b0C (de Wit and Swinkels, 1980) whereupon the protein dimer dissociates and the constituent molecules begin to unfold. This reveals the free thiol of Cys121 (located at the C-terminal end of the H strand; see Fig. 7.1) and a patch of hydrophobic residues, leading to the possibility of both covalent and hydrophobic intermolecular association (Qi et al., 1995; Iametti et al., 1996). The ensuing disulfide interchange reactions lead to the formation of a variety of mixed disulfide-bonded polymeric species (Creamer et al., 2004). Genetically engineered mutants with an extra cysteine positioned to allow a third disulfide bond to be formed to Cys121 have been shown to both retard thermal denaturation by 8\u201310 \u00b0C and to resist heat-induced aggregation (Cho et al., 1994). In mixtures of bovine \u03b2-Lg, \u03b1-La, and BSA, or of \u03b2-Lg and one or other of \u03b1-La and BSA at pH 6.8 subjected to high temperatures, homo- and heteropolymeric disulfide-bridged species were observed (Havea et al., 2001). The formation of \u03b1-La \u2212 \u03b1-La disulfide links (\u03b1-La has no free cysteine; see below) is attributed to catalysis by BSA or \u03b2-Lg (Havea et al., 2001). At low pH where the protein is monomeric, denaturation is largely reversible at temperatures below 70 \u00b0C (Pace and Tanford, 1968; Alexander and Pace, 1971; Mills, 1976; Edwards et al., 2002). Heating above this temperature leads to the formation of large aggregates, but in contrast to the behavior at neutral pH, the species are predominantly noncovalently bonded (Schokker et al., 2000).\n\nThe precise denaturation process is complex and is influenced by factors such as pH, protein concentration, ionic environment, genetic variant, and presence of ligands. Lowering the pH (Kella and Kinsella 1988; Relkin et al. 1992), or adding calyx-bound ligands (Puyol et al., 1994; Considine et al., 2005a; Busti et al., 2006) can make the protein more resistant to thermal unfolding. The stability of the genetic variants (at pH 6.7) appears to decrease in the order C > A > B, with the A variant showing the least cooperative unfolding transition (Manderson et al., 1997). The protein's susceptibility to thermal denaturation at pH 6.7\u20138 is strongly concentration dependent up to about 6 mM, being most susceptible to unfolding at a concentration of \u223c1.4 mM (Qi et al., 1995). It is possible that at high protein concentration (\u223c6 mM) tertiary structure is lost directly from the native dimer state (Qi et al., 1995).\n\nThere is some evidence that the thermal unfolding occurs in more than one step. Kaminogawa et al. (1989) used antibody binding affinities to propose that thermal unfolding of variant A of \u03b2-Lg occurs in at least two stages, starting with conformational changes near the N terminus followed by changes in the region of the 3-turn \u03b1-helix. FTIR measurements by Casal et al. have also indicated a loss of helical content early in the denaturation process (using variant B of \u03b2-Lg in 50 mM phosphate buffer at pH 7) (Casal et al., 1988). Qi et al. used FTIR and CD measurements to propose that variant A of \u03b2-Lg forms a molten globule with reduced \u03b2 structure when heated above 65 \u00b0C in 30\u201360 mM NaCl at pH 6.5 (Qi et al., 1997). NMR studies, observing hydrogen\/deuterium (H-D) exchange of the backbone amide protons of \u03b2-Lg A at pH 2\u20133, have revealed a stable core comprising the FG and H strands, possibly stabilized by the Cys106\u2013Cys119 disulfide bond between strands F and G (Belloque and Smith, 1998; Edwards et al., 2002). The observation of significant secondary structure even at a temperature as high as 90 \u00b0C has been reported (Casal et al., 1988; Qi et al., 1997; Bhattacharjee et al., 2005).\n\n### Effect of Pressure on Bovine \u03b2-Lactoglobulin\n\nHigh-pressure treatment of food is of increasing commercial importance due to increasing consumer demand for products that have been subjected to minimal processing damage. Pressure treatment as part of the processing regime has greater potential to produce dairy products with improved functional and organoleptic properties than those produced by thermal treatment alone (Messens et al., 2003).\n\nOf the major whey proteins \u03b2-Lg is the most susceptible to pressure-induced change (Stapelfeldt et al., 1996; Patel et al., 2005). Presumably this is due to its relatively inefficient packing caused by the presence of the \u03b2-barrel, with its large solvent-exposed hydrophobic pocket and the lower number of disulfide bonds (two compared to four in, for example, the similar-sized \u03b1-lactalbumin). A reduction in the molar volume of bovine \u03b2-Lg has been detected at pressures as low as 10 MPa, possibly due to a contraction of the calyx (Vant et al., 2002). A number of studies have shown that \u03b2-Lg becomes more susceptible to enzymatic cleavage when exposed to pressure, possibly due to pressure-induced conformational change. The free cysteine has been shown to become exposed at between 50 and 100 MPa (Stapelfeldt et al., 1996; Tanaka and Kunugi,1996; M\u00f8ller et al., 1998). Exposure of the protein to pressures in excess of \u223c300 MPa causes irreversible changes to \u03b2-Lg tertiary and quaternary structure. A combination of CD and fluorescence spectroscopy of \u03b2-Lg at neutral pH exposed to pressures as high as 900 MPa indicated that pressure induces monomer formation with subsequent aggregation, but with only small irreversible effects on \u03b2-Lg tertiary structure (Iametti et al., 1997). However, more recent results from tryptic hydrolysis suggest that while exposure to pressures below 150 MPa has no detectable permanent effect on \u03b2-Lg A's conformation, pressures above 300 MPa lead to the detachment of strands D and G from the \u03b2-barrel together with the formation of disulfide-bonded oligomers (Knudsen et al., 2002).\n\nIn mixtures of bovine \u03b2-Lg with either \u03b1-La or BSA at pH 6.6 subjected to high pressures, intermolecular disulfide-bridged aggregates form only between \u03b2-Lg and itself. No \u03b2-Lg \u2212 \u03b1-La or \u03b2-Lg \u2212 BSA disulfide-bridged species are detected (Patel et al., 2005), in contrast to heat-treated mixtures where such species are observed (Havea et al., 2001).\n\nIn order to correlate the pressure-induced conformational changes with the protein's primary sequence, Belloque et al. have made NMR amide H-D exchange observations of \u03b2-Lg A and B following exposure of solutions at neutral pH to pressures of up to 400 MPa (Belloque et al., 2000). Little H-D exchange was reported at 100 MPa, which indicates that any conformational change that occurs is not increasing exposure of most amide protons to the solvent compared to their exposure in the native conformation at ambient pressure. A large increase in the extent of H-D substitution at 200 MPa and above indicated increased conformational flexibility, but the similarity of the spectra of control samples recorded in H2O rather than D2O before and after pressurization demonstrated that any pressure-induced conformational changes were largely reversible up to 400 MPa. The authors proposed that the A variant's structure was more sensitive to changes in pressure than that of the B variant and that the F, G, and H strands of the protein's \u03b2-barrel were the most resistant to conformational change, the latter conclusion paralleling the effects of temperature (Belloque and Smith 1998; Edwards et al., 2002).\n\nFTIR and SAXS experiments suggest that even at 1 GPa the unfolded state contains significant secondary structure (Panick et al., 1999). Combined application of pressure and heat has shown that changing the temperature over the range 5 to 37 \u00b0C has a negligible effect on the susceptibility of \u03b2-Lg to pressures up to 200 MPa (Skibsted et al., 2007). However, combined application of pressure and moderate temperature at 600 MPa\/50 \u00b0C (Yang et al., 2001) and 294 MPa\/ 62 \u00b0C (Aouzelleg et al., 2004) has indicated the formation of a molten globule with an \u03b1-helical structure on the basis of results obtained from CD spectroscopy.\n\nIt should be noted, therefore, that the potential for temperature increases induced by rapid pressurization of the sample needs to be considered when studying the effects of pressure on protein conformation and stability.\n\nEnzymatic proteolysis observations indicate that \u03b2-Lg is less susceptible to pressure- induced change at acidic pH (Dufour et al., 1995) than at neutral or basic pH, but this result may be confounded by pressure-induced changes in the proteinases, thermolysin and pepsin. Nevertheless, NMR measurements of monomeric \u03b2-Lg at pH 2 while under pressure of up to 200 MPa, have shown that the two \u03b2 sheets unfold independently to form two intermediates to an unfolded state that still appears to contain significant secondary structure (Kuwata et al., 2001).\n\nA three-step mechanism has been proposed for \u03b2-Lg denaturation at neutral pH and ambient temperature, which broadly encompasses the above observations: A pressure of 50 MPa causes partial collapse of the calyx (with concomitant reduction in ligand-binding capacity) together with exposure of Cys121. Increasing the pressure to 200 MPa causes further (partially reversible) disruption to the hydrophobic structure, together with a decrease in the molecular volume. Higher pressures cause irreversible aggregation reactions involving disulfide interchange reactions (Stapelfeldt and Skibsted, 1999; Considine et al., 2005b).\n\n### Effect of Chemical Denaturants on Bovine \u03b2-Lactoglobulin\n\nChemical denaturants are often used to unfold proteins and to characterize mechanisms and transition states of protein-folding processes. Commonly used denaturants include alcohols, particularly 2,2,2-trifluoroethanol (TFE), urea, and guanidinium chloride (GdmCl).\n\nTheoretical calculations predict a significantly higher amount of \u03b1-helical secondary structure than is actually observed in native \u03b2-Lg (Creamer et al., 1983; Nishikawa and Noguchi, 1991). That is, the native structure is the result of competition between \u03b1-helix favoring local interactions and \u03b2-sheet forming long-range interactions. However, addition of alcohols such as TFE can disturb this balance by weakening the hydrophobic interactions and strengthening the helical propensity of the peptide chain (Thomas and Dill, 1993). The ability to increase the \u03b1-helical content of bovine \u03b2-Lg by the addition of alcohols (ethanol, 1-propanol, 2-chloroethanol) was first demonstrated by Tanford using optical rotary dispersion measurements (Tanford et al., 1960). Contemporary studies tend to favor the use of TFE, where the \u03b2-Lg \u03b2-sheet-to-\u03b1-helix transition has been shown to be highly cooperative, occurring over the range \u223c15\u201320% v\/v of cosolvent (Shiraki et al., 1995; Hamada and Goto, 1997; Kuwata et al., 1998). The higher proportion of \u03b1-helical structures in the so-called TFE-state is found in the N-terminal half of the molecule (Kuwata et al., 1998). Magnetic relaxation dispersion measurements of the solvent nuclei have shown that this state is an open, solvent-permeated structure (unlike the collapsed state of a molten globule), and its formation is accompanied by a progressive swelling of the protein with increasing TFE concentration (Kumar et al., 2003). High-protein and TFE concentration (8% v\/w and 50% v\/v, respectively) can lead to fibrillar aggregation (see below) and gel formation of bovine \u03b2-Lg at both acid and neutral pH (Gosal et al., 2002).\n\nThe ability of urea to induce protein unfolding is thought to be via a combination of hydrogen-bond formation with the protein backbone and a reduction in the magnitude of the hydrophobic effect (Bennion and Daggett, 2003). Therefore, in contrast to TFE, both the helical propensity and the hydrophobic effect are reduced. Urea-induced unfolding of bovine \u03b2-Lg at acidic pH was first reported as a two-state process (Pace and Tanford, 1968). Subsequent NMR H-D exchange measurements of bovine \u03b2-Lg B at pH 2.1 also allowed the urea-induced unfolding to be well approximated as a two-state transition between folded protein and unfolded state via a cooperative unfolding of the \u03b2-barrel and the C-terminus of the major \u03b1-helix (Ragona et al., 1999). However, Dar et al. have provided evidence that urea also causes unfolding via an intermediate, albeit with structural properties between those of the native and unfolded states (Dar et al., 2007). Addition of anionic amphiphiles, sodium dodecyl sulfate (SDS), or palmitate, causes \u03b2-Lg to resist urea-induced unfolding due to binding inside the calyx (Creamer, 1995).\n\nGuanidinium chloride is often used as an alternative to urea in studies of protein stability. At the neutral or acidic pH of most stability studies, GdmCl will be fully dissociated. At low GdmCl concentration (<\u223c1 M), chloride ions screen the electrostatic repulsion between positively charged groups of the protein (Hagihara et al., 1993). The result is that the additional electrostatic interactions of GdmCl compared to the neutral urea molecule GdmCl have the potential to both stabilize and destabilize protein structure depending on the concentration of GdmCl (Hagihara et al., 1993). D'Alfonso et al. have compared denaturation of bovine \u03b2-Lg B with both GdmCl and urea between pH 2 and pH 8, as monitored by CD, UV differential absorption, and fluorescence measurements (D'Alfonso et al., 2002). Discrepancies between unfolding free energies obtained using the two denaturants could be reconciled if GdmCl denaturation was assumed to occur via an intermediate state. The secondary structure of this state is similar to that of the native protein, but with greater rigidity in the vicinity of the Trp residues consistent with the screening of electrostatic repulsion between charged residues (D'Alfonso et al., 2002). The GdmCl-induced unfolding intermediate of bovine \u03b2-Lg A at pH 2 has been reported to have increased \u03b1-helical structure (Dar et al., 2007).\n\nPorcine \u03b2-Lg has also been shown to unfold via an intermediate state on addition of GdmCl. The stability of the porcine protein was lower than that of its bovine counterpart, and the intermediate state was richer in \u03b1-helical structure. Most of the hydrophobic\u2013hydrophobic interactions of the buried core of the native state are conserved between bovine and porcine \u03b2-Lg. However, four pairwise interactions of the Phe105 side chain of bovine \u03b2-Lg are lost on the change to Leu in the porcine protein. This indicates that the presence of the aromatic residue may play an important role in the increased stability of the bovine protein (D'Alfonso et al. 2004). This residue is particularly resistant to H-D exchange in heated \u03b2-Lg solutions (Edwards et al., 2002).\n\n### Fibrillar Formation from Bovine \u03b2-Lactoglobulin\n\nAt high temperatures (>80 \u00b0C), low pH (typically pH \u223c2) and low ionic strength (<\u223c 20 mM), \u03b2-Lg self-assembles into fibrils (Aymard et al., 1999; Gosal et al., 2002; Arnaudov et al., 2003). These structures have some characteristics of classic \u03b2-amyloid fibrils, such as binding the fluorophores thioflavin-T and Congo red and showing x-ray diffraction consistent with a cross-\u03b2-sheet structure (Bromley et al., 2005). However, they differ substantially in that they are easily dissociated (Akkermans et al., 2008; Jordens et al., 2011). The fibrils have contour lengths in the order of \u03bcm (Aymard et al., 1999) and diameters in the range 4\u20138 nm (Gosal et al., 2004).\n\nResults from the combined use of AFM, PAGE, and in-situ FTIR suggest that the formation of heat-induced \u03b2-Lg fibrils involves its partial unfolding, self-hydrolysis, and self-assembly of some of the resulting species (Oboroceanu et al., 2010). It has been proposed that each fibril is typically composed of several protofilaments that grow to a length of \u223c0.5\u20131 \u03bcm before aligning and twisting together (Adamcik et al., 2010; Bolisetty et al., 2011; Adamcik and Mezzenga, 2012). Hydrolysis of \u03b2-Lg appears to play an important role in fibril formation (Akkermans et al., 2008), but it is likely to also be responsible for their disintegration if the temperature is raised to 120 \u00b0C for extended periods (Loveday et al., 2012).\n\nFibrils have been shown to contain peptide fragments in the range of 2\u20138 kDa, with sequences weighted toward \u03b2-Lg's termini (Akkermans et al., 2008), and those toward the N terminus being the more abundant (Dave et al., 2013). 2-D gel electrophoresis indicates the presence of disulfide-linked fragments, possibly involving the Cys66-Cys160 linkage found in the native protein (Dave et al., 2013). Recently, irradiation with microwaves followed by sample storage has been shown to be a particularly efficient method of fibril formation. Notably, the resulting fibrils contain not only peptides, but sequences consistent with intact \u03b2-Lg monomers (Hettiarachchi et al., 2012).\n\nAlthough fibril formation in food products has yet to be utilized, there is potential for their application as functional ingredients to control such properties as viscosity, gelation propensity, and emulsion and foam stability (Kroes-Nijboer et al., 2012; Nicolai and Durand, 2013).\n\n## \u03b1-Lactalbumin\n\n\u03b1-Lactalbumin (\u03b1-La) is a 123 amino acid, 14.2 kDa globular protein found in the milk of all mammals. The bovine protein binds Ca2+ with the holo form, being the more abundant form in milk. Within the Golgi apparatus of the mammary epithelial cell, \u03b1-La is the regulatory component of the lactose synthase complex (in which it combines with N-acetyl galactosamine synthase, now named \u03b2-1,4-galactosyltransferase-I); its role is to transfer galactose from UDP-galactose to glucose (Brew, 2003). The structure of human \u03b1-La in a 1:1 complex with \u03b2-1,4-galactosyltransferase-I has been determined (Ramakrishnan and Qasba, 2001; Ramakrishnan et al., 2001; Ramakrishnan et al., 2006), and a 2:1 structure has recently become available (Ramakrishnan, 2013, unpublished data).\n\nIn the absence of \u03b1-La and in the presence of a transition metal ion such as manganese(II), the catalytic domain of bovine \u03b2-1,4-galactosyltransferase-1 (residues 130\u2013402) transfers galactose (Gal) to N-acetylglucosamine (GlcNAc), which may be either free or linked to an oligosaccharide, generating a disaccharide unit, Gal-\u03b2-1,4-GlcNAc (N-acetyllactosamine). The calcium-ion binding site is remote from the active site of the \u03b1-La \u2212 \u03b2-1,4-galactosyltransferase-I complex (Brew, 2003). \u03b1-Lactalbumin has been studied extensively, largely due to its formation of a molten globule state under mild denaturing conditions (Dolgikh et al., 1981).\n\n### Molecular Structure of Bovine \u03b1-Lactalbumin\n\nThe tertiary structure of bovine \u03b1-La is typical of that of the protein from other mammalian species (Acharya et al., 1991; Calderone et al., 1996; Pike et al., 1996) and is similar to that of lysozyme, with which it shares significant homology. As illustrated in Figure 7.3a, \u03b1-La is made up of two lobes: the \u03b1-lobe contains residues 1\u201334 and 86\u2013123, and the smaller \u03b2-lobe spans residues 35\u201385. The \u03b1-lobe contains three \u03b1-helices (residues 5\u201311, 23\u201334, and 86\u201398) and two short 310-helices (residues 18\u201320 and 115\u2013118). A small, three-stranded \u03b2-sheet (residues 41\u201344, 47\u201350, and 55\u201356) and a short 310-helix (residues 77\u201380) make up the \u03b2-lobe (Calderone et al., 1996; Pike et al., 1996). The structure is stabilized by four disulfide bonds (Cys6\u2013Cys120 and Cys28\u2013Cys111 in the \u03b1-lobe, Cys60\u2013Cys77 in the \u03b2-sheet, and Cys73\u2013Cys90 tethering the two lobes together) (Brew, 2003). Unlike \u03b2-Lg, \u03b1-La has no free thiol. A calcium ion binds with a submicromolar dissociation constant at the so-called binding elbow formed by residues 79\u201388 located in a cleft between the two lobes, with the metal ion coordinated in a distorted pentagonal bipyramidal configuration by the side-chain carboxylate groups of Asp82, Asp87, and Asp88, the carbonyl oxygens of Lys79 and Asp84, and the oxygen atoms of two water molecules (Calderone et al., 1996; Pike et al., 1996).\n\nFigure 7.3 (a) Structure of bovine \u03b1-lactalbumin showing the Ca2+ binding site (PDB code: 1f6s). The peptide chain is rainbow colored, beginning at the N-terminus in blue and progressing to the C-terminus in red, in order to show the assembly of the subdomains. The Ca2+ ion is seven-coordinate. Loop 79\u201384 provides three ligands, two from main-chain carbonyl oxygen atoms of Lys79 and Asp84 and one from the side chain of Asp82. Coordination about the Ca2+ is completed by carboxylate oxygen atoms from Asp87 and Asp88 at the N-terminal end of the main 4-turn helix, and by two water molecules. The four disulfide bonds are shown in ball-and-stick representation (one in the helical domain is obscured, and the two linking the helical domain and calcium-ion binding loop to the \u03b2 domain are on the left half of the panel). (b) The lactose synthase complex formed from bovine \u03b1-lactalbumin (yellow) with \u03b2-1,4-galactosyltransferase (gray) (PDB code 2fyd). Several substrate molecules are observed, together with the cleaved nucleotide-sugar moiety (cyan sticks). The MnII ion is shown as a pink sphere and the Ca2+ ion as a gray sphere. For clarity, loop regions are given a smoothed representation. \u03b2-1,4-galactosyltransferase is not present in milk, and the lactose synthase complex is present only in the milk-producing cells of the alveoli. Figure drawn with PyMOL (Delano, 2002).\n\nThe structure of the apo form of bovine \u03b1-La is similar to that of the holo protein. The largest changes involve the movement of the Tyr103 side chain in the interlobe cleft with little change in the vicinity of the Ca2+ binding site (Chrysina et al., 2000). The salient features of the structure of the holo protein are depicted in Figure 7.3, together with its complex with \u03b2-1,4-galactosyltransferase-I with bound substrates and, interestingly, a trapped intermediate species.\n\nThe structures of \u03b1-La from several other species, including goat, baboon, human, guinea pig, and buffalo, have also been characterized by x-ray diffraction methods (Acharya et al., 1989; Acharya et al., 1991; Pike et al., 1996; Makabe et al., 2012; Calderone et al., 1996). Consistent with high-sequence identity, there are no significant differences among these structures, except for a flexible loop at residues 105\u2013110 implicated in formation of the lactose synthase complex (Acharya et al., 1989; Calderone et al., 1996; Pike et al., 1996). The recombinant goat protein, which has an added methionine at the N-terminus, is markedly less stable, by \u223c14 kJ mol\u22121, than the native protein, mostly the result of an increased rate of unfolding (but preserved rate of refolding) (Chaudhuri et al., 1999). Similar observations have been made on recombinant bovine \u03b1-La (Acharya et al., 1989). Thus, native protein functionality and stability should not in general be inferred from measurements of recombinant proteins heterologously expressed in bacterial systems (which generally add an N-terminal methionine residue).\n\n### Effect of Temperature on Bovine \u03b1-Lactalbumin\n\nIn general, holo \u03b1-La undergoes a thermal unfolding at a lower temperature than \u03b2-Lg (Ruegg et al., 1977). The role of bound calcium ions appears to confer stability to the tertiary structure: With less than equimolar amounts of bound calcium, the thermal unfolding transition is lowered substantially, decreasing to about 35 \u00b0C for the apo form (Relkin, 1996; Ishikawa et al., 1998). The presence of calcium ions also accelerates the rate of refolding of \u03b1-La by more than two orders of magnitude (Wehbi et al., 2005). The presence of calcium ions also aids in the refolding and formation of the correct disulfide linkages of denatured reduced protein (Wehbi et al., 2005). The structurally closely related, but functionally unrelated, enzyme lysozyme can be subdivided into two classes, a noncalcium-binding subclass, typified by egg-white lysozyme (Grobler et al., 1994; Steinrauf, 1998) and a calcium-binding subclass, including equine and echidna lysozyme (Tsuge et al., 1992; Guss et al., 1997).\n\nThe thermal denaturation behavior of bovine \u03b1-La from three different sources has been studied; significant differences were reported (McGuffey et al., 2005), thereby providing some resolution of the apparently discordant denaturation data from different groups. In the presence of \u03b2-Lg or BSA, each of which has an unpaired cysteine, \u03b2-Lg \u2212 \u03b1-La, \u03b1-La \u2212 BSA and even \u03b1-La \u2212 \u03b1-La oligomers form at high temperature. Since \u03b1-La (which lacks a free thiol) by itself fails under similar conditions to form disulfide-linked oligomers, intermolecular disulfide-sulfhydryl interchange reactions appear to play a role in forming \u03b1-La \u2212 \u03b1-La oligomers (Havea et al., 2001; Hong and Creamer, 2002).\n\n### Effect of Pressure on Bovine \u03b1-Lactalbumin\n\nAgain the absence of free thiol groups renders \u03b1-La intrinsically less susceptible to irreversible structural and functional change induced by high pressure. Reversible unfolding to a molten globule state begins at 200 MPa, and loss of native structure becomes irreversible beyond 400 MPa (McGuffey et al., 2005) (corresponding numbers for \u03b2-Lg are 50 and 150 MPa (Stapelfeldt and Skibsted, 1999; Considine et al., 2005b)). In the presence of calcium ions, denaturation pressure increases by 200 MPa for \u03b1-La (Dzwolak et al., 1999). Only in the presence of thiol reducers does oligomerization of \u03b1-La occur at high pressures (Jegouic et al., 1996).\n\n### Effect of Denaturants on Bovine \u03b1-Lactalbumin\n\nAt neutral pH, the calcium-depleted, or apo form of \u03b1-La, reversibly denatures to a variety of partially folded or molten globule states upon moderate heating (45 \u00b0C), or, at room temperature, by dissolving the protein in aqueous TFE (15% TFE) or by adding oleic acid (7.5 equivalents) (Svensson et al., 2000; de Laureto et al., 2002). Under these various conditions, the UV-CD spectra of apo-\u03b1-La are essentially identical to those of the most studied molten globule form of \u03b1-La, the A-state found at pH 2.0 (Kuwajima, 1996). At 4 \u00b0C and pH 8.3, proteolysis of apo-\u03b1-La by proteinase-K occurs slowly and nonspecifically, leading to small peptides only. On the other hand, at 37 \u00b0C, preferential cleavage by proteinase-K is observed at peptide bonds located in loop regions of the \u03b2-sheet subdomain of the \u03b2 domain of the protein (residues 35\u201385), creating peptides in which disulfide bridges link N-terminal residues 1\u201334 to C-terminal fragments, residues 54\u2013123 or 57\u2013123. Preferential cleavage at similar sites and similar disulfide-bridged fragments has also been observed for proteolysis of the molten globule states induced by TFE and oleic acid. Therefore, polypeptides formed from the molten globule A-state of \u03b1-La comprise a well-structured native-like conformation of the \u03b1-domain and a disordered conformation of the \u03b2-subdomain, residues 34\u201357 (de Laureto, et al., 2002).\n\nThe oleic acid treatment leads to a kinetically trapped folding variant of the protein, which can also bind calcium ions, called HAMLET (human \u03b1-lactalbumin made lethal to tumor cells, and its bovine analogue BAMLET), which has been shown to induce apoptosis in tumor cells (Svensson et al., 2000). Under conditions where thermal denaturation of \u03b1-La is reversible, thermal denaturation of HAMLET is irreversible, with respect to loss of its apoptotic effect on tumor cells (Fast et al., 2005).\n\nHuman and bovine \u03b1-La also weakly bind a second calcium ion, structurally characterized for human \u03b1-La (Chandra et al., 1998). In addition, zinc-ion binding to possibly structurally inequivalent sites has been characterized for human and bovine \u03b1-La by fluorescence spectroscopic techniques (Permyakov and Berliner, 2000). The binding of zinc ions to calcium-loaded \u03b1-La has been shown to destabilize the native structure to heat denaturation (Permyakov and Berliner, 2000). However, the weak binding of zinc (submillimolarity dissociation constant) means that this binding is probably not physiologically relevant.\n\n## Serum albumin\n\nSerum albumin (SA) is an approximately 580-residue protein found in both the blood serum and milk of all mammals and appears to function as a promiscuous transporter of hydrophobic molecules. However, as with many proteins, this transport role appears not to be the only physiological function for serum albumins. The structure of human serum albumin (HSA), described in more detail in the next subsection, is notable also for the number of disulfide bridges, 17 in total. There is one unpaired cysteine, Cys34, in HSA (highlighted in Figure 7.4), also Cys34 in BSA. This cysteine is part of a highly conserved QQCP(F\/Y) motif and is susceptible to various oxidations, including a two-electron oxidation to sulfenic acid (-SOH) (Carballal et al., 2007).\n\nFigure 7.4 Structure of human serum albumin (HSA) complexed with halothane (slate\/purple) partially occupying seven distinct sites and myristic acid (yellow\/red) occupying fully five distinct sites (PDB code: 1e7c). Domain IA (residues 5\u2013107) is shown in blue; domain IB (108\u2013196) in light blue; domain IIA (197\u2013297) in green; domain IIB (residues 297\u2013383) in light green; domain IIIA (residues 384\u2013497) in red; and domain IIIB (residues 498\u2013582) in light red. The single cysteine, Cys34, is arrowed (Bhattacharya et al., 2000). The 17 disulfide bonds, which tie together individual subdomains, are represented in stick format. Figure drawn with PyMOL (Delano, 2002).\n\nEvidence has been produced that, at least in blood serum, this cysteine is involved in HSA's role in the control of redox properties (Kawakami et al., 2006). A similar role can be postulated for bovine serum albumin (BSA) in blood serum, but in both human milk and bovine milk, this redox role has not been established (or even investigated). In terms of milk flavor and the flavor of milk products, control of redox states of milk components is obviously of importance. It appears also that in blood serum, where HSA is the major protein component, present at a concentration of 0.6 mM, HSA is the first line of defense against radicals, including reactive oxygen species and nitric oxide.\n\nIn vitro studies showing the reactivity of Cys34 in HSA have been complemented by in vivo studies showing that in primary nephrotic syndrome, Cys34 is oxidized to sulfonate, \u2013SO3- (Musante et al., 2006). Again, in milk, defenses against reactive oxygen species are essential to preserve milk quality, but HSA and BSA are at much lower concentrations in milk than in blood. HSA has also been shown to have esterase activity (Sakurai et al., 2004). Whether this is physiologically important in blood (or in milk) has not been established for either BSA or HSA. Finally, an active role for HSA in transport of fatty acids across membranes has been characterized (Cupp et al., 2004). It is in this process that serum albumins are introduced into mammalian milks.\n\n### Structure of Serum Albumins\n\nThe structure of HSA, with which BSA shares 75% sequence identity, has been well characterized for apo protein, as well as for a variety of complexes with a variety of long-chain fatty acids and other more compact hydrophobic molecules. The structure of HSA complexed with the anesthetic halothane (C2F3Cl2Br) and myristic acid (CH3(CH2)12COOH) is shown in Figure 7.4.\n\nThe structure of HSA comprises three structurally homologous domains, each of just under 200 residues, denoted I-III (Curry et al., 1998) and involving residues 5\u2013196, 197\u2013383, and 384\u2013582, respectively. Each domain has two subdomains, each of \u223c100 residues, denoted A and B. The structure lacks \u03b2-strands and is predominantly (68%) \u03b1-helical, with several lengthy loops connecting A and B subdomains. On ligand binding, there is substantial movement of the domains with respect to each other, but the tertiary structure of each domain undergoes only small changes (Curry et al., 1998). Medium- and long-chain fatty acids occupy five distinct sites (dissociation constants 0.05\u20131 \u03bcM) (Spector, 1975), one in domain I, a second between domains I and II, and the remaining three in domain III, as characterized by x-ray techniques for HSA. In the case of halothane binding, two sites are located in domain I and five in domain II.\n\nA comprehensive study of the binding of 17 distinct drugs to HSA, in the presence and absence of myristate, has been published (Ghuman et al., 2005). Whereas binding of steroids to BSA is influenced by binding of fatty acids, for HSA, there is much less influence (Watanabe and Sato, 1996). NMR titrations have shown that BSA, like HSA, binds five myristates; four of the five sites appear to be in structurally homologous sites to those identified crystallographically for HSA (Hamilton et al., 1984; 1991; Cistola et al., 1987; Simard et al., 2005). The x-ray structure of equine serum albumin (ESA) is very similar to that of HSA, consistent with \u223c75% sequence identity between these two proteins (Ho et al., 1993).\n\nThe structures of bovine, equine, and leporine (rabbit) SA have been recently published by two independent groups (Bujacz, 2012; Majorek et al., 2012; Sekula et al., 2013). HSA has also been heterologously expressed in rice and structurally characterized (He et al., 2011), finessing problems associated with availability and disease risks when sourced for clinical and cell-culture applications from human blood sources.\n\n### Effect of Temperature on Serum Albumins\n\nCareful differential scanning calorimetry (DSC) measurements have been made of defatted HSA and its binding to short- to medium-chain fatty acids. In the absence of fatty acids, a single sharp endotherm is observed, yielding a midpoint temperature for denaturation, T m, of 64.7 \u00b0C, consistent with a concerted unfolding. In the presence of fatty acids, the endotherm broadens, and there is a steady increase in T m as the chain length of the fatty acid increases from n-butanoate (T m = 77.6 \u00b0C) to n-octanoate (T m = 87.2 \u00b0C); T m for n-nonanoate and n-decanoate are very similar to that for n-octanoate. The short-chain fatty acids, formate, acetate, and n-propionate show evidence for inducing increased stability in HSA through binding of the fatty acids at secondary sites inaccessible to the longer-chain fatty acids. The concentration of fatty acid at which maximum stability of HSA is achieved decreases from more than 2900 mM for formate to less than 15 mM for a 30 mg\/mL (\u223c0.5 mM) solution of HSA at pH 7.0 (Shrake et al., 2006).\n\nFor the native protein, DSC data could be fitted to a two-state model with \u223c7 more or less equivalent binding sites for n-decanoate. This number may be contrasted with the value of 5 observed by x-ray and NMR methods for the binding of myristate (tetradecanoate acid) to HSA and also BSA (see above) (Simard et al., 2005).\n\n### Effect of Pressure on Serum Albumins\n\nBSA, despite the unpaired cysteine, is relatively stable to high pressures (800 MPa) (L\u00f3pez-Fandi\u00f1o, 2006). BSA undergoes substantial secondary structure changes, but unlike the case with \u03b2-Lg, these changes are reversible. It appears that the large number of disulfide bonds protects the hydrophobic core of the protein, including the largely buried Cys34 in subdomain IA, which is held together by three disulfide bonds (L\u00f3pez-Fandi\u00f1o, 2006). The effects of binding partners, such as fatty acids or other whey components, such as \u03b2-Lg, on structure and stability of BSA at high pressure remain uncharacterized.\n\nCombined pressure-temperature infrared studies of the amide vibrational modes of ESA have shown very recently that high pressure (400 MPa) can convert an intermolecular \u03b2-sheet aggregate formed by heating ESA to 60 \u00b0C at 0.1 MPa (i.e., ambient pressure) to a disordered structure, which reverts to the native structure upon release of pressure. The activation volume of \u223c+92 mLmol\u22121 and partial molar volume difference between native and heat-denatured states of (\u0394 V N\u2192HA = +32 mLmol\u22121) are consistent with the decreasing stability of the heat-denatured intermolecular \u03b2-sheet with increasing pressure (Okuno et al., 2007).\n\n### Effect of Chemical Denaturants on Serum Albumins\n\nThe denaturation of BSA and HSA by urea and GdmCl has been extensively studied (Lapanje and Skerjanc, 1974; Khan et al., 1987; Guo and Qu, 2006). In the presence of fatty acids and other molecules, especially molecules that bind to domain III (e.g., diazepam), denaturation of BSA by urea changes from a three-step process to a two-step process, indicating that the initial denaturation involves changes in tertiary and secondary structure of domain III (Ahmad and Qasim, 1995; Tayyab et al., 2000). For HSA, denaturation appears to be an intrinsically two-step process with fatty acids converting denaturation to a one-step process (Muzammil et al., 2000; Shrake et al., 2006). In both cases, the binding of ligands to BSA or HSA stabilizes the protein against urea-induced denaturation. There is evidence that the urea-induced denatured state and that induced by high pressure are similar, at least for HSA (Tanaka et al., 1997).\n\nIn the presence of cations, such as guanidinium and cetylpyridinium salts, denaturation of SA again features domain III as being the most susceptible to denaturation (Ahmad et al., 2005; Sun et al., 2005). Consistent with the high helical content of SA, perfluorinated alcohols and alcohols with bulky hydrophobic heads stabilize HSA (and presumably BSA) against denaturation by both urea and GdmCl (Kumar et al., 2005).\n\nThe stability of HSA in the presence of polyethylene glycols (PEGs) has also been examined by Farruggia et al. (1999); low-molecular-weight PEGs affect ionization of surface tyrosines, and high-molecular-weight PEGs lower the thermal transition temperatures.\n\n## Immunoglobulins\n\nThe immunoglobin proteins (Ig) form a diverse family whose members, when in milk, protect the gut mucosa against pathogenic microorganisms. In bovine milk, the predominant species of Ig proteins are members of the IgG subfamily, in particular the IgG1. Colostrum contains 40\u2013300 times the concentration of IgG proteins than does milk; its role is to confer passive immunity to the neonate while its own immune system is developing (Gapper et al., 2007).\n\nIgG proteins have multiple functions, including complement activation, bacterial opsonization (rendering bacterial cells susceptible to immune response), and agglutination. They inactivate bacteria by binding to specific sites on the bacterial surface. Given the significance attributed to bovine milk and milk products in human nutrition and health, it is important to note that there are significant differences in the levels of the various subfamilies of immunoglobins in milk from different species. Human colostrum and milk contain relatively low levels of the IgG subfamily compared to bovine milk; the opposite is the case with respect to the IgA subfamily. The properties and accurate quantitation of bovine Ig proteins have been reviewed in detail by Gapper et al. (2007).\n\n### Structure of Immunoglobulin G\n\nThe structure of IgG is illustrated schematically in Figure 7.5a and as revealed by x-ray techniques in Figure 7.5b. Both the heavy chain and light chain are predominantly \u03b2-sheet structures. Disulfide bridges link pairs of molecules, as well as the heavy chain to the light chain. The protein is generally glycosylated at a number of sites. However, the actual structure is much less tidy than the schematic Y-shaped figure; in particular, the disulfide bonds are at the base of the light chain\u2013heavy chain associations.\n\nFigure 7.5 (a) Schematic of the general structure of immunoglobulins. Reproduced from Gapper et al. (2007) with permission. The different classes are distinguished by the constant or Fc regions of the heavy and light chains. Reproduced from Gapper et al. (2007) with permission. (b) The x-ray structure of the human IgG1 molecule (PDB code 1hzh). The heavy chains are in blue and green, the light chains are in magenta and pink. The asparagine N-linked glycan is shown in stick representation. The lack of two-fold symmetry indicates the extreme flexibility of the domains with respect to one another and consequent sensitivity to crystal-packing effects. Disulfide bridges are shown as spheres. Each domain has a disulfide bridge joining the two sheets; additional disulfide bridges link the two heavy chains and the light chains to the heavy chains. The N-termini of each chain is at the top left and top right of the diagram; the C-termini of the heavy chains are at the base of the molecule. Structures (e.g., PDB code: 1wej, 3hfm) where antigens are bound at the light chain\u2013heavy chain interface indicate that binding of antigen occurs across the top of the molecule with little embedding of antigen between the domains, contrary to the mode implied by (a). Figure drawn with PyMOL (Delano, 2002).\n\nBovine \u03b2-Lg has long been recognized for eliciting strong immunological responses in selected susceptible individuals (Wal, 1998). The structure of bovine \u03b2-Lg in complex with a recombinant human IgE Fab fragment, that is, an IgE epitope of bovine \u03b2-Lg, has been reported (Niemi et al., 2007).\n\n### Effects of Temperature, Pressure, and Chemical Denaturants on Ig Structure and Stability\n\nThe response of bovine IgG (isoform not specified) to temperature and chemical denaturants, urea, and guanidinium hydrochloride has been reported (Ye et al., 2005). Thermal denaturation and thermal denaturation in the presence of denaturants was irreversible, producing via a series of steps an incompletely unfolded aggregate. Isothermal chemical denaturation produced, also by a series of steps, a completely unfolded random-coil state (Ye et al., 2005). The response of IgG to high pressure (200\u2013700 MPa) in the presence of the kosmotrope sucrose has also been reported (Zhang et al., 1998).\n\nA comparative study of the thermal denaturation of bovine IgG, IgA, and IgM has been published, with stability in the order just given (Mainer et al., 1997). As retention of immunological properties on standard milk-processing techniques was of interest, the activity of heat-treated protein was determined by an immunological assay using antibodies raised against these Ig proteins. The response of IgG to pulsed electric fields (and to heat) was reported (Li et al., 2005). Little change in secondary structure or in immunoactivity was reported for samples subjected to a pulsed electric field of \u223c41 kV\/cm.\n\n## Lactoferrin\n\nLactoferrin (Lf) is a monomeric, globular, Fe3+-binding glycoprotein comprising \u223c680 amino acids giving a molecular mass of \u223c80 kDa. It is a member of the transferrin family, but unlike the eponymous protein, to date there is no strong evidence that lactoferrin is involved in iron transport or metabolism under normal circumstances. Indeed, the protein is only lightly loaded with iron(III) in milk, allowing it to perform a major bacteriostatic role by sequestering iron(III) despite bacteria-producing iron(III)-sequestering agents (siderophores) that have affinities for iron(III) many orders of magnitude higher than that for lactoferrin. Lf is, however, a multifunctional protein, with evidence to suggest that it, and especially its N-terminal Arg-rich fragments, called lactoferricin(s), obtained by pepsin hydrolysis of the entire protein, have active antimicrobial activity as well as activity as antiviral and antiparasitic agents (Str\u00f8m et al., 2000; ).\n\nAccordingly, commercial applications utilizing bovine Lf and its partially digested peptides are appearing as nutraceuticals in infant formulas, health supplements, oral care products, and animal feeds. In addition, reputed antioxidant properties are being utilized in cosmetics (van Hooijdonk and Steijns, 2002). For a review on the remarkable properties of Lf and its commercial applications, see Brock (2002). The protein is synthesized in the mammary gland, but it is also found in other exocrine fluids besides milk. Bovine Lf is sometimes used as a supplement in bovine milk-based infant formulas (to offset the lower abundance relative to that found in human milk) and is potentially useful as a constituent in functional foods, with marked effects on bone-cell activity (Cornish et al., 2004).\n\n### Bovine Lactoferrin Structure\n\nBovine Lf has a tertiary structure (Moore et al., 1997) very similar to that of the Lfs of other species determined so far: human (Anderson et al., 1987; ; Haridas et al., 1995), buffalo (Karthikeyan et al., 1999), horse (Sharma et al., 1999), and camel (Khan et al., 2001). All Lfs and transferrins contain two lobes, which share internal homology (\u223c40% sequence identity between the N- and C- terminal lobes) and a common fold (see Fig. 7.6). The homology between lobes for a given species is less than that between corresponding lobes from different species, indicating that the gene duplication event is of ancient origin. Each lobe contains two \u03b1\/\u03b2 domains divided by a cleft that incorporates an iron-binding site. Huge structural changes accompany iron binding: Each lobe closes over the iron(III), encapsulating a synergistic bicarbonate anion. Remarkably, for camel lactoferrin, the N-terminal lobe appears to have sequence and iron-binding properties similar to those of other lactoferrins, whereas the C-terminal lobe resembles more closely in sequence and iron-binding properties of transferrins, hen ovo-transferrin in particular (Khan et al., 2001).\n\nFigure 7.6 Structure of lactoferrin. (a) Human apo-lactoferrin (PDB code: 1cb6), showing the domain structure. The N lobe (blue for subdomain N-IN and cyan for subdomain N-IC) is in the open conformation, the C-lobe (magenta for subdomain C-IN and pink for subdomain C-IC), despite no metal ion present, is in the closed conformation. The helix connecting the two lobes is shown in yellow. Metal-binding ligands are shown as spheres. (b) Holo-(FeIII)-bovine lactoferrin (PDB code: 1blf; the human analogue, 1b0l, is structurally very similar). The polypeptide is rainbow colored, blue at the N-terminus to red at the C-terminus, to highlight the manner in which the subdomains N-IN and C-IN are formed from residues \u223c1\u201390 and \u223c250\u2013320 (N-IN) and \u223c350\u2013440 and \u223c600\u2013680 (C-IN); for reference, these subdomains are shown in approximately the same orientation in frames (a) and (b). The iron-binding ligands, including the synergistic bicarbonate ion in (b), are shown in stick form, the iron atom as a red sphere and the cysteines as orange spheres (16 Cys forming 8 disulfides for human Lf and 34 Cys forming 17 disulfides for bovine Lf). Loops are not smoothed. Figure drawn with PyMOL (Delano 2002).\n\nNotwithstanding these structural changes, for the apo protein, at least for human Lf, there is little difference in free energy between open and closed forms (indeed, the structure of human apo-Lf has one lobe open, the other closed). This delicate balance is achieved by both open and closed conformations of the protein having, remarkably, the same surface area exposed to solvent. The same applies, but by a different structural mechanism, to open and closed forms of the N-terminal recombinantly produced half molecule of human lactoferrin (Jameson et al. 1998; ; 2002b;). Like \u03b2-Lg, holo-Lf undergoes a pH-dependent conformational change (in this case as low as pH \u223c3) that releases the bound ferric ion; the structurally related, but genetically and functionally distinct, serum transferrin releases its cargo at significantly higher pH (pH \u223c5.5) (Baker and Baker, 2004).\n\n### Effects of Temperature, Pressure, and Chemical Denaturants on Lactoferrin Structure and Stability\n\nAs a minor component of milk proteins, Lf seems largely to have escaped detailed study of its intrinsic response to temperature, pressure, and chemical denaturants or of its influence, if any, on stability of either itself, or other whey proteins, in the presence of other whey proteins. Calcium ions have been reported to bind to bovine Lf, probably to the sialic acid groups of the asparagine N-linked glycan, with micromolar dissociation constants; both apo- and holo forms of calcium-bound bovine Lf are more stable to heat and chemical denaturants (Rossi et al., 2002). The calcium-bound forms appear to reduce the Lf-induced release of lipopolysaccharide moieties from bacterial membranes (Rossi et al., 2002).\n\nThe heat stability of bovine Lf in isolation (S\u00e1nchez et al., 1992) and in association with the major bovine whey proteins, \u03b2-Lg (weak association), \u03b1-La (no detectable association), and BSA (weak 1:1 association) (Lampreave et al., 1990) has also been studied. The stability of bovine Lf is such that standard pasteurization conditions (but not ultra-high-temperature treatment) are likely to have little effect on structure and properties, especially immunological properties (Oria et al., 1993).\n\n## Concluding remarks\n\nThree-dimensional structure determines physiological function as well as the functionality of whey proteins in their varied applications, both in whole milk and in whey itself. In this review we have tried to look beyond individual proteins to see what structural features may be important to protein\u2013protein interactions under stresses of temperature, pressure, and chemical denaturants. In particular, we have attempted to uncover more recent results that challenge existing paradigms or offer new perspectives. In general, changes that are brought about as a consequence of pH, heat, pressure, chaotropes, and so on, shift equilibrium points (e.g., between monomer and dimer, native and unfolded states, etc.), and it is only when a new covalent bond is formed (or removed) that there is irreversible change.\n\nOne key interaction between like and unlike proteins arises from disulfide bond interchange, generally facilitated by a single cysteine residue. For this reason, we have focused, especially in the figures, on the observed locations of cysteines. However, detailed structural information, at a level comparable to that of individual partners, is generally lacking in intermolecular assemblies, even when only pairwise associations are formed and isolated.\n\nGiven sufficient time at elevated temperatures and low pH, proteins, in particular \u03b2-Lg, are observed to hydrolyze and reassemble in fibrillar species, for which signatures of extended \u03b2 sheet structures are seen, but the extreme chemical and thermal stability that characterizes true \u03b2-amyloid aggregates is absent.\n\nWhereas considerable attention has been directed toward understanding interactions among components of milk and of whey, relatively little attention has been given to date to the redox state and redox changes during storage and processing of milk proteins and their effects on milk processing and flavors of milk-derived products. This remark originates from recent studies on blood sera, where serum albumins play key roles in redox state and protection against reactive oxygen species. It is important, then, that these structures and functional states are characterized and understood, especially if milk processing, particularly by means of relatively new methods such as pressure treatment, results in refolded proteins that may have altered properties and functionality, such as processability and digestibility.\n\n## Acknowledgments\n\nThe authors would like to acknowledge illuminating discussions with Lindsay Sawyer over many years, and long-standing collaboration with Lawrie Creamer, facilitated by Mike Boland.\n\n# References\n\nAcharya KR ,  Ren JS ,  Stuart DI ,  Phillips DC ,  Fenna RE .  Crystal structure of human \u03b1-lactalbumin at 1.7 \u00c5 resolution .  _Journal of Molecular Biology_.  1991 ;221 (2) : 571 \u2013 581 .\n\nAcharya KR ,  Stuart DI ,  Walker NPC ,  Lewis M ,  Phillips DC .  Refined structure of baboon \u03b1-lactalbumin at 1.7 \u00c5 resolution. Comparison with C-type lysozyme .  _Journal of Molecular Biology_.  1989 ;208 (1) : 99 \u2013 127 .\n\nAdamcik J ,  Jung JM ,  Flakowski J ,  De Los Rios P ,  Dietler G ,  Mezzenga R .  Understanding amyloid aggregation by statistical analysis of atomic force microscopy images .  _Nature Nanotechnology_.  2010 ;5 (6) : 423 \u2013 428 .\n\nAdamcik J ,  Mezzenga R .  Proteins fibrils from a polymer physics perspective .  _Macromolecules_.  2012 ;45 (3) : 1137 \u2013 1150 .\n\nAdams JJ ,  Anderson BF ,  Norris GE ,  Creamer LK ,  Jameson GB .  Structure of bovine \u03b2-lactoglobulin (variant A) at very low ionic strength .  _Journal of Structural Biology_.  2006 ;154 (3) : 246 \u2013 254 .\n\nAhmad B ,  Ahmed Md Z ,  Haq Soghra K ,  Khan Rizwan H .  Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III .  _Biochimica et Biophysica Acta_.  2005 ;1750 (1) : 93 \u2013 102 .\n\nAhmad N ,  Qasim MA .  Fatty acid binding to bovine serum albumin prevents formation of intermediate during denaturation .  _European Journal of Biochemistry_.  1995 ;227 (1\/2) : 563 \u2013 565 .\n\nAkkermans C ,  Venema P ,  van der Goot AJ ,  Gruppen H ,  Bakx EJ ,  Boom RM ,  van der Linden E .  Peptides are building blocks of heat-induced fibrillar protein aggregates of \u03b2-lactoglobulin formed at pH 2 .  _Biomacromolecules_.  2008 ;9 (5) : 1474 \u2013 1479 .\n\nAlexander SS ,  Pace CN .  A comparison of the denaturation of bovine \u03b2-lactoglobulins A and B and goat \u03b2-lactoglobulin .  _Biochemistry_.  1971 ;10 (14) : 2738 \u2013 2743 .\n\nAnderson BF ,  Baker HM ,  Dodson EJ ,  Norris GE ,  Rumball SV ,  Waters JM ,  Baker EN .  Structure of human lactoferrin at 3.2 \u00c5 resolution .  _Proceedings of the National Academy of Sciences of the USA_.  1987 ;84 (7) : 1769 \u2013 1773 .\n\nAnderson BF ,  Baker HM ,  Norris GE ,  Rice DW ,  Baker EN .  Structure of human lactoferrin\u2014crystallographic structure-analysis and refinement at 2.8 \u00c5 resolution .  _Journal of Molecular Biology_.  1989 ;209 (4) : 711 \u2013 734 .\n\nAouzelleg A ,  Bull L-A ,  Price NC ,  Kelly SM .  Molecular studies of pressure\/temperature-induced structural changes in bovine \u03b2-lactoglobulin .  _Journal of the Science of Food and Agriculture_.  2004 ;84 (5) : 398 \u2013 404 .\n\nArnaudov LN ,  de Vries R ,  Ippel H ,  van Mierlo CP .  Multiple steps during the formation of \u03b2-lactoglobulin fibrils .  _Biomacromolecules_.  2003 ;4 (6) : 1614 \u2013 1622 .\n\nAymard P ,  Nicolai T ,  Durand D ,  Clark A .  Static and dynamic scattering of \u03b2-lactoglobulin aggregates formed after heat-induced denaturation at pH 2 .  _Macromolecules_.  1999 ;32 (8) : 2542 \u2013 2552 .\n\nBaker BM ,  Murphy KP .  Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry .  _Biophysical Journal_.  1996 ;71 (4) : 2049 \u2013 2055 .\n\nBaker HM ,  Baker EN .  Lactoferrin and Iron: Structural and dynamic aspects of binding and release .  _BioMetals_.  2004 ;17 (3) : 209 \u2013 216 .\n\nBanaszak L ,  Winter N ,  Xu Z ,  Bernlohr DA ,  Cowan S ,  Jones TA .  Lipid-binding proteins: A family of fatty acid and retinoid transport proteins .  _Advances in Protein Chemistry_.  1994 ;45 : 89 \u2013 151 .\n\nBello M ,  Portillo-T\u00e9llez MC ,  Garcia-Hern\u00e1ndez E .  Energetics of ligand recognition and self-association of bovine \u03b2-lactoglobulin: Differences between variants A and B .  _Biochemistry_.  2011 ;50 (1) : 151 \u2013 161 .\n\nBello M ,  Guti\u00e9rrez G ,  Garcia-Hern\u00e1ndez E .  Structure and dynamics of \u03b2-lactoglobulin in complex with dodecyl sulfate and laurate: A molecular dynamics study .  _Biophysical Chemistry_.  2012 ; : 165 \u2013 166 : 79-86 .\n\nBelloque J ,  L\u00f3pez-Fandi\u00f1o R ,  Smith GM .  A 1H-NMR study on the effect of high pressures on \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 (9) : 3906 \u2013 3912 .\n\nBelloque J ,  Smith GM .  Thermal denaturation of \u03b2-lactoglobulin. A 1H NMR study .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 (5) : 1805 \u2013 1813 .\n\nBennion BJ ,  Daggett V .  The molecular basis for the chemical denaturation of proteins by urea .  _Proceedings of the National Academy of Sciences of the USA_.  2003 ;100 (9) : 5142 \u2013 5147 .\n\nBerman HM ,  Westbrook J ,  Feng Z ,  Gilliland G ,  Bhat TN ,  Weissig H ,  Shindyalov IN ,  Bourne PE .  The Protein Data Bank .  _Nucleic Acids Res_.  2000 ;28 (1) : 235 \u2013 242 .\n\nBewley MC ,  Qin BY ,  Jameson GB ,  Sawyer L ,  Baker EN .  Bovine \u03b2-lactoglobulin and its variants: A three-dimensional structural perspective .  _International Dairy Federation_.  1997 ;9702 (Special Issue) : 100 \u2013 109 : Milk Protein Polymorphism .\n\nBhattacharjee C ,  Saha S ,  Biswas A ,  Kundu M ,  Ghosh L ,  Das KP .  Structural changes of \u03b2-lactoglobulin during thermal unfolding and refolding\u2014An FT-IR and circular dichroism study .  _Protein Journal_.  2005 ;24 (1) : 27 \u2013 35 .\n\nBhattacharya AA ,  Curry S ,  Franks NP .  Binding of the general anesthetics propofol and halothane to human serum albumin\u2014High resolution crystal structures .  _Journal of Biological Chemistry_.  2000 ;275 (49) : 38731 \u2013 38738 .\n\nBolisetty S ,  Adamcik J ,  Mezzenga R .  Snapshots of fibrillation and aggregation kinetics in multistranded amyloid \u03b2-lactoglobulin fibrils .  _Soft Matter_.  2011 ;7 (2) : 493 .\n\nBrew K .  \u03b1-Lactalbumin. Advanced Dairy Chemistry ,  1 : 387-419 .\n\nBrock JH .  The physiology of lactoferrin .  _Biochemistry and Cell Biology_.  2002 ;80 (1) : 1 \u2013 6 .\n\nBromley EHC ,  Krebs MRH ,  Donald AM .  Aggregation across the length-scales in \u03b2-lactoglobulin .  _Faraday Discussions_.  2005 ;128 : 13 .\n\nBrownlow S ,  Cabral JHM ,  Cooper R ,  Flower DR ,  Yewdall SJ ,  Polikarpov I ,  North AC ,  Sawyer L .  Bovine \u03b2-lactoglobulin at 1.8 \u00c5 resolution\u2014still an enigmatic lipocalin .  _Structure (London)_.  1997 ;5 (4) : 481 \u2013 495 .\n\nBujacz A .  Structures of bovine, equine and leporine serum albumin .  _Acta Crystallographica, Section D: Biological Crystallography_.  2012 ;68 (10) : 1278 \u2013 1289 .\n\nBurova TV ,  Grinberg NV ,  Visschers RW ,  Grinberg VY ,  de Kruif CG .  Thermodynamic stability of porcine \u03b2-lactoglobulin\u2014A structural relevance .  _European Journal of Biochemistry_.  2002 ;269 (16) : 3958 \u2013 3968 .\n\nBusti P ,  Gatti CA ,  Delorenzi NJ .  Binding of alkylsulfonate ligands to bovine \u03b2-lactoglobulin: Effects on protein thermal unfolding .  _Food Research International_.  2006 ;39 (4) : 503 \u2013 509 .\n\nCalderone V ,  Giuffrida MG ,  Viterbo D ,  Napolitano L ,  Fortunato D ,  Conti A ,  Acharya KR .  Amino acid sequence and crystal structure of buffalo \u03b1-lactalbumin .  _FEBS Letters_.  1996 ;394 (1) : 91 \u2013 95 .\n\nCarballal S ,  Alvarez B ,  Turell L ,  Botti H ,  Freeman BA ,  Radi R .  Sulfenic acid in human serum albumin .  _Amino Acids_.  2007 ;32 (4) : 543 \u2013 551 .\n\nCasal HL ,  Kohler U ,  Mantsch HH .  Structural and conformational changes of \u03b2-lactoglobulin B: An infrared spectroscopic study of the effect of pH and temperature .  _Biochimica et Biophysica Acta_.  1988 ;957 (1) : 11 \u2013 20 .\n\nCavanagh J ,  Fairbrother W ,  Palmer III AG ,  Skelton N .  _Protein NMR Spectroscopy: Principles and Practice_ .  San Diego :  Academic Press ;  1995 .\n\nChamani J ,  Moosavi-Movahedi AA ,  Rajabi O ,  Gharanfoli M ,  Momen-Heravi M ,  Hakimelahi GH ,  Neamati-Baghsiah A ,  Varasteh AR .  Cooperative \u03b1-helix formation of \u03b2-lactoglobulin induced by sodium n-alkyl sulfates .  _Journal of Colloid and Interface Science_.  2006 ;293 (1) : 52 \u2013 60 .\n\nChandra N ,  Brew K ,  Acharya KR .  Structural evidence for the presence of a secondary calcium binding site in human \u03b1-lactalbumin .  _Biochemistry_.  1998 ;37 (14) : 4767 \u2013 4772 .\n\nChatterton DEW ,  Nguyen DN ,  Bering SB ,  Sangild PT .  Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns .  _International Journal of Biochemistry and Cell Biology_.  2013 ;45 (8) : 1730 \u2013 1747 .\n\nChatterton DEW ,  Smithers G ,  Roupas P ,  Brodkorb A .  Bioactivity of \u03b2-lactoglobulin and \u03b1-lactalbumin\u2014Technological implications for processing .  _International Dairy Journal_.  2006 ;16 (11) : 1229 \u2013 1240 .\n\nChaudhuri TK ,  Horii K ,  Yoda T ,  Arai M ,  Nagata S ,  Terada TP ,  Uchiyama H ,  Ikura T ,  Tsumoto K ,  Kataoka H ,  Matsushima M ,  Kuwajima K ,  Kumagai I .  Effect of the extra N-terminal methionine residue on the stability and folding of recombinant \u03b1-lactalbumin expressed in Escherichia coli .  _Journal of Molecular Biology_.  1999 ;285 (3) : 1179 \u2013 1194 .\n\nCherrier MV ,  Engilberge S ,  Amara P ,  Chevalley A ,  Salmain M ,  Fontecilla-Camps JC .  Structural basis for enantioselectivity in the transfer hydrogenation of a ketone catalyzed by an artificial metalloenzyme .  _European Journal of Inorganic Chemistry_.  2013 ;2013 (21) : 3596 \u2013 3600 .\n\nCho Y ,  Gu W ,  Watkins S ,  Lee SP ,  Kim TR ,  Brady JW ,  Batt CA .  Thermostable variants of bovine \u03b2-lactoglobulin .  _Protein Engineering_.  1994 ;7 (2) : 263 \u2013 270 .\n\nChrysina ED ,  Brew K ,  Acharya KR .  Crystal structures of apo- and holo-bovine \u03b1-lactalbumin at 2.2\u00c5 resolution reveal an effect of calcium on inter-lobe interactions .  _Journal of Biological Chemistry_.  2000 ;275 (47) : 37021 \u2013 37029 .\n\nCistola DP ,  Small DM ,  Hamilton JA .  Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin.1. The filling of individual fatty-acid binding sites .  _Journal of Biological Chemistry_.  1987 ;262 (23) : 10971 \u2013 10979 .\n\nConsidine T ,  Patel HA ,  Singh H ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (8) : 3197 \u2013 3205 .\n\nConsidine T ,  Singh H ,  Patel HA ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (20) : 8010 \u2013 8018 .\n\nCornish J ,  Callon KE ,  Naot D ,  Palmano KP ,  Banovic T ,  Bava U ,  Watson M ,  Lin J-M ,  Tong PC ,  Chen Q ,  Chan VA ,  Reid HE ,  Fazzalari N ,  Baker HM ,  Baker EN ,  Haggarty NW ,  Grey AB ,  Reid IR .  Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo .  _Endocrinology_.  2004 ;145 (9) : 4366 \u2013 4374 .\n\nCreamer LK .  Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine \u03b2-lactoglobulin .  _Biochemistry_.  1995 ;34 (21) : 7170 \u2013 7176 .\n\nCreamer LK ,  Bienvenue A ,  Nilsson H ,  Paulsson M ,  Van Wanroij M ,  Lowe EK ,  Anema SG ,  Boland MJ ,  Jimenez-Flores R .  Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 (25) : 7660 \u2013 7668 .\n\nCreamer LK ,  Blair B ,  Korte R ,  Jameson GB .  _Unpublished results. Abstract 424: Binding of small amphipathic molecules to \u03b2-lactoglobulin_ .  Baltimore, Maryland :  American Dairy Science Association and American Society of Animal Science Joint Meeting ;  2000 .\n\nCreamer LK ,  Parry DAD ,  Malcolm GN .  Secondary structure of bovine \u03b2-lactoglobulin B .  _Archives of Biochemistry and Biophysics_.  1983 ;227 (1) : 98 \u2013 105 .\n\nCupp D ,  Kampf JP ,  Kleinfeld AM .  Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes .  _Biochemistry_.  2004 ;43 (15) : 4473 \u2013 4481 .\n\nCurry S ,  Mandelkow H ,  Brick P ,  Franks N .  Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites .  _Nature Structural Biology_.  1998 ;5 (9) : 827 \u2013 835 .\n\nD'Alfonso L ,  Collini M ,  Baldini G .  Does \u03b2-lactoglobulin denaturation occur via an intermediate state? .  _Biochemistry_.  2002 ;41 (1) : 326 \u2013 333 .\n\nD'Alfonso L ,  Collini M ,  Ragona L ,  Ugolini R ,  Baldini G ,  Molinari H .  Porcine \u03b2-lactoglobulin chemical unfolding: Identification of a non-native \u03b1-helical intermediate .  _Proteins: Structure, Function, and Bioinformatics_.  2004 ;58 (1) : 70 \u2013 79 .\n\nDar TA ,  Singh LR ,  Islam A ,  Anjum F ,  Moosavi-Movahedi AA ,  Ahmad F .  Guanidinium chloride and urea denaturations of \u03b2-lactoglobulin A at pH 2.0 and 25 \u00b0C: The equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches) .  _Biophysical Chemistry_.  2007 ;127 (3) : 140 \u2013 148 .\n\nDave AC ,  Loveday SM ,  Anema SG ,  Loo TS ,  Norris GE ,  Jameson GB ,  Singh H .  \u03b2-lactoglobulin self-assembly: Structural changes in early stages and disulfide bonding in fibrils .  _Journal of Agricultural and Food Chemistry_.  2013 ;61 (32) : 7817 \u2013 7828 . \nde Laureto PP ,  Frare E ,  Gottardo R ,  Fontana A .  Molten globule of bovine \u03b1-lactalbumin at neutral pH induced by heat, trifluoroethanol, and oleic acid: A comparative analysis by circular dichroism spectroscopy and limited proteolysis .  _Proteins: Structure, Function, and Genetics_.  2002 ;49 (3) : 385 \u2013 397 .\n\nde Wit JN ,  Swinkels GAM .  A differential scanning calorimetric study of the thermal denaturation of bovine \u03b2-lactoglobulin. Thermal behavior at temperatures up to 100 \u00b0C .  _Biochimica et Biophysica Acta_.  1980 ;624 (1) : 40 \u2013 50 .\n\nDelano WL .  _PyMOL_ .  Palo Alto, CA, USA :  Delano Scientific ;  2002 .\n\nDenton H ,  Smith M ,  Husi H ,  Uhr\u00edn D ,  Barlow PN ,  Batt CA ,  Sawyer L .  Isotopically labeled bovine \u03b2-lactoglobulin for NMR studies expressed in Pichia pastoris .  _Protein Expression and Purification_.  1998 ;14 (1) : 97 \u2013 103 .\n\nDolgikh DA ,  Gilmanshin RI ,  Brazhnikov EV ,  Bychkova VE ,  Semisotnov GV ,  Venyaminov SY ,  Ptitsyn OB .  \u03b1-Lactalbumin: Compact state with fluctuating tertiary structure? .  _FEBS Letters_.  1981 ;136 (2) : 311 \u2013 315 .\n\nDufour E ,  Genot C ,  Haertl\u00e9 T .  \u03b2-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy .  _Biochimica et Biophysica Acta_.  1994 ;1205 (1) : 105 \u2013 112 .\n\nDufour E ,  Herv\u00e9 G ,  Haertl\u00e9 T .  Hydrolysis of \u03b2-lactoglobulin by thermolysin and pepsin under high hydrostatic pressure .  _Biopolymers_.  1995 ;35 (5) : 475 \u2013 483 .\n\nDzwolak W ,  Kato M ,  Shimizu A ,  Taniguchi Y .  Fourier-transform infrared spectroscopy study of the pressure-induced changes in the structure of the bovine \u03b1-lactalbumin: The stabilizing role of the calcium ion .  _Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology_.  1999 ;1433 (1-2) : 45 \u2013 55 .\n\nEdwards PJB ,  Jameson GB ,  Palmano KP ,  Creamer LK .  Heat-resistant structural features of bovine \u03b2-lactoglobulin A revealed by NMR H\/D exchange observations .  _International Dairy Journal_.  2002 ;12 (4) : 331 \u2013 344 .\n\nFarrell Jr HM ,  Jimenez-Flores R ,  Bleck GT ,  Brown EM ,  Butler JE ,  Creamer LK ,  Hicks CL ,  Hollar CM ,  Ng-Kwai-Hang KF ,  Swaisgood HE .  Nomenclature of the proteins of cows' milk\u2014Sixth revision .  _Journal of Dairy Science_.  2004 ;87 (6) : 1641 \u2013 1674 .\n\nFarruggia B ,  Nerli B ,  Di Nuci H ,  Rigatusso R ,  Pico G .  Thermal features of the bovine serum albumin unfolding by polyethylene glycols .  _International Journal of Biological Macromolecules_.  1999 ;26 (1) : 23 \u2013 33 .\n\nFast J ,  Mossberg A-K ,  Svanborg C ,  Linse S .  Stability of HAMLET \u2013 a kinetically trapped \u03b1-lactalbumin oleic acid complex .  _Protein Science_.  2005 ;14 (2) : 329 \u2013 340 .\n\nFlower DR .  The lipocalin protein family: Structure and function .  _Biochemical Journal_.  1996 ;318 (1) : 1 \u2013 14 .\n\nFogolari F ,  Ragona L ,  Zetta L ,  Romagnoli S ,  de Kruif KG ,  Molinari H .  Monomeric bovine \u03b2-lactoglobulin adopts a \u03b2-barrel fold at pH 2 .  _FEBS Letters_.  1998 ;436 (2) : 149 \u2013 154 .\n\nFrapin D ,  Dufour E ,  Haertl\u00e9 T .  Probing the fatty acid binding site of \u03b2-lactoglobulins .  _Journal of Protein Chemistry_.  1993 ;12 (4) : 443 \u2013 449 .\n\nGapper L ,  Copestake D ,  Otter D ,  Indyk H .  Analysis of bovine immunoglobulin G in milk, colostrum and dietary supplements: A review .  _Analytical and Bioanalytical Chemistry_.  2007 ;389 (1) : 93 \u2013 109 .\n\nGhuman J ,  Zunszain PA ,  Petitpas I ,  Bhattacharya AA ,  Otagiri M ,  Curry S .  Structural basis of the drug-binding specificity of human serum albumin .  _Journal of Molecular Biology_.  2005 ;353 (1) : 38 \u2013 52 .\n\nGlasgow BJ ,  Abduragimov AR ,  Farahbakhsh ZT ,  Faull KF ,  Hubbell WL .  Tear lipocalins bind a broad array of lipid ligands .  _Current Eye Research_.  1995 ;14 (5) : 363 \u2013 372 .\n\nGoldberg RN ,  Kishore N ,  Lennen RM .  Thermodynamic quantities for the ionization reactions of buffers .  _Journal of Physical and Chemical Reference Data_.  2002 ;31 (2) : 231 \u2013 370 .\n\nGosal WS ,  Clark AH ,  Pudney PDA ,  Ross-Murphy SB .  Novel amyloid fibrillar networks derived from a globular protein: \u03b2-lactoglobulin .  _Langmuir_.  2002 ;18 (19) : 7174 \u2013 7181 .\n\nGosal WS ,  Clark AH ,  Ross-Murphy SB .  Fibrillar \u03b2-lactoglobulin gels: Part 1 .  _Fibril formation and structure. Biomacromolecules_.  2004 ;5 (6) : 2408 \u2013 2419 .\n\nGottschalk M ,  Nilsson H ,  Roos H ,  Halle B .  Protein self-association in solution: the bovine \u03b2-lactoglobulin dimer and octamer .  _Protein Science_.  2003 ;12 (11) : 2404 \u2013 2411 .\n\nGreen DW ,  Aschaffenburg R ,  Camerman A ,  Coppola JC ,  Dunnill P ,  Simmons RM ,  Komorowski ES ,  Sawyer L ,  Turner EMC ,  Woods KF .  Structure of bovine \u03b2-lactoglobulin at 6\u00c5 resolution .  _Journal of Molecular Biology_.  1979 ;131 (2) : 375 \u2013 397 .\n\nGrobler JA ,  Rao KR ,  Pervaiz S ,  Brew K .  Sequences of two highly divergent canine type c lysozymes: Implications for the evolutionary origins of the lysozyme\/\u03b1-lactalbumin superfamily .  _Archives of Biochemistry and Biophysics_.  1994 ;313 (2) : 360 \u2013 366 .\n\nGroves ML ,  Hipp NJ ,  McMeekin TL .  Effect of pH on the denaturation of \u03b2-lactoglobulin and its dodecyl sulfate derivative .  _Journal of the American Chemical Society_.  1951 ;73 : 2790 \u2013 2793 .\n\nGuo L-H ,  Qu N .  Chemical-induced unfolding of cofactor-free protein monitored by electrochemistry .  _Analytical Chemistry_.  2006 ;78 (17) : 6275 \u2013 6278 .\n\nGuss JM ,  Messer M ,  Costello M ,  Hardy K ,  Kumar V .  Structure of the calcium-binding echidna milk lysozyme at 1.9 \u00c5 resolution .  _Acta Crystallographica, Section D: Biological Crystallography_.  1997 ;D53 (4) : 355 \u2013 363 .\n\nGuti\u00e9rrez-Magdaleno G ,  Bello M ,  Portillo-T\u00e9llez MC ,  Rodr\u00edguez-Romero A ,  Garcia-Hern\u00e1ndez E .  Ligand binding and self-association cooperativity of \u03b2-lactoglobulin .  _Journal of Molecular Recognition_.  2013 ;26 (2) : 67 \u2013 75 .\n\nHagihara Y ,  Aimoto S ,  Fink AL ,  Goto Y .  Guanidine hydrochloride-induced folding of proteins .  _Journal of Molecular Biology_.  1993 ;231 (2) : 180 \u2013 184 .\n\nHamada D ,  Goto Y .  The equilibrium intermediate of \u03b2-lactoglobulin with non-native \u03b1-helical structure .  _Journal of Molecular Biology_.  1997 ;269 (4) : 479 \u2013 487 .\n\nHamada D ,  Kuroda Y ,  Tanaka T ,  Goto Y .  High helical propensity of the peptide fragments derived from \u03b2-lactoglobulin, a predominantly \u03b2-sheet protein .  _Journal of Molecular Biology_.  1995 ;254 (4) : 737 \u2013 746 .\n\nHambling SG ,  McAlpine AS ,  Sawyer L .  \u03b2-Lactoglobulin .  _Advanced Dairy Chemistry_.  1992 ;1 : 141 \u2013 190 .\n\nHamilton JA ,  Cistola DP ,  Morrisett JD ,  Sparrow JT ,  Small DM .  Interactions of myristic acid with bovine serum albumin: A 13C NMR study .  _Proceedings of the National Academy of Sciences of the USA_.  1984 ;81 (12) : 3718 \u2013 3722 .\n\nHamilton JA ,  Era S ,  Bhamidipati SP ,  Reed RG .  Locations of the 3 primary binding sites for long-chain fatty acids on bovine serum albumin .  _Proceedings of the National Academy of Sciences of the USA_.  1991 ;88 (6) : 2051 \u2013 2054 .\n\nHaridas M ,  Anderson BF ,  Baker EN .  Structure of human diferric lactoferrin refined at 2.2 \u00c5 resolution .  _Acta Crystallographica Section D: Biological Crystallography_.  1995 ;51 (5) : 629 \u2013 646 .\n\nHavea P ,  Singh H ,  Creamer LK .  Characterization of heat-induced aggregates of \u03b2-lactoglobulin, \u03b1-lactalbumin and bovine serum albumin in a whey protein concentrate environment .  _Journal of Dairy Research_.  2001 ;68 (3) : 483 \u2013 497 .\n\nHe Y ,  Ning T ,  Xie T ,  Qiu Q ,  Zhang L ,  Sun Y ,  Jiang D ,  Fu K ,  Yin F ,  Zhang W ,  Shen L ,  Wang H ,  Li J ,  Lin Q ,  Sun Y ,  Li H ,  Zhu Y ,  Yang D .  Large-scale production of functional human serum albumin from transgenic rice seeds .  _Proceedings of the National Academy of Sciences of the USA_.  2011 ;108 (47) : 19078 \u2013 19083 .\n\nHettiarachchi CA ,  Melton LD ,  Gerrard JA ,  Loveday SM .  Formation of \u03b2-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conventional heating .  _Biomacromolecules_.  2012 ;13 (9) : 2868 \u2013 2880 .\n\nHo JX ,  Holowachuk EW ,  Norton EJ ,  Twigg PD ,  Carter DC .  X-ray and primary structure of horse serum-albumin (Equus caballus) at 0.27 nm resolution .  _European Journal of Biochemistry_.  1993 ;215 (1) : 205 \u2013 212 .\n\nHoedemaeker FJ ,  Visschers RW ,  Alting AC ,  de Kruif KG ,  Kuil ME ,  Abrahams JP .  A novel pH-dependent dimerization motif in \u03b2-lactoglobulin from pig (Sus scrofa) .  _Acta Crystallographica, Section D: Biological Crystallography_.  2002 ;D58 (3) : 480 \u2013 486 .\n\nHong Y-H ,  Creamer LK .  Changed protein structures of bovine \u03b2-lactoglobulin B and \u03b1-lactalbumin as a consequence of heat treatment .  _International Dairy Journal_.  2002 ;12 (4) : 345 \u2013 359 .\n\nIametti S ,  De Gregori B ,  Vecchio G ,  Bonomi F .  Modifications occur at different structural levels during the heat denaturation of \u03b2-lactoglobulin .  _European Journal of Biochemistry_.  1996 ;237 (1) : 106 \u2013 112 .\n\nIametti S ,  Transidico P ,  Bonomi F ,  Vecchio G ,  Pittia P ,  Rovere P ,  Dall'Aglio G .  Molecular modifications of \u03b2-lactoglobulin upon exposure to high pressure .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 (1) : 23 \u2013 29 .\n\nInvernizzi G ,  Samalikova M ,  Brocca S ,  Lotti M ,  Molinari H ,  Grandori R .  Comparison of bovine and porcine \u03b2-lactoglobulin: A mass spectrometric analysis .  _Journal of Mass Spectrometry_.  2006 ;41 (6) : 717 \u2013 727 .\n\nIshikawa N ,  Chiba T ,  Chen LT ,  Shimizu A ,  Ikeguchi M ,  Sugai S .  Remarkable destabilization of recombinant \u03b1-lactalbumin by an extraneous N-terminal methionyl residue .  _Protein Engineering_.  1998 ;11 (5) : 333 \u2013 335 .\n\nJameson GB ,  Adams JJ ,  Creamer LK .  Flexibility, functionality and hydrophobicity of bovine \u03b2-lactoglobulin .  _International Dairy Journal_.  2002 ;12 (4) : 319 \u2013 329 .\n\nJameson GB ,  Anderson BF ,  Breyer WA ,  Day CL ,  Tweedie JW ,  Baker EN .  Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form .  _Acta Crystallographica, Section D: Biological Crystallography_.  2002 ;58 (6\/2) : 955 \u2013 962 .\n\nJameson GB ,  Anderson BF ,  Norris GE ,  Thomas DH ,  Baker EN .  Structure of human apolactoferrin at 2.0 \u00c5 resolution. Refinement and analysis of ligand-induced conformational change .  _Acta Crystallographica, Section D: Biological Crystallography_.  1998 ;54 (6\/2) : 1319 \u2013 1335 .\n\nJameson, G.B., Anderson, B.F., Norris, G.E., Thomas, D.H., Baker, E.N., 1999. Structure of human apolactoferrin at 2.0 \u00c5 resolution. Refinement and analysis of ligand-induced conformational change. (corrigendum: vol 54, pg 1319, 1998). Acta Crystallographica, Section D: Biological Crystallography 55 (5), 1108.\n\nJegouic M ,  Grinberg VY ,  Guingant A ,  Haertl\u00e9 T .  Thiol-induced oligomerization of \u03b1-lactalbumin at high pressure .  _Journal of Protein Chemistry_.  1996 ;15 (6) : 501 \u2013 509 .\n\nJordens S ,  Adamcik J ,  Amar-Yuli I ,  Mezzenga R .  Disassembly and reassembly of amyloid fibrils in water-ethanol mixtures .  _Biomacromolecules_.  2011 ;12 (1) : 187 \u2013 193 .\n\nKaminogawa S ,  Shimizu M ,  Ametani A ,  Hattori M ,  Ando O ,  Hachimura S ,  Nakamura Y ,  Totsuka M ,  Yamauchi K .  Monoclonal antibodies as probes for monitoring the denaturation process of bovine \u03b2-lactoglobulin .  _Biochimica et Biophysica Acta_.  1989 ;998 (1) : 50 \u2013 56 .\n\nKarthikeyan S ,  Paramasivam M ,  Yadav S ,  Srinivasan A ,  Singh TP .  Structure of buffalo lactoferrin at 2.5 \u00c5 resolution using crystals grown at 303 K shows different orientations of the N and C lobes .  _Acta Crystallographica, Section D: Biological Crystallography_.  1999 ;55 : 1805 \u2013 1813 .\n\nKawakami A ,  Kubota K ,  Yamada N ,  Tagami U ,  Takehana K ,  Sonaka I ,  Suzuki E ,  Hirayama K .  Identification and characterization of oxidized human serum albumin. A slight structural change impairs its ligand-binding and antioxidant functions .  _FEBS Journal_.  2006 ;273 (14) : 3346 \u2013 3357 .\n\nKella NK ,  Kinsella JE .  Enhanced thermodynamic stability of \u03b2-lactoglobulin at low pH. A possible mechanism .  _Biochemical Journal_.  1988 ;255 (1) : 113 \u2013 118 .\n\nKhan JA ,  Kumar P ,  Paramasivam M ,  Yadav RS ,  Sahani MS ,  Sharma S ,  Srinivasan A ,  Singh TP .  Camel lactoferrin, a transferrin-cum-lactoferrin: Crystal structure of camel apolactoferrin at 2.6 \u00c5 resolution and structural basis of its dual role .  _Journal of Molecular Biology_.  2001 ;309 (3) : 751 \u2013 761 .\n\nKhan MY ,  Agarwal SK ,  Hangloo S .  Urea-induced structural transformations in bovine serum albumin .  _Journal of Biochemistry (Tokyo, Japan)_.  1987 ;102 (2) : 313 \u2013 317 .\n\nKim T-R ,  Goto Y ,  Hirota N ,  Kuwata K ,  Denton H ,  Wu S-Y ,  Sawyer L ,  Batt CA .  High-level expression of bovine \u03b2-lactoglobulin in Pichia pastoris and characterization of its physical properties .  _Protein Engineering_.  1997 ;10 (11) : 1339 \u2013 1345 .\n\nKnudsen JC ,  Otte J ,  Olsen K ,  Skibsted LH .  Effect of high hydrostatic pressure on the conformation of \u03b2-lactoglobulin A as assessed by proteolytic peptide profiling .  _International Dairy Journal_.  2002 ;12 (10) : 791 \u2013 803 .\n\nKobayashi T ,  Ikeguchi M ,  Sugai S .  Molten globule structure of equine \u03b2-lactoglobulin probed by hydrogen exchange .  _Journal of Molecular Biology_.  2000 ;299 (3) : 757 \u2013 770 .\n\nKobayashi T ,  Ikeguchi M ,  Sugai S .  Construction and characterization of \u03b2-lactoglobulin chimeras .  _Proteins: Structure, Function, and Genetics_.  2002 ;49 (3) : 297 \u2013 301 .\n\nKontopidis G ,  Holt C ,  Sawyer L .  The ligand-binding site of bovine \u03b2-lactoglobulin: evidence for a function? .  _Journal of Molecular Biology_.  2002 ;318 (4) : 1043 \u2013 1055 .\n\nKontopidis G ,  Holt C ,  Sawyer L .  Invited Review: \u03b2-lactoglobulin: Binding properties, structure, and function .  _Journal of Dairy Science_.  2004 ;87 (4) : 785 \u2013 796 .\n\nKonuma T ,  Sakurai K ,  Goto Y .  Promiscuous binding of ligands by \u03b2-lactoglobulin involves hydrophobic interactions and plasticity .  _Journal of Molecular Biology_.  2007 ;368 (1) : 209 \u2013 218 .\n\nKroes-Nijboer A ,  Venema P ,  van der Linden E .  Fibrillar structures in food .  _Food Function_.  2012 ;3 (3) : 221 \u2013 227 .\n\nKumar S ,  Modig K ,  Halle B .  Trifluoroethanol-induced \u03b2 \u2192 \u03b1 transition in \u03b2-lactoglobulin: Hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion .  _Biochemistry_.  2003 ;42 (46) : 13708 \u2013 13716 .\n\nKumar Y ,  Muzammil S ,  Tayyab S .  Influence of fluoro, chloro and alkyl alcohols on the folding pathway of human serum albumin .  _Journal of Biochemistry (Tokyo, Japan)_.  2005 ;138 (4) : 335 \u2013 341 .\n\nKumosinski TF ,  Timasheff SN .  Molecular interactions in \u03b2-lactoglobulin. X. The stoichiometry of the \u03b2-lactoglobulin mixed tetramerization .  _Journal of the American Chemical Society_.  1966 ;88 (23) : 5635 \u2013 5642 .\n\nKuroda Y ,  Hamada D ,  Tanaka T ,  Goto Y .  High helicity of peptide fragments corresponding to \u03b2-strand regions of \u03b2-lactoglobulin observed by 2D-NMR spectroscopy .  _Folding & Design_.  1996 ;1 (4) : 255 \u2013 263 .\n\nKuwajima K .  The molten globule state of \u03b1-lactalbumin .  _FASEB Journal_.  1996 ;10 (1) : 102 \u2013 109 .\n\nKuwata K ,  Hoshino M ,  Era S ,  Batt CA ,  Goto Y .  \u03b1 \u2192 \u03b2 Transition of \u03b2-lactoglobulin as evidenced by heteronuclear NMR .  _Journal of Molecular Biology_.  1998 ;283 (4) : 731 \u2013 739 .\n\nKuwata K ,  Hoshino M ,  Forge V ,  Era S ,  Batt CA ,  Goto Y .  Solution structure and dynamics of bovine \u03b2-lactoglobulin A .  _Protein Science_.  1999 ;8 (11) : 2541 \u2013 2545 .\n\nKuwata K ,  Li H ,  Yamada H ,  Batt CA ,  Goto Y ,  Akasaka K .  High pressure NMR reveals a variety of fluctuating conformers in \u03b2-lactoglobulin .  _Journal of Molecular Biology_.  2001 ;305 (5) : 1073 \u2013 1083 .\n\nLampreave F ,  Pi\u00f1eiro A ,  Brock JH ,  Castillo H ,  S\u00e1nchez L ,  Calvo M .  Interaction of bovine lactoferrin with other proteins of milk whey .  _International Journal of Biological Macromolecules_.  1990 ;12 (1) : 2 \u2013 5 .\n\nLange DC ,  Kothari R ,  Patel RC ,  Patel SC .  Retinol and retinoic acid bind to a surface cleft in bovine \u03b2-lactoglobulin: A method of binding site determination using fluorescence resonance energy transfer .  _Biophysical Chemistry_.  1998 ;74 (1) : 45 \u2013 51 .\n\nLapanje S ,  Skerjanc J .  Dilatometric study of the denaturation of bovine serum albumin by guanidine hydrochloride .  _Biochemical and Biophysical Research Communications_.  1974 ;56 (2) : 338 \u2013 342 .\n\nLi SQ ,  Bomser JA ,  Zhang QH .  Effects of pulsed electric fields and heat treatment on stability and secondary structure of bovine immunoglobulin G .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (3) : 663 \u2013 670 .\n\nLoch J ,  Polit A ,  G\u00f3recki A ,  Bonarek P ,  Kurpiewska K ,  Dziedzicka-Wasylewska M ,  Lewinski K .  Two modes of fatty acid binding to bovine \u03b2-lactoglobulin\u2014crystallographic and spectroscopic studies .  _Journal of Molecular Recognition_.  2011 ;24 (2) : 341 \u2013 349 .\n\nLoch JI ,  Bonarek P ,  Polit A ,  Ri\u00e8s D ,  Dziedzicka-Wasylewska M ,  Lewinski K .  Binding of 18-carbon unsaturated fatty acids to bovine \u03b2-lactoglobulin-structural and thermodynamic studies .  _International Journal of Biological Macromolecules_.  2013 ;57 : 226 \u2013 231 .\n\nLoch JI ,  Bonarek P ,  Polit A ,  Swiatek S ,  Dziedzicka-Wasylewska M ,  Lewinski K .  The differences in binding 12-carbon aliphatic ligands by bovine \u03b2-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography .  _Journal of Molecular Recognition_.  2013 ;26 (8) : 357 \u2013 367 .\n\nLoch JI ,  Polit A ,  Bonarek P ,  Olszewska D ,  Kurpiewska K ,  Dziedzicka-Wasylewska M ,  Lewinski K .  Structural and thermodynamic studies of binding saturated fatty acids to bovine \u03b2-lactoglobulin .  _International Journal of Biological Macromolecules_.  2012 ;50 (4) : 1095 \u2013 1102 .\n\nL\u00f3pez-Fa\u00f1dino R .  High pressure-induced changes in milk proteins and possible applications in dairy technology .  _International Dairy Journal_.  2006 ;16 (10) : 1119 \u2013 1131 .\n\nLoveday SM ,  Wang XL ,  Rao MA ,  Anema SG ,  Singh H .  \u03b2-Lactoglobulin nanofibrils: Effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions .  _Food Hydrocolloids_.  2012 ;27 (1) : 242 \u2013 249 .\n\nLuebke M ,  Guichard E ,  Tromelin A ,  Le Quere JL .  Nuclear magnetic resonance spectroscopic study of \u03b2-lactoglobulin interactions with two flavor compounds, \u03b3-decalactone and \u03b2-ionone .  _Journal of Agricultural and Food Chemistry_.  2002 ;50 (24) : 7094 \u2013 7099 .\n\nMainer G ,  Sanchez L ,  Ena JM ,  Calvo M .  Kinetic and thermodynamic parameters for heat denaturation of bovine milk IgG, IgA and IgM .  _Journal of Food Science_.  1997 ;62 (5) : 1034 \u2013 1038 .\n\nMajorek KA ,  Porebski PJ ,  Dayal A ,  Zimmerman MD ,  Jablonska K ,  Stewart AJ ,  Chruszcz M ,  Minor W .  Structural and immunologic characterization of bovine, horse, and rabbit serum albumins .  _Molecular Immunology_.  2012 ;52 (3-4) : 174 \u2013 182 .\n\nMakabe, K., Nakamura, T., Kuwajima, K., 2012. Structural insights into the stability perturbations by N-terminal variations in human and goat alpha-lactalbumin www.rcsb.org. (PDB code: 3b0k (goat) and 3b0o. (human)).\n\nManderson GA ,  Hardman MJ ,  Creamer LK .  Spectroscopic examination of the heat-induced changes in \u03b2-lactoglobulin A, B and C .  _International Dairy Federation [Special Issue]_.  1997 ;9702 (Milk Protein Polymorphism) : 204 \u2013 211 .\n\nMcGuffey MK ,  Epting KL ,  Kelly RM ,  Foegeding EA .  Denaturation and aggregation of three \u03b1-lactalbumin preparations at neutral pH .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (8) : 3182 \u2013 3190 .\n\nMcKenzie HA ,  Sawyer WH .  Effect of pH on \u03b2-lactoglobulins .  _Nature (London)_.  1967 ;214 (5093) : 1101 \u2013 1104 .\n\nMercadante D ,  Melton LD ,  Norris GE ,  Loo TS ,  Williams MAK ,  Dobson RCJ ,  Jameson GB .  Bovine \u03b2-lactoglobulin is dimeric under imitative physiological conditions: dissociation equilibrium and rate constants over the pH range of 2.5-7.5 .  _Biophysical Journal_.  2012 ;103 (2) : 303 \u2013 312 .\n\nMessens W ,  Van Camp J ,  Dewettinck K .  High-pressure processing to improve dairy product quality .  _Dairy Processing: Improving Quality_ .  Boca Raton, FL :  CRC Press LLC ;  2003 : 310 \u2013 332 .\n\nMills OE .  Effect of temperature on tryptophan fluorescence of \u03b2-lactoglobulin B .  _Biochimica et Biophysica Acta_.  1976 ;434 (2) : 324 \u2013 332 .\n\nM\u00f8ller RE ,  Stapelfeldt H ,  Skibsted LH .  Thiol reactivity in pressure-unfolded \u03b2-lactoglobulin. Antioxidative properties and thermal refolding .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 (2) : 425 \u2013 430 .\n\nMonaco HL ,  Zanotti G ,  Spadon P ,  Bolognesi M ,  Sawyer L ,  Eliopoulos EE .  Crystal structure of the trigonal form of bovine \u03b2-lactoglobulin and of its complex with retinol at 2.5 \u00c5 resolution .  _Journal of Molecular Biology_.  1987 ;197 (4) : 695 \u2013 706 .\n\nMoore SA ,  Anderson BF ,  Groom CR ,  Haridas M ,  Baker EN .  Three-dimensional structure of diferric bovine lactoferrin at 2.8 \u00c5 resolution .  _Journal of Molecular Biology_.  1997 ;274 (2) : 222 \u2013 236 .\n\nMusante L ,  Bruschi M ,  Candiano G ,  Petretto A ,  Dimasi N ,  Del Boccio P ,  Urbani A ,  Rialdi G ,  Ghiggeri GM .  Characterization of oxidation end product of plasma albumin 'in vivo' .  _Biochemical and Biophysical Research Communications_.  2006 ;349 (2) : 668 \u2013 673 .\n\nMuzammil S ,  Kumar Y ,  Tayyab S .  Anion-induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation .  _Proteins: Structure, Function, and Genetics_.  2000 ;40 (1) : 29 \u2013 38 .\n\nNarayan M ,  Berliner LJ .  Mapping fatty acid binding to \u03b2-lactoglobulin: Ligand binding is restricted by modification of Cys 121 .  _Protein Science_.  1998 ;7 (1) : 150 \u2013 157 .\n\nNicolai T ,  Durand D .  Controlled food protein aggregation for new functionality .  _Current Opinion in Colloid and Interface Science_.  2013 ;18 (4) : 249 \u2013 256 .\n\nNiemi M ,  J\u00e4nis J ,  Jylh\u00e4 S ,  Kallio JM ,  Hakulinen N ,  Laukkanen M-L ,  Takkinen K ,  Rouvinen J .  Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine \u03b2-lactoglobulin allergen .  _Acta Crystallographica, Section F: Structural Biology Crystallization Communications_.  2008 ;64 : 25 \u2013 28 .\n\nNiemi M ,  Jylh\u00e4 S ,  Laukkanen ML ,  S\u00f6derlund H ,  M\u00e4kinen-Kiljunen S ,  Kallio JM ,  Hakulinen N ,  Haahtela T ,  Takkinen K ,  Rouvinen J .  Molecular interactions between a recombinant IgE antibody and the \u03b2-lactoglobulin allergen .  _Structure_.  2007 ;15 (11) : 1413 \u2013 1421 .\n\nNishikawa K ,  Noguchi T .  Predicting protein secondary structure based on amino acid sequence .  _Methods in Enzymology_.  1991 ;202 : 31 \u2013 44 .\n\nOboroceanu D ,  Wang L ,  Brodkorb A ,  Magner E ,  Auty MA .  Characterization of \u03b2-lactoglobulin fibrillar assembly using atomic force microscopy, polyacrylamide gel electrophoresis, and in situ fourier transform infrared spectroscopy .  _Journal of Agricultural and Food Chemistry_.  2010 ;58 (6) : 3667 \u2013 3673 .\n\nOhtomo H ,  Konuma T ,  Utsunoiya H ,  Tsuge H ,  Ikeguchi M .  Structure and stability of Gyuba, a \u03b2-lactoglobulin chimera .  _Protein Science_.  2011 ;20 (11) : 1867 \u2013 1875 .\n\nOksanen E ,  Jaakola VP ,  Tolonen T ,  Valkonen K ,  Aakerstr\u00f6m B ,  Kalkkinen N ,  Virtanen V ,  Goldman A .  Reindeer \u03b2-lactoglobulin crystal structure with pseudo-body-centered noncrystallographic symmetry .  _Acta Crystallographica, Section D: Biological Crystallography_.  2006 ;D62 (11) : 1369 \u2013 1374 .\n\nOkuno A ,  Kato M ,  Taniguchi Y .  Pressure effects on the heat-induced aggregation of equine serum albumin by FT-IR spectroscopic study: Secondary structure, kinetic and thermodynamic properties .  _Biochimica et Biophysica Acta_.  2007 ;1774 (5) : 652 \u2013 660 .\n\nOria R ,  Ismail M ,  S\u00e1nchez L ,  Calvo M ,  Brock JH .  Effect of heat treatment and other milk proteins on the interaction of lactoferrin with monocytes .  _Journal of Dairy Research_.  1993 ;60 (3) : 363 \u2013 369 .\n\nPace CN ,  Tanford C .  Thermodynamics of the unfolding of \u03b2-lactoglobulin A in aqueous urea solutions between 5 and 55\u00b0 .  _Biochemistry_.  1968 ;7 (1) : 198 \u2013 208 .\n\nPanick G ,  Malessa R ,  Winter R .  Differences between the pressure and temperature induced denaturation and aggregation of \u03b2-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle x-ray scattering .  _Biochemistry_.  1999 ;38 (20) : 6512 \u2013 6519 .\n\nPapiz MZ ,  Sawyer L ,  Eliopoulos EE ,  North ACT ,  Findlay JBC ,  Sivaprasadarao R ,  Jones TA ,  Newcomer ME ,  Kraulis PJ .  The structure of \u03b2-lactoglobulin and its similarity to plasma retinol-binding protein .  _Nature (London)_.  1986 ;324 (6095) : 383 \u2013 385 .\n\nPatel HA ,  Singh H ,  Havea P ,  Considine T ,  Creamer LK .  Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (24) : 9590 \u2013 9601 .\n\nPedersen KO .  Ultracentrifugal and electrophoretic studies on the milk proteins: The lactoglobulin of Palmer .  _Biochemical Journal_.  1936 ;30 (6) : 961 \u2013 970 .\n\nPermyakov EA ,  Berliner LJ .  \u03b1-Lactalbumin: Structure and function .  _FEBS Letters_.  2000 ;473 (3) : 269 \u2013 274 .\n\nPike ACW ,  Brew K ,  Acharya KR .  Crystal structures of guinea pig, goat and bovine \u03b1-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase .  _Structure (London)_.  1996 ;4 (6) : 691 \u2013 703 .\n\nPuyol P ,  P\u00e9rez MD ,  Peiro JM ,  Calvo M .  Effect of binding of retinol and palmitic acid to bovine \u03b2-lactoglobulin on its resistance to thermal denaturation .  _Journal of Dairy Science_.  1994 ;77 (6) : 1494 \u2013 1502 .\n\nQi XL ,  Brownlow S ,  Holt C ,  Sellers P .  Thermal denaturation of \u03b2-lactoglobulin: effect of protein concentration at pH 6.75 and 8.05 .  _Biochimica et Biophysica Acta_.  1995 ;1248 (1) : 43 \u2013 49 .\n\nQi XL ,  Holt C ,  McNulty D ,  Clarke DT ,  Brownlow S ,  Jones GR .  Effect of temperature on the secondary structure of \u03b2-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis .  _Biochemical Journal_.  1997 ;324 (1) : 341 \u2013 346 .\n\nQin BY ,  Bewley MC ,  Creamer LK ,  Baker EN ,  Jameson GB .  Functional implications of structural differences between variants A and B of bovine \u03b2-lactoglobulin .  _Protein Science_.  1999 ;8 (1) : 75 \u2013 83 .\n\nQin BY ,  Bewley MC ,  Creamer LK ,  Baker HM ,  Baker EN ,  Jameson GB .  Structural basis of the Tanford transition of bovine \u03b2-lactoglobulin .  _Biochemistry_.  1998 ;37 (40) : 14014 \u2013 14023 .\n\nQin BY ,  Creamer LK ,  Baker EN ,  Jameson GB .  12-Bromododecanoic acid binds inside the calyx of bovine \u03b2-lactoglobulin .  _FEBS Letters_.  1998 ;438 : 272 \u2013 278 .\n\nRagona L ,  Fogolari F ,  Catalano M ,  Ugolini R ,  Zetta L ,  Molinari H .  EF Loop conformational change triggers ligand binding in \u03b2-lactoglobulins .  _Journal of Biological Chemistry_.  2003 ;278 (40) : 38840 \u2013 38846 .\n\nRagona L ,  Fogolari F ,  Romagnoli S ,  Zetta L ,  Maubois JL ,  Molinari H .  Unfolding and refolding of bovine \u03b2-lactoglobulin monitored by hydrogen exchange measurements .  _Journal of Molecular Biology_.  1999 ;293 (4) : 953 \u2013 969 .\n\nRagona L ,  Fogolari F ,  Zetta L ,  Perez DM ,  Puyol P ,  de Kruif KG ,  Lohr F ,  Ruterjans H ,  Molinari H .  Bovine \u03b2-lactoglobulin: Interaction studies with palmitic acid .  _Protein Science_.  2000 ;9 (7) : 1347 \u2013 1356 .\n\nRagona L ,  Pusterla F ,  Zetta L ,  Monaco HL ,  Molinari H .  Identification of a conserved hydrophobic cluster in partially folded bovine \u03b2-lactoglobulin at pH 2 .  _Folding and Design_.  1997 ;2 (5) : 281 \u2013 290 .\n\nRamakrishnan B ,  Qasba PK .  Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the \u03b21,4-galactosyltransferase-I .  _Journal of Molecular Biology_.  2001 ;310 (1) : 205 \u2013 218 .\n\nRamakrishnan, B., and P. K. Qasba., 2013. Crystal structure of human lactose synthase forms a novel 12 complex between \u03b2-1,4-galactosyltransferase 1 and \u03b1-lactalbumin proteins. (www.rcsb.org. PDSB code: 4L41).\n\nRamakrishnan B ,  Ramasamy V ,  Qasba PK .  Structural snapshots of \u03b2-1,4-galactosyltransferase-I along the kinetic pathway .  _Journal of Molecular Biology_.  2006 ;357 (5) : 1619 \u2013 1633 .\n\nRamakrishnan B ,  Shah PS ,  Qasba PK .  \u03b1-Lactalbumin (LA) stimulates milk \u03b2-1,4-galactosyltransferase I (\u03b24Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of \u03b24Gal-T1\u00b7LA complex with UDP-Glc .  _Journal of Biological Chemistry_.  2001 ;276 (40) : 37665 \u2013 37671 .\n\nRelkin P .  Thermal unfolding of \u03b2-lactoglobulin, \u03b1-lactalbumin, and bovine serum albumin. A thermodynamic approach .  _Critical Reviews in Food Science and Nutrition_.  1996 ;36 (6) : 565 \u2013 601 .\n\nRelkin P ,  Eynard L ,  Launay B .  Thermodynamic parameters of \u03b2-lactoglobulin and \u03b1-lactalbumin. A DSC study of denaturation by heating .  _Thermochimica Acta_.  1992 ;204 (1) : 111 \u2013 121 .\n\nRossi P ,  Giansanti F ,  Boffi A ,  Ajello M ,  Valenti P ,  Chiancone E ,  Antonini G .  Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide .  _Biochemistry and Cell Biology_.  2002 ;80 (1) : 41 \u2013 48 .\n\nRuegg M ,  Moor U ,  Blanc B .  Calorimetric study of thermal-denaturation of whey proteins in simulated milk ultrafiltrate .  _Journal of Dairy Research_.  1977 ;44 (3) : 509 \u2013 520 .\n\nSakai K ,  Sakurai K ,  Sakai M ,  Hoshino M ,  Goto Y .  Conformation and stability of thiol-modified bovine \u03b2-lactoglobulin .  _Protein Science_.  2000 ;9 (9) : 1719 \u2013 1729 .\n\nSakurai K ,  Goto Y .  Manipulating monomer-dimer equilibrium of bovine \u03b2-lactoglobulin by amino acid substitution .  _Journal of Biological Chemistry_.  2002 ;277 (28) : 25735 \u2013 25740 .\n\nSakurai K ,  Goto Y .  Dynamics and mechanism of the Tanford transition of bovine \u03b2-lactoglobulin studied using heteronuclear NMR spectroscopy .  _Journal of Molecular Biology_.  2006 ;356 (2) : 483 \u2013 496 .\n\nSakurai K ,  Konuma T ,  Yagi M ,  Goto Y .  Structural dynamics and folding of \u03b2-lactoglobulin probed by heteronuclear NMR .  _Biochimica et Biophysica Acta_.  2009 ;1790 (6) : 527 \u2013 537 .\n\nSakurai K ,  Oobatake M ,  Goto Y .  Salt-dependent monomer-dimer equilibrium of bovine \u03b2-lactoglobulin at pH 3 .  _Protein Science_.  2001 ;10 (11) : 2325 \u2013 2335 .\n\nSakurai Y ,  Ma SF ,  Watanabe H ,  Yamaotsu N ,  Hirono S ,  Kurono Y ,  Kragh-Hansen U ,  Otagiri M .  Esterase-like activity of serum albumin: Characterization of its structural chemistry using p-nitrophenyl esters as substrates .  _Pharmaceutical Research_.  2004 ;21 (2) : 285 \u2013 292 .\n\nS\u00e1nchez L ,  Peir\u00f3 JM ,  Castillo H ,  P\u00e9rez MD ,  Ena JM ,  Calvo M .  Kinetic parameters for denaturation of bovine milk lactoferrin .  _Journal of Food Science_.  1992 ;57 (4) : 873 \u2013 879 .\n\nSawyer L ,  Brownlow S ,  Polikarpov I ,  Wu S-Y .  \u03b2-Lactoglobulin: structural studies, biological clues .  _International Dairy Journal_.  1998 ;8 (2) : 65 \u2013 72 .\n\nSchokker EP ,  Singh H ,  Pinder DN ,  Creamer LK .  Heat-induced aggregation of \u03b2-lactoglobulin AB at pH 2.5 as influenced by ionic strength and protein concentration .  _Journal of Agricultural and Food Chemistry_.  2000 ;10 : 233 \u2013 240 .\n\nSekula B ,  Zielinski K ,  Bujacz A .  Crystallographic studies of the complexes of bovine and equine serum albumin with 3,5-diiodosalicylic acid .  _International Journal of Biological Macromolecules_.  2013 ;60 : 316 \u2013 324 .\n\nSharma AK ,  Paramasivam M ,  Srinivasan A ,  Yadav MP ,  Singh TP .  Three-dimensional structure of mare diferric lactoferrin at 2.6 \u00c5 resolution .  _Journal of Molecular Biology_.  1999 ;289 (2) : 303 \u2013 317 .\n\nShiraki K ,  Nishikawa K ,  Goto Y .  Trifluoroethanol-induced stabilization of the \u03b1-helical structure of \u03b2-lactoglobulin: Implication for non-hierarchical protein folding .  _Journal of Molecular Biology_.  1995 ;245 (2) : 180 \u2013 194 .\n\nShrake A ,  Frazier D ,  Schwarz FP .  Thermal stabilization of human albumin by medium- and short-chain n-alkyl fatty acid anions .  _Biopolymers_.  2006 ;81 (4) : 235 \u2013 248 .\n\nSimard JR ,  Zunszain PA ,  Ha CE ,  Yang JS ,  Bhagavan NV ,  Petitpas I ,  Curry S ,  Hamilton JA .  Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy .  _Proceedings of the National Academy of Sciences of the USA_.  2005 ;102 (50) : 17958 \u2013 17963 .\n\nSkibsted LH ,  Orlien V ,  Stapelfeldt H .  Temperature effects on pressure denaturation of \u03b2-lactoglobulin .  _Milchwissenschaft_.  2007 ;62 (1) : 13 \u2013 15 .\n\nSpector AA .  Fatty-acid binding to plasma albumin .  _Journal of Lipid Research_.  1975 ;16 (3) : 165 \u2013 179 .\n\nStapelfeldt H ,  Petersen PH ,  Kristiansen KR ,  Qvist KB ,  Skibsted LH .  Effect of high hydrostatic pressure on the enzymic hydrolysis of \u03b2-lactoglobulin B by trypsin, thermolysin and pepsin .  _Journal of Dairy Research_.  1996 ;63 (1) : 111 \u2013 118 .\n\nStapelfeldt H ,  Skibsted LH .  Pressure denaturation and aggregation of \u03b2-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing .  _Journal of Dairy Research_.  1999 ;66 (4) : 545 \u2013 558 .\n\nSteinrauf LK .  Structures of monoclinic lysozyme iodide at 1.6 \u00c5 and of triclinic lysozyme nitrate at 1.1 \u00c5 .  _Acta Crystallographica, Section D: Biological Crystallography_.  1998 ;D54 (5) : 767 \u2013 779 .\n\nStr\u00f8m MB ,  Haug BE ,  Rekdal O ,  Skar ML ,  Stensen W ,  Svendsen JS .  Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity .  _Biochemistry and Cell Biology_.  2002 ;80 (1) : 65 \u2013 74 .\n\nStr\u00f8m MB ,  Rekdal O ,  Svendsen JS .  Antibacterial activity of 15-residue lactoferricin derivatives .  _Journal of Peptide Research_.  2000 ;56 (5) : 265 \u2013 274 .\n\nSun C ,  Yang J ,  Wu X ,  Huang X ,  Wang F ,  Liu S .  Unfolding and refolding of bovine serum albumin induced by cetylpyridinium bromide .  _Biophysical Journal_.  2005 ;88 (5) : 3518 \u2013 3524 .\n\nSvensson M ,  H\u00e5kansson A ,  Mossberg AK ,  Linse S ,  Svanborg C .  Conversion of alpha-lactalbumin to a protein inducing apoptosis .  _Proceedings of the National Academy of Sciences of the USA_.  2000 ;97 (8) : 4221 \u2013 4226 .\n\nTanaka N ,  Kunugi S .  Effect of pressure on the deuterium exchange reaction of \u03b1-lactalbumin and \u03b2-lactoglobulin .  _International Journal of Biological Macromolecules_.  1996 ;18 (1,2) : 33 \u2013 39 .\n\nTanaka N ,  Nishizawa H ,  Kunugi S .  Structure of pressure-induced denatured state of human serum albumin: a comparison with the intermediate in urea-induced denaturation .  _Biochimica et Biophysica Acta_.  1997 ;1338 (1) : 13 \u2013 20 .\n\nTanford C ,  Bunville LG ,  Nozaki Y .  Reversible transformation of \u03b2-lactoglobulin at pH 7.5 .  _Journal of the American Chemical Society_.  1959 ;81 : 4032 \u2013 4036 .\n\nTanford C ,  De PK ,  Taggart VG .  The role of the \u03b1-helix in the structure of proteins. Optical rotatory dispersion of \u03b2-lactoglobulin .  _Journal of the American Chemical Society_.  1960 ;82 : 6028 \u2013 6034 .\n\nTanford C ,  Nozaki Y .  Physico-chemical comparison of \u03b2-lactoglobulins A and B .  _Journal of Biological Chemistry_.  1959 ;234 : 2874 \u2013 2877 .\n\nTayyab S ,  Sharma N ,  Mushahid Khan M .  Use of domain specific ligands to study urea-induced unfolding of bovine serum albumin .  _Biochemical and Biophysical Research Communications_.  2000 ;277 (1) : 83 \u2013 88 .\n\nThomas PD ,  Dill KA .  Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation .  _Protein Science_.  1993 ;2 (12) : 2050 \u2013 2065 .\n\nTromelin A ,  Guichard E .  Interaction between flavor compounds and \u03b2-lactoglobulin: Approach by NMR and 2D\/3D-QSAR studies of ligands .  _Flavour and Fragrance Journal_.  2006 ;21 (1) : 13 \u2013 24 .\n\nTsuge H ,  Ago H ,  Noma M ,  Nitta K ,  Sugai S ,  Miyano M .  Crystallographic studies of a calcium binding lysozyme from equine milk at 2.5 \u00c5 resolution .  _Journal of Biochemistry_.  1992 ;111 (2) : 141 \u2013 143 .\n\nUgolini R ,  Ragona L ,  Silletti E ,  Fogolari F ,  Visschers RW ,  Alting AC ,  Molinari H .  Dimerization, stability and electrostatic properties of porcine \u03b2-lactoglobulin .  _European Journal of Biochemistry_.  2001 ;268 (16) : 4477 \u2013 4488 .\n\nUhr\u00ednov\u00e1 S ,  Smith MH ,  Jameson GB ,  Uhr\u00edn D ,  Sawyer L ,  Barlow PN .  Structural changes accompanying pH-induced dissociation of the \u03b2-lactoglobulin dimer .  _Biochemistry_.  2000 ;39 (13) : 3565 \u2013 3574 .\n\nUhr\u00ednov\u00e1 S ,  Uhr\u00edn D ,  Denton H ,  Smith M ,  Sawyer L ,  Barlow PN .  Complete assignment of 1H, 13C and 15N chemical shifts for bovine \u03b2-lactoglobulin: Secondary structure and topology of the native state is retained in a partially unfolded form .  _Journal of Biomolecular NMR_.  1998 ;12 (1) : 89 \u2013 107 .\n\nvan Hooijdonk T ,  Steijns J .  Incorporation of bioactive substances into products: technological challenges .  _Bulletin of the International Dairy Federation_.  2002 ;375 : 65 \u2013 69 .\n\nVant SC ,  Glen NF ,  Kontopidis G ,  Sawyer L ,  Schaschke CJ .  Volumetric changes to the molecular structure of \u03b2-lactoglobulin processed at high pressure .  _High Temperatures \u2013High Pressures_.  2002 ;34 (6) : 705 \u2013 712 .\n\nWal JM .  Allergy review series II: An update on allergens: cow's milk allergens .  _Allergy (Copenhagen)_.  1998 ;53 (11) : 1013 \u2013 1022 .\n\nWatanabe S ,  Sato T .  Effects of free fatty acids on the binding of bovine and human serum albumin with steroid hormones .  _Biochimica et Biophysica Acta_.  1996 ;1289 (3) : 385 \u2013 396 .\n\nWehbi Z ,  P\u00e9rez M-D ,  Sanchez L ,  Pocovi C ,  Barbana C ,  Calvo M .  Effect of heat treatment on denaturation of bovine \u03b1-lactalbumin: Determination of kinetic and thermodynamic parameters .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (25) : 9730 \u2013 9736 .\n\nWu SY ,  P\u00e9rez MD ,  Puyol P ,  Sawyer L .  \u03b2-Lactoglobulin binds palmitate within its central cavity .  _Journal of Biological Chemistry_.  1999 ;274 (1) : 170 \u2013 174 .\n\nYamamoto M ,  Nakagawa K ,  Fujiwara K ,  Shimizu A ,  Ikeguchi M ,  Ikeguchi M .  A native disulfide stabilizes non-native helical structures in partially folded states of equine \u03b2-lactoglobulin .  _Biochemistry_.  2011 ;50 (49) : 10590 \u2013 10597 .\n\nYang J ,  Dunker AK ,  Powers JR ,  Clark S ,  Swanson BG .  \u03b2-lactoglobulin molten globule induced by high pressure .  _Journal of Agricultural and Food Chemistry_.  2001 ;49 (7) : 3236 \u2013 3243 .\n\nYang M-C ,  Guan H-H ,  Liu M-Y ,  Lin Y-H ,  Yang J-M ,  Chen W-L ,  Chen C-J ,  Mao SJT .  Crystal structure of a secondary vitamin D3 binding site of milk \u03b2-lactoglobulin .  _Proteins: Structure, Function, and Bioinformatics_.  2008 ;71 (30) : 1197 \u2013 1210 .\n\nYe M-Q ,  Yi T-Y ,  Li H-P ,  Guo L-L ,  Zou G-L .  Study on thermal and thermal chemical denaturation of bovine immunoglobulin G .  _Huaxue Xuebao_.  2005 ;63 (22) : 2047 \u2013 2054 .\n\nZhang H ,  Deligeersang J Guo ,  Mu Z ,  Zhang Y ,  Zhu H .  Denaturation of bovine milk IgG at high pressure and its stabilization .  _Shipin Kexue (Beijing)_.  1998 ;19 (4) : 10 \u2013 12 .\n\nZsila F .  A new ligand for an old lipocalin: Induced circular dichroism spectra reveal binding of bilirubin to bovine \u03b2-lactoglobulin .  _FEBS Letters_.  2003 ;539 (1-3) : 85 \u2013 90 .\n\nZsila F ,  Bikadi Z ,  Simonyi M .  Retinoic acid binding properties of the lipocalin member \u03b2-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modeling methods .  _Biochemical Pharmacology_.  2002 ;64 (11) : 1651 \u2013 1660 .\n\nZsila F ,  Hazai E ,  Sawyer L .  Binding of the pepper alkaloid piperine to bovine \u03b2-lactoglobulin: Circular dichroism spectroscopy and molecular modeling study .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (26) : 10179 \u2013 10185 . \nChapter 8\n\n# Effects of High-pressure Processing on Structure and Interactions of Milk Proteins\n\nHasmukh A. Patel*\n\nThom Huppertz**\n\n* Dairy Science Department, South Dakota State University, South Dakota, USA  \n** NIZO food research BV, Ede, the Netherlands\n\n## Abstract\n\nHigh-pressure processing (HPP) is a rapidly growing nonthermal preservation technology that can potentially be used to create novel protein structures by bringing about particular changes in the molecular structure of proteins and thus may give rise to innovative, new generation value-added food products with new properties that are not possible through conventional methods of protein modification. Compared to heat treatment, HPP has been reported to have some similar and some different effects on the structure of milk proteins. Knowledge of the effects of HPP on the structure and interactions of milk proteins, therefore, can be used to develop food products with novel textures.\n\nIn this chapter, we review recent literature reports on how HPP can affect the structure of casein micelles and individual casein fractions, as well as denaturation and aggregation of whey proteins and their interactions with caseins. The mechanisms and pathways underlying pressure-induced denaturation and aggregation of whey proteins and their interactions with casein micelles are explained in various systems, including model systems, commercial whey protein solutions, and skim milk.\n\n## Keywords\n\nHigh pressure\n\nhigh-pressure processing\n\nheat, milk proteins\n\ncasein\n\nwhey proteins\n\nprotein structure\n\ndenaturation\n\naggregation\n\nprotein interactions\n\nOutline\n\nIntroduction 244\n\nHigh-pressure-induced changes in caseins 244\n\nEffects on Individual Casein Fractions 244\n\nEffects on Casein Micelles 245\n\nEffects of HPP on Caseinates 247\n\nEffects of high pressure on interactions of milk proteins involving whey proteins 247\n\nDenaturation and Aggregation of Pure Whey Protein Fractions in Model Systems 248\n\n\u03b2-Lactoglobulin (\u03b2-LG) 248\n\n\u03b1-Lactalbumin (\u03b1-LA) 250\n\nBovine Serum Albumin (BSA) 251\n\nImmunoglobulins (Igs) and Lactoferrin (LF) 251\n\nMixtures of \u03b1-LA and \u03b2-LG 252\n\nMixtures of \u03b2-LG, \u03b1-LA, and BSA 252\n\nCommercial Whey Protein Solutions 252\n\nPressure-induced Gelation of Whey Proteins 253\n\nHPP-induced Changes in Milk 256\n\nDenaturation of Whey Proteins in the Milk System 257\n\nInteractions of Whey Proteins with Casein Micelles 258\n\nConcluding remarks 261\n\nAcknowledgment 261\n\n## Introduction\n\nProcessing treatments such as heat and high pressure are normally applied in the food industry, for the purpose of microbial destruction, shelf-life extension, or achievement of desired functionality in the final product (Jelen & Rattray, 1995; Singh, 1995). It is well known that the heat-induced interactions of milk proteins have a marked impact on the functionality of the final products and such interactions are of considerable commercial importance in the dairy and food industries. Therefore, considerable research efforts have been directed to studying the detailed pathways\/mechanisms of heat-induced functionality and the effects of heat treatments on milk proteins (denaturation, aggregation, and gelation of whey proteins) and protein\u2013protein interactions, including interactions of caseins and whey proteins, have been studied in great detail over five to six decades (e.g., Havea et al. 1998; ; ; ; Manderson et al., 1998; Schokker et al. 1999; ; Anema & Li, 2003a,b; Cho et al., 2003; Livney & Dalgleish, 2004; Patel et al. 2004; ; ; ), and the subject has often been reviewed (e.g., De la Fuente et al., 2002; O'Connell & Fox, 2003; Singh & Havea, 2003; Singh, 2004).\n\nMore than a century ago, pioneering work by Hite (1899) showed the potential of high-pressure processing (HPP) as a nonthermal (alternative) preservation process, and Bridgman (1914) demonstrated the effects of HPP on the denaturation and functional properties of egg proteins. The recent interest in the HPP of food materials as an alternative to or in addition to temperature treatment led to many fundamental studies on the behavior of proteins under HPP. It is also evident from the literature that, in many cases, heat treatment and high-pressure treatment have different effects on different milk proteins. This suggests that HPP has the potential for both preservation and modification of the structure of proteins, alteration to their functional properties, and the creation of value-added products (e.g., L\u00f3pez-Fandi\u00f1o et al., 1996; Garc\u00eda-Risco et al., 2000; ; Patel et al. 2004; ; ).\n\nToday, many high-pressure-treated products such as fresh fruit jams, jellies, juices, salad dressings, rice, cakes, and guacamole are commercially available (L\u00f3pez-Fandi\u00f1o, 2006a,b). As there is increased interest in the high-pressure (HP) treatment of dairy products, it is currently a major focus of investigation. This subject has also been extensively reviewed (e.g., Datta & Deeth, 1999; ; Farkas & Hoover, 2000; Boonyaratanakornkit et al., 2002; Huppertz et al., 2002; Lullien-Pellerin & Balny, 2002; Trujillo et al., 2002; Claeys et al., 2003; Huppertz et al., 2006a,b; L\u00f3pez-Fandi\u00f1o, 2006a,b; Considine et al., 2007b; Rastogi et al., 2007; Patel and Creamer, 2008; Patel et al., 2008).\n\nThis chapter focuses on the effects of HPP on structures of individual whey proteins and caseins and their interactions in various systems. Where possible, the discussion is extended to describe pathways and mechanisms of pressure-induced denaturation, aggregation, and interactions of milk proteins.\n\n## High-pressure-induced changes in caseins\n\n### Effects on Individual Casein Fractions\n\nResearchers have found very different effects of HPP on \u03b1s1-casein and \u03b2-casein fractions (Schmidt & Payens, 1972; Payens & Heremans, 1969). For \u03b1s1-casein, the intensity of light scattered at an angle of 90\u00b0 (I90\u00b0) under pressure decreased with increasing pressure in the range of 0.1\u2013300 MPa. Reductions in I90\u00b0 indicate a reduction in the size and molar mass of the \u03b1s1-casein aggregates present in the suspension. In contrast, a reduction in I90\u00b0 was observed for \u03b2-casein with increasing pressure up to 150 MPa at 20\u201325 \u00b0C, but further increase in pressure resulted in increase in I90\u00b0, similar to that of original suspension (Schmidt & Payens, 1972). However, such changes were completely reversible upon pressure release (Schmidt and Payens, 1972). On the other hand, when \u03b2-casein was pressurized at 5 \u00b0C rather than 20\u201325 \u00b0C, pressures up to 150 MPa had little effect on I90\u00b0, whereas higher pressures caused a progressive increase in I90\u00b0 (Payens & Heremans, 1969).\n\nDifferences in the behavior of \u03b1s1\\- and \u03b2-casein under pressure can readily be related to their differences in primary structure and self-association. The type of self-association displayed by \u03b1s1-casein is best described as consecutive self-association in the form of dimers, trimers, tetramers, and the like (Huppertz, 2013). In contrast, due to its highly amphipathic structure, with a hydrophilic and highly charged N-terminus and a hydrophobic C-terminus with only few charged amino acid residues, \u03b2-casein is prone to micellization above critical micellar concentration (CMC) (Huppertz, 2013). Similar minima for I90\u00b0 at 100\u2013200 MPa are also observed for a wide-range of surfactant molecules and thus cannot be related directly to specific properties of \u03b2-casein. It is more likely that HPP-induced changes in water structure contributed to the observed effects of HPP on micellization of \u03b2-casein. Moreover, at low pressures, micellization may be reduced due to reduced hydrophobic interactions or increased electrostatic repulsion as a result of enhanced dissociation under high pressure (Morild, 1981). At higher pressures, such effects are overruled and micellization is promoted. Comparatively smaller reductions in I90\u00b0 were observed for \u03b1s1-casein, indicating a shift of the association equilibrium toward particles of lower molecular mass and enhanced dissociation of side-groups of ionizable amino acid residues, which are more randomly distributed on \u03b1s1-casein and thus may have contributed to such phenomena (Huppertz, 2013). No studies have been reported on the effects of HPP on \u03b1s2-casein and \u03ba-casein.\n\n### Effects on Casein Micelles\n\nAs for individual caseins, the behavior of casein micelles under high pressure has also been studied in detail, largely by determination of light transmission, rather than I90\u00b0. Light transmission of skim milk or other suspensions containing micellar casein increased with increase in pressure, indicating disruption of casein micelles (Huppertz et al., 2006c; Huppertz & De Kruif, 2006; Orlien et al., 2006; Gebhart et al., 2005). At micellar casein concentrations comparable to those in skim milk, that is, 2.5\u20132.8%, light transmission increased with increase in pressure up to \u223c400 MPa, above which no further increase in light transmission was observed (Huppertz et al., 2006c; Orlien et al., 2006). The increase in the light transmission of skim milk or micellar casein suspensions at this pressure indicated significant disruption of casein micelles. The extent of micellar disruption under pressure also decreased with increase in pH (Huppertz & De Kruif, 2006), increase in concentration of micellar casein (Huppertz & De Kruif, 2006), or increase in temperature of pressurization (Orlien et al., 2006). Moreover, addition of calcium to milk prior to HPP also resulted in increased stability of casein micelles to HPP-induced disruption (Huppertz & De Kruif, 2006).\n\nWhen considering the factors governing the stability of casein micelles to dissociation, two primary factors can be considered: (1) the weak interactions (such as hydrophobic interactions, electrostatic interactions, and hydrogen bonding) which collectively facilitate the association of casein micelles; and (2) the calcium phosphate nanoclusters, whose stability is primarily governed by the solubility of calcium phosphate in the serum phase of the product. At atmospheric pressure, micellar disruption as a result of the disruption of the weak cohesive protein\u2013protein interactions can be achieved by the addition of urea or sodium dodecyl sulfate (SDS), whereas the addition of calcium-chelating agents such as citrate or ethylene diamine tetraacetic acid (EDTA) results in the disruption of micelles through solubilization of the micellar calcium present in the calcium phosphate nanoclusters. Considering this theory, association of \u03b1s1-casein is reduced, whereas that of \u03b2-casein is actually enhanced at pressures where extensive micellar disruption is observed. Therefore, explaining HPP-induced disruption of casein micelles solely on the basis of reduced interactions between caseins appears unrealistic. Contributions from solubilization of micellar calcium phosphate (MCP) arise due to the 'electrostrictive' effect; that is, water molecules arrange in a more compact structure around ions than salts (Stippl et al., 2005). As a result, ionization reactions and solubilization of minerals are promoted under pressure. Studies on the solubilization of MCP using NMR (Hubbard et al., 2002) or light scattering from casein nanogel particles (Huppertz & De Kruif, 2007a) have reported that MCP is progressively solubilized with increasing pressure. At a micellar casein concentration of 2.5%, full solubilization of MCP was achieved at \u223c400 MPa (Huppertz & De Kruif, 2007a), which coincides with the pressure at which the highest extent of micellar disruption under HPP was achieved (Huppertz et al., 2006c; Orlien et al., 2006). While the initial phase of HPP treatment results in disruption of casein micelles, prolonged holding of the samples under pressure can result in partial reversal in light transmission effects, indicating some reformation of particles from the micellar fragments (Huppertz et al., 2006c; Orlien et al., 2006). This was observed primarily at pressures in the range 200\u2013300 MPa, but was not observed at >350 MPa, where solubilization of MCP and micellar disruption were nearly complete. Furthermore, this process was more extensive when HPP was performed at a higher temperature (Orlien et al., 2006) and at lower pH (Huppertz & De Kruif, 2006). This process was not influenced by the presence of whey proteins (Huppertz & De Kruif, 2007a,b), suggesting that it is primarily driven by casein\u2013casein interactions.\n\nWhen the pressure is released, a subsequent reduction in light transmission is observed, indicating further reformation of casein particles. This reduction in light transmission coincided with the reversal in the solubility of MCP upon pressure release (Hubbard et al., 2002), to the extent that the distribution of calcium and inorganic phosphate between the micellar and serum phase of milk was identical to that of untreated samples (Regnault et al., 2006). However, when treatment was carried out at pressures >300 MPa, initial light transmission values were not restored, indicating that casein micelles were not reformed in their native state (Keenan et al., 2001; Huppertz et al., 2006c; ). Another factor to take into consideration is the pressure-release rate, which effectively controls the rate of reformation of casein micelles upon pressure release (Merel-Rausch et al., 2006).\n\nAs a result of the above-mentioned changes, the properties of casein micelles in high-pressure-treated milk can differ strongly from those in untreated milk, for example, in terms of particle size, turbidity and distribution. It has been reported that HPP treatment of milk for up to 10 min at 250 MPa causes a reduction in particle size, whereas treatment for >15 min at this pressure causes increases in particle size (Huppertz et al., 2004a). Treatment at pressures \u2264200 MPa showed little effect on size of casein micelle and turbidity, whereas in milk treated at >300 MPa, particle size and turbidity were reduced considerably (Gaucheron et al., 1997; Huppertz et al., 2004a,b; Regnault et al., 2004; Anema et al., 2005b; Anema, 2008). Such HP-induced changes were also dependent on pressurization temperature (Huppertz et al., 2004a; Anema et al., 2005b), pH (Huppertz et al., 2004a) and the solids content of the milk (Anema, 2008). However, such changes were independent of treatment time.\n\nThe increase in the particle size was accompanied by reductions in turbidity and light-scattering intensity suggesting that increase in average particle size was most likely due to the presence of a small proportion of large particles, as was also apparent from the electron micrographs of Knudsen and Skibsted (2009). The increase in particle size at 250 MPa was reversible on subsequent storage of the milk, with the greater reversibility observed upon storage at 20 \u00b0C than at 5 \u00b0C. In contrast, HPP-induced reductions in particle size were largely irreversible on subsequent storage (Huppertz et al., 2004a).\n\n### Effects of HPP on Caseinates\n\nCompared to milk, fewer reports are available on HPP-induced changes in caseinates. High-pressure treatment of caseinate suspensions at 200 MPa caused strong reductions in the turbidity (Lee et al., 1996; Anema et al., 1997). The reduction in turbidity was more extensive with increase in pH or when 5 or 10 mM CaCl2 was added, although the pH at which large reductions in turbidity occurred shifted to a higher pH with increasing amounts of added CaCl2. Unlike milk, the reductions in the turbidity of calcium caseinate suspensions were largely irreversible upon storage and also occurred when whey proteins were present in the calcium caseinate suspension (Lee et al., 1996; Anema et al., 1997).\n\n## Effects of high pressure on interactions of milk proteins involving whey proteins\n\nProteins are composed of amino acids, and the particular sequence of amino acids in a protein determines its structure (primary, secondary, tertiary, and quaternary structures), conformation, and properties. The native three-dimensional structure of a protein is maintained by a variety of noncovalent interactions (such as hydrogen bonding, electrostatic, van der Waals, and hydrophobic interactions) between amino acid residues within the polypeptide chain and between residues and solvent molecules (Singh, 1995). Three-dimensional structure has a very important role to play in the stability and functional properties of a protein.\n\nAccording to Le Chatelier-Braun's principle, under pressure, reactions with a negative volume change are enhanced and reactions with a positive volume change are suppressed (Buchheim & Prokopek, 1992; Johnston, 1995; Balci & Wilbey, 1999). Applications of high pressure cause proteins to lose their native three-dimensional structure and lead to denaturation and aggregation of whey proteins and\/or their interactions with each other (whey protein \u2212 whey protein interactions) or with the caseins (casein \u2212 whey protein interactions).\n\nIn contrast to heat treatments, where covalent bonds and noncovalent bonds are affected, it has been reported that HPP at room temperature (\u224820 \u00b0C) disrupts only relatively weak bonding such as intramolecular hydrophobic and electrostatic interactions (Balny & Masson, 1993; Silva & Weber, 1993), whereas hydrogen bonds are relatively insensitive to pressure, suggesting that high pressure affects the tertiary (three-dimensional configuration held together mainly by hydrophobic and ionic interactions) and quaternary (the spatial arrangement by noncovalent interactions into a multimeric protein) structures of globular proteins and has little effect on their secondary structure. There are views that covalent bonds are largely insensitive to pressure treatment at relatively low temperature (Hayakawa et al., 1996), which means that the primary protein structure (the amino acid sequence) will remain intact. This partly explains why HPP has been reported to have slightly different effects on protein structure compared with heat treatments.\n\nThe effects of high pressure on the denaturation, aggregation, and interactions of whey proteins have been studied extensively under various conditions and in different systems such as milk (e.g., Felipe et al., 1997; Law et al., 1998; Arias et al., 2000; Scollard et al., 2000; Huppertz et al., 2002; 2004a,b; Anema et al., 2005a; Patel et al., 2005), whey protein concentrate (WPC) (e.g., Van Camp et al., 1997a,b) and whey protein isolate (WPI) (Hinrichs et al., 1996a,b; Michel et al., 2001), and using pure whey proteins (Dumay et al. 1994; ; Funtenberger et al., 1995; Galazka et al., 1996a; Jegouic et al., 1996; Olsen et al., 1999). It has been reported that pressure-induced reactions of whey proteins lead to the unfolding of monomeric proteins, aggregation, and gelation (Van Camp & Huyghebaert, 1995a,b; Van Camp et al., 1997a,b; Balci & Wilbey, 1999; Tedford et al., 1999a,b; Huppertz et al., 2002; Fertsch et al., 2003), by reformation of intra- and intermolecular bonds within or between the molecules, linked by hydrophobic interactions and disulfide bridges (e.g., Cheftel, 1992; Masson, 1992; Hoover, 1993; Galazka et al., 1996a; Messens et al., 1997; Trujillo et al., 2002), depending on the type of protein, protein concentration, pH, ionic strength, applied pressure, pressurizing temperature and duration of the pressure treatment, and so on (Messens et al., 1997; Fertsch et al., 2003; Huppertz et al., 2004a). Also, different pressurizing temperatures may have different effects on the denaturation and aggregation of proteins (e.g., Huppertz et al., 2004a; Patel, 2007), because of the combined effects of pressure and temperature, which can have different effects on the interactions that maintain protein structures. Many studies related to the combined effects of pressure and temperature on milk proteins have been published (e.g., Tedford et al., 1999b; Huppertz et al., 2004a; Patel, 2007). However, the present review focuses mainly on the HPP- induced changes in milk proteins that occur at ambient temperature, unless specified otherwise.\n\nSelected reports on the effects of high pressure on individual whey protein fractions such as \u03b2-lactoglobulin (\u03b2-LG), \u03b1-lactalbumin (\u03b1-LA), bovine serum albumin (BSA), or their combinations in various systems are summarized in the following sections.\n\n### Denaturation and Aggregation of Pure Whey Protein Fractions in Model Systems\n\n#### \u03b2-Lactoglobulin (\u03b2-LG)\n\nWith increasing pressure, protein molecules undergo a sequence of conformational changes because of alterations in stabilizing interactions (Johnston et al., 1992). Different effects of high pressure on proteins have been observed when the samples are analyzed under high pressure (in situ analysis) and when analyzed after pressure release. Many reports have suggested that \u03b2-LG is the most sensitive of the major whey proteins to high pressure and that it dominates the pressure-induced denaturation, aggregation, and gelation of the whey protein system (Stapelfeldt et al., 1996; Van Camp et al., 1996; 1997a,b; Belloque et al., 2000; Patel et al. 2004; ; L\u00f3pez-Fandi\u00f1o, 2006a,b; Considine et al., 2005a,b; 2007b). Therefore, the majority of studies have concentrated on the effects of high pressure on \u03b2-LG in order to gain insight into the mechanisms of unfolding and aggregation that occur during pressurization or after pressure release. During the pressure release phase and after pressure treatment, new intermolecular interactions are formed, and the proteins may be newly structured (Fertsch et al., 2003). The majority of the reports included in the present review deal with the interactions of proteins after pressure release.\n\nAt relatively low pressures (50 MPa), analysis of thiol reactivity (M\u00f8ller et al., 1998; Stapelfeldt et al., 1999) and NMR studies (Tanaka & Kunugi, 1996) suggested the existence of a 'pre-denatured' state of \u03b2-LG. This pre-denatured form corresponded to a not-completely-unfolded structure, which preceded reversible denaturation. Belloque et al. (2000), using1H NMR, showed that the degree of deuterium exchange was very small at 100 MPa and that there were no variations in the resonances belonging to the strongly bonded 'core' of \u03b2-LG. These observations might suggest that the regions of \u03b2-LG affected by the pre-denatured state are likely to be different from the core, as the core was still tight and remained unaltered at 100 MPa. Whereas pressures ranging between 0 and 140 MPa did not affect \u03b2-sheets (Subirade et al., 1998), the reactivity of the free sulfhydryl group of \u03b2-LG increased with pressure up to 150 MPa (Tanaka et al., 1996a,b). These results suggested that, in spite of having a similar overall conformation, the architectures of \u03b2-LG before and after dynamic high pressure were stabilized by slightly different interactions (Subirade et al., 1998).\n\nThe pressure-induced denaturation of \u03b2-LG was believed to be a simple two-step mechanism until Jonas and Jonas (1994) reported pressure-induced pre-denaturation transitions and thus demonstrated that the pressure-induced denaturation of \u03b2-LG could be a stepwise process. A few years later, Stapelfeldt and Skibsted (1999) proposed a three-step denaturation process, and recently Considine et al. (2005b) published a three-stage model of the pressure denaturation of \u03b2-LG (Fig. 8.1; for a detailed description, also refer to Considine et al., 2007b). It has also been reported that addition of hydrophobic ligands such as all-trans-retinol, palmitic acid, SDS, and 8-anilino-1-naphthalenesulfonate (ANS) to \u03b2-LG solution before pressure treatment (see Fig. 8.1) affects the pathways of denaturation (Considine et al., 2005b, 2007b).\n\nFigure 8.1 Proposed three-stage model of the pressure denaturation of \u03b2-LG A and \u03b2-LG B with added ANS, retinol or SDS. Reproduced with the permission of [Considine et al. (2005b), copyright 2005 Journal of Agricultural and Food Chemistry.]\n\nThe overall major changes that occur to the structure of \u03b2-LG include monomerization of the dimeric state (Iametti et al., 1997), a decrease in \u03b1-helix and \u03b2-sheet content (Hayakawa et al., 1996; Panick et al., 1999), and irreversible changes involving the formation of intermolecular disulfide bonds (Funtenberger et al., 1997; Iametti et al., 1997; L\u00f3pez-Fandi\u00f1o et al., 1997; M\u00f8ller et al., 1998). In the pressure-induced mechanism proposed (Iametti et al., 1997), release of monomers represents one of the earliest events, whereas association of transiently modified monomers stabilizes the denatured forms of the protein. In addition, it has been reported that inter- and intramolecular reactions of sulfhydryl groups can occur (Tanaka et al., 1996a,b), leading to the formation of new disulfide bonds through sulfhydryl\u2013disulfide interchange reactions (Funtenberger et al., 1997) when samples are pressure treated at 450 MPa. At 800 MPa, most of the \u03b2-LG present in the system becomes involved in hydrophobic and disulfide-linked aggregates (Patel, 2007); this behavior is quite similar to the effects of heat treatment on \u03b2-LG (Havea et al. 1998; ; ).\n\nMoreover, it has been reported that factors, such as protein concentration (Dumay et al., 1994), pH, ionic strength, type, and molarity of the buffer used for preparation of the protein solutions (Funtenberger et al., 1995; Cheftel & Dumay, 1996), pressure intensity, pressurizing time and pressurizing temperature (Yang et al., 2001; Patel, 2007), and binding of hydrophobic ligands (Considine et al., 2005b, 2007a) and small molecules such as sucrose (Dumay et al., 1994) can affect the pressure-induced denaturation and aggregation of \u03b2-LG. It has been found that high-pressure-induced denaturation is partially reversible at lower (2.5%) protein concentration but that the denaturation is irreversible and that aggregation occurs at higher (5.0%) concentration (Dumay et al., 1994). The progressive formation of intermolecular disulfide-bonded dimers to hexamers or higher polymers of \u03b2-LG (pH 7.0) has been reported to be a function of the pressure level and of the buffer type and molarity (Funtenberger et al., 1995). It was suggested that high pressure induced the formation of intermolecular disulfide bonds, especially at neutral pH. When the combined effects of pressure, temperature, and time were evaluated, the pressure intensity was found to have major effects on the structure of \u03b2-LG (Aouzelleg et al., 2004), which is somewhat different from the finding that the combined effects of pressure intensity and temperature have the greatest effect on the denaturation of \u03b2-LG (Patel, 2007).\n\nDifferent aspects of the pressure-induced unfolding and aggregation of \u03b2-LG have also been reviewed in detail (see L\u00f3pez-Fandi\u00f1o, 2006b; Considine et al., 2007b), including the effects of high pressure on the functional properties of \u03b2-LG (L\u00f3pez-Fandi\u00f1o, 2006b).\n\n#### \u03b1-Lactalbumin (\u03b1-LA)\n\nSeveral studies reported that, compared with \u03b2-LG, \u03b1-LA is resistant to pressure denaturation (L\u00f3pez-Fandi\u00f1o et al., 1996; Tanaka & Kunugi, 1996; Scollard et al., 2000; Huppertz et al., 2004a; Patel et al. 2004; ). A comparison of pressure-induced changes of two major whey proteins, \u03b1-LA and \u03b2-LG, at neutral pH showed that the reversible unfolding to a molten globule state of \u03b1-LA begins at 200 MPa and loss of native structure becomes irreversible only beyond 400 MPa, as compared with 50 and 150 MPa, respectively, for \u03b2-LG (Tanaka & Kunugi, 1996; Tanaka et al., 1996c; Stapelfeldt & Skibsted, 1999; McGuffey et al., 2005). Various explanations have been provided for such differences in the stability of these two proteins, including the differences in their secondary structures (which lead to a higher effective hydrophobicity in \u03b2-LG) and\/or in the number of disulfide bonds (four in \u03b1-LA and two in \u03b2-LG) and also the Ca2+ binding sites (Tanaka & Kunugi, 1996). In fact, the binding of calcium is reported to remarkably stabilize \u03b1-LA to pressure, by a 200 MPa increase in the pressure value at which denaturation occurs (Dzwolak et al., 1999; Hosseini-nia et al., 2002). This observation is partly supported by the finding that there was a difference in \u0394 V values between apo- and holo-\u03b1-LA (Kobashigawa et al., 1999).\n\nFluorescent measurement of dansylated (prepared at atmospheric pressure) proteins, especially the energy transfer from the intrinsic tryptophan residue to the dansyl group, showed that the protein structure was deformed by pressure and that the energy transfer mechanisms of the two proteins were differently affected by high pressure, probably reflecting the degree of compactness of their pressure-perturbed structures (Tanaka et al., 1996c). It has been reported that \u03b1-LA is present in a molten globule state beyond 200 MPa and up to 400 MPa (Jonas, 2002) and that \u03b1-LA changes its conformation from the molten globule state to the unfolded state without volume changes (Kobashigawa et al., 1999). The volume of \u03b1-LA changes only at the transition from the native state to the molten globule state (Kobashigawa et al., 1999). Lasselle et al. (2003) reported that heat and high pressure had similar effects, supporting the view that the molten globule state is stabilized by hydrophobic interactions.\n\nIn samples of severely heated solutions of \u03b1-LA, dimers and larger aggregates of \u03b1-LA were formed (Lyster, 1970; Havea et al., 2001). However, no effects on monomeric \u03b1-LA were noticeable when pure \u03b1-LA was pressure treated at 800 MPa (Patel, 2007), except that some changes in the structure of \u03b1-LA were found when \u03b1-LA samples were pressure treated at 1000 MPa (Jegouic et al., 1996).\n\n#### Bovine Serum Albumin (BSA)\n\nBSA has been found to be quite resistant to pressure treatment up to 400 MPa (Hayakawa et al., 1992; L\u00f3pez-Fandi\u00f1o et al., 1996; Patel et al. 2004; ; ; L\u00f3pez-Fandi\u00f1o, 2006b). Several reports explaining the pressure stability of BSA are available. There are views that pressure-induced changes in the secondary structure of BSA are mainly reversible (Hosseini-nia et al., 2002) and that the greater stability of BSA is probably related to the fact that this molecule, through its 17 intramolecular disulfide bonds and the presence of several separate domains, has an extremely rigid structure (Hayakawa et al., 1992; L\u00f3pez-Fandi\u00f1o et al., 1996; L\u00f3pez-Fandi\u00f1o, 2006b). It is possible that the relatively high number of disulfide linkages in BSA may impede pressure-induced aggregation by protecting the hydrophobic core\/groups present inside the molecule from exposure to the solvent (Hosseini-nia et al., 2002).\n\nCeol\u00edn (2000) studied the hydrodynamic behavior of BSA, using a perturbed angular correlation technique, as a function of high pressure up to 410 MPa. It was reported that, at moderate pressure (\u2248150 MPa), the BSA molecule suffers structural modifications that produce an increase in the molecular volume and the rotational correlation time of the molecule. However, it may be possible that, unlike \u03b2-LG, the changes in the secondary structure of BSA are largely reversible (L\u00f3pez-Fandi\u00f1o, 2006b). However, processing at 800 MPa was reported to have a substantial effect on the secondary structure of BSA, and BSA was polymerized through disulfide bonding involving the free sulfhydryl residue (Galazka et al. 1996b; ; Patel, 2007).\n\n#### Immunoglobulins (Igs) and Lactoferrin (LF)\n\nBoth IgG and LF are more stable under pressure than under heat (Patel et al. 2005; ; Carroll et al., 2006; Palmano et al., 2006). This finding has great commercial significance for using HPP in the manufacture of nutritional products containing IgG and LF. It was reported that the pressure stability of IgG was better in colostrum solutions than in pure IgG solutions (Indyk et al., 2008), suggesting that some other colostrum milk components had protective effects on the denaturation of IgG. A study on the response of IgG to high pressure (200\u2013700 MPa) in the presence of the kosmotrope sucrose has been reported (Zhang et al., 1998). Brisson et al. (2007) studied the effects of iron saturation on the thermal aggregation of LF at neutral pH and found that iron saturation markedly increased the thermal stability of LF and decreased aggregation. Palmano et al. (2006) made a similar observation for pressure-treated iron-saturated LF solutions.\n\n#### Mixtures of \u03b1-LA and \u03b2-LG\n\n\u03b1-LA does not form aggregates when pressure treated alone at 800 MPa (Patel, 2007), but it forms high-molecular-weight disulfide-bonded oligomers at high pressure in the presence of thiol reducers (Jegouic et al., 1996). The oligomers of \u03b1-LA are stabilized mainly by non-native interchain disulfide bridges. As reported for heat-treated mixtures of \u03b1-LA and \u03b2-LG (e.g., Havea et al., 2001; Hong & Creamer, 2002), mixed aggregates of denatured \u03b1-LA and \u03b2-LG were readily formed in pressure-treated whey protein solutions (Jegouic et al., 1997). This observation supports the view that the presence of reactive thiol groups is a prerequisite for pressure-induced denaturation, aggregation, and oligomerization of \u03b1-LA (Jegouic et al., 1997; Grinberg & Haertl\u00e9, 2000).\n\nYet another possibility is that the interactions of \u03b1-LA and \u03b2-LG occur in a hydrophobic environment. However, it has been reported that, under pressure, the volume change of \u03b1-LA is much less (Lassalle et al., 2003) than that of \u03b2-LG (Royer, 2002) and therefore there is little possibility for such interactions to take place in a hydrophobic environment. This partly explains why \u03b1-LA retains most of its structure under high pressure. At very high pressure (e.g., 800 MPa), the irreversible denaturation of \u03b1-LA was much less than that of \u03b2-LG, which was assigned to the difference in the number of bonds stabilizing the structure of each protein (Hinrichs et al., 1996b; Messens et al., 1997).\n\n#### Mixtures of \u03b2-LG, \u03b1-LA, and BSA\n\nComparatively little information is available on the effects of high pressure on mixtures of \u03b2-LG, \u03b1-LA, and BSA in pure protein systems. Recently, Patel (2007) reported that, when mixtures of \u03b2-LG, \u03b1-LA, and BSA were pressure treated, a somewhat similar aggregation trend was observed to that reported for heat-treated mixtures (Gezimati et al. 1996; ; Havea et al., 2001). However, in the pressure-treated samples, it appeared that \u03b2-LG, being the most pressure-sensitive whey protein, formed early aggregates prior to the unfolding of either \u03b1-LA or BSA.\n\nPressure treatment of a ternary mixture of BSA, \u03b2-LG, and \u03b1-LA generated aggregates comprising a mixture of hydrophobically linked and disulfide-linked aggregates (Patel, 2007), whereas, when pure BSA solutions or combinations of BSA and other whey proteins (e.g., a binary mixture of BSA and \u03b1-LA) were pressure treated, almost all the aggregates were disulfide-linked and only a small proportion of the aggregates were hydrophobically linked (Patel, 2007). This may be due to structural differences in each of these proteins, and\/or the effects of high-pressure treatment on the structure of each of these proteins.\n\n### Commercial Whey Protein Solutions\n\nIn addition to the above studies using pure protein systems, several studies have been conducted on heat- and pressure-induced denaturation, aggregation, and gelation of whey proteins using commercial whey protein ingredients, such as WPC or WPI.\n\nPatel et al. (2004; 2005); reported that the sensitivities of each of the whey proteins to heat treatment (Ig > LF > BSA > \u03b2-LG B > \u03b2-LG A > \u03b1-LA) and pressure treatment (\u03b2-LG B > \u03b2-LG A > IgG > LF > BSA > \u03b1-LA) were considerably different. Also, high-pressure treatment generated a comparatively greater proportion of smaller aggregates than did heat treatment (Patel et al., 2004). These results confirmed the view that there are some similarities and some differences between the heat- and high-pressure-induced aggregation and gelation of whey proteins (Van Camp & Huyghebaert, 1995a,b; Van Camp et al., 1996; Dumay et al., 1998). It was concluded that the large internal hydrophobic cavity of \u03b2-LG may have been partially responsible for its sensitivity to high-pressure treatment. Conversely, \u03b1-LA responds to pressure by modifying its structure to be more molten globule and does not fully unfold at very high pressures (Patel et al., 2006).\n\nCharacterization of pressure-treated WPC solutions using 2D PAGE (Patel et al. 2004; ) suggested that HPP generated both hydrophobically bonded and disulfide-bonded aggregates consisting of all whey proteins, including \u03b2-LG, IgG, LF, BSA, and \u03b1-LA (Fig. 8.2), fairly similar to those reported by Havea et al. (1998) for heat-treated WPC solutions. Almost all of the \u03b2-LG was incorporated into the aggregates via disulfide bonds and to a lesser extent via hydrophobic interactions (Havea et al., 1998). However, when similar samples were pressure treated, the \u03b2-LG dimer was predominant (Patel et al. 2004; ). The detailed characterization and identification of the disulfide-linked aggregates formed in pressure-treated WPC solution are shown in Figure 8.3, which clearly shows that severe pressure treatment of WPC solutions generated disulfide-bonded dimer, trimer, tetramer, 1:1 complexes of \u03b2-LG:\u03b1-LA, and the like, as well as forming higher-molecular-weight disulfide-linked aggregates consisting of BSA, LF, Ig, \u03b2-LG, and \u03b1-LA.\n\nFigure 8.2 2D PAGE patterns of control and pressure-treated WPC solutions (12% w\/v). Native- and then non-reduced SDS-PAGE patterns of (A) the control and (B) a sample pressure treated for 20 min at 800 MPa. Similarly, SDS- and then reduced SDS-PAGE patterns of (C) the control and (D) a sample pressure treated for 20 min at 800 MPa. Gel strips marked as a\u2032 and a\u2033 represent the sample strip and the stained strip, respectively. X2 and X3 are dimer and trimer of \u03b2-LG, respectively, and X4, X5, and X6 are high-molecular-weight aggregates, which were caught up at the beginning of the resolving gel, caught up within the stacking gel and could not enter the gel, respectively. For a detailed description, refer to Patel et al. (2005). Reproduced with permission from [Patel et al. (2005), copyright 2005 Journal of Agricultural and Food Chemistry.]\n\nFigure 8.3 Detailed identification of the disulfide-linked aggregates and protein interactions on a 2D SDS- and then reduced SDS-PAGE pattern of a pressure-treated (800 MPa for 30 min) WPC solution.\n\nHinrichs et al. (1996b) determined orders of reaction of n = 2.0 for \u03b1-LA and n = 2.5 for \u03b2-LG in a WPI solution. These reaction rate constants were found to vary slightly at higher protein concentrations (Keim & Hinrichs, 2004). \u03b2-LG, \u03b1-LA, and BSA participate in pressure-induced aggregation and gelation through disulfide bonding. Moreover, it has been reported that the number of stabilizing disulfide bonds directly influences the texture properties of pressure-induced whey protein gels. At high-protein concentration (10%), intermolecular interactions and irreversible aggregation are favored (Wong & Heremans, 1988; Dumay et al., 1994). High-pressure treatment of concentrated (80\u2013160 g\/kg) \u03b2-LG isolate solutions (pH 7.0) prepared in water or various buffers induces \u03b2-LG gelation at low temperature (Zasypkin et al., 1996; Dumay et al., 1998). The decreasing solubility (in various dissociating media) of the protein constituents of pressure-induced gels as a function of storage time after pressure release suggests that the aggregation and gelation result from hydrophobic interactions and also disulfide bonds and that a progressive build-up of these interactions takes place after pressure release (Dumay et al., 1998).\n\n### Pressure-induced Gelation of Whey Proteins\n\nThe effects of protein concentration, intensity of pressure treatment, holding time, and pressurizing temperature on whey protein aggregation in WPC solutions have also been investigated (Patel, 2007). It was reported that the rate of aggregation of the whey proteins increased with an increase in the concentration of protein in the WPC solution and the pressurizing temperature. The combination of low-protein concentration, mild pressure treatment (200 MPa), and low pressurizing temperature (20 \u00b0C) led to minimal loss of native-like and SDS-monomeric \u03b2-LG, whereas the combination of high-protein concentration, severe pressure treatment (600 MPa), and higher pressurizing temperature (40 \u00b0C and higher) led to significant loss of both native-like and SDS-monomeric \u03b2-LG. The sensitivity of the pressure-resistant whey proteins, such as \u03b1-LA and BSA, to aggregation was significantly increased at pressurizing temperatures of 40 \u00b0C and higher. Self-supporting gels were formed when 8 or 12% (w\/v) WPC solutions were pressure treated at 600\u2013800 MPa and 20 \u00b0C. At protein concentrations sufficiently high for gel formation, WPC was found to produce pressure-induced gels in the pressure range 200\u2013400 MPa (Van Camp & Huyghebaert, 1995a,b; Van Camp et al., 1996). Also, the WPC gels produced by high pressure (400 MPa for 30 min) at protein concentrations ranging from 110 g\/L up to 183 g\/L differed significantly from heat-induced protein gels (80 \u00b0C for 30 min) with respect to gel strength and appearance (Van Camp & Huyghebaert, 1995a,b).\n\nAs discussed earlier, significant differences in protein denaturation and aggregation induced by heat compared with high pressure have been demonstrated (Heremans et al., 1997; Patel et al. 2004; ). This might suggest that the gels produced from whey proteins by high-pressure treatment may have different properties from those made by heat treatment. For example, HPP generated gels that had a more porous structure and lower firmness (Van Camp & Huyghebaert, 1995b; Zasypkin et al., 1996; Dumay et al., 1998), and that were weaker, less elastic, and more exudative than heat-induced gels (Cheftel & Dumay, 1996; Dumay et al., 1998). In contrast to heat-induced gels, pressure-induced gels of \u03b2-LG underwent mechanical and protein solubility changes when stored at 4 \u00b0C following pressure release, clearly indicating a time-dependent strengthening of protein\u2013protein interactions, probably because the primary aggregates of \u03b2-LG further aggregated during storage through hydrophobic interactions and disulfide bonds (Dumay et al., 1998).\n\nA recent study on characterization of protein\u2013protein interactions during pressure-induced gel formation using combinations of techniques such as transmission electron microscopy (TEM), size exclusion chromatography (SEC), and 1D and 2D PAGE (Patel et al., 2006) clearly showed a time-dependent loss of native whey proteins and a corresponding increase in non-native proteins and protein aggregates of different sizes. Using 1D PAGE (native, SDS, and SDSR PAGE) and 2D PAGE (native: SDS and SDS:SDSR PAGE), these aggregates were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions (e.g., hydrophobic bonds). These aggregates altered the viscosity and opacity of the samples.\n\nVarious possible hypotheses in support of pressure-induced gel formation have been discussed (Patel et al., 2006). It was proposed that at 800 MPa, the formation of a \u03b2-LG disulfide-bonded network precedes the formation of disulfide bonds between \u03b1-LA or BSA and \u03b2-LG to form multiprotein aggregates, possibly because the disulfide bonds of \u03b1-LA and BSA are less exposed than those of \u03b2-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurs at high pressure and that hydrophobic association becomes important after the high-pressure treatment, that is, a novel pathway of whey protein gel formation using high pressure. It was postulated that \u03b2-LG plays a major role in the aggregation and gel formation of WPC under pressure (Van Camp et al., 1997a,b; Patel et al., 2006), which suggested that the major whey protein component in WPC primarily determines its functional behavior under high pressure. However, some additional studies will be needed in model systems to confirm this hypothesis, as well as to deduce the role of other whey proteins (i.e., \u03b1-LA, BSA, and Ig) in gel formation. Similar to heat-induced gelation of whey proteins, it has been reported that factors such as protein concentration, applied pressure, holding time, and pressurizing temperature (Van Camp & Huyghebaert, 1995a; Walkenstr\u00f6m & Hermansson, 1997), pHs (Van Camp & Huyghebaert, 1995b; Arias et al., 2000), and calcium contents (Van Camp et al., 1997b) affect the aggregation behavior, pressure-induced functionality such as gel formation, and physical, rheological, and microstructural properties of whey proteins. Protein\u2013protein interactions are favored near the isoelectric point of the whey proteins, and neutral and alkaline pHs stimulate the formation of intermolecular disulfide bonds (Van Camp & Huyghebaert, 1995b). Pressure-induced \u03b2-LG denaturation increases considerably at alkaline pH and decreases at acidic pH (Arias et al., 2000). Longer pressure holding times improve the strength of the gel network, stimulating the formation of more intensive intermolecular interactions (Van Camp & Huyghebaert, 1995a). Further, it has been reported that the role of calcium in the aggregation and gelation of whey proteins under pressure may be explained in a similar manner to the heat-induced effects on whey proteins (Mulvihill & Kinsella, 1988; Kinsella & Whitehead, 1989; Van Camp et al., 1997b). A combination of pressure and higher pressurizing temperature (up to 70 \u00b0C) has been recommended for inactivating microbial spores, and therefore it is important to determine its effects on the proteins in food systems.\n\n### HPP-induced Changes in Milk\n\nStudies on the effects of high pressure on milk can be broadly grouped into several topics, including the effects of high pressure on casein micelle size and its dissociation, changes in the appearance of pressure-treated milks, denaturation of whey proteins and their interaction with the casein micelles in milk, and effects of high pressure on milk from various species. Some of these topics have been reviewed recently (Huppertz et al., 2002; 2006a,b; L\u00f3pez-Fandi\u00f1o, 2006a,b; Considine et al., 2007b), including the effects of HPP on technological properties including rennet coagulation and cheese-making properties, and acid coagulation properties. The main focus of this section is on the main aspects of pressure-induced denaturation of whey proteins and their interactions with casein in the milk system.\n\n#### Denaturation of Whey Proteins in the Milk System\n\nConsiderable differences in the sensitivities of the different proteins to heat (LF > Ig > BSA > \u03b2-LG > \u03b1-LA) and pressure (\u03b2-LG> LF > Ig > BSA > \u03b1-LA) have been reported (Patel et al., 2006), showing that \u03b2-LG is the most pressure sensitive among all the whey proteins. About 70\u201380% denaturation of \u03b2-LG occurs at 400 MPa (L\u00f3pez-Fandi\u00f1o et al. 1996; L\u00f3pez-Fandi\u00f1o & Olano, 1998; Arias et al., 2000; Garc\u00eda-Risco et al., 2000; Scollard et al., 2000). Relatively little further denaturation of \u03b2-LG occurs at 400\u2013800 MPa (Scollard et al., 2000). Compared with \u03b2-LG, \u03b1-LA is stable to pressures up to about 400\u2013500 MPa in the milk environment at ambient temperature (Hinrichs et al., 1996a,b; L\u00f3pez-Fandi\u00f1o et al., 1996; Felipe et al., 1997; Gaucheron et al., 1997; L\u00f3pez-Fandi\u00f1o & Olano, 1998; Arias et al., 2000; Garc\u00eda-Risco et al., 2000; Needs et al., 2000; Scollard et al., 2000; Huppertz et al. 2002; 2004b).\n\nDifferences in the pressure stabilities of \u03b1-LA and \u03b2-LG may be linked to the more rigid molecular structure of the former (L\u00f3pez-Fandi\u00f1o et al., 1996; Gaucheron et al., 1997), caused probably by differences in the secondary structure and in the number of disulfide bonds and Ca2+ binding sites. The pressure resistance of \u03b1-LA is partially caused by the different numbers of intramolecular disulfide bonds in the two proteins (Hinrichs et al., 1996a,b; Gaucheron et al., 1997) or by the lack of a free sulfhydryl group in \u03b1-LA (L\u00f3pez-Fandi\u00f1o et al., 1996; Funtenberger et al., 1997). It has also been reported that the molecular structure of \u03b1-LA is more stable than that of \u03b2-LG, and that oligomerization takes place only if, during unfolding, free sulfhydryl groups from other molecules are available (Hinrichs et al., 1996b; L\u00f3pez-Fandi\u00f1o et al., 1996; Gaucheron et al., 1997; Jegouic et al., 1997). This difference in pressure sensitivity can also be explained by the types of bonds stabilizing the conformational structures of \u03b2-LG and \u03b1-LA (Hinrichs et al., 1996b; Messens et al., 1997).\n\nBSA has also been found to be resistant to pressures up to 400 MPa in raw milk (Hinrichs et al., 1996b; L\u00f3pez-Fandi\u00f1o et al., 1996) or 600 MPa (Hayakawa et al., 1992). The high stability of BSA can be explained by the fact that BSA carries one sulfhydryl group and 17 disulfide bonds. The energy received under pressure treatment is too small to disrupt all the disulfide bonds and to change the molecular structure of BSA. IgG in caprine milk (Felipe et al., 1997) and bovine milk (Carroll et al., 2006; Patel et al., 2006) has been reported to be more resistant to pressure denaturation than to heat denaturation.\n\nVarious studies have reported different extents of denaturation of \u03b2-LG following high-pressure treatment at 600 MPa of pasteurized milk (Needs et al., 2000) or reconstituted skim milk powder (Gaucheron et al., 1997). This finding may be attributed to the level of denaturation caused by treatments before pressurization, which may influence the amount of denaturation measured afterward. The pressure intensity and the holding time have also been reported to affect the level of denaturation of whey proteins in milk (L\u00f3pez-Fandi\u00f1o & Olano, 1998; Huppertz et al., 2004a; Anema et al., 2005b; Hinrichs & Rademacher, 2005). The reaction order of pressure- induced denaturation of \u03b2-LG is 2.5 (Hinrichs et al., 1996b), indicating that the denaturation process is concentration dependent and that a lower initial concentration of native \u03b2-LG should reduce the extent of denaturation of \u03b2-LG under pressure. Also, \u03b2-LG and \u03b1-LA are reported to be comparatively more pressure resistant in whey than in milk, which may be attributed to the absence of casein micelles and colloidal calcium phosphate in whey (Huppertz et al., 2004b).\n\n#### Interactions of Whey Proteins with Casein Micelles\n\nFor reasons of functionality, one of the major reactions of interest in the heat-treated and pressure-treated milk systems is the interaction between the denatured whey proteins and the casein micelles. Unlike the heat-treated milk system, comparatively little information is available on the interactions of casein and whey proteins in high-pressure treated milk systems.\n\nOn high-pressure treatment of milk at 300\u2013600 MPa, \u03b2-LG may form small aggregates (Felipe et al., 1997) or may interact with the casein micelles (Needs et al., 2000; Scollard et al., 2000; Huppertz et al. 2004c). It was reported that, when mixtures of \u03ba-CN and \u03b2-LG were pressure treated at 400 MPa, the presence of \u03b2-LG reduced the susceptibility of \u03ba-CN to subsequent hydrolysis by chymosin, indicating interactions between the proteins (L\u00f3pez-Fandi\u00f1o et al., 1997). SDS-PAGE analysis of pressure-treated and untreated milks or solutions containing \u03ba-CN or \u03b2-LG or both in the presence or absence of denaturing agents showed evidence of the formation of aggregates linked by intermolecular disulfide bonds (L\u00f3pez-Fandi\u00f1o et al., 1997). Interestingly, \u03b1s2-casein (\u03b1s2-CN) occurs at the same concentration as \u03ba-CN and has one disulfide bond, but it has not normally been reported to interact with \u03b2-LG in milk systems heated at 85\u221290 \u00b0C. In contrast, Patel et al. (2006) reported that the effects of heat treatment and high-pressure treatment on the interactions of the caseins and whey proteins in milk were significantly different by demonstrating the formation of disulfide-linked complexes involving \u03b1s2-CN, \u03ba-CN, and whey proteins in heat- and pressure-treated milks. The results have been explained using modified 2D SDS- and then reduced SDS-PAGE and by proposing the possible reactions of the caseins and whey proteins in heat- and pressure-treated milk (Figs 8.4 and 8.5). The virtual absence of \u03b1s2-CN from the heat-induced aggregates formed at 85\u201390 \u00b0C in milk, as reported in previous studies, might be because \u03b1s2-CN is not a surface component of the micelle and therefore its disulfide bond(s) are inaccessible to the denatured \u03b2-LG. On the other hand, \u03ba-CN is on the surface of the micelles, and its disulfide bond(s) could be readily accessible to a thiol group of \u03b2-LG. Moreover, it has been reported that large quantities of very large aggregates that cannot enter the gel are present to a greater extent in heat-treated milk than in pressure-treated milk (Fig. 8.4; Patel et al., 2006), indicating that the sizes of the aggregates are comparatively smaller in pressure-treated milks than in heat-treated milks. Such differences can be attributed to different effects of heat treatment and pressure treatment on the structure of the proteins, which may ultimately lead to different textures of the final products.\n\nFigure 8.4 2D SDS- and then reduced SDS-PAGE patterns of the control sample (A) and samples heat treated at 72 \u00b0C for 15 s (B) and 140 \u00b0C for 5 s (C). Similarly, 2D PAGE patterns of samples pressure treated at 200 MPa for 30 min (D) and 800 MPa for 30 min (E). Gel strips marked as a\u2032 and a\u2033 represent the sample strip and the stained strip, respectively, and X4, X5, and X6 are high-molecular-weight aggregates, which were caught up at the beginning of the resolving gel, caught up within the stacking gel, and could not enter the gel, respectively. For a detailed description, refer to Patel et al. (2006). Reproduced in part with the permission of [Patel et al. (2006), copyright 2006 Journal of Agricultural and Food Chemistry.]\n\nFigure 8.5 Pictorial representation of the likely effects of medium (\u2248250 MPa) and high (>600 MPa) pressure treatment at \u224822 \u00b0C. The casein micelle swells at \u2248250 MPa and the \u03b2-LG unfolds and aggregates via disulfide bonds. \u03b2-LG forms disulfide-bonded dimers at lower pressure and probably aggregates with \u03ba-CN, but does not form larger \u03b2-LG aggregates. The proportion of \u03b1-LA that is included in the aggregates is less than that of \u03b2-LG because it does not readily unfold. At pressures >600 MPa, \u03b1s2-CN becomes available for thiol interchange reactions, assisted by the permeation of water into the micelle and the dissolution of the calcium phosphate. Also the \u03b2-LG molecules can polymerize into larger aggregates than dimers. Redrawn with the permission of [Patel et al. (2006), copyright 2006 Journal of Agricultural and Food Chemistry; and Considine et al. (2007b), copyright Elsevier.]\n\nUpon HPP treatment of milk serum depleted of casein micelles, no sedimentable whey proteins were observed despite high levels of whey protein denaturation, indicating that sedimentable whey proteins in HPP-treated milk are mostly associated with the casein micelles (Huppertz et al, 2004a). The level of denatured \u03b2-LG associated with the casein micelles increased with increase in pressure intensity, treatment time, and pressurization temperature (Huppertz et al., 2004a; Zobrist et al., 2005; Anema 2008; ). No effects of \u03b2-LG or the solids content of milk were observed (Anema, 2008). However, the association of \u03b2-LG with casein micelles increased with increase in pH of the milk before HP treatment (Huppertz et al., 2004a; Anema 2010), whereas the addition of KIO3 to milk prior to HPP treatment resulted in a lower level of denatured \u03b2-LG associated with casein micelles in HPP-treated milk, probably because the formation of disulfide bridges through thiol\u2013thiol interactions rather than thiol\u2013disulfide interchange reactions is favored in the oxidizing environment (Zobrist et al., 2005). In contrast to \u03b2-LG, most denatured \u03b1-LA was found in the serum phase of HPP-treated milk.\n\nThe 2D-PAGE results of Patel et al, (2006) also showed that pressure treatment of milk at 200 MPa (Fig. 8.4D) caused \u03b2-LG to form disulfide-bonded dimers and incorporated \u03b2-LG into aggregates, probably disulfide bonded to \u03ba-CN, suggesting that preferential reaction occurred at this pressure. The other whey proteins appeared to be less affected at 200 MPa. In contrast, pressure treatment at 800 MPa incorporated \u03b2-LG and most of the minor whey proteins (including Ig and LF), as well as \u03ba-CN and much of the \u03b1s2-CN, into large aggregates (Fig. 8.4E). However, only a proportion of the \u03b1-LA was denatured or incorporated into the large aggregates. Relatively lower degree of \u03b1-LA reactivity at high pressures is probably related to the relative stability of this protein compared with \u03b2-LG, as discussed earlier, and is based on the unusual pressure-dependent behavior of \u03b1-LA (Kuwajima et al., 1990; Kobashigawa et al., 1999; Lasalle et al., 2003). At higher pressures (>400 MPa), the polymerization of \u03b2-LG becomes the norm, and pressure-induced \u03b2-LG aggregation becomes similar to heat-induced \u03b2-LG aggregation (Fig. 8.5). The \u03b2-LG in WPC or in milk is not significantly modified by the other components; that is, \u03b2-LG dominates the denaturation and aggregation pathway during pressure (>400 MPa) treatment, as it has been shown to dominate the reaction at high-temperature heat treatments.\n\nAll these results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that result in differences among the compositions of resultant aggregation or interaction products (including their sizes) that ultimately may affect product functionalities.\n\n## Concluding remarks\n\nIn the present chapter, we reviewed the mechanisms and pathways of pressure-induced denaturation and aggregation of whey proteins and their interactions with casein in various systems. It has been reported that compared to heat treatment, HPP has many different effects on denaturation, aggregation, and interactions of milk proteins. Such differences can be attributed to different effects of heat treatment and pressure treatment on the structure of the proteins. In view of this consideration, HPP has potential to improve rennet, acid coagulation, and many other functional properties of milk and milk products. HPP can be applied to develop dairy and food products with novel texture and unique functional properties that are inaccessible via conventional methods.\n\n## Acknowledgment\n\nThe authors are grateful to Lawrence Creamer and Harjinder Singh.\n\n# References\n\nAnema SG .  Effect of milk solids concentration on whey protein denaturation, particle size changes and solubilization of casein in high-pressure-treated skim milk .  _International Dairy Journal_.  2008 ;18 : 228 \u2013 235 .\n\nAnema SG .  Effect of pH at pressure treatment on the acid gelation of skim milk .  _Innovative Food Science and Emerging Technologies_.  2010 ;11 : 265 \u2013 273 .\n\nAnema SG ,  Li Y .  Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size .  _Journal of Dairy Research_.  2003 ;70 : 73 \u2013 83 .\n\nAnema SG ,  Li Y .  Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 1640 \u2013 1646 .\n\nAnema SG ,  Lee SK ,  Schrader K ,  Buchheim W .  Effect of pH on the turbidity of pressure treated calcium caseinate suspensions and skim milk .  _Milchwissenschaft_.  1997 ;52 : 141 \u2013 146 .\n\nAnema SG ,  Stockmann R ,  Lowe EK .  Denaturation of \u03b2-lactoglobulin in pressure-treated skim milk .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 7783 \u2013 7791 .\n\nAnema SG ,  Lowe EK ,  Stockmann R .  Particle size changes and casein solubilisation in high-pressure-treated skim milk .  _Food Hydrocolloids_.  2005 ;19 : 257 \u2013 267 .\n\nAouzelleg A ,  Bull LA ,  Price NC ,  Kelly SM .  Molecular studies of pressure\/temperature-induced structural changes in bovine \u03b2-lactoglobulin .  _Journal of the Science of Food and Agriculture_.  2004 ;84 : 398 \u2013 404 .\n\nArias M ,  L\u00f3pez-Fandi\u00f1o R ,  Olano A .  Influence of pH on the effects of high pressure on milk proteins .  _Milchwissenschaft_.  2000 ;55 : 191 \u2013 194 .\n\nBalci AT ,  Wilbey RA .  High pressure processing of milk\u2014the first 100 years in the development of new technology .  _International Journal of Dairy Technology_.  1999 ;52 : 149 \u2013 155 .\n\nBalny C ,  Masson P .  Effects of high pressure on proteins .  _Food Reviews International_.  1993 ;9 : 611 \u2013 628 .\n\nBelloque J ,  L\u00f3pez-Fandi\u00f1o R ,  Smith GM .  A 1H-NMR study on the effect of high pressures on \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 3906 \u2013 3912 .\n\nBoonyaratanakornkit BB ,  Park CB ,  Clark DS .  Pressure effects on intra- and intermolecular interactions within proteins .  _Biochimica et Biophysica Acta_.  2002 ;1595 : 235 \u2013 249 .\n\nBridgman PW .  The coagulation of albumen by pressure .  _Journal of Biological Chemistry_.  1914 ;19 : 511 \u2013 512 .\n\nBrisson G ,  Britten M ,  Pouliot Y .  Heat-induced aggregation of bovine lactoferrin at neutral pH: Effect of iron saturation .  _International Dairy Journal_.  2007 ;17 : 617 \u2013 624 .\n\nBuchheim W ,  Prokopek D .  Die Hochdruckbehandlung .  _Deutsche Milchwirtschaft_.  1992 ;43 : 1374 \u2013 1378 .\n\nCarroll, T.J., Patel, H.A., Gonzalez, M.A., Dekker, J.W., Collett, M.A. & Lubbers, M.W., 2006. High pressure processing of metal ion lactoferrin. International Patent Publication Number WO 2006\/096073 A1.\n\nCeol\u00edn M .  Perturbed angular correlation experiments on the pressure-induced structural modification of bovine serum albumin .  _Journal of Biochemical and Biophysical Methods_.  2000 ;45 : 117 \u2013 125 .\n\nCheftel JC ,  Effects of high hydrostatic pressure on food constituents: an overview .  Balny C ,  Hayashi R ,  Heremans K ,  Masson P ,  eds.  _High Pressure and Biotechnology_ ,  224 .  Montrouge, France :  John Libbey Eurotext ;  1992 : 195 \u2013 209 : Colloque INSERM .\n\nCheftel J ,  Dumay E .  Effects of high pressure on dairy proteins: A review .  In:  Hayashi R ,  Balny C ,  eds.  _High Pressure Bioscience and Biotechnology_ .  Amsterdam :  Elsevier Science ;  1996 : 299 \u2013 308 .\n\nCho Y ,  Singh H ,  Creamer LK .  Heat-induced interactions of \u03b2-lactoglobulin A and \u03ba-casein B in a model system .  _Journal of Dairy Research_.  2003 ;70 : 61 \u2013 71 .\n\nClaeys WL ,  Indrawati O ,  Van Loey AM ,  Hendrickx ME .  Review: are intrinsic TTIs for thermally processed milk applicable for high-pressure processing assessment? .  _Innovative Food Science and Emerging Technologies_.  2003 ;4 : 1 \u2013 14 .\n\nConsidine T ,  Patel HA ,  Singh H ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 3197 \u2013 3205 .\n\nConsidine T ,  Singh H ,  Patel HA ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on high-pressure-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 8010 \u2013 8018 .\n\nConsidine T ,  Patel HA ,  Singh H ,  Creamer LK .  Influence of binding of conjugated linoleic acid and myristic acid on the heat- and high-pressure-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Food Chemistry_.  2007 ;102 : 1270 \u2013 1280 .\n\nConsidine T ,  Patel HA ,  Anema SG ,  Singh H ,  Creamer LK .  Interactions of milk proteins during heat and high hydrostatic pressure treatments\u2014a review .  _Innovative Food Science and Emerging Technologies_.  2007 ;8 : 1 \u2013 23 .\n\nDatta N ,  Deeth HC .  High pressure processing of milk and dairy products .  _Australian Journal of Dairy Technology_.  1999 ;54 : 41 \u2013 48 .\n\nDatta N ,  Deeth HC .  High pressure processing .  In:  Roginski H ,  Fuquay JW ,  Fox PF ,  eds.  _Encyclopedia of Dairy Sciences_ .  London :  Academic Press ;  2003 : 1327 \u2013 1333 .\n\nDe la Fuente MA ,  Singh H ,  Hemar Y .  Recent advances in the characterization of heat-induced aggregates and intermediates of whey proteins .  _Trends in Food Science and Technology_.  2002 ;13 : 262 \u2013 274 .\n\nDumay EM ,  Kalichevsky MT ,  Cheftel JC .  High-pressure unfolding and aggregation of \u03b2-lactoglobulin and the baroprotective effects of sucrose .  _Journal of Agricultural and Food Chemistry_.  1994 ;42 : 1861 \u2013 1868 .\n\nDumay EM ,  Kalichevsky MT ,  Cheftel JC .  Characteristics of pressure-induced gels of \u03b2-lactoglobulin at various times after pressure release .  _Lebensmittel-Wissenschaft und -Technologie_.  1998 ;31 : 10 \u2013 19 .\n\nDzwolak W ,  Kato M ,  Shimizu A ,  Taniguchi Y .  Fourier-transform infrared spectroscopy study of the pressure-induced changes in the structure of the bovine \u03b1-lactalbum in the stabilizing role of the calcium ion .  _Biochimica et Biophysica Acta_.  1999 ;1433 : 45 \u2013 55 .\n\nFarkas DF ,  Hoover DG .  High pressure processing. Kinetics of microbial inactivation for alternative food processing technologies .  _Journal of Food Science_.  2000 ;65 (Suppl) : 47 \u2013 64 .\n\nFelipe X ,  Capellas M ,  Law AJR .  Comparison of the effects of high-pressure treatments and heat pasteurization on the whey proteins in goat's milk .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 627 \u2013 631 .\n\nFertsch B ,  M\u00fcller M ,  Hinrichs J .  Firmness of pressure-induced casein and whey protein gels modulated by holding time and rate of pressure release .  _Innovative Food Science and Emerging Technologies_.  2003 ;4 : 143 \u2013 150 .\n\nFuntenberger S ,  Dumay E ,  Cheftel JC .  Pressure-induced aggregation of \u03b2-lactoglobulin in pH 7.0 buffers .  _Lebensmittel-Wissenschaft und -Technologie_.  1995 ;28 : 410 \u2013 418 .\n\nFuntenberger S ,  Dumay E ,  Cheftel JC .  High pressure promotes \u03b2-lactoglobulin aggregation through SH\/S \u2212 S interchange reactions .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 912 \u2013 921 .\n\nGalazka VB ,  Dickinson E ,  Ledward DA .  Effect of high pressure on the emulsifying behaviour of \u03b2-lactoglobulin .  _Food Hydrocolloids_.  1996 ;10 : 213 \u2013 219 .\n\nGalazka VB ,  Sumner IG ,  Ledward DA .  Changes in protein\u2013protein and protein\u2013polysaccharide interactions induced by high pressure .  _Food Chemistry_.  1996 ;57 : 393 \u2013 398 .\n\nGalazka VB ,  Ledward DA ,  Sumner IG ,  Dickinson E .  Influence of high pressure on bovine serum albumin and its complex with dextran sulfate .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 3465 \u2013 3471 .\n\nGarc\u00eda-Risco MR ,  Olano A ,  Ramos M ,  L\u00f3pez-Fandi\u00f1o R .  Micellar changes induced by high pressure. Influence in the proteolytic activity and organoleptic properties of milk .  _Journal of Dairy Science_.  2000 ;83 : 2184 \u2013 2189 .\n\nGaucheron F ,  Famelart MH ,  Mariette F ,  Raulot K ,  Michel F ,  Le Graet Y .  Combined effects of temperature and high pressure treatments on physicochemical characteristics of skim milk .  _Food Chemistry_.  1997 ;59 : 439 \u2013 447 .\n\nGebhart R ,  Doster W ,  Kulozik U .  Pressure-induced dissociation of casein micelles: size distribution and effects of temperature .  _Brazilian Journal of Medical and Biological Research_.  2005 ;38 : 1209 \u2013 1214 .\n\nGezimati J ,  Singh H ,  Creamer LK .  Heat-induced interactions and gelation of mixtures of bovine \u03b2-lactoglobulin and serum albumin .  _Journal of Agricultural and Food Chemistry_.  1996 ;44 : 804 \u2013 810 .\n\nGezimati J ,  Creamer LK ,  Singh H .  Heat-induced interactions and gelation of mixtures of \u03b2-lactoglobulin and \u03b1-lactalbumin .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 1130 \u2013 1136 .\n\nGrinberg VY ,  Haertl\u00e9 T .  Reducer driven baric denaturation and oligomerisation of whey proteins .  _Journal of Biotechnology_.  2000 ;79 : 205 \u2013 209 .\n\nHavea P ,  Singh H ,  Creamer LK ,  Campanella OH .  Electrophoretic characterization of the protein products formed during heat treatment of whey protein concentrate solutions .  _Journal of Dairy Research_.  1998 ;65 : 79 \u2013 91 .\n\nHavea P ,  Singh H ,  Creamer LK .  Formation of new protein structures in heated mixtures of BSA and \u03b1-lactalbumin .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 1548 \u2013 1556 .\n\nHavea P ,  Singh H ,  Creamer LK .  Characterization of heat-induced aggregates of \u03b2-lactoglobulin, \u03b1-lactalbumin and bovine serum albumin in a whey protein concentrate environment .  _Journal of Dairy Research_.  2001 ;68 : 483 \u2013 497 .\n\nHavea P ,  Carr AJ ,  Creamer LK .  The roles of disulfide and non-covalent bonding in the functional properties of heat-induced whey protein gels .  _Journal of Dairy Research_.  2004 ;71 : 330 \u2013 339 .\n\nHayakawa I ,  Kajihara J ,  Morikawa K ,  Oda M ,  Fujio Y .  Denaturation of bovine serum albumin (BSA) and ovalbumin by high pressure, heat and chemicals .  _Journal of Food Science_.  1992 ;57 : 288 \u2013 292 .\n\nHayakawa I ,  Linko Y-Y ,  Linko P .  Mechanism of high pressure denaturation of proteins .  _Lebensmittel-Wissenschaft und -Technologie_.  1996 ;29 : 756 \u2013 762 .\n\nHeremans K ,  Van Camp J ,  Huyghebaert A ,  High-pressure effects on proteins .  Damodaran S ,  Paraf A ,  eds.  _Food Proteins and their Applications_ New York :  Marcel Dekker ;  1997 : 473 \u2013 502 .\n\nHinrichs J ,  Rademacher B .  Kinetics of combined thermal and pressure-induced whey protein denaturation in bovine skim milk .  _International Dairy Journal_.  2005 ;15 : 315 \u2013 323 .\n\nHinrichs J ,  Rademacher B ,  Kessler HG .  Food processing of milk products with ultra high pressure .  _Heat Treatments and Alternative Methods_ .  Brussels :  International Dairy Federation ;  1996 : 185 \u2013 201 : IDF Special Issue No 9602 .\n\nHinrichs J ,  Rademacher B ,  Kessler HG .  Reaction kinetics of pressure-induced denaturation of whey proteins .  _Milchwissenschaft_.  1996 ;51 : 504 \u2013 509 .\n\nHite BH .  The effect of pressure in the preservation of milk .  _Bulletin of the West Virginia University Agricultural Experimental Station_.  1899 ;58 : 15 \u2013 35 .\n\nHong Y-H ,  Creamer LK .  Changed protein structures of bovine \u03b2-lactoglobulin B and \u03b1-lactalbumin as a consequence of heat treatment .  _International Dairy Journal_.  2002 ;12 : 345 \u2013 359 .\n\nHoover DG .  Pressure effects on biological systems .  _Food Technology_.  1993 ;47 (6) : 150 \u2013 155 .\n\nHosseini-nia T ,  Ismail AA ,  Kubow S .  Effect of high hydrostatic pressure on the secondary structures of BSA and apo- and holo-\u03b1-lactalbumin employing Fourier transform infrared spectroscopy .  _Journal of Food Science_.  2002 ;67 : 1341 \u2013 1347 .\n\nHubbard CD ,  Caswell D ,  L\u00fcdemann HD ,  Arnold M .  Characterization of pressure-treated skimmed milk powder dispersions: Application of NMR spectroscopy .  _Journal of the Science of Food and Agriculture_.  2002 ;82 : 1107 \u2013 1114 .\n\nHuppertz T .  Chemistry of Caseins .  In:  McSweeney PLH ,  Fox PF ,  eds.  _Advanced Dairy Chemistry 1 \u2013 Proteins \u2013 Volume 1A: Basic Aspects_ .  New York :  Springer ;  2013 : 135 \u2013 160 .\n\nHuppertz T ,  Kelly AL ,  Fox PF .  Effects of high pressure on constituents and properties of milk .  _International Dairy Journal_.  2002 ;12 : 561 \u2013 572 .\n\nHuppertz T ,  Fox PF ,  Kelly AL .  High pressure treatment of bovine milk: Effects of casein micelles and whey proteins .  _Journal of Dairy Research_.  2004 ;71 : 97 \u2013 106 .\n\nHuppertz T ,  Fox PF ,  Kelly AL .  High pressure-induced denaturation of \u03b1-lactalbumin and \u03b2-lactoglobulin in bovine milk and whey: a possible mechanism .  _Journal of Dairy Research_.  2004 ;71 : 489 \u2013 495 .\n\nHuppertz T ,  Fox PF ,  Kelly AL .  Properties of casein micelles in high pressure-treated bovine milk .  _Food Chemistry_.  2004 ;87 : 103 \u2013 110 .\n\nHuppertz T ,  De Kruif CG .  Disruption and reassociation of casein micelles under high pressure: influence of milk serum composition and casein micelle concentration .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 5903 \u2013 5909 .\n\nHuppertz T ,  Fox PF ,  de Kruif KG ,  Kelly AL .  High pressure-induced changes in bovine milk proteins: A review .  _Biochimica et Biophysica Acta_.  2006 ;1764 : 593 \u2013 598 .\n\nHuppertz T ,  Smiddy MA ,  Upadhyay VK ,  Kelly AL .  High pressure-induced changes in bovine milk: A review .  _International Journal of Dairy Technology_.  2006 ;59 : 58 \u2013 66 .\n\nHuppertz T ,  Kelly AL ,  de Kruif CG .  Disruption and reassociation of casein micelles under high pressure .  _Journal of Dairy Research_.  2006 ;73 : 294 \u2013 298 .\n\nHuppertz T ,  De Kruif CG .  High pressure-induced solubilisation of micellar calcium phosphate from cross-linked casein micelles .  _Colloids and Surfaces A_.  2007 ;295 : 264 \u2013 268 .\n\nHuppertz T ,  De Kruif CG .  Disruption and reassociation of casein micelles during high pressure treatment: influence of whey proteins .  _Journal of Dairy Research_.  2007 ;74 : 194 \u2013 197 .\n\nHuppertz T ,  Smiddy MA ,  Goff HD ,  Kelly AL .  Effects of high pressure treatment of mix on ice cream manufacture .  _International Dairy Journal_.  2011 ;21 : 718 \u2013 726 .\n\nIametti S ,  Transidico P ,  Bonomi F ,  Vecchio G ,  Pittia P ,  Rovere P ,  Dall'Aglio G .  Molecular modifications of \u03b2-lactoglobulin upon exposure to high pressure .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 23 \u2013 29 .\n\nIndyk HE ,  Williams JW ,  Patel HA .  Analysis of denaturation of bovine IgG by heat and high pressure using an optical biosensor .  _International Dairy Journal_.  2008 ;18 : 359 \u2013 366 .\n\nJegouic M ,  Grinberg VY ,  Guingant A ,  Haertl\u00e9 T .  Thiol-induced oligomerization of \u03b1-lactalbumin at high pressure .  _Journal of Protein Chemistry_.  1996 ;15 : 501 \u2013 509 .\n\nJegouic M ,  Grinberg VY ,  Guingant A ,  Haertl\u00e9 T .  Baric oligomerization in \u03b1-lactalbumin\/\u03b2-lactoglobulin mixtures .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 19 \u2013 22 .\n\nJelen P ,  Rattray W .  Thermal denaturation of whey proteins .  In:  Fox PF ,  ed.  _Heat-induced Changes in Milk_ .  Brussels :  International Dairy Federation ;  1995 : 66 \u2013 85 : IDF Special Issue No 9501 .\n\nJohnston DE .  High pressure effects on milk and meat .  In:  Ledward DA ,  Johnston DE ,  Earnshaw RG ,  Hasting APM ,  eds.  _High Pressure Processing of Foods_ .  Nottingham :  Nottingham University Press ;  1995 : 99 \u2013 121 .\n\nJohnston DE ,  Austin BA ,  Murphy RJ ,  The effects of high pressure treatment of skim milk .  Balny C ,  Hayashi R ,  Heremans K ,  Masson P ,  eds.  _High Pressure and Biotechnology_ ,  224 .  Montrouge, France :  John Libbey Eurotext ;  1992 : 243 \u2013 247 : Colloque INSERM .\n\nJonas J .  High-resolution nuclear magnetic resonance studies of proteins .  _Biochimica et Biophysica Acta_.  2002 ;1595 : 145 \u2013 159 .\n\nJonas J ,  Jonas A .  High-pressure NMR spectroscopy of proteins and membranes .  _Annual Review of Biophysics and Biomolecular Structure_.  1994 ;23 : 287 \u2013 318 .\n\nKeenan RD ,  Young DJ ,  Tier CM ,  Jones AD ,  Underdown J .  Mechanism of pressure-induced gelation of milk .  _Journal of Agricultural and Food Chemistry_.  2001 ;49 : 3394 \u2013 3402 .\n\nKeim S ,  Hinrichs J .  Influence of stabilizing bonds on the texture properties of high-pressure-induced whey protein gels .  _International Dairy Journal_.  2004 ;14 : 355 \u2013 363 .\n\nKinsella JE ,  Whitehead DM .  Proteins in whey: chemical, physical, and functional properties .  _Advances in Food and Nutrition Research_.  1989 ;33 : 343 \u2013 438 .\n\nKnudsen JC ,  Skibsted LH .  High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy .  _Food Chemistry_.  2009 ;119 : 202 \u2013 208 .\n\nKobashigawa Y ,  Sakurai M ,  Nitta K .  Effect of hydrostatic pressure on unfolding of \u03b1-lactalbum in volumetric equivalence of the molten globule and unfolded state .  _Protein Science_.  1999 ;8 : 2765 \u2013 2772 .\n\nKuwajima K ,  Ikeguchi M ,  Sugawara T ,  Hiraoka Y ,  Sugai S .  Kinetics of disulfide bond reduction in \u03b1-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond .  _Biochemistry_.  1990 ;29 : 8240 \u2013 8249 .\n\nLassalle MW ,  Li H ,  Yamada H ,  Akasaka K ,  Redfield C .  Pressure-induced unfolding of the molten globule of all-Ala \u03b1-lactalbumin .  _Protein Science_.  2003 ;12 : 66 \u2013 72 .\n\nLaw AJR ,  Leaver J ,  Felipe X ,  Ferragut V ,  Pla R ,  Guamis B .  Comparison of the effects of high pressure and thermal treatments on the casein micelles in goat's milk .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 2523 \u2013 2530 .\n\nLee SK ,  Anema SG ,  Schrader K ,  Buchheim W .  Effect of high hydrostatic pressure on Ca-caseinate systems .  _Milchwissenschaft_.  1996 ;51 : 17 \u2013 21 .\n\nLivney YD ,  Dalgleish DG .  Specificity of disulfide bond formation during thermal aggregation in solutions of \u03b2-lactoglobulin B and \u03ba-casein A .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 5527 \u2013 5532 .\n\nL\u00f3pez-Fandi\u00f1o R .  High pressure-induced changes in milk proteins and possible applications in dairy technology .  _International Dairy Journal_.  2006 ;16 : 1119 \u2013 1131 .\n\nL\u00f3pez-Fandi\u00f1o R .  Functional improvement of milk whey proteins induced by high hydrostatic pressure .  _Critical Reviews in Food Science and Nutrition_.  2006 ;48 : 351 \u2013 363 .\n\nL\u00f3pez-Fandi\u00f1o R ,  Olano A .  Effects of high pressures combined with moderate temperatures on the rennet coagulation properties of milk .  _International Dairy Journal_.  1998 ;8 : 623 \u2013 627 .\n\nL\u00f3pez-Fandi\u00f1o R ,  Carrascosa AV ,  Olano A .  The effects of high pressure on whey protein denaturation and cheese-making properties of raw milk .  _Journal of Dairy Science_.  1996 ;79 : 929 \u2013 936 .\n\nL\u00f3pez-Fandi\u00f1o R ,  Ramos M ,  Olano A .  Rennet coagulation of milk subjected to high pressures .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 3233 \u2013 3237 .\n\nLullien-Pellerin V ,  Balny C .  High-pressure as a tool to study some proteins' properties: Conformational modification, activity and oligomeric dissociation .  _Innovative Food Science and Emerging Technologies_.  2002 ;3 : 209 \u2013 221 .\n\nLyster RLJ .  The denaturation of \u03b1-lactalbumin and \u03b2-lactoglobulin in heated milk .  _Journal of Dairy Research_.  1970 ;37 : 233 \u2013 243 .\n\nManderson GA ,  Hardman MJ ,  Creamer LK .  Effect of heat treatment on the conformation and aggregation of \u03b2-lactoglobulin A, B, and C .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 5052 \u2013 5061 .\n\nMasson P ,  Pressure denaturation of proteins .  Balny C ,  Hayashi R ,  Heremans K ,  Masson P ,  eds.  _High Pressure and Biotechnology_ ,  224 .  Montrouge, France :  John Libbey Eurotext ;  1992 : 89 \u2013 99 : Colloque INSERM .\n\nMcGuffey MK ,  Epting KL ,  Kelly RM ,  Foegeding EA .  Denaturation and aggregation of three \u03b1-lactalbumin preparations at neutral pH .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 3182 \u2013 3190 . \nMerel-Rausch E ,  Duma I ,  Hinrichs J .  Pressure-induced modification of casein micelles\u2014Influence of pressure built-up rate, pressure level, release rate and temperature on viscosity and particle size .  _Michwissenschaft_.  2006 ;61 : 255 \u2013 259 .\n\nMessens W ,  Van Camp J ,  Huyghebaert A .  The use of high pressure to modify the functionality of food proteins .  _Trends in Food Science and Technology_.  1997 ;8 : 107 \u2013 112 .\n\nMichel M ,  Leser ME ,  Syrbe A ,  Clerc M-F ,  Bauwens I ,  Bovetto L ,  von Schack M-L ,  Watzke HJ .  Pressure effects on whey protein-pectin mixtures .  _Lebensmittel-Wissenschaft und -Technologie_.  2001 ;34 : 41 \u2013 52 .\n\nM\u00f8ller RE ,  Stapelfeldt H ,  Skibsted LH .  Thiol reactivity in pressure-unfolded \u03b2-lactoglobulin. Antioxidative properties and thermal refolding .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 425 \u2013 430 .\n\nMorild E .  The theory of pressure effects on enzymes .  _Advances in Protein Chemistry_.  1981 ;34 : 93 \u2013 166 .\n\nMulvihill DM ,  Kinsella JE .  Gelation of \u03b2-lactoglobulin: effects of sodium chloride and calcium chloride on rheological and structural properties of gels .  _Journal of Food Science_.  1988 ;53 : 231 \u2013 236 .\n\nNeeds EC ,  Capellas M ,  Bland AP ,  Manoj P ,  MacDougal D ,  Paul G .  Comparison of heat and pressure treatments of skim milk, fortified with whey protein concentrate, for set yogurt preparation: Effects of milk proteins and gel structure .  _Journal of Dairy Research_.  2000 ;67 : 329 \u2013 348 .\n\nO'Connell JE ,  Fox PF .  Heat-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 879 \u2013 930 .\n\nOlsen K ,  Ipsen R ,  Otte J ,  Skibsted LH .  Effect of high pressure on aggregation and thermal gelation of \u03b2-lactoglobulin .  _Milchwissenschaft_.  1999 ;54 : 543 \u2013 545 .\n\nOrlien V ,  Knudsen JC ,  Colon M ,  Skibsted LH .  Dynamics of casein micelles in skim milk during and after high pressure treatment .  _Food Chemistry_.  2006 ;98 : 513 \u2013 521 .\n\nPalmano KP ,  Patel HA ,  Carroll TJ ,  Elgar DF ,  Gonzalez-Martin MA .  _High pressure processing of bioactive compositions_ .  International Patent Publication ;  2006 : Number WO 2006\/096074 A1 .\n\nPanick G ,  Malessa R ,  Winter R .  Differences between the pressure- and temperature-induced denaturation and aggregation of \u03b2-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering .  _Biochemistry_.  1999 ;38 : 6512 \u2013 6519 .\n\nPatel HA .  _Studies on heat- and pressure-induced interactions of milk proteins_ .  Palmerston North, New Zealand :  PhD Thesis, Massey University ;  2007 .\n\nPatel HA ,  Creamer LK .  High pressure-induced interaction involving whey proteins .  In:  Thompson A ,  Boland M ,  Singh H ,  eds.  _Milk Proteins: From Expression to Food_ .  San Diego :  Academic Press ;  2008 : 205 \u2013 227 .\n\nPatel HA ,  Singh H ,  Anema SG ,  Creamer LK .  Effects of heat and high-hydrostatic pressure treatments on the aggregation of whey proteins in whey protein concentrate solutions .  _Food New Zealand_.  2004 ;4 (3) : 29 \u2013 35 .\n\nPatel HA ,  Singh H ,  Havea P ,  Considine T ,  Creamer LK .  Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 9590 \u2013 9601 .\n\nPatel HA ,  Singh H ,  Anema SG ,  Creamer LK .  Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 3409 \u2013 3420 .\n\nPatel HA ,  Carroll T ,  Kelly AL .  Nonthermal preservation technologies for dairy applications .  In:  Chandan RC ,  Kilara A ,  Shah NP ,  eds.  _Dairy Processing and Quality Assurance_ .  Ames, Iowa :  Blackwell Publishing ;  2008 : Chapter 21 .\n\nPayens TJ ,  Heremans K .  Effect of pressure on the temperature-dependent association of \u03b2-casein .  _Biopolymers_.  1969 ;8 : 335 \u2013 345 .\n\nRegnault S ,  Thiebaud M ,  Dumay E ,  Cheftel JC .  Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9 \u00b0C or 20 \u00b0C: Effects on casein micelle size distribution .  _International Dairy Journal_.  2004 ;14 : 55 \u2013 68 .\n\nRegnault S ,  Dumay E ,  Cheftel JC .  Pressurisation of raw skim milk and of a dispersion of phosphocaseinate at 9 \u00b0C or 20 \u00b0C: Effects on the distribution of minerals and proteins between colloidal and soluble phases .  _Journal of Dairy Research_.  2006 ;73 : 91 \u2013 100 .\n\nRastogi NK ,  Raghavarao KSMS ,  Balasubramaniam VM ,  Niranjan K ,  Knorr D .  Opportunities and challenges in high pressure processing of foods .  _Critical Reviews in Food Science and Nutrition_.  2007 ;47 : 69 \u2013 112 .\n\nRoyer CA .  Revisiting volume changes in pressure-induced protein unfolding .  _Biochimica et Biophysica Acta_.  2002 ;1595 : 201 \u2013 209 .\n\nSchmidt DG ,  Payens TAJ .  The evaluation of positive and negative contributions to the second virial coefficient of some milk proteins .  _Journal of Colloid and Interface Science_.  1972 ;39 : 655 \u2013 662 .\n\nSchokker EP ,  Singh H ,  Pinder DN ,  Norris GE ,  Creamer LK .  Characterization of intermediates formed during heat-induced aggregation of \u03b2-lactoglobulin AB at neutral pH .  _International Dairy Journal_.  1999 ;9 : 791 \u2013 800 .\n\nSchokker EP ,  Singh H ,  Creamer LK .  Heat-induced aggregation of \u03b2-lactoglobulin A and B with \u03b1-lactalbumin .  _International Dairy Journal_.  2000 ;10 : 843 \u2013 853 .\n\nScollard PG ,  Beresford TP ,  Needs EC ,  Murphy PM ,  Kelly AL .  Plasmin activity, \u03b2-lactoglobulin denaturation and proteolysis in high pressure treated milk .  _International Dairy Journal_.  2000 ;10 : 835 \u2013 841 .\n\nSilva JL ,  Weber G .  Pressure stability of proteins .  _Annual Review of Physical Chemistry_.  1993 ;44 : 89 \u2013 113 .\n\nSingh H .  Heat-induced changes in casein, including interactions with whey proteins .  In:  Fox PF ,  ed.  _Heat-induced Changes in Milk_ .  Brussels :  International Dairy Federation ;  1995 : 86 \u2013 104 .\n\nSingh H .  Heat stability of milk .  _International Journal of Dairy Technology_.  2004 ;57 : 111 \u2013 119 .\n\nSingh H ,  Havea P .  Thermal denaturation, aggregation and gelation of whey proteins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1, Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1257 \u2013 1283 .\n\nStapelfeldt H ,  Skibsted LH .  Pressure denaturation and aggregation of \u03b2-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing .  _Journal of Dairy Research_.  1999 ;66 : 545 \u2013 558 .\n\nStapelfeldt H ,  Petersen PH ,  Kristiansen KR ,  Qvist KB ,  Skibsted LH .  Effect of high hydrostatic pressure on the enzymic hydrolysis of \u03b2-lactoglobulin B by trypsin, thermolysin and pepsin .  _Journal of Dairy Research_.  1996 ;63 : 111 \u2013 118 .\n\nStapelfeldt H ,  Olsen CE ,  Skibsted LH .  Spectrofluorometric characterization of \u03b2-lactoglobulin B covalently labeled with 2-(4'-maleimidylanilino) naphthalene-6-sulfonate .  _Journal of Agricultural and Food Chemistry_.  1999 ;47 : 3986 \u2013 3990 .\n\nStippl VM ,  Delgado A ,  Becker TM .  Ionization equilibria at high pressure .  _European Food Research and Technology_.  2005 ;221 : 151 \u2013 156 .\n\nSubirade M ,  Loupil F ,  Allain A-F ,  Paquin P .  Effect of dynamic high pressure on the secondary structure of \u03b2-lactoglobulin and on its conformational properties as determined by Fourier transform infrared spectroscopy .  _International Dairy Journal_.  1998 ;8 : 135 \u2013 140 .\n\nTanaka N ,  Kunugi S .  Effect of pressure on the deuterium exchange reaction of \u03b1-lactalbumin and \u03b2-lactoglobulin .  _International Journal of Biological Macromolecules_.  1996 ;18 : 33 \u2013 39 .\n\nTanaka N ,  Koyasu A ,  Kobayashi I ,  Kunugi S .  Pressure-induced change in proteins studied through chemical modifications .  _International Journal of Biological Macromolecules_.  1996 ;18 : 275 \u2013 280 .\n\nTanaka N ,  Nakajima K ,  Kunugi S .  The pressure-induced structural change of bovine \u03b1-lactalbumin as studied by a fluorescence hydrophobic probe .  _International Journal of Peptide and Protein Research_.  1996 ;48 : 259 \u2013 264 .\n\nTanaka N ,  Tsurui Y ,  Kobayashi I ,  Kunugi S .  Modification of the single unpaired sulfhydryl group of \u03b2-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation [single SH of \u03b2-lactoglobulin and pressure denaturation] .  _International Journal of Biological Macromolecules_.  1996 ;19 : 63 \u2013 68 .\n\nTedford L-A ,  Smith D ,  Schaschke CJ .  High pressure processing effects on the molecular structure of ovalbumin, lysozyme and \u03b2-lactoglobulin .  _Food Research International_.  1999 ;32 : 101 \u2013 106 .\n\nTedford L-A ,  Kelly SM ,  Price NC ,  Schaschke CJ .  Interactive effects of pressure, temperature and time on molecular structure of \u03b2-lactoglobulin .  _Journal of Food Science_.  1999 ;64 : 396 \u2013 399 .\n\nTrujillo AJ ,  Capellas M ,  Saldo J ,  Gervilla R ,  Guamis B .  Applications of high-hydrostatic pressure on milk and dairy products: a review .  _Innovative Food Science and Emerging Technologies_.  2002 ;3 : 295 \u2013 307 .\n\nVan Camp J ,  Huyghebaert A .  A comparative rheological study of heat and high pressure induced whey protein gels .  _Food Chemistry_.  1995 ;54 : 357 \u2013 364 .\n\nVan Camp J ,  Huyghebaert A .  High pressure-induced gel formation of a whey protein and haemoglobin protein concentrate .  _Lebensmittel-Wissenschaft und -Technologie_.  1995 ;28 : 111 \u2013 117 .\n\nVan Camp J ,  Feys G ,  Huyghebaert A .  High pressure induced gel formation of haemoglobin and whey proteins at elevated temperatures .  _Lebensmittel-Wissenschaft und -Technologie_.  1996 ;29 : 49 \u2013 57 .\n\nVan Camp J ,  Messens W ,  Cl\u00e9ment J ,  Huyghebaert A .  Influence of pH and sodium chloride on the high pressure-induced gel formation of a whey protein concentrate .  _Food Chemistry_.  1997 ;60 : 417 \u2013 424 .\n\nVan Camp J ,  Messens W ,  Cl\u00e9ment J ,  Huyghebaert A .  Influence of pH and calcium chloride on the high-pressure-induced aggregation of a whey protein concentrate .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 1600 \u2013 1607 .\n\nWalkenstr\u00f6m P ,  Hermansson A-M .  High-pressure treated mixed gels of gelatin and whey proteins .  _Food Hydrocolloids_.  1997 ;11 : 195 \u2013 208 .\n\nWong PTT ,  Heremans K .  Pressure effects on protein secondary structure and hydrogen deuterium exchange in chymotrypsinogen: A Fourier transform infrared spectroscopic study .  _Biochimica et Biophysica Acta_.  1988 ;956 : 1 \u2013 9 .\n\nYang J ,  Dunker AK ,  Powers JR ,  Clark S ,  Swanson BG .  \u03b2-Lactoglobulin molten globule induced by high pressure .  _Journal of Agricultural and Food Chemistry_.  2001 ;49 : 3236 \u2013 3243 .\n\nZasypkin DV ,  Dumay E ,  Cheftel JC .  Pressure- and heat-induced gelation of mixed \u03b2-lactoglobulin\/xanthan solutions .  _Food Hydrocolloids_.  1996 ;10 : 203 \u2013 211 .\n\nZhang H ,  Deligeersang ,  Guo J ,  Mu Z ,  Zhang Y ,  Zhu H .  Denaturation of bovine milk IgG at high pressure and its stabilization .  _Shipin Kexue (Beijing)_.  1998 ;19 : 10 \u2013 12 .\n\nZobrist MR ,  Huppertz T ,  Uniacke T ,  Fox PF ,  Kelly AL .  High pressure-induced changes in rennet-coagulation properties of bovine milk .  _International Dairy Journal_.  2005 ;15 : 655 \u2013 662 . \nChapter 9\n\n# The Whey Proteins in Milk: Thermal Denaturation, Physical Interactions, and Effects on the Functional Properties of Milk\n\nSkelte G. Anema Fonterra Research and Development Centre, Palmerston North, New Zealand\n\n## Abstract\n\nThis chapter reviews the literature on the denaturation of the whey proteins in milk, examines their interaction with other milk protein components, and provides some examples on the relationships between denaturation\/interaction reactions of the whey proteins and the functional behavior of the milk in selected applications. Early studies on whey protein denaturation in milk were aimed at developing methods to assess denaturation levels, and determining the relationships between the denaturation of the whey proteins and the functional behavior of milk products in bakery and other applications. Subsequent studies were directed toward modeling the whey protein denaturation processes through kinetic and thermodynamic evaluations, and determining the role of various milk components on these denaturation processes. Although the denaturation of whey proteins is critical in modifying the functional behavior of dairy products, it has become increasingly apparent that a measure of the denaturation level alone is not in itself a good predictor of functional performance. Therefore further studies have investigated the interactions of the denatured whey proteins with other milk protein components, in particular the interactions between the denatured whey proteins and the casein micelles (including identification of the specific disulfide bonds involved in complex formation between the denatured \u03b2-lactoglobulin and \u03ba-casein). A limited number of recent studies have indicated that manipulation of the interactions of the denatured whey proteins with the other milk protein components may provide a significant tool for modifying or controlling the functional performance of milk protein products in some applications.\n\nOutline\n\nIntroduction 270\n\nThe casein micelle 270\n\nThe heat treatment of milk 273\n\nWhey Protein Denaturation 274\n\nAssessment of the Denaturation of Whey Proteins in Milk 274\n\nKinetic Evaluation and Modeling of Whey Protein Denaturation 276\n\nInteractions between Denatured Whey Proteins and \u03ba-Casein\/Casein Micelles 279\n\nInteractions between Denatured Whey Proteins and \u03ba-Casein in Model Systems 279\n\nInteractions between Denatured Whey Proteins and \u03ba-Casein\/Casein Micelles in Milk Systems 280\n\nDetermination of the Specific Disulfide Bonds Formed between \u03ba-Casein and \u03b2-Lactoglobulin 290\n\nRelationships between denaturation\/interactions of the whey proteins in heated milk and the functional properties of milk 295\n\nExamples of the Relationships between Whey Protein Denaturation and the Functional Properties of Milk 295\n\nExamples of the Relationships between the Level of Interactions of Whey Proteins with \u03ba-Casein\/Casein Micelles and the Functional Properties of Milk 297\n\nAcid-induced Aggregation\/Gelation of Heated Milk 298\n\nChymosin-induced Aggregation\/Gelation of Heated Milk 306\n\nExamples of the Effect of Denaturing Whey Proteins Separately from Casein Micelles on the Functional Properties of Milk 310\n\nConclusion 311\n\n## Introduction\n\nMilk is produced in the mammary gland of female mammals and is intended for the feeding of the neonate from birth to weaning. Milk is a highly nutritious, readily digested food, rich in protein, minerals, and energy in an aqueous solution. It also provides the neonate with many other essential compounds such as protective agents, hormones, and growth factors. Milk is a highly perishable fluid and was intended by nature to be consumed soon after production. However, humans have used milk and dairy-derived foods to supplement the diet for centuries, and dairy products are still a major food source. Because of the commercial and nutritional significance of dairy products, manufacturing processes to preserve the food value of milk long after its initial production have been developed.\n\nOver the last century, modern dairy milk processing has been transformed from an art into a science. Traditional products such as cheeses and yogurts combine centuries-old knowledge with modern science, technology, and processing techniques. In contrast, more recently developed products (such as spray-dried milk products, milk protein concentrates, and whey protein concentrates) have been based on modern technologies of the time. The majority of the milk processed is of bovine (cow) origin; however, significant quantities of buffalo, goat, and sheep milk are also manufactured into dairy products (Fox, 2003).\n\nIn milk, the lactose, some of the mineral components, and the native whey proteins are in true molecular solution. However, the casein and most of the calcium and phosphate are found in large macromolecular assemblies called casein micelles. The colloidal suspension of casein micelles in milk serum is a remarkably stable food protein system. Milk can be subjected to high temperatures and pressures, high shear, and variations in concentration without appreciable damage to the casein micelle system. Even the extreme action of drying milk to a powder does not significantly alter the milk system, as milk powders can be reconstituted to produce liquid milks that have many properties similar to those of the milk from which they were derived (Kelly et al., 2003; Nieuwenhuijse & van Boekel, 2003; O'Connell & Fox, 2003; Singh & Newstead, 1992).\n\n## The casein micelle\n\nIn order to understand and rationalize any changes to the properties and stability of milk, it is necessary to have some knowledge of the casein micelle structure. Despite extensive research efforts, the detailed structure and assembly of the casein micelle have not been unequivocally established. Several models have been proposed over the years, and these models have been progressively updated or modified as more information on the casein micelle has become available (Fig. 9.1; Dalgleish, 2011; Holt, 1992; Holt & Horne, 1996; Horne, 1998; Schmidt, 1982; Walstra, 1990; ). Although there is some agreement on some aspects of the various models, there is still considerable debate over the detailed structure of the casein micelle, as evidenced from the numerous recent papers and reviews devoted to this subject\u2014see Chapter 6 (Dalgleish, 2011; Dalgleish & Corredig, 2012; de Kruif et al., 2012; Farrell et al., 2006; Qi 2007; ; Trejo et al., 2011).\n\nFigure 9.1 Recent models of the casein micelle. (a): Original 'hairy' submicelle model of the casein micelle. Adapted with permission from Walstra & Jenness (1984). Copyright (1984) John Wiley & Sons. (b): modified hairy submicelle model of the casein micelle. Adapted with permission from Walstra (1999). Copyright (1999) Elsevier. (c): Nanocluster model of the casein micelle. Original depiction used with permission from Holt (1992). Copyright (1992) Elsevier. (d): The dual-binding model of the casein micelle. Adapted with permission from Horne (1998). Copyright (1998) Elsevier. (e) Dalgleish model of the casein micelle. Reproduced with permission from Dalgleish (2012). Copyright (2012) Annual Reviews.\n\nEvidence from early electron microscopy and light-scattering studies suggested that the casein micelle was assembled from smaller subunits and, as a consequence, submicelle models of the casein micelle structure were developed. In the later iterations of these submicelle models, the casein proteins were hydrophobically aggregated to form the submicelle units, and these submicelle units were linked by colloidal calcium phosphate (CCP) to form the casein micelle. The distribution of \u03ba-casein between submicelles was heterogeneous, and submicelles with high levels of \u03ba-casein were located at the micelle surface, whereas those with low levels of \u03ba-casein were in the interior, thus giving a surface location to \u03ba-casein that was consistent with experiments (Fig. 9.1a; Schmidt, 1982; Walstra, 1990).\n\nSubsequent experimental evidence did not support the existence of submicelles, and therefore the validity of the submicelle model of the casein micelles was questioned (Holt, 1992; Holt & Horne, 1996; Horne 1998; ; ;; Walstra, 1999). In particular, there was evidence showing that the CCP was uniformly distributed through the casein micelle, which precluded submicelles being linked by CCP to form the micelle. In addition, it was considered unlikely that there would be heterogeneous populations of casein submicelles with different levels of \u03ba-casein, or that assembly into casein micelles via calcium phosphate would occur only after the casein submicelles had been formed.\n\nIn addition, electron micrograph images of casein micelles using modern techniques did not display the internal substructure expected for casein submicelles, and it was considered that the appearance of submicelles in earlier micrographs were artifacts of the early preparation techniques for electron microscopy (Horne, 2006; McMahon & McManus, 1998). In an attempt to reconcile this new evidence, the submicelle model of the casein micelle was refined to change the role of CCP from that of linking the submicelles to a charge-neutralizing agent to allow for a uniform distribution of CCP, and the submicelles were now linked together via hydrophobic interactions (Fig. 9.1b; Walstra, 1999).\n\nHowever, new models for the casein micelle that do not rely on the formation of submicelles have been proposed (Dalgleish, 2011; de Kruif & Holt, 2003; Holt, 1992; Holt & Horne, 1996; Horne, 1998). Recent models include the nanocluster model (Fig. 9.1c; de Kruif & Holt, 2003; Holt, 1992; Holt & Horne, 1996) and the dual-binding model (Fig. 9.1d; Horne, 1998). There has been some convergence of these two models, as the combined results of detailed experiments on micelle structure using small-angle x-ray\/neutron scattering and static light scattering were only consistent with calculation if weak interactions (hydrophobic interactions, hydrogen bonding, ion pairing, etc.) were incorporated into the nanocluster model (de Kruif et al., 2012).\n\nThe dual-binding model proposed by Horne (1998) describes the types of interactions involved in the assembly of casein micelles and demonstrates that micelles with a consistent arrangement of casein proteins and CCP can be achieved. However, this dual-binding model does not give a detailed description of the surface or internal structure of the casein micelle. The most recent model of the casein micelle, proposed by Dalgleish (2011; Fig. 9.1e), has a relatively sparse hairy layer of \u03ba-casein on the surface, dense enough to stabilize against approach by other casein micelles or other large colloidal particles, but sufficiently diffuse to allow denatured whey proteins to interact with the para-\u03ba-casein region of \u03ba-casein or to allow \u03b2-casein to dissociate and reassociate with the micelles on cooling and subsequent warming. In addition, this model attempts to reconcile the structural arrangement of the interior of the micelles with its high hydration by giving a specific role to \u03b2-casein. It was proposed that some of the \u03b2-casein acts as a surfactant in stabilizing the hydrated internal channels of the micelle. This \u03b2-casein may be loosely bound to the micelles so that it dissociates and reassociates during cold and warm temperature cycling.\n\nDespite the emergence of new models without submicelles, there is still no universal agreement on casein micelle structure. There are proponents of submicelle models, and convincing arguments based on a structural biology perspective have been presented (Farrell et al., 2006; Qi, 2007). Even within the groups that support the models without submicelles, diverse views on the structural arrangements and relative importance of different types of bonding still exist (Dalgleish, 2011; Dalgleish & Corredig, 2012; de Kruif et al., 2012; Horne, 2006; McMahon & Oommen, 2008).\n\nAlthough various models of the casein micelles have been proposed, these have largely been derived from the same pool of research data and are therefore different depictions or interpretations of similar information. As a consequence, many of the salient features of the structure, assembly, and stability of the different models are similar (Dalgleish, 2011; Dalgleish & Corredig, 2012; de Kruif et al., 2012; Holt & Horne, 1996; Horne, 1998; Schmidt, 1982; Walstra 1990; ;). Hydrophobic interactions and CCP are important in maintaining micelle integrity. Therefore, micelle integrity can be modified or destroyed by disruption to hydrophobic interactions or by the dissolution of the CCP. In all recent models, \u03ba-casein has a preferential surface location, with the C-terminal region protruding from the surface layer as a flexible hair. These models of the casein micelles, with the surface layers of \u03ba-casein and an internal structure maintained by hydrophobic interactions and CCP, have been used to explain micelle stability and the destabilization by the enzymes in rennet, by acidification, or by the addition of alcohol (Holt & Horne, 1996; Horne 1998; ; Walstra, 1990). However, less studied and less well understood are the mechanisms responsible for the changes that occur to the casein micelles during the heating of milk, in particular the interactions with the denatured whey proteins, the heat-induced, pH-dependent dissociation of the casein (especially \u03ba-casein) from the micelles, and the eventual heat-induced coagulation of the casein micelles.\n\n## The heat treatment of milk\n\nThe effect of heat on the milk system is an important consideration in dairy chemistry, as a heat treatment is involved in the manufacture of almost all milk products. The heat treatment may range from thermization (about 65 \u00b0C for 15 s) to sterilization (about 120 \u00b0C for 10\u201320 min) or ultra-high-temperature (UHT) treatment (typically 138\u2013142 \u00b0C for several seconds). As the thermal history of milk influences the behavior of the milk in subsequent applications, the effects of heat on milk have been the subject of intensive, if somewhat intermittent, research and many reviews and books on the subject are available (IDF 1995; ; O'Connell & Fox, 2003; Singh & Creamer, 1992; Singh, 2004).\n\nWhen milk is heated, a number of competitive and often interdependent reactions occur; the importance of each reaction is determined by the heating conditions as well as by factors such as milk composition or concentration. When considering the protein components of milk, reactions of particular importance are whey protein denaturation, the interactions of denatured whey proteins with other proteins (including those of the casein micelles), and casein micelle dissociation. These three reaction processes can markedly modify the physicochemical properties of milk and may play a major role in determining the stability of milk and the functional performance of heated milk products.\n\n### Whey Protein Denaturation\n\nThe whey proteins are typical globular proteins with well-defined secondary and tertiary structures. The whey proteins (especially \u03b1-lactalbumin and \u03b2-lactoglobulin) retain their native conformations only within relatively limited temperature ranges. Exposing the whey proteins to extremes of temperature results in the denaturation and aggregation of the proteins; this process can be expressed using the simple reaction scheme as shown in Equation 9.1.\n\nFor protein species where the native protein is in the form of noncovalently linked oligomers (such as dimeric \u03b2-lactoglobulin), the first step in the denaturation process is the reversible dissociation of the oligomer into monomeric species (Equation 9.1a). The monomeric protein can then unfold, disrupting the native conformation (Equation 9.1b). In principle, this unfolding step is reversible; however, in complex mixtures such as milk, the unfolding process is accompanied by the exposure of reactive amino side-chain groups, which allows irreversible aggregation reactions to occur. The unfolded whey protein can undergo aggregation reactions with other (unfolded) whey proteins, with aggregates or with the casein micelles (represented by A in Equation 9.1c).\n\nAt a fundamental level, protein denaturation is often defined as any noncovalent change to the secondary or tertiary structure of the protein molecule (Equation 9.1b). From this denatured state, the protein can revert to its native state (refold) or interact with other components in the system (aggregate). Under this definition, \u03b1-lactalbumin is generally regarded as one of the most heat-labile whey proteins, whereas \u03b2-lactoglobulin is one of the most heat-stable whey proteins (Ruegg et al., 1977).\n\nHowever, for the dairy industry, it is the irreversible aggregation processes that largely determine the functional properties of dairy products. Hence, it is common practice to define whey protein denaturation as the formation of irreversibly denatured and aggregated whey proteins (Kelly et al., 2003; Sanderson, 1970b; Singh & Newstead, 1992); therefore this encompasses only the irreversible process shown in Equation 9.1c. Unless otherwise stated, the irreversible denaturation process is the definition used in this chapter. Using this definition for the denaturation of whey proteins in milk, the immunoglobulins are the most heat labile and \u03b1-lactalbumin is the most heat stable of the whey proteins, with \u03b2-lactoglobulin and bovine serum albumin being intermediate (Larson & Rolleri, 1955). In general, significant denaturation of the major whey proteins, \u03b1-lactalbumin and \u03b2-lactoglobulin, occurs only on heating milk at temperatures above about 70 \u00b0C.\n\n(PN)n\u21c6nPN\n\n (9.1a)\n\nPN\u21c6PU\n\n (9.1b)\n\nPU+A\u2192(P\u2212A)\n\n (9.1c)\n\n#### Assessment of the Denaturation of Whey Proteins in Milk\n\nA considerable amount of research has been directed toward determining and understanding the denaturation processes of the major whey proteins when milk is heated. In early studies, the casein and denatured whey proteins were precipitated by adjustment of the pH to the isoelectric point of the casein (about pH 4.6). The supernatant was analyzed from the unheated and heated milks for protein nitrogen, which gave estimates of initial native whey protein levels and levels following heat treatment (Rowland, 1933).\n\nA rapid method for determining the native whey protein levels was required for assessing milk powders for suitability in applications in the bakery industry (Harland & Ashworth, 1947), and also for categorizing milk powders based on the heat treatments received during the powder manufacturing process (Sanderson, 1970a,c). From these requirements, the whey protein nitrogen index (WPNI) method was developed.\n\nIn the WPNI method, the casein and the denatured whey proteins were precipitated and separated from the native whey proteins by saturating the milk with salt, and the supernatant containing the native whey proteins was analyzed for protein content. This was originally achieved by dilution and pH adjustment of the supernatant to produce a turbid solution, with the turbidity proportional to the level of native whey protein originally present (Harland & Ashworth, 1947; Kuramoto et al., 1959; Leighton, 1962). However, the WPNI method displayed considerable variability in the degree of turbidity developed for samples with similar levels of whey protein denaturation. To overcome this problem, Sanderson (1970b) combined a dye-binding method for determining the total protein content of milk with the original WPNI method, thus giving a more accurate and reliable method for determining the WPNI.\n\nThe original WPNI method or one of its variants is still the industry standard for determining the native whey protein levels of milk powder products. It is still widely used to classify milk powders according to the heat treatments received (Kelly et al., 2003; Singh & Newstead, 1992). However, a recent report indicates that Fourier transform near-infrared spectroscopy may have potential as a rapid method for determining the WPNI of milk powders in the dry state, eliminating the necessity of reconstitution, precipitation, and filtration (Patel et al., 2007).\n\nAlthough the WPNI method can give an estimate of the level of whey protein denaturation, research into the denaturation and interactions of the individual whey proteins requires more accurate separation and analysis procedures. There are numerous quantitative methods for separating and determining the level of the individual whey proteins in milk, and these methods can be used directly or adapted to determine the level of denaturation after defined heat treatments. The methods that have been used include polyacrylamide gel electrophoresis (PAGE; e.g., Anema & McKenna, 1996; Dannenberg & Kessler, 1988a; Hillier & Lyster, 1979; Kessler & Beyer, 1991), capillary electrophoresis (e.g., Butikofer et al., 2006; Fairise & Cayot, 1998), differential scanning calorimetry (e.g., Manji & Kakuda, 1987; Ruegg et al., 1977), high-performance liquid chromatography (HPLC; e.g., Kessler & Beyer, 1991), and various immune-based assays (e.g., Lyster, 1970). In recent years, miniaturized and\/or rapid automated methods have been adapted for determining whey protein denaturation and protein compositions, such as PAGE techniques on microfluidic chips (Anema, 2009; Wu et al., 2008) or optical biosensor-based assays (Dupont & Muller-Renaud, 2006; Indyk, 2009).\n\nIn general, good correlations have been observed when the various methods for determining whey protein denaturation have been compared (Anema & Lloyd, 1999; Anema, 2009; Indyk, 2009; Kessler & Beyer, 1991; Manji & Kakuda, 1987; Patel et al., 2007). These methods are more time consuming than the traditional WPNI methods and therefore cannot be used for routine analysis and classification of milk products. However, they have higher accuracy and reproducibility, and can be used to determine the denaturation behavior of the individual whey proteins. In addition, variations of the techniques or coupling to additional detection devices can provide further information on the interactions of the denatured whey proteins with other components in the milk (e.g., Donato & Guyomarc'h, 2009; Lowe et al., 2004; Patel et al., 2006; ).\n\n#### Kinetic Evaluation and Modeling of Whey Protein Denaturation\n\nEarly studies showed that the denaturation of whey proteins was a kinetic phenomenon, and was therefore dependent on both the temperature and duration of the heat treatment (Harland & Ashworth, 1945; Rowland, 1933). Although these early studies considered the whey protein components as a single entity, it was noted that the denaturation process did not follow a simple exponential law and was not a first-order (uni-molecular) process. In addition, there was a change in temperature dependence above about 80 \u00b0C, which was probably the first indication of the complex nature of the irreversible denaturation of the whey proteins in milk (Rowland, 1933).\n\nAlthough early studies on the effect of temperature and heating time on the denaturation of the individual whey proteins had been performed (e.g., Gough & Jenness, 1962; Harland & Ashworth, 1945), it was the kinetic study of Lyster (1970) over a wide temperature range (68\u2013155 \u00b0C) that conclusively demonstrated the complexity of the denaturation process of the whey proteins. Lyster (1970) found that the denaturation of \u03b1-lactalbumin appeared to follow first-order kinetics and that the denaturation of \u03b2-lactoglobulin was second-order. Arrhenius plots for both \u03b1-lactalbumin and \u03b2-lactoglobulin indicated that the irreversible denaturation reaction was not a simple process, as a change in temperature dependence was observed at about 80\u201390 \u00b0C for both \u03b1-lactalbumin and \u03b2-lactoglobulin (Fig. 9.2). The rate constants increased more rapidly with an increase in temperature in the low-temperature ranges than at higher temperatures.\n\nFigure 9.2 Effect of milk concentration on the Arrhenius plot for the thermal denaturation of \u03b2-lactoglobulin (a) and \u03b1-lactalbumin (b) over a 75\u2013100 \u00b0C temperature range.  , 9.6% total solids milk;  , 19.2% total solids milk;  , 28.8% total solids milk;  , 38.4% total solids milk. Part (a) was adapted with permission from Anema (2000). Copyright (2000) American Chemical Society. Part (b) was adapted with permission from Anema (2001). Copyright (2001) Blackwell Publishing.\n\nFurther studies confirmed the complexity of the denaturation process and provided relationships between compositional aspects and the rate of denaturation (Hillier & Lyster, 1979; Lyster, 1970; Manji & Kakuda, 1986). The kinetic and thermodynamic studies of Dannenberg and Kessler (1988a) provided insights into the possible mechanisms responsible for the complex temperature dependences of the denaturation of \u03b1-lactalbumin and \u03b2-lactoglobulin. Dannenberg and Kessler (1988a) found that, in milk, the denaturation of \u03b2-lactoglobulin had an order of about 1.5, which is now generally accepted, and that the denaturation of \u03b1-lactalbumin was pseudo first-order.\n\nFrom thermodynamic evaluations of the denaturation reactions of \u03b2-lactoglobulin and \u03b1-lactalbumin in the two temperature ranges (i.e., at temperatures above and below the marked change in temperature dependence for the denaturation reactions; Fig. 9.2), information on the possible rate-determining steps in the denaturation reactions was obtained. At temperatures below about 90 \u00b0C for \u03b2-lactoglobulin and 80 \u00b0C for \u03b1-lactalbumin, the high values for the activation energies and enthalpies indicated that a large number of bonds were disrupted, and the positive activation entropies indicated a lower state of order of the reaction products. These kinetic and thermodynamic parameters were interpreted as indicating that the unfolding (reversible denaturation) of the whey proteins was the rate-determining step in the lower temperature ranges.\n\nAt higher temperatures, above 80 \u00b0C for \u03b1-lactalbumin and above 90 \u00b0C for \u03b2-lactoglobulin, the considerably lower activation energies and enthalpies were typical of chemical reactions and the negative activation entropies indicated a higher state of order. These parameters suggested that chemical (aggregation) reactions were the rate-determining step in the higher temperature ranges. Subsequent studies have supported these interpretations in skim and whole milk under industrial processing conditions (Anema & McKenna, 1996; Oldfield et al., 1998a).\n\nThe denaturation reactions of both \u03b2-lactoglobulin and \u03b1-lactalbumin are enhanced when the pH of the milk is increased from the natural pH and are retarded when the pH is decreased (Law & Leaver, 2000). The denaturation of \u03b2-lactoglobulin was retarded when all components in the milk were concentrated, although the effect was less pronounced as the temperature was increased (Fig. 9.2a; Anema, 2000). In contrast, the denaturation of \u03b1-lactalbumin was hardly affected by milk concentration, with similar rates of denaturation at all milk concentrations regardless of the heating temperature (Fig. 9.2b; Anema, 2001).\n\nThe seemingly contrasting effects of milk concentration on the denaturation of \u03b1-lactalbumin and \u03b2-lactoglobulin have been explained through detailed studies on the effect of the concentrations of the individual components of milk on the denaturation reactions. Increasing the protein concentration of milk while maintaining essentially constant concentrations of nonprotein soluble components increased the rate of denaturation of both \u03b1-lactalbumin and \u03b2-lactoglobulin (Fig. 9.3; Anema et al., 2006; Law & Leaver, 1997), with a similar effect at all temperatures (Anema et al., 2006).\n\nFigure 9.3 Comparison of the effects of the concentrations of protein ( ,  ), nonprotein soluble components ( ,  ), lactose ( ,  ), and total solids ( ,  ) on the rate constants for the denaturation of \u03b2-lactoglobulin (a) and \u03b1-lactalbumin (b) at 80 \u00b0C (filled symbols) and 95 \u00b0C (open symbols). Reproduced with permission from Anema et al. (2006) Copyright (2006) American Chemical Society.\n\nIncreasing the concentration of nonprotein soluble components while maintaining constant protein concentrations retarded the denaturation of both \u03b2-lactoglobulin and \u03b1-lactalbumin. However, the effects on these two proteins were somewhat different (Fig. 9.3; Anema et al., 2006). For \u03b2-lactoglobulin, increasing the nonprotein soluble components caused a substantial retardation of denaturation in the lower temperature range, and this effect became less pronounced at higher temperatures. In contrast, the effect of increasing the nonprotein soluble components on \u03b1-lactalbumin denaturation was less pronounced than for \u03b2-lactoglobulin and was similar at all temperatures investigated (Fig. 9.3). The increase in lactose concentration, the major component of the nonprotein soluble components, explained much of the effect of increasing nonprotein soluble components. Clearly, however, other compositional factors such as pH and ionic components also have an effect (Fig. 9.3; Anema et al., 2006).\n\nFrom these results, it was possible to explain the effects of milk concentration on the denaturation of \u03b2-lactoglobulin and \u03b1-lactalbumin. For \u03b1-lactalbumin, on increasing the total solids concentration of the milk (both protein and nonprotein soluble components), the retardation of the reaction rate by increasing the nonprotein soluble components concentration was almost exactly offset by the increase in the denaturation rate on increasing the protein concentration. As this effect was similar at all temperatures, increasing total solids appeared to have no effect on the rate of denaturation of \u03b1-lactalbumin (Figs 9.2b and 9.3b ; Anema, 2001; ).\n\nFor \u03b2-lactoglobulin, the retardation in the rate of denaturation on increasing the concentration of the nonprotein soluble components was not completely offset by the increasing rate of denaturation on increasing the protein concentration. Therefore \u03b2-lactoglobulin denaturation was retarded by increasing the total solids concentration of the milk. However, the nonprotein soluble components were less effective in retarding the denaturation of \u03b2-lactoglobulin at higher temperatures and, as a consequence, the increase in total solids concentration appeared to have a smaller effect on the denaturation of \u03b2-lactoglobulin at the higher temperatures, and particularly above about 90 \u00b0C (Figs 9.2a and 9.3a ; Anema, 2000; Anema et al., 2006). The effects of the nonprotein soluble components concentration or the lactose concentration on the denaturation of \u03b2-lactoglobulin and \u03b1-lactalbumin have been discussed in terms of the preferential hydration theory (Anema, 2000; Anema et al., 2006).\n\n### Interactions between Denatured Whey Proteins and \u03ba-Casein\/Casein Micelles\n\nAn understanding of the denaturation reactions of the whey proteins provides information on the initial steps of a complex series of aggregation reactions that can occur when milk is heated. This aggregation process can involve other milk protein components and may involve numerous reaction pathways or interaction processes. Although the reactions of the denatured whey proteins with other milk protein components are important, these types of reactions are considerably more difficult to measure than the irreversible denaturation processes, particularly in a complex mixture of components such as is found in (skim) milk.\n\n#### Interactions between Denatured Whey Proteins and \u03ba-Casein in Model Systems\n\nOne of the major reactions of interest is the interaction between the denatured whey proteins and the casein micelles, particularly interactions of denatured \u03b2-lactoglobulin with \u03ba-casein at the micelle surface. Early studies on model systems indicated that there was an interaction between \u03b2-lactoglobulin and \u03ba-casein when these components were heated together (Long et al., 1963; Sawyer et al., 1963; Zittle et al., 1962). These conclusions were drawn from electrophoretic studies, which showed that the discrete bands assigned to \u03ba-casein and \u03b2-lactoglobulin observed in unheated solutions produced species of intermediate mobility when the solutions were heated together. Sedimentation velocity experiments also confirmed complex formation, as the \u03b2-lactoglobulin\u2013 \u03ba-casein complex formed on heating had markedly higher sedimentation coefficients than did the individual proteins when heated separately (Zittle et al., 1962).\n\nOnce interactions between \u03ba-casein and denatured \u03b2-lactoglobulin had been confirmed, subsequent investigations in heated model systems were aimed at determining the types of bonds involved in complex formation, the stoichiometry of the complexes formed, and the involvement of other whey proteins (particularly \u03b1-lactalbumin). It was shown that reducing agents dissociated the heat-induced complexes and that thiol-blocking agents prevented the formation of the complexes (Sawyer et al., 1963). These results supported earlier suggestions that the free thiol group of \u03b2-lactoglobulin was involved in the interactions (Trautman & Swanson, 1958; Zittle et al., 1962), and it was suggested that intermolecular disulfide bonds were formed between \u03ba-casein and denatured \u03b2-lactoglobulin (Sawyer et al., 1963). This has been corroborated by numerous subsequent studies (e.g., Grindrod & Nickerson, 1967; Purkayastha et al., 1967; Sawyer, 1969; Tessier et al., 1969).\n\nSome studies indicated that the heat-induced self-aggregation of \u03b2-lactoglobulin was limited when \u03ba-casein was present, which suggested that \u03ba-casein formed complexes with intermediate species of aggregated \u03b2-lactoglobulin (McKenzie et al., 1971; Sawyer, 1969). In contrast, other studies indicated that the aggregation of \u03b2-lactoglobulin was not a prerequisite for interaction with \u03ba-casein (Euber & Brunner, 1982). The reason for these apparently conflicting observations may have been resolved through the detailed study of Cho et al. (2003) in which many of the possible pathways involved in the aggregation of \u03b2-lactoglobulin with \u03ba-casein in heated model systems were elucidated. Cho et al. (2003) proposed that, when mixtures of \u03b2-lactoglobulin and \u03ba-casein were heated, the free thiol of \u03b2-lactoglobulin was exposed and this initiated a series of thiol\u2013disulfide exchange reactions of \u03b2-lactoglobulin with other denatured \u03b2-lactoglobulin molecules or with \u03ba-casein. The products formed ranged from 1:1 \u03b2-lactoglobulin\u2013 \u03ba-casein complexes to large heterogeneous aggregates, and the product mix was dependent on the ratio of \u03ba-casein to \u03b2-lactoglobulin. The aggregate species were held together by either or both disulfide bonds and hydrophobic interactions.\n\nAlthough there have been some indications of interactions between \u03b1-lactalbumin and \u03ba-casein on heating (Doi et al., 1983; Shalabi & Wheelock, 1976), others have reported that interactions between these proteins do not occur (Baer et al., 1976; Elfagm & Wheelock, 1978). It is now generally believed that interactions between \u03b1-lactalbumin and \u03ba-casein will occur only if \u03b2-lactoglobulin (or another whey protein with a free thiol) is present during heating, and this may require the initial formation of a \u03b2-lactoglobulin\u2013 \u03b1-lactalbumin complex, which subsequently interacts with \u03ba-casein (Baer et al., 1976; Elfagm & Wheelock, 1978).\n\nThere is considerable evidence to show that disulfide bonds are involved in the aggregates formed between the denatured whey proteins and \u03ba-casein; however, there are reports suggesting that noncovalent bonding may be important in these interactions, particularly in the early stages of heating and at lower heating temperatures (Haque et al., 1987; Haque & Kinsella, 1988; Hill, 1989; Sawyer, 1969). Other studies have shown that, although a substantial part of the denatured whey proteins in heated milk are involved in disulfide-bonded aggregates, a significant proportion can be recovered as monomeric protein under dissociating but nonreducing conditions, indicating that noncovalent interactions are also involved (Anema, 2000; Oldfield et al., 1998b). As Cho et al. (2003) have suggested, it is likely that both hydrophobic and disulfide interactions are important in the early stages of aggregate formation, with the interaction mechanism dependent on the composition of the system and the conditions of heating.\n\n#### Interactions between Denatured Whey Proteins and \u03ba-Casein\/Casein Micelles in Milk Systems\n\nMost of the early studies examining the heat-induced interactions between denatured whey proteins and \u03ba-casein involved model systems using purified proteins in buffer solutions. Milk is considerably more complex, with numerous protein species that could potentially interact upon heating. A number of the milk proteins have free thiol groups and\/or disulfide bonds. Although \u03b2-lactoglobulin is the major whey protein component, denatured \u03b1-lactalbumin and bovine serum albumin can also be involved in thiol\u2013disulfide exchange reactions and therefore can be incorporated in the aggregated products. For the caseins, both \u03ba-casein and \u03b1S2-casein have disulfide bonds; therefore, both could participate in thiol\u2013disulfide exchange reactions with denatured \u03b2-lactoglobulin or other denatured thiol-bearing whey proteins. As a consequence of this complexity, there are numerous potential thiol\u2013disulfide interaction pathways, as well as noncovalent interactions, and therefore the separation and analysis of the reaction products can be difficult.\n\nStudies on the interactions between the proteins in heated milk suggest that, despite the complexity of the system, the reactions between \u03b2-lactoglobulin and \u03ba-casein may be similar to those occurring in the model systems. In early electrophoretic studies on heated milk, it was noted that the bands corresponding to \u03b2-lactoglobulin disappeared, along with a reduction in the intensity of the bands corresponding to casein. This was accompanied by the formation of bands corresponding to new (heterogeneous) components (Slatter & van Winkle, 1952; Tobias et al., 1952). When thiol-blocking agents were added, the band pattern was comparable with that of the original skim milk, indicating that thiol\u2013disulfide exchange reactions were involved in the interaction mechanisms (Trautman & Swanson, 1958). Subsequent studies confirmed that an interaction between denatured \u03b2-lactoglobulin and \u03ba-casein on the casein micelles occurred on heating milk although, as expected, the other denatured whey proteins were also involved in the interactions (Corredig & Dalgleish, 1996a,b, ; Elfagm & Wheelock, 1978; Noh et al., 1989a,b; Oldfield et al., 1998b; Smits & van Brouwershaven, 1980; Snoeren & van der Spek, 1977).\n\nUnlike \u03ba-casein, \u03b1S2-casein does not readily interact with denatured whey proteins when milk is heated, although some interactions in UHT milks have been reported (Patel et al., 2006; Snoeren & van der Spek, 1977). This low reactivity may be due to the location of \u03b1S2-casein in the interior of the casein micelles, which makes it less accessible for interaction, whereas \u03ba-casein is located at the casein micelle surface and is therefore more accessible for interaction (Horne, 1998; Walstra, 1990). Interestingly, in pressure-treated skim milk, disulfide-bonded aggregates between \u03b1S2-casein and the denatured whey proteins are observed, suggesting that the disulfide bonds of \u03b1S2-casein may become accessible to thiol groups of the denatured whey proteins when the casein micelle structure is disrupted under pressure (Patel et al., 2006).\n\nThe degree of interaction of the denatured whey proteins with the casein micelles is dependent on many variables, including the time, temperature, and rate of heating, the milk and individual protein concentrations, the milk pH, and the concentration of the milk salts (Anema & Li, 2003a; Corredig & Dalgleish, 1996a,b; Oldfield et al., 2000; Oldfield, et al., 2005; Smits & van Brouwershaven, 1980). For example, when the temperature of milk is gradually increased above 70 \u00b0C, as in indirect heating systems, most of the denatured \u03b2-lactoglobulin and \u03b1-lactalbumin associates with the casein micelles, presumably as disulfide-bonded complexes with \u03ba-casein at the micelle surface (Corredig & Dalgleish, 1996a; Smits & van Brouwershaven, 1980). In contrast, when milk is heated rapidly, as in direct heating systems, only about half of the denatured \u03b2-lactoglobulin and \u03b1-lactalbumin associates with the casein micelles, with the rest remaining in the milk serum (Corredig & Dalgleish, 1996b; Oldfield, et al., 1998b; Singh & Creamer, 1991a).\n\nCorredig and Dalgleish (1999) suggested that, on heating milk, \u03b1-lactalbumin and \u03b2-lactoglobulin initially aggregate in the serum phase at a ratio dependent on the initial individual whey protein concentrations. These complexes subsequently associate with \u03ba-casein at the casein micelle surface on prolonged heating. However, Oldfield et al. (1998b) proposed that, under rapid heating rates, \u03b2-lactoglobulin forms aggregates in the serum before interacting with the casein micelles and this limits the level of association with the casein micelles, whereas, at slower heating rates, monomers or smaller aggregates of \u03b2-lactoglobulin may interact with the micelles and this may allow higher association with the casein micelles.\n\nThe pH of the milk at heating is important in determining the level of interaction between the denatured whey proteins and the casein micelles. When milk is heated at high temperatures (about 140 \u00b0C), the heat coagulation time\/pH profiles of most milks show increasing heat stability with increasing pH to a maximum at about pH 6.7, followed by decreasing stability to a minimum at about pH 6.9, and increasing stability again as the pH is increased further (Rose, 1961). Considerable research has been undertaken over decades in an attempt to explain this unusual pH-dependent heat stability of milk, and numerous factors are known to influence the heat stability behavior. Many review papers on the heat stability of milk are available (IDF, 1995; O'Connell & Fox, 2003; Singh & Creamer, 1992; Singh, 2004).\n\nThe results from the studies on the heat stability of milk have influenced the direction of future research on the effects of heat on milk, and in particular the interactions between denatured whey proteins and \u03ba-casein\/casein micelles. Therefore, it is appropriate to briefly review aspects of the pH-dependence of heat stability that are relevant to understanding the interactions between denatured whey proteins and \u03ba-casein\/casein micelles.\n\nElectron microscopic studies showed that when milk was heated at high temperatures (90\u2013140 \u00b0C) for long periods (30 min) at pH below 6.7, the denatured whey proteins complexed on to the micelle surfaces as filamentous appendages. However, when the milk was heated at higher pH, the denatured whey proteins were found in the serum phase as aggregated complexes (Creamer et al., 1978; Creamer & Matheson, 1980). These were the first indications that the pH at heating the milk may influence the interactions between the denatured whey proteins and the casein micelles.\n\nKudo (1980) showed that the amount of nonsedimentable protein in milk heated at pH 6.5 was lower than that in unheated milk; however, the level of nonsedimentable protein increased with the pH at heating so that, above pH 6.7, the level was markedly higher than in the unheated milk and increased with increasing pH. Kudo (1980) concluded that the denatured whey proteins co-sedimented with the casein micelles at low pH (about pH 6.5), whereas most of the denatured whey proteins along with some casein (particularly \u03ba-casein) was released from the casein micelles at pH above 6.8. It was also proposed that the transition from whey-protein-coated casein micelles to protein-depleted forms with changing pH at heating could explain the pH-dependence of the heat stability of milk at high temperatures.\n\nSingh and Fox (1985a,b; ; 1987a,b,c), in a series of extensive studies, showed that the dissociation of \u03ba-casein-rich protein on heating was dependent on the pH at heating. At pH below about 6.8, little dissociation of micellar \u03ba-casein occurred, whereas at higher pH, particularly above pH 6.9, high levels of \u03ba-casein dissociated from the micelles, with the level increasing proportionally with increased pH. The whey proteins, particularly \u03b2-lactoglobulin, played an important role in the heat-induced pH-dependent dissociation of \u03ba-casein (Singh & Fox, 1987b,c), as did mineral components such as calcium and phosphate (Singh & Fox, 1987a). The results from these studies have been used to develop detailed mechanisms for the pH-dependent heat stability of milk and concentrated milk systems (O'Connell & Fox, 2003; Singh & Creamer, 1992; Singh, 2004).\n\nInitially, it was reported that the dissociation of \u03ba-casein from the casein micelles only occurred when milk at high pH (above about pH 6.8) was heated at high temperatures, particularly 90 \u00b0C or above (Singh & Fox, 1985b). However, subsequent studies demonstrated that, at these pH values, the dissociation of \u03ba-casein occurred as soon as the temperature was raised above ambient, with the level of dissociated \u03ba-casein increasing proportionally with temperature up to 90 \u00b0C. In these studies, the dissociation of \u03b1S-casein (\u03b1S1-casein and \u03b1S2-casein combined), and \u03b2-casein showed unusual temperature dependence. Increasing levels of these caseins dissociated as the temperature was increased up to about 70 \u00b0C, with the levels then decreasing again at higher temperatures (Fig. 9.4; Anema & Klostermeyer, 1997; Anema, 1998).\n\nFigure 9.4 Effect of temperature and pH on the level of protein in the supernatants obtained from 10% total solids reconstituted skim milk samples heated for 30 min: \u03ba-casein (a); \u03b1s-casein (b); \u03b2-casein (c).  , pH 6.3;  , pH 6.5;  , pH 6.7;  , pH 6.9;  , pH 7.1. Adapted with permission from Anema & Klostermeyer (1997). Copyright (1997) American Chemical Society.\n\nThe unusual temperature dependence of \u03b1S-casein and \u03b2-casein was a consequence of the whey proteins, particularly \u03b2-lactoglobulin. When whey-protein-depleted milk was heated, the levels of \u03b1S-casein and \u03b2-casein dissociating from the casein micelles increased with increasing temperature up to 90 \u00b0C. When compared with heating standard milk, this indicated that higher levels of \u03b1S-casein and \u03b2-casein dissociated from the micelles in the whey-protein-depleted milks at temperatures above about 70 \u00b0C (Anema & Li, 2000).\n\nIt was postulated that all the caseins dissociated from the micelles on heating. On subsequent cooling, the dissociated \u03ba-casein stabilized the dissociated \u03b1S-casein and \u03b2-casein as small serum-phase aggregates if the heating temperature was below about 70 \u00b0C. However, above about 70 \u00b0C, \u03ba-casein associated with denatured whey proteins. It was already known that the complex formed between \u03ba-casein and denatured \u03b2-lactoglobulin was less effective at stabilizing \u03b1S-casein and \u03b2-casein in the presence of calcium ions than uncomplexed \u03ba-casein (Zittle et al., 1962). Therefore, this interaction may have prevented \u03ba-casein from stabilizing the other caseins, and they either reassociated with the casein micelles or formed larger aggregates on subsequent cooling (Anema & Li, 2000).\n\nEarly studies on the effect of the pH at heating on the interaction of denatured whey proteins with the casein micelles tended to use relatively large pH steps. In a model milk system containing casein micelles and \u03b2-lactoglobulin, about 80% of the denatured \u03b2-lactoglobulin associated with the casein micelles when the milk was heated at pH 5.8 or pH 6.3, whereas only about 20% associated with the casein micelles at pH 6.8 or pH 7.1 (Smits & van Brouwershaven, 1980).\n\nThe studies on the heat-induced, pH-dependent dissociation of \u03ba-casein from the casein micelles showed that this dissociation was accompanied by increases in the levels of denatured whey proteins remaining in the serum (Singh & Creamer, 1991b). This finding was confirmed by Anema and Klostermeyer (1997) and Oldfield et al. (2000), who reported that 80\u201390% of the denatured whey proteins associated with the casein micelles when milk was heated at pH below 6.7, whereas only about 20% of the denatured whey proteins was associated with the casein micelles at pH above 6.8. Corredig and Dalgleish (1996a) measured the ratio of \u03b2-lactoglobulin or \u03b1-lactalbumin to \u03ba-casein in the colloidal phase obtained from heated milk adjusted to pH 5.8, 6.2, or 6.8. Although the denatured whey proteins interacted with the casein micelles at a faster rate at lower pH and at higher temperatures, the ratios of denatured whey proteins to \u03ba-casein on the casein micelles were not markedly different under the different heating conditions.\n\nFurther studies demonstrated the extreme importance of pH on the association of denatured whey proteins (\u03b1-lactalbumin and \u03b2-lactoglobulin) with the casein micelles when milk was heated above 70 \u00b0C, particularly at pH 6.7 or below, where differences in association behavior could be measured at pH differences as small as 0.05 pH units (Anema & Li, 2003a,b; Vasbinder & de Kruif, 2003). From these studies, it was shown that about 80% of the denatured whey protein associated with the casein micelles at pH 6.5 and that this level of association decreased linearly as the pH at heating was increased, so that only about 30% was associated at pH 6.7. At higher pH (above pH 6.7), very low levels of denatured whey proteins associated with the casein micelles on heating milk (Fig. 9.5).\n\nFigure 9.5 Level of whey proteins associated with the casein micelles\/nonsedimentable whey proteins in skim milk samples that were heated at 90 \u00b0C for various times. The pH values of the milk samples prior to heating were:  , pH 6.5;  , pH 6.55;  , pH 6.6;  , pH 6.65;  , pH 6.7;  , pH 6.9;  , pH 7.1. Adapted with permission from Anema et al. (2004a). Copyright (2004) American Chemical Society.\n\nAlthough the heat-induced pH-dependent dissociation of \u03ba-casein from the casein micelles could explain the low levels of denatured whey proteins interacting with the casein micelles at pH above 6.8, it had been reported that very little \u03ba-casein dissociated from the casein micelles at pH below 6.8 (Nieuwenhuijse et al., 1991; Singh & Fox, 1985b; Singh, 2004). Therefore, it was initially unknown why small shifts in pH between pH 6.5 and pH 6.7 affected the association of denatured whey proteins with the casein micelles when milk was heated. The level of \u03ba-casein in the serum phase was low; therefore, it was initially believed that \u03ba-casein was not involved in this partition of the whey proteins between the serum and colloidal phases (Anema & Li, 2003a; Oldfield et al., 1998b; Vasbinder & de Kruif, 2003).\n\nSubsequent studies, however, showed that the heat-induced dissociation of \u03ba-casein was pH dependent from pH 6.5 to pH 7.1, with a linear increase in serum-phase \u03ba-casein as the pH was increased throughout the pH range from 6.5 to 7.1 (Fig. 9.6a), and that the level of serum-phase \u03ba-casein was correlated with the level of serum-phase denatured whey protein (Fig. 9.6b; Anema, 2007). A similar pH-dependent dissociation of \u03ba-casein and formation of serum-phase denatured whey proteins was observed when concentrated milks were heated at different pH (Anema, 2008b). The differences in the level of dissociated \u03ba-casein between the earlier and later studies may be related to the centrifuging conditions, which may have masked the effects at the lower pH, especially under conditions where the particles are less hydrated and more readily deposited (Anema, 2007; Parker et al., 2005; Rodriguez del Angel & Dalgleish, 2006).\n\nFigure 9.6 (a) Effect of the pH at heating on the level of nonsedimentable \u03ba-casein in milk.  , serum-phase \u03ba-casein in unheated milk;  , serum-phase \u03ba-casein in milk heated at 90 \u00b0C for 20 min;  , serum-phase \u03ba-casein in milk heated at 90 \u00b0C for 25 min;  , serum-phase \u03ba-casein in milk heated at 90 \u00b0C for 30 min. (b) Relationship between the serum-phase denatured whey protein and the level of serum-phase \u03ba-casein for the heated milk samples.  , milk heated at 90 \u00b0C for 20 min;  , milk heated at 90 \u00b0C for 25 min;  , milk heated at 90 \u00b0C for 30 min. Adapted with permission from Anema (2007). Copyright (2007) American Chemical Society.\n\nAlthough the level of \u03ba-casein in the serum phase at pH below 6.7 was relatively low (less than about 30% of the total \u03ba-casein), the ratio of denatured whey protein to \u03ba-casein was high and relatively constant (about 2.5 whey proteins to each monomeric \u03ba-casein) for the serum-phase proteins at all pH. In contrast, the ratio of denatured whey protein to \u03ba-casein was only about 1:1 for the whey protein associated with the casein micelles at pH 6.5, and this decreased to about 0.5:1 at pH 7.1 (Anema, 2007). Intensive studies on the soluble whey protein\u2013 \u03ba-casein complexes formed when milk was heated at the natural pH also showed that \u03ba-casein was intimately involved in the serum-phase aggregates, and that a high ratio of denatured whey proteins to \u03ba-casein was observed (Guyomarc'h et al., 2003). Electron micrographs of the serum-phase whey protein\u2013 \u03ba-casein aggregates indicated that these particles were roughly spherical, with a relatively uniform size of about 20\u201350 nm (Parker et al., 2005; Rodriguez del Angel & Dalgleish, 2006).\n\nThere is still some debate over the sequence of events in the interaction reactions between the denatured whey proteins and \u03ba-casein. Some reports suggest that \u03ba-casein dissociates from the micelles early in the heating process and that the denatured whey proteins subsequently interact with the \u03ba-casein either in the serum phase or on the micelles, with a preferential serum-phase reaction (Anema & Li, 2000; Anema 2007; 2008a;). This proposal was supported by the observations that the dissociation of \u03ba-casein is a rapid process and that significant dissociation of \u03ba-casein can occur at temperatures below those where the denaturation of whey proteins occurs (Anema & Klostermeyer, 1997). In addition, significant dissociation of \u03ba-casein occurs in systems that have been depleted of whey proteins (Anema & Li, 2000). The higher ratio of denatured whey protein to \u03ba-casein for the serum phase regardless of the pH at heating or the level of dissociated \u03ba-casein may also suggest a preferential serum-phase reaction between the denatured whey proteins and \u03ba-casein (Anema, 2007).\n\nHowever, other reports suggest that, on heating milk, the denatured whey proteins first interact with the casein micelles and that the whey protein\u2013 \u03ba-casein complexes subsequently dissociate from the casein micelles (Donato & Dalgleish, 2006; Donato et al., 2007b; Donato & Guyomarc'h, 2009; Parker et al., 2005). This proposal was supported by the observation that the addition of sodium caseinate to milk did not increase the level of serum-phase complexes between the denatured whey proteins and \u03ba-casein, which was interpreted as indicating that the complexes between the denatured whey proteins and \u03ba-casein were formed on the casein micelle surface regardless of the pH at heating (Parker et al., 2005).\n\nMilks with added \u03ba-casein were analyzed by size exclusion chromatography (Donato et al., 2007b). The difference profiles between the sera from unheated and heated milk with added \u03ba-casein produced a negative peak in the region of the native whey proteins and a positive peak in the region of the whey protein\u2013 \u03ba-casein aggregates. As the difference spectra was the same as for milks without added \u03ba-casein, this was also interpreted as indicating that the added \u03ba-casein was not involved in the formation of the serum-phase aggregates, and therefore the denatured whey proteins only interacted with micelle bound \u03ba-casein, with the complexes subsequently dissociating from the casein micelles (Donato et al., 2007b; Donato & Guyomarc'h, 2009).\n\nThe partial hydrolysis of \u03ba-casein by chymosin prevented dissociation of unhydrolyzed \u03ba-casein from the casein micelles. This in turn prevented the formation of serum-phase whey protein\u2013 \u03ba-casein complexes and therefore increased the level of denatured whey proteins associated with the casein micelles (Renan et al., 2007). This observation was also used as evidence to support the initial interaction of denatured whey proteins with the casein micelles, with the subsequent dissociation of whey protein\u2013 \u03ba-casein complexes as it was suggested that the unhydrolyzed \u03ba-casein should still be able to dissociate and interact with serum-phase denatured whey proteins if this was the preferential reaction pathway (Renan et al., 2007). However, this proposal did not take into account the polymeric nature of \u03ba-casein (Holland et al., 2008), and therefore the partial hydrolysis of a \u03ba-casein would substantially increase the hydrophobicity of the polymer even when it contained some unhydrolyzed \u03ba-casein. This increased hydrophobicity may account for the reduced dissociation of \u03ba-casein from the micelles and the increased interaction of denatured whey proteins with the casein micelles (Donato & Guyomarc'h, 2009).\n\nIt was also suggested that two mechanisms occur depending on the pH at heating. This suggestion was based on observations that the protein composition of the serum phase appeared to vary markedly depending on whether the milk was heated at pH above or below the natural pH of the milk (Donato & Dalgleish, 2006). However, other studies did not display a marked difference in composition of serum-phase proteins with pH (Anema, 2007).\n\nA detailed study was conducted aimed specifically at elucidating the sequence of events occurring when denatured whey proteins interacted with \u03ba-casein in heated milks (Anema, 2008a). It was shown that \u03ba-casein could dissociate from the casein micelles at temperatures that were below those where the whey proteins denatured (Fig. 9.7a). When heated at temperatures at which the whey proteins could denature, it was found that \u03ba-casein dissociated from the casein micelles in the early stages of heating and before significant levels of whey proteins were denatured. In addition, the maximum level of serum-phase \u03ba-casein was obtained when less than half the whey proteins were denatured. Once this maximum level of \u03ba-casein was dissociated, any additional denatured whey proteins formed on prolonged heating were predominantly found in the serum phase (Fig. 9.7b), indicating a preferential interaction of the denatured whey proteins with the serum-phase whey protein\u2013 \u03ba-casein complexes.\n\nFigure 9.7 (a) Level of serum-phase whey protein (filled symbols) and \u03ba-casein (open symbols) in milk samples heated for 15 min at different temperatures. (b) Level of denatured (filled symbols) and micelle-bound (open symbols) whey proteins in milk samples heated at 90 \u00b0C for different times. (c) Level of serum-phase \u03ba-casein in milk samples heated at 90 \u00b0C for different times. The skim milks were adjusted to pH 6.5 ( ,  ), pH 6.7 ( ,  ), and pH 6.9 ( ,  ) before heating. Adapted with permission from Anema (2008a). Copyright (2008) Cambridge University Press.\n\nWhen \u03ba-casein was added to the milk prior to heating, the denatured whey proteins preferentially interacted with the added serum-phase \u03ba-casein, regardless of the pH at heating (Table 9.1). Taken together, these results provide unequivocal evidence that \u03ba-casein dissociation from the micelles can precede the interaction of denatured whey proteins with the \u03ba-casein, and that denatured whey proteins will preferentially interact with serum-phase \u03ba-casein (Anema, 2008a).\n\nTable 9.1\n\nSerum-phase denatured whey proteins from skim milk with different levels of added \u03ba-casein that were adjusted to pH 6.5, 6.7, and 6.9 before heating at 90 \u00b0C for 15 min.\n\nAdded \u03ba-casein (%) | pH at heating | Serum-phase denatured whey protein  \n(% of total)  \n---|---|---  \n0 | 6.5 | 35 \u00b1 1a  \n0.1 | 6.5 | 67 \u00b1 2b  \n0.2 | 6.5 | 84 \u00b1 3c  \n|  |   \n0 | 6.7 | 71 \u00b1 2a  \n0.1 | 6.7 | 78 \u00b1 3b  \n0.2 | 6.7 | 92 \u00b1 3c  \n|  |   \n0 | 6.9 | 86 \u00b1 3a  \n0.1 | 6.9 | 91 \u00b1 3a,b  \n0.2 | 6.9 | 95 \u00b1 3b\n\nThe numbers represent the average and standard deviation of triplicate measurements.\n\nData at a given pH with the same letters are not significantly different from each other at P <0.05.\n\nReproduced with permission from Anema (2008a). Copyright (2008) Cambridge University Press.\n\nThe pH-dependent changes in the association of the denatured whey proteins with the casein micelles, and the dissociation of \u03ba-casein from the micelles can have an effect on some of the physical properties of the milk. A marked increase in casein micelle size was observed when high levels of denatured whey protein were associated with the colloidal phase, as is observed on heating milk at pH 6.5. This change in size was less pronounced as the pH at heating the milk was increased to pH 6.7, and a decrease in casein micelle size was observed when significant levels of \u03ba-casein were dissociated from the colloidal phase, as is seen on heating milk at pH above 6.7 (Fig. 9.8a; Anema & Li, 2003a,b). Similar changes in viscosity (Fig. 9.8b) and turbidity (Fig. 9.8c) with the pH at heating were also observed (Anema et al., 2004c).\n\nFigure 9.8 Effects of the pH at heating on the changes in the size of casein micelles (a), the viscosity of the milk (b), and the turbidity of the milk (c). The milk samples were heated at 90 \u00b0C for various times and the pH values of the milk samples prior to heating were:  , pH 6.5;  , pH 6.55;  , pH 6.6;  , pH 6.65;  , pH 6.7;  , pH 6.9;  , pH 7.1. Some of the particle size and viscosity results were adapted with permission from Anema et al. (2004c). Copyright (2004) Elsevier.\n\nThe difficulty in interpreting these changes in size, viscosity, and turbidity is determining (1) whether the association of the denatured whey proteins with the casein micelles is directly responsible for the change in size\/volume of the casein micelles by increasing the diameters of the individual particles as the proteins interact, or (2) whether there is some associated phenomenon, such as aggregation of the casein micelles, that is related to the level of whey protein or \u03ba-casein that is in the serum phase or associated with the casein micelles. The strong relationship between the level of whey protein associating with the colloidal phase and the size\/volume of the casein micelles, the observation that the size change plateaus on prolonged heating, and the relationships between the protein composition of the micelles and size, viscosity, and turbidity seem to suggest that the size changes are a direct consequence of the distribution of protein between the colloidal and serum phases, rather than an associated aggregation reaction (Anema & Li, 2003a,b; Anema et al., 2004c).\n\n#### Determination of the Specific Disulfide Bonds Formed between \u03ba-Casein and \u03b2-Lactoglobulin\n\nAlthough many types of bonding may be involved in the early stages of interactions between the denatured whey proteins and \u03ba-casein, there is clear evidence that disulfide bonds are involved in complex formation when model systems and milk are heated. Recent studies have focused on determining the specific thiol groups of \u03ba-casein and, in particular, \u03b2-lactoglobulin that are involved in the disulfide bonding between these two protein species when they are heated in model systems or milk (Creamer et al., 2004; Henry et al., 2002; Livney & Dalgleish, 2004; Lowe et al., 2004). Understanding the specific disulfide bonds involved in the interaction process may provide useful insights into the mechanisms for the denaturation and subsequent aggregation reactions of the whey proteins in milk.\n\nNative \u03b2-lactoglobulin has two disulfide bonds and one free thiol group at Cys121 (Qin et al., 1999), whereas \u03ba-casein is found as a heterogeneous polymeric protein cross-linked in a random manner by disulfide bonding via the two Cys groups in the monomer protein (Holland et al., 2008; Rasmussen et al., 1999). It was believed that the formation of disulfide bonds between the denatured \u03b2-lactoglobulin and other milk proteins, including \u03ba-casein, during heating first involved the dissociation of the \u03b2-lactoglobulin dimer to monomer species, followed by the unfolding of the native structure, exposing the buried side groups including the free thiol at Cys121. The exposure of this free Cys121 then initiated a series of intermolecular thiol\u2013disulfide exchange reactions with other denatured whey proteins and with \u03ba-casein on the casein micelle surface (Creamer et al., 2004; Hoffmann & van Mil, 1997; Iametti et al., 1996; Snoeren & van der Spek, 1977; Vasbinder & de Kruif, 2003; Verheul et al., 1998).\n\nStudies on pure \u03b2-lactoglobulin, however, indicated that in the early stages of the denaturation process, non-native monomeric \u03b2-lactoglobulin species are formed, which may be intermediates in the intermolecular aggregation processes. These non-native monomeric species are stable upon subsequent cooling and can be separated by alkaline PAGE techniques (Hong & Creamer, 2002; Manderson et al., 1998) or by gel permeation chromatography (Croguennec et al., 2003; ; Iametti et al., 1996). It was hypothesized that the non-native monomers were formed from intramolecular thiol\u2013disulfide exchange reactions between the free Cys121 of \u03b2-lactoglobulin and the Cys106\u2013Cys119 and\/or Cys66\u2013Cys160 disulfide bonds within the same \u03b2-lactoglobulin monomer (Croguennec et al. 2003; ; Hong & Creamer, 2002; Iametti et al., 1996; Manderson et al., 1998).\n\nWith the advent of sensitive mass spectrometric techniques, the identification of the Cys residues involved in disulfide-bonded protein species was possible. The strategy for identifying specific disulfide bonds involves a number of steps (Gillece Castro & Stults, 1996; Gorman et al., 2002; Kehoe et al., 2007; Lowe et al., 2004). For the sample under analysis, the protein species are first hydrolyzed under conditions where no further thiol\u2013disulfide bond exchange reactions are likely to occur. The peptides formed from this hydrolysis are separated, usually by reverse-phase HPLC, and the mass of individual peptides is determined by mass spectrometry (MS). The identification of individual peptides can be achieved by comparing the measured masses with those of expected peptides for the hydrolysis of the protein under study. For the disulfide-bonded peptides, usually other criteria also need to be satisfied, such as the peptides being present in nonreduced hydrolysates but absent in the reduced system.\n\nFurther confirmation can be gained by the use of tandem MS, where single molecular ions are isolated and analyzed in the first mass analyzer and then passed into a collision cell where fragmentation of the peptide is induced by collision with an inert gas collision-induced dissociation (CID)] and the fragments are characterized in the second mass analyzer. From the mass of the fragments, the sequence of the amino acids in the peptides can be determined, providing conclusive characterization of the peptides ([Gorman et al., 2002; Kehoe et al., 2007; Lowe et al., 2004).\n\nUsing these types of mass spectrometric techniques, researchers found a stable non-native \u03b2-lactoglobulin monomer with a free thiol group at position Cys119 rather than the natural position of Cys121 in heated \u03b2-lactoglobulin solutions (Croguennec et al., 2003). This finding confirmed that intramolecular thiol\u2013disulfide exchange within monomeric \u03b2-lactoglobulin could occur. It was suggested that this \u03b2-lactoglobulin with the free thiol at Cys119 may be the activated monomer that was proposed as the starting point for intermolecular aggregation reactions leading to large polymers. It was equally possible, however, that unfolded protein with a free thiol at the natural position of Cys121 was that activated monomer (Creamer et al., 2004; Croguennec et al. 2003; ;).\n\nA more recent investigation on the disulfide bonding patterns in heated \u03b2-lactoglobulin found that a significant proportion of Cys160 was in the reduced form after heating \u03b2\\- lactoglobulin in solution (Creamer et al., 2004; Kehoe et al., 2007), indicating that the Cys66\u2013Cys160 disulfide bond was broken during the early stages of heating. This may occur concurrently with the interchange of the free thiol from Cys121 to Cys119. It was suggested that a monomeric \u03b2-lactoglobulin species with a free thiol at Cys160 may be (one of) the reactive species involved in the intermolecular thiol\u2013disulfide bonding responsible for cross-linking in heat-induced whey protein aggregates because of its position near the C-terminal end of the protein.\n\nAttempts have been made to identify the specific Cys residues involved in disulfide bonds formed between \u03ba-casein and \u03b2-lactoglobulin when these proteins are heated together. Livney and Dalgleish (2004) compared masses of peptides from tryptic digests of heated \u03ba-casein\/\u03b2-lactoglobulin mixtures with theoretical values and concluded that Cys106\/119\/121 of \u03b2-lactoglobulin were involved in disulfide bonds with both Cys11 and Cys88 of \u03ba-casein. (Note that the hydrolysis pattern does not allow the separation of the three Cys106\/119\/121 residues of \u03b2-lactoglobulin unless CID is used for sequencing.) Although some peptides involving Cys66 and Cys160 of \u03b2-lactoglobulin and the two Cys residues of \u03ba-casein were also identified based on mass comparisons, the high abundance of disulfide-bonded peptides containing Cys106\/119\/121 led these authors to conclude that \u03b2-lactoglobulin with a free thiol at Cys119\/121 was the predominant species involved in intermolecular disulfide bonding. The potential disulfide-bonded species were characterized based on mass analysis alone; no confirmatory experiments were performed, such as comparing reduced with nonreduced systems to ensure that the proposed intermolecular peptides were disulfide bonded, or confirming sequences by CID\u2013MS to preclude misidentification of similarly massed peptides.\n\nIn a novel study, Lowe et al. (2004) used an activated monomeric \u03ba-casein with reduced thiol groups that were blocked with thionitrobenzoate (TNB). \u03b2-lactoglobulin was added to the mixture, and the system was heated under very mild conditions (60 \u00b0C). The TNB groups on the thiols of \u03ba-casein are good leaving groups and, when a reactive thiol from \u03b2-lactoglobulin is exposed, it is capable of interacting with the activated TNB groups on \u03ba-casein in a specific 1:1 oxidative reaction, forming a disulfide-bonded complex and releasing the TNB as a brightly colored compound. This approach allowed the formation of specific disulfide bonds between \u03ba-casein and \u03b2-lactoglobulin under mild heating conditions. Because of the chemical nature of the reaction, it limited further thiol\u2013disulfide exchange reactions, which allowed specific interactions between \u03b2-lactoglobulin and \u03ba-casein to be monitored during the early stages of the denaturation of \u03b2-lactoglobulin.\n\nThe interacted \u03b2-lactoglobulin\u2013 \u03ba-casein complexes were hydrolyzed with trypsin and separated by reverse-phase HPLC followed by MS. In addition, disulfide bonding was confirmed by comparing HPLC traces of nonreduced systems with those of reduced systems, and the identities of some peptides were confirmed by sequencing using CID\u2013MS. Although it was possible to identify disulfide bonds between Cys106\/119\/121 of \u03b2-lactoglobulin and Cys88 of \u03ba-casein, Cys160 of \u03b2-lactoglobulin was found to have formed disulfide bonds with both Cys11 and Cys88 of \u03ba-casein as major products (Fig. 9.9). This supported the earlier findings on pure \u03b2-lactoglobulin that intramolecular thiol\u2013disulfide exchange may precede the intermolecular reactions and that the non-native monomeric form of \u03b2-lactoglobulin species with a free thiol at Cys160 is likely to be one of the reactive monomer species that initiates intermolecular thiol\u2013disulfide exchange reactions (Creamer et al., 2004; Kehoe et al., 2007).\n\nFigure 9.9 Diagram indicating the identified peptides on the linear sequences of \u03ba-casein and \u03b2-lactoglobulin, and the intermolecular disulfide bonds formed between \u03ba-casein and \u03b2-lactoglobulin on heating model systems and milk. The horizontal box lines represent the protein sequence; the lines over the boxes represent the peptides; and S\u2013S indicates the presence of a disulfide bond. Arrows indicate the major proteolytic sites, the chymosin site for \u03ba-casein, and the rapid tryptic sites for \u03b2-lactoglobulin. Potential glycosylation and phosphorylation sites are indicated for \u03ba-casein. Reproduced with permission from Lowe et al. (2004). Copyright (2004) American Chemical Society.\n\nLowe et al. (2004), using the techniques developed for the model system, expanded the study to examine the specific disulfide bonds involved in aggregation between \u03b2-lactoglobulin and \u03ba-casein in heated milk systems. Interestingly, no disulfide bonds between Cys106\/119\/121 of \u03b2-lactoglobulin and either of the Cys residues of \u03ba-casein could be found, even though the disulfide bond between Cys88 of \u03ba-casein and Cys106\/119\/121 of \u03b2-lactoglobulin was readily identified in the model system. In the heated milk system, it was found that Cys160 of \u03b2-lactoglobulin formed disulfide bonds with both Cys88 and Cys11 of \u03ba-casein, as was found in the model system (Fig. 9.9). In independent studies, a similar disulfide bond between Cys160 of \u03b2-lactoglobulin and Cys88 of \u03ba-casein was identified in a heated model goat milk system consisting of isolated casein micelles and \u03b2-lactoglobulin suspended in milk ultrafiltrate, although it appears that no attempts were made to isolate and characterize other intermolecular disulfide bonds between these protein species (Henry et al., 2002).\n\nFrom these observations, it was concluded that, in the model system of \u03b2-lactoglobulin and activated \u03ba-casein, non-native monomeric \u03b2-lactoglobulin species with a free thiol at either Cys119 or Cys121 (but probably not Cys106) could be the reactive monomer involved in intermolecular thiol\u2013disulfide exchange reactions, as Cys119\/121 was involved in disulfide bonds with \u03ba-casein. However, further intramolecular thiol\u2013disulfide exchange reactions in \u03b2-lactoglobulin must precede or occur concurrently with the intermolecular reactions, as disulfide bonds between Cys160 of \u03b2-lactoglobulin and the two Cys residues of \u03ba-casein were also observed as major products in the model system (Fig. 9.9).\n\nIn the heated milk system, no peptides involving Cys106\/119\/121 and the two Cys residues of \u03ba-casein were isolated. Only peptides involving Cys160 and Cys66 with both Cys residues of \u03ba-casein were found (Fig. 9.9). As Cys160 and Cys66 are involved in a disulfide bond in native \u03b2-lactoglobulin, this indicates that intramolecular thiol\u2013disulfide exchange reactions in \u03b2-lactoglobulin precede the intermolecular thiol\u2013disulfide exchange reactions, and that a \u03b2-lactoglobulin (monomeric) species with a free thiol group at Cys160 may play a significant role in the interprotein disulfide bonding that occurs in heated milk or whey protein systems.\n\nThe differences in reaction products between the model systems and milk may be a consequence of factors such as the heating conditions, the nature of the reactions (oxidative interaction compared with thiol\u2013disulfide interchange reactions), and the fact that the \u03ba-casein in milk is found on the casein micelles, whereas in the model system it is not (Lowe et al., 2004). Because of the C-terminal location of Cys160 in \u03b2-lactoglobulin, when this Cys is in the free thiol form and not linked to Cys66, it may be able to productively react with the disulfide bonds of \u03ba-casein to give stable \u03ba-casein\u2013whey protein aggregates (Lowe et al., 2004).\n\n## Relationships between denaturation\/interactions of the whey proteins in heated milk and the functional properties of milk\n\nWhen milk is heated, numerous changes take place, including changes to the proteins, the milk salts (such as changes in the mineral equilibria between the colloidal and serum phases), and lactose. Many of the changes can involve more than one of the milk constituents (IDF, 1995). The changes can be irreversible or reversible to various extents, depending on the changes being monitored and the conditions of the heat treatment. Although the changes to the protein system are an important determinant of the functional properties of milk products, all other changes to the milk system should also be considered to obtain a full understanding of the relationships between heat treatments, interactions, and functional performance. However, there are limited examples of changes to components other than the proteins, and the functional behavior of milk products, and therefore this review, are restricted to some examples of the relationships between the changes in the milk protein system and the functional performance of the milk.\n\n### Examples of the Relationships between Whey Protein Denaturation and the Functional Properties of Milk\n\nIn the early days of milk powder manufacture, it was recognized that the level of whey protein denaturation could be used as an index for the extent of heat treatment the milk had received during the manufacture of the milk powders. The functional properties of the milk products were related to some extent to the heat treatment that the milk had received during processing and therefore the level of whey protein denaturation (Harland & Ashworth, 1947; Harland et al., 1952; Larson et al., 1951).\n\nEven as early as 1952, the concept of 'tailor-made' milk powders was discussed, where powders were processed to provide specific requirements, such as low-heat powders for beverage applications and cottage cheese manufacture, and high-heat powders for bakery applications (Harland et al., 1952). Although there were no standards of quality or processing at this time, it was recognized that the proper control of processing conditions, particularly preheating of the milk, was necessary to produce satisfactory products. Measurement of the level of whey protein denaturation could be used as an objective method for determining the suitability of milk (powder) products for particular commercial and functional applications.\n\nThe heat treatment of milk, whether in liquid milk applications or prior to drying for milk powder manufacture, remains one of the major processes for manipulating the functional properties of milk products. Products such as milk powders are still generally classified according to the heat treatments received during manufacture using one of the derivatives of the WPNI test (Kelly et al., 2003; Singh & Newstead, 1992). With the extensive research on the denaturation of the whey proteins and the ability to predict the denaturation levels after defined heat treatments, it would be envisaged that the level of denaturation of the whey proteins could be used as an indicator of the functional properties of milk products. In a broad sense, this is true. For example, certain heat classifications of milk powders will give improved functionality for particular applications over other classes of milk powders. Some of the general applications of different heat-classified milks and their functional uses, in particular for milk powder products, have been summarized in numerous publications (IDF 1995; ; Kelly et al., 2003; Singh & Newstead, 1992).\n\nHowever, an infinite range of temperature and heating time combinations are available to denature the whey proteins when milk is heated. As a consequence, specific correlations between the level of whey protein denaturation and the functional properties of milk across all possible heating conditions and milk sources do not exist. For example, the WPNI method was developed for assessing the suitability of milk powders for use in bakery applications; however, it was noted that a powder with a low WPNI did not always correspond to good baking qualities (Harland & Ashworth, 1947). Some of these variations are due to factors such as natural variations in the initial whey protein levels in the milk (Harland et al., 1955; Sanderson, 1970a). Others, however, are due to the methods of heat treatment during milk processing. As such, the WPNI or level of whey protein denaturation is, at best, a guide for the suitability of powders for specific applications, or an in-factory guide on processing conditions. Many manufacturers impose additional specifications on the milk powders to ensure suitability in their specific applications (Sanderson, 1970c; Singh & Creamer, 1991a).\n\nSome of the most detailed studies on the relationship between the functional performance of milk and the heat treatment conditions or whey protein denaturation levels have been reported for acid gel or yogurt systems. Parnell-Clunies et al. (1986) showed correlations between the level of whey protein denaturation and the firmness and apparent viscosity of yogurt, regardless of the method used to heat the milk [batch (85 \u00b0C), high-temperature short-time (98 \u00b0C), and ultra-high-temperature (140 \u00b0C) heating systems for different holding times]. However, other properties such as water-holding capacity\/syneresis were more dependent on the heating system used, and it was concluded that high levels of whey protein denaturation in milk were not necessarily associated with an improved water-holding capacity in yogurt.\n\nIn extensive studies, Dannenberg and Kessler (1988b,c) examined the relationship between the denaturation level of whey proteins in milk and the functional performance (firmness, flow properties, and syneresis) of the milk in set yogurt applications. There was a clear relationship between whey protein denaturation and yogurt firmness, with a higher firmness at higher levels of whey protein denaturation. Similar results were obtained for the flow properties of the yogurt. However, very high levels of whey protein denaturation appeared to be detrimental, with a decrease in the firmness and flow properties at denaturation levels above about 95% (Dannenberg & Kessler, 1988c).\n\nFor syneresis of the yogurt, a negative relationship between whey protein denaturation and the level of serum expelled from the yogurt was observed (Dannenberg & Kessler, 1988b). Despite the apparent correlations between denaturation and firmness, flow properties, or syneresis, there were significant variations at each denaturation level, indicating that the temperature of heating used to denature the whey proteins, rather than just the whey protein denaturation level, may be an important factor in determining the functional performance in acid gels.\n\nIn a study on reconstituted whole milk, McKenna and Anema (1993) also observed a positive correlation between the denaturation of the whey proteins in the milk and the firmness of the yogurt made from the milk, regardless of whether the heat treatment was performed before or after powder manufacture (Fig. 9.10a). However, when individual heating conditions were examined, it was also noted that excessive heat treatment of the milk could be detrimental to the firmness of the set yogurt (McKenna & Anema, 1993). A less clear relationship between syneresis and the level of whey protein denaturation was observed, with the level of syneresis appearing to have a greater dependence on the heating conditions (temperature, time, and before\/after reconstitution) than on the level of denaturation itself (Fig. 9.10b; McKenna & Anema, 1993), which supports the findings of Parnell-Clunies et al. (1986).\n\nFigure 9.10 Relationship between the level of whey protein denaturation in reconstituted whole milk and the firmness (a) and syneresis (b) of acid gels prepared from the heated milks. The milks were heated only before powder manufacture ( ), heated only after reconstitution ( ) or heated both before powder manufacture and after reconstitution ( ). Adapted with permission from the results of McKenna & Anema (1993). Copyright (1993) International Dairy Federation.\n\n### Examples of the Relationships between the Level of Interactions of Whey Proteins with \u03ba-Casein\/Casein Micelles and the Functional Properties of Milk\n\nA major limitation in using whey protein denaturation as an index of the functional properties of milk is that it does not consider the subsequent interaction reactions of the denatured whey proteins. These interactions will be dependent on the conditions of denaturation such as temperature and time as well as on the properties of the milk such as pH, concentration, and composition. It is more complex to investigate these aggregation reactions, as there are potentially numerous pathways, and there is great difficulty in isolating and characterizing the specific reaction products. However, in recent years some effort has been made in identifying the interaction reactions of the denatured whey proteins with other components in milk, and in some cases their effects on the functional properties of the milk.\n\n#### Acid-induced Aggregation\/Gelation of Heated Milk\n\nAnema et al. (2004a) showed that small changes in pH of milk from the natural value at the time of heating markedly affected the properties of acid gels prepared from these heated milks. During acidification of the heated pH-adjusted milks, the acid gelation profiles were progressively shifted to higher firmness as the pH at heating was increased (Fig. 9.11a), so that the final firmness of the acid gels (measured as the storage modulus, G\u2032, at pH 4.2) was almost doubled as the pH at heating was increased from pH 6.5 to pH 7.1 (Fig. 9.11). The effect was particularly pronounced in the milks that were heated for times sufficient to fully denature the whey proteins (Fig. 9.11b). This effect of small changes in the pH of the milk at the time of heating on the firmness of acid gels prepared from the heated milk has been independently confirmed (Lakemond & van Vliet, 2005; 2008b; Rodriguez del Angel & Dalgleish, 2006).\n\nFigure 9.11 (a) Changes in firmness (G\u2032) with time after the addition of glucono-\u03b4-lactone (GDL) for heated (90 \u00b0C\/30 min) skim milk samples. (b) Changes in the final firmness (final G\u2032) for acid gels prepared from milk samples heated for various times at 90 \u00b0C. The pH values of the milk samples prior to heating were  , pH 6.5;  , pH 6.55;  , pH 6.6;  , pH 6.65;  , pH 6.7;  , pH 6.9;  , pH 7.1. In all samples, the pH was readjusted back to pH 6.7 before addition of GDL, and this pH reduced to about pH 4.2 after 5.5 h. Reproduced with permission from Anema et al. (2004a). Copyright (2004) American Chemical Society.\n\nLarge strain deformation experiments were also conducted on the set gels. In these experiments, the strain was increased at a constant rate and the stress was monitored until the gel structure yielded and the stress decreased. The maximum in the strain versus stress curves was considered to be the point at which the gel structure broke (Fig. 9.12). The yield properties of the final set acid gels were affected by the pH at which the milk was heated. The yield stresses of the gels increased markedly, and the yield strains of the gels decreased slightly as the pH at which the milk was heated was increased (Fig. 9.12; Anema, 2008b; Lakemond & van Vliet, 2008b).\n\nFigure 9.12 Stress versus strain curves for acid gels prepared from heated (90 \u00b0C\/15 min) skim milk samples. The pH values of the milk samples prior to heating were  , pH 6.5;  , pH 6.6;  , pH 6.7;  , pH 6.9;  , pH 7.1. The maximum in the stress represents the breaking point of the gel, and the stress and strain at this point are considered to be the breaking stress and breaking strain, respectively.\n\nIn addition to influencing the final firmness of the acid gels, the pH at the heat treatment of the milk also influenced the pH at which the milk started gelling\/aggregating during acidification (Anema et al., 2004a; 2004b; Lakemond & van Vliet, 2005; 2008b; Rodriguez del Angel & Dalgleish, 2006; Vasbinder & de Kruif, 2003). On subsequent acidification of milk samples that were heated over a pH range from pH 6.5 to 7.1, samples heated at higher pH (pH 7.1) started gelling at significantly higher pH than samples heated at a lower pH. These effects were very dependent on the temperature at which the milks were acidified (Anema et al., 2004b).\n\nIn further detailed studies on the properties of the acid gels prepared from milks heated at different pH (Lakemond & van Vliet, 2008b), it was shown that the permeability coefficients of the gels increased as the pH of the milks at heating increased. In addition, adding thiol blocking agents to the milks during acid gelation did not affect the firmness of gels prepared from milks heated at low pH (about pH 6.20), but markedly reduced the firmness of the gels prepared from milks heated at higher pH (about pH 6.90). Based on the permeability, effects of thiol blocking agents and the small and large strain rheological results of the acid gels prepared from pH adjusted heated milks, it was concluded that acid gels from milks heated at low pH had finer stranded structure with a higher strand curvature and the gel contained fewer (intermolecular) disulfide bonds. These differences in gel properties accounted for the lower firmness of the gels prepared from milks heated at lower pH (Lakemond & van Vliet, 2008b).\n\nIn concentrated milk, a similar effect of milk pH at heating on acid gel firmness and yield stress was observed, with a marked increase in final gel firmness (Fig. 9.13a and b) and yield stress (Fig. 9.14a and b) as the pH of the milk at heating was increased from the natural pH, and a marked decrease in firmness when the pH of the milk at heating was decreased (Anema, 2008b). As the natural pH of milk decreases when milk is concentrated, the percentage change in firmness or yield stress of the gels from that obtained at the natural pH was plotted against the relative change in pH, and it was found that the firmness (Fig. 9.13c) or yield stresses (Fig. 9.14c) of the gels at all milk concentrations fell on a single curve. This indicates that changing the pH of milk at heating had the same effect on the final firmness or yield stresses of the acid gels at all milk concentrations (Anema, 2008b).\n\nFigure 9.13 (a) Changes in storage modulus, G\u2032, with time after GDL addition for heated (80 \u00b0C\/30 min) 10% TS skim milk samples.  , pH 6.48;  , pH 6.55;  , pH 6.59;  , pH 6.67;  , pH 6.90;  , pH 7.10. (b) Changes in storage modulus, G\u2032, with time after GDL addition for heated (80 \u00b0C\/30 min) 20% TS skim milk samples.  , pH 6.28;  , pH 6.39;  , pH 6.48;  , pH 6.75;  , pH 6.95. (c) Percentage change in final G\u2032 versus change in pH at heating from the natural pH of the milk.  ,  : 10% TS milk samples;  ,  : 15% TS milk samples;  ,  : 20% TS milk samples;  ,  : 25% TS milk samples. Open symbols: samples at the natural pH; filled symbols: samples that were adjusted in pH before heating. Reproduced with permission from Anema et al. (2008b). Copyright (2008) Elsevier.\n\nFigure 9.14 (a) Stress versus strain curves for acid gels prepared from heated (80 \u00b0C\/30 min) 10% TS skim milk samples.  , pH 6.48;  , pH 6.55;  , pH 6.59;  , pH 6.67;  , pH 6.90;  , pH 7.10. (b) Stress versus strain curves for acid gels prepared from heated (80 \u00b0C\/30 min) 20% TS skim milk samples.  , pH 6.28;  , pH 6.39;  , pH 6.48;  , pH 6.75;  , pH 6.95. (c) Percentage change in breaking stress versus change in pH at heating from the natural pH of the milk.  : 10% TS milk samples;  : 15% TS milk samples;  : 20% TS milk samples;  : 25% TS milk samples. Reproduced with permission from Anema et al. (2008b). Copyright (2008) Elsevier.\n\nWhen low levels of starch (up to 1% w\/w) were added to milks prior to heating and acidification, the pH effect on gel firmness was similar to that of milk without starch addition; however, the firmness of the gels increased as the starch level increased (Oh et al., 2007). The gelatinized starch absorbed the aqueous phase on the milk and consequently increased the density of the protein network in the acid gels, increasing the firmness in a similar fashion to increasing the concentration of the milk. However, when higher levels of starch were added to the milk (>1% w\/w), the pH effect diminished, although the gels were still firmer as the starch level increased (Oh et al., 2007). At these high levels of starch, the viscosity of the milk markedly increased after heating due to the gelatinization of the starch and the leaching of amylose into the continuous phase. It was proposed that the high viscosity of the continuous phase may affect the diffusion of protein components during heating and subsequent acidification. This in turn changed the network structure formed in the acid gel, diminishing the importance of serum-phase components (Oh et al., 2007).\n\nThe changes in acid gel firmness or yield stress on changing the pH at heating of the milk could not be related solely to the level of whey protein denaturation (Fig. 9.11a). Small changes in the pH of the milk before heating markedly affected the distribution of the denatured whey proteins and \u03ba-casein between the colloidal and serum phases in milk at its natural concentration (Figs 9.4 and 9.5 ; Anema & Klostermeyer, 1997; Anema & Li, 2003a,b; Lakemond & van Vliet, 2008a; Rodriguez del Angel & Dalgleish, 2006; Vasbinder & de Kruif, 2003) and similar effects were observed in concentrated milks (Anema, 2008b; Chandrapala et al., 2010).\n\nAlthough heating milk prior to acidification markedly increased the firmness of the acid gels, i.e., acid gels prepared from heated milks always had a considerably higher firmness than acid gels prepared from unheated milks (Dannenberg & Kessler, 1988c; Lucey et al., 1997; Lucey & Singh, 1998), the distribution of the denatured whey proteins and \u03ba-casein between the colloidal and serum phases also appeared to influence the firmness of the acid gels. When the final firmness of the acid gels was plotted against the level of nonsedimentable denatured whey proteins in the milk, the results for all pH values fell on a single curve for milk at its natural concentration (Fig. 9.11b). Similarly for concentrated milks, when the percentage change in final gel firmness (Fig. 9.15a) or yield stress (Fig. 9.15b) was plotted against the level of nonsedimentable denatured whey proteins in the milk, the results for all pH and milk concentrations fell onto a single curve (Anema et al., 2004a; Anema, 2008b).\n\nFigure 9.15 (a) Relationship between the change in final G\u2032 for acid skim milk gels and the level of nonsedimentable denatured whey protein in heated (80 \u00b0C\/30 min), pH-adjusted skim milk. (b) Relationship between the change in breaking stress for acid skim milk gels and the level of nonsedimentable denatured whey protein in heated (80 \u00b0C\/30 min), pH-adjusted skim milk.  : 10% TS milk samples;  : 15% TS milk samples;  : 20% TS milk samples;  : 25% TS milk samples. Reproduced with permission from Anema et al. (2008b). Copyright (2008) Elsevier.\n\nFrom these results it was concluded that, although the denatured whey proteins that associated with the micelles have a significant effect on the final firmness of the acid gels, those denatured whey proteins that remain in the serum appear to have a more dominant influence over the final firmness than those associated with the casein micelles (Anema et al., 2004a; Anema, 2008b). For samples where virtually all the whey proteins were denatured, the final gel strength for acid gels prepared from milks in which all the denatured whey proteins were in the serum phase was found to be essentially a factor of two higher than that for acid gels prepared from milks in which all the whey proteins were associated with the casein micelles (Fig. 9.16; Anema et al., 2004a; Anema, 2008b). This effect was observed over a wide range of milk concentrations (Anema, 2008b).\n\nFigure 9.16 Comparison between the final firmness (final G\u2032) and the level of denatured whey protein (open symbols) and the level of soluble denatured whey protein (filled symbols) for acid gels prepared from heated (90 \u00b0C\/30 min) skim milk samples. The pH values at heating of the milks were ( ,  ): pH 6.5; ( ,  ): pH 6.55; ( ,  ): pH 6.6; ( ,  ): pH 6.65; ( ,  ): pH 6.7; ( ,  ): pH 6.9; dotted ( ,  ): pH 7.1. Adapted with permission from Anema et al. (2004a). Copyright (2004) American Chemical Society.\n\nRodriguez del Angel and Dalgleish (2006) separated the nonsedimentable whey protein\u2013 \u03ba-casein aggregates from milks heated at different pH using size exclusion chromatography, and related the peak area of these aggregates to the firmness of the acid gels. They also concluded that the gel firmness appeared to be strongly dependent on the formation of soluble complexes in the milks, and that there appeared to be a linear relationship between the level of soluble aggregates in the heated milk and the final strength of the acid gels.\n\nBased on these results, a hypothesis on the roles of the nonsedimentable and micelle-bound denatured whey protein\u2013 \u03ba-casein aggregates has been developed (Anema et al., 2004a; 2004b; Anema, 2008b; Donato et al., 2007a; Lakemond & van Vliet, 2008a,b; Rodriguez del Angel & Dalgleish, 2006). The increased pH of gelation and the increased acid gel strength of heated milk when compared with unheated milk has been attributed to the incorporation of the whey proteins as well as casein (micelles) in the acid gel structure during the acidification of milk (Graveland-Bikker & Anema, 2003; Lucey et al., 1997; Lucey, 2002).\n\nIn milk, the casein is insoluble at its isoelectric point (about pH 4.6), whereas the native whey proteins remain soluble at all pH. Therefore, unheated milk starts aggregating when the milk pH approaches the isoelectric point of casein, and visible gelation is observed at about pH 4.9. However, for heated milk, the denatured whey proteins are insoluble at their isoelectric points (about pH 5.3 for \u03b2-lactoglobulin, the major whey protein). Therefore, on acidification of heated milk, the proteins will start aggregating at a much higher pH, closer to the isoelectric points of the whey proteins. As a consequence, the contribution of the denatured whey proteins to the acid gel structure and the firmness of the acid gels is markedly higher than that observed for unheated milk (Graveland-Bikker & Anema, 2003; Lucey et al., 1997).\n\nThe pH at heating the milk will produce casein micelle particles with markedly different compositions (Figs. 9.5 and 9.6 ; Anema & Li, 2003a; Anema, 2008b; Vasbinder & de Kruif, 2003). Therefore, on acidification of milks heated at different pH values, different aggregation and gelation behavior is observed. For the milks heated at high pH, the serum-phase denatured whey proteins\/\u03ba-casein complexes may aggregate separately and at a higher pH than the casein micelles. As the isoelectric point of these serum-phase protein components will be higher than that of the casein micelles, the pH at which aggregation occurs will be progressively shifted to higher pH as the heating pH and the concentration of the serum-phase denatured whey proteins\/\u03ba-casein are increased (Anema et al., 2004a; Guyomarc'h et al., 2009; Rodriguez del Angel & Dalgleish, 2006).\n\nIn addition, the dissociation of \u03ba-casein from the casein micelles may also contribute to the higher aggregation pH as the pH at heating is increased, particularly above pH 6.7. Lower levels of \u03ba-casein on the micelles will reduce the density of the surface hairy layer. This may cause the surface hairy layer to collapse at a higher pH, or this layer may have a reduced efficiency in stabilizing the casein micelles. Either effect will allow the \u03ba-casein-depleted micelles to aggregate at a pH that is markedly higher than that observed for the native casein micelles or for casein micelles in milk heated at a lower pH.\n\nIn a study where the serum-phase and colloidal-phase protein aggregates were labeled with different fluorescent dyes before remixing and acidification, it was not possible to identify separate aggregation stages of the different fractions at the early stages of gelation. In addition, the final gel had co-localized serum and colloidal-phase aggregates (Guyomarc'h et al., 2009). This suggests that there may not be a two-stage gelation process and that when gelation starts, both serum-phase and colloidal-phase components are involved in the aggregation process. However, it is still possible that either the serum phase denatures whey proteins\/\u03ba-casein complexes, or the \u03ba-casein-depleted micelles begin aggregating at higher pH and incorporate all phases in the gelling matrix.\n\nThe firmness of acid gels can be related to the number and properties of the contact points between the protein components in the acid gel (Lakemond & van Vliet, 2008b; Lucey et al., 1997; Mellema et al., 2002; van Vliet & Keetals, 1995). As the pH at heating of the milk is increased, the level of serum-phase denatured whey protein\u2013 \u03ba-casein complexes increases, and therefore there are a greater number of particles to aggregate during the subsequent acidification to form the acid gels. There is also the potential for the formation of a more complex acid gel structure when the milk is heated at high pH, where there are high levels of serum-phase denatured whey protein\u2013 \u03ba-casein complexes than when the milk is heated at low pH, where most of the denatured whey protein and \u03ba-casein are associated with the casein micelles. In the latter case, the acid gel process will probably involve only entire whey protein\u2013casein micelle complexes. Therefore, there may be fewer contact points in the acid gels formed from milk with the denatured whey proteins associated with the micelles than in those formed from milk with soluble denatured whey proteins; hence a gel with a lower firmness is observed (Anema et al., 2004a,b; Lakemond & van Vliet, 2008b).\n\nThe large-strain deformation properties also give some indication of the types of bonds involved in the acid gel network. As the pH at heating was increased, the breaking stress of the acid gels prepared from the heated milks was found to increase markedly. However, the breaking strain was virtually unchanged (Figs. 9.12 and 9.14 ; Anema, 2008b; Lakemond & van Vliet, 2008b). For a gel to break on increasing the strain, the strands within the gel network are first straightened and then stretched until the strands, or the bonds within the strands, rupture (Lakemond & van Vliet, 2008b; Mellema et al., 2002; vanVliet & Walstra, 1995). Therefore, the breaking strain is dependent on factors such as the degree of curvature of the strands, with a higher breaking strain when the strands have greater curvature. As the breaking strain of the acid gels only changed slightly with the pH at heating of the milk, despite the marked change in final firmness (Figs. 9.12 and 9.14), this indicates that the relative curvature of the individual strands within the gel network was similar for all acid gel samples.\n\nThe types of bonds involved in the acid gel network will have an influence on the breaking stress (Lakemond & van Vliet, 2008b; Mellema et al., 2002; van Vliet & Walstra, 1995; van Vliet, 1996). The breaking of strands containing covalent bonds requires a greater force than the breaking of strands held together by noncovalent bonds, as covalent bonds have higher bond energies. Therefore, a change in the number or distribution of covalent bonds within the gel network may explain the differences in breaking stress as the pH of the milk at heating was changed (Figs. 9.12 and 9.13). It seems unlikely that the difference in breaking stress can be due to a greater degree of disulfide bonding within the gelled sample; although continuing thiol\u2013disulfide exchange reactions may be occurring during acidification (Vasbinder et al., 2003), the physical number of disulfide bonds is unlikely to be markedly different between the samples.\n\nThe denatured whey proteins, along with some of the \u03ba-casein, are progressively transferred to the serum phase when the pH of the milk is increased before heating (Figs. 9.5 and 9.6). As these interactions involve disulfide bonding, this indicates that the interaction between the denatured whey proteins and \u03ba-casein is transferred from the colloidal phase (casein micelle) to the serum phase as the pH of the milk at heating is increased. On subsequent acidification, both nonsedimentable and colloidal-phase denatured whey proteins are incorporated in the acid gel structure. The nonsedimentable denatured whey protein\u2013 \u03ba-casein complexes can form strands that may be involved in interconnecting the colloidal particles. As the nonsedimentable aggregates are disulfide bonded, those samples heated at high pH and with high levels of nonsedimentable whey protein\u2013 \u03ba-casein aggregates will have a greater number of these strands interconnecting the residual casein micelles.\n\nIn contrast, the samples heated at lower pH will have the denatured whey proteins predominantly associated with the casein micelles and therefore fewer of the whey protein\u2013 \u03ba-casein aggregates interconnecting the colloidal particles. Therefore, the samples heated at higher pH may have a greater number of disulfide bonds interconnecting the colloidal particles and therefore a higher breaking stress, whereas for the samples heated at lower pH, most of the disulfide bonds are on the colloidal particles and fewer disulfide bonds interconnect the colloidal particles; this may explain the lower breaking stress (Figs. 9.12 and 9.14).\n\n#### Chymosin-Induced Aggregation\/Gelation of Heated Milk\n\nNote: The pH of milk has a marked effect on the action of chymosin in destabilizing the system (Walstra & Jenness, 1984; Walstra et al., 1999). In all studies where the pH of milk at heating is being discussed in relation to chymosin treatment, after the heat treatment the pH of the milks were readjusted back to the natural pH, so that pH effects on the enzyme activity were eliminated.\n\nChanging the pH of milk at heating changed the interactions between denatured whey proteins and the casein micelles; however, this did not appear to influence the gelation behavior of heated milk by chymosin to any great extent (Anema et al. 2007; ; Kethireddipalli et al. 2010; ;). The rate of release of glycomacropeptide (GMP) was similar in all milks regardless of whether they were unheated or heated, and the pH at heating had almost no effect on GMP release (Anema et al., 2007; Kethireddipalli et al., 2011; Vasbinder et al., 2003). When looking at gelation profiles by chymosin, all heated milks had very long gelation times and formed very weak gels compared with unheated milks (Fig. 9.17; Anema et al. 2007; ; Kethireddipalli et al., 2010).\n\nFigure 9.17 Changes in storage modulus (G\u2032) with time after addition of rennet to unheated milks (solid symbols) and skim milk samples heated at 90 \u00b0C for 30 min (open symbols). The pH values at heating of the milks were ( ,  ): pH 6.5; ( ,  ): pH 6.7; ( ,  ): pH 6.9; ( ,  ): pH 7.1. All samples were readjusted back to the natural pH (pH 6.67) before addition of rennet (40 \u03bcL of 1:3 diluted rennet per 1.3 mL of milk). Reproduced with permission from Anema et al. (2011). Copyright (2011) American Chemical Society.\n\nThese results indicated that there was a retardation in the chymosin-induced gelation regardless of whether the denatured whey proteins were associated with the caseins micelles (as observed on heating milk at low pH) or in the serum phase associated with \u03ba-casein that had dissociated from the casein micelles (as is observed on heating milk at high pH). The inhibition of the gelation process was therefore not due to steric or charge effects of the denatured whey proteins that are associated with \u03ba-casein at the casein micelle surface. The denatured whey proteins, whether as serum-phase complexes with \u03ba-casein or associated with \u03ba-casein at the casein micelle surface, interfere with the aggregation process and therefore increase the gelation time (Anema et al., 2007; Kethireddipalli et al., 2010).\n\nThis effect was confirmed by experiments in which serum and colloidal phases from unheated and heated milks were exchanged, as it was shown that denatured whey proteins, whether in the serum phase or associated with casein micelles, inhibited the gelation of milk by chymosin. However, this inhibition may be more complex, as it was also shown that heated casein micelles in the absence of whey proteins and nonprotein serum components from heated milks also inhibited gelation (Kethireddipalli et al., 2010). From these observations it was concluded that the inhibition of gelation by chymosin for heated milks was complex and may be due to the combined effects of heat on casein micelles, denatured whey proteins (both serum and colloidal phase), and nonprotein serum components (Kethireddipalli et al., 2010).\n\nGelation of pH adjusted, heated milks by chymosin did not reveal differences; however, this is not an objective measure of the destabilization of the system, as it requires the formation of an interconnected network structure, and it is possible for the casein micelles to be destabilized without forming a gel. By monitoring the particle size changes of milk during chymosin treatment, it was possible to examine the early stages of the aggregation process (Anema et al., 2011). For all milk samples, after adding chymosin, the size initially decreased slightly due to the cleavage of GMP from \u03ba-casein (lag phase), and then increased as the destabilized particles aggregated (aggregation phase; Fig. 9.18).\n\nFigure 9.18 Changes in particle size on the rennet treatment of (a) unheated skim milk, (b) skim milk heated at 90 \u00b0C for 2.5 min, and (c) skim milk heated at 90 \u00b0C for 30 min. Milk samples (20 \u03bcL) were diluted in Ca-imidazole buffer (1 mL) before the addition of rennet (10 \u03bcL of 1:120 diluted rennet). The pHs of the milk samples were ( ) 6.5, ( ) 6.6, ( ) 6.7, ( ) 6.9, and ( ) 7.1. (d) Effect of pH on the time taken for the particle size to increase by 50 nm from the initial size (T50) after the addition of rennet to the milk samples. The milk samples were unheated ( ) or heated at 90 \u00b0C for ( ) 1 min, ( ) 2.5 min, ( ) 5 min, ( ) 10 min, ( ) 15 min, or ( ) 30 min. Reproduced with permission from Anema et al. (2011). Copyright (2011) American Chemical Society.\n\nMilks heated at temperatures below those where whey proteins denatured (\u2264 60\u00b0C) had short lag phases and rapid aggregation phases; these were similar to those from unheated milks and were only slightly affected by the pH of the milk at heating. However, milks heated at higher temperatures (> 60 \u00b0C) and at low pH (e.g., pH 6.5) had extremely long lag phases and once destabilized, the particles aggregated at a slow rate. Increasing the pH at heating shortened the lag phase and increased the rate at which the particles aggregated. In fact, samples heated at pH 7.1 had lag phases and aggregation rates not dissimilar to those of unheated milks (Anema et al., 2011).\n\nSimilarly, for milks heated at 90 \u00b0C for different times, the lag phase decreased, and the rate of aggregation increased markedly as the pH of the milk at heating was increased from pH 6.5 to pH 7.1. In fact, the lag phases and aggregation rates for the milks heated at pH 7.1 were not substantially different from those from unheated milks (Fig. 9.18). The effect of pH was greater as the heating time was increased, predominantly as a result of an increased lag phase and decreased aggregation rate of the samples heated at lower pH when compared with those heated at a higher pH (Anema et al., 2011; Fig. 9.18).\n\nFor the milk samples heated at high temperatures, there were positive correlations between the aggregation time and the level of whey protein or \u03ba-casein that was associated with the casein micelles (Fig. 9.19). It was proposed that casein micelles that were substantially coated in denatured whey proteins and \u03ba-casein (obtained on heating milks at low pH) were more resistant to chymosin-induced aggregation than casein micelles with low levels of denatured whey proteins and \u03ba-casein on the casein micelle surface (as observed on heating milk at high pH; Anema et al., 2011). It was also proposed that, although casein micelles in heated milks aggregated on chymosin treatment, the denatured whey proteins, whether associated with the micelles or in the serum phase, inhibited the gelation of the milk by stabilizing the surface of aggregated particles and preventing or slowing the formation of an interconnected network structure (Anema et al., 2011).\n\nFigure 9.19 Relationship between the time taken for the particle size to increase by 50 nm and (a) the percentage of micelle-bound \u03b2-lactoglobulin, (b) the percentage of micelle-bound \u03b1-lactalbumin, (c) the percentage of micelle-bound \u03ba-casein (all data), and (d) the percentage of micelle-bound \u03ba-casein (selected data with high levels of denatured whey proteins). The milk samples were heated either at 90 \u00b0C for various times (open symbols) or at different temperatures for 30 min (filled symbols). The pHs of the samples were ( ,  ) 6.5, ( ) 6.55, ( ) 6.6, ( ) 6.65, ( ,  ) 6.7, ( ,  ) 6.9, and ( ,  ) 7.1. Reproduced with permission from Anema et al. (2011). Copyright (2011) American Chemical Society.\n\n### Examples of the Effect of Denaturing Whey Proteins Separately from Casein Micelles on the Functional Properties of Milk\n\nCompared with heated milks, different effects on functional properties can be observed when the whey proteins are denatured and aggregated separately from the casein micelles and then remixed. However, the effects of adding pre-denatured whey proteins to casein micelle suspensions or milk on the functional properties are dependent on the conditions under which the whey proteins are denatured. Lucey et al. (1998) showed that acid gels prepared from milk samples where the whey proteins were denatured in the presence of casein micelles had a markedly higher firmness than acid gels prepared from milk samples where the whey proteins in milk serum were pre-denatured and then added back to the casein micelle suspensions. In many cases, the samples with denatured whey proteins added back to the casein micelles produced acid gels with firmness similar to or only slightly higher than those prepared from unheated milks.\n\nIn another study, Schorsch et al. (2001) prepared model milk systems in which the whey proteins in a simulated milk serum were either heated in the presence of casein micelles or heated separately and added back to the casein micelles. Acid gels were prepared from these model milk systems. It was shown that the acid-induced gelation occurred at a higher pH and in a shorter time when the whey proteins were denatured separately from the casein micelles than when they were heated in the presence of casein micelles. However, the gels formed were weaker and more heterogeneous because of the particulate nature of the denatured whey proteins.\n\nIt was suggested that the large denatured whey protein aggregates, as formed when the whey proteins in milk serum were heated separately from the casein micelles, hinder the formation of a casein gel network when the milk is subsequently acidified, and that a weak acid gel with a heterogeneous structure results. When the whey proteins are heated in the presence of the casein micelles, the denatured whey proteins interact with the \u03ba-casein at the casein micelle surface. On subsequent acidification, the denatured whey protein\u2013casein micelle complexes aggregate to form a firmer acid gel with a more homogeneous structure (Schorsch et al., 2001). This proposal is supported by early studies, which showed that the aggregation of the denatured whey proteins, and in particular \u03b2-lactoglobulin, formed large aggregate species when heated in the absence of \u03ba-casein, whereas aggregation was limited when the whey proteins were heated in the presence of \u03ba-casein (McKenzie et al., 1971).\n\nIn contrast, if the whey proteins are denatured at relatively low protein concentrations, at low ionic strengths, and at a pH far from the isoelectric point (>pH 6.5), then soluble denatured whey protein polymers can be formed. The polymers are linear and can be induced to gel when salt is added or the pH is reduced (Britten & Giroux, 2001; Gustaw et al., 2006; ). When these whey protein polymers are added to heated skim milk, and the preparations are acidified, the acid gels formed had markedly higher firmness and water-holding capacities than those from the skim milk or milks heated with equivalent levels of native whey proteins (Britten & Giroux, 2001; Gustaw et al. 2006; ;). The firmness and water-holding capacities were markedly higher than those where the whey proteins were denatured in milk serum and then added back to casein micelle suspensions or milk before acidification (Lucey et al., 1998; Schorsch et al., 2001).\n\nThe isoelectric points of denatured whey protein complexes were chemically modified through succinylation or methylation (Morand et al., 2012b). These modified denatured whey protein complexes were added to whey protein-free milk suspensions to produce model heated milk systems, and the milks were subsequently acidified to form gels. The gelation pH of these milks increased markedly as the isoelectric point of the whey protein complexes increased, supporting the proposition that it is the higher isoelectric point of the whey proteins that causes heated milks to gel at markedly higher pH than unheated milks (Anema et al., 2004a; Lucey et al., 1997). Interestingly, the final firmness of the gels was not markedly affected by the pI of the complexes (Morand et al., 2012b).\n\nIn similar studies, the hydrophobicity of denatured whey protein complexes was altered by acylation of lysine amino acids using anhydrides of various carbon chain lengths (Morand et al., 2012a). These complexes were also added to whey protein-free milk suspensions, and the milks were acidified to form gels. Increasing the hydrophobicity of the whey protein complexes also increased the pH at which the milks gelled as well as the maximum firmness of the gels, although this maximum was not always at the same pH. Taken together, these results indicate that both the isoelectric properties and the hydrophobicity of the serum-phase complexes influence the acid gelation properties of heated milk systems (Morand et al., 2011; 2012a, b).\n\n## Conclusion\n\nA considerable amount of work has gone into understanding the irreversible denaturation reactions of the whey proteins in heated milk systems. These detailed studies have produced models that allow reasonably accurate prediction of the level of whey protein denaturation in milks under a wide range of heating conditions, even in milk samples with markedly modified concentrations and compositions. However, with a few exceptions, monitoring of the whey protein denaturation levels provides only a crude indication of the functionality of the milk system. As a consequence, more recent research efforts have focused on trying to understand the specific interaction reactions of the denatured whey proteins with other proteins in the milk system. Early indications suggest that these types of studies on the interactions of denatured whey proteins may provide greater insights into the functional properties of heated milk products than can be obtained by monitoring just whey protein denaturation levels. These initial studies on protein interactions have been conducted under relatively narrowly defined conditions (temperatures, heating times, pH, milk concentrations, and milk compositions). It is likely that changes to these variables will markedly influence the interaction behavior and will explain the changes in functional behavior when the heating conditions are changed (even though the whey protein denaturation levels may be similar). Although studies on understanding the specific interactions between milk proteins, particularly in complex systems such as milk, are extremely difficult, these types of studies should continue to give useful insights into the behavior of milk proteins during heating and the functional behavior of the heated milk products.\n\n# References\n\nAnema SG ,  McKenna AB .  Reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk .  _Journal of Agricultural and Food Chemistry_.  1996 ;44 : 422 \u2013 428 .\n\nAnema SG ,  Klostermeyer H .  Heat-induced, pH-dependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100 \u00b0C .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 1108 \u2013 1115 .\n\nAnema SG .  Effect of milk concentration on heat-induced, pH-dependent dissociation of casein from micelles in reconstituted skim milk at temperatures between 20 and 120 \u00b0C .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 2299 \u2013 2305 .\n\nAnema SG ,  Lloyd RJ .  Analysis of whey protein denaturation: A comparative study of alternative methods .  _Milchwissenschaft_.  1999 ;54 : 206 \u2013 210 .\n\nAnema SG .  Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 4168 \u2013 4175 .\n\nAnema SG ,  Li Y .  Further studies on the heat-induced, pH-dependent dissociation of casein from the micelles in reconstituted skim milk .  _Lebensmittel Wissenschaft und Technologie_.  2000 ;33 : 335 \u2013 343 .\n\nAnema SG .  Kinetics of the irreversible thermal denaturation and disulfide aggregation of \u03b1-lactalbumin in milk samples of various concentrations .  _Journal of Food Science_.  2001 ;66 : 2 \u2013 9 .\n\nAnema SG ,  Li Y .  Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 1640 \u2013 1646 .\n\nAnema SG ,  Li Y .  Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size .  _Journal of Dairy Research_.  2003 ;70 : 73 \u2013 83 .\n\nAnema SG ,  Lee SK ,  Lowe EK ,  Klostermeyer H .  Rheological properties of acid gels prepared from heated pH-adjusted skim milk .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 337 \u2013 343 .\n\nAnema SG ,  Lowe EK ,  Lee SK .  Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk .  _Lebensmittel Wissenschaft und Technologie_.  2004 ;37 : 779 \u2013 787 .\n\nAnema SG ,  Lowe EK ,  Li Y .  Effect of pH on the viscosity of heated reconstituted skim milk .  _International Dairy Journal_.  2004 ;14 : 541 \u2013 548 .\n\nAnema SG ,  Lee SK ,  Klostermeyer H .  Effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible thermal denaturation of \u03b2-lactoglobulin and \u03b1-lactalbumin in skim milk .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 7339 \u2013 7348 .\n\nAnema SG .  Role of \u03ba-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk .  _Journal of Agricultural and Food Chemistry_.  2007 ;55 : 3635 \u2013 3642 .\n\nAnema SG ,  Lee SK ,  Klostermeyer H .  Effect of pH at heat treatment on the hydrolysis of \u03ba-casein and the gelation of skim milk by chymosin .  _LWT - Food Science and Technology_.  2007 ;40 : 99 \u2013 106 .\n\nAnema SG .  On heating milk, the dissociation of \u03ba-casein from the casein micelles can precede interactions with the denatured whey proteins .  _Journal of Dairy Research_.  2008 ;75 : 415 \u2013 421 .\n\nAnema SG .  Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk .  _Food Chemistry_.  2008 ;108 : 110 \u2013 118 .\n\nAnema SG .  The use of \"lab-on-a-chip\" microfluidic SDS electrophoresis technology for the separation and quantification of milk proteins .  _International Dairy Journal_.  2009 ;19 : 198 \u2013 204 .\n\nAnema SG ,  Lee SK ,  Klostermeyer H .  Rennet-induced aggregation of heated pH-adjusted skim milk .  _Journal of Agricultural and Food Chemistry_.  2011 ;59 : 8413 \u2013 8422 .\n\nBaer A ,  Oroz M ,  Blanc B .  Serological studies on heat-induced interactions of \u03b1-lactalbumin and milk-proteins .  _Journal of Dairy Research_.  1976 ;43 : 419 \u2013 432 .\n\nBritten M ,  Giroux HJ .  Acid-induced gelation of whey protein polymers: Effects of pH and calcium concentration during polymerization .  _Food Hydrocolloids_.  2001 ;15 : 609 \u2013 617 .\n\nButikofer U ,  Meyer J ,  Rehberger B .  Determination of the percentage of \u03b1-lactalbumin and \u03b2-lactoglobulin of total milk protein in raw and heat treated skim milk .  _Milchwissenschaft_.  2006 ;61 : 263 \u2013 266 .\n\nChandrapala J ,  Augustin MA ,  McKinnon I ,  Udabage P .  Effects of pH, calcium-complexing agents and milk solids concentration on formation of soluble protein aggregates in heated reconstituted skim milk .  _International Dairy Journal_.  2010 ;20 : 777 \u2013 784 .\n\nCho Y ,  Singh H ,  Creamer LK .  Heat-induced interactions of \u03b2-lactoglobulin A and \u03ba-casein B in a model system .  _Journal of Dairy Research_.  2003 ;70 : 61 \u2013 71 .\n\nCorredig M ,  Dalgleish DG .  Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk .  _Food Research International_.  1996 ;29 : 49 \u2013 55 .\n\nCorredig M ,  Dalgleish DG .  The binding of \u03b1-lactalbumin and \u03b2-lactoglobulin to casein micelles in milk treated by different heating systems .  _Milchwissenschaft_.  1996 ;51 : 123 \u2013 127 .\n\nCorredig M ,  Dalgleish DG .  The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk .  _International Dairy Journal_.  1999 ;9 : 233 \u2013 236 .\n\nCreamer LK ,  Berry GP ,  Matheson AR .  Effect of pH on protein aggregation in heated skim milk .  _New Zealand Journal of Dairy Science and Technology_.  1978 ;13 : 9 \u2013 15 .\n\nCreamer LK ,  Matheson AR .  Effect of heat-treatment on the proteins of pasteurized skim milk .  _New Zealand Journal of Dairy Science and Technology_.  1980 ;15 : 37 \u2013 49 .\n\nCreamer LK ,  Bienvenue A ,  Nilsson H ,  Paulsson M ,  van Wanroij M ,  Lowe EK ,  Anema SG ,  Boland MJ ,  Jimenez-Flores R .  Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 7660 \u2013 7668 .\n\nCroguennec T ,  Bouhallab S ,  Molle D ,  O'Kennedy BT ,  Mehra R .  Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of \u03b2-lactoglobulin .  _Biochemical and Biophysical Research Communications_.  2003 ;301 : 465 \u2013 471 .\n\nCroguennec T ,  Molle D ,  Mehra R ,  Bouhallab S .  Spectroscopic characterization of heat-induced nonnative \u03b2-lactoglobulin monomers .  _Protein Science_.  2004 ;13 : 1340 \u2013 1346 .\n\nDalgleish DG .  On the structural models of bovine casein micelles\u2014review and possible improvements .  _Soft Matter_.  2011 ;7 : 2265 \u2013 2272 .\n\nDalgleish DG ,  Corredig M .  The structure of the casein micelle of milk and its changes during processing .  _Annual Review of Food Science and Technology_.  2012 ;3 : 449 \u2013 467 .\n\nDannenberg F ,  Kessler HG .  Reaction kinetics of the denaturation of whey proteins in milk .  _Journal of Food Science_.  1988 ;53 : 258 \u2013 263 .\n\nDannenberg F ,  Kessler HG .  Effect of denaturation of \u03b2-lactoglobulin on texture properties of set-style nonfat yoghurt. 1. Syneresis .  _Milchwissenschaft_.  1988 ;43 : 632 \u2013 635 .\n\nDannenberg F ,  Kessler HG .  Effect of denaturation of \u03b2-lactoglobulin on texture properties of set-style nonfat yoghurt. 2. Firmness and flow properties .  _Milchwissenschaft_.  1988 ;43 : 700 \u2013 704 .\n\nde Kruif CG ,  Holt C .  Casein micelle structure, functions and interactions .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1: Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 233 \u2013 276 .\n\nde Kruif CG ,  Huppertz T ,  Urban VS ,  Petukhov AV .  Casein micelles and their internal structure .  _Advances in Colloid and Interface Science_.  2012 ; : 171 \u2013 172 : 36\u201352 .\n\nDoi H ,  Tokuyama T ,  Kuo FH ,  Ibuki F ,  Kanamori M .  Heat-induced complex-formation between \u03ba-casein and \u03b1-lactalbumin .  _Agricultural and Biological Chemistry_.  1983 ;47 : 2817 \u2013 2824 .\n\nDonato L ,  Dalgleish DG .  Effect of the pH of heating on the qualitative and quantitative compositions of the sera of reconstituted skim milks and on the mechanisms of formation of soluble aggregates .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 7804 \u2013 7811 .\n\nDonato L ,  Alexander M ,  Dalgleish DG .  Acid gelation in heated and unheated milks: Interactions between serum protein complexes and the surfaces of casein micelles .  _Journal of Agricultural and Food Chemistry_.  2007 ;55 : 4160 \u2013 4168 .\n\nDonato L ,  Guyomarc'h F ,  Amiot S ,  Dalgleish DG .  Formation of whey protein\/\u03ba-casein complexes in heated milk: Preferential reaction of whey protein with \u03ba-casein in the casein micelles .  _International Dairy Journal_.  2007 ;17 : 1161 \u2013 1167 .\n\nDonato L ,  Guyomarc'h F .  Formation and properties of the whey protein\/\u03ba-casein complexes in heated skim milk\u2014A review .  _Dairy Science and Technology_.  2009 ;89 : 3 \u2013 29 .\n\nDupont D ,  Muller-Renaud S .  Quantification of proteins in dairy products using an optical biosensor .  _Journal of AOAC International_.  2006 ;89 : 843 \u2013 848 .\n\nElfagm AA ,  Wheelock JV .  Heat interaction between \u03b1-lactalbumin, \u03b2-lactoglobulin and casein in bovine milk .  _Journal of Dairy Science_.  1978 ;61 : 159 \u2013 163 .\n\nEuber JR ,  Brunner JR .  Interaction of \u03ba-casein with immobilized \u03b2-lactoglobulin .  _Journal of Dairy Science_.  1982 ;65 : 2384 \u2013 2387 .\n\nFairise JF ,  Cayot P .  New ultrarapid method for the separation of milk proteins by capillary electrophoresis .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 2628 \u2013 2633 .\n\nFarrell HM ,  Malin EL ,  Brown EM ,  Qi PX .  Casein micelle structure: What can be learned from milk synthesis and structural biology? .  _Current Opinion in Colloid and Interface Science_.  2006 ;11 : 135 \u2013 147 .\n\nFox PF .  Milk proteins: General and historical aspects .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 1: Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1 \u2013 48 .\n\nGillece Castro BL ,  Stults JT ,  Peptide characterization by mass spectrometry .  _High Resolution Separation and Analysis of Biological Macromolecules, Pt B_ ,  271 .  427 \u2013 448 .\n\nGorman JJ ,  Wallis TP ,  Pitt JJ .  Protein disulfide bond determination by mass spectrometry .  _Mass Spectrometry Reviews_.  2002 ;21 : 183 \u2013 216 .\n\nGough P ,  Jenness RJ .  Heat denaturation of \u03b2-lactoglobulins A and B .  _Journal of Dairy Science_.  1962 ;45 : 1033 \u2013 1039 .\n\nGraveland-Bikker JF ,  Anema SG .  Effect of individual whey proteins on the rheological properties of acid gels prepared from heated skim milk .  _International Dairy Journal_.  2003 ;13 : 401 \u2013 408 .\n\nGrindrod J ,  Nickerson TA .  Changes in milk proteins treated with hydrogen peroxide .  _Journal of Dairy Science_.  1967 ;50 : 142 \u2013 146 .\n\nGustaw W ,  Glibowski P ,  Mleko S .  The rheological properties of yoghurt with incorporated whey protein aggregates\/polymers .  _Milchwissenschaft \u2013Milk Science International_.  2006 ;61 : 415 \u2013 419 .\n\nGustaw W ,  Szwajgier D ,  Mleko S .  The rheological properties of yoghurt with the addition of lyophilized polymerized whey protein .  _Milchwissenschaft \u2013Milk Science International_.  2009 ;64 : 60 \u2013 64 .\n\nGuyomarc'h F ,  Law AJR ,  Dalgleish DG .  Formation of soluble and micelle-bound protein aggregates in heated milk .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 4652 \u2013 4660 .\n\nGuyomarc'h F ,  Jemin M ,  Le Tilly V ,  Madec MN ,  Famelart MH .  Role of the heat-induced whey protein-\u03ba-casein complexes in the formation of acid milk gels: A kinetic study using rheology and confocal microscopy .  _Journal of Agricultural and Food Chemistry_.  2009 ;57 : 5910 \u2013 5917 .\n\nHaque Z ,  Kristjansson MM ,  Kinsella JE .  Interaction between \u03ba-casein and \u03b2-lactoglobulin\u2014possible mechanism .  _Journal of Agricultural and Food Chemistry_.  1987 ;35 : 644 \u2013 649 .\n\nHaque Z ,  Kinsella JE .  Interaction between heated \u03ba-casein and \u03b2-lactoglobulin\u2014predominance of hydrophobic interactions in the initial stages of complex formation .  _Journal of Dairy Research_.  1988 ;55 : 67 \u2013 80 .\n\nHarland HA ,  Ashworth US .  The preparation and effect of heat treatment on the whey proteins of milk .  _Journal of Dairy Science_.  1945 ;28 : 879 \u2013 886 .\n\nHarland HA ,  Ashworth US .  A rapid method for estimation of whey proteins as an indication of baking quality of nonfat dry milk solids .  _Food Research_.  1947 ;12 : 247 \u2013 251 .\n\nHarland HA ,  Coulter ST ,  Jenness R .  The effect of the various steps in the manufacture on the extent of serum protein denaturation in nonfat dry milk solids .  _Journal of Dairy Science_.  1952 ;35 : 363 \u2013 368 .\n\nHarland HA ,  Coulter ST ,  Jenness R .  Natural variation of milk serum proteins as a limitation of their use in evaluating the heat treatment of milk .  _Journal of Dairy Science_.  1955 ;38 : 858 \u2013 869 .\n\nHenry G ,  Molle D ,  Morgan F ,  Fauquant J ,  Bouhallab S .  Heat-induced covalent complex between casein micelles and \u03b2-lactoglobulin from goat's milk: Identification of an involved disulfide bond .  _Journal of Agricultural and Food Chemistry_.  2002 ;50 : 185 \u2013 191 .\n\nHill AR .  The \u03b2-lactoglobulin-\u03ba-casein complex .  _Canadian Institute of Food Science and Technology Journal_.  1989 ;22 : 120 \u2013 123 .\n\nHillier RM ,  Lyster RLJ .  Whey protein denaturation in heated milk and cheese whey .  _Journal of Dairy Research_.  1979 ;46 : 95 \u2013 102 .\n\nHoffmann MAM ,  van Mil PJJM .  Heat-induced aggregation of \u03b2-lactoglobul in: Role of the free thiol group and disulfide bonds .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 2942 \u2013 2948 .\n\nHolland JW ,  Deeth HC ,  Alewood PF .  Analysis of disulfide linkages in bovine \u03ba-casein oligomers using two-dimensional electrophoresis .  _Electrophoresis_.  2008 ;29 : 2402 \u2013 2410 .\n\nHolt C .  Structure and stability of bovine casein micelles .  _Advances in Protein Chemistry_.  1992 ;43 : 63 \u2013 151 .\n\nHolt C ,  Horne DS .  The hairy casein micelle: Evolution of the concept and its implications for dairy technology .  _Netherlands Milk and Dairy Journal_.  1996 ;50 : 85 \u2013 111 .\n\nHong YH ,  Creamer LK .  Changed protein structures of bovine \u03b2-lactoglobulin B and \u03b1-lactalbumin as a consequence of heat treatment .  _International Dairy Journal_.  2002 ;12 : 345 \u2013 359 .\n\nHorne DS .  Casein interactions: Casting light on the black boxes, the structure in dairy products .  _International Dairy Journal_.  1998 ;8 : 171 \u2013 177 .\n\nHorne DS .  Casein structure, self-assembly and gelation .  _Current Opinion in Colloid and Interface Science_.  2002 ;7 : 456 \u2013 461 .\n\nHorne DS .  Casein micelles as hard spheres: Limitations of the model in acidified gel formation .  _Colloids and Surfaces A: Physicochemical and Engineering Aspects_.  2003 ;213 : 255 \u2013 263 .\n\nHorne DS .  Casein micelle structure: Models and muddles .  _Current Opinion in Colloid and Interface Science_.  2006 ;11 : 148 \u2013 153 .\n\nIametti S ,  Gregori B ,  Vecchio G ,  Bonomi F .  Modifications occur at different structural levels during the heat denaturation of \u03b2-lactoglobulin .  _European Journal of Biochemistry_.  1996 ;237 : 106 \u2013 112 .\n\nIDF .  Heat-induced changes in milk .  In:  Fox PF ,  ed.  _International Dairy Federation Special Issue 9501_ .  2nd ed. Brussels, Belgium :  International Dairy Federation ;  1995 .\n\nIDF .  Heat treatments and alternative methods .  In:  Fox PF ,  ed.  _International Dairy Federation Special Issue 9602_ .  2nd ed. Brussels, Belgium :  International Dairy Federation ;  1995 .\n\nIndyk HE .  Development and application of an optical biosensor immunoassay for \u03b1-lactalbumin in bovine milk .  _International Dairy Journal_.  2009 ;19 : 36 \u2013 42 .\n\nKehoe JJ ,  Brodkorb A ,  Molle D ,  Yokoyama E ,  Famelart MH ,  Bouhallab S ,  Morris ER ,  Croguennec T .  Determination of exposed sulfhydryl groups in heated \u03b2-lactoglobulin A using IAEDANS and mass spectrometry .  _Journal of Agricultural and Food Chemistry_.  2007 ;55 : 7107 \u2013 7113 .\n\nKelly AL ,  O'Connell JE ,  Fox PF .  Manufacture and properties of milk powders .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry-1: Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1027 \u2013 1061 .\n\nKessler HG ,  Beyer HJ .  Thermal denaturation of whey proteins and its effect in dairy technology .  _International Journal of Biological Macromolecules_.  1991 ;13 : 165 \u2013 173 .\n\nKethireddipalli P ,  Hill AR ,  Dalgleish DG .  Protein interactions in heat-treated milk and effect on rennet coagulation .  _International Dairy Journal_.  2010 ;20 : 838 \u2013 843 .\n\nKethireddipalli P ,  Hill AR ,  Dalgleish DG .  Interaction between casein micelles and whey protein\/\u03ba-casein complexes during renneting of heat-treated reconstituted skim milk powder and casein micelle\/serum mixtures .  _Journal of Agricultural and Food Chemistry_.  2011 ;59 : 1442 \u2013 1448 .\n\nKudo S .  The heat stability of milk: Formation of soluble proteins and protein-depleted micelles at elevated temperatures .  _New Zealand Journal of Dairy Science and Technology_.  1980 ;15 : 255 \u2013 263 .\n\nKuramoto S ,  Jenness R ,  Coulter ST ,  Choi RP .  Standardization of the Harland-Ashworth test for whey protein nitrogen .  _Journal of Dairy Science_.  1959 ;42 : 28 \u2013 38 .\n\nLakemond CMM ,  van Vliet T .  Rheology of acid skim milk gels .  In:  Dickinson E ,  ed.  _Food Colloids: Interactions, microstructure and processing_ .  Cambridge :  The Royal Society of Chemistry ;  2005 : 26 \u2013 36 : (Special Publication 298) .\n\nLakemond CMM ,  van Vliet T .  Acid skim milk gels: The gelation process as affected by preheating pH .  _International Dairy Journal_.  2008 ;18 : 574 \u2013 584 .\n\nLakemond CMM ,  van Vliet T .  Rheological properties of acid skim milk gels as affected by the spatial distribution of the structural elements and the interaction forces between them .  _International Dairy Journal_.  2008 ;18 : 585 \u2013 593 .\n\nLarson BL ,  Jenness R ,  Geddes WF ,  Coulter ST .  An evaluation of the methods used for determining the baking quality of nonfat dry milk solids .  _Cereal Chemistry_.  1951 ;28 : 351 \u2013 370 .\n\nLarson BL ,  Rolleri GD .  Heat denaturation of the specific serum proteins in milk .  _Journal of Dairy Science_.  1955 ;38 : 351 \u2013 360 .\n\nLaw AJR ,  Leaver J .  Effect of protein concentration on rates of thermal denaturation of whey proteins in milk .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 4255 \u2013 4261 .\n\nLaw AJR ,  Leaver J .  Effect of pH on the thermal denaturation of whey proteins in milk .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 672 \u2013 679 .\n\nLeighton FR .  Determination of the whey protein index of skim milk powder .  _Australian Journal of Dairy Technology_.  1962 ; : 166 \u2013 169 .\n\nLivney YD ,  Dalgleish DG .  Specificity of disulfide bond formation during thermal aggregation in solutions of \u03b2-lactoglobulin B and \u03ba-casein A .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 5527 \u2013 5532 .\n\nLong JE ,  Gould IA ,  van Winkle Q .  Heat-induced interaction between crude \u03ba-casein and \u03b2-lactoglobulin .  _Journal of Dairy Science_.  1963 ;46 : 1329 \u2013 1334 .\n\nLowe EK ,  Anema SG ,  Bienvenue A ,  Boland MJ ,  Creamer LK ,  Jimenez-Flores R .  Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine \u03b2-lactoglobulin and \u03ba-casein .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 7669 \u2013 7680 .\n\nLucey JA ,  Teo CT ,  Munro PA ,  Singh H .  Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk .  _Journal of Dairy Research_.  1997 ;64 : 591 \u2013 600 .\n\nLucey JA ,  Singh H .  Formation and physical properties of acid milk gels: a review .  _Food Research International_.  1998 ;30 : 529 \u2013 542 .\n\nLucey JA ,  Tamehana M ,  Singh H ,  Munro PA .  Effect of interactions between denatured whey proteins and casein micelles on the formation and rheological properties of acid skim milk gels .  _Journal of Dairy Research_.  1998 ;65 : 555 \u2013 567 .\n\nLucey JA .  Formation and physical properties of milk protein gels .  _Journal of Dairy Science_.  2002 ;85 : 281 \u2013 294 .\n\nLyster LJ .  The denaturation of \u03b1-lactalbumin and \u03b2-lactoglobulin in heated milk .  _Journal of Dairy Research_.  1970 ;37 : 233 \u2013 243 .\n\nManderson GA ,  Hardman MJ ,  Creamer LK .  Effect of heat treatment on the conformation and aggregation of \u03b2-lactoglobulin A, B, and C .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 5052 \u2013 5061 .\n\nManji B ,  Kakuda Y .  Thermal denaturation of whey proteins in skim milk .  _Canadian Institute of Food Science and Technology Journal_.  1986 ;19 : 161 \u2013 166 .\n\nManji B ,  Kakuda Y .  Determination of whey protein denaturation in heat-processed milks: Comparison of three methods .  _Journal of Dairy Science_.  1987 ;70 : 1355 \u2013 1361 .\n\nMcKenna AB ,  Anema SG .  The effect of thermal processing during whole milk powder manufacture and after its reconstitution on set-yoghurt properties .  _International Dairy Federation Special Issue 9303_ .  Belgium :  Brussels ;  1993 : 307 \u2013 316 .\n\nMcKenzie GH ,  Norton RS ,  Sawyer WH .  Heat-induced interaction of \u03b2-lactoglobulin and \u03ba-casein .  _Journal of Dairy Research_.  1971 ;38 : 343 \u2013 351 .\n\nMcMahon DJ ,  McManus WR .  Rethinking casein micelle structure using electron microscopy .  _Journal of Dairy Science_.  1998 ;81 : 2985 \u2013 2993 .\n\nMcMahon DJ ,  Oommen BS .  Supramolecular structure of the casein micelle .  _Journal of Dairy Science_.  2008 ;91 : 1709 \u2013 1721 .\n\nMellema M ,  van Opheusden JHJ ,  van Vliet T .  Categorization of rheological scaling models for particle gels applied to casein gels .  _Journal of Rheology_.  2002 ;46 : 11 \u2013 29 .\n\nMorand M ,  Guyomarc'h F ,  Famelart M-H .  How to tailor heat-induced whey protein\/\u03ba-casein complexes as a means to investigate the acid gelation of milk\u2014a review .  _Dairy Science and Technology_.  2011 ;91 : 97 \u2013 126 .\n\nMorand M ,  Dekkari A ,  Guyomarc'h F ,  Famelart M-H .  Increasing the hydrophobicity of the heat-induced whey protein complexes improves the acid gelation of skim milk .  _International Dairy Journal_.  2012 ;25 : 103 \u2013 111 .\n\nMorand M ,  Guyomarc'h F ,  Legland D ,  Famelart M-H .  Changing the isoelectric point of the heat-induced whey protein complexes affects the acid gelation of skim milk .  _International Dairy Journal_.  2012 ;23 : 9 \u2013 17 .\n\nNieuwenhuijse JA ,  van Boekel MAJS ,  Walstra P .  On the heat-induced association and dissociation of proteins in concentrated skim milk .  _Netherlands Milk and Dairy Journal_.  1991 ;45 : 3 \u2013 22 .\n\nNieuwenhuijse JA ,  van Boekel MAJS .  Protein stability in sterilised milk and milk products .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry-1: Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 947 \u2013 974 .\n\nNoh B ,  Richardson T ,  Creamer LK .  Radiolabelling study of the heat-induced interactions between \u03b1-lactalbumin, \u03b2-lactoglubulin and \u03ba-casein in milk and in buffer solutions .  _Journal of Food Science_.  1989 ;54 : 889 \u2013 893 .\n\nNoh BS ,  Creamer LK ,  Richardson T .  Thermally induced complex-formation in an artificial milk system .  _Journal of Agricultural and Food Chemistry_.  1989 ;37 : 1395 \u2013 1400 .\n\nO'Connell JE ,  Fox PF .  Heat-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry-1: Proteins_ .  3rd ed. New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 879 \u2013 945 .\n\nOh HE ,  Wong M ,  Pinder DN ,  Hemar Y ,  Anema SG .  Effect of pH adjustment at heating on the rheological properties of acid skim milk gels with added potato starch .  _International Dairy Journal_.  2007 ;17 : 1384 \u2013 1392 .\n\nOldfield DJ ,  Singh H ,  Taylor M ,  Pearce K .  Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot plant .  _International Dairy Journal_.  1998 ;8 : 311 \u2013 318 .\n\nOldfield DJ ,  Singh H ,  Taylor MW .  Association of \u03b2-lactoglobulin and \u03b1-lactalbumin with the casein micelles in skim milk heated in an ultra-high temperature plant .  _International Dairy Journal_.  1998 ;8 : 765 \u2013 770 .\n\nOldfield DJ ,  Singh H ,  Taylor MW ,  Pearce KN .  Heat-induced interactions of \u03b2-lactoglobulin and \u03b1-lactalbumin with the casein micelle in pH-adjusted skim milk .  _International Dairy Journal_.  2000 ;10 : 509 \u2013 518 .\n\nOldfield DJ ,  Singh H ,  Taylor MW .  Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration .  _Journal of Dairy Research_.  2005 ;72 : 369 \u2013 378 .\n\nParker EA ,  Donato L ,  Dalgleish DG .  Effects of added sodium caseinate on the formation of particles in heated milk .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 8265 \u2013 8272 .\n\nParnell-Clunies EM ,  Kakuda Y ,  Mullen K ,  Arnott DR ,  Deman JM .  Physical properties of yogurt\u2014a comparison of vat versus continuous heating systems of milk .  _Journal of Dairy Science_.  1986 ;69 : 2593 \u2013 2603 .\n\nPatel HA ,  Singh H ,  Anema SG ,  Creamer LK .  Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 3409 \u2013 3420 .\n\nPatel HA ,  Anema SG ,  Holroyd SE ,  Singh H ,  Creamer LK .  Methods to determine denaturation and aggregation of proteins in low-, medium- and high-heat skim milk powders .  _Lait_.  2007 ;87 : 251 \u2013 268 .\n\nPurkayastha R ,  Tessier H ,  Rose D .  Thiol-disulfide interchange in formation of \u03b2-lactoglobulin-\u03ba-casein complex .  _Journal of Dairy Science_.  1967 ;50 : 764 \u2013 766 .\n\nQi PX .  Studies of casein micelle structure: The past and the present .  _Lait_.  2007 ;87 : 363 \u2013 383 .\n\nQi PX .  Studies of the biological function and structure of casein micelles, and future implications .  In:  Corredig M ,  ed.  _Dairy Derived Ingredients, Food and Nutraceutical Uses_ .  Cambridge :  Woodhead Publishing Limited ;  2009 : 147 \u2013 169 .\n\nQin BY ,  Bewley MC ,  Creamer LK ,  Baker EN ,  Jameson GB .  Functional implications of structural differences between variants A and B of bovine \u03b2-lactoglobulin .  _Protein Science_.  1999 ;8 : 75 \u2013 83 .\n\nRasmussen LK ,  Johnsen LB ,  Tsiora A ,  Sorensen ES ,  Thomsen JK ,  Nielsen NC ,  Jakobsen HJ ,  Petersen TE .  Disulfide-linked caseins and casein micelles .  _International Dairy Journal_.  1999 ;9 : 215 \u2013 218 .\n\nRenan M ,  Guyomarc'h F ,  Chatriot M ,  Gamerre V ,  Famelart MH .  Limited enzymatic treatment of skim milk using chymosin affects the micelle\/serum distribution of the heat-induced whey protein\/\u03ba-casein aggregates .  _Journal of Agricultural and Food Chemistry_.  2007 ;55 : 6736 \u2013 6745 .\n\nRodriguez del Angel C ,  Dalgleish DG .  Structure and some properties of soluble protein complexes formed by the heating of reconstituted skim milk powder .  _Food Research International_.  2006 ;39 : 472 \u2013 479 .\n\nRose D .  Variations in the heat stability and composition of milk from individual cows during lactation .  _Journal of Dairy Science_.  1961 ;44 : 430 \u2013 441 .\n\nRowland SJ .  The heat denaturation of albumin and globulin in milk .  _Journal of Dairy Research_.  1933 ;5 : 46 \u2013 53 .\n\nRuegg M ,  Moor U ,  Blanc B .  A calorimetric study of thermal denaturation of whey proteins in simulated milk ultrafiltrate .  _Journal of Dairy Research_.  1977 ;44 : 509 \u2013 520 .\n\nSanderson WB .  Seasonal variations affecting the determination of the whey protein nitrogen index of skim milk powder .  _New Zealand Journal of Dairy Science and Technology_.  1970 ; : 48 \u2013 52 .\n\nSanderson WB .  Determination of undenatured whey protein nitrogen in skim milk powder by dye binding .  _New Zealand Journal of Dairy Science and Technology_.  1970 ;5 : 46 \u2013 48 .\n\nSanderson WB .  Reconstituted and recombined dairy products .  _New Zealand Journal of Dairy Science and Technology_.  1970 ; : 139 \u2013 143 .\n\nSawyer WH ,  Coulter ST ,  Jenness R .  Role of sulfhydryl groups in the interaction of \u03ba-casein and \u03b2-lactoglobulin .  _Journal of Dairy Science_.  1963 ;46 : 564 \u2013 565 .\n\nSawyer WH .  Complex between \u03b2-lactoglobulin and \u03ba-casein. A review .  _Journal of Dairy Science_.  1969 ;52 : 1347 \u2013 1355 .\n\nSchmidt DG .  Association of caseins and casein micelle structure .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry-1. Proteins_ .  London :  Elsevier Applied Science Publishers ;  1982 : 61 \u2013 86 .\n\nSchorsch C ,  Wilkins DK ,  Jones MG ,  Norton IT .  Gelation of casein-whey mixtures: Effects of heating whey proteins alone or in the presence of casein micelles .  _Journal of Dairy Research_.  2001 ;68 : 471 \u2013 481 .\n\nShalabi SI ,  Wheelock JV .  Role of \u03b1-lactalbumin in the primary phase of chymosin action on heated casein micelles .  _Journal of Dairy Research_.  1976 ;43 : 331 \u2013 335 .\n\nSingh H ,  Fox PF .  Heat-stability of milk\u2014the mechanism of stabilization by formaldehyde .  _Journal of Dairy Research_.  1985 ;52 : 65 \u2013 76 .\n\nSingh H ,  Fox PF .  Heat stability of milk: pH-dependent dissociation of micellar \u03ba-casein on heating milk at ultra high temperatures .  _Journal of Dairy Research_.  1985 ;52 : 529 \u2013 538 .\n\nSingh H ,  Fox PF .  Heat-stability of milk\u2014further studies on the pH-dependent dissociation of micellar \u03ba-casein .  _Journal of Dairy Research_.  1986 ;53 : 237 \u2013 248 .\n\nSingh H ,  Fox PF .  Heat-stability of milk: influence of colloidal and soluble salts and protein modification on the pH-dependent dissociation of micellar \u03ba-casein .  _Journal of Dairy Research_.  1987 ;54 : 523 \u2013 534 .\n\nSingh H ,  Fox PF .  Heat-stability of milk\u2014role of \u03b2-lactoglobulin in the pH-dependent dissociation of micellar \u03ba-casein .  _Journal of Dairy Research_.  1987 ;54 : 509 \u2013 521 .\n\nSingh H ,  Fox PF .  Heat-stability of milk\u2014influence of modifying sulfhydryl-disulfide interactions on the heat coagulation time pH profile .  _Journal of Dairy Research_.  1987 ;54 : 347 \u2013 359 .\n\nSingh H ,  Creamer LK .  Denaturation, aggregation and heat-stability of milk protein during the manufacture of skim milk powder .  _Journal of Dairy Research_.  1991 ;58 : 269 \u2013 283 .\n\nSingh H ,  Creamer LK .  Influence of concentration of milk solids on the dissociation of micellar \u03ba-casein on heating reconstituted milk at 120 \u00b0C .  _Journal of Dairy Research_.  1991 ;58 : 99 \u2013 105 .\n\nSingh H ,  Creamer LK .  Heat stability of milk .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry-1: Proteins_ .  London :  Elsevier Applied Science ;  1992 : 621 \u2013 656 .\n\nSingh H ,  Newstead DF .  Aspects of proteins in milk powder manufacture .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry-1: Proteins_ .  London :  Elsevier Applied Science ;  1992 : 735 \u2013 765 .\n\nSingh H .  Heat stability of milk .  _International Journal of Dairy Technology_.  2004 ;57 : 111 \u2013 119 .\n\nSlatter WL ,  van Winkle Q .  An electrophoretic study of the protein in skim milk .  _Journal of Dairy Science_.  1952 ;35 : 1083 \u2013 1093 .\n\nSmits P ,  van Brouwershaven JH .  Heat-induced association of \u03b2-lactoglobulin and casein micelles .  _Journal of Dairy Research_.  1980 ;47 : 313 \u2013 325 .\n\nSnoeren THM ,  van der Spek CA .  Isolation of a heat-induced complex from UHTST Milk .  _Netherlands Milk and Dairy Journal_.  1977 ;31 : 352 \u2013 355 .\n\nTessier H ,  Yaguchi M ,  Rose D .  Zonal ultracentrifugation of \u03b2-lactoglobulin and \u03ba-casein complexes induced by heat .  _Journal of Dairy Science_.  1969 ;52 : 139 \u2013 145 .\n\nTobias J ,  Whitney RM ,  Tracy PH .  Electrophoretic properties of milk proteins, II. Effect of heating to 300 \u00b0F by means of the Mallory small-tube heat exchanger on skim milk proteins .  _Journal of Dairy Science_.  1952 ;35 : 1036 \u2013 1045 .\n\nTrautman JC ,  Swanson AM .  Additional evidence of a stable complex between \u03b2-lactoglobulin and \u03b1-casein .  _Journal of Dairy Science_.  1958 ;41 : 715 \u2013 1715 .\n\nTrejo R ,  Dokland T ,  Jurat-Fuentes J ,  Harte F .  Cryo-transmission electron tomography of native casein micelles from bovine milk .  _Journal of Dairy Science_.  2011 ;94 : 5770 \u2013 5775 .\n\nvan Vliet T ,  Keetals CJAM .  Effect of preheating of milk on the structure of acidified milk gels .  _Netherlands Milk and Dairy Journal_.  1995 ;49 : 27 \u2013 35 .\n\nvan Vliet T ,  Walstra P .  Large deformation and fracture behaviour of gels .  _Faraday Discussions_.  1995 ;101 : 359 \u2013 370 .\n\nvan Vliet T .  Large deformation and fracture behaviour of gels .  _Current Opinion in Colloid and Interface Science_.  1996 ;1 : 740 \u2013 745 .\n\nVasbinder AJ ,  Alting AC ,  Visschers RW ,  de Kruif CG .  Texture of acid milk gels: Formation of disulfide cross-links during acidification .  _International Dairy Journal_.  2003 ;13 : 29 \u2013 38 .\n\nVasbinder AJ ,  de Kruif CG .  Casein-whey protein interactions in heated milk: The influence of pH .  _International Dairy Journal_.  2003 ;13 : 669 \u2013 677 .\n\nVasbinder AJ ,  Rollema HS ,  de Kruif CG .  Impaired rennetability of heated milk: Study of enzymatic hydrolysis and gelation kinetics .  _Journal of Dairy Science_.  2003 ;86 : 1548 \u2013 1555 .\n\nVerheul M ,  Roefs SPFM ,  de Kruif KG .  Kinetics of heat-induced aggregation of \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 896 \u2013 903 .\n\nWalstra P ,  Jenness R .  _Dairy Chemistry and Physics_ .  New York :  John Wiley and Sons ;  1984 .\n\nWalstra P .  On the stability of casein micelles .  _Journal of Dairy Science_.  1990 ;73 : 1965 \u2013 1979 .\n\nWalstra P .  _Casein sub-micelles: Do they exist? International Dairy Journal_.  1999 ;9 : 189 \u2013 192 .\n\nWalstra P ,  Geurts TJ ,  Noomen A ,  Jellema A ,  van Boekel MAJS .  _Dairy Technology: Principles of Milk Properties and Processes_ .  New York :  Marcel Dekker ;  1999 .\n\nWu D ,  Qin J ,  Lin B .  Electrophoretic separations on microfluidic chips .  _Journal of Chromatography A_.  2008 ;1184 : 542 \u2013 559 .\n\nZittle CA ,  Custer JH ,  Cerbulis J ,  Thompson MP .  \u03ba-Casein\u2013 \u03b2-lactoglobulin interaction in solution when heated .  _Journal of Dairy Science_.  1962 ;45 : 807 \u2013 810 . \nChapter 10\n\n# Effects of Drying on Milk Proteins\n\nPierre Schuck*\n\n* INRA, UMR 1253, STLO, Rennes, France\n\n## Abstract\n\nDehydration by spray drying is a valuable technique for water evaporation, using hot air to stabilize the majority of dairy ingredients. In view of the increased development of filtration processes, the dairy industry requires greater understanding of the effects of spray drying on the quality of protein powders. Several publications have reported that the proteins have an important role in the mechanisms of water transfer during drying and rehydration. The residence time of the droplet is so short that it is very difficult to study the mechanisms of the structural change in the protein without fundamental research into relationships with the process\/product interactions. Following an introduction to spray drying, this chapter on the effects of drying on milk proteins covers five areas: the world dairy powder situation, the properties of spray-dried milk products, the principles of spray drying, the drying of proteins, and the rehydration of dried protein powders.\n\n## Keywords\n\nspray-dried milk products\n\ndairy powders\n\nwhole milk powder\n\nskim milk powder\n\nwhey products\n\nOutline\n\nIntroduction 319\n\nWorld Dairy Powder Situations 321\n\nWhole Milk Powder 321\n\nSkim Milk Powder 322\n\nWhey Products, Casein, and Other Dairy Ingredients 323\n\nProperties of spray-dried milk products 323\n\nPrinciples of spray drying 324\n\nProcess improvement 328\n\nDrying of proteins 328\n\nExamples of Dairy Protein Concentrates and Powders 329\n\nResearch Approach Using Drying by Desorption 330\n\nDesorption Results 331\n\nIndustrial Implications 333\n\nIntroduction 333\n\nAvailability of Water 333\n\nWhey Proteins 335\n\nCaseins 335\n\nRehydration of Protein Powders 335\n\nConclusions 339\n\n## Introduction\n\nThe purpose of the dehydration of milk and whey is to stabilize these products for their storage and later use. Milk and whey powders are used mostly in animal feed. With changes in agricultural policies, such as the implementation of the quota system and the dissolution of the price support system in the European Union (EU), the dairy industry was forced to look for better uses for the dairy surplus and for the by-products of cheese (whey) produced from milk and buttermilk produced from cream. Studies on the reuse of protein fractions with their nutritional qualities and functional properties led us to believe that they could have several applications.\n\nMainly because of the emergence of filtration technology (microfiltration, ultrafiltration, nanofiltration, and reverse osmosis), in the past 30 years the dairy industry has developed new technological processes for extracting and purifying proteins (casein, caseinates, whey proteins, etc.) (Kjaergaard et al., 1987; Maubois, 1991), such as:\n\n\u2022 dairy proteins and whey concentrates (Le Gra\u00ebt & Maubois, 1979; Goud\u00e9dranche et al., 1980; Madsen & Bjerre, 1981; Maubois et al., 1987; Caron et al., 1997);\n\n\u2022 micellar casein concentrates (Fauquant et al., 1988; Schuck et al., 1994a);\n\n\u2022 micellar casein (MC) (Pierre et al., 1992; Schuck et al., 1994b);\n\n\u2022 whey concentrates, selectively demineralized concentrates (Jeantet et al., 1996); and\n\n\u2022 super-clean skim milk concentrates (Piot et al., 1987; Vincens & Tabard, 1988; Trouv\u00e9 et al., 1991; Schuck et al., 1994a)\n\nUsed as either nutritional or functional ingredients, most of these proteins are marketed in dehydrated form (Fig. 10.1). The application of different processing steps allows the production of a wide range of different dried and stable intermediate dairy products. Many new uses for these constituents have emerged with the manufacture of formula products, substitutes, and adapted raw materials.\n\nFigure 10.1 Fractionation of milk.\n\nThe most frequently used technique for the dehydration of dairy products is spray drying. It became popular in the dairy industry in the 1970s, but at that time there were few scientific or technical studies on spray drying and, in particular, there were none on the effects of spray drying parameters or the effects of the physicochemical composition and microbiology of the concentrates on powder quality. Manufacturers acquired expertise in milk drying and eventually in whey-drying processes through trial and error. Because of the variety and complexity of the mixes to be dried, a more rigorous method based on physicochemical and thermodynamic properties has become necessary. Greater understanding of the biochemical properties of milk products before drying, water transfer during spray drying, and the properties of powders and influencing factors is now essential in the production of milk powders. A lack of technical and economic information and of scientific methods prevents the manufacturer from optimizing the powder plant in terms of energy costs and powder quality.\n\nThe aim of this chapter is to provide a brief summary of the process of spray drying of dairy products and to review current knowledge on the properties of spray-dried milk products, the modeling and simulation of water-transfer processes (drying and rehydration) and dairy powders, spray drying equipment, and energy consumption.\n\n### World Dairy Powder Situations\n\nThe nature of dairy powders has changed over the last 20\u201325 years (CNIEL 1991; 2005). There has been a decrease in production, mainly of skim and fat-filled milk powders, although the production of whole milk powder and whey powder increased between 1986 and 2004. This increase was reflected in the types of whey and derived powders (protein concentrates) produced. Cheese production from cow's milk increased between 1986 and 2004, with a corresponding increase in whey production. Having fallen in 2004, the production of dry milk products did not recover significantly in the first months of 2005, the decline being due mainly to slower increase in milk supplies in many parts of the world.\n\nAccording to the International Dairy Federation (2012), production increased worldwide in 2011 for every dairy product, but growth was especially strong for butter and milk powder. In 2011 SMP production recovered strongly after a fall in 2010. In contrast to the previous year, stocks were fairly low in 2011 while sustained international demand stimulated production. World butter production continued to grow in 2011, reaching levels substantially above previous years owing to exceptional growth in output from the United States and New Zealand. The industrial output of cow's milk cheese and whole milk powder was broadly in line with levels observed in the preceding ten years.\n\n### Whole Milk Powder\n\nAccording to the International Dairy Federation (2012), world production of whole milk powder (WMP) is estimated at around 4.5 million tons. The two main players, China (+4.5%) and New Zealand (+5.6%), increased their production strongly, but the most impressive growth occurred in Argentina (+35.8%), where the high additional volumes of milk produced in 2011 were mainly processed into WMP. The structural decline in WMP production in Europe continued (\u20132.4%).\n\nWorld trade in WMP increased by 7%, up to 2.2 million tons. While more than half of this was channeled into Asia and the Middle East, market growth focused on Latin America and Africa, where key markets such as Algeria, Venezuela, Brazil, and Mexico all stepped up their imports significantly. New Zealand strengthened its position as the leading WMP supplier to the world market (49%), achieving another milestone and for the first time breaking through the barrier of one million tons of WMP exports. The geographical focus for New Zealand remained the Middle East and Asia. Exports to all major destinations in these regions expanded, including those to China, despite the fact that the enormous increase in WMP flow to that country came to an end in 2011. China alone absorbed about 28% of New Zealand's total WMP exports. Meanwhile, New Zealand also benefited from better export opportunities in Algeria. On the other hand, it lost share in a key market, Venezuela, because of renewed competition from within South America, in particular Argentina.\n\nArgentina experienced a most remarkable increase in export volume in 2011. With a massive additional 75,000 tons, Argentina was the largest contributor to world trade expansion after New Zealand. Spurred on by favorable production circumstances and high price levels on international markets, the country's dairy sector more than recovered from the previous year's export slump, reaching a level that remained not far from its historical height of five years before. Besides New Zealand and Argentina, of all the major exporters of WMP only Australia and the Philippines managed to increase export volumes in 2011. By contrast, exports from the EU dropped for the third consecutive year, as a result of lack of competiveness in the international markets. The fact that the EU lost ground to New Zealand, Argentina, and Australia in a number of its traditional key markets, such as Algeria, Nigeria, and the Middle East, is illustrative of this decline. Unlike its neighbor, Uruguay experienced a decline in WMP exports after years of expansion. This was apparently due to a priority switch in the export range in favor of product categories such as butter, cheese, and skim milk powder (SMP), which absorbed the additional milk available in 2011 (International Dairy Federation, 2012).\n\n### Skim Milk Powder\n\nWorld production of skim milk powder (SMP) is estimated at around 4 to 4.5 million tons. Stimulated by firm demand, the output of SMP increased in most parts of the world between 2010 and 2011. SMP had an increasing role over these two years in exports from the EU and the United States in that there is no longer a price gap between these two blocks and New Zealand. Since 2010, North American and European processors have been competitive throughout the year in the world SMP market, which is not the case for butter and WMP. This convergence in terms of price is corroborated by the presence of American and European companies in the SMP auction on the Global Dairy Trade platform (International Dairy Federation, 2012).\n\nWorld trade in SMP in 2011 reflected very dynamic development. Worldwide exports for this category soared to over 1.7 million tons (+19%). After a period of weaker interest, this was the fourth consecutive year that the world trade volume increased. All major exporters expanded their trade, benefiting from growth in demand in various regions. The main reason for this was both developments in Southeast Asia and increased trade to various key markets in the Middle East, South America, and Africa. Mexico remained the world's largest single SMP market, but the expanding export volume entering Mexico was of benefit mainly to American exporters, as they were facilitated by NAFTA arrangements, and this is why the United States now retains nearly 90% of that market. The SMP export market is rather concentrated on the supply side, since effectively 75% of world trade volume is supplied by only three exporters: the EU, the United States, and New Zealand. This situation was reaffirmed in 2011, when the EU reestablished its position as the leading exporter. International price developments in combination with internal dairy policy in recent years have contributed to the EU's competitiveness in milk protein in the world market.\n\nDestinations in Northern Africa remained very important to EU exporters, as did several key destinations in Asia, such as Indonesia and China. They also seized opportunities in India, South Korea, and Mexico, while on the other hand the 2010 surge in exports to the Russian market was reversed. Though not expanding to the extent of the EU, the United States remained by far the second largest global SMP supplier. As already mentioned, its exports are closely linked to trade with Mexico, which took an additional 59,000 tons of U.S. milk powder in 2011 and absorbed nearly 40% of total U.S. exports. Moreover, U.S. exporters developed substantial markets in Asia, which also allowed them to benefit from the growth in that region. Of the minor suppliers, Australia, Uruguay, and Ukraine all made significant progress, recovering from weak export results in 2010. At the same time, exports from Belarus and Argentina were down; for Belarus this was a reaffirmation of a downward trend since the previous year, closely related to reduced demand from Russia (International Dairy Federation, 2012).\n\n### Whey Products, Casein, and Other Dairy Ingredients\n\nIn 2011 casein production increased in most countries where statistics are available. Output in the EU was estimated at around 145,000 tons\u2014that is, 15,000 tons more than in 2010. Liquid whey production results primarily from the industrial production of cheese, which generates more than 80% of the total whey available, and secondarily from casein output. The major processors of whey are therefore located in Europe, North America, and the South Sea Islands, corresponding to the major cheese production areas. Compared to 2010, no significant changes occurred in 2011 in the United States; production remained fairly stable at 500,000 tons of whey powder and condensed whey, as well as 195,000 tons of whey protein concentrates and almost 30,000 tons of whey protein isolates. The production of whey powder within the EU is estimated at around 1.9 million tons. It increased slightly (+1.6%) in 2011 (International Dairy Federation, 2012).\n\nWorld trade in whey powder and whey products in 2011 continued its dynamic development, increasing to over 1.5 million tons (+11%). This trade remained dominated by exports from the EU and the United States, which represented two-thirds of total world trade. While exports from the former increased substantially, mainly on the wave of expanding export opportunities in China and other Asian destinations, exports from the latter stabilized as a result of reduced exports to neighboring markets and mixed results in Asia. China is by far the largest and the most dynamic market, absorbing 22% of world trade after a 36% growth.\n\nExcept for Switzerland and Australia, all other significant whey exporters increased their exports. In absolute terms, Argentina expanded the most, after the EU, mainly due to imports to Asia in general and China in particular. In relative terms, Belarus showed the highest export increase, which reflected recent investments in whey processing. As usual, most of its products went to Russia, ousting competitors from a contracting market (International Dairy Federation, 2012).\n\n## Properties of spray-dried milk products\n\nA dairy powder is characterized not only by its composition (proteins, carbohydrates, fats, minerals, and water) but also by its microbiological and physical properties (bulk and particle density, instant characteristics, flowability, floodability, hygroscopicity, degree of caking, whey protein nitrogen index, thermostability, insolubility index, dispersibility index, wettability index, sinkability index, free fat, occluded air, interstitial air, and particle size), which form the basic elements of quality specifications. There are well-defined test methods for their determination according to international standards (Pisecky, 1986; 1990; 1997; American Dairy Products Institute 1990; Masters, 1991). These characteristics depend on drying parameters (type of tower spray drier, nozzles\/wheels, pressure, agglomeration, and thermodynamic conditions of the air, such as temperature, relative humidity, and velocity), and the characteristics of the concentrate before spraying (composition\/physicochemical characteristics, viscosity, thermosensitivity, and availability of water). Several scientific papers have been published on the effects of technological parameters on these properties (Hall & Hedrick, 1966; De Vilder et al., 1979; Baldwin et al., 1980; Pisecky, 1980; 1981; 1986; Kessler 1981; Bloore & Boag, 1982; De Vilder, 1986; Tuohy, 1989; Ilari & Loisel, 1991; Masters, 1991; Mahaut et al., 2000).\n\nWater content, water dynamics, and water availability are among the most important biochemical properties of dairy powders (Fig. 10.2).\n\nFigure 10.2 Properties and qualities of powders.\n\nThe nutritional quality of dairy powders depends on the intensity of the thermal processing during the technological process. The thermal processing induces physicochemical changes that tend to decrease the availability of the nutrients (loss of vitamins, reduction of available lysine content, and whey protein denaturation) or to produce nutritional compounds such as lactulose (Straatsma et al., 1999a,b).\n\n## Principles of spray drying\n\nAccording to Pisecky (1997), spray drying is an industrial process for the dehydration of a liquid containing dissolved and\/or dispersed solids (e.g., dairy products) by transforming the liquid into a spray of small droplets and exposing these droplets to a flow of hot air. The very large surface area of the spray droplets causes evaporation of the water to take place very quickly, converting the droplets into dry powder particles.\n\nIndeed, when a wet droplet is exposed to hot dry gas, variations in the temperature and the partial pressure of the water vapor are established spontaneously between the droplet and the air:\n\n\u2022 Heat transfer from the air to the droplet occurs under the influence of the temperature variation.\n\n\u2022 Water transfer occurs in the opposite direction, explained by variation in the partial pressure of water vapor between the air and the droplet surface.\n\nAir is thus used both for fluid heating and as a carrier gas for the removal of water. The air enters the spray drier hot and dry and leaves wet and cool. Spray drying is a phenomenon of surface water evaporation maintained by the movement of capillary water from the interior to the surface of the droplet. As long as the average moisture is sufficient to feed the surface regularly, the evaporation rate is constant; if not, it decreases.\n\nThe drying kinetics are related to three factors.\n\n\u2022 Evaporation surface created by the diameter of the particles. Spraying increases the exchange surface: 1 L of liquid sprayed as particles of 100 \u03bcm diameter develops a surface area of 60 m2, whereas the surface area is only approximately 0.05 m2 for one sphere of the same volume.\n\n\u2022 Difference in the partial pressure of water vapor between the particle and the drying air. A decrease in the absolute humidity of the air and\/or an increase in the air temperature tend to increase the difference in the partial pressure of water vapor between the particle and the drying air.\n\n\u2022 Rate of water migration from the center of the particle toward its surface. This parameter is essential for the quality of dairy powders. Indeed, it is important that there is always water on the surface of the product so that the powder surface remains at the wet bulb temperature for as long as possible. The rate of water migration depends on the water diffusion coefficient, which varies according to the biochemical composition, water content, and droplet temperature. Calculation of this coefficient is therefore complex and the mathematical models suggested are not easily exploitable by the dairy industry.\n\nAccording to Masters (1991) and Pisecky (1997), the main components of the spray drier, as shown in Figure 10.3, are as follows:\n\n\u2022 A drying chamber (Fig. 10.3, point 7). The chamber can be horizontal (box drier), although in the dairy industry the chamber design is generally vertical with a conical or flat base.\n\n\u2022 An air disperser with a hot air supply system such as a main air filter, supply fan, air heater, and air disperser (Fig. 10.3, point 3). The air aspiration is performed through filters, the type depending on the local conditions and the nature of the product to be treated. The air can be heated in two different ways: by direct heating (gas) and\/or by indirect heating (vapor, gas, oil, or electricity). The air flow chamber can be in co-current, countercurrent, or mixed mode.\n\n\u2022 An atomizing device with a feed supply system such as a feed tank, feed pump, water tank, concentrate heater, and atomizing device. There are three types of atomizing device: rotary (wheel or disc), nozzle (pressure, pneumatic or sonic), and combined (rotary and pneumatic) (Fig. 10.3, point 3).\n\n\u2022 A powder recovery system. Separation of the dried product can be achieved by a primary discharge from the drying chamber followed by a secondary discharge from a particulate collector (using a cyclone, bag filter, or electrostatic precipitation), followed by total discharge from the particulate collector and finishing with final exhaust air cleaning in a wet scrubber and dry filter (Fig. 10.3, points 8 and 9).\n\nFigure 10.3 Multiple effect spray dryer.\n\nAccording to Sougnez (1983), Masters (1991), and Pisecky (1997), the simplest types of installation are single-stage systems with a very short residence time (20\u201360 s). Thus, there is no real balance between the relative humidity of the air and the moisture content of the powder. The outlet temperature of the air must therefore be higher, and the thermal efficiency of the single-stage spray drier is then reduced. This type of drying chamber was the standard equipment for drying milk in the 1960s. Space requirements were small and building costs were low. Generally, installations without any post-treatment system are suitable only for nonagglomerated powders that do not require cooling. If necessary, a pneumatic conveying system could be added to cool the powder while transporting the chamber fraction and the cyclone fraction to a single discharge point.\n\nThe two-stage drying system consists of limiting the spray drying process to a process with a longer residence time (several minutes) to provide a better thermodynamic balance. This involves a considerable reduction in the outlet air temperature, as well as an increase in the inlet air temperature. A second final drying stage is necessary to optimize the moisture content by using an integrated (static) fluid bed or an external (vibrating) fluid bed, the air temperatures of which are 15\u201325 \u00b0C lower than with a single-stage system to improve and\/or preserve the quality of the dairy powder (Fig. 10.3, points 11 and 14). Consequently, the surrounding air temperature at the critical drying stage and the particle temperature are also correspondingly lower, thus contributing to further economic improvement. The integrated fluid bed can be either circular (e.g., the Multi Stage Drier (MSD\u2122) chamber) or annular (e.g., the Compact Drier (CD) chamber). Two-stage drying has its limitations, but it can be applied to products such as skim milk, whole milk, precrystallized whey, caseinates, whey proteins, and derivatives. The moisture content of the powder leaving the first stage is limited by the thermoplasticity of the wet powder\u2014that is, by its stickiness in relation to the water activity and the glass transition temperature (Roos, 2002). The moisture content must be close to 7\u20138%, 9\u201310%, and 2\u20133% for skim\/whole milk, caseinate\/whey protein, and precrystallized whey powders, respectively. The two-stage drying techniques can be applied to the production of both nonagglomerated and agglomerated powders, but this technique is very suitable for the production of agglomerated powders, by separating the nonagglomerated particles from the agglomerates [i.e., collecting the cyclone fractions and reintroducing these fine fractions (called fines) into the wet zone around the atomizer of the chamber].\n\nThe three-stage drying systems, with an internal fluid bed as a second stage in combination with an external vibrating fluid bed as a third-stage drier, first appeared at the beginning of the 1980s and were called the Compact Drier Instantization (CDI) or MSD\u2122. Today, they dominate the dairy powder industry (Fig. 10.3). Three-stage systems combine all the advantages of extended two-stage drying, using spray drying as the primary stage, fluid bed drying of a static fluid as the second drying stage, and drying on an external vibrating fluid bed as the third drying stage. The final drying stage terminates with cooling to under the glass transition temperature. Evaporation performed at each stage can be optimized to achieve both gentle drying conditions and good thermal economy.\n\nThe compact dryer (CD) is suitable for producing both nonagglomerated and agglomerated powders of practically any kind of dried dairy product. It can also cope successfully with whey powders, fat-filled milk, and whey products as well as caseinates, both nonagglomerated and agglomerated. It has a fat content limit of about 50% fat in total solids. Powder quality and appearance are comparable to those of products from two-stage drying systems, but they have considerably better flowability and the process is more economical. In comparison with the CD, the MSD\u2122 can process an even wider range of products and can handle an even higher fat content. The main characteristics of MSD\u2122 powder are very good agglomeration and mechanical stability, low particle size fractions (below 125 \u03bcm), and very good flowability.\n\nOptimization of the process has allowed considerable improvement in the drying efficiency, and the quality of the product obtained is generally better. The various advantages are:\n\n\u2022 Improved thermal efficiency: significant reduction in the outlet air temperature, permitting an increase in the inlet air temperature;\n\n\u2022 Reduction in material obstruction: the capacity in one volume is two or three times higher than that for a traditional unit;\n\n\u2022 Considerable reduction in powder emission to the atmosphere: a reduction in the drying air flow and an increase in powder moisture content reduce the loss of fine particles in the outlet air;\n\n\u2022 Improved powder quality in relation to the agglomeration level, solubility, dispersibility, wettability, particle size, density, and so on\n\nThere are other examples of drying equipment such as the 'tall form drier,' the 'Filtermat\u00ae drier,' the 'Paraflash\u00ae drier,' and the 'Tixotherm\u00ae drier.' All these towers have characteristics related to the specific properties of the product being dried (e.g., high fat content, starch, maltodextrin, egg, and hygroscopic products).\n\n## Process improvement\n\nThis section shows the use of a thermohygrometric sensor, with some examples of such measurements (temperature, absolute humidity (AH), and relative humidity (RH), dry air flow rate, water activity), through calculation of mass and AH to prevent sticking in the drying chamber and to optimize powder moisture and water activity in relation to the RH of the outlet air.\n\nSchuck et al. (2005) demonstrated that a thermohygrometer can be used to avoid sticking and to optimize water content and water activity in dairy powders. These results demonsrate that the calculated AH is systematically higher than the measured AH because the calculated AH corresponds to the maximum theoretical value that can be reached. Calculation of AH by means of the mass balance is based on the hypothesis that the air circulating in the spray drier removes all the water from the concentrate. Thus, if the difference between the calculated and the measured AH of the outlet air is below 2 g of water kg-1 dry air (depending on the spray drier with regard to measurement accuracy), there is no problem of sticking in the spray dryer chambers, whatever the dairy concentrate used. On the other hand, sticking was observed in this study for differential AH above 2 g of water kg-1 dry air, corresponding to lower water removal and consequently to favorable sticking conditions. The operator can follow the AH and anticipate a variation in drying parameters according to the differences between the calculated and the measured AH.\n\nThe operator can also follow the relative humidity in the outlet air. To achieve a dairy powder with the same water activity and moisture content, the same RH in the outlet air using the previous equations according to each dairy product, whatever the spray drying conditions (inlet air temperature, RH and AH), must always be maintained.\n\nThe changes in RH and AH (resulting from variations in AH of inlet air, total solid content of concentrate, crystallization rate, outlet air temperature, etc.) can be rapidly observed in the outlet air using a thermohygrometer before such changes significantly affect powder moisture, water activity, and powder behavior with regard to sticking.\n\n## Drying of proteins\n\nThe native properties of milk components are substantially unaffected by moderate drying conditions. Depending on the preheating conditions, drier design, and temperature operation, the properties of spray-dried powder may vary significantly. An evaporating milk droplet in a spray drier in co-current air flow does not initially appreciably exceed the wet bulb temperature and can be held effectively at temperatures below 60 \u00b0C. As the falling temperature period is approached in the course of further evaporation, the temperature rises to a final value determined by the final temperature of the drying gas and the residence time in the drier. Under properly controlled spray drying conditions, the changes in milk protein structure and solubility are minor. Spray drying does not denature the whey protein significantly, and the levels of denatured whey protein in dairy powders are more or less equal to those of condensed milk and heated milk, which is substantially more denatured than during spray drying. An excellent example is in relation to the whey protein nitrogen index (WPNI). According to Pisecky (1997), the WPNI expresses the amount of undenatured whey protein (milligrams of whey protein nitrogen per gram of powder). It is a measure of the sum of heat treatments to which the milk has been subjected prior to evaporation and spray drying. The heat treatment of a concentrate, and subsequently of a powder, has only a negligible effect on the WPNI. The main operation to achieve the required value is the pasteurization process, that is, time\/temperature combination. However, many other factors influence the WPNI, including the total amount of whey protein and the overall composition of the processed milk as influenced by animal breed and seasonal variations. The individual design of the processing equipment, that is, the pasteurizer and holding tubes, is also very important. It is therefore difficult to predict the conditions required to achieve the required WPNI on a general basis. The main purpose of heat pretreatment is obviously to ensure the microbiological quality of dairy products.\n\nThe influence of the heat treatment on the denaturation of whey proteins to achieve the desired properties of the final products is just as important in milk powder production. SMP for cheese manufacture should have as much undenatured protein as possible; that is, it should be low heat-produced (WPNI >6), whereas, for bakery products, high heat-produced powder with high denaturation is required (WPNI <1.5). For ice cream, chocolate, and confectionery, medium heat-produced powder is required. According to Schuck et al. (1994a), the use of microfiltration (pore diameter, 1.4 \u03bcm), combined with low-heat treatment during vacuum evaporation, allows the production of a low low heat SMP with a WPNI close to 9 mg of whey protein nitrogen\/g of powder, bacterial count <1000 CFU\/g powder, solubility index >99.5%, dispersability index >98.5%, and wettability index <15 s. After water rehydration, such a powder has the same renneting time as the original raw milk and can be used as a reference powder for both industrial and scientific purposes.\n\nThe stability of protein powders during storage is critically affected by the moisture content and the storage temperature. More precisely, such stability is governed by the water activity (aw) and the glass transition (Tg) temperature. The aw should be close to 0.2 at 25 \u00b0C for optimal preservation, with an ideal moisture content determined by using the sorption isotherm of some dairy powders. For example, the corresponding moisture content for skim milk, whey, and protein powders must be close to 4%, 2\u20133%, and 6%, respectively. The optimal storage temperature must be below the Tg temperature, which is close to 40\u201350 \u00b0C at 0.2 aw.\n\n### Examples of Dairy Protein Concentrates and Powders\n\nTo illustrate this chapter through examples, micellar casein concentrate (MCC) was prepared by microfiltration and diafiltration (pore diameter, 0.1 \u03bcm) on an MFS 19 (Tetra Laval, \u00c5arhus, Denmark; 4.6 m2) at 50 \u00b0C, at 200 g\/kg total solids, according to Fauquant et al. (1988) and Pierre et al. (1992). The whey protein concentrate (WPC) was obtained by ultrafiltration and diafiltration of the microfiltrate (0.1 \u03bcm) using a DDS module (GEA, Soeborg, Denmark) with the plane membrane (10,000 g\/mol molecular weight cut-off, 9 m2, 50 \u00b0C) at 200 g.kg-1 total solids. The sodium, calcium, and potassium caseinate concentrates were reconstituted from sodium, calcium, and potassium caseinate powder at 190 g.kg-1 total solids. The microfiltration retentate (R4 MF) was obtained by microfiltration (0.1 \u03bcm) on an MFS 19 (Tetra Laval, \u00c5arhus, Denmark; 4.6 m2) at 50 \u00b0C. The volume reduction ratio was 4. The ultrafiltration retentate (R4 UF) was manufactured by ultrafiltration on a 2-S37 module with M1 membranes (Tech Sep, Rh\u00f4ne Poulenc, St Maurice de Beynost, France; 100,000 g\/mol nominal molecular weight cut-off, 6.8 m2) at 50 \u00b0C. The volume reduction ratio was 4. NaCl solution, CaCl2 solution, sodium phosphate solution at pH 7.1 (for MCC) or pH 6.6 (for WPC) and sodium citrate solution at pH 7.1 (for MCC) or pH 6.6 (for WPC) in 205 5 g.kg-1. Total solids were added to the MCC or the WPC to obtain a concentrate with 12% (w\/w) (NaCl, CaCl2, and sodium phosphate solution) and 30% (w\/w) (sodium citrate) of mineral salts\/total solids. After the addition of salt, the pH was adjusted to 7.1 (for MCC) or 6.6 (for WPC) with 1 N KOH (NaCl, CaCl2, and sodium phosphate solution) or 1 N HCl (sodium citrate) at 20 \u00b0C.\n\nSpray drying of the concentrates was performed at Bionov (Rennes, France) in a three-stage pilot plant spray drier (GEA, Niro Atomizer, St Quentin en Yvelines, France) according to Schuck et al. (1998a) and Bimbenet et al. (2002) to obtain a micellar casein powder (MCP) or a whey protein powder (WPP). The temperature of the concentrate before drying was 40 \u00b0C for MCC and 20 \u00b0C for WPC. The atomizer was equipped with a pressure nozzle (0.73 mm diameter orifice) and a four-slot core (0.51 mm nominal width), providing a 60\u00b0 spray angle. The evaporation capacity was 70\u2013120 kg.h-1 (depending on the inlet and outlet air temperatures and the air flow). The pressure in the nozzle was 16 MPa. The inlet temperature was 208 \u00b0C for WPC and 215 \u00b0C for MCC; the integrated fluid bed air temperature was 70 \u00b0C for MCC and WPC; and the outlet temperature was 80 \u00b0C for WPC and 70 \u00b0C for MCC. The inlet air humidity was controlled and adjusted by a dehumidifier (Munters, Sollentuna, Sweden). Two granulation states were obtained for each MCP or WPP, i.e., nongranulated (NG) and granulated (G) powders, by reintroduction of the fine particles after the cyclones at the top of the spray drier.\n\n#### Research Approach Using Drying by Desorption\n\nThe concentrates were dried in a water activity meter (Novasina RTD 200\/0, Pf\u00e4ffikon, Switzerland) at 20 \u00b0C (constant temperature). The concentrate (100 mg) was placed in a plastic support (area, 95 mm2) with a zeolite WE 291 dryer below (7 g) (Bayer, Puteaux, France). This method was used to simulate the conditions of spray drying by establishing a difference in vapor pressure equilibrium between the dairy concentrate and the drying air and to determine the water transfer from inside the dairy concentrate to the surface. The RH was measured in relation to time following water transfer from the dairy concentrate to the zeolite. The final slope of the absolute value of the decrease in RH (\u03b2) represented the ability to remove bound water from the solute at the end of the drying phase (Schuck et al. 1998b; 1999); the lower the \u03b2 slope, the greater the difficulty of removing water at the end of drying and the higher the bound water content.\n\n#### Desorption Results\n\nThe drying slopes (\u03b2) of the various dairy products tested are shown in Table 10.1. The dairy products, ranging from the highest to the lowest absolute value of the slope, were skim milk (\u03b2 = 0.90%.min-1), R4 UF (\u03b2 = 0.75%.min-1), R4 MF (\u03b2 = 0.70%.min-1), and MCC (\u03b2 = 0.34%.min-1). These results could be explained by a decrease in water diffusion through the dried product, that is, the final residue obtained at the end of drying, when the micellar casein concentration increased. Water transport was probably affected by the high micellar casein content of the sprayed droplet in the atomization tower, and similarly when the powder granule was dissolved in water. These results are in accordance with the results of Schuck et al. (1994a, b).\n\nTable 10.1\n\nDrying of Dairy Concentrates by Desorption\n\nDairy concentrate | \u03b2 slope (%.min-1)  \n---|---  \nSkim milk | 0.90  \nR4 UF | 0.75  \nR4 MF | 0.70  \nMCC | 0.34  \nWPC | 0.68  \nSodium caseinate | 0.64  \nPotassium caseinate | 0.65  \nCalcium caseinate | 0.51  \nWPC + NaCl | 0.24  \nWPC + CaCl2 | 0.46  \nWPC + Phosphate | 0.41  \nWPC + Citrate | 0.48  \nMCC + NaCl | 0.19  \nMCC + CaCl2 | 0.36  \nMCC + Phosphate | 0.49  \nMCC + Citrate | 0.45\n\nR: retentate; UF: ultrafiltration; MF: microfiltration; MCC: micellar casein concentrate; WPC: whey protein concentrate\n\nDrying of the caseinates showed that sodium caseinate and potassium caseinate dried more easily (\u03b2 = 0.64% and 0.65%\/min, respectively) than calcium caseinate (\u03b2 = 0.51%\/min) (Table 10.1). The limitation of water diffusion through the calcium caseinate may be explained by the structure of this colloidal dispersion. In calcium caseinate, the casein subunits are more aggregated because of calcium binding, whereas in sodium and potassium caseinates, the caseins are more soluble. The above results showed that the water bound in a micellar structure (e.g., MCC) was more strongly bound than that bound to the soluble caseins in sodium caseinate. The situation was intermediate for calcium caseinate. We assumed that these differences in water transfer during drying could be explained by the casein structure. Therefore the decrease in water inside the dairy concentrate led to a decrease in the water concentration on the surface of the concentrate in the water activity meter or on the surface of the droplet during spray drying and thus decreased the drying kinetics. These results were confirmed by the desorption drying of two different classes of proteins, that is, MCC (micellar structure), with a \u03b2 value of 0.34%\/min, and WPC (globular structure), with a \u03b2 value of 0.68%\/min (Table 10.1). These two different types of protein had the same protein content (89% of total solids) and the same water content before desorption drying, but not the same drying time or \u03b2 value. All these results show that the drying rate is dependent on the nature and structure of the casein. Water may be less available during the drying of a protein with a micellar structure than during the drying of a protein with a globular structure.\n\nAddition of minerals to WPC decreased the \u03b2 value, the smallest decrease being with citrate (29% reduction) and the greatest decrease being with NaCl (65% reduction) (Table 10.1). A decrease in the \u03b2 value means a lower rate of water transfer at the end of drying (Schuck et al., 1998b). The decrease in water transfer during the drying of modified WPC could be explained by the high hygroscopicity of the added mineral salts. This result suggested that the water is more closely bound to the mineral salts than to the whey proteins at the end of drying. The addition of minerals to WPC under the test conditions had little effect on the whey protein structure but probably had some effect on the increase in bound water in the modified WPC.\n\nAddition of NaCl to MCC decreased the \u03b2 value (0.19%\/min) (Table 10.1). Water in a casein system containing NaCl is more rotationally mobile than water in a casein model system without NaCl, with the same water activity (Curme et al., 1990). For high electrolyte concentrations, the amount of bound water decreases because of the suppression of the electrical double layer surrounding the protein molecule. This is directly related to the hydration of ions (Na+, Cl\u2013) and hence to the ability to separate the water molecules from the protein molecules (Robin et al., 1993). Water is less closely bound to micellar casein in the presence of NaCl (Cayot & Lorient, 1998). The decrease in water transfer at the end of drying can be explained by the hygroscopicity of NaCl. The water is more closely bound to NaCl than to the micellar caseins.\n\nAddition of CaCl2 to MCC increased the \u03b2 value (0.36%\/min) (Table 10.1). Moreover, addition of CaCl2 to milk decreases micellar solvation (Tarodo de la Fuente, 1985; Van Hooydonk et al., 1986; Jeurnink & de Kruif, 1995; Le Ray et al., 1998). First, the water inside the micellar structure in the case of MCC without addition of salt might be less available than the water inside the micellar structure of MCC with CaCl2. Second, the lower water content inside micellar structures with additional calcium might lead to an increase in water transfer during drying.\n\nAddition of phosphate ions to MCC increased the \u03b2 value (0.49%\/min) (Table 10.1). The increase in water transfer was explained by the partial solubilization of caseins (Le Ray et al., 1998), although an increase in casein micelle solvation and an increase in viscosity were observed. These results may be discussed in terms of the strength of water binding to caseins, either in a soluble form as in sodium caseinate (Schuck et al., 1998b) or in the micellar structure (MCC) as in the experiments reported here. The water bound to micellar caseins was probably less easy to remove than the water bound to soluble caseins. Partial solubilization of caseins improved water transfer during dehydration of phosphate solution + MCC.\n\nAddition of citrate solution to MCC at 30%\/total solids increased the \u03b2 value (0.45%\/min) (Table 10.1). Addition of citrate induces the release of large amounts of soluble casein from the micellar phase because of solubilization of colloidal calcium phosphate (Le Ray et al., 1998). Similar results occurred with the addition of phosphate ions. Solubilization of the micellar casein improved the water transfer during dehydration of citrate solution + MCC.\n\n### Industrial Implications\n\n#### Introduction\n\nSeveral studies (Masters, 1991; Pisecky, 1997) have reported that the moisture content can vary for the same outlet air temperature in the spray drier according to the product. For example, Pisecky (1997) reported that the moisture content is close to 4% and 5% for WMP and SMP, respectively, for an outlet air temperature close to 85 \u00b0C. On the other hand, to produce WMP and SMP at the same moisture content (4%, for example), the outlet air temperatures must be different (80 \u00b0C and 90 \u00b0C, respectively). All these differences can be explained by the effects of the biochemical composition of the ingredients on the availability of the water to be transferred from the droplet to the drying air.\n\n#### Availability of Water\n\nConcerning the availability of the water, Schuck et al. (1998a,b; 2009) have developed a new method (drying by desorption, using a thermohygrometer sensor) in order to determine major drying parameters of food components in relation to their interactions with water (bound and free water) and linked to water-transfer kinetics. Studies by Schuck et al. (1998b; 2009) and Zhu et al. (2011a,b) have shown that drying by desorption is an excellent tool to determine and optimize the major spray-drying parameters in relation to biochemical composition according to water availability and desorption behavior (calculation of extra energy, \u0394E). The experimental device proposed by the authors differs from spray-drying equipment in terms of duration of drying, drying temperature, surface\/volume ratio, and so on, because the concentrate is dried in a cup and not in a droplet. Computational tools have been developed to improve the method by taking these factors into account such as the 'Spray Drying Parameter Simulation and Determination Software' (SD2P\u00ae) registered under the following identification: IDDN.FR.001.480002.003.R.P.2005.000.30100. For reasons of calculation speed and reliability, this method has been computerized, and it can already be used in determining the parameters of spray drying for food products. Validation tests (>80 products) have indicated that this method could be applied to a wide range of food products and spray-dryer types.\n\nCombined with knowledge of the temperature, the software provides analysis of the desorption curve (measured relative humidity versus time), total solids, density, and specific heat capacity of the concentrate, air flow rates, water content, RH of the outlet air, current weather conditions, and cost per kWh. The percentage of drying in the integrated fluid allows determination of enthalpy H, T, RH (including \u0394E) for each inlet air, concentrate and powder flow rate, specific energy consumption, energy and mass balance, yield of the dryer, and cost (in \u20ac or in $) to remove 1 kg of water or to produce 1 kg of powder (summarized in Fig. 10.4). This figure is a representation of the software delivery:\n\n\u2022 air characteristics at the dryer (with or without integrated fluid bed) inlet and outlet (upper part)\n\n\u2022 flow, energy, and cost calculations (lower part) (Schuck et al., 2009)\n\nFigure 10.4 Parameters of spray drying calculated by SD2P\u00ae software.\n\nThus, the interests of the desorption curves lay in evaluatimg water transfer during spray drying of various dairy concentrates using thermodynamic and biochemical approaches. Whey protein concentrates and isolates (WPC35, WPC50, WPC70, WPI90) with or without heat denaturation, MC, sodium caseinate (NaCas), and milk with and without whey protein enrichment were dried in a three-stage pilot plant spray drier. When the concentrate temperature, air flow rate, concentrate flow rate, total solids content of the concentrate, inlet air temperature absolute humidity, inlet air temperature before and after heating, and outlet air temperature after drying are known, it is possible to determine the specific energy consumption (SEC)\u2014that is, the ratio of the energy consumed to the evaporation of 1 kg of water (measured in kJ.kg-1 water) (Schuck et al., 1998a; Bimbenet et al., 2002). Thus, if you spray dry only free water, the energy used in terms of SEC would be close to 2500 kJ.kg-1 water. If the concentrate amounts of bound water to free water increase, the SEC increases (up to 10,000 kJ.kg-1 water, for example). The significance of a very high SEC relates to the decreasing availability of the water, limiting water transfer, and thus increasing both the surface temperature of the droplet and the risk of protein denaturation of the powder.\n\n#### Whey Proteins\n\nThe results presented in Table 10.2 show that water transfer during spray drying decreased when the whey proteins were native proteins. For the same moisture content, the SEC for drying was higher when (a) the native whey protein content increased in WPC and in milk and (b) the whey proteins were heat denatured in WPC35. However, the SEC was lower when (c) the whey proteins were heat denatured in WPC50, WPC70, and WPI90. These results can be explained by the availability of the water (bound or unbound) in the concentrate in relation to the nature and content of the whey proteins.\n\nTable 10.2\n\nSpecific Energy Consumption at 4% Moisture Content for the Drying of Dairy Proteins\n\n| SM | SM + WPI | WPC | WPI | MC | NaCas  \n---|---|---|---|---|---|---  \nProtein content (%) | 34 | 50 | 35 |  | 50 | 70 | 90 |  | 90 | 90  \nHeat treatment (72 \u00b0C\/4 min) | N | N | N | Y | N | Y | N | Y | N | Y | N | N  \nSEC (\u00b13%) (kJ.kg-1 water) | 5900 | 6400 | 5950 | 7700 | 6800 | 6550 | 7050 | 6600 | 7200 | 6500 | 6900 | 5900\n\nY: heat treatment; N: no heat treatment; SM: skim milk; WPI: whey protein isolate; WPC: whey protein concentrate; MC: micellar casein; NaCas: sodium caseinate; SEC: specific energy consumption at 4% of moisture content.\n\n#### Caseins\n\nThe results presented in Table 10.2 also show that water transfer during spray drying decreased when the micellar casein content increased. For the same moisture content, the SEC for drying was higher when (a) the micellar casein content increased in MC compared with skim milk and (b) casein remained in a micellar state (as in MC) compared with a soluble state (e.g,. in NaCas). These results can be explained by the availability of the water in the concentrate in relation to the content and the structure of the caseins. Water is more available when the caseins are soluble than when they are in a micellar state.\n\nAll these results also show that water transfer depends on the relationship between the water and the protein components and that these components should be taken into account when optimizing spray drying parameters for proteins.\n\n#### Rehydration of Protein Powders\n\nMost food additives are prepared in powder form and need to be dissolved before use. Water interactions in dehydrated products and dissolution are thus important factors in food development and formulation (Hardy et al., 2002). Dissolution is an essential quality attribute of a dairy powder as a food ingredient (King, 1966). Many sensors and analytical methods such as the insolubility index (ISI, International Dairy Federation, 1988; American Dairy Products Institute, 1990), nuclear magnetic resonance (NMR) spectroscopy (Davenel et al., 1997), turbidity, viscosity, and particle size distribution (Gaiani et al., 2006) can now be used to study water transfer in dairy protein concentrates during rehydration. Using combinations of these methods, it is very easy to determine the different stages of the rehydration process, that is, wettability, swellability, sinkability, dispersibility, and solubility.\n\nThe ISI (in %) described by the IDF standard (International Dairy Federation, 1988; Schuck et al., 2012) for skim milk is the volume of sediment (for 50 mL) after rehydration (10 g of powder in 100 mL of distilled water, at 25 \u00b0C), mixing (for 90 s, at 4000 rev\/min) and centrifugation (for 300 s, at 160 g). With this method, the quantity of insoluble material (whether true or false) can be determined.\n\nNMR spectroscopy is a technique for determining the rate of solution, the time required for complete reconstitution of powders, and the transverse relaxation rate of reconstituted solutions. The method was first described by Davenel et al. (1997). A 40 mm diameter glass tube filled with 20 mL of water at 40 \u00b0C was placed in the gap of the magnet of a Minispec Bruker PC 10 NMR spectrometer operating at a resonance frequency of 10 MHz. A suitably designed funnel and an electric stirrer (glass spatula) were inserted into the tube. They showed that the solubilization rate was independent of the quantity of powder poured (up to 20 g powder\/100 mL water) and increased with the stirring rate. In subsequent experiments, the rotation rate of the stirrer was adjusted after starting at 1150 rev.min-1 for spray-dried powders, and 1 g of powder was poured into the water. The NMR measurements were generally continued until the solution was completely reconstituted, except if insoluble material was formed. Each decay curve was obtained by sampling a maximum of 845 spin echoes of a Carr-Purcell-Meiboom-Gill (CPMG) sequence every 20 s during the reconstitution period. Interpulse spacing between 180\u00b0 pulses was fixed at 2 ms to limit the diffusion effect caused by stirring. The NMR kinetics method was used in triplicate. The CPMG curves were well approximated by the sum of two exponential curves to determine the protons attributed to water protons in fast exchange with exchangeable protons of nondissolved powder particles, as well as the protons attributed to water protons and exchangeable protons in the reconstituted phase (Davenel et al., 1997). With this method, it is possible to differentiate between truly insoluble material and falsely insoluble material. The falsely insoluble material can be explained by the low water transfer during rehydration and not by denatured protein, which is truly insoluble (Schuck et al., 1994b).\n\nFor viscosity measurement, a rheometer can be used to obtain viscosity profiles. In the studies of Gaiani et al. (2005; 2006), the blades were placed at right angles to each other to provide good homogenization. Industrial dissolution processes usually include stirring at a constant speed, and the experiments were therefore designed to provide a constant shear rate (100 s\u20131). MCP was added to the rheometer cup manually. The aqueous phase used was distilled water at a volume of 18 mL. The powder was dispersed in the rheometer cup 50 s after starting the rheometer. Dissolution was highly dependent on temperature and concentration. The total nitrogen concentration employed to study these effects was about 5% (w\/v), and the temperature was about 24 \u00b0C.\n\nThe experiments to provide the turbidity profiles were carried out in a 2 L vessel equipped with a four-blade 45\u00b0 impeller rotating at 400 rev\/min. A double-walled jacket vessel maintained the temperature at 24 \u00b0C. The turbidity sensor was placed 3 cm below the surface of the water and was positioned through the vessel wall to avoid disturbance during stirring. Turbidity changes accompanying powder rehydration were followed using a turbidity meter. The apparatus used light in the near-infrared region (860 nm), the incident beam being reflected back at 180\u00b0 by any particle in suspension in the fluid to a sensitive electronic receptor (Gaiani et al., 2005).\n\nA laser light diffraction apparatus with a 5 mW He\u2013Ne laser operating at a wavelength of 632.8 nm can be used to record particle-size distributions. The particle-size distribution of dried particles was determined by using a dry powder feeder attachment, and the standard optical model presentation for particles dispersed in air was used. To measure the particle size distribution of micellar casein in concentrates, 0.5 mL of suspension was taken from the rheometer cup and introduced into 100 mL of prefiltered distilled water (membrane diameter, 0.22 \u03bcm) to reach the correct obscuration. The results obtained corresponded to average diameters calculated according to the Mie theory. The criterion selected was d(50), meaning that 50% of the particles had diameters lower than this criterion (midpoint of cumulative volume distribution) (Gaiani et al. 2005; 2006).\n\nBy using this combination of three methods, it was possible to follow the water transfer during rehydration and obtain the wetting time, determined using the first peak of increased viscosity and turbidity, and the swelling time, determined using the second peak of viscosity in relation to the increase in particle size. The rehydration time was then determined according to stabilization of the viscosity, turbidity, and particle-size values.\n\nThe results in Table 10.3 show that rehydration of MCP occurs in different stages: First wetting and swelling of the particles take place, followed by slow dispersion to reach a homogeneous fluid, in agreement with Gaiani et al. (2005; 2006). Using an NMR method, Davenel et al. (1997) also demonstrated two stages during MC rehydration, attributed to water absorption by powder and solubilization of particles (i.e., swelling and dispersion stages). They estimated the water uptake by the powder at around 5 g water\/g powder during the first 20 min of rehydration but could not identify a wetting stage with this method.\n\nTable 10.3\n\nReconstitution Period, Insolubility Index, and Rehydration Time of Dairy Protein Powders\n\nPowders | RP using NMR (min) | ISI using IDF Standard (mL) | WT (min) | ST (min) | DT + SolT (min) | RT (min)  \n---|---|---|---|---|---|---  \nMCP G | 22 | 14.5 | 1 | 2 | 804 | 807  \nMCP NG | 8 | 3.5 | 3 | 17 | 551 | 571  \nMCP + Carbohydrate, G | 18 | 5.0 | 1 | nm | nm | 116  \nMCP + Carbohydrate, NG | nm | nm | 2 | 0 | 95 | 97  \nMCP + NaCl, G | 9.5 | 0.9 | nm | nm | nm | nm  \nMCP + CaCl2, G | \u221e | 14.5 | nm | nm | nm | nm  \nMCP + SCS \/ SPS, G | 6 \/5 | <0.5 | nm | nm | nm | nm  \nWPP, G | 5 | <0.5 | 4 | 0 | 0 | 4  \nWPP, NG | 15 | <0.5 | 17 | 0 | 0 | 17\n\nMCP: micellar casein powder; G: granulate; NG: nongranulate; SCS: sodium citrate solution; SPS: sodium phosphate solution; WPP: whey protein powder; RP: reconstitution period; ISI: insolubility index; WT: wetting time; ST: swelling time; DT: dispersability time; SolT: solubility time; RT: rehydration time = WT + ST + DT + SolT; \u221e: infinite delay; nm: not measured.\n\nMCPs with a high ISI (14.5 mL) are generally considered to be poorly soluble powders in which rehydration of the micelle remains incomplete (Jost, 1993). Addition of NaCl to the MC concentrate before spray drying considerably reduced the ISI and reconstitution period (RP) values (ISI 0.9 mL; RP 9.5 min) (Table 10.3). It has been hypothesized that the significant decrease in the RP value is probably related to the hygroscopic strength of NaCl.\n\nAddition of sodium citrate solution (SCS) or sodium phosphate solution (SPS) resulted in fast solubilization, as shown by the very low RP value and by the lower than 0.5 mL ISI value (Table 10.3). The resulting solution consisted of changing casein micelles in sodium caseinate form, associated with the occurrence of a single proton population, characterized by NMR relaxation rates that were much lower than the relaxation rate measured with reconstituted MC. This could be attributed to a decrease in the amount of hydration water induced by the change in micelle structure. The transparency of the solution indicated the formation of soluble caseins related to greater quantities of calcium complexes.\n\nReconstitution of MCP in the presence of CaCl2 led to considerable changes in protein structure, associated with instability of the casein micelles, which began to precipitate just after mixing, as shown by the high ISI and the nonmeasurable RP value due to experimental delay. This precipitate probably resulted from aggregation of casein micelles or submicelles through a decreasingly negative charge on the protein by additional calcium binding, leading to a reduction in electrostatic repulsion (Dalgleish, 1982). In this case, the high ISI of these solutions was related to the presence of insoluble substances, whereas in the case of rehydration of MCP the high ISI represented only the low water transfer rate in the casein (Table 10.3).\n\nOn the other hand, the rehydration of whey powders was totally different (Table 10.3). As the wettability of whey powders is poor, the turbidity instability at the beginning of the profile could be due to lump formation going past the sensor, as reported by Freudig et al. (1999). For nongranulated (NG) WPI powder, the very long signal instability could be explained by a tendency for the lumps to be stuck together in a thick layer of wet particles, due to the small size of the particles (Kinsella, 1984). Powder swelling has not been reported for WPI powders, probably because globular protein powders bind less water than intact casein micelle powders (Kinsella, 1984; Robin et al., 1993). De Moor and Huyghebaert (1983) also reported that whey powders have a lower water-holding capacity than casein powder.\n\nAs expected, granulation had a positive effect on wetting. The wetting time was systematically better for granulated particles. This phenomenon is well known, as fast wetting is enhanced, with large particles forming large pores, high porosity, and small contact angles between the powder surface and the penetrating water (Pisecky, 1986; Freudig et al., 1999; Gaiani et al., 2005). A surprising influence of granulation on the rehydration time was observed. Depending on the nature of the protein, the granulation influence involved opposite effects. WPI rehydration was enhanced for granulated particles, whereas the rehydration time was shorter for nongranulated particles of MCP. This was unexpected and could be explained by the controlling stage rate. The controlling stage for whey proteins is wetting (Baldwin & Sanderson, 1973; Schubert, 1993). As granulation improves the wetting stage, the rehydration of whey powders is enhanced for granulated particles. In contrast, the controlling stage for casein proteins is dispersion. Indeed, even with a shorter wetting time, a granulated powder is slower to rehydrate than a nongranulated powder (Gaiani et al., 2005).\n\nThe above results are not compatible with those of other studies, in which it was generally accepted that a single particle size around 200 \u03bcm (Neff & Morris, 1968) or 400 \u03bcm (Freudig et al., 1999) represented optimal dispersibility and sinkability. In fact, the optimal particle size depends on the composition of the dairy powder. As shown in Table 10.3, if the industry wishes to optimize powder rehydration, it seems to be better to rehydrate granulated powders if the protein is whey and to rehydrate nongranulated powders if the protein is casein.\n\n## Conclusions\n\nThe aim of this chapter was to explain the process of dehydration, that is, spray drying, in order to understand the effects of spray drying on the quality of protein powders (micellar caseins and globular proteins) during drying and rehydration. We then demonstrated that the quality of these powders depends on the biochemical environment.\n\nIt is very important for the dairy industry to understand that enrichment of milk in micellar casein (by ultrafiltration or microfiltration) decreases water transfer during the drying and rehydration processes. Insolubility (International Dairy Federation, 1988) is related to the decrease in water transfer required for rehydration and not to thermal denaturation, and decrease in water transfer is related to the micellar structure. The destabilization of the micellar structure induced by the addition of phosphate or citrate solution to MC increases water transfer during drying and during rehydration. Water transfer in WPC or MC containing added carbohydrates is improved during rehydration. Addition of NaCl to MC decreases water transfer during drying but increases water transfer during rehydration, and thus is related to the hygroscopicity of the carbohydrate and the NaCl.\n\nThe industrial requirement for protein powders with specific properties is expanding. As powder is the easiest way to carry and store milk derivatives, an understanding of the rehydration behavior of a dairy powder will become more and more important in the future.\n\nMoreover, it is essential for both dairy powder producers and dairy powder users to have a method to evaluate the rehydration behavior of dairy powders. As demonstrated in several studies, the industry should take into account certain technological factors such as granulation and the incorporation mode, and also the nature of the protein being rehydrated, to optimize the rehydration of a dairy powder. In contrast to other studies, we found that improving the wetting stage by using granulated powders did not systematically improve total rehydration. Depending on the nature of the protein, it seems to be better to work with granulated (for whey) or nongranulated (for micellar casein) powders to obtain more rapid rehydration (Gaiani et al., 2007).\n\nIn conclusion, water transfer in dairy protein concentrates during dehydration and during rehydration depends on the aqueous environment, the nature of the mineral salts, and the structure of the dairy proteins (MC or WPC). The water\u2013protein interaction requires further study, to understand the effects of preheat treatment and spray drying on the functional properties of protein powders.\n\n# References\n\nAmerican Dairy Products Institute .  _Standards for Grades of Dry Milk Including Methods of Analysis_ .  Chicago, Illinois :  American Dairy Products Institute ;  1990 .\n\nBaldwin AJ ,  Sanderson WB .  Factors affecting the reconstitution properties of whole milk powder .  _New Zealand Journal of Dairy Science and Technology_.  1973 ;8 : 92 \u2013 100 .\n\nBaldwin AJ ,  Baucke AG ,  Sanderson WB .  The effect of concentrate viscosity on the properties of spray dried skim milk powder .  _New Zealand Journal of Dairy Science and Technology_.  1980 ;15 : 289 \u2013 297 .\n\nBimbenet JJ ,  Schuck P ,  Roignant M ,  Brul\u00e9 G ,  M\u00e9jean S .  Heat balance of a multistage spray-dryer: Principles and example of application .  _Lait_.  2002 ;82 : 541 \u2013 551 .\n\nBloore CG ,  Boag IF .  The effect of processing variables on spray dried milk powder .  _New Zealand Journal of Dairy Science and Technology_.  1982 ;17 : 103 \u2013 120 .\n\nCaron A ,  St-Gelais D ,  Pouliot Y .  Coagulation of milk enriched with ultrafiltered or diafiltered microfiltered milk retentate powders .  _International Dairy Journal_.  1997 ;7 : 445 \u2013 451 .\n\nCayot P ,  Lorient D .  Modifications chimiques (biochimiques) de l'environnement des prot\u00e9ines du lait .  _Structures et Technofonctions des Prot\u00e9ines du Lait_ .  Paris :  Technique et Documentation Lavoisier ;  1998 : 159 \u2013 178 .\n\nCNIEL .  _Centre National Interprofessionnel de l'Economie Laiti\u00e8re_ .  Paris :  L'\u00e9conomie laiti\u00e8re en chiffres ;  1991 .\n\nCNIEL .  _Centre National Interprofessionnel de l'Economie Laiti\u00e8re_ .  Paris :  L'\u00e9conomie laiti\u00e8re en chiffres ;  2005 .\n\nCurme AG ,  Schmidt SJ ,  Steinberg MP .  Mobility and activity of water in casein model systems as determined by 2H NMR and sorption isotherms .  _Journal of Food Science_.  1990 ;55 : 430 \u2013 433 .\n\nDalgleish DG .  Milk proteins: chemistry and physics .  In:  Fox PF ,  Condon JJ ,  eds.  _Food Proteins_ .  London :  Applied Science Publishers ;  1982 : 155 \u2013 178 .\n\nDavenel A ,  Schuck P ,  Marchal P .  A NMR relaxometry method for determining the reconstitutability and the water-holding capacity of protein-rich milk powders .  _Milchwissenschaft_.  1997 ;52 : 35 \u2013 39 .\n\nDe Moor H ,  Huyghebaert A .  Functional properties of dehydrated protein-rich milk products .  _Physico-chemical Aspects of Dehydrated Protein-rich Milk Products_ .  Helsingor, Denmark :  Proceedings of IDF Symposium ;  1983 : 276 \u2013 301 : Statens Forsogsmejeri, Hillerod .\n\nDe Vilder J .  La fabrication de poudre de lait \u00e9cr\u00e9m\u00e9 instantan\u00e9e .  _I. Les caract\u00e9ristiques physiques et chimiques. Revue Agricole_.  1986 ;39 : 865 \u2013 877 .\n\nDe Vilder J ,  Martens R ,  Naudts M .  The influence of the dry matter content, the homogenization and the heating of concentrate on physical characteristics of whole milk powder .  _Milchwissenschaft_.  1979 ;34 : 78 \u2013 84 .\n\nFauquant J ,  Maubois JL ,  Pierre A .  Microfiltration du lait sur membrane min\u00e9rale .  _Techniques Laiti\u00e8res_.  1988 ;1028 : 21 \u2013 23 .\n\nFreudig B ,  Hogekamp S ,  Schubert H .  Dispersion of powders in liquid in a stirred vessel .  _Chemical Engineering and Processing_.  1999 ;38 : 525 \u2013 532 .\n\nGaiani C ,  Banon S ,  Scher J ,  Schuck P ,  Hardy J .  Use of a turbidity sensor to characterize casein powders rehydration: Influence of some technological effects .  _Journal of Dairy Science_.  2005 ;88 : 2700 \u2013 2706 .\n\nGaiani C ,  Scher J ,  Schuck P ,  Hardy J ,  Desobry S ,  Banon S .  The dissolution behaviour of native phosphocaseinate as a function of concentration and temperature using a rheological approach .  _International Dairy Journal_.  2006 ;16 : 1427 \u2013 1434 .\n\nGaiani C ,  Schuck P ,  Scher J ,  Hardy J ,  Desobry S ,  Banon S .  Dairy powder rehydration: influence of proteins and some technological effects .  _Journal of Dairy Science_.  2007 ;90 : 570 \u2013 581 .\n\nGoud\u00e9dranche H ,  Maubois JL ,  Ducruet P ,  Mahaut M .  Utilization of the new mineral UF membrane for making semi-hard cheeses .  _Desalination_.  1980 ;35 : 243 \u2013 258 .\n\nHall CW ,  Hedrick TI .  Quality control and sanitation .  In:  Hall CW ,  Hedrick TI ,  eds.  _Drying Milk and Milk Products_ .  Westport, CT :  AVI Publishing Co ;  1966 : 197 \u2013 231 .\n\nHardy J ,  Scher J ,  Banon S .  Water activity and hydration of dairy powders .  _Lait_.  2002 ;82 : 441 \u2013 452 .\n\nIlari JL ,  Loisel C .  La ma\u00eetrise de la fonctionnalit\u00e9 des poudres .  _Process_.  1991 ;1063 : 39 \u2013 43 .\n\nInternational Dairy Federation .  _Dried milk and milk products\u2014determination of insolubility index_ .  Brussels :  International Dairy Federation ;  1988 .\n\nInternational Dairy Federation .  _World Dairy Situation_ .  Brussels :  CD-Rom. International Dairy Federation ;  2012 .\n\nJeantet R ,  Schuck P ,  Famelart MH ,  Maubois JL .  Int\u00e9r\u00eat de la nanofiltration dans la production de poudres de lactos\u00e9rum d\u00e9min\u00e9ralis\u00e9es .  _Lait_.  1996 ;76 : 283 \u2013 301 .\n\nJeurnink TJM ,  de Kruif KG .  Calcium concentration in milk in relation to heat stability and fouling .  _Netherlands Milk and Dairy Journal_.  1995 ;49 : 151 \u2013 165 .\n\nJost R .  Functional characteristics of dairy proteins .  _Trends in Food Science and Technology_.  1993 ;4 : 283 \u2013 288 .\n\nKessler H-G .  Drying\u2014instantizing .  _Food Engineering and Dairy Technology_ .  Freising :  Verlag A Kessler ;  1981 : 269 \u2013 328 .\n\nKing N .  Dispersibility and reconstitutability of dried milk .  _Dairy Science Abstracts_.  1966 ;28 (3) : 105 \u2013 118 .\n\nKinsella JE .  Milk proteins: physicochemical and functional properties .  _Critical Reviews in Food Science and Nutrition_.  1984 ;21 : 197 \u2013 262 .\n\nKjaergaard JG ,  Ipsen RH ,  Ilsoe C .  Functionality and application of dairy ingredients in dairy products .  _Food Technology_.  1987 ;41 : 66 \u2013 71 .\n\nLe Gra\u00ebt Y ,  Maubois JL .  Fabrication de fromages \u00e0 p\u00e2te fra\u00eeche \u00e0 partir de poudres de r\u00e9tentat et de pr\u00e9fromage .  _Revue Laiti\u00e8re Fran\u00e7aise_.  1979 ;373 : 23 \u2013 26 .\n\nLe Ray C ,  Maubois JL ,  Gaucheron F ,  Brul\u00e9 G ,  Pronnier P ,  Garnier F .  Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition .  _Lait_.  1998 ;78 : 375 \u2013 390 .\n\nMadsen RF ,  Bjerre P .  Production of cheese-base .  _Nordeuropaeisk Mejeri Tidsskrift_.  1981 ;5 : 135 \u2013 139 .\n\nMahaut M ,  Jeantet R ,  Brul\u00e9 G ,  Schuck P .  _Les Produits Industriels Laitiers_ .  Paris :  Technique et Documentation Lavoisier ;  2000 .\n\nMasters K .  _Spray Drying_ .  Essex :  Longman Scientific & Technical ;  1991 .\n\nMaubois JL .  New applications of membrane technology in the dairy industry .  _Australian Journal of Dairy Technology_.  1991 ;46 : 91 \u2013 95 .\n\nMaubois JL ,  Pierre A ,  Fauquant J ,  Piot M .  Industrial fractionation of main whey proteins .  _Bulletin of the International Dairy Federation_.  1987 ;212 : 154 \u2013 159 .\n\nNeff E ,  Morris HA .  Agglomeration of milk powder and its influence on reconstitution properties .  _Journal of Dairy Science_.  1968 ;51 : 330 \u2013 338 .\n\nPierre A ,  Fauquant J ,  Le Gra\u00ebt Y ,  Piot M ,  Maubois JL .  Pr\u00e9paration de phosphocas\u00e9inate natif par microfiltration sur membrane .  _Lait_.  1992 ;72 : 461 \u2013 474 .\n\nPiot M ,  Vachot JC ,  Veaux M ,  Maubois JL ,  Brinkman GE .  Ecr\u00e9mage et \u00e9puration bact\u00e9rienne du lait entier cru par microfiltration sur membrane en flux tangentiel .  _Technique Laiti\u00e8re and Marketing_.  1987 ;1016 : 42 \u2013 46 .\n\nPisecky J .  Bulk density of milk powders .  _Australian Journal of Dairy Technology_.  1980 ;35 : 106 \u2013 111 .\n\nPisecky J .  Technology of skimmed milk drying .  _Journal of the Society of Dairy Technology_.  1981 ;34 : 57 \u2013 62 .\n\nPisecky J .  Standards, specifications and test methods for dry milk products .  In:  MacCarthy D ,  ed.  _Concentration and Drying of Food_ .  London :  Elsevier ;  1986 : 203 \u2013 220 .\n\nPisecky J .  20 years of instant whole milk powder .  _Scandinavian Dairy Information_.  1990 ;4 : 74 .\n\nPisecky J .  _Handbook of Milk Powder Manufacture_ .  Copenhagen :  Niro A\/S ;  1997 .\n\nRobin O ,  Turgeon S ,  Paquin P .  Functional proteins of milk proteins .  _Dairy Science and Technology Handbook. 1. Principles and Properties_ .  New York :  VCH Publishers ;  1993 : 277 \u2013 353 .\n\nRoos YH .  Importance of glass transition and water activity to spray drying and stability of dairy powders .  _Lait_.  2002 ;82 : 478 \u2013 484 .\n\nSchubert H .  Instantization of powdered food products .  _International Chemical Engineering_.  1993 ;33 (1) : 28 \u2013 45 .\n\nSchuck P ,  Piot M ,  M\u00e9jean S ,  Fauquant J ,  Brul\u00e9 G ,  Maubois JL .  D\u00e9shydratation des laits enrichis en cas\u00e9ine micellaire par microfiltration; comparaison des propri\u00e9t\u00e9s des poudres obtenues avec celles d'une poudre de lait ultra-propre .  _Lait_.  1994 ;74 : 47 \u2013 63 .\n\nSchuck P ,  Piot M ,  M\u00e9jean S ,  Le Gra\u00ebt Y ,  Fauquant J ,  Brul\u00e9 G ,  Maubois JL .  D\u00e9shydratation par atomisation de phosphocas\u00e9inate natif obtenu par microfiltration sur membrane .  _Lait_.  1994 ;74 : 375 \u2013 388 .\n\nSchuck P ,  Roignant M ,  Brul\u00e9 G ,  M\u00e9jean S ,  Bimbenet JJ .  Caract\u00e9risation \u00e9nerg\u00e9tique d'une tour de s\u00e9chage par atomisation multiple effet .  _Industries Alimentaires et Agricoles_.  1998 ;115 : 9 \u2013 14 .\n\nSchuck P ,  Roignant M ,  Brul\u00e9 G ,  Davenel A ,  Famelart MH ,  Maubois JL .  Simulation of water transfer in spray drying .  _Drying Technology_.  1998 ;16 : 1371 \u2013 1393 .\n\nSchuck P ,  Briard V ,  M\u00e9jean S ,  Piot M ,  Famelart MH ,  Maubois JL .  Dehydration by desorption and by spray drying of dairy proteins: influence of the mineral environment .  _Drying Technology_.  1999 ;17 : 1347 \u2013 1357 .\n\nSchuck P ,  M\u00e9jean S ,  Dolivet A ,  Jeantet R .  Thermohygrometric sensor: a tool for optimizing the spray drying process .  _Innov. Food Sci. and Emerg. Technol_.  2005 ;6 : 45 \u2013 50 .\n\nSchuck P ,  Dolivet A ,  M\u00e9jean S ,  Zhu P ,  Blanchard E ,  Jeantet R .  Drying by desorption: A tool to determine spray drying parameters .  _Journal of Food Engineering_.  2009 ;94 : 199 \u2013 204 .\n\nSchuck P ,  Dolivet A ,  Jeantet R .  _Analytical Methods for Food and Dairy Powders_ .  Oxford :  John Wiley & Sons ;  2012 .\n\nSougnez M .  L'\u00e9volution du s\u00e9chage par atomisation .  _Chimie Magazine_.  1983 ;1 : 1 \u2013 4 .\n\nStraatsma J ,  Vanhouwelingen G ,  Steenbergen AE ,  Dejong P .  Spray drying of food products. 1. Simulation model .  _Journal of Food Engineering_.  1999 ;42 : 67 \u2013 72 .\n\nStraatsma J ,  Vanhouwelingen G ,  Steenbergen AE ,  Dejong P .  Spray drying of food products. 2. Prediction of insolubility index .  _Journal of Food Engineering_.  1999 ;42 : 73 \u2013 77 .\n\nTarodo de la Fuente B .  Solvation of casein in bovine milk .  _Journal of Dairy Science_.  1985 ;58 : 293 \u2013 300 .\n\nTrouv\u00e9 E ,  Maubois JL ,  Piot M ,  Madec MN ,  Fauquant J ,  Rouault A ,  Tabard J ,  Brinkman G .  R\u00e9tention de diff\u00e9rentes esp\u00e8ces microbiennes lors de l'\u00e9puration du lait par microfiltration en flux tangentiel .  _Lait_.  1991 ;71 : 1 \u2013 13 .\n\nTuohy JJ .  Some physical properties of milk powders .  _Irish Journal of Food Science and Technology_.  1989 ;13 : 141 \u2013 152 .\n\nVan Hooydonk ACM ,  Hagedoorn HG ,  Boerrigter IJ .  The effect of various cations on the renneting of milk .  _Netherlands Milk and Dairy Journal_.  1986 ;40 : 369 \u2013 390 .\n\nVincens D ,  Tabard J .  L'\u00e9limination des germes bact\u00e9riens sur membranes de microfiltration .  _Techniques Laiti\u00e8res_.  1988 ;1033 : 62 \u2013 64 .\n\nZhu P ,  Patel K ,  Lin S ,  M\u00e9jean S ,  Blanchard E ,  Chen XD ,  Schuck P ,  Jeantet R .  Simulating industrial spray drying operations using a reaction engineering approach and a modified desorption method .  _Drying Technology_.  2011 ;29 : 419 \u2013 428 .\n\nZhu P ,  M\u00e9jean S ,  Blanchard B ,  Jeantet R ,  Schuck P .  Prediction of dry mass glass transition temperature and the spray drying behaviour of a concentrate using a desorption method .  _Journal of Food Engineering_.  2011 ;105 : 460 \u2013 467 . \nChapter 11\n\n# Changes in Milk Proteins during Storage of Dry Powders\n\nKerianne Higgs*\n\nMike J. Boland**\n\n* Fonterra Research and Development Centre, Palmerston North, New Zealand  \n** Riddet Institute, Palmerston North, New Zealand\n\n## Abstract\n\nMilk proteins undergo chemical changes, even in dried powders. This chapter reviews the changes undergone by caseins and whey proteins in milk powders and in purified protein products. Maillard compounds are of particular importance in milk powders, milk protein concentrates, and whey protein concentrate products, where lactose is present. Caseins undergo isopeptide bond formation, as the result of dephosphorylation of phosphoserine residues and subsequent reactions of the dehydroalanine produced.\n\nWe discuss the nutritional significance of these changes. Both the Maillard reaction and the formation of isopeptides lead to loss of bioavailable lysine. This is not a problem in dairy proteins, which contain an excess of lysine, but it can affect the nutritional value of protein blends which may be limiting in lysine.\n\n## Keywords\n\nchanges in milk proteins\n\nstorage of dry powders\n\ndephosphorylation\n\nphosphoserine residues\n\ndehydroalanine\n\nOutline\n\nIntroduction 343\n\nThe formation of Maillard and pre-Maillard compounds 345\n\nMeasuring Lactulosyl Lysine Levels 346\n\nRates of Formation of Lactulosyl Lysine 346\n\nFormation of isopeptide bonds 349\n\nRates of Formation of Lysinoalanine 350\n\nAmino acids other than lysine 351\n\nImplications for nutritional value of milk proteins 352\n\nLysine 352\n\nSulfur Amino Acids 354\n\nOther Amino Acids 355\n\nProduct-specific storage trials 355\n\nConclusions 356\n\n## Introduction\n\nMilk is an unstable foodstuff, prone in particular to microbiological degradation, but also to long-term chemical change. A large part of the world's dairy production occurs in areas remote from the markets in which it is consumed, and production is often seasonal, requiring storage to smooth out supply. The production of milk powders and other dried milk protein-containing products has been the method of choice for over 100 years for the storage and shipping of milk over long distances and\/or times, as it confers stability and massively reduces weight and bulk.\n\nMilk powders were known to the Chinese and were described by Marco Polo. The production of milk powders was described by Nicolas Appert in the early nineteenth century, and commercial processes for the spray drying of milk were patented in the United States in 1872 and 1905. This opened the way for large-scale industrial production of milk powders throughout the twentieth century. Milk powder production is covered in Chapter 10.\n\nTable 11.1 summarizes the biggest exporters and importers of milk powders in 2011. In the same period, somewhere between 8 and 9 million tons of milk powder was produced globally (IDF, 2012).\n\nTable 11.1\n\nTop Six Exporters and Importers of Milk Powders in 2011 (000 tons)\n\nExport | New Zealand | European Union | United States | Australia | Argentina | Belarus  \n---|---|---|---|---|---|---  \nSMP | 350 | 518 | 436 | 166 | 18 | 55  \nWMP | 1081 | 390 | 22 | 143 | 199 | 27  \nTotal | 1431 | 908 | 458 | 309 | 217 | 82  \nImport | China | Algeria | Mexico | Indonesia | Singapore | Philippines  \n---|---|---|---|---|---|---  \nSMP | 130 | 125 | 191 | 128 | 61 | 111  \nWMP | 320 | 203 | 31 | 68 | 81 | 28  \nTotal | 450 | 328 | 222 | 195 | 142 | 139\n\nSMP, skim milk powder; WMP, whole milk powder\n\nSource: World Dairy Situation, International Dairy Federation, Brussels, 2012.\n\nIn addition to milk powders, dried dairy protein products that are traded on the world market, largely as food ingredients, include casein and caseinate, whey powders, whey protein concentrates, whey protein isolates, milk protein concentrates, milk protein isolates, and specialist nutritional powders and blends, which may also contain hydrolyzed dairy proteins.\n\nMilk powders are used primarily for making reconstituted and recombined milks and are usually sold to consumers in UHT format, although substantial amounts are used to make other dairy products such as yogurt and ice cream as well as being minor ingredients in a wide range of nondairy foods.\n\nThe main uses for dried milk protein products are nutritional, and products include infant formulas, medical foods, specialist foods for weight management, and foods for muscle building (where high protein and high levels of branched-chain amino acids are desirable). They are also used in non-nutritional applications, including desserts, confectionery, toppings, imitation cheeses, sauces and dressings, and coffee whiteners, where their functional properties are important.\n\nThese products, especially those from New Zealand and Australia, often have long storage times because of geographic distance to market and seasonal production. Research from our own and other laboratories has shown that a range of reactions can occur in the dry powders and that these are known to affect powder functionality and nutritional value. The quality of dried milk protein products deteriorates upon storage at ambient temperatures mainly because of two reactions: the Maillard reaction and isopeptide bond formation. There are also some other minor reactions, which are covered later in this chapter.\n\nThe standard abbreviations used throughout the world for many of the dried milk products are as follows: SMP = skim milk powder; WMP = whole milk powder; WPC = whey protein concentrate; WPI = whey protein isolate (usually >85% protein); MPC = milk protein concentrate; and MPI = milk protein isolate. Milk powders sold internationally must conform to standard protein levels and fat levels for WMP. A number following the abbreviation for the other products is the percentage of protein by weight in the dry powder. Caseins and caseinates are not usually abbreviated and are typically >90% protein by dry weight.\n\nA range of chemical reactions that modify proteins can occur in dried milk products, particularly at elevated temperatures. Both of the most important of these reactions involve lysyl side chains: they are the formation of Maillard and pre-Maillard compounds, in the presence of sugars, and the formation of isopeptide bonds, particularly in products containing phosphoseryl residues, such as casein.\n\n## The formation of maillard and pre-maillard compounds\n\nThe Maillard reaction occurs when lysine-containing proteins interact with reducing sugars. The first stable compound formed during the Maillard reaction is the Amadori product (so-called because it is the result of a class of reaction called the Amadori rearrangement), shown in Figure 11.1. These compounds block the \u025b-amino groups of lysine residues, reducing the bioavailability of that essential amino acid. This reaction is dependent on a reducing sugar, usually lactose, being present as the co-reactant (Erbersdobler, 1986).\n\nFigure 11.1 Formation of lactulosyl lysine.\n\nPure protein products such as casein and caseinate do not suffer significantly from this reaction, as not enough lactose is present. The reaction is particularly important in WMPs, SMPs, WPCs, and MPCs, and can occur to a limited extent in some MPIs and WPIs. The rate of reaction is critically dependent on the level of moisture (water activity) and the temperature as well as the lactose content.\n\nAdvanced Maillard reaction products are partly responsible for the development of aromas and color during food processing and preparation.\n\n### Measuring Lactulosyl Lysine Levels\n\nLactulosyl lysine formation is conveniently monitored by measuring furosine concentrations (Erbersdobler, 1987) and can be indirectly measured by monitoring the amount of available amine (Hern\u00e1ndez et al, 1991). Lactulosyl lysine in WPCs can be measured directly by mass spectrometry where the addition of one lactose molecule increases the molecular weight of the protein by 324 Da. Figure 11.2 shows the mass spectrum of a mixture of the native A and B variants of \u03b2-lactoglobulin and their mono-, di-, and trilactosylated derivatives. The results from all three methods have been shown to correlate well (Fig. 11.3).\n\nFigure 11.2 Mass spectrum of native and lactosylated \u03b2-lactoglobulin.\n\nFigure 11.3 (a) Correlation between available amine and furosine values for stored samples of a WPC56. (b) Average number of lactose molecules bound, determined by mass spectrometry, against furosine for a WPC56. K. Higgs, unpublished data.\n\n### Rates of Formation of Lactulosyl Lysine\n\nWe studied the rate of lactosylation for WPC products as dry powders. The rates were found to be dependent on the lactose concentration (a consequence of processing to reach desired protein levels), water activity (aW), and temperature (T). The trials lasted only a few months, and extrapolation beyond that timeframe cannot be done with confidence.\n\nDetailed kinetics developed using WPC80 for a range of aW and T values allowed the rates of lactosylation to be predicted for periods of up to 4 months.\n\nFor the kinetic evaluation, Figure 11.1 can be simplified to\n\nreactants \u27f6 k 1 lactulosyl lysine \u27f6 k 2 advanced Maillard products\n\nwhere k 1 and k 2 are the rate constants for the formation and degradation of lactulosyl lysine.\n\nThe rate equation for the formation of lactulosyl lysine is\n\nd L d t = k 1 [ reactants ] n 1 \u2212 k 2 [ L ] n 2\n\n (11.1)\n\nwhere L represents lactulosyl lysine and n 1 and n 2 are the rate orders for the formation and degradation of lactulosyl lysine respectively.\n\nBecause the formation of lactulosyl lysine in dairy powders uses only a fraction of the available reactants, the first reaction can be considered to be zero order, and the degradation of lactulosyl lysine is a first-order process. This simplifies the equation to\n\nd L d t = k 1 \u2212 k 2 [ L ]\n\n (11.2)\n\nRearrangement of this equation and solving for [L]t gives\n\n[ L ] t = k 1 k 2 \u2212 k 1 k 2 \u2212 [ L ] 0 ) exp ( \u2212 k 2 t )\n\n (11.3)\n\nFurosine is a hydrolysis product of lactulosyl lysine and gives a direct measure of its concentration (Fig. 11.3). Therefore, furosine can be substituted for lactulosyl lysine in the rate equations.\n\nThe samples used were individual samples that were removed from controlled storage at seven time points (2, 6, 12, 24, 40, 78, and 116 days). They were analyzed for furosine, and the values were plotted against time. The rates were determined using nonlinear regression with Sigma Plot 8.0. At 40 \u00b0C, the samples at time points after 40 days for water activities of 0.54 and 0.80 showed advanced Maillard browning and were not included in the regression analysis. R2 values for the regressions were between 0.95 and 0.99.\n\nThe lactosylation rate constants were greater at higher temperatures and at higher water activities. The rates were low at low water activity (aW = 0.33), with small increases with temperature; however, at higher water activities, the rates increased substantially with increasing temperature (Fig. 11.4).\n\nFigure 11.4 Rate constants for the formation of furosine in a WPC80.\n\nIt was observed for a number of WPC80 specifications that lactosylation appeared to stop when only 2.5 (average) or 3 lactose molecules had been bound per \u03b2-lactoglobulin molecule. This corresponded to about 20% of the total lysine being blocked. This condition was specific to the 80% protein products, which contained about 12% lactose. WPC56 products showed a much greater degree of lactosylation. However, much higher levels of lactosylation have reportedly been seen in overseas laboratories in WPC80 samples stored for long periods (W. J. Harper, Ohio State University, 2003, personal communication).\n\nSimilar kinetic work has been reported for SMPs stored at a range of temperatures (37, 50, and 60 \u00b0C), with a series of initial water activities (0.33, 0.43, 0.52, 0.69, 0.85, and 0.98) (Pereyra Gonzales et al., 2010). As with WPCs, there were significant increases in Maillard reaction rates with temperature, but they did not see an increase related to the powder's initial water activity.\n\n## Formation of isopeptide bonds\n\nIsopeptide bonds are formed largely by the breakdown of the phosphoseryl side chains that are present in products containing casein during processing or storage to form dehydroalanyl side chains (Fig.11.5) (Friedman, 1999). The side chains are reactive and will form cross-links, mainly with adjacent lysyl (but also with histidinyl or cysteinyl) side chains to form lysinoalanyl, histidinoalanyl, or lanthionyl isopeptides, respectively. The reaction during milk powder processing is considered to be an alkali-catalyzed reaction that is accelerated by heat treatment. This reaction is not known to be significant in whey products, which do not contain significant amounts of phosphoseryl residue.\n\nFigure 11.5 Formation of lysinoalanine.\n\nThe main isopeptide product in both acid and gastrointestinal digestion gives lysinoalanine, which renders the lysine nonbioavailable. Additional minor reactions form histidinoalanine and lanthionine on digestion. Although lanthionine is only a minor component, it is important because it renders cysteine partially nonbioavailable, and the sulfur amino acids are often nutritionally limiting in milk proteins. (Note: Lanthionine formation blocks the bioavailability of cysteine, which is not normally considered to be an essential amino acid, because it can be synthesized from methionine; however, methionine is itself a nutritionally limiting amino acid in casein.) Studies have indicated that, although lanthionine and histidinoalanine linkages are formed under the alkaline conditions encountered during processing, it is only lysinoalanine that is formed in the neutral conditions encountered in powders.\n\nLysinoalanine formation in \u03b2-casein has been found to be enhanced by pressure treatment under alkaline conditions (Schwarzenbolz & Henle, 2010). Still to be investigated is whether pressure treatment also affects the rate at which lysinoalanine is formed during subsequent storage.\n\nLysinoalanine is usually measured directly in protein hydrolysates as part of an extended amino acid analysis (Henle et al., 1991). In casein products, measurement of available amine is a useful and simpler, though less specific, alternative.\n\n### Rates of Formation of Lysinoalanine\n\nThe rates of formation of lysinoalanine in caseinates and proteinates (TMPates) have been investigated by W. Thresher (1996; 1997; personal communications) for a range of temperatures and water activities. The rate constants for lysinoalanine formation as a function of temperature are shown in Figure 11.6.\n\nFigure 11.6 Rate constants for lysinoalanine formation in caseinates stored at various temperatures. W. Thresher, 1996, personal communication.\n\nThe rate of lysinoalanine formation increased with increasing temperature and water activity for both caseinates and TMPates. Calcium TMPates and caseinates were found to have lower rates of lysinoalanine formation than potassium or sodium TMPates and caseinates.\n\n## Amino acids other than lysine\n\nCysteine, methionine, and tryptophan are other essential amino acids that can be rendered nonbioavailable by reacting during processing and\/or storage. Cysteine undergoes \u03b2-elimination to give dehydroalanine when treated with alkali. This can then react with a lysine residue to give lysinoalanine. Cysteine can also react with dehydroalanine to give lanthionine (Friedman, 1999). Chemically determined values for cysteine and lysine availability have been found to correlate well with rat protein efficiency ratios for heat- and alkali-treated caseinates (W. Thresher, 1996, personal communication).\n\nTryptophan residues are relatively stable during processing and storage. They are not easily oxidized and have been found to be relatively resistant to oxidizing lipids, alkali, quinines, and reducing sugars (Nielsen et al., 1985). Any losses are small and not significant when compared with the losses of other amino acids such as methionine and lysine. However, we have seen small amounts of oxidized tryptophan residues in digests of skim milk purchased from the supermarket (Fig. 11.7).\n\nFigure 11.7 Time-of-flight mass spectrometry of the M2+ ion from the tryptophan-containing \u03ba-casein peptide SPAQILQWQVLSNTVPAK. The chemical structures in the figure show the various levels of oxidation found.\n\nMethionine is relatively easily oxidized to the sulfoxide, but methionine in the sulfoxide form is still bioavailable (Nielsen et al., 1985).\n\nTable 11.2 indicates levels of key essential amino acids following either alkali treatment of casein or extensive lactosylation of WPC. Note that the losses of amino acids other than lysine are considerably lower than the losses of lysine. The conditions used for the casein are well beyond any normal exposure during processing or storage.\n\nTable 11.2\n\nAmino Acid Concentrations of a Control and an Alkali-treated Casein, and a Control and a Lactosylated WPC56\n\nAmino Acid | Concentration (mg\/g crude protein) | Concentration (mg\/g sample)  \n---|---|---  \n| Control casein | Alkali-treated casein a | Control WPC | Lactosylated WPC b  \nTryptophan | 13.4 | 12.1 (90%) | 8.4 | 8.1 (96%)  \nLysine | 91.0 | 60.8 (67%) | 50.3 | 44.5 (88%)  \nMethionine | 31.4 | 25.3 (80%) | 12.3 | 12.5 (102%)  \nCysteine | 4.4 | 3.3 (75%) | 14.9 | 14.8 (99%)\n\na Casein was heated for 4 h at 80 \u00b0C in 0.15 M NaOH (Nielsen et al., 1985).\n\nb WPC was heated at 40 \u00b0C, aW 0.75, for 100 h. The median number of lactosyl groups on \u03b2-lactoglobulin was 5 (K Higgs, unpublished results).\n\nSource: Nielsen et al., 1985; K Higgs, unpublished results.\n\n## Implications for nutritional value of milk proteins\n\nMilk protein is rich in essential amino acids, with many, including lysine, well exceeding recommended requirements (Table 11.3). Use of these proteins as the predominant nutritional source thus poses few problems if some of the lysine is nonbioavailable.\n\nTable 11.3\n\nEssential Amino Acid Content of Milk and Other Proteins\n\nEssential amino acid (AA) | Recommended requirementa (mg\/g protein) | Caseinate | TMP, MPI | WPC | Soy | Wheat  \n---|---|---|---|---|---|---  \nIsoleucine | 28 | 46 | 44 | 54 | 47 | 33  \nLeucine | 66 | 91 | 103 | 119 | 85 | 68  \nLysine | 58 | 77 | 81 | 94 | 63 | 27  \nSulfur AAb | 25 | 33 | 39 | 52 | 24 | 39  \nAromatic AA | 63 | 106 | 102 | 68 | 97 | 78  \nThreonine | 34 | 43 | 45 | 66 | 8 | 29  \nTryptophan | 11 | 12 | 14 | 20 | 11 | 11  \nValine | 35 | 57 | 57 | 51 | 49 | 43  \nHistidine | 19 | 29 | 27 | 21 | 29 | \u2013\n\na For 2- to 5-year-olds.\n\nb Includes cysteine, cystine, and methionine.\n\n### Lysine\n\nThe main concern during the storage of most nutritional proteins is the loss of lysine as a result of Maillard reactions or isopeptide bond formation.\n\nLactulosyl lysine renders lysine nonbioavailable. The protein efficiency ratio (PER) was found to be decreased in a WPC56, with an average of five lactulosyl lysine residues per protein molecule (Fig. 11.8). A more detailed study using skim milk diets with pigs confirmed that lactulosyl lysine was nonbioavailable (Rerat et al., 2002). That study also found a decrease in the digestibility of lysine, phenylalanine, valine, cystine, aspartic acid, glycine, and methionine residues. This decrease suggests that lactulosyl lysine residues hinder the release and therefore the utilization of adjacent amino acids.\n\nFigure 11.8 Plot of lactosylation level against PER. The PER value for ANRC casein was used for zero lactosylation. PER, protein efficiency ratio.\n\nLimited human studies have been conducted on Maillard reaction products that include lactulosyl lysine. One study looked at adolescent males on diets containing different levels of Maillard reaction products, which principally showed that the Maillard reaction reduces protein digestibility (Gilani et al., 2012).\n\nStudies on infant formula have shown that products with an increased amount of Maillard reaction products had decreased PER values. Those formulas with lower PER values also resulted in the rats having lower levels of plasma lysine.\n\nLysinoalanine is not a bioavailable source of lysine (Robbins et al., 1980; Friedman, 1999; Gilani et al. 2012). Alkali treatment of casein with 0.2 N NaOH at 80 \u00b0C for 1 h reduced the PER of casein from 3.09 to 0.02 for a diet containing 10% casein (Possompes et al., 1989; cited in Friedman, 1999).\n\nMilk proteins are unusually rich in lysine (Table 11.3), and can stand to lose a significant proportion of the lysine before it becomes limiting (around 50% in the case of whey proteins and 25% in the case of casein). The real concern arises when milk proteins, and particularly whey proteins, are being added to a mixture to provide a source of lysine supplementation. When this is the case, it is particularly important to ensure that the lysine content has not been compromised after long storage. Steps to ensure the integrity of the lysine content include keeping the product at temperatures less than 25 \u00b0C for most of the time and ensuring that the water activity in the product is kept low, preferably at or below 0.3. Experiments carried out in our laboratories to determine bioavailable lysine showed that loss of lysine upon storage was negligible at 30 \u00b0C, whereas losses were severe at 40 \u00b0C (Fig. 11.9).\n\nFigure 11.9 Loss of bioavailable lysine with time for SMP, WMP, and ALACEN 421 with a water activity (aW) of 0.3 during storage at (a) 30 \u00b0C and (b) 40 \u00b0C. The solid and dashed lines on the graphs show the levels at which lysine becomes limiting in WPC, SMP, and WMP, respectively. K. Higgs, unpublished results.\n\n### Sulfur Amino Acids\n\nModification of sulfur amino acids is possible through loss of cysteine via lanthionine formation and through oxidation of methionine to methionine sulfoxide. Whey proteins are relatively rich in sulfur amino acids, and casein has more than adequate quantities. It should be noted that these scores are relative to World Health Organization (WHO) requirements for children aged 2\u20135 years. The requirement for rat diets is higher, probably because of the rat's requirement for hair production (hair is rich in sulfur amino acids).\n\nLanthionine is a problem only for caseinates, and lanthionine formation is known to occur under the conditions used in the caseinating process (Aymard et al., 1978). The rates of lanthionine formation in dry powder have not been studied.\n\nOxidation of methionine to the sulfoxide does not alter its bioavailability per se. It has been claimed, however, that the presence of the sulfoxide side chain in intact proteins may hamper digestion of the protein, thus affecting its overall bioavailability (Anon., 1973).\n\n### Other Amino Acids\n\nOne essential amino acid known to be destroyed during some processes is tryptophan. Extensive investigations by Nielsen et al. (1985) on whey proteins, casein, and WMP showed that tryptophan remained relatively intact even when substantial lysine modification had occurred. Tryptophan analysis gave a result of 100% (within experimental error) and a bioavailability of >90% in milk powder that had been stored at 60 \u00b0C for 5 weeks, which resulted in a loss of 80% of available amine. This powder was considered to show 'advanced' Maillard browning.\n\n## Product-specific storage trials\n\nSamples of WPC80 powders that had earlier been shipped from New Zealand to the United States or Europe were obtained and analyzed for lactosylation levels. The powders were at the time less than 2 years old and thus were considered to be current stock. The powders had average bound lactose levels of 0.7 to 1.2. This correlates to between 87% and 95% of the lysine in the products being bioavailable (estimated by extrapolation from the remaining lysine in \u03b2-lactoglobulin). This compares well with freshly produced WPC80 powders, which had an average number of lactose bound of 0.6, or 96% of available lysine remaining (Fig. 11.10).\n\nFigure 11.10 Levels of lactosylation in market samples of a WPC80 compared with levels in freshly produced WPC80 in a subsequent season.\n\nWe carried out a two-year storage study on >80% protein powders to determine changes, if any, in nutritional properties, but these powders were kept at constant temperature and were not exposed to any of the temporal variations possible during shipping and storage in overseas warehouses.\n\nA 5% decrease in available amine was seen in MPC85 when stored at 20 \u00b0C for 2 years; this increased to 10% when the storage temperature was increased to 30 \u00b0C. A single MPI stored for 2 years gave consistent available amine results over the storage period.\n\nThe caseinates and caseins in the 2-year study showed no definitive trend in available amine values. Most values remained consistent over the 2 years. This was expected as a previous 3-month study showed that storage temperatures in excess of 30 \u00b0C were required for significant levels of lysinoalanine formation.\n\n## Conclusions\n\nMilk proteins do undergo change when dry powders are stored. Powders containing appreciable amounts of lactose (milk powders and WPCs) form pre-Maillard reaction products, rendering lysine nonbioavailable, whereas those containing casein undergo formation of isopeptide bonds, reducing the availability of lysine and sulfur amino acids. Because milk proteins are rich in lysine, loss of some lysine will not be a significant problem. However, when milk proteins are used as 'balancers' in formulations with other proteins that are poor in lysine, attention should be given to the storage history of the protein.\n\nBoth the Maillard reaction and the formation of isopeptide bonds are undesirable, and are best avoided by ensuring that shipping and storage temperatures do not exceed 30 \u00b0C for significant periods. This should not be a problem in temperate climates; however, in climates where high temperatures routinely occur, consideration should be given to storage in a cool store.\n\n# References\n\nAnon .  Nutritional implications of sulfur amino acid oxidation .  _Nutrition Reviews_.  1973 ;31 : 220 \u2013 221 .\n\nAymard C ,  Cuq JL ,  Cheftel JC .  Formation of lysino-alanine and lanthionine in various food proteins, heated at neutral or alkaline pH .  _Food Chemistry_.  1978 ;3 : 1 \u2013 5 .\n\nErbersdobler H .  Twenty years of furosine\u2014better knowledge about the biological significance of the Maillard reaction in food and nutrition .  In:  Fujimaki M ,  Namiki M ,  Kato H ,  eds.  _Amino-Carbonyl Reactions in Food and Biological Systems. Proceedings of the 3rd International Symposium on the Maillard Reaction_ .  Susono, Shizuoka, Japan, 1\u20135 July 1985 .  Amsterdam :  Elsevier ;  1986 : 481 \u2013 491 .\n\nErbersdobler H ,  Dehn B ,  Nangpal A ,  Reuter H .  Determination of furosine in heated milk as a measure of heat intensity during processing .  _Journal of Dairy Research_.  1987 ;54 : 147 \u2013 151 .\n\nFriedman M .  Chemistry, biochemistry, nutrition and microbiology of lysinoalanine, lanthionine, and histidinoalanine in food and other proteins .  _Journal of Agricultural and Food Chemistry_.  1999 ;47 : 1295 \u2013 1319 .\n\nGilani GS ,  Xiao CW ,  Cockell KA .  Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality .  _British J Nutrition_.  2012 ;108 : S315 \u2013 S332 .\n\nHenle T ,  Walter H ,  Krause I ,  Klostermeyer H .  Efficient determination of individual maillard compounds in heat-treated milk products by amino acid analysis .  _International Dairy Journal_.  1991 ;1 : 125 \u2013 135 .\n\nHern\u00e1ndez MJM ,  Domingo EB ,  Cama\u0148as RMV ,  Alvarez-Coque MCG .  Use of the \u03bf-phthalaldehyde and N-acetyl-L-cysteine in the evaluation of milk proteins .  _Journal of Dairy Science_.  1991 ;74 : 1779 \u2013 1785 .\n\nIDF, 2012. The world dairy situation, 2012. Bulletin of the International Dairy Federation, 458\/2012, Brussels.\n\nNielsen HK ,  de Weck D ,  Finot PA ,  Liardon R ,  Hurrell RF .  Stability of tryptophan during food processing and storage. 1. Comparative losses of tryptophan, lysine and methionine in different model systems .  _British Journal of Nutrition_.  1985 ;53 : 281 \u2013 292 .\n\nPereyra Gonzales AS ,  Naranjo GB ,  Leiva GE ,  Malec LS .  Maillard reaction kinetics in milk powder: Effect of water activity at mild temperatures .  _International Dairy Journal_.  2010 ;20 : 40 \u2013 45 .\n\nRerat A ,  Calmes R ,  Vaissade P ,  Finot P-A .  Nutritional and metabolic consequences of the early Maillard reaction of heat treated milk in the pig .  _Significance for man. European Journal of Nutrition_.  2002 ;41 : 1 \u2013 11 .\n\nRobbins KR ,  Baker DH ,  Finley JW .  Studies on the utilization of lysinoalanine and lanthionine .  _Journal of Nutrition_.  1980 ;110 : 907 \u2013 915 .\n\nSchwarzenbolz U ,  Henle T .  Non-enzymatic modifications of proteins under high-pressure treatment .  _High Pressure Research_.  2010 ;30 : 458 \u2013 465 . \nChapter 12\n\n# Interactions and Functionality of Milk Proteins in Food Emulsions\n\nHarjinder Singh\n\nAiqian Ye*\n\n* Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nBecause of their high nutritional quality and versatile functional properties, milk proteins are widely used as ingredients in many manufactured food colloids, e.g., dairy desserts, nutritional beverages, ice cream, yogurt, spreads, confectionery, and baked goods. Milk proteins perform a wide range of key functions in prepared foods, including emulsification, thickening, gelling, and foaming. An important functionality of milk proteins in food colloids is their ability to facilitate the formation and stabilization of oil droplets in emulsions. The ability of milk protein products to adsorb at the oil\u2013water interface and to stabilize emulsions is influenced by the structures, flexibility, and aggregation state of the constituent proteins. This chapter deals mainly with the properties and functionalities of food emulsions formed with a range of milk protein products and how they are influenced by different environmental and processing conditions. Of particular importance are the effects of pH, calcium ions, and protein content and the influences of thermal and high-pressure processing. The chapter focuses on the structure and composition of adsorbed protein layers, competition between proteins, and the creaming and flocculation behaviors of emulsion droplets. Recent work on the influence of the emulsion structure, in particular of the adsorbed layers, on lipid oxidation and lipid digestion behavior is also reviewed briefly.\n\n## Keywords\n\nmilk proteins\n\nfood emulsions\n\nemulsifying abilities of milk proteins\n\noral behaviour\n\nOutline\n\nIntroduction 359\n\nAdsorption of milk proteins during the formation of emulsions 361\n\nStability of milk protein-based emulsions 366\n\nHeat-induced changes in milk protein-based emulsions 370\n\nPressure-induced changes in milk protein-based emulsions 372\n\nMilk protein hydrolysates and oil-in-water emulsions 373\n\nLactoferrin-based oil-in-water emulsions 374\n\nLipid oxidation in milk protein-based emulsions 376\n\nBehavior of milk protein-stabilized emulsions under physiological conditions 378\n\nConclusions 380\n\n## Introduction\n\nMilk proteins possess functional properties that provide desirable textural and other attributes to the final product. For this reason, they have found numerous applications in traditional dairy products and other foods. The functional properties of milk proteins, such as emulsification, thickening, gelling, flavor binding, and foaming, contribute to the sensory characteristics and the stability of the manufactured foods (Table 12.1). Several types of milk protein products (e.g., caseins and caseinates, whey protein concentrates (WPCs) and whey protein isolates (WPIs), milk protein concentrate (MPC) powders, and hydrolyzed proteins) are manufactured from milk by the dairy industry.\n\nTable 12.1\n\nFunctional Properties of Milk Proteins in Food Systems\n\nFunctional property | Food system  \n---|---  \nSolubility  \nEmulsification  \nFoaming  \nWater binding  \nHeat stability  \nGelation  \nAcid stability | Beverages  \nCoffee whiteners, cream liqueurs, salad dressings, desserts  \nWhipped toppings, shakes, mousses, cakes, meringues  \nBreads, meats, snack bars, custard, soups, sauces, cultured foods  \nUHT- and retort-processed beverages, soups and sauces, custard  \nMeats, curds, cheese, surimi, yogurt  \nAcid beverages, fermented drinks\n\nCaseinates are produced from skim milk by adding acid (hydrochloric acid or lactic acid) or microbial cultures to precipitate the casein from the whey at pH 4.6. The acid-precipitated casein can then be re-solubilized with alkali or an alkaline salt (using calcium, sodium, potassium, or magnesium hydroxide) to about pH 6.7 and spray dried to form caseinate. Caseinates have exceptional water-binding capacity, fat emulsification properties and whipping ability, and a bland flavor. Emulsion-type products, including coffee whiteners, whipped toppings, cream liqueurs, and low-fat spreads, are an important application of caseinates in the food industry. In recent years, the use of casein and caseinate in dietary preparations, nutritional products, and medical applications has increased; many of these preparations are also oil-in-water emulsions containing relatively small amounts of fat.\n\nWPC and WPI are concentrated forms of whey protein components. Ultrafiltration, diafiltration, and ion-exchange technology are used to concentrate and separate the protein from other components. The whey protein is then dried to obtain WPC or WPI, both of which are highly soluble in water, with protein levels ranging from 80% to 95%. Both WPC and WPI have a wide range of food applications and, because of their high protein content, can function as water-binding, gelling, emulsifying, and foaming agents. Processing treatments used in the manufacture of WPC and WPI may sometimes cause some protein denaturation, which tends to affect their functionality.\n\nMPCs are processed directly from skim milk by a combination of ultrafiltration\/diafiltration (Mulvihill, 1992). The protein content of MPCs can vary from 56% to 82%; the caseins are in a micellar form, similar to that found in milk, and the whey proteins are also in their native state. Recently, new MPC products in which the casein micelles have been dissociated to some extent by reducing the colloidal calcium content have been developed. MPCs have been used as ingredients in many food applications, such as milk extension in cheese, yogurt manufacture, and nutritional beverages.\n\nThe functionality of milk proteins in processed foods is determined by their molecular structures and interactions with other food components, such as fats, sugars, polysaccharides, salts, flavors, and aroma compounds. The type and strength of various interactions determine the structure, texture, rheology, sensory properties, and shelf life of manufactured food products. Much knowledge on the structure and properties of individual milk protein components has been gained, but less is known about interactions between different components that occur in a food system as a result of processing and formulation. Controlling these interactions is of key significance for development of novel products and processes as well as for improvement of conventional products and processes. In recent years, there has been an increased interest in the understanding of how the interactions of food components and food structure design influence the rates of nutrient digestion and bioavailability. This research is aimed at developing new foods that regulate calorie intake, provide increased satiety responses, provide controlled digestion, and\/or deliver bioactive molecules (Singh & Sarkar, 2011). Most of this research effort focuses on understanding the changes in physical and biochemical structures of food emulsions during digestion (Singh et al., 2009; McClements et al., 2009; Le R\u00e9v\u00e9rend et al., 2010; Golding & Wooster, 2010).\n\nThis chapter focuses on the emulsifying properties of milk proteins, as this functional property is very important in all the food applications of milk protein products. The adsorption behavior of different milk protein products at oil-in-water interfaces and the stability of the resulting emulsions in different environments are considered. Recent advances in understanding the behavior of milk protein-based emulsions under physiological conditions are discussed briefly.\n\n## Adsorption of Milk Proteins During the Formation of Emulsions\n\nEmulsions are composed of oil droplets (average range 0.5\u20135 \u03bcm diameter) enveloped by a continuous film of surfactant material that stabilizes the droplets. In the food industry, homogenization is widely used for finely dispersing oils in food products, and proteins are most commonly used as emulsifying agents. The state of the droplet size distribution after homogenization reflects the emulsifying capacity of the proteins, the energy input during formation, as well as the effects of various factors, such as pH, temperature, ionic strength, and ratio of the two phases, on the surface activity of the proteins (Walstra, 1993; Dickinson 1998a). In addition, the droplet size distribution influences markedly the properties of food emulsions, such as stability, viscosity, texture, and mouthfeel.\n\nDuring homogenization, the milk protein, in the form of individual molecules or protein aggregates, becomes rapidly adsorbed at the surface of the newly formed oil droplets. The amount of protein present at the interface per unit surface of dispersed phase is defined as the protein load, which is usually expressed as milligrams of protein per unit area of the dispersed phase (mg\/m2). The protein load determines the amount of protein required to make an emulsion with a desired oil volume and droplet size and is dependent on the concentration and type of protein as well as on the conditions used for emulsion formation. The factors that affect the protein load include protein concentration, volume of oil, energy input, state of protein aggregation, pH, ionic strength, temperature, and calcium ions (Dickinson & Stainsby, 1988; Walstra, 1993).\n\nThe properties of the adsorbed layers depend on the amounts and structures of the proteins present during homogenization. Proteins are amphipathic molecules containing both polar and nonpolar parts and orientate at the interface in such a way that a substantial proportion of the nonpolar amino acids remains in contact with the oil phase, and the polar groups are in contact with the aqueous phase (Dickinson 1992; 1998a). The main thermodynamic driving force for the adsorption of proteins is the removal of hydrophobic residues from the unfavorable environment of the bulk aqueous phase by displacement of structured water molecules from the close vicinity of the interface. An additional important driving force is the unfolding and reorganization of the native protein structure, which is due to interaction with the interface. By adsorbing at the interface, the protein reduces the free energy of the system and hence the interfacial tension. The effectiveness of any particular protein in lowering the surface tension depends on the number and type of contacts it makes with the interface (Dickinson et al., 1988a; Dickinson, 1999). A protein molecule that spreads out a lot, and thus has a substantial proportion of its nonpolar residues in contact with the surface, is also very effective in reducing the interfacial tension. Flexible proteins (caseins) with a higher proportion of nonpolar groups are more effective in reducing the interfacial tension than rigid proteins with fewer nonpolar groups (Dickinson & McClements, 1995). The order of surface activity that has been reported for the individual milk proteins is: \u03b2-casein > monodispersed casein micelles > serum albumin > \u03b1-lactalbumin > \u03b1s-caseins = \u03ba-casein > \u03b2-lactoglobulin (Mulvihill & Fox, 1989).\n\nOnce a protein is adsorbed at an interface, it undergoes unfolding and rearrangement to form a stabilizing adsorbed layer (Dickinson, 1992; Dalgleish 1996) and the extent of unfolding depends on the flexibility of the protein molecule, that is, on the strength of the forces maintaining the secondary and tertiary structures. Because the caseins have flexible structures, they unfold rapidly at the interface and may form extended layers up to about 10 nm thick (Dalgleish, 1990). Dalgleish (1999) suggested that casein molecules are stretched to their maximum extent when their overall surface coverage is less than about 1 mg\/m2. Conversely, the presence of excess casein increases the monolayer coverage to a maximum value of 3 mg\/m2, the parts of the molecules in contact with the interface adopt a more compact conformation, and the hydrophilic moieties protrude further from the interface.\n\nWhey proteins (such as \u03b2-lactoglobulin), which give adsorbed layers that are only about 2 nm thick, change conformation and unfold their structure to some extent at the surface (Dalgleish & Leaver, 1993; Mackie et al., 1993; Dalgleish, 1995; Dickinson & McClements, 1995; Dalgleish, 1996; Fang & Dalgleish, 1998). The adsorbed whey protein structure lies somewhere in between the native structure and the fully denatured state, which may have a native-like secondary structure and an unfolded tertiary structure (Dickinson, 1998a). Additionally, the partial unfolding of the globular whey protein structure following adsorption causes exposure of the reactive sulfhydryl group, leading to slow polymerization of the adsorbed protein in the aged layer via sulfydryl\u2013disulfide interchange (Dickinson & Matsumura, 1991; McClements et al., 1993).\n\nThe amount of protein adsorbed on the interface of an emulsion droplet suggests the state of the protein adsorbed at the interface. If the protein load is < 1mg\/m2, it suggests that the protein molecules are fully unfolded. If the protein load is 1\u20133 mg\/m2, a monolayer of globular proteins may be present, or unfolded molecules may be adsorbed in the conformation of trains, loops, and tails. Protein load values above 5 mg\/m2 suggest the adsorption of aggregates of proteins or multilayers of proteins. Some proteins of higher molecular weight may also give higher protein loads (Phillips, 1981; Hunt & Dalgleish, 1994; Dam et al., 1995; Srinivasan et al., 1996).\n\nExtensive studies on purified milk protein systems show that a disordered casein monomer may be regarded as a complex linear copolymer that adsorbs to give an entangled monolayer of flexible chains, with some sequences of segments in direct contact with the surface (trains) and other sequences of segments protruding into the aqueous phase (loops and tails) (Dickinson, 1998a). Based on various experimental studies and molecular modeling, the \u03b2-casein molecule has been shown to adsorb with an extensive hydrophobic region anchored directly at the surface and a hydrophilic region (40\u201350 residues at the N-terminus) protruding extensively into the aqueous phase. This is probably also the portion of the molecule that forms the hydrodynamically thick layer. In contrast to the dangling tail predicted for \u03b2-casein, a loop-like conformation has been predicted for \u03b1s1-casein, and it does not have such a pronounced inequality in the distribution of hydrophobic and hydrophilic residues in its primary structure. It has been suggested that \u03b1s1-casein adsorbs to the oil\u2013water interface via peptides toward the middle of its sequence, rather than the end as in \u03b2-casein, and it may be this that causes the protein to form a thinner adsorbed layer than \u03b2-casein (Dalgleish, 1996). The simple train\u2013loop\u2013tail model is not adequate to describe the molecular configuration of adsorbed \u03b2-lactoglobulin. A closely packed, dense, and rather thin (2\u20133 nm at neutral pH) layer of \u03b2-lactoglobulin is formed, which can be modeled as a dense two-dimensional assembly of highly interacting deformable particles.\n\nMilk proteins used by the food industry contain complex mixtures of proteins in various states of aggregation. The structures of the adsorbed layers formed with these complex mixtures are not understood in molecular detail. The most commonly used milk proteins, (i.e., sodium caseinate and whey proteins) show excellent emulsifying ability, and it is possible to make stable emulsions at a relatively low protein-to-oil ratio (about 1:60). In emulsions formed with sodium caseinate or whey proteins, the protein load increases with an increase in protein concentration until it reaches a plateau value of about 2.0\u20133.0 mg\/m2 (Singh, 2005) (Fig. 12.1). The emulsifying ability of 'aggregated' milk protein products, such as MPC and calcium caseinate, is much lower than that of whey protein or sodium caseinate; that is, much higher concentrations of protein are required to make stable emulsions, and larger droplets are formed under similar homogenization conditions. The surface protein concentration of emulsions formed with MPC is in the range 5\u201320 mg\/m2, depending on the protein concentration used in making the emulsions (Euston & Hirst, 1999). At low protein-to-oil ratios, protein aggregates are shared by adjacent droplets, resulting in bridging flocculation and consequently a marked increase in droplet size. In addition, the spreading of protein at the interface is limited in these emulsions, because the aggregates are held together by calcium bonds and\/or colloidal calcium phosphate. These bonds are unlikely to be affected during the emulsification process. The higher conformational stability of these aggregates will also contribute to their reduced emulsifying ability (Euston & Hirst, 1999; Srinivasan et al., 1999). New MPC products called calcium-depleted MPCs that show improved emulsifying ability have been developed recently (Ye, 2011). In these MPCs, the depletion of colloidal calcium results in disintegration of the casein micelle structure that consequently produces smaller casein particles. These smaller casein particles are adsorbed more efficiently at the droplet surface than the native casein micelles, resulting in more stable emulsions with smaller droplets. The surface protein concentration in emulsions formed with MPC with 80% calcium removal is very similar to that of sodium caseinate (Ye, 2011).\n\nFigure 12.1 Influence of protein concentration on average droplet size d 43 (left) and surface protein coverage (right) in emulsions (30% soya oil) made with sodium caseinate ( ), calcium caseinate ( ), WPC ( ), or MPC (\u2666). From Singh, 2005, reproduced with the permission of The Royal Society of Chemistry.\n\nThe composition of the interfacial layer is determined by the quantities and structures of the proteins present at the moment the emulsion is formed. If proteins are the only emulsifiers present, they will adsorb to the oil\u2013water interface, generally in proportion to their concentration in the aqueous phase (Dalgleish, 1997). However, certain mixtures of caseins show competition during adsorption at oil\u2013water interfaces and rapid exchanges between adsorbed and unadsorbed caseins after emulsion formation. Studies have demonstrated that \u03b2-casein, because of its greater surface activity, adsorbs in preference to \u03b1s1-casein in emulsions stabilized by mixtures of these proteins and that \u03b2-casein displaces \u03b1s1-casein rapidly from the droplet surface (Dickinson et al., 1988b). In binary mixtures containing \u03b2-lactoglobulin and \u03b1-lactalbumin, some limited competitive adsorption does occur, but little exchange between the adsorbed and unadsorbed protein occurs. The protein that arrives at the interface first during homogenization is the protein that predominates there afterward.\n\nIn contrast to model systems, no competitive adsorption has been observed in emulsions stabilized by more complex casein mixtures, such as sodium caseinate (Hunt & Dalgleish, 1994). Interestingly, this behavior appears to be related to the ratio of protein to oil in the emulsions (Euston et al., 1995; Srinivasan et al. 1999). Srinivasan et al. (1999) has shown that in sodium caseinate emulsions when the ratio of protein to oil is very low (about 1:60), \u03b2-casein is preferentially adsorbed at the droplet surface. However, when the total amount of protein is greatly in excess of the amount needed for full surface coverage, \u03b1s1-casein is adsorbed in preference to the other caseins. At all concentrations, \u03ba-casein from sodium caseinate appears to be less readily adsorbed (Fig. 12.2). The concentration dependence of the competitive adsorption of \u03b1s1-casein and \u03b2-casein in sodium caseinate emulsions may be a consequence of the different complexes that can be formed by caseins in solution (Rollema, 1992). The preferential adsorption of \u03b2-casein, because of its high surface activity, appears to exist only at low concentrations where caseins may exist as monomers. With increasing protein concentration, caseins aggregate to form various complexes (Lucey et al., 2000) and it is likely that \u03b2-casein loses its competitive ability because of its self-aggregation to form micelles or through the formation of complexes with other caseins. Therefore, the surface composition of emulsions formed using a relatively high sodium caseinate concentration is likely to be determined by the surface activities and flexibilities of the casein aggregates and complexes. Although extensive information on the surface activity and hydrophobicity of individual caseins is available, little is known about how these characteristics are modified when casein molecules undergo self-association under different environmental conditions.\n\nFigure 12.2 Surface concentrations of \u03b1s1-casein ( ), \u03b2-casein ( ) and \u03ba-casein ( ) in sodium caseinate emulsions (30% oil). From Srinivasan et al., 1999, reproduced with the permission of Elsevier Inc.\n\nWhen the casein is in the highly aggregated form of casein particles, as in calcium caseinate or MPC, very little competitive adsorption and protein exchange take place (Euston & Hirst, 1999; Srinivasan et al., 1999). In these systems, the average surface composition is probably determined by the adsorption of protein aggregates of fixed composition. For instance, calcium caseinate solution consists of large \u03b1s1-casein-rich aggregates, which appear to dominate the droplet surface after emulsification (Srinivasan et al., 1999). When WPCs or WPIs are used to make emulsions, there is no preferential adsorption between \u03b2-lactoglobulin and \u03b1-lactalbumin regardless of the protein-to-oil ratio in the emulsion (Euston et al., 1996; Ye & Singh, 2000; 2006a).\n\nThe aggregation state and the flexibility of protein molecules can be altered by changes in pH, addition of divalent cations, and various processing treatments prior to emulsification. These changes will inevitably influence the adsorption behavior of milk proteins at the oil\u2013water interface. For example, addition of CaCl2 at above a certain critical concentration to a sodium caseinate or whey protein solution before homogenization increases the droplet size, increases the surface protein coverage, and, in sodium caseinate emulsions, also affects the competition between different proteins (Ye & Singh, 2000; 2001). The proportions of \u03b2-lactoglobulin and \u03b1-lactalbumin at the droplet surface remain unaffected by the addition of CaCl2 to a whey protein solution prior to emulsification. In contrast, addition of CaCl2 to a sodium caseinate solution markedly enhances the adsorption of \u03b1s1-casein at the droplet surface, with a much smaller effect on \u03b2-casein adsorption. The effects of calcium on surface coverage and composition can be explained by the binding of the ions to the negatively charged amino acid residues on the protein. This reduces electrostatic repulsions between the protein molecules and increases the potential for intermolecular associations. Because of the presence of clusters of phosphoserine residues, the caseins have stronger affinity than the whey proteins to bind calcium. Consequently, the caseins (except \u03ba-casein) are precipitated by calcium, with \u03b1s1-casein being the most sensitive to aggregation and precipitation by calcium. In sodium caseinate emulsions, the increased surface coverage upon addition of calcium prior to emulsification is probably due to adsorption of casein aggregates onto the droplet surface (Ye & Singh, 2001). Greater \u03b1s1-casein adsorption reflects its stronger tendency to be aggregated by calcium ions in solution or at the interface.\n\nThe native whey proteins do not bind much calcium and are not precipitated in the presence of calcium (Baumy &; Brule, 1988a,b), although heat-denatured whey proteins are able to bind considerable amounts of calcium and undergo aggregation (Pappas & Rothwell, 1991). The increase in surface protein coverage suggests the formation of aggregates of whey proteins in the presence of calcium, which subsequently become adsorbed during emulsification (Ye & Singh, 2000). This has been attributed to a decrease in the denaturation temperature of the whey proteins in the presence of calcium.\n\nAll of these results confirm that, under a given set of homogenization conditions, the surface composition is largely dependent on the protein-to-oil ratio and the aggregation state of the proteins in solution. It appears that the structure of the interfacial layer in emulsions can be manipulated by controlling the protein concentration, the protein type, and the ionic environment. Because of their different interfacial structures, these droplets would be expected to exhibit different reactivities that could be exploited to develop new food textures. Further studies are required for an understanding of the relationship between droplet surface structures and the sensitivity of the droplets to different environments and processing conditions.\n\n## Stability of Milk Protein-Based Emulsions\n\nThe term emulsion stability refers to the ability of an emulsion to resist any alteration in its properties over the timescale of observation (McClements, 1999; Dickinson, 2003; McClements 2005;). An emulsion is thermodynamically unstable, as the free energy of mixing is positive because of the large interfacial area between the oil and the aqueous phase. Therefore, the kinetic stability\u2014that is, the time period for which the emulsion is stable\u2014is important (Damodaran, 1997; McClements, 1999; Dickinson, 2003; McClements 2005;). For instance, an emulsion can be considered to be 'stable' if the inevitable process of separation has been slowed to an extent that it is not of practical importance during the shelf life of the product. An emulsion may become unstable because of a number of different types of physical and chemical processes. Physical instability refers to the change in spatial arrangement or size distribution of emulsion droplets, such as creaming, flocculation, or coalescence, whereas chemical instability includes change in the composition of the emulsion droplet itself, such as oxidation and hydrolysis (McClements & Decker, 2000; McClements, 2005). Creaming is the movement of oil droplets, under gravity or in a centrifuge, to form a concentrated layer at the top of an oil-in-water emulsion sample, with no accompanying change in the droplet size distribution. Creaming is reversible, and the original uniform distribution of droplets can usually be obtained by gentle mixing. The creaming process can be explained by Stokes's Law (Hunter, 1986; McClements 2005):\n\n\u03bd s t o k e s = 2 \u03b3 2 \u03c1 1 \u2212 \u03c1 2 9 \u03b7\n\n (12.1)\n\nwhere \u03bd stokes = velocity of creaming, \u03b3 = emulsion droplet radius, \u03c1 1 and \u03c1 2 = density of the continuous phase, and the dispersed phase, respectively, and \u03b7 = shear viscosity of the continuous phase. The creaming rate can be reduced by lowering the radius, increasing the continuous phase viscosity, or decreasing the difference in density between the two phases. However, this law often fails to define the rate of creaming due to flocculation or coalescence.\n\nCoalescence, that is, an increase in droplet size by accretion, gradually results in separation of the oil and the aqueous phase and is always irreversible. Coalescence requires rupture of the stabilizing film at the oil\u2013water interface, but this occurs only when the layer of continuous phase between the droplets has thinned to a certain critical thickness (Dickinson & Stainsby, 1988; Britten & Giroux, 1991; Das & Kinsella, 1993; Walstra, 1993).\n\nFlocculation has been defined as the reversible aggregation mechanism that arises when droplets associate as a result of unbalanced attractive and repulsive forces (Dalgleish, 1997). Generally, two types of flocculation are distinguished: depletion flocculation and bridging flocculation (Dickinson, 2003). The type of mechanism prevailing depends upon the interaction between the interfacial layer and the emulsion droplets.\n\nBridging flocculation normally occurs when a high-molecular-weight biopolymer at a significantly low concentration adsorbs to two or more emulsion droplets, forming bridges (Dickinson & Pawlowsky, 1998; Dickinson, 1998b; 2003; McClements, 1999; 2005; Fellows &; Doherty 2006;). Depletion flocculation occurs as a result of the presence of unadsorbing biopolymer in the continuous phase, which can promote association of oil droplets by inducing an osmotic pressure gradient within the continuous phase surrounding the droplets (de Hek & Vrij, 1981; Dickinson, 1999; Tuinier & de Kruif, 1999; McClements, 2005).\n\nIf the added biopolymer is either unadsorbed or poorly adsorbed, it is squeezed out of the area between two approaching emulsion droplets. The concentration of biopolymer between the emulsion droplets becomes less than its overall solution concentration, resulting in osmotic imbalance. The net effect is that the particles are attracted to each other, resulting in flocculation. The attraction energy is determined by the concentration of the polymer, and the range of interaction depends on the radius of gyration of the polymer molecule. The bonds formed through the depletion flocculation mechanism are generally weak, flexible, and reversible.\n\nThe ability of proteins to stabilize emulsions is the most important criterion besides the emulsion formation in most food applications. The forces involved in stabilizing and destabilizing emulsions include van der Waals's attractive forces, electrostatic interactions, and steric factors. At pH values away from their isoelectric point, as proteins are electrically charged, there is an electrostatic repulsion, which prevents dispersed droplets from closely approaching one another. With the possible exception of highly charged proteins, a predominant contribution to emulsion stabilization by protein comes from the steric stabilization mechanism. Interactions between droplets stabilized by proteins may be influenced by the presence of certain ions, particularly calcium, as proteins are capable of binding ions.\n\nAs long as sufficient protein is present during homogenization to cover the oil droplets, emulsions stabilized by milk proteins are generally very stable to coalescence over prolonged storage. However, these emulsions are susceptible to different types of flocculation, which in turn leads to enhanced creaming or serum separation. At low protein-to-oil ratios, there is insufficient protein to fully cover the oil\u2013water interface during homogenization, and this results in bridging flocculation. Another consequence of insufficient protein is coalescence of droplets during or immediately after emulsion formation. Bridging flocculation is commonly observed in emulsions formed with aggregated milk protein products, such as calcium caseinate or MPC, in which the droplets are bridged by casein aggregates or micelles. Optimum stability can generally be attained at protein concentrations high enough to allow full saturation coverage at the oil\u2013water interface. However, at very high protein-to-oil ratios, the presence of excess, unadsorbed protein may lead to depletion flocculation in some emulsions. Both depletion flocculation and bridging flocculation cause an emulsion to cream more rapidly.\n\nDepletion flocculation has been observed in sodium-caseinate-based emulsions but not in emulsions formed with calcium caseinate, MPC, or whey proteins (Dickinson & Golding, 1997; Euston & Hirst, 1999; Srinivasan et al., 2001; Singh, 2005) (Fig. 12.3). In sodium-caseinate-based emulsions, it was shown that at a protein content of nearly 2.0 wt%, the emulsion droplets were protected from flocculation by a thick steric-stabilizing layer of sodium caseinate. The emulsion was stable against flocculation, coalescence, and creaming for several weeks. However, when the protein content was increased to above 3.0 wt%, unadsorbed protein gave rise to depletion flocculation. Because of this depletion flocculation, the effective diameter of the droplets increased, resulting in a marked decrease in creaming stability, with an increase in the caseinate concentration from 3 to 5 wt%. Further increasing the protein content to 6.0 wt% and above resulted in very high depletion flocculation, leading to a strong emulsion droplet network that was stable to creaming.\n\nFigure 12.3 Creaming stability and microstructure of emulsions made with sodium caseinate ( ) or WPC ( ) (30% oil). Scale bar represents 10 \u03bcm. From Singh, 2005, reproduced with the permission of The Royal Society of Chemistry.\n\nThe differences in the creaming stabilities of emulsions made with different kinds of milk protein products are largely related to depletion flocculation effects (Singh, 2005). The depletion interaction free energy (G DEP), of the order of a few kT, can be estimated using Equation 12.2 (Walstra, 1993):\n\n\u0394 G D E P = \u2212 2 \u03c0 \u03b3 2 \u220f \u03b3 d \u2212 2 \u03b3 m \/ 3\n\n (12.2)\n\nwhere \u03a0 is the osmotic pressure of the polymer solution, represented as a fluid of hard spheres of radius \u03b3 m, and \u03b3 d is the mean droplet radius. The osmotic pressure under ideal conditions is given by the following equation:\n\n\u220f = c R T \/ M\n\n (12.3)\n\nwhere R is the molar gas constant, T is the temperature, M is the molecular mass of the polymer, and c is the number concentration of the polymer.\n\nFor depletion flocculation to occur, the polymer has to have a fairly high M, so that the \u03b3 m is relatively large. However, at a given c, M is inversely proportional to \u03c0. Therefore, an increase in the polymer molecular mass will reduce the osmotic pressure driving the depletion interaction. Hence, at a given concentration, the depletion interaction free energy is low for a polymer of low molecular mass, increases with an increase in molecular mass until it reaches a maximum, and then decreases with a further increase in molecular mass. Similarly, a reduction in the polymer number concentration will reduce the osmotic pressure.\n\nAlthough the exact state of the casein molecules in concentrated sodium caseinate solutions is unknown, a sodium caseinate solution has been reported to have a radius of gyration of about 20\u201330 nm, as determined by static light scattering (Lucey et al., 2000). Depletion flocculation in sodium caseinate emulsions is likely caused by the presence of these casein aggregates formed from self-assembly of sodium caseinate in the aqueous phase of the emulsion at concentrations above 2 wt% (Dickinson & Golding, 1998). Theoretically, a casein aggregate with a radius of approximately 20 nm causes the strongest depletion flocculation of fine emulsion droplets (mean diameter \u223c0.4 \u03bcm), that is, corresponding to a size ratio of about 10:1 (Radford et al., 2004). The estimated optimum size of casein particles for inducing depletion flocculation is similar to the size of small casein aggregates actually found in sodium caseinate dispersions at low ionic strength (Lucey et al., 2000).\n\nEmulsions formed with whey proteins, MPC, and calcium caseinate do not show depletion flocculation, probably because there are no suitably sized protein particles at the required concentrations in the aqueous phase. The molecular size of whey proteins is less than the optimum, whereas the casein micelles in MPC are too large to induce depletion flocculation. Calcium caseinate consists of mixtures of casein aggregates of different sizes, but the concentration of aggregates capable of inducing depletion flocculation is probably too low. The extent of creaming in these emulsions is largely determined by the particle size of the droplets. Generally, in these emulsion systems, the creaming stability increases with increasing protein concentration up to a certain concentration and then remains almost constant (Euston & Hirst, 1999; Srinivasan et al., 2001). However, the creaming stability of emulsions formed with calcium caseinate or MPC at relatively high protein concentration tends to be higher than that of whey-protein-stabilized emulsions. This can be attributed to an increase in the droplet density as a result of the presence of a much thicker and denser adsorbed protein layer at the droplet surface.\n\nThe addition of moderate amounts of CaCl2 to emulsions containing excess sodium caseinate has been shown to eliminate depletion flocculation and to improve the creaming stability (Ye & Singh, 2001). This effect appears to be due to an increase in the average size of the casein aggregates in the aqueous phase, resulting in a large increase in the molecular mass of the caseins (Dickinson et al., 2001). In addition, there is a reduction in the concentration of unadsorbed caseinate. Both of these effects are expected to cause a substantial reduction in the concentration of small particles, which are assumed to be the main depleting species responsible for inducing reversible flocculation in the calcium-free systems. In contrast, depletion of calcium from MPC causes dissociation of casein micelles, resulting in much smaller casein aggregates that induce depletion flocculation in the MPC-stabilized emulsions (Ye, 2011). Presumably, the substantial reduction in osmotic pressure makes the magnitude of G DEP predicted from Equation 12.2 too small to cause depletion flocculation. Similarly, addition of NaCl at above a certain concentration reduces the extent of depletion flocculation of sodium caseinate emulsions and improves the creaming stability (Srinivasan et al., 2000). This effect is due to increased adsorption of protein at the droplet surface and hence a lower concentration of unadsorbed protein remaining in the solution. Decreasing the pH of emulsions formed with excess sodium caseinate also gradually eliminates depletion flocculation, through aggregation of adsorbed protein and a transfer of more protein to the droplet surface (Singh, 2005). Therefore, it seems to be possible to switch depletion flocculation off and on by controlling the concentration and the aggregation state of the casein molecules in the aqueous phase.\n\n## Heat-Induced Changes in Milk Protein-Based Emulsions\n\nFood emulsions are often heat treated at relatively high temperatures to provide a long shelf life to the product via microbial sterility. These heat treatments can cause denaturation and aggregation of adsorbed and unadsorbed proteins, resulting in aggregation or coalescence of droplets and gel formation. Emulsions formed with whey proteins at neutral pH are stable against heating when the ionic strength and\/or the concentration of protein in the emulsions are low. Addition of KCl at 100 mM or above has been shown to cause destabilization of whey protein emulsions, leading to gel formation (Hunt & Dalgleish, 1995).\n\nBoth the unadsorbed and the adsorbed proteins are necessary for the heat-induced aggregation of whey-protein-stabilized emulsions. Aggregation of emulsion droplets is more extensive and proceeds more rapidly as the concentration of protein in the emulsion is increased, whereas removal of unadsorbed protein from the emulsion decreases the rate of droplet aggregation (Euston et al., 2000). During heat treatment, the protein-covered droplet appears to interact more readily with the unadsorbed protein than with another emulsion droplet. This has been explained by assuming that the relative surface hydrophobicities of the emulsion droplet and the unadsorbed denatured whey proteins are different. Interaction of two emulsion droplets through their respective adsorbed protein layers will have a relatively low probability because the surface hydrophobicity is likely to be relatively low. When an emulsion droplet and an unadsorbed protein molecule aggregate, at least one of them (the denatured protein molecule) has a relatively high surface hydrophobicity, and this will increase the probability of interaction and aggregation (Euston et al., 2000).\n\nIn emulsions made with 3.0% WPI and 25% soya oil, the amount of adsorbed protein was shown to increase from 2.9 to 3.7 mg\/m2 within the first 10 min of heating at 75 \u00b0C, but further heating had no effect (Sliwinski et al., 2003). At 90 \u00b0C, the plateau value of about 4 mg\/m2 was reached within 5 min of heating. Studies on the effects of heating temperature in the range 50\u201390 \u00b0C on WPI emulsions (pH 7.0) (Monohan et al., 1996; Demetriades & McClements, 1998) show that droplet aggregation occurs on heating in the range 75\u201380 \u00b0C, which causes an increase in viscosity and a loss of creaming stability, but the degree of aggregation and the susceptibility to creaming decrease on heating at temperatures above 80 \u00b0C. It has been suggested that in the temperature range 75\u201380 \u00b0C, the whey protein molecules at the droplet surface are only partly unfolded and that not all of the hydrophobic amino acid residues are directed toward the oil phase. Consequently, the surface of the droplet is more hydrophobic, making it susceptible to droplet aggregation. At higher temperatures, the proteins become fully unfolded, with all of the hydrophobic residues being directed into the oil phase, which makes the droplets less prone to aggregation. The role of sulfhydryl\u2013disulfide interchange reactions in droplet aggregation is not clear. It has been suggested that disulfide-mediated interactions during heat treatment are not critical during the initial stages of aggregation, but they tend to strengthen the aggregates (Demetriades & McClements, 1998).\n\nDickinson and Parkinson (2004) and Parkinson and Dickinson (2004) reported that the addition of a very small proportion of caseinate (0.03\u20130.15% of the total emulsion) can stabilize a whey-protein-based emulsion against heat treatment. The magnitude of the effect is dependent on the type of casein, with the order of effectiveness being \u03b2-casein > sodium caseinate > \u03b1s1-casein. The stabilizing effect of the casein in these mixed milk protein systems is strongly synergistic. The casein polymer appears to be acting in a colloidal stabilizing capacity at a surface concentration very much lower than that at which it could be used as an emulsifying or stabilizing agent simply on its own. It has been suggested that adsorbed casein molecules keep the emulsion droplet surfaces sufficiently far apart to prevent the normal cross-linking processes that occur between whey-protein-coated droplets during heat-induced aggregation and gelation, because of the steric hindrance from the loops and tails of the disordered casein polymers (Parkinson & Dickinson, 2004).\n\nIn contrast to whey proteins, emulsions formed with sodium caseinate (2 wt% protein, 20% soya oil) are stable to heating at 90 \u00b0C for 30 min or 121 \u00b0C for 15 min, as determined by droplet size analysis (Hunt & Dalgleish, 1995; Srinivasan et al., 2002). However, the protein coverage and the adsorbed casein composition change upon heat treatment, indicating that interactions between unadsorbed caseinate molecules and caseinate at the droplet surface may occur during heating (Srinivasan et al., 2002). Analysis of adsorbed caseins isolated from emulsions heated at 121 \u00b0C for 15 min has shown that a substantial proportion of the adsorbed caseinate is polymerized to form high-molecular-weight aggregates (Srinivasan et al., 2002), held together through covalent bonds other than disulfide bonds. These covalent bonds appear to form mainly between caseinate molecules at the surface of the same droplet because of the higher local concentrations of casein molecules at the droplet surface. Interestingly, the adsorbed caseins also appear to undergo thermal degradation, resulting in the formation of low-molecular-weight species. Relatively high proportions of casein degradation products present at the droplet surface indicate that the adsorbed caseinate molecules are more susceptible to fragmentation during heating than those in solution and that these peptides remain adsorbed. This is probably due to different structures and conformations of the caseins at the droplet surface than of those in the solution.\n\nThe creaming stability of sodium caseinate emulsions has been found to improve upon heating, with the onset of depletion flocculation occurring at higher protein concentration than in unheated emulsions (Srinivasan et al., 2002). This can be attributed to a reduction in the number of unadsorbed caseinate molecules\/aggregates in the aqueous phase as a result of increased surface coverage and heat-induced polymerization and degradation of the casein molecules. The improvement in the creaming stability in heated emulsions at low protein concentrations may be attributed to an increase in droplet density because of the presence of greater amounts of polymerized protein at the droplet surface.\n\nThe surface protein composition of emulsion droplets may also change during heat treatment in emulsions formed with whey proteins. For WPI-stabilized emulsions, the amount of \u03b2-lactoglobulin at the droplet surface was found to increase during heat treatment, whereas the amount of adsorbed \u03b1-lactalbumin decreased markedly (Ye & Singh, 2006a; Ye, 2010). It seems that \u03b2-lactoglobulin displaces \u03b1-lactalbumin from the interface on heating at temperatures up to 90 \u00b0C, but the reason for this is not clear. Similar phenomena were observed in studies of exchanges of caseins and whey proteins at the interfaces of oil-in-water emulsion droplets (Dalgleish et al., 2002). It was found that, at temperatures above 40 \u00b0C, addition of WPI to the aqueous phase of caseinate-stabilized emulsions caused a displacement of adsorbed caseins. As the \u03b2-lactoglobulin and \u03b1-lactalbumin adsorbed, \u03b1s1\\- and \u03b2-caseins were desorbed, principally the \u03b1s1\\- caseins, whereas the \u03b1s2\\- and \u03ba-caseins were not displaced. The rate of the displacement or exchange reaction was temperature dependent, being almost undetectable at room temperature, but complete within 2 min at 80 \u00b0C. The displacement reaction was not affected by ionic strength; neither were any of the reactions apparently dependent on sulfhydryl exchange reactions (Dalgleish et al., 2002). However, no exchange of proteins occurred when an emulsion prepared with WPI was treated with caseinate and heat treated at 80 \u00b0C for 2 min (Brun & Dalgleish, 1999). This was surprising in view of the known interactions of whey proteins with \u03b1s2\\- and \u03ba-caseins, involving sulfhydryl\u2013disulfide interchange reactions.\n\n## Pressure-Induced Changes in Milk Protein-Based Emulsions\n\nThe effect of ultra-high pressure (100\u20131000 MPa) on the structures of milk proteins in aqueous solution has received considerable attention over the last few years (see Chapter 8). High pressure can disrupt the quaternary and tertiary structures of globular proteins with relatively little influence on their secondary structure. In addition, the proteinaceous colloidal aggregates (e.g., casein micelles), which are held together by ionic and hydrophobic interactions, can be dissociated by high-pressure treatment (Gaucheron et al., 1997; Huppertz et al. 2004). Whey proteins are sensitive to high-pressure treatments (L\u00f3pez-Fandin\u00f3 et al., 1996; Anema et al., 2005). Solution studies (Patel et al., 2005) of native \u03b2-lactoglobulin and whey protein products have shown that high-pressure treatment has a marked effect on the protein's conformation and consequently its aggregation behavior; the aggregation is more extensive at high protein concentrations (Patel et al., 2005). The formation of aggregates is most probably due to the generation of intermolecular disulfide bridges through sulfhydryl\u2013disulfide interchange reactions (Patel et al., 2006).\n\nIn model oil-in-water emulsions, high-pressure treatment has been shown to have no effect on the droplet size distribution or the emulsion viscosity of sodium-caseinate-based emulsions at pH 7 (Dumay et al., 1996). However, high-pressure treatment significantly induced flocculation of emulsion droplets and increased the emulsion viscosity of oil-in-water emulsions stabilized by \u03b2-lactoglobulin or WPC at neutral pH (Dumay et al., 1996; Dickinson & James, 1998). The unfolded unadsorbed whey proteins in the emulsion treated by high pressure appear to be the major contributor to the cross-linking or flocculation of emulsion droplets because greater emulsion flocculation was observed in emulsions with higher proportions of unadsorbed protein in the aqueous phase. As in the case of emulsions treated by heat processing, whey-protein-stabilized emulsions are more sensitive to pressure and temperature at pH values closer to the isoelectric point and at high ionic strength. In terms of the change in emulsion rheology, severe high-pressure treatment (800 MPa for 60 min) is equivalent to relatively mild thermal treatment (65 \u00b0C for 5 min) (Dickinson & James, 1998). In a concentrated emulsion formed with \u03b2-lactoglobulin (1% protein and 40% vol% n-tetradecane), an emulsion gel was produced following high-pressure treatment. When \u03b2-lactoglobulin or WPC solution was treated by high pressure before emulsion formation, the emulsions had larger droplet sizes than emulsions made with the native protein (Galazka et al., 1995). The results indicated a modification of protein structure, leading to the loss of emulsifying efficiency as a result of protein aggregation, despite an increase in surface hydrophobicity. After adsorption on the surface, the protein probably became partially unfolded at the interface, and subsequent pressure treatment caused no further conformational change. No studies on the behavior of emulsions formed with aggregated milk proteins, such as micellar casein, upon high-pressure treatment have been reported.\n\n## Milk Protein Hydrolysates and Oil-In-Water Emulsions\n\nMilk protein hydrolysates have been used extensively in infant and specialized adult nutritional formulations. Extensively hydrolyzed proteins are more easily digested and have substantially reduced immunological reactivities. These formulations are essentially multicomponent emulsion systems, and therefore the emulsifying properties of protein hydrolysates are important.\n\nThe flexibility and thus the availability of hydrophobic and hydrophilic segments within the protein chain can be improved by moderate enzymatic hydrolysis of globular proteins (e.g., whey proteins), thus improving the emulsifying properties of the protein. However, extensive hydrolysis (above 20% degree of hydrolysis), because of the production of many short peptides, has been found to be detrimental to the emulsifying and stabilizing properties of whey proteins (Singh & Dalgleish, 1998). The main form of instability in emulsions formed with highly hydrolyzed whey proteins is the coalescence that arises because of the inability of the predominantly short peptides to adequately stabilize the large oil surface generated during homogenization (Singh &; Dalgleish, 1998; Agboola et al., 1998ab). Nevertheless, it seems to be possible to make a fairly stable emulsion using highly hydrolyzed whey proteins at high peptide concentrations (protein-to-oil ratio about 1:1) and at low homogenization pressures as the sole emulsifier (Agboola et al., 1998a,b). Under these conditions, there is a sufficient amount of high-molecular-weight peptides (>5000 Da) in the emulsion to cover and stabilize the emulsion droplets.\n\nAddition of calcium or magnesium at above 20 mM has been shown to reduce the emulsion stability of emulsions formed with whey protein hydrolysates (Ramkumar et al., 2000). This instability arises mainly from the binding of calcium to the adsorbed peptides, leading to a reduction in the charge density at the droplet surface, which would reduce the inter-droplet repulsion and enhance the likelihood of droplet flocculation. The formation of calcium bridges between peptides present on two different emulsion droplets would also enhance flocculation.\n\nIn these emulsions, some very large droplets, apparently formed by coalescence, are also formed in the presence of calcium. This is likely to be due to the binding of calcium ions to the negatively charged peptides, causing aggregation of larger, more surface-active peptides. This situation would reduce the effective concentrations of emulsifying peptides available during emulsion formation.\n\nHeat treatment of emulsions stabilized by highly hydrolyzed whey proteins at 121 \u00b0C for 16 min results in destabilization of the emulsions, which appears to occur mainly via a coalescence mechanism (Agboola et al., 1998b). As the adsorbed peptide layers in these emulsions lack the cohesiveness of the parent proteins and have poor ability to provide steric or charge stabilization, increased collisions between the droplets during heating would cause droplet aggregation, leading to coalescence. It is also possible that desorption of some loosely adsorbed peptides occurs during heating, as indicated by the decrease in the amount of peptides associated with the oil surface after heating, which would also enhance coalescence.\n\n## Lactoferrin-Based Oil-In-Water Emulsions\n\nBovine milk contains low levels of lactoferrin, an iron-binding glycoprotein with about 700 amino acid residues and a molecular weight of about 80,000 Da (Baker & Baker, 2005). The polypeptide is folded into two globular lobes, representing its N- and C-terminal halves, commonly referred to as the N-lobe and the C-lobe. The surface of the lactoferrin molecule has several regions with high concentrations of positive charge, giving it a high isoelectric point (pI \u22489). This positive charge is one of the features that distinguishes lactoferrin from other milk proteins, such as \u03b2-lactoglobulin, which have isoelectric points in the range 4.5\u20135.5 and are negatively charged at neutral pH. This unique difference could allow the formation of oil-in-water emulsions containing cationic emulsion droplets, through adsorption of lactoferrin, over a wider pH range.\n\nYe and Singh (2006b) showed that, similar to other milk proteins (e.g., caseinate and \u03b2-lactoglobulin), lactoferrin adsorbs onto the interface of oil-in-water emulsion droplets and forms stable emulsions, but emulsion droplets with an overall positive surface charge are produced. In contrast to caseinate- and whey-protein-stabilized emulsions, the cationic emulsion droplets formed by lactoferrin are stable against a change in the pH from 7.0 to 3.0. For emulsions prepared under the same conditions (concentrations of oil and protein, pH, homogenization pressure), the droplet sizes in the lactoferrin emulsions are similar to those in \u03b2-lactoglobulin-stabilized emulsions, but the surface protein coverage (mg\/m2) of the emulsions made at pH 7.0 is higher in lactoferrin emulsions possibly because of its higher molecular weight.\n\nThe formation of a positively charged adsorbed layer in lactoferrin-stabilized emulsions over a wide pH range provides an opportunity for electrostatic interactions with other milk proteins that are mostly negatively charged around neutral pH. In aqueous solutions, lactoferrin tends to form a complex with \u03b2-lactoglobulin via electrostatic interactions (Wahlgren et al., 1993). Adsorption of such a complex onto the droplet surface during emulsion formation results in greater amounts of protein at the droplet surface and the formation of thick interfacial layers. It is interesting to note that oil-in-water emulsions formed using a binary mixture of \u03b2-lactoglobulin and lactoferrin are very stable, even though the overall charge (\u03b6-potential) of the emulsion droplets is close to zero. This suggests that steric repulsion plays an important role in this binary protein-stabilized emulsion.\n\nMultilayered emulsions can be produced by interactions of oppositely charged milk proteins, that is, lactoferrin and \u03b2-lactoglobulin or caseinate at neutral pH (Ye & Singh, 2007). A primary emulsion, containing either anionic droplets coated with \u03b2-lactoglobulin or cationic droplets coated with lactoferrin, can be produced. A secondary emulsion can then be made by mixing either \u03b2-lactoglobulin solution or lactoferrin solution with the primary emulsion (Ye & Singh, 2007). For example, when the emulsions formed with lactoferrin (1 wt%, pH 7.0) were diluted with aqueous phase containing a range of \u03b2-lactoglobulin concentrations, the adsorption of \u03b2-lactoglobulin increased considerably with an increase in the \u03b2-lactoglobulin concentration up to 0.42 wt%, with very little change above this concentration. This increase in \u03b2-lactoglobulin on the surface of emulsions formed with lactoferrin was further confirmed by the change in the \u03b6-potential. In the absence of \u03b2-lactoglobulin, the \u03b6-potential of the emulsion droplets was around +50 mV, because the lactoferrin used to stabilize the droplets has a net positive charge at pH 7.0. The \u03b6-potential became less positive, and eventually changed from positive to negative, as the \u03b2-lactoglobulin concentration in the emulsion was increased (Fig. 12.4).\n\nFigure 12.4 Influence of addition of \u03b2-lactoglobulin into emulsions formed with 1 wt% lactoferrin (30 wt% soya oil, pH 7.0) on the \u03b6-potential of the emulsion droplets. From Ye & Singh, 2007, reproduced with the permission of Springer.\n\nRecently, it was reported that the multilayered protein emulsions are more stable against various environmental condition such as Ca2+, high ionic strength, and heat treatment than the standard protein emulsions (Schmelz et al., 2011; Ye et al. 2012). For example, the addition of Ca2+ to the casein- or whey protein-stabilized emulsions at neutral pH causes droplet aggregation (Agboola & Dalgleish, 1995; Ye & Singh, 2000; Dickinson & Davies, 1999; Ye & Singh, 2001). Addition of lactoferrin to the caseinate or whey protein-stabilized emulsions results in the association of lactoferrin with adsorbed caseins or whey proteins via electrostatic interactions (Ye et al., 2012). This multisurface layer significantly reduces the calcium-induced destabilization of the emulsions, even when a small amount of lactoferrin is involved in the surface layer. Steric repulsion interactions produced by the large lactoferrin molecules on the surface were considered to contribute to this stabilizing effect (Ye et al., 2012).\n\n## Lipid Oxidation in Milk Protein-Based Emulsions\n\nIn addition to the physical properties of emulsions in foods, lipid oxidation is one of the major issues in food storage and consumption as it greatly influences the flavor, odor, and color of foods. Similar to the physical properties of emulsions, lipid oxidation in oil-in-water emulsions is influenced by the droplet size, interfacial characteristics of the lipid droplets, and the type of emulsifying agent (Dickinson & Stainsby, 1982; McClements & Decker, 2000). In addition to their remarkable emulsifying properties, both WPI and caseinate have been shown to inhibit the oxidative deterioration of unsaturated fatty acids, either as part of triacylglycerols or in free form (Hu et al., 2003; Djordjevic et al., 2004; Kiokias et al., 2006; Ries et al. 2010). WPI and caseinate therefore appear to be useful for the design of emulsions that serve as delivery systems for omega-3 fatty acids because of their dual functionality as emulsifiers and antioxidants (Singh et al., 2006). Such emulsions may be incorporated into real food emulsion systems, notably milk, yogurt, mayonnaise, ice cream, and cheese (Ye et al., 2009).\n\nRecently, the oxidation stability of the emulsions made with various milk protein products and linoleic acid was evaluated by determining the formation of lipid hydroperoxides and hexanal (Ries et al., 2010). The oxidative stability of both WPI- and sodium-caseinate-stabilized linoleic acid emulsions with smaller droplet size was greater than that of emulsions with larger droplet size. Other studies have reported contradictory results; some found greater lipid oxidation in emulsions with small droplets (Gohtani et al., 1999; Jacobsen et al., 2000; Lethuaut et al. 2002), whereas others found greater lipid oxidation for large droplets (Nakaya et al., 2005; Let et al. 2007).\n\nCaseinate appears to be a better antioxidant than WPI in the emulsions (Hu et al., 2003; Djordjevic et al., 2004; Kiokias et al., 2006; Ries et al. 2010). The inhibition of lipid oxidation by proteins in emulsions is considered to be mostly due to metal ion chelation and free radical scavenging (Benjelloun et al., 1991). In general, the specific antioxidative activity of caseinate appears to be due to its chelating capacity owing to its phosphoseryl groups (Baumy &; Brule 1988ab; Gaucheron et al., 1996; Bennett et al., 2000; Sugiarto et al. 2010), and that of whey protein appears to be due to its free radical scavenging activity as a result of free sulfhydryl groups (McClements & Decker 2000; Tong et al., 2000; Hu et al., 2003; Kiokias et al., 2007). As caseins do not possess a free sulfhydryl group, their free radical scavenging activity would be expected to be lower than that of whey proteins. On the other hand, whey proteins have a limited ability to chelate metal ions, due to lack of phosphoseryl groups. However, phosphoseryl groups and free sulfhydryl groups do not contribute solely to the total antioxidative capacity of the respective protein. It has been shown that the dephosphorylation of \u03b1s1\\- and \u03b2-casein only partially suppresses their antioxidative activity in a liposome system (Cervato et al., 1999) and that blocking of sulfhydryl groups of whey protein with N-ethylmaleimide in aqueous solution reduces its free radical scavenging activity by only 20% (Tong et al., 2000).\n\nRies et al. (2010) reported that the extent of lipid oxidation decreased with an increase in the protein concentration (Fig. 12.5). Furthermore, an increase in protein concentration led to a decrease in the difference in lipid hydroperoxide production between large- and small-droplet-sized emulsions. At high protein concentrations, the antioxidative effect of the protein in the emulsions appeared to offset the effects of emulsion droplet size and protein type. In addition to the physical barrier of the interfacial protein layer and the antioxidative effect of protein on the interface of emulsion droplets, unadsorbed protein in the continuous phase played an important role in the oxidative stability of emulsions.\n\nFigure 12.5 Lipid hydroperoxide concentration after 4 h (top) and hexanal concentration after 24 h (bottom) of storage at 50 \u00b0C in WPI-stabilized emulsions with average droplet sizes of 0.65 \u03bcm ( ) and 0.31 \u03bcm (\u02c6), and in sodium-caseinate-stabilized emulsions with average droplet sizes of 0.65 \u00b1 0.03 \u03bcm ( ) and 0.31 \u00b1 0.03 \u03bcm ( ). Each data point is the average of determination on two separate emulsions. Bars indicate standard errors. From, Ries et al., 2010 reproduced with the permission of Elsevier.\n\nThe experiments involving the replacement of the continuous phase of the emulsions with water or protein solutions showed that, compared with the control emulsion, the replacement of the continuous phase with water increased the production of lipid hydroperoxides. Replacement of the continuous phase with protein solution decreased the production of lipid hydroperoxides (Ries et al., 2010). In addition, the lipid hydroperoxide concentration was lower in the aqueous phase of emulsions containing caseinate than that containing WPI solution. Furthermore, it has been shown that the oxidative stability increased with increasing protein concentration in the continuous phase. This suggests that the antioxidative mechanism of protein at the interfacial region, such as binding trace metal ions from the lipid phase and free radical scavenging activity, may involve a dynamic exchange process with protein molecules from the continuous phase.\n\nThe antioxidative properties of the milk proteins are also influenced by processing and environmental conditions\u2014that is, heat treatment and change in the pH. When whey proteins that had been heated at temperatures higher than 80 \u00b0C were added to the fish oil emulsions, the oxidation stability of fish oil improved significantly compared to the control samples, as assessed by the lipid hydroperoxide formation and TBARS (Tong et al., 2000; Elias et al. 2007). It has been suggested that the increased oxidative stability could have been due to a greater exposure of free radical scavenging amino acid residues (e.g., tryptophan, tyrosine, phenylalanine, or methionine) to whey proteins and\/or greater interfacial contact of the protein because of increased hydrophobicity after heat treatment, both leading to improved effectiveness of the free radical scavenging process.\n\n## Behavior of Milk Protein-Stabilized Emulsions Under Physiological Conditions\n\nIn recent years, there has been considerable research on the physicochemical and structural changes in food emulsions during oral and gastrointestinal processing. These studies have focused mainly on understanding the role of emulsion structure on the lipolysis of emulsified triacylglycerols, with a view to developing emulsion systems with a controlled rate of lipid digestion and delivery of lipid soluble nutrients. Several studies have shown that the ability of lipases to digest emulsified oil droplets is affected by the composition of the interfacial layer and droplet size of emulsions (see reviews by Singh, 2011; Singh & Ye, 2013). Digestion involves complex mechanical, physicochemical, and physiological processes. Different parts of the human gastrointestinal tract, including the mouth, stomach, small intestine, and large intestine, work in a highly cooperative manner in the overall digestion and absorption of food. Here we provide a brief overview of the basic physicochemical and physiological processes that occur during digestion of milk protein-based emulsions.\n\nThe behavior of milk protein-based emulsions in the oral cavity is largely driven by the interactions of saliva with the adsorbed layer on emulsion droplets (van Aken et al., 2005; Vingerhoeds et al. 2008). Emulsions formed with WPI, sodium caseinate, or lysozyme showed flocculation of droplets when mixed with human saliva. This flocculation was considered to be driven by depletion, van der Waals's forces, and\/or electrostatic interactions between emulsion droplets and salivary mucins, and was largely dependent on the initial charge of the emulsion droplets (Vingerhoeds et al., 2005; Sarkar et al. 2009; Silletti et al., 2007a,b). For example, negatively charged protein-stabilized emulsions (i.e., \u03b2-lactoglobulin emulsions at neutral pH) did not interact with the artificial saliva because of strong repulsive forces between anionic mucin and the anionic \u03b2-lactoglobulin interfacial layer at neutral pH, but underwent depletion flocculation on the addition of higher concentrations of mucin (\u22651.0 wt%). In contrast, positively charged lactoferrin-stabilized emulsions interacted with mucin via electrostatic interaction and resulted in the formation of a secondary layer around the lactoferrin-stabilized droplets, with some bridging-type flocculation. These interactions could have an impact on sensorial and textural perceptions of food emulsions in vivo. For instance, Vingerhoeds et al. (2008) showed that positively charged lysozyme-stabilized emulsions, which underwent irreversible flocculation with saliva, were perceived to be dry and astringent in the mouth.\n\nThe biochemical conditions prevailing in the stomach have a major impact on the structure and stability of protein-based emulsions. The stomach has highly acidic pH (between 1 and 3 for a fasting stomach) and contains various minerals and both proteolytic and lipolytic enzymes. There is also some mechanical agitation because of peristalsis in the stomach (Ekmekcioglu, 2002; Kalantzi et al., 2006; Pal et al. 2007). As most protein-based emulsions are negatively charged at neutral pH, the decrease in the pH to below 2.0 causes substantial changes in the droplet charge, as well as some droplet aggregation around the isoelectric point. The action of pepsin is most critical as it causes major modifications of the adsorbed protein layers and the droplet characteristics, affecting the stability of the emulsion and the digestibility of its components. Because of their highly disordered structures, caseins undergo rapid hydrolysis by pepsin, but \u03b2-lactoglobulin shows some resistance to gastric digestion owing to its highly folded conformation (Reddy et al., 1988; Schmidt & van Markwijk, 1993). However, the rate of hydrolysis of \u03b2-lactoglobulin by pepsin increases when this protein is present as the adsorbed layer in an emulsion (Macierzanka et al., 2009; Sarkar et al. 2009), possibly due to change in the conformation of the \u03b2-lactoglobulin molecules upon adsorption at the oil\u2013water interface (Macierzanka et al., 2009). Surprisingly, adsorbed \u03b1-lactalbumin in oil-in-water emulsions appears to be more resistant to hydrolysis by pepsin, compared with native \u03b1-lactalbumin in solution (Nik et al., 2010). Casein and bovine serum albumin adsorbed at the surface of emulsion droplets have been shown to be readily hydrolyzed by pepsin after mixing with the gastric fluid (Li et al., 2012; Kenmogne-Domguia et al. 2013).\n\nBecause of the hydrolysis of interfacial protein by pepsin (Macierzanka et al., 2009; Sarkar et al., 2009; Nik et al., 2010; Li et al., 2012; Kenmogne-Domguia et al. 2013), the emulsions stabilized by milk proteins (such as WPI, sodium caseinate, \u03b2-lactoglobulin, or \u03b2-casein) undergo flocculation and coalescence of the droplets. In WPI-stabilized emulsions, the presence of excess unadsorbed protein appears to significantly improve the stability of the oil droplets during gastric digestion (Nik et al., 2010).\n\nThe human stomach also contains gastric lipase that is able to penetrate the adsorbed layer and act on the triglyceride core, preferentially cleaving at the sn-3 ester bonds of triglycerides. This lipolysis leads to the accumulation of protonated free fatty acids at the oil\u2013water interface, which competitively displace the proteins and peptides from the interface (Armand et al., 1994; 1996; Pafumi et al. 2002). However, the effects of gastric lipase on the stability of protein-stabilized emulsions have not been studied in any detail, partly because of the nonavailability of commercial gastric lipase for in vitro experiments. Several other materials, such as mucins and phospholipids, that are present in the stomach could alter the physicochemical properties of emulsions.\n\nThe small intestine is the main site for digestion and absorption, and it contains various salts, pancreatic enzymes, coenzymes, bile salts, and phospholipids. The pH of the partly digested\/modified emulsions entering the small intestine increases (to between 6 and 7), because of the secretion of sodium bicarbonate, which allows pancreatic enzymes to act efficiently (Bauer et al., 2005; Krondahl et al. 1997). The change in pH and ionic strength affect the stability of protein-stabilized oil droplets via electrostatic interactions. The pancreatic proteinases (i.e., trypsin and chymotrypsin) cause further hydrolysis of the adsorbed and unadsorbed proteins\/peptides, although the mechanisms of complex interactions of these proteinases with the adsorbed proteins\/peptides are not known. Pancreatic lipase adsorbs to the droplet interface, usually by complexation with co-lipase and\/or bile salts, and then cleaves triglycerides to form 2-monoglycerides and free fatty acids. Bile salts are highly surface-active and are able to displace any protein or peptide material remaining on the droplet surface. Partial or complete displacement of protein from the droplet surface after the introduction of bile salts into the simulated intestinal fluid for emulsions formed with caseins, WPI, and lactoferrin has been demonstrated (Maldonado-Valderrama et al., 2008; Torcello-Gomez et al., 2011; Hur et al., 2009; Mun et al., 2007; Sarkar et al., 2010; Klinkerson &; McClements 2010;). Whey proteins appeared to be more readily displaced than caseinates from the emulsion droplet surface (Mun et al., 2007). \u03b2-Lactoglobulin was rapidly displaced from the oil\u2013water interface compared to lactoferrin, possibly because of differences in droplet charges. Bile salts appeared to bind to the positively charged lactoferrin emulsion droplet, forming a mixed lactoferrin\/bile salt interfacial layer (Sarkar et al., 2010). The exposure of protein-stabilized emulsions to in vitro intestinal conditions has been shown to cause coalescence of some droplets initially (Golding & Wooster, 2010; Sarkar et al., 2010), but all aggregated\/flocculated droplets are broken down, resulting in uniform dispersions of small droplets. The negative charge imparted by adsorbing bile salts\/other surface-active molecules is thought to provide electrostatic repulsions between the droplets and to prevent their further aggregation.\n\n## Conclusions\n\nMilk proteins in soluble and dispersed forms have excellent surface-active and emulsion-stabilizing properties. Differences in the emulsifying abilities of milk proteins arise largely from the differences in structure, flexibility, state of aggregation, and composition of the proteins. These attributes of milk proteins (and hence their emulsifying abilities) are modified through various interactions occurring during the processing required to isolate the protein components, as well as during the manufacture of prepared foods. Emulsions with different surface compositions and structures can be made using different kinds of milk proteins; these emulsions exhibit different sensitivities to solution conditions, such as pH and ionic strength, and processing conditions, such as heat and high-pressure treatments. This could offer possibilities for the formation of emulsions with a range of functionalities for different food applications.\n\nMost of the research during the last 20 years has been performed on oil-in-water emulsions using purified or simple mixtures of caseins and whey proteins. A great deal of information is now available on the conformation of proteins at oil\u2013water interfaces, competitive exchange reactions between adsorbed and unadsorbed proteins, protein\u2013polysaccharide interactions, and factors controlling the rheology and stability of emulsions. In addition, some understanding of how processing conditions (heat treatments, high-pressure treatments) influence interfacial structures and emulsion properties has been achieved. There is much less understanding of the behavior of more complex mixtures of proteins in emulsions and the stability behavior of emulsions under processing environments commonly encountered in the food industry. In addition, there is a lack of understanding of the behavior of emulsions during oral processing in the mouth, as well as during digestion processes. It is critical to understand the oral behavior of emulsions, as common sensorial attributes (e.g., creaminess, smoothness) and the release of fat-soluble flavors are based on interfacial structures and rheological parameters. There is some evidence to show that the behavior of emulsions in the gastrointestinal tract is affected by their physicochemical properties, and that the properties of the interface modulate fat digestion and consequently influence the bioavailability of lipid nutrients. This is the emerging area of emulsion science which may contribute to the development of novel products with health and sensory attributes.\n\n# References\n\nAgboola SO ,  Dalgleish DG .  Calcium-induced destabilization of oil-in-water emulsions stabilized by caseinate or by \u03b2-lactoglobulin .  _Journal of Food Science_.  1995 ;60 : 399 \u2013 403 .\n\nAgboola SO ,  Singh H ,  Munro PA ,  Dalgleish DG ,  Singh AM .  Destabilization of oil-in-water emulsions formed using highly hydrolyzed whey proteins .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 84 \u2013 90 .\n\nAgboola SO ,  Singh H ,  Munro PA ,  Dalgleish DG ,  Singh AM .  Stability of emulsions formed using whey protein hydrolysate: Effects of lecithin addition and retorting .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 1814 \u2013 1819 .\n\nAnema SG ,  Stockmann R ,  Lowe EK .  Denaturation of \u03b2-lactoglobulin in pressure treated skim milk .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 7783 \u2013 7791 .\n\nArmand M ,  Borel P ,  Dubois C ,  Senft M ,  Peyrot J ,  Salducci J .  Characterization of emulsions and lipolysis of dietary lipids in the human stomach .  _American Journal of Physiology\u2014Gastrointestinal and Liver Physiology_.  1994 ;266 : 372 \u2013 381 .\n\nArmand M ,  Borel P ,  Pasquier B ,  Dubois C ,  Senft M ,  Andre M .  Physicochemical characteristics of emulsions during fat digestion in human stomach and duodenum .  _American Journal of Physiology\u2014Gastrointestinal and Liver Physiology_.  1996 ;271 : 172 \u2013 183 .\n\nBaker EN ,  Baker HM .  Molecular structure, binding properties and dynamics of lactoferrin .  _Cellular and Molecular Life Sciences_.  2005 ;62 : 2531 \u2013 2539 .\n\nBauer E ,  Jakob S ,  Mosenthin R .  Principles of physiology of lipid digestion .  _Asian Australian Journal of Animal Science_.  2005 ;18 : 282 \u2013 295 .\n\nBaumy JJ ,  Brule G .  Binding of bivalent cations to \u03b1-lactalbumin and \u03b2-lactoglobulin: Effect of pH and ionic strength .  _Lait_.  1988 ;68 : 33 \u2013 48 .\n\nBaumy JJ ,  Brule G .  Effect of pH and ionic strength on the binding of bivalent cations to \u03b2-casein .  _Lait_.  1988 ;68 (4) : 409 \u2013 417 .\n\nBenjelloun B ,  Talou T ,  Delmas M ,  Gaset A .  Oxidation of rapeseed oil\u2014effect of metal traces .  _Journal of the American Oil Chemists Society_.  1991 ;68 (3) : 210 \u2013 211 .\n\nBennett T ,  Desmond A ,  Harrington M ,  McDonagh D ,  FitzGerald R ,  Flynn A ,  Cashman KD .  The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat .  _British Journal of Nutrition_.  2000 ;83 (6) : 673 \u2013 680 .\n\nBritten M ,  Giroux HJ .  Emulsifying properties of whey protein and casein composite blends .  _Journal of Dairy Science_.  1991 ;74 : 3318 \u2013 3325 .\n\nBrun JM ,  Dalgleish DG .  Some effects of heat on the competitive adsorption of caseins and whey proteins in oil-in-water emulsions .  _International Dairy Journal_.  1999 ;9 : 323 \u2013 327 .\n\nCervato G ,  Cazzola R ,  Cestaro B .  Studies on the antioxidant activity of milk caseins .  _International Journal of Food Sciences and Nutrition_.  1999 ;50 (4) : 291 \u2013 296 .\n\nDalgleish DG .  The conformation of proteins on solid\/water interfaces\u2014caseins and phosvitin on polystyrene lattices .  _Colloids and Surfaces_.  1990 ;46 : 141 \u2013 155 .\n\nDalgleish DG .  Structures and properties of adsorbed layers in emulsions containing milk proteins .  In:  Dickinson E ,  Lorient D ,  eds.  _Food Macromolecules and Colloids_ .  Cambridge :  The Royal Society of Chemistry ;  1995 : 23 \u2013 33 .\n\nDalgleish DG .  Conformations and structures of milk proteins adsorbed to oil\u2013water interfaces .  _Food Research International_.  1996 ;29 : 541 \u2013 547 .\n\nDalgleish DG .  Adsorption of protein and the stability of emulsions .  _Trends in Food Science and Technology_.  1997 ;8 : 1 \u2013 6 .\n\nDalgleish DG .  Interfacial structures and colloidal interactions in protein-stabilised emulsions .  In:  Dickinson E ,  Rodriguez-Patino JM ,  eds.  _Food Emulsions and Foams: Interfaces, Interactions and Stability_ .  Cambridge :  The Royal Society of Chemistry ;  1999 : 1 \u2013 16 .\n\nDalgleish DG ,  Leaver J .  Dimensions and possible structures of milk proteins at oil\u2013water interfaces .  In:  Dickinson E ,  Walstra P ,  eds.  _Food Colloids and Polymers: Stability and Mechanical Properties_ .  Cambridge :  The Royal Society of Chemistry ;  1993 : 113 \u2013 122 .\n\nDalgleish DG ,  Goff HD ,  Brun JM ,  Luan BB .  Exchange reactions between whey proteins and caseins in heated soya oil-in-water emulsion systems\u2014overall aspects of the reaction .  _Food Hydrocolloids_.  2002 ;16 : 303 \u2013 311 .\n\nDam BV ,  Watts K ,  Campbell I ,  Lips A .  On the stability of milk protein-stabilized concentrated oil-in-water food emulsions .  In:  Dickinson E ,  Lorient D ,  eds.  _Foods Macromolecules and Colloids_ .  Cambridge :  The Royal Society of Chemistry ;  1995 : 215 \u2013 222 .\n\nDamodaran S .  Food proteins: an overview .  In:  Damodaran S ,  ed.  _Food Proteins and Their Applications_ .  London :  CRC Press ;  1997 : 1 \u2013 24 .\n\nDas KP ,  Kinsella JE .  Droplet size and coalescence stability of whey protein stabilized milkfat peanut oil emulsions .  _Journal of Food Science_.  1993 ;58 : 439 \u2013 444 .\n\nde Hek H ,  Vrij A .  Interactions in mixtures of colloidal silica spheres and polystyrene molecules in cyclohexane .  _Journal of Colloid and Interface Science_.  1981 ;84 : 409 \u2013 422 .\n\nDemetriades K ,  McClements DJ .  Influence of pH and heating on physicochemical properties of whey protein-stabilized emulsions containing a nonionic surfactant .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 3936 \u2013 3942 .\n\nDickinson E .  Structure and composition of adsorbed protein layers and the relationship to emulsion stability .  _Journal of the Chemical Society\u2014Faraday Transactions_.  1992 ;88 : 2973 \u2013 2983 .\n\nDickinson E .  Proteins at interfaces and in emulsions: Stability, rheology and interactions .  _Journal of the Chemical Society\u2014Faraday Transactions_.  1998 ;94 : 1657 \u2013 1669 .\n\nDickinson E .  Stability and rheological implications of electrostatic milk protein-polysaccharide interactions .  _Trends in Food Science and Technology_.  1998 ;9 : 347 \u2013 354 .\n\nDickinson E .  Adsorbed protein layers at fluid interfaces: Interactions, structure and surface rheology .  _Colloids and Surfaces B: Biointerfaces_.  1999 ;15 : 161 \u2013 176 .\n\nDickinson E .  Hydrocolloids at interfaces and the influence on the properties of dispersed systems .  _Food Hydrocolloids_.  2003 ;17 : 25 \u2013 39 .\n\nDickinson E ,  Davies E .  Influence of ionic calcium on stability of sodium caseinate emulsions .  _Colloids and Surfaces B-Biointerfaces_.  1999 ;2 : 203 \u2013 212 .\n\nDickinson E ,  Golding M .  Depletion flocculation of emulsions containing unadsorbed sodium caseinate .  _Food Hydrocolloids_.  1997 ;11 : 13 \u2013 18 .\n\nDickinson E ,  Golding M .  Influence of alcohol on stability of oil-in-water emulsions containing sodium caseinate .  _Journal of Colloid and Interface Science_.  1998 ;197 : 133 \u2013 141 .\n\nDickinson E ,  James JD .  Rheology and flocculation of high pressure-treated \u03b2-lactoglobulin-stabilized emulsions: comparison with thermal treatment .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 : 2565 \u2013 2571 .\n\nDickinson E ,  Matsumura Y .  Time-dependent polymerization of \u03b2-lactoglobulin through disulfide bonds at the oil\u2013water interface in emulsions .  _International Journal of Biological Macromolecules_.  1991 ;13 : 26 \u2013 30 .\n\nDickinson E ,  McClements DJ ,  eds.  _Advances in Food Colloids_ .  London :  Blackie Academic & Professional ;  1995 .\n\nDickinson E ,  Parkinson EL .  Heat-induced aggregation of milk protein-stabilized emulsions: sensitivity to processing and composition .  _International Dairy Journal_.  2004 ;14 : 635 \u2013 645 .\n\nDickinson E ,  Pawlowsky K .  Influence of \u03ba-carrageenan on the properties of a protein-stabilized emulsion .  _Food Hydrocolloids_.  1998 ;12 : 417 \u2013 423 .\n\nDickinson E ,  Stainsby G .  _Colloids in Food_ .  London :  Applied Science Publishers ;  1982 .\n\nDickinson E ,  Stainsby G .  Emulsion stability .  In:  Dickinson E ,  Stainsby G ,  eds.  _Advances in Food Emulsion and Foams_ .  London :  Elsevier Applied Science ;  1988 : 1 \u2013 44 .\n\nDickinson E ,  Murray BS ,  Stainsby G .  Protein adsorption at air\u2013water and oil\u2013water interfaces .  In:  Dickinson E ,  Stainsby G ,  eds.  _Advances in Food Emulsion and Foams_ .  London :  Elsevier Applied Science ;  1988 : 123 \u2013 162 .\n\nDickinson E ,  Rolfe S ,  Dalgleish DG .  Competitive adsorption of \u03b1s1-casein and \u03b2-casein in oil-in-water emulsions .  _Food Hydrocolloids_.  1988 ;2 : 193 \u2013 203 .\n\nDickinson E ,  Semenova MG ,  Belyakova LE ,  Antipova AS ,  Il'in MM ,  Tsapkina EN ,  Ritzoulis C .  Analysis of light scattering data on the calcium ion sensitivity of caseinate solution thermodynamics: relationship to emulsion flocculation .  _Journal of Colloid and Interface Science_.  2001 ;239 : 87 \u2013 97 .\n\nDjordjevic D ,  McClements DJ ,  Decker EA .  Oxidative stability of whey protein-stabilized oil-in-water emulsions at pH 3: Potential omega-3 fatty acid delivery systems (part B) .  _Journal of Food Science_.  2004 ;69 : C356 \u2013 C362 .\n\nDumay E ,  Lambert C ,  Funtenberger S ,  Cheftel JC .  Effects of high pressure on the physicochemical characteristics of dairy creams and model oil\/water emulsions .  _Lebensmittel-Wissenschaft und -Technologie_.  1996 ;29 : 606 \u2013 625 .\n\nEkmekcioglu C .  A physiological approach for preparing and conducting intestinal bioavailability studies using experimental systems .  _Food Chemistry_.  2002 ;76 : 225 \u2013 230 .\n\nElias RJ ,  McClements DJ ,  Decke EA .  Impact of thermal processing on the antioxidant mechanisms of continuous phase \u03b2-lactoglobulin in oil-in-water emulsions .  _Food Chemistry_.  2007 ;104 (4) : 1402 \u2013 1409 .\n\nEuston SE ,  Singh H ,  Munro PA ,  Dalgleish DG .  Competitive adsorption between sodium caseinate and oil-soluble and water-soluble surfactants in oil-in-water emulsions .  _Journal of Food Science_.  1995 ;60 : 1124 \u2013 1131 .\n\nEuston SE ,  Singh H ,  Munro PA ,  Dalgleish DG .  Oil-in-water emulsions stabilized by sodium caseinate or whey protein isolate as influenced by glycerol monostearate .  _Journal of Food Science_.  1996 ;61 : 916 \u2013 920 .\n\nEuston SR ,  Hirst RL .  Comparison of the concentration-dependent emulsifying properties of protein products containing aggregated and non-aggregated milk protein .  _International Dairy Journal_.  1999 ;9 : 693 \u2013 701 .\n\nEuston SR ,  Finnigan SR ,  Hirst RL .  Aggregation kinetics of heated whey protein-stabilized emulsions .  _Food Hydrocolloids_.  2000 ;14 : 155 \u2013 161 .\n\nFang Y ,  Dalgleish DG .  The conformation of \u03b1-lactalbumin as a function of pH, heat treatment and adsorption at hydrophobic surfaces studied by FTIR .  _Food Hydrocolloids_.  1998 ;12 : 121 \u2013 126 .\n\nFellows CM ,  Doherty WOS .  Insights into bridging flocculation .  _Macromolecular Symposia_.  2006 ;231 : 1 \u2013 10 .\n\nGalazka VB ,  Ledward DA ,  Dickinson E ,  Langley KR .  High pressure effects on emulsifying behaviour of whey protein concentrate .  _Journal of Food Science_.  1995 ;60 : 1341 \u2013 1343 .\n\nGaucheron F ,  Famelart MH ,  LeGraet Y .  Iron-supplemented caseins: preparation, physicochemical characterization and stability .  _Journal of Dairy Research_.  1996 ;63 (2) : 233 \u2013 243 .\n\nGaucheron F ,  Famelart MH ,  Mariette F ,  Raulok K ,  Michel F ,  Le Graet Y .  Combined effects of temperature and high-pressure treatments on physicochemical characteristics of skim milk .  _Food Chemistry_.  1997 ;59 : 439 \u2013 447 .\n\nGohtani S ,  Sirendi M ,  Yamamoto N ,  Kajikawa K ,  Yamano Y .  Effect of droplet size on oxidation of docosahexaenoic acid in emulsion system .  _Journal of Dispersion Science and Technology_.  1999 ;20 (5) : 1319 \u2013 1325 .\n\nGolding M ,  Wooster J .  The influence of emulsion structure and stability on lipid digestion .  _Current Opinion in Colloid and Interface Science_.  2010 ;15 : 90 \u2013 101 .\n\nHu M ,  McClements DJ ,  Decker EA .  Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 1435 \u2013 1439 .\n\nHunt JA ,  Dalgleish DG .  Adsorption behaviour of whey protein isolate and caseinate in soya oil-water emulsions .  _Food Hydrocolloids_.  1994 ;8 : 175 \u2013 187 .\n\nHunt JA ,  Dalgleish DG .  Heat stability of oil-in-water emulsions containing milk proteins: Effect of ionic strength and pH .  _Journal of Food Science_.  1995 ;60 : 1120 \u2013 1123 .\n\nHunter RJ ,  ed.  _Foundations of Colloid Science_ ,  Volume 1 .  Oxford :  Oxford University Press ;  1986 : 674 .\n\nHuppertz T ,  Fox PF ,  Kelly AL .  Dissociation of caseins in high-pressure treated bovine milk .  _International Dairy Journal_.  2004 ;14 : 675 \u2013 680 .\n\nHur SJ ,  Decker EA ,  McClements DJ .  Influence of initial emulsifier type on microstructural changes occurring in emulsified lipids during in vitro digestion .  _Food Chemistry_.  2009 ;114 : 253 \u2013 262 .\n\nJacobsen C ,  Hartvigsen K ,  Lund P ,  Thomsen MK ,  Skibsted LH ,  Adler-Nissen J ,  Holmer G ,  Meyer AS .  Oxidation in fish oil-enriched mayonnaise 3 .  _Assessment of the influence of the emulsion structure on oxidation by discriminant partial least squares regression analysis. European Food Research and Technology_.  2000 ;211 (2) : 86 \u2013 98 .\n\nKalantzi L ,  Goumas K ,  Kalioras V ,  Abrahamsson B ,  Dressman J ,  Reppas C .  Characterization of the human upper gastrointestinal contents under conditions simulating bioavailability\/bioequivalence studies .  _Pharmaceutical Research_.  2006 ;23 : 165 \u2013 176 .\n\nKenmogne-Domguia HB ,  Meynier A ,  Viau M ,  Llamas G ,  Genot C .  Gastric conditions control both the evolution of the organization of protein-stabilized emulsions and the kinetic of lipolysis during in vitro digestion .  _Journal of Functional Foods_.  2013 ;3 : 1302 \u2013 1309 .\n\nKiokias S ,  Dimakou C ,  Oreopoulou V .  Effect of heat treatment and droplet size on the oxidative stability of whey protein emulsions .  _Food Chemistry_.  2007 ;105 (1) : 94 \u2013 100 .\n\nKiokias SN ,  Dimakou CP ,  Tsaprouni IV ,  Oreopoulou V .  Effect of compositional factors against the thermal oxidative deterioration of novel food emulsions .  _Food Biophysics_.  2006 ;1 : 115 \u2013 123 .\n\nKlinkerson U ,  McClements DJ .  Impact of lipase, bile salts, and polysaccharides on properties and digestibility of tuna oil multilayer emulsions stabilized by lecithin\u2013chitosan .  _Food Biophysics_.  2010 ;5 : 73 \u2013 81 .\n\nKrondahl E ,  Orzechowski A ,  Ekstrom G ,  Lennernas H .  Rat jejunal permeability and metabolism of mu-selective tetrapeptides in gastrointestinal fluids from humans and rats .  _Pharmaceutical Research_.  1997 ;14 : 1780 \u2013 1785 .\n\nLe R\u00e9v\u00e9rend BJD ,  Norton IT ,  Cox PW ,  Spyropoulos F .  Colloidal aspects of eating .  _Current Opinion in Colloid and Interface Science_.  2010 ;15 : 84 \u2013 89 .\n\nLet MB ,  Jacobsen C ,  Meyer AS .  Lipid oxidation in milk, yoghurt, and salad dressing enriched with neat fish oil or pre-emulsified fish oil .  _Journal of Agricultural and Food Chemistry_.  2007 ;55 (19) : 7802 \u2013 7809 .\n\nLethuaut L ,  Metro F ,  Genot C .  Effect of droplet size on lipid oxidation rates of oil-in-water emulsions stabilized by protein .  _Journal of the American Oil Chemists Society_.  2002 ;79 (5) : 425 \u2013 430 .\n\nLi J ,  Ye A ,  Lee SJ ,  Singh H .  Influence of gastric digestive reaction on subsequent in vitro intestinal digestion of sodium caseinate-stabilized emulsions .  _Journal of Functional Foods_.  2012 ;3 : 320 \u2013 326 .\n\nL\u00f3pez-Fandin\u00f3 R ,  Carrascosa AV ,  Olano A .  The effects of high pressure on whey protein denaturation and cheese-making properties of raw milk .  _Journal of Dairy Science_.  1996 ;79 : 929 \u2013 936 .\n\nLucey JA ,  Srinivasan M ,  Singh H ,  Munro P .  Characterization of commercial and experimental sodium caseinates by multiangle laser light scattering and size-exclusion chromatography .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 1610 \u2013 1616 .\n\nMacierzanka A ,  Sancho AI ,  Mills ENC ,  Rigby NM ,  Mackie AR .  Emulsification alters simulated gastrointestinal proteolysis of \u03b2-casein and \u03b2-lactoglobulin .  _Soft Matter_.  2009 ;5 : 538 \u2013 550 .\n\nMackie AR ,  Mingins J ,  Dann R .  Preliminary studies of \u03b2-lactoglobulin adsorbed on polystyrene latex .  In:  Dickinson E ,  Walstra P ,  eds.  _Food Colloids and Polymers: Stability and Mechanical Properties_ .  Cambridge :  The Royal Society of Chemistry ;  1993 : 96 \u2013 112 .\n\nMaldonado-Valderrama J ,  Woodward NC ,  Gunning AP ,  Ridout MJ ,  Husband FA ,  Mackie AR .  Interfacial characterization of \u03b2-lactoglobulin networks: displacement by bile salts .  _Langmuir_.  2008 ;24 : 6759 \u2013 6767 .\n\nMcClements DJ ,  Decker EA .  Lipid oxidation in oil-in-water emulsions: impact of molecular environment on chemical reactions in heterogeneous food systems .  _Journal of Food Science_.  2000 ;65 : 1270 \u2013 1282 .\n\nMcClements DJ .  Theoretical analysis of factors affecting the formation and stability of multilayered colloidal dispersions .  _Langmuir_.  2005 ;21 : 9777 \u2013 9785 .\n\nMcClements DJ ,  ed.  _Food Emulsions: Principles, Practice, and Techniques_ .  London :  CRC Press ;  1999 .\n\nMcClements DJ ,  Decker EA .  Lipid oxidation in oil-in-water emulsions: Impact of molecular environment on chemical reactions in heterogeneous food systems .  _Journal of Food Science_.  2000 ;65 (8) : 1270 \u2013 1282 .\n\nMcClements DJ ,  Decker EA ,  Park Y .  Controlling lipid bioavailability through physicochemical and structural approaches .  _Critical Reviews in Food Science and Nutrition_.  2009 ;49 : 48 \u2013 67 .\n\nMcClements DJ ,  Monahan FJ ,  Kinsella JE .  Disulfide bond formation affects stability of whey protein isolate emulsions .  _Journal of Food Science_.  1993 ;58 : 1036 \u2013 1039 .\n\nMonahan FJ ,  McClements DJ ,  German JB .  Disulfide-mediated polymerization reactions and physical properties of heated WPI-stabilized emulsions .  _Journal of Food Science_.  1996 ;61 : 504 \u2013 509 .\n\nMulvihill DM ,  Fox PF .  Physico-chemical and functional properties of milk proteins .  In:  Fox PF ,  ed.  _Developments in Dairy Chemistry\u20144. Functional Milk Proteins_ .  London :  Elsevier Applied Science ;  1989 : 131 \u2013 172 .\n\nMulvihill DM .  Production, functional properties and utilization of milk protein products .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry\u20131. Proteins_ .  London :  Elsevier Applied Science ;  1992 : 369 \u2013 405 .\n\nMun S ,  Decker EA ,  McClements DJ .  Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase .  _Food Research International_.  2007 ;40 : 770 \u2013 781 .\n\nNakaya K ,  Ushio H ,  Matsukawa S ,  Shimizu M ,  Ohshima T .  Effects of droplet size on the oxidative stability of oil-in-water emulsions .  _Lipids_.  2005 ;40 (5) : 501 \u2013 507 .\n\nNik AM ,  Wright AJ ,  Corredig M .  Surface adsorption alters the susceptibility of whey proteins to pepsin-digestion .  _Journal of Colloid and Interface Science_.  2010 ;344 : 372 \u2013 381 .\n\nPafumi Y ,  Lairon D ,  de la Porte PL ,  Juhel C ,  Storch J ,  Hamosh M .  Mechanisms of inhibition of triacylglycerol hydrolysis by human gastric lipase .  _Journal of Biological Chemistry_.  2002 ;277 : 28070 \u2013 28079 .\n\nPal A ,  Brasseur JG ,  Abrahamsson B .  A stomach road or \"magenstrasse\" for gastric emptying .  _Journal of Biomechanics_.  2007 ;40 : 1202 \u2013 1210 .\n\nPappas CP ,  Rothwell J .  The effect of heating, alone or in the presence of calcium or lactose, on calcium binding to milk proteins .  _Food Chemistry_.  1991 ;42 : 183 \u2013 201 .\n\nParkinson EL ,  Dickinson E .  Inhibition of heat-induced aggregation of a \u03b2-lactoglobulin-stabilized emulsion by very small additions of casein .  _Colloids and Surfaces B: Biointerfaces_.  2004 ;39 : 23 \u2013 30 .\n\nPatel HA ,  Singh H ,  Anema SG ,  Creamer LK .  Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 3409 \u2013 3420 .\n\nPatel HA ,  Singh H ,  Havea P ,  Considine T ,  Creamer LK .  Pressure-induced unfolding and aggregation of the protein in whey protein concentrate solutions .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 9590 \u2013 9601 .\n\nPhillips MC .  Protein conformation at liquid interfaces and its role in stabilizing emulsions and foams .  _Food Technology_.  1981 ;35 : 424 \u2013 427 .\n\nRadford SJ ,  Dickinson E ,  Golding M .  Stability and rheology of emulsions containing sodium caseinate: Combined effects of ionic calcium and alcohol .  _Journal of Colloid and Interface Science_.  2004 ;274 : 673 \u2013 686 .\n\nRamkumar C ,  Singh H ,  Munro PA ,  Dalgleish DG ,  Singh AM .  Influence of calcium, magnesium, or potassium ions on the formation and stability of emulsions prepared using highly hydrolyzed whey proteins .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 : 1598 \u2013 1604 .\n\nReddy M ,  Kella NKD ,  Kinsella JE .  Structural and conformational basis of the resistance of \u03b2-lactoglobulin to peptic and chymotryptic digestion .  _Journal of Agriculture and Food Chemistry_.  1988 ;36 : 737 \u2013 741 .\n\nRies D ,  Ye A ,  Haisman D ,  Singh H .  Antioxidant properties of caseins and whey proteins in model oil-in-water emulsions .  _International Dairy Journal_.  2010 ;20 : 72 \u2013 78 .\n\nRollema HS .  Casein association and micelle formation .  In:  Fox PF ,  ed.  _Advanced Dairy Chemistry\u2014 1: Proteins_ .  London :  Elsevier Applied Science ;  1992 : 111 \u2013 140 .\n\nSarkar A ,  Goh KKT ,  Singh RP ,  Singh H .  Behaviour of an oil-in-water emulsion stabilized by \u03b2-lactoglobulin in an in vitro gastric model .  _Food Hydrocolloids_.  2009 ;23 : 1563 \u2013 1569 .\n\nSarkar A ,  Horne DS ,  Singh H .  Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model .  _International Dairy Journal_.  2010 ;20 : 589 \u2013 597 .\n\nSchmelz T ,  Lesmes U ,  Weiss J ,  McClements DJ .  Modulation of physicochemical properties of lipid droplets using \u03b2-lactoglobulin and\/or lactoferrin interfacial coatings .  _Food Hydrocolloids_.  2011 ;25 : 1181 \u2013 1189 .\n\nSchmidt DG ,  van Markwijk BW .  Enzymatic hydrolysis of whey proteins .  _Influence of heat treatment of \u03b1-lactalbumin and \u03b2-lactoglobulin on their proteolysis by pepsin and papain. Netherlands Milk Dairy Journal_.  1993 ;47 : 15 \u2013 22 .\n\nSilletti E ,  Vingerhoeds MH ,  Norde W ,  van Aken GA .  Complex formation in mixtures of lysozyme- stabilized emulsions and human saliva .  _Journal of Colloid and Interface Science_.  2007 ;313 : 485 .\n\nSilletti E ,  Vingerhoeds MH ,  Norde W ,  van Aken GA .  The role of electrostatics in saliva-induced emulsion flocculation .  _Food Hydrocolloids_.  2007 ;21 : 596 .\n\nSingh AM ,  Dalgleish DG .  The emulsifying properties of hydrolyzates of whey proteins .  _Journal of Dairy Science_.  1998 ;81 : 918 \u2013 924 .\n\nSingh H .  Milk protein functionality in food colloids .  In:  Dickinson E ,  ed.  _Food Colloids: Interactions, Microstructure and Processing_ .  Cambridge :  The Royal Society of Chemistry ;  2005 : 179 \u2013 193 .\n\nSingh H .  Aspects of milk-protein-stabilised emulsions .  _Food Hydrocolloids_.  2011 ;25 : 1938 \u2013 1944 .\n\nSingh H ,  Sarkar A .  Behaviour of protein-stabilised emulsions under various physiological conditions .  _Advances in Colloid and Interface Science_.  2011 ;165 : 47 \u2013 57 .\n\nSingh H ,  Ye A .  Structural and biochemical factors affecting the digestion of protein-stabilized emulsions .  _Current Opinion in Colloid and Interface Science_.  2013 ;18 (4) : 360 \u2013 370 .\n\nSingh H ,  Ye A ,  Horne D .  Structuring food emulsions in the gastrointestinal tract to modify lipid digestion .  _Progress in Lipid Research_.  2009 ;48 : 92 \u2013 100 .\n\nSingh, H., Zhu, X.Q., Ye, A., 2006. Lipid encapsulation. W.I.P.O. Patent No. WO2006\/115420.\n\nSliwinski EL ,  Roubos PJ ,  Zoet FD ,  van Boekel MAJS ,  Wouters JTM .  Effects of heat on physicochemical properties of whey protein-stabilised emulsions .  _Colloids and Surfaces B: Biointerfaces_.  2003 ;31 : 231 \u2013 242 .\n\nSrinivasan M ,  Singh H ,  Munro PA .  Sodium caseinate-stabilized emulsions: Factors affecting coverage and composition of surface proteins .  _Journal of Agricultural and Food Chemistry_.  1996 ;44 : 3807 \u2013 3811 .\n\nSrinivasan M ,  Singh H ,  Munro PA .  Adsorption behaviour of sodium and calcium caseinates in oil-in-water emulsions .  _International Dairy Journal_.  1999 ;9 : 337 \u2013 341 .\n\nSrinivasan M ,  Singh H ,  Munro PA .  The effect of sodium chloride on the formation and stability of sodium caseinate emulsions .  _Food Hydrocolloids_.  2000 ;14 : 497 \u2013 507 .\n\nSrinivasan M ,  Singh H ,  Munro PA .  Creaming stability of oil-in-water emulsions formed with sodium and calcium caseinates .  _Journal of Food Science_.  2001 ;66 : 441 \u2013 446 .\n\nSrinivasan M ,  Singh H ,  Munro PA .  Formation and stability of sodium caseinate emulsions: influence of retorting (121 degrees C for 15 min) before or after emulsification .  _Food Hydrocolloids_.  2002 ;16 : 153 \u2013 160 .\n\nSugiarto M ,  Ye A ,  Taylor MW ,  Singh H .  Milk protein-iron complexes: Inhibition of lipid oxidation in an emulsion .  _Dairy Science and Technology_.  2010 ;90 : 87 \u2013 98 .\n\nTong LM ,  Sasaki S ,  McClements DJ ,  Decker EA .  Antioxidant activity of whey in a salmon oil emulsion .  _Journal of Food Science_.  2000 ;65 (8) : 1325 \u2013 1329 .\n\nTorcello-Gomez A ,  Maldonado-Valderrama J ,  de Vicente J .  Investigating the effect of surfactants on lipase interfacial behaviour in the presence of bile salts .  _Food Hydrocolloids_.  2011 ;25 : 809 \u2013 816 .\n\nTuinier R ,  de Kruif CG .  Phase behavior of casein micelles\/exocellular polysaccharide mixtures: experiment and theory .  _Journal of Colloid and Interface Science_.  1999 ;110 : 9296 \u2013 9304 .\n\nvan Aken GA ,  Vingerhoeds MH ,  De Hoog EHA .  Colloidal behaviour of food emulsions under oral conditions .  In:  Dickinson E ,  ed.  _Food Colloids: Interactions, Microstructure and Processing_ .  Cambridge :  Royal Society of Chemistry ;  2005 : 356 \u2013 366 .\n\nVingerhoeds MH ,  Blijdenstein TBJ ,  Zoet FD ,  van Aken GA .  Emulsion flocculation induced by saliva and mucin .  _Food Hydrocolloids_.  2005 ;19 : 915 \u2013 922 .\n\nVingerhoeds MH ,  Silletti E ,  de Groot J ,  Schipper RG ,  van Aken GA .  Relating the effect of saliva-induced emulsion flocculation on rheological properties and retention on the tongue surface with sensory perception .  _Food Hydrocolloids_.  2008 ;23 : 773 \u2013 785 .\n\nWahlgren MC ,  Arnebrant T ,  Paulsson MA .  The adsorption from solutions of \u03b2-lactoglobulin mixed with lactoferrin or lysozyme onto silica and methylated silica surfaces .  _Journal of Colloid and Interface Science_.  1993 ;158 : 46 \u2013 53 .\n\nWalstra P .  Introduction to aggregation phenomena in food colloids .  In:  Dickinson E ,  Walstra P ,  eds.  _Food Colloids and Polymers: Stability and Mechanical Properties_ .  Cambridge :  The Royal Society of Chemistry ;  1993 : 3 \u2013 15 .\n\nYe A .  Surface protein composition and concentration of whey-protein-isolate-stabilized oil-in-water emulsions: effect of heat treatment .  _Colloids and Surfaces B: Biointerfaces_.  2010 ;78 : 24 \u2013 29 .\n\nYe A .  Functional properties of milk protein concentrates: Emulsifying properties, adsorption and stability of emulsions .  _International Dairy Journal_.  2011 ;21 : 14 \u2013 20 .\n\nYe A ,  Singh H .  Influence of calcium chloride addition on the properties of emulsions stabilized by whey protein concentrate .  _Food Hydrocolloids_.  2000 ;14 : 337 \u2013 346 .\n\nYe A ,  Singh H .  Interfacial composition and stability of sodium caseinate emulsions as influenced by calcium ions .  _Food Hydrocolloids_.  2001 ;15 : 195 \u2013 207 .\n\nYe A ,  Singh H .  Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: Influence of polysaccharides .  _Food Hydrocolloids_.  2006 ;20 : 269 \u2013 276 .\n\nYe A ,  Singh H .  Adsorption behaviour of lactoferrin in oil-in-water emulsions as influenced by interaction with \u03b2-lactoglobulin .  _Journal of Colloid and Interface Science_.  2006 ;295 : 249 \u2013 254 .\n\nYe A ,  Singh H .  Formation of multilayers at the interface of oil-in-water emulsion via interactions between lactoferrin and \u03b2-lactoglobulin .  _Food Biophysics_.  2007 ;2 : 125 \u2013 132 .\n\nYe A ,  Cui J ,  Taneja A ,  Zhu X ,  Singh H .  Evaluation of processed cheese fortified with fish oil emulsion .  _Food Research International_.  2009 ;42 : 1093 \u2013 1098 .\n\nYe A ,  Lo J ,  Singh H .  Formation of interfacial milk protein complexation to stabilize oil-in-water emulsions against calcium .  _Journal of Colloid and Interface Science_.  2012 ;378 : 184 \u2013 190 . \nChapter 13\n\n# Milk Protein\u2013Polysaccharide Interactions\n\nKelvin K.T. Goh*\n\nAnwesha Sarkar**\n\nHarjinder Singh***\n\n* Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand  \n** Nestec Ltd., Vevey, Switzerland  \n*** Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nProteins and polysaccharides are common ingredients present in many food formulations. They are generally responsible for imparting key sensory attributes (e.g., textural attributes, controlled flavor release) and are capable of modifying phase stability in food colloidal systems. Their physicochemical properties depend not only on the molecular parameters of the individual biopolymers but also on the nature of interactions between the protein and polysaccharide molecules. This chapter provides an overview of the possible types and nature of interactions that can occur between protein and polysaccharide molecules in aqueous solutions and at interfaces. Extensive research carried out in this field over the last few decades outlining different milk protein polysaccharide interactions is summarized in tables. The last section attempts to categorize the different types of interactions and their impact on microstructures and rheological properties of the systems. The chapter concludes by stressing the importance of understanding these interactions, which potentially provide food scientists with the opportunities to modify or create novel food structures and functionalities.\n\n## Keywords\n\nMicrostructures\n\nbiopolymers\n\nphase separation\n\ndepletion flocculation\n\nthermodynamic incompatibility\n\nco-solubility\n\ncoacervates\n\nemulsion\n\nMaillard\n\ninterface\n\nOutline\n\nIntroduction 388\n\nMixing behavior of biopolymers 388\n\nPhase diagram 390\n\nNature of interactions in protein\u2013polysaccharide systems 392\n\nRepulsive Interactions 392\n\nAttractive Interactions 393\n\nCovalent Bonds 394\n\nMilk protein\u2013polysaccharide interactions in the aqueous phase 395\n\nMilk protein\u2013polysaccharide interactions at the interface 398\n\nRheological properties and microstructures of protein\u2013polysaccharide systems 401\n\nNoninteracting Protein\u2013Polysaccharide Mixtures 402\n\nNongelling Phase-separated System 403\n\nCasein Micelles and Galactomannans 403\n\nMilk Proteins and Xanthan 404\n\nGelling Phase-separated System 404\n\nWhey Protein and Galactomannans 405\n\nWhey Protein Isolate and Xanthan 405\n\n\u03b2-Lactoglobulin and Pectin 406\n\n\u03ba-Carrageenan and \u03b2-Lactoglobulin 406\n\nInteracting Protein\u2013Polysaccharide Mixtures 406\n\nNongelling Phase-separated System 407\n\n\u03b2-Lactoglobulin and Chitosan 407\n\nWhey Proteins and Exopolysaccharides 407\n\nWhey Proteins and Gum Arabic 407\n\nSodium Caseinate and Gum Arabic 408\n\nCasein Micelle and Pectin 408\n\nGelling Phase-separated System 409\n\nSodium Caseinate and Pectin 409\n\nCasein Micelles and Iota-carrageenan 409\n\nConcluding remarks 410\n\n## Introduction\n\nProteins and polysaccharides are broadly classified as biopolymers because of their large molecular structures. These macromolecules are known to possess important physicochemical roles, such as imparting thickening, stabilizing, gelling, and emulsifying properties in food products (Dickinson, 2003; Dickinson et al., 2003; Hemar et al., 2001a,b). The physicochemical properties of individual proteins and polysaccharides have been studied extensively in the last several decades. It is well established that the factors influencing the physicochemical properties of these macromolecules in solution include molar mass, molecular conformation, polydispersity, charged density, concentration, pH, ionic strength, temperature, solvent quality, and the nature of molecular (intra-\/inter-) interactions (de Kruif & Tuinier, 2001; Doublier et al., 2000; Tolstoguzov, 1997). In many food systems, their physical properties become more complex, as both proteins and polysaccharides are present (either naturally or added as ingredients) among the complex multicomponent mixtures. The overall stability and microstructure of these food systems depend not only on the physicochemical properties of proteins or polysaccharides alone, but also on the nature and strength of interaction between protein and polysaccharide (Dickinson, 1998b; Dickinson et al., 1998). This chapter reviews a number of studies carried out in the field of protein\u2013polysaccharide interactions, with a particular focus on milk proteins and a diverse range of polysaccharides in aqueous systems.\n\n## Mixing behavior of biopolymers\n\nWhen aqueous solutions of proteins and polysaccharides are mixed, one of four phenomena can arise: (1) co-solubility, (2) thermodynamic incompatibility, (3) depletion interaction, or (4) complex coacervation (Fig. 13.1) (Benichou et al., 2002; de Kruif & Tuinier, 2001; de Kruif et al., 2004; Dickinson, 2003; Martinez et al., 2005; Schmitt et al., 1998; Syrbe et al., 1998; Tolstoguzov, 1991; ; ). These phenomena can be explained as follows:\n\nFigure 13.1 Different types of interactions (co-solubility, thermodynamic incompatibility, depletion interaction, and complex coacervation) between protein and polysaccharide in aqueous solutions.\n\nCo-solubility refers to the creation of a stable homogeneous solution, that is, the generation of one phase in which the two macromolecular species either do not interact or exist as soluble complexes in the aqueous medium. When intermolecular attraction is absent, macromolecules are only co-soluble in dilute solutions where the entropy of mixing favors more randomness in the system (Tolstoguzov, 2003). To achieve co-solubility from a thermodynamic angle, the Gibbs free energy of mixing (\u0394Gmixing) given in Equation 13.1 must be negative. This means that the entropy of mixing should favorably exceed the enthalpy term. (Note: The highest level of entropy is achieved when the different kinds of molecules are randomly distributed throughout the system) (McClements, 2005a). The expression for Gibbs free energy accompanying mixing in standard conditions is given by\n\n\u0394Gmixing=\u0394Hmixing\u2212T\u0394mixing\n\n (13.1)\n\nwhere \u0394Gmixing, \u0394Hmixing, and T\u0394Smixing are the free energy, enthalpy (interaction energy), and entropy changes between the mixed and unmixed states, respectively.\n\nWhen the size of the molecules is small, as it is in the case of monomer sugars and hydrophilic amino acids, mixing the two species results in a co-soluble system. However, with increasing molecular weight and concentration of the polymers, the system tends to become less co-soluble due to thermodynamic incompatibility (Tolstoguzov, 1991; ). This is because the entropy of mixing of biopolymers is significantly lower than that of the monomers. The bulky size and rigid structure of biopolymer molecules decrease the entropy of mixing, resulting in a higher free energy. For a mixed biopolymer solution, the enthalpy\u2013entropy balance generally results in mutual exclusion of one biopolymer from the local vicinity of the other. This means that biopolymers in mixed solution show a preference to be surrounded by their own type; otherwise, consequently, their mixtures tend to separate into liquid phases, as described below (Grinberg & Tolstoguzov, 1972; ; Polyakov et al., 1997; Tolstoguzov, 1988; ; Tolstoguzov et al., 1985).\n\nThermodynamic incompatibility occurs when the two dissimilar noninteracting macromolecular species separate into two different phases as enthalpy of mixing exceeds the entropy difference (Benichou et al., 2002; Grinberg & Tolstoguzov, 1997; Schmitt et al., 1998; Tolstoguzov, 2002). The driving force to segregation is the enthalpic advantage of molecules being surrounded by others of the same type. For small molecules, this is normally outweighed by the entropic advantage of both species being free to move throughout the entire volume. However, for polymer solutions, where there are far fewer individual molecules, the entropy of mixing is much smaller, which can allow phase separation to occur. Of the two distinct immiscible aqueous phases formed, each phase is mainly loaded with only one biopolymer species, that is, a protein-rich phase and a polysaccharide-rich phase. Phase separation due to incompatibility can also occur if each biopolymer shows varying affinity toward the solvent (Piculell & Lindman, 1992; Tolstoguzov, 1991). In this case, solvent\u2013protein (or solvent\u2013polysaccharide) interactions are favored over protein\u2013polysaccharide interactions and solvent\u2013solvent interactions, leading to two phases, one enriched in protein and the other in polysaccharides (Doublier et al., 2000). Thermodynamic incompatibility can also arise within a mixture of polysaccharides or proteins. Some examples include polysaccharides with different structures; proteins of different classes like water-soluble albumins with salt-soluble globulins; native and denatured forms of the same protein, as well as aggregated and nonaggregated forms of the same protein (Tolstoguzov, 2002).\n\nThermodynamic incompatibility is highly dependent on pH and ionic strength, and is prevalent when protein and neutral polysaccharide are present or when both protein and polysaccharide carry the same negative charge at neutral pH (Doublier et al., 2000). Although thermodynamic incompatibility is prevalent in mixed-polymer systems, some of these systems do not achieve thermodynamic equilibrium within a limited timescale due to the presence of kinetic energy barriers. When the kinetic energy exceeds the thermal energy of the system, the molecules become 'trapped' in a metastable state (McClements, 2005a). Some examples of kinetic energy barriers include the formation of a gel network within an incompatible system or a highly viscous continuous phase that slows down the phase separation process. The choice of which phase to gel and the component used to promote gelation depends on the type of biopolymers used in the system. (Bryant & McClements, 2000a,b; Kim et al., 2006; Norton & Frith, 2001).\n\nDepletion flocculation usually involves spherical particles in the presence of macromolecules (Asakura & Oosawa, 1954; ; Bourriot et al., 1999a). Phase separation of particulate suspension is enhanced by the addition of a polymer. This phenomenon usually occurs in the colloidal dispersion in the presence of noninteracting polymers (e.g., polysaccharides in an emulsion, polysaccharides, and colloidal casein micelles). The higher osmotic pressure of the polymer molecules surrounding the colloidal particles (as compared to the interparticle region) causes an additional attractive force between particles leading to the flocculation of particles. The attractive force depends on the size, shape, and concentration of the polymer molecules and the colloidal particles (Hemar et al., 2001b). When colloidal particles approach each other, the excluded (or depleted) layer starts to overlap, allowing more space for the polymer molecules. The increase in volume causes the total entropy of the system to increase (i.e., free energy to decrease), which in turn encourages attraction between the colloidal particles (de Bont et al., 2002). In a mixed protein\u2013polysaccharide system containing casein micelles, phase separations are often attributed to depletion flocculation phenomena (Bourriot et al., 1999a; Tuinier & De Kruif, 1999; Tuinier et al., 2000). This is because of the large colloidal particle size of casein micelles and because increasing the concentration of polysaccharides results in greater attraction between the casein micelles (Doublier et al., 2000).\n\nComplex coacervation is the formation of electrostatic complexes between the protein and polysaccharide molecules, leading to a two-phase system. One phase has both the biopolymers in a complex matrix, while the other phase contains mainly the solvent water and is depleted in both biopolymers. Complex coacervation commonly occurs between oppositely charged biopolymers. Complex coacervation between oppositely charged proteins and polysaccharides was first reported by mixing gelatin and gum arabic in acetic acid solution (Tiebackx, 1911). The term coacervation was first introduced in 1929 to describe a process in which aqueous colloidal solutions separate into two liquid phases, one rich in colloid, that is, the coacervate, and the other containing little colloid (Bungenberg de Jong & Kruyt, 1929). If the two biopolymers are present in equal proportions by weight at a pH such that they carry net equal but opposite charges, the yield of coacervates will be at its maximum (Schmitt et al., 1998). The size and morphology of these structures may be exploited to bring about new functionalities and textural changes in processed foods.\n\n## Phase diagram\n\nMixing two aqueous solutions of proteins and polysaccharides may give rise to a one-phase or two-phase system depending on the solution composition and environmental conditions, as depicted in Figure 13.1 (Benichou et al., 2002; de Kruif & Tuinier, 2001; de Kruif et al., 2004; Dickinson, 2003; Martinez et al., 2005; Schmitt et al., 1998b; Syrbe et al., 1998; Tolstoguzov, 1991;1997).\n\nIn a one-phase system, protein and polysaccharide can exist either as individual molecules or as soluble complexes that are uniformly dispersed throughout the entire system. However, with increasing molecular weight and concentration of biopolymers, the system tends to become less co-soluble and to give rise to a two-phase system; that is, the system separates into two distinct phases that have different biopolymer concentrations.\n\nFor a system with relatively strong net repulsion between protein and polysaccharide in aqueous solution, the two biopolymers move into two different phases due to thermodynamic incompatibility. Two distinct immiscible aqueous phases are formed, and each of them is mainly loaded with only one-biopolymer species, that is, one phase protein-rich and the other phase polysaccharide-rich. A typical phase diagram for segregating the biopolymer system is shown in Figure 13.2 which has been explained by many researchers (Antonov et al., 1982; Bourriot et al., 1999a; Clark, 2000; Closs et al., 1999; Ercelebi & Ibanoglu, 2007; Grinberg & Tolstoguzov, 1972; ; Lundin et al., 2003; Polyakov, et al., 1980; Thaiudom & Goff, 2003; Tolstoguzov, 2003; Tolstoguzov et al., 1985). The phase diagram consists of a typical binodal curve (the solid line curve), which divides the single-phase miscible region (below the curve) from the two-phase immiscible region (shaded region). The binodal branches exhibit the points of limited co-solubility. The points of the binodal curve connected by the tie line represent the composition of the coexisting equilibrium phases. From the phase diagram, it is possible to determine the effective concentrations of biopolymers in the two phases and the concentrations at which maximal co-solubility of the biopolymers is achieved. In addition, it helps to establish which of the two biopolymers forms the continuous phase.\n\nFigure 13.2 A typical phase diagram showing a protein\u2013polysaccharide solution, with water as the solvent at a particular pH, temperature, and ionic strength. A sample of composition O (which was initially made with A% of protein and B% of polysaccharide) separates out into two bulk polymer-rich phases. The protein-enriched phase will have a composition C% protein, whereas the polysaccharide-enriched phase will have composition D% polysaccharide. The binodal (solid curve) separates the single-phase region from the two-phase domain (which can be obtained by direct observation of the phase separation in test tubes). The % protein in the polysaccharide phase will be negligible and vice versa. The tie-line can be calculated. The points on the tie-line have the same effective concentration of the phases at equilibrium even though their phase volume ratios differ. The ratio of DO\/OC represents the volume ratio of protein-rich phase C and polysaccharide-rich phase D, respectively, by inverse-lever rule. If O is shifted along the tie-line to O1, the new phase volume ratio will be DO1\/ O1C. The line obtained by joining the midpoints (+) of two or more tie-lines gives the rectilinear diameter. The coordinates of the critical point E (obtained from the intersection of the binodal to the rectilinear diameter) show the composition of a system separating into two phases of the same volume and composition, which means the separated-phase systems will have 50% protein and 50% polysaccharide in the same phase-volume ratio. Point F represents the separation threshold that is the minimum critical concentration required for the biopolymers to separate into two phases.\n\n## Nature of interactions in protein\u2013polysaccharide systems\n\nThe interactions responsible for complex formation between biopolymers (Fig. 13.1) can be classified as weak or strong, specific or nonspecific, attractive or repulsive (Dickinson, 1993). The overall interaction between protein and polysaccharides is the average of the following different intermolecular forces arising between the various segments and chains of the two biopolymers (Dickinson, 1998b; Schmitt et al., 1998).\n\n### Repulsive Interactions\n\nRepulsive interactions are always nonspecific and of transient duration. They usually arise from excluded volume effects and\/or electrostatic interactions and tend to be weak, except at very close range or very low ionic strength.\n\nThe excluded volume or steric exclusion effects are the nonspecific and transient interactions. This arises when proteins and polysaccharides are non-ionic and noninteracting. As a result, the volume surrounding one polymer molecule becomes unavailable to the other polymer molecule in the aqueous solution (Polyakov et al., 1997; Schmitt et al., 1998; Tolstoguzov 1991; ; ). Excluded volume effects exhibit mutual spatial restrictions and competition between the biopolymers for solution space; that is, there is a reduction in the mixing entropy of the system due to the reduction in the volume available for the biopolymer molecules to occupy.\n\nNet repulsive interactions, due to electrostatic effects, depend largely on the pH and ionic strength of the background electrolyte concentration. The electrostatic repulsive interactions are commonly found in mixtures of proteins and anionic polysaccharides under conditions where both the biopolymers carry the same net charge\u2014for example, pH is above the isoelectric point (pI) of the protein.\n\n### Attractive Interactions\n\nAttractive interactions between proteins and polysaccharides may be weak or strong and either specific or nonspecific. Nonspecific attractive interactions arise as a result of a multitude of weak interactions between groups on the biopolymers, such as electrostatic, van der Waals, hydrogen bonding, and hydrophobic interactions. Hydrogen bonding and hydrophobic interactions are actually collective interactions (e.g., electrostatic, van der Waals, and steric overlap), including some entropy effects (McClements, 2005a).\n\nElectrostatic interactions are the most important forces involved in the complex formation between proteins and ionic polysaccharides. These interactions between charged biopolymers lead to a decrease in the electrostatic free energy of the system. Moreover, the enthalpy contribution due to interactions of oppositely charged biopolymers and liberation of counter-ions along with water molecules often compensates for the loss of configurational entropy of mixing rigid biopolymers (Piculell & Lindman, 1992; Tolstoguzov, 1997). Strong electrostatic attractive interactions occur between positively charged proteins (pH < pI) and anionic polysaccharides, especially at low ionic strengths. Generally, two types of complexes are formed by electrostatic interactions (Schmitt et al., 1998; Tolstoguzov, 1997; ; ). Soluble complexes are obtained when opposite charges carried by the two biopolymers are not equal in number, whereas insoluble complexes result when the net charge on the complex is close to zero.\n\nVan der Waals forces are extremely weak electrical attractions arising because of temporary dipole interactions (Dickinson, 1998b; Sherony & Kintner, 1971; Stainsby, 1980). Basically, every atom has an electron cloud that can yield a temporary electric dipole. The dipole in one atom can induce a corresponding dipole in another atom. This is possible only if the atoms are close. However, if they are too close, repulsive forces between the adjacent negatively charged electron clouds may not allow these van der Waals attractions. Although these transient electrical attractive forces are very weak, they can influence macromolecular interactions, together with other noncovalent forces described above (Damodaran, 1997).\n\nHydrophobic bonding is an entropy-driven long-range interaction between nonpolar groups, and it is promoted by conformational and structural modifications of biopolymers, mostly by the unfolding of polymeric chains exposing hydrophobic groups. These kinds of interactions are promoted by an increase in temperature (Antonov et al., 1996a; Piculell & Lindman, 1992; Samant et al., 1993; Stainsby, 1980; Tolstoguzov, 1997).\n\nHydrogen bonding is a moderately strong bond that becomes relatively insignificant at high temperatures. These bonds are ionic in nature and refer to the interaction between hydrogen atoms attached to an electronegative atom (oxygen, sulfur) with another electronegative atom (e.g., sulfur of sulfate group), that is, \u2013O-H\u03b4+...\u03b4 \u2212O <. A classical example of hydrogen bonding has been shown in the complex coacervation of gelatin and pectin (Braudo & Antonov, 1993), which is obtained over a wide range of pH, including the isoelectric pH (4.8) of gelatin. Protein\u2013polysaccharide hydrogen bonding between gelatin\u2013pectin, gelatin\u2013alginate, and chitosan\u2013collagen has been well established by various studies over a wide range of pH values (Antonov et al., 1996a; Taravel & Domard, 1995).\n\n### Covalent Bonds\n\nCovalent bonds are very strong, specific, non-electrostatic, and permanent linkages. Two principal methods can be used to generate a covalent linkage between proteins and polysaccharides. The most commonly used method utilizes the chemical reaction between amino groups of proteins and carboxylic group of polysaccharides (the Maillard reaction) to give an amide covalent bond (Stainsby, 1980). Covalent bonds can also be generated enzymatically using the oxidoreductase family of enzymes (E.C. 1.XXX) that catalyze the oxidation of the phenolic group of tyrosine residues with carbohydrate groups containing phenolic residues, such as cereal arabinoxylans (Boeriu et al., 2004). Tyrosine-containing peptides have also been conjugated with ferulic acid (Oudgenoeg et al., 2001) and with whey proteins through the use of three different oxidoreductases (Faergemand et al., 1998).\n\nThe potential for cross-linking proteins and polysaccharides using transglutaminase has been suggested (E.C. 2.3.2.13) (Flanagan & Singh, 2006). Many polysaccharides contain residual protein, for example, gum arabic, guar gum, and locust bean gum (LBG), all containing low levels of protein. Gum arabic (approximately 2% protein, depending on source) consists of, among other subunits, a glycoprotein and an arabinogalactan protein. Provided the residual protein in these polysaccharides contains lysine and\/or glutamine residues, the treatment of protein and polysaccharide mixtures with transglutaminase could theoretically lead to the formation of heteropolymers (i.e., protein\u2013polysaccharide conjugates) in addition to homopolymers (cross-linked protein or cross-linked polysaccharide). Flanagan and Singh (2006) demonstrated that sodium caseinate\u2013gum arabic conjugates catalyzed by transglutaminase can be produced.\n\nThese kinds of interactions are generally very stable to pH and ionic strength. Because of their stable properties, this kind of bonding has been intentionally used to produce conjugated emulsifiers (Akhtar & Dickinson, 2003; ; Benichou et al., 2007; Dunlap & C\u00f4t\u00e9, 2005; Neirynck et al., 2004; Shepherd et al., 2000; Song et al., 2002). In most of these studies, the covalent conjugation between proteins and polysaccharides has been achieved through the Maillard reaction.\n\nApart from these major interactions, ion-bridging involving the binding of cations like Ca2+ may also contribute to protein\u2013polysaccharide interactions to some extent, although they do not have the predominant influence (Antonov et al., 1996a; Dickinson, 1998b; Stainsby, 1980). For example, firm sodium caseinate gels (G \u2032 > 100 Pa) were formed with pectin concentrations \u22650.6% at one particular degree of methylation (\u223c31%) and amidation (\u223c17%) in the presence of Ca2+ ions (1.8 mM) at pH \u223c3.6 (Matia-Merino et al., 2004).\n\n## Milk protein\u2013polysaccharide interactions in the aqueous phase\n\nMilk proteins and polysaccharides dissolved in the aqueous phase form a pseudoternary system of milk protein\u2013polysaccharide water. Various interactions in these systems could lead to complex formation or bulk-phase separation. Extensive studies have been carried out in areas of protein\u2013polysaccharide interactions, particularly using well-studied milk proteins and commercially available polysaccharides (Dickinson, 1998b). Table 13.1 and 13.2 show a compilation (non-exhaustive) of various milk proteins (casein and\/or whey proteins) and polysaccharide mixtures in aqueous systems, and the conditions in which different kinds of interactions occur. Following this section, we describe the microstructure and rheological properties of some of these systems.\n\nTable 13.1\n\nCasein\u2013Polysaccharide Interactions in Aqueous Systems\n\nSL. NO. | Casein\u2013polysaccharide aqueous systems | Conditions | Interactions | References  \n---|---|---|---|---  \n1. | Milk proteins (Casein micelle + Whey proteins) \\+ Pectin (High methoxyl-62.7% methylated) | 20 \u00b0C, pH 6.0\u201310.5, 0\u20130.5 M NaCl | Thermodynamic incompatibility | (Antonov et al., 1982)  \nMilk proteins (Casein micelle + Whey proteins) \\+ Gum arabic  \nMilk proteins (Casein micelle + Whey proteins) \\+ Arabinogalactan  \n2. | Casein micelle + Alginate | 25 \u00b0C, pH 7.2 | Thermodynamic incompatibility | (Suchkov et al., 1988; Suchkov et al., 1981)  \n3. | Casein micelle (2.5%) \\+ Pectin (Low methoxyl- 35%, High methoxyl- 73%, Low methoxyl amidated- 35% methylated & 20% amidated) (0.1\u20130.2%) | 60 \u00b0C, pH 6.7\/ 5.3 | pH 6.7: Depletion interaction Methoxylation affects interaction | (Maroziene & de Kruif, 2000)  \n4. | Casein micelle (0.8-4%) \\+ Galactomannans (Guar Gum, LBG) (0.09\u20130.3%) | 5\/20 \u00b0C,  \npH 6.8\/ 7.0, 0.08\/0.25 M NaCl, Sucrose  \n(10\u201340 wt%) | Depletion interaction Sucrose affects interaction | (Bourriot et al., 1999a; Schorsch et al., 1999)  \n5. | Casein micelle (1.0%) \\+ Carrageenan (\u03b9-, \u03ba-, \u03bb-forms) (0.12%) | 60\/ 50\/ 20 \u00b0C, pH 6.7\/ pH 7.0, 0.25 M NaCl\/ 0.05 M NaCl\u20130.01 M KCl | Depletion interaction  \n| (Bourriot et al., 1999c; Dalgleish & Morris, 1988; Langendorff et al., 1997; ; )  \n6. | Sodium caseinate (0.1\u20130.5%) \\+ Gum arabic (0.01\u20135%) | pH 2.0\u20137.0, 0.5 M NaCl, slow acidification with glucono-\u03b4-lactone | Soluble electrostatic complexation | (Ye, Flanagan, & Singh, 2006)  \n7. | Casein micelle (0.1%) \\+ Exopolysaccharide (5.0%) (Lactococcus lactis subsp. cremoris B40) | 25 \u00b0C, pH 6.6 | Depletion interaction | (Tuinier & De Kruif, 1999; Tuinier et al., 1999)  \n8. | Sodium caseinate + Maltodextrin  \n(2:1, 1:1, and 1:4) | 60 \u00b0C, 2\u20134 days | Covalent conjugate via Maillard reaction. No phase separation | (Morris et al., 2004; Shepherd et al., 2000)  \n9. | Casein (\u03b2-casein, \u03b1s-casein) \\+ Polysaccharide  \n(Dextran, Galactomannan)  \n1:1) | 60 \u00b0C, 24 hours | Covalent conjugate via Maillard reaction. No phase separation | (Dickinson & Semenova, 1992; Kato et al., 1992)  \n10. | Sodium caseinate (6.0%) \\+ Sodium alginate (1%) | 23 \u00b0C, pH 7.0, | Thermodynamic incompatibility | (Guido et al., 2002; Simeone et al., 2002)\n\nTable 13.2\n\nWhey Protein\u2013Polysaccharide Interactions in Aqueous Systems\n\nSL. NO. | Whey protein\u2013polysaccharide aqueous systems | Conditions | Interactions | References  \n---|---|---|---|---  \n1. | \u03b2 -lactoglobulin ( \u03b2 -lg) (0.5%) \\+ Chitosan (Degree of Deacetylation: 85%) (0\u20130.1%) | pH 3.0\u20137.0, 5 mM phosphate buffer | pH dependent \u03b2-lg-chitosan Soluble\/insoluble complex coacervation | (Guzey & McClements, 2006a, 2006b)  \n2. | Heat denatured whey protein isolate (HD-WPI) (8.0%) \\+ Pectin (28, 35, 40, 47, and 65% methylation) (0.1\u20131.5%) | 80 \u00b0C\/85 \u00b0C, pH 6.0\/ 7.0, 5.0\/10.0 mM CaCl2 | Thermodynamic incompatibility | (Beaulieu et al., 2001; Kim et al., 2006)  \n3. | \u03b2 -lg (12.0%) \\+ Alginate (0.1\u20131.0%) | 87 \u00b0 C\/30 \u00b0C, pH 7.0\/ (3.0\u20137.0),  \nHigh pressure | pH dependent \u03b2-lg-chitosan Soluble\/insoluble complex coacervation | (Dumay et al., 1999; Harnsilawat et al., 2006)  \n4. | \u03b2 -lg (0.05%) \\+ Pectin (Low methoxyl- 28.3\/42.6%, High methoxyl- 71.3\/73.4%) (0.0125%) | 4\u201340 \u00b0C\/ 25 \u00b0C\/87 \u00b0C, pH 4.0\u20137.5\/ 6.5, 0.11\/ 0.1\u20131.0 M NaCl, High pressure | pH, ionic strength, & temp. Complex coacervation. Precipitation for modified pectin. Methylation affects complexation | (Dumay et al., 1999; Girard et al., 2002; 2003a; 2004; Maude et al., 2003b; Kazmiersi et al., 2003; Wang & Qvist, 2000)  \n5. | Whey protein isolate (WPI) (5.0%) \\+ Galactomannans (LBG) (0\u20130.4%) | pH 5.0\u20137.0 | pH and concentration: Biphasic gel | (Tavares & Lopes da Silva, 2003)  \n6. | HD-WPI (8.5%) \\+ Xanthan gum (0\u20130.2%) | 25\u201390 \u00b0C\/ 75\u201380 \u00b0C, pH 7.0\/ 5.4, 0.2 M NaCl, High pressure treatment | Native WPI: Co-solubility.  \nHD-WPI: Thermodynamic incompatibility | (Bryant & McClements, 2000b; Li et al., 2006)  \n7. | WPI (4\u201312.5%) \\+ Xanthan gum (0.01\u20131.0%) | pH 5.5\/ 6.0\/ 6.5\/7.0, 0.1\/0.5 M NaCl, high-pressure treatment | Depletion interaction, pH-dependent electrostatic complexation | (Benichou et al., 2007; Bertrand & Turgeon, 2007; Hemar et al., 2001b; Laneuville et al., 2000; Zasypkin et al., 1996)  \n8. | Bovine serum albumin (BSA) + Sulfated polysaccharides (\u03b9-, \u03ba-carrageenan, dextran sulfate) (2.5:1 and 5:1) | pH 6.5\u20138.0, High-pressure treatment | Complex coacervation | (Galazka et al., 1996; ; )  \n9. | HD-WPI (10.0%) + \u03ba -Carageenan (0.5%) | 80 \u00b0C,  \npH 1.0\u201312.0 | Complex coacervation | (Mleko et al., 1997)  \n10. | \u03b2 -lg (0.5\u201310.0%) + \u03ba- Carageenan (1.0%)  \n(1:2, 5:1, and 10:1) | 45\u201380 \u00b0C, pH 7.0, 0.1 M NaCl\/ 0.01 M CaCl2 | Temp., pH, & concentration dependent phase separated bi-continuous gel formation | (Capron et al., 1999; Ould Eleya & Turgeon, 2000)  \n11. | \u03b2 -lg + Gum arabic  \n(2:1) | pH 3.6\u20135.0, 0.005\u201310.7 mM NaCl | Complex coacervation | (Sanchez et al., 2002; ; Sanchez & Renard, 2002; Schmitt et al. 1998; ; )  \n12. | WPI + \u03bb -Carrageenan  \n(1:1 to 150:1) | pH over a wide range, 0\u20130.1 M (NaCl\/ CaCl2) | Electrostatic complexation Precipitation | (Weinbreck et al., 2004a)  \n13. | WPI + Gum arabic  \n(2:1) | pH 4.0\u20137.0, 0\u20130.1 M NaCl | Complex coacervation Glassy state | (Weinbreck et al., 2003a; 2004b; 2004c)  \n14. | \u03b2 -lg + Carboxymethyl dextran  \n(1:1 and 7:2) | 4 \u00b0C\/ 25 \u00b0C, pH 5.5\/ 4.75 | \u03b2-lg-Carboxymethyl dextran covalent conjugate. No phase separation | (Hattori et al., 1994)  \n15. | WPI + Carboxymethyl potato starch  \n(2:1) | 24 \u00b0C, pH 7.0 | WPI-Carboxymethyl starch covalent conjugate | (Hattori et al., 1995)  \n16. | WPI + Exopolysaccharide (Lactococcus lactis subsp. cremoris B40)  \n(2:1) | 25 \u00b0C, pH over a wide range, 0\u20130.1 M (NaCl\/CaCl2), Heat treatment of WPI | Electrostatic complexation Precipitation HD-WPI: Depletion interaction | (de Kruif & Tuinier, 1999; Tuinier & de Kruif, 1999; Weinbreck et al., 2003b)  \n17. | \u03b2 -lg + Pullulan | 4 \u00b0C, 0.01 M NaCl | Depletion interaction | (Wang et al., 2001)  \n18. | \u03b2 -lg + Carboxymethyl cellulose  \n(CMC) | 60 \u00b0C, pH 2.5\u20137.0, 0.05\u20130.2 M | Insoluble electrostatic complex Sedimentation | (Hansen et al., 1974; Hidalgo & Hansen, 1969)  \n19. | WPI + Maltodextrin  \n(1:2 and 1:3) | 80 \u00b0C, 2 hours, 79% RH | Covalent conjugation No phase separation | (Akhtar & Dickinson, 2007)  \n20. | WPI\/ Whey protein concentrate (WPC) + Pectin  \n(4:1, 2:1, 1:1, and 1:2) | 60 \u00b0C, pH 7.0, 14 days, | Covalent conjugation No phase separation | (Mishra et al., 2001; Neirynck et al., 2004)\n\n## Milk protein\u2013polysaccharide interactions at the interface\n\nIn an emulsion system containing both milk protein and polysaccharide, protein generally forms the primary interfacial layer by directly adsorbing to the oil surface. The hydrophilic polysaccharide possibly forms a thick secondary steric-stabilizing layer on the outside of protein-adsorbed emulsion droplets, providing the protein\u2013polysaccharide interaction is sufficiently attractive (Dickinson, 1994). Generally, strong electrostatic interaction between the oppositely charged adsorbed protein and added polysaccharide leads to the formation of multilayered interfacial membranes stabilizing emulsion droplets (Dickinson & James, 2000; Guzey et al., 2004; G\u00fczey & McClements, 2006a,b; Hong & McClements, 2007; Laplante, et al., 2005; Moreau et al., 2003; Mun et al., 2006).\n\nTo date, much research has been done on protein\u2013polysaccharide interactions in emulsion systems under different conditions of temperature, pH, ionic strength, concentration of protein and polysaccharide, pressure treatment, and so on. In most of these cases, the presence of polysaccharides creates flocculation by bridging interactions at lower concentrations, followed by emulsion stabilization at sufficiently high concentrations to enable the saturation of the protein-adsorbed emulsion droplets. However, in systems where polysaccharides do not interact with the proteins, depletion flocculation can be expected. Protein\u2013polysaccharide interactions are sensitive to details of protein structure as well as to the charge density on the biopolymers. For example, partial denaturation of globular proteins can result in increased complexation with hydrocolloids at the interface as compared to that of native proteins in an aqueous solution at the same pH and ionic strength (Dickinson, 2003). Generally, strong electrostatic interaction between the adsorbed protein and the added polysaccharide leads to the formation of a stabilizing layer. At sufficiently high hydrocolloid concentrations, emulsion stability is increased by immobilizing the emulsion droplets in a gelled protein\u2013polysaccharide network. However, the same polysaccharide can induce irreversible bridging flocculation of the protein-coated emulsion droplets if an insufficient amount of polysaccharides is present for surface coverage.\n\nTable 13.3 summarizes some of the recent investigations on potential milk\u2013protein polysaccharide interactions in the emulsion system (non-exhaustive).\n\nTable 13.3\n\nMilk Protein\u2013Polysaccharide Interactions in Emulsion Systems\n\nSL. NO. | Milk protein\u2013polysaccharide in emulsion systems | Oil phase | Conclusion | References  \n---|---|---|---|---  \n1. | Bovine serum albumin (BSA) + Carrageenan  \n(\u03b9-and \u03ba\\- forms)  \n| n-Tetradecane  \n(20, 40 vol%) | Strong bridging flocculation, BSA-\u03ba-interaction weaker than BSA-\u03b9-carrageenan at same pH & ionic strength | (Dickinson & Pawlowsky, 1997; )  \n2. | Caseins (\u03b1s1-, \u03b2-casein and caseinate) + High-methoxy Pectin | Sunflower oil  \n(11, 40 vol%) | Steric stabilization at low pH, depletion flocculation at neutral pH, salt-induced destabilization for \u03b1s1-stabilized emulsion only | (Dickinson et al., 1998)  \n3. | \u03b2-lactoglobulin + Low-methoxy pectin | Soybean oil (20 vol%) | Gelation at high-pressure treatment (400 MPa): | (Dickinson & James, 2000)  \n4. | WPI (85% \u03b2-lg) + Low-methoxy pectin | Soybean oil (20 wt%) | Covalent conjugation, better emulsion stability at pH 5.5 | (Neirynck et al., 2004)  \n5. | WPI + Dextran sulfate | Medium-chain triglyceride oil, Silicone oil, Orange oil &  \nn-Tetradecane (20 vol%) | Covalent conjugation, long-term emulsion stability at low concentration, steric stabilization | (Akhtar & Dickinson, 2003)  \n6. | Casein + Maltodextrin | Soybean oil (30 wt%) | Covalent conjugation, better emulsion stability | (Shepherd et al., 2000)  \n7. | Hydrolyzed WPI + Hydrocolloid (Xanthan gum, Guar gum, \u03ba-Carrageenan) | Corn oil  \n(4wt%) | Coalescence rate: guar gum > xanthan gum > \u03ba-carrageenan, depletion interaction induced coalescence at high-pressure treatment | (Ye et al., 2004; Ye & Singh, 2006)  \n8. | WPC + Hydrocolloids (Xanthan gum, Polypropylene glycol alginate (PGA), Carrageenan) | Soybean oil  \n(20 wt%) | Droplet aggregation by depletion mechanism (xanthan gum) on heating. Better creaming stability with PGA-WPC complexation | (Euston et al., 2002)  \n9. | WPI + Maltodextrin | Medium-chain triglyceride oil & orange oil  \n(20 vol%) | Covalent conjugation, no creaming over 40 days experimental period | (Akhtar & Dickinson, 2007)  \n10. | WPC + CMC | Corn oil  \n(10 vol%) | WPC-CMC complex formation inducing bridging flocculation at pH5, 0.3 M NaCl | (Damianou & Kiosseoglou, 2006)  \n11. | Sodium caseinate + Pectin (High-methoxyl: 59% DE, Low-methoxyl: 32% DE) | Corn oil  \n(10 wt%) | Bridging flocculation\/depletion interaction depending on pH and pectin type | (Surh et al., 2006)  \n12. | WPI + Chitosan | Canola oil  \n(10 vol%) | Electrostatic interaction and stable emulsion at pH > 5, depletion flocculation at pH < 5 | (Laplante et al., 2005)  \n13. | \u03b2-lactoglobulin + Carrageenan | Corn oil  \n(5 wt%) | Stable emulsion on thermal processing at ionic strength (< 500 mM NaCl, < 2 mM CaCl2) | (Gu et al., 2005a, 2005b)  \n14. | \u03b2-lactoglobulin + Dextran | Sunflower oil  \n(10 wt%) | Depletion flocculation but stability obtained by sucrose (> 20%) addition | (Blijdenstein et al., 2004)  \n15. | Sodium caseinate + \u03ba-Carrageenan | Soybean oil  \n(30 wt%) | Electrostatic repulsion; depletion flocculation at 55\u00b0 C, pH 7.0 | (Singh et al., 2003)  \n16. | Sodium caseinate + Apple pectin | Corn oil  \n(25 vol%) | Covalent conjugation, better emulsion stability than that of gum arabic and commercial emulsifiers | (Al-Hakkak & Kavale, 2002)  \n17. | Sodium caseinate + Xanthan gum | Soybean oil  \n(30 wt%) | Depletion flocculation, creaming stability obtained by flocculated droplet network | (Hemar et al., 2001a)  \n18. | Sodium caseinate + Portulaca oleracea gum | Medium-chain triglycerides (5wt%) | Caseinate-portulaca gum electrostatic complex providing better emulsion stability | (Garti et al., 1999)  \n19. | \u03b2-lactoglobulin + Pectin (59% DE) | Corn oil  \n(10 wt%) | Stable emulsion at pH (3\u20134), 0.1 M NaCl | (Guzey et al., 2004)  \n20. | \u03b2-lactoglobulin + Pectin (59% DE) + Chitosan | Corn oil  \n(2.5 wt%) | Stable tertiary emulsion at pH (3\u20135), 0.1 M NaCl | G\u00fczey & McClements, 2006a, 2006b)  \n21. | \u03b2-lactoglobulin + Sodium alginate | Hydrogenated palm oil  \n(5 wt%) | Electrostatic attraction at pH (3\u20135), bridging flocculation at pH (6\u20137), improved emulsion stability with sonication | (Pongsawatmanit et al., 2006)  \n22. | Lysozymes + Dextran | Corn oil  \n(25.0\u2013vol%) | Covalent conjugation and improved emulsion stability | (Nakamura et al., 1991; Scaman et al., 2006)  \n23. | Lysozymes + Galactomannan | Corn oil  \n(25.0-vol%) | Covalent conjugation and cationic electrostatic repulsion providing emulsion stability | (Nakamura et al., 1994; Scaman et al., 2006)  \n24. | \u03b2-lactoglobulin + Dextran | Sunflower oil  \n(20.0-wt%) | Covalent conjugation providing better emulsion stability | (Dunlap & C\u00f4t\u00e9, 2005)  \n25. | Lysozymes + Chitosan | Corn oil  \n(25.0-vol%) | Covalent conjugate: Emulsion stability\u2013High-molecular-type chitosan > Low molecular type | (Song et al., 2002)\n\nThe interaction of polysaccharides with proteins is not limited to electrostatic interactions. Apart from the use of transglutaminase as cross-linkers between proteins, covalent conjugates formed via Maillard reactions between milk proteins and polysaccharides have gained much interest due to their improved emulsification abilities compared with the biopolymer alone. These conjugates are stable over a wide range of temperature, pH, and ionic strength. The conjugates with a high molecular weight possess both the properties of a hydrophobic protein being adsorbed to the surface of the oil droplet and the properties of a hydrophilic polysaccharide being highly hydrated by the aqueous phase. Although these conjugates possess both a hydrophobic and hydrophilic group and are effective surface active polymers, the presence of excess unreacted hydrocolloid may lead to depletion effects (Syrbe et al., 1998). Consequently, interfacial layers made up of different structures, thicknesses, compositions, and charges require knowledge of the functionality of different protein\u2013polysaccharide combinations to meet the structural demands, environmental challenges, and stability of food emulsions.\n\n## Rheological properties and microstructures of protein\u2013polysaccharide systems\n\nThe rheological properties of a solution containing only protein are expected to be different from those of a pure polysaccharide solution. Polysaccharide molecules generally have a greater effect in causing a significant increase in solution viscosity than proteins. This is because polysaccharide molecules are usually much larger and more extended (\u223c5.0 \u00d7 105 to 2.0 \u00d7 106 Da) than globular proteins (\u223c1.0 \u00d7 104 to 1.0 \u00d7 105 Da). Hence, polysaccharide molecules generally occupy larger hydrodynamic volumes that give rise to higher solution viscosity. The above assumes that intermolecular interactions are absent or negligible (e.g., in dilute solution). When intermolecular interactions are present among neighboring polymer molecules (i.e., polysaccharide\u2013polysaccharide or protein\u2013protein), the rheological properties of many systems are expected to change significantly. The changes in rheological properties may arise as a result of an increase in the size of particles (e.g., protein\u2013polysaccharide complexes) or when depletion interactions occur in the mixed system or if one or more polymer species form continuous network structures. The overall effect results in the formation of different microstructures. Schematic illustrations of some possible microstructures formed from mixtures of protein and polysaccharides under some specific conditions (e.g., pH, ionic strength, heat treatment, etc.) are shown in Figures 13.3a and 13.3b.\n\nFigure 13.3a Schematic diagrams of some possible microstructures formed between noninteracting protein\u2013polysaccharide mixtures. Circle (\u2022) represents protein; coil structure represents polysaccharide molecules. (a) Flocculated protein network is formed, with polysaccharide filling the space in the network; (b) polysaccharide molecules overlap and form continuous 'network,' with protein filling the space; (c) particulate protein gel network formed, with polysaccharide filling the space; (d) polysaccharide gel network formed, with protein filling the space; (e) bi-continuous network formed from protein and polysaccharide; (f) polysaccharide gels dispersed among weakly flocculated protein network; and (g) protein gels dispersed among entangled polysaccharide molecules.\n\nFigure 13.3b Schematic diagrams of some possible microstructures formed between interacting protein\u2013 polysaccharide mixtures. Circle (\u2022) represents protein; coil structure represents polysaccharide molecules. (a) Protein\u2013polysaccharide complexes formed; (b) protein interacting with gelling polysaccharides helices; (c) polysaccharide interacting with protein particulate gel network; and (d) polysaccharide gel helices interacting with protein particulate gel network.\n\nTo characterize the physicochemical properties of protein\u2013polysaccharide systems, various rheological techniques have been employed. Generally, if the mixtures are liquid-like, viscosity measurements using rotational viscometers are commonly used to obtain steady-state viscosity curves, yield stress, and the like. Other simpler methods include the use of a kinematic viscometer (e.g., the Ubbelodhe capillary viscometer) to obtain a single point relative viscosity measurement. If the samples are viscoelastic (e.g., gels), rheometers are widely used to obtain rheological data (e.g., loss and storage moduli obtained within the linear viscoelastic region) by performing small deformation oscillatory measurements. The rheological data yield information on the viscosity and viscoelastic properties of the mixed systems. Knowledge of the rheological properties of mixed protein\u2013polysaccharide systems is essential to gain insights into the nature of the interactions and the resulting microstructure of the system. A fundamental understanding of the interactions at the molecular and colloidal levels will provide a strong foundation in exploiting the physical functionality of such complex systems in different applications (e.g., microencapsulation technology, imparting specific sensory characteristics, time\/temperature\/pH\/ionic control-release, emulsion stability, etc.).\n\nIn the following sections, we provide various examples of mixed systems involving different milk proteins and polysaccharides. An attempt was made to classify these mixed systems into two broad categories (i.e., interacting and noninteracting). Under each of these headings, they are further grouped according to whether the systems form or do not form gels (i.e., gelling or nongelling). The discussion focuses mainly on the techniques used and the rheological properties of the systems.\n\n### Noninteracting Protein\u2013Polysaccharide Mixtures\n\nNoninteracting protein\u2013polysaccharide mixtures existing as one phase are rare, but may take place when the two different molecular species have good chemical resemblance in terms of hydrophilicity and conformation (Tolstoguzov, 1991; ). Many polymer mixtures are thermodynamically incompatible, and segregative interactions often occur in the absence of electrostatic interaction or in the presence of electrostatic repulsion (Neiser et al., 1998). Protein\u2013polysaccharide mixtures that commonly exist as two separate phases are the result of either thermodynamic incompatibility or depletion phenomenon (Doublier et al., 2000).\n\n### Nongelling Phase-separated System\n\nThe following are examples of noninteracting protein and polysaccharide mixtures. Both proteins and polysaccharides were mixed under conditions where the mixtures did not form gels. The rheological properties of these systems are discussed in relation to their interactions and the microstructures formed.\n\n#### Casein Micelles and Galactomannans\n\nA noninteracting protein\u2013polysaccharide mixture wherein phase separation occurs has been reported in the case of a mixed system consisting of micellar casein (3%) and guar gum (0.2%) at pH 7 (Bourriot et al., 1999b). The rheological properties showed a significant change in the flow and viscoelastic properties compared to the individual biopolymer system. With the mixed system, an increase in the apparent viscosity was reported. Furthermore, the mechanical spectra (elastic modulus G', viscous modulus G\") of the frequency sweeps showed slightly higher values of the moduli, which were less frequency dependent. The results suggested the formation of a weak network structure within the system due to the flocculation of casein micelles as the polysaccharide molecules were excluded from the protein phase. The appearance of a slightly thixotropic behavior indicated that the network can be easily broken under shear because the network formed by the micellar casein was weakly flocculated and reversible, presumably attributable to the depletion\u2013flocculation mechanism. The study also showed that the lower the intrinsic viscosity of the polysaccharide, the higher the concentration of the polysaccharide required before phase separation occurred (Bourriot et al., 1999b). An increase in the concentration of polysaccharide resulted in stronger flocculation of the casein micelles as the volume occupied and the osmotic pressure from the surrounding polysaccharides increased.\n\nSimilar thixotropic behavior has been reported for a ternary solution consisting of micellar casein\/LBG\/sucrose (Schorsch et al., 1999). The results from the ternary solution showed that at pH \u223c6.8, casein micelles and LBG were thermodynamically incompatible, behaving as a water-in-water emulsion. The presence of sucrose even at high concentration (40%) did not significantly improve the compatibility of the biopolymers (Schorsch et al., 1999).\n\n#### Milk Proteins and Xanthan\n\nAnother study investigated the interaction between xanthan gum (0\u20131% w\/w, a polysaccharide known to have 'weak gel' properties) and different types of milk proteins 5% w\/w, sodium caseinate (Na-CN), skim milk powder (SMP), whey protein isolate (WPI), and milk protein concentrate (MPC)] in an aqueous solution at neutral pH ([Hemar et al., 2001b). Depending on the xanthan gum concentrations and the protein type, the microstructures of the mixtures were different. In the case of xanthan mixtures with either MPC or SMP, depletion flocculation of casein micelles took place. The size of the depleted protein aggregates decreased with increasing xanthan concentration (microstructures resembled a particulate network). In the case of xanthan solutions containing either Na-CN or ultracentrifuged WPI, no phase separation occurred within the timescale of the experiment. This was attributed to the larger size of casein micelles (average diameter \u223c0.2 \u03bcm) compared to the nanometer-size scale of WPI and Na-CN (0.05 \u03bcm) (Lucey et al., 2000). However, the rheological behavior of the mixtures was found to be very similar to the rheological behavior of xanthan. The differences in microstructures of the mixtures observed by the confocal laser scanning microscope (CLSM) were not detected by viscosity measurements probably because the weakly flocculated proteins were easily re-dispersed by the shearing action of the viscometer during measurement.\n\n### Gelling Phase-separated System\n\nIn a system where two biopolymer species (e.g., proteins and polysaccharides) do not interact, gelation of one or more of the components in a thermodynamically incompatible system will cause competition between phase separation and gelation (Neiser et al., 1998). Gelation basically means the formation of a three-dimensional aggregated network structure, which is generally induced by heating, cooling, acidification, enzymatic treatments, high-pressure processing, and so on. Generally, heating enhances hydrophobic and covalent interactions. In the case of whey protein, unfolded proteins interact to give rise to aggregates (Boye et al., 1997; Kinsella, 1984). In mixed systems, the microstructure will depend on the rates of phase separation and gel formation (Tavares et al., 2005). The gel may appear homogeneous at a macroscopic level but heterogeneous at the microscopic level. However, the rheological properties of such gels depend on the concentration and arrangement of each species in the different phases. If the gelling species is in the continuous phase, the gel strength is higher than one in the dispersed phase where the network is disrupted (Neiser et al., 1998).\n\n#### Whey Protein and Galactomannans\n\nOne such study was based on a mixture of LBG (a nongelling neutral polysaccharide) and whey protein at neutral pH and pH 5 (close to the pI of whey proteins) (Tavares & Lopes da Silva, 2003). At neutral pH, it is known that whey protein forms clear fine-stranded gels (protein aggregation is hindered by electrostatic repulsion), while at lower pH (e.g., pH 5), an opaque course particulate gel is formed (Aguilera, 1995; Langton & Hermansson, 1992). Rheological measurements showed that whey protein isolate gel (13% w\/w) had a stronger and more elastic character at pH 5 than at pH 7 because of the thick particulate network formed (Bertrand & Turgeon, 2007; Stading et al., 1993). For the protein gels at pH 7, increasing LBG concentration (>0.25%) decreased both the onset temperature for gelation and the gelation time. The presence of LBG was also found to increase gel rigidity. The authors attributed this effect to a decrease in macromolecular mobility within the network in the presence of LBG, due to segregative interactions and the 'local' concentration of each polymer species. The LBG molecules acted as fillers in the continuous protein network. At pH 5, the elastic character of the particulate gel network was shown to decrease in the presence of LBG, especially at low protein concentration (5%). It was suggested that LBG chains hampered protein\u2013protein interactions and were detrimental to the protein gel development. However, at a higher protein concentration (13%) where sufficient particulate gel network was formed, LBG acted as fillers within the network, improving the gel strength.\n\nIn a subsequent study carried out using whey protein isolate and guar gum at pH 7, an increase in protein gel strength was found with a decreasing degree of branching of the galactomannans (Tavares et al., 2005). Like LBG, the guar gum was dispersed as droplets among the whey protein network at low concentration (0.2%). However, at higher gum concentration (0.6%), the dispersed droplets joined to form a continuous polysaccharide-rich phase. Despite the different microstructures observed, the linear viscoelastic profiles were rather similar, indicating that viscoelasticity was fairly insensitive to microstructural changes of this nature.\n\n#### Whey Protein Isolate and Xanthan\n\nA very similar trend was observed for whey protein and xanthan mixtures after heat treatment (Bertrand & Turgeon, 2007). The microstructures and rheological properties of the gels were highly dependent on pH and salt concentration. At pH 6.5, the presence of xanthan improved the elastic modulus of the WPI gel. This was attributed to segregative phase separation, where xanthan was dispersed among the protein gel network. However, upon lowering the pH to 5.5 (close to the pI of WPI), the addition of xanthan decreased the elastic modulus of the gel. It was suggested that the possible formation of WPI\u2013xanthan complexes decreased protein\u2013protein interactions, producing a weaker gel network.\n\n#### \u03b2-Lactoglobulin and Pectin\n\nA different type of network was formed in mixtures of \u03b2-lactoglobulin (8% w\/w) and low methoxyl (LM) pectin (0.85% w\/w) after thermal treatment at pH 6.8. The storage modulus of the mixed gel system was significantly lower than the protein gel alone. Microstructure observed by CLSM revealed phase separation, with \u03b2-lactoglobulin appearing as spherical colloidal particles distributed in a continuous pectin network (Donato et al., 2005). A similar type of protein depletion-induced phase separation was reported for a mixed system containing aggregated whey protein and an exopolysaccharide (EPS) from lactic acid bacteria (Tuinier et al., 2000).\n\n#### \u03ba-Carrageenan and \u03b2-Lactoglobulin\n\nIf two gelling species are present in a binary system, the mixed gels may form interpenetrating, coupled, or phase-separated networks (Morris, 1986). Interpenetrating networks are the result of two independent continuous networks formed throughout the gel, and only topological interactions exist between the networks. Coupled networks (ordered into junction zones like those of a polysaccharide gel) are formed when favorable interactions between the two molecular species exist. However, such systems involving protein\u2013polysaccharide interactions are uncommon (Rao, 1999).\n\nPhase-separated networks are formed when one polymer species is incompatible with the other, forming phase-separated regions within the network gel (Piculell & Lindman, 1992; Turgeon & Beaulieu, 2001). An example of a phase-separated gel can be found with \u03ba-carrageenan and \u03b2-lactoglobulin (Capron et al., 1999). The mixed polymer formed a weaker gel than the carrageenan gel alone when the protein was in its native state. Upon heating the mixture to 90 \u00b0C, holding for 30 min and then cooling to 20 \u00b0C, the gel rheology indicated the melting of \u03ba-carrageenan and the gelation of \u03b2-lactoglobulin above 65 \u00b0C. There was no aggregation of \u03ba-carrageenan with \u03b2-lactoglobulin upon heating. The gelation time of \u03b2-lactoglobulin was reduced in the presence of \u03ba-carrageenan, which was attributed to micro phase separation, which caused an increase in local concentration of the \u03b2-lactoglobulin (Capron et al., 1999). Upon cooling, the mixed gel system formed a phase-separated bicontinuous network (Ould Eleya & Turgeon, 2000).\n\n### Interacting Protein\u2013Polysaccharide Mixtures\n\nAnother phase separation phenomenon is the associative phase separation where associative interactions are present. Associative interactions between protein and polysaccharide can occur as a result of electrostatic interactions, hydrogen bonding, hydrophobic interactions, or poor solvent conditions (Antonov et al., 1996b; de Kruif et al., 2004; Doublier et al., 2000; Gao & Dubin, 1999). In some cases, complexes known as coacervates are formed via electrostatic interactions. Coacervates of protein\u2013polysaccharide can occur when the pH of the mixture is lower than the pI of the protein. At this pH, protein possesses a net positive charge while the polysaccharides still possess a negative charge. The result of the complexation is the formation of a solvent-rich phase and a coacervate-rich phase (Doublier et al., 2000; Ould Eleya & Turgeon, 2000). The rheological properties of milk\u2013protein polysaccharide complexes are related to the interaction between the complexes and the water molecules, which forms soluble (or liquid coacervate phase) or insoluble (or precipitate) complexes. The solubility of complexes is based on the energetic difference between biopolymer\u2013biopolymer and biopolymer\u2013solvent interactions (Damodaran, 1997). The main parameters that affect the solubility of biopolymer complexes are charge density, pH, ionic strength, and protein:polysaccharide (PP:PS) ratio (Schmitt et al., 1998). It has been suggested that a complex involving a strong polyelectrolyte will form a precipitate rather than a liquid coacervate. A number of protein\u2013polysaccharide systems where complex coacervations occur have been reviewed by several authors (de Kruif et al., 2004; Schmitt et al., 1998; Turgeon et al., 2003). The following section presents some examples of interacting polymers in mixed systems and the effect on rheological properties.\n\n### Nongelling Phase-separated System\n\n#### \u03b2-Lactoglobulin and Chitosan\n\nIt has been reported that protein solubility increases below its pI when it complexes with an anionic polysaccharide (Tolstogusov, 1986; Tolstoguzov et al., 1985). A recent study of the \u03b2-lactoglobulin-chitosan complex has shown that depending on the pH, the complex is either soluble or insoluble (Guzey & McClements, 2006a, 2006b). The interaction of soluble chitosan (MW = 15,000 Da, DD = 85%, 0\u20130.1 wt%, 5mM phosphate buffer) with \u03b2-lactoglobulin (0.5 wt% \u03b2-lg, 5mM phosphate buffer) in aqueous solutions studied at pH 3\u20137 showed that at pH 3, 4, and 5, the majority of the \u03b2-lactoglobulin\u2013chitosan complex in the solutions was soluble, but at pH 6 and 7 a significant fraction of the two biopolymers was insoluble.\n\n#### Whey Proteins and Exopolysaccharides\n\n'Soluble complexes' formed via electrostatic interactions were reported for EPS B40 (an exopolysaccharide from Lactococcus lactis subsp cremoris NIZO B40) and whey proteins (PP:PS = 2:1) under specific pH and ionic conditions (with no macroscopic phase separation) (Weinbreck et al., 2003b). Decreasing the pH of the mixtures increased further aggregation of the complexes, which led to phase separation. In addition, increasing the ionic strength of the solution caused a shift to lower pH value for the onset of complexation. In this study, complexation in this system led to a decrease in solution viscosity as intramolecular repulsion of the EPS was reduced in the presence of whey proteins. The decrease in viscosity was attributed to a reduction of the quantity of dispersed phase, that is, water present within the complexes. Consequently, it was suggested that dilute solution viscosity measurement (which is related to the size of complexes) could be used to determine the optimum conditions for complexation (Weinbreck et al., 2003b). A potential benefit of this complexation is that it protects the protein from loss of solubility due to aggregation during thermal or high-pressure treatments (Galazka et al., 1997; Imeson, 1977).\n\n#### Whey Proteins and Gum Arabic\n\nViscosity curves were obtained to evaluate the 'strength' of electrostatic interactions of whey protein\/gum arabic coacervates (Weinbreck & Wientjes, 2004). This study showed that the stronger the interaction, the greater the shear-thinning behavior and the slower the reformation of the complexes after shearing. The highly viscous coacervates at pH 4 were considered to be due to electrostatic interactions. At pH above the pI (without electrostatic interactions), the mixtures appeared to be more elastic than viscous.\n\n#### Sodium Caseinate and Gum Arabic\n\nIn contrast to whey proteins, sodium caseinate and gum arabic mixtures show some peculiar behavior (Ye et al., 2006) as no coacervation is observed in these systems. Below a certain pH (pH 5.4), electrostatic interactions between sodium caseinate and gum arabic leads to the formation of stable composite particles in the size range 100\u2013200 nm. Over a pH range of 3.2\u20135.4, the particle complexes were consistent in size and remained stable and soluble. This pH range could shift depending on the ratio of sodium caseinate to gum arabic and ionic strength in the mixtures. The sodium caseinate\/gum arabic particles associated to form larger particles, which resulted in phase separation when the pH was lower than 3.0. A mechanism for the formation of these particles based around the self-aggregation of casein and the electrostatic interaction between the aggregated particles of casein and gum arabic molecules has been proposed. As the pH of the mixture decreases below pH 5.4, the caseinate molecules tend toward small-scale aggregation, prior to large-scale aggregation and precipitation at pH values closer to their pI (pH 4.6). In this case, in the early stages of aggregation the gum arabic molecules may attach to the outside of these small-scale aggregates through electrostatic interactions between negatively charged gum arabic and exposed positive patches on the surface of the caseinate aggregates. The presence of hydrophilic gum arabic molecules on the outside of the caseinate aggregate may be enough to sterically stabilize these nanoparticles and consequently prevent self-aggregation. As the charge on these particles is quite low (\u223c15 mV at pH 4.0), steric stabilization is probably important. In a recent study, the formation of sodium casein\u2013gum arabic complexes was reported to occur at temperatures above 60 \u00b0C at a certain mass ratio of protein to gum arabic (e.g., 1:5) and pH (maximum complexation at pH 6.5) (Ye et al., 2012). Interestingly, the complex formation is reversible when the temperature is decreased to below 60 \u00b0C (although not in the case of pH 5.0). The temperature-dependent complexation between sodium caseinate and gum arabic was attributed to hydrophobic interactions between the two polymer molecules. These unique complexes can potentially be used to form interfacial layers of emulsion droplets that can be altered by temperature.\n\n#### Casein Micelle and Pectin\n\nProtein\u2013polysaccharide interactions have been shown to be pH-dependent, as in the case of pectin and casein micelles (Ambjerg & J\u00f8rgensen, 1991; Maroziene & de Kruif, 2000). At pH 6.7, pectin did not adsorb onto casein micelles. With sufficient pectin present (0.1\u20130.2%), phase separation occurred due to depletion interactions of the casein micelles (\u223c0.1%). However, adsorption of pectin onto the casein micelles did occur at pH 5.3. Viscosity measurements were employed to study the changes that occurred at different polymer concentrations. At low pectin concentrations (\u223c0.1%) and at pH 5.3, a maximum viscosity was reached that was attributed to bridging flocculation. Bridging among the casein particles was interpreted as having a larger effective volume. As pectin concentration increased (>0.1%), the casein micelles became fully covered, and interactions between casein particles were reduced. This caused a decline in viscosity, but the solution viscosity remained higher than the pure milk samples (without added pectin). The amount of pectin required for full coverage of the casein micelles differed depending on the type of pectin in the sequence high methoxyl (HM) < low methoxyl amidated (LMA) < LM pectin. Adding pectin at levels beyond the concentration required for full coverage led to phase separation due to depletion interactions. A further increase in pectin reduced the thickness of the casein depleted layer, as the viscosity of the continuous phase became very high and gelled polymer networks were formed (Maroziene & de Kruif, 2000). When the pH of the mixture was increased from 5.3 back to 6.7, desorption of pectin from the casein occurred, but over a much longer timescale (\u223c10\u201315 min) than the adsorption process (Maroziene & de Kruif, 2000).\n\n### Gelling Phase-separated System\n\n#### Sodium Caseinate and Pectin\n\nThe dynamic rheological properties of glucono-\u03b4-lactone (GDL) acidified protein gels (2% w\/v Na-CN) were studied in the presence of LMA pectin (0.01\u20131%w\/v) at pH\u223c4 (Matia-Merino et al., 2004). The presence of pectin (0.01\u20130.05% w\/v) decreased the storage modulus and increased the gelation time as pectin adsorped onto the casein particles. At pectin concentrations >0.08% w\/v, acid-induced gelation appeared to be completely inhibited over \u223c9 hr at 25 \u00b0C.\n\n#### Casein Micelles and Iota-Carrageenan\n\nIn the case of casein\u2013carrageenan mixed systems, the attractive interactions involved the negatively charged sulfated groups of the polysaccharides and the positive 'patches' between residues 97 and 112 of \u03ba-caseins (Snoeren, 1975), despite a pH above the pI isoelectric point and an overall net negative charge of the casein micelles. The interaction between \u03b9-carrageenan (0.5%) and skim milk (based on 3.3% protein) mixtures was studied above and below carrageenan's coil-helix transition temperature (Langendorff et al., 1999). Above this temperature, carrageenan did not adsorb to casein micelles, resulting in depletion flocculation, while below this temperature, attractive interactions between carrageenan and casein micelles occurred. The higher charge density of the double-helix form as compared to the coil conformation of carrageenan probably explained the stronger attractive interaction between casein micelles and carrageenan. The presence of casein micelles increased the gel strength (indicated by higher G' and G\") and the gelation temperature (from 39 \u00b0C to 47 \u00b0C) when the mixtures were heated to 65 \u00b0C and cooled down to 25 \u00b0C. Depending on the concentration of carrageenan, different types of gel network were deduced from the frequency sweep. At low carrageenan concentrations (<0.2%), one type of network was formed on cooling. This was probably due to the bridging of casein micelles by the adsorbed carrageenan helical chains. The network was much more thermally stable than the pure carrageenan gels. At carrageenan concentrations above 0.2%, in addition to the formation of a network as described above, a second network was formed, similar to that of carrageenan gel in the absence of proteins. This was attributed to interactions between carrageenan chains (Langendorff et al., 1999). For the different types of carrageenans, the amount required for full coverage increased from \u03ba< \u03b9< \u03bb (Langendorff et al., 1997) as the charge density of the polymer determined the strength of adsorption (Maroziene & de Kruif, 2000; Pereyra et al., 1997).\n\nFrom the above examples, it is clear that mixed protein and polysaccharide systems can provide very different physicochemical properties. The properties of these systems are the results of cumulative effects from the molecular parameters of the macromolecules (e.g., size, conformation, charged density, concentration, polysaccharide:protein ratio), the conditions the mixed systems are subjected to (e.g., pH, temperature, ionic strength), and the resulting interactions among the macromolecules (e.g., type of interaction, strength of interactions, gels, or aggregates). Understanding the physicochemical properties of these systems may help in the development of novel food structures with unique sensory properties and functionalities, including microencapsulation and controlled-release applications.\n\n## Concluding remarks\n\nProteins and polysaccharides are the two main structural entities in foods, and a great deal of work has been published on their interactions over the last few decades. The following highlights some of the current and future key areas of research in protein\u2013polysaccharide interactions.\n\nProtein\u2013polysaccharide interactions using plant proteins (e.g., soy and pea) and egg proteins are gaining increasing interest among researchers. Proteins from different sources differ in their amino acid sequence, tertiary structures, molar mass, and distribution of reactive groups. All of these features can contribute to different functionalities in different environmental conditions when these proteins interact with polysaccharides (Andrade et al., 2010; Elmer et al., 2011; Mession et al., 2012; Miquelim et al., 2010; Souza et al., 2013; Tran & Rousseau, 2013; Yin et al., 2012).\n\nResearch on novel polysaccharides from new sources will continue, as the structural complexity of these materials continues to intrigue researchers. Their interactions with proteins depend largely on their chemical composition, chain length, chain conformation, and reactive groups present (Ettelaie et al., 2012; Yadav et al., 2010; Yin et al., 2012).\n\nProtein\u2013polysaccharide conjugates formed by Maillard-type reactions with different protein and polysaccharide combinations continue to be effective in improving emulsion stability. The area of research is envisaged to continue, with better understanding of the functionality of new protein and polysaccharide sources (Kasran et al., 2013; Markman & Livney, 2012; O'Regan & Mulvihill, 2010; Xu et al., 2012; Zhang et al., 2012).\n\nProtein\u2013polysaccharide interactions based on different ratios of proteins to polysaccharides over a range of pH, temperature, and ionic strengths have been shown to produce complexes that differ in functionality. Thus, further exploration of the impact of environmental conditions is likely. There is much room for continued research due to wide permutation of proteins and polysaccharides (Liu et al., 2012; Paraskevopoulou et al., 2013; Ru et al., 2012; Souza et al., 2013; van Gruijthuijsen et al., 2012).\n\nModifications of existing polysaccharides and proteins by chemical, enzymatic, and physical techniques have also drawn interest among scientists. Among these techniques, physical modification techniques such as ultra-high pressure, ultrasonication, electric-pulsed, and microwave irradiation appear to be favored because of their environmentally friendly nature (Bigikocin et al., 2011; Ma & Wang, 2013; Panteloglou et al., 2010; Singh et al., 2012; Wong et al., 2010).\n\nParticles of nanometric size formed by a combination of proteins and polysaccharides through various interactions such as hydrogen, electrostatic, hydrophobic, and covalent bonds will continue to offer opportunities to create new structures with desired functionalities. For instance, such particles are used to form and stabilize emulsions and dispersed particles. The physicochemical properties of emulsions stabilized by nanoparticles are substantially different from protein-stabilized emulsions. This provides opportunities for texture modification and controlled delivery of nutrients (Dickinson 2012; ).\n\nIn recent years, excellent progress has been made on understanding the key variables and interactions that control the physical stability, rheology, and microstructure of protein\u2013 polysaccharide mixtures. Although milk proteins, particularly whey proteins, have formed the basis of the most widely studied protein\u2013polysaccharide systems, most work has dealt with relatively simple binary combinations of one protein and one polysaccharide. More complex systems, including ternary mixtures, still remain to be investigated in detail. At a practical level, it seems possible to manipulate these interactions and produce different microstructures by controlling the internal (pH, ionic strength, biopolymer ratio, molecular weight, and charge of the biopolymer) and external (temperature, pressure, and shear rate) parameters. However, there is a considerable challenge in understanding how different microstructures relate to the sensory and nutritional properties of food products, such as mouthfeel and nutrient release.\n\nThe formation of complexes through hydrogen, electrostatic, hydrophobic, and covalent interactions has been the subject of intensive studies. This is mainly because of the potential for better functionality of the composites compared with the protein or polysaccharide alone, for example, in terms of rheology, gelation, and interfacial properties. Protein\u2013 polysaccharide complexes can serve as texturing agents, encapsulating agents, fat replacers, and stabilizers of emulsions and other dispersed systems. However, information is still lacking on the detailed molecular structures of protein\u2013polysaccharide complexes, and describing the experimental observations within the known theoretical frameworks remains a challenge.\n\nIt is now becoming apparent that the modification of food structure through modulation of macromolecular interactions can also be used to control the release of nutrients and bioactive components during digestion, and to target where and how such components are released. The basic science underpinning these functions is largely unknown. New knowledge in this area will enable development of composite food systems and ingredients that are superior in nutritional value and textural characteristics.\n\n# References\n\nAguilera JM .  Gelation of whey proteins .  _Food Technology_.  1995 ;49 : 83 \u2013 89 .\n\nAkhtar M ,  Dickinson E .  Emulsifying properties of whey protein-dextran conjugates at low pH and different salt concentrations .  _Colloids and Surfaces B: Biointerfaces_.  2003 ;31 (1\u20134) : 125 \u2013 132 .\n\nAkhtar M ,  Dickinson E .  Whey protein-maltodextrin conjugates as emulsifying agents: An alternative to gum arabic .  _Food Hydrocolloids_.  2007 ;21 (4) : 607 \u2013 616 .\n\nAl-Hakkak J ,  Kavale S .  Improvement of emulsification properties of sodium caseinate by conjugating to pectin through the Maillard reaction .  _International Congress Series_.  2002 ;1245 : 491 \u2013 499 .\n\nAmbjerg PHC ,  J\u00f8rgensen BB .  Influence of pectin on the stability of casein solutions studied in dependence of varying pH and salt concentration .  _Food Hydrocolloids_.  1991 ;5 : 323 \u2013 328 .\n\nAndrade RJ ,  Azevedo AG ,  Musampa RM ,  Maia JM .  Thermo-rheological behavior of model protein-polysaccharide mixtures .  _Rheologica Acta_.  2010 ;49 (4) : 401 \u2013 410 .\n\nAntonov YA ,  Grinberg VY ,  Zhuravskaya NA ,  Tolstoguzov VB .  Concentration of the proteins of skimmed milk by membraneless, isobaric osmosis .  _Carbohydrate Polymers_.  1982 ;2 (2) : 81 \u2013 90 .\n\nAntonov YA ,  Lashko NP ,  Glotova YA ,  Malovikova A ,  Markovich O .  Effect of the structural features of pectins and alginates on their thermodynamic compatibility with gelatin in aqueous media .  _Food Hydrocolloids_.  1996 ;10 (1) : 1 \u2013 9 .\n\nAntonov YA ,  Lashko NP ,  Glotova YK ,  Malovikova A ,  Markovich O .  Effect of the structural features of pectins and alginates on their thermodynamic compatibility with gelatin in aqueous media .  _Food Hydrocolloids_.  1996 ;10 (1) : 1 \u2013 9 .\n\nAsakura S ,  Oosawa F .  On interaction between 2 bodies immersed in a solution of macromolecules .  _Journal of Chemical Physics_.  1954 ;22 (7) : 1255 \u2013 1256 .\n\nAsakura S ,  Oosawa F .  Interaction between particles suspended in solutions of macromolecules .  _Journal of Polymer Science_.  1958 ;33 (126) : 183 \u2013 192 .\n\nBeaulieu M ,  Turgeon SL ,  Doublier J-L .  Rheology, texture and microstructure of whey proteins\/low methoxyl pectins mixed gels with added calcium .  _International Dairy Journal_.  2001 ;11 (11-12) : 961 \u2013 967 .\n\nBenichou A ,  Aserin A ,  Garti N .  Protein-polysaccharide interactions for stabilization of food emulsions .  _Journal of Dispersion Science and Technology_.  2002 ;23 (1-3) : 93 \u2013 123 .\n\nBenichou A ,  Aserin A ,  Lutz R ,  Garti N .  Formation and characterization of amphiphilic conjugates of whey protein isolate (WPI)\/xanthan to improve surface activity .  _Food Hydrocolloids_.  2007 ;21 (3) : 379 \u2013 391 .\n\nBertrand M-E ,  Turgeon SL .  Improved gelling properties of whey protein isolate by addition of xanthan gum .  _Food Hydrocolloids_.  2007 ;21 (2) : 159 \u2013 166 .\n\nBigikocin E ,  Mert B ,  Alpas H .  Effect of high hydrostatic pressure and high dynamic pressure on stability and rheological properties of model oil-in-water emulsions .  _High Pressure Research_.  2011 ;31 (3) : 462 \u2013 474 .\n\nBlijdenstein TBJ ,  Zoet FD ,  van Vliet T ,  van der Linden E ,  van Aken GA .  Dextran-induced depletion flocculation in oil-in-water emulsions in the presence of sucrose .  _Food Hydrocolloids_.  2004 ;18 (5) : 857 \u2013 863 .\n\nBoeriu CG ,  Oudgenoeg G ,  Spekking WTJ ,  Berendsen L ,  Vancon L ,  Boumans H ,  Voragen AGJ .  Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with beta-casein .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 (21) : 6633 \u2013 6639 .\n\nBourriot S ,  Garnier C ,  Doublier JL .  Phase separation, rheology and microstructure of micellar casein-guar gum mixtures .  _Food Hydrocolloids_.  1999 ;13 (1) : 43 \u2013 49 .\n\nBourriot S ,  Garnier C ,  Doublier JL .  Phase separation, rheology and structure of micellar casein-galactomannan mixtures .  _International Dairy Journal_.  1999 ;9 (3\u20136) : 353 \u2013 357 .\n\nBourriot S ,  Garnier C ,  Doublier JL .  Micellar-casein-k-carrageenan mixtures. I. Phase separation and ultrastructure .  _Carbohydrate Polymers_.  1999 ;40 (2) : 145 \u2013 157 .\n\nBoye JI ,  Ma CY ,  Harwalker VR .  Thermal denaturation and coagulation of proteins .  In:  Damodaran S ,  Paraf A ,  eds.  _Food proteins and their applications_ .  New York :  Marcel Dekker ;  1997 : 25 \u2013 56 .\n\nBraudo EE ,  Antonov YA .  Non-coulombic complex formation of protein as a structure forming factor in food systems .  In:  Schwenke KD ,  Mothes R ,  eds.  _Proteins, Structure and Functionality_ .  VCH :  Weinheim ;  1993 .\n\nBryant CM ,  McClements DJ .  Optimizing preparation conditions for heat-denatured whey protein solutions to be used as cold-gelling ingredients .  _Journal of Food Science_.  2000 ;65 (2) : 259 \u2013 263 .\n\nBryant CM ,  McClements DJ .  Influence of xanthan gum on physical characteristics of heat-denatured whey protein solutions and gels .  _Food Hydrocolloids_.  2000 ;14 (4) : 383 \u2013 390 .\n\nBungenberg de Jong HG ,  Kruyt HR .  Coacervation (partial miscibility in colloid systems .  _Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen_.  1929 ;32 : 849 \u2013 856. .\n\nCapron I ,  Nicolai T ,  Durand D .  Heat induced aggregation and gelation of \u03b2-lactoglobulin in the presence of k-carrageenan .  _Food Hydrocolloids_.  1999 ;13 (1) : 1 \u2013 5 .\n\nClark AH .  Direct analysis of experimental tie line data (two polymer-one solvent systems) using Flory-Huggins theory .  _Carbohydrate Polymers_.  2000 ;42 (4) : 337 \u2013 351 .\n\nCloss CB ,  Conde-Petit B ,  Roberts ID ,  Tolstoguzov VB ,  Escher F .  Phase separation and rheology of aqueous starch\/galactomannan systems .  _Carbohydrate Polymers_.  1999 ;39 (1) : 67 \u2013 77 .\n\nDalgleish DG .  The conformations of proteins on solid\/water interfaces\u2014caseins and phosvitin on polystyrene lattices .  _Colloids and Surfaces_.  1990 ;46 (2) : 141 \u2013 155 .\n\nDalgleish DG .  Structures and properties of adsorbed layers in emulsions containing milk proteins .  In:  Dickinson E ,  Lorient D ,  eds.  _Food macromolecules and colloids_ .  Cambridge, UK :  The Royal Society of Chemistry ;  1995 : 23 \u2013 34 .\n\nDalgleish DG .  Conformations and structures of milk proteins adsorbed to oil-water interfaces .  _Food Research International_.  1996 ;29 (5-6) : 541 \u2013 547 .\n\nDalgleish DG .  Food emulsions\u2014their structures and structure-forming properties .  _Food Hydrocolloids_.  2006 ;20 (4) : 415 \u2013 422 .\n\nDalgleish DG ,  Leaver J .  Dimensions and possible structures of milk proteins at oil-water interfaces .  In:  Dickinson E ,  Walstra P ,  eds.  _Food colloids and polymers: Stability and mechanical properties_ .  Cambridge, UK :  Royal Society of Chemistry ;  1993 : 113 \u2013 122 .\n\nDalgleish DG ,  Morris ER .  Interactions between carrageenans and casein micelles: electrophoretic and hydrodynamic properties of the particles .  _Food Hydrocolloids_.  1988 ;2 (4) : 311 \u2013 320 .\n\nDamianou K ,  Kiosseoglou V .  Stability of emulsions containing a whey protein concentrate obtained from milk serum through carboxymethylcellulose complexation .  _Food Hydrocolloids_.  2006 ;20 (6) : 793 \u2013 799 .\n\nDamodaran S .  Food proteins: An overview .  In:  Damodaran S ,  Paraf A ,  eds.  _Food proteins and their applications_ .  New York :  Marcel Dekker ;  1997 : 1 \u2013 24 .\n\nde Bont PW ,  van Kempen GMP ,  Vreeker R .  Phase separation in milk protein and amylopectin mixtures .  _Food Hydrocolloids_.  2002 ;16 (2) : 127 \u2013 138 .\n\nde Kruif CG ,  Tuinier R .  Whey protein aggregates and their interaction with exo-polysaccharides .  _International Journal of Food Science and Technology_.  1999 ;34 (5-6) : 487 \u2013 492 .\n\nde Kruif CG ,  Tuinier R .  Polysaccharide protein interactions .  _Food Hydrocolloids_.  2001 ;15 (4-6) : 555 \u2013 563 .\n\nde Kruif CG ,  Weinbreck F ,  de Vries R .  Complex coacervation of proteins and anionic polysaccharides .  _Current Opinion in Colloid and Interface Science_.  2004 ;9 (5) : 340 \u2013 349 .\n\nDickinson E .  Protein-stabilized emulsions .  _Journal of Food Engineering_.  1994 ;22 (1-4) : 59 \u2013 74 .\n\nDickinson E .  Proteins at interfaces and in emulsions: Stability, rheology and interactions .  _Journal of the Chemical Society, Faraday Transactions_.  1998 ;94 (12) : 1657 \u2013 1669 .\n\nDickinson E .  Stability and rheological implications of electrostatic milk protein-polysaccharide interactions .  _Trends in Food Science and Technology_.  1998 ;9 (10) : 347 \u2013 354 .\n\nDickinson E .  Caseins in emulsions: Interfacial properties and interactions .  _International Dairy Journal_.  1999 ;9 (3-6) : 305 \u2013 312 .\n\nDickinson E .  Milk protein interfacial layers and the relationship to emulsion stability and rheology .  _Colloids and Surfaces B: Biointerfaces_.  2001 ;20 (3) : 197 \u2013 210 .\n\nDickinson E .  Hydrocolloids at interfaces and the influence on the properties of dispersed systems .  _Food Hydrocolloids_.  2003 ;17 (1) : 25 \u2013 39 .\n\nDickinson E .  Use of nanoparticles and microparticles in the formation and stabilization of food emulsions. [Review] .  _Trends in Food Science and Technology_.  2012 ;24 (1) : 4 \u2013 12 .\n\nDickinson E .  Stabilising emulsion-based colloidal structures with mixed food ingredients .  _Journal of the Science of Food and Agriculture_.  2013 ;93 (4) : 710 \u2013 721 .\n\nDickinson E ,  James JD .  Influence of high-pressure treatment on \u03b2-lactoglobulin-pectin associations in emulsions and gels .  _Food Hydrocolloids_.  2000 ;14 (4) : 365 \u2013 376 .\n\nDickinson E ,  McClements DJ .  _Advances in Food Colloids_ .  London, UK :  Blackie Academic and Professional ;  1995 : pp. 27-80 .\n\nDickinson E ,  Pawlowsky K .  Effect of \u03b9-carrageenan on flocculation, creaming, and rheology of a protein-stabilized emulsion .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 (10) : 3799 \u2013 3806 .\n\nDickinson E ,  Pawlowsky K .  Influence of \u03b9-carrageenan on the properties of a protein-stabilized emulsion .  _Food Hydrocolloids_.  1998 ;12 (4) : 417 \u2013 423 .\n\nDickinson E ,  Radford SJ ,  Golding M .  Stability and rheology of emulsions containing sodium caseinate: Combined effects of ionic calcium and non-ionic surfactant .  _Food Hydrocolloids_.  2003 ;17 (2) : 211 \u2013 220 .\n\nDickinson E ,  Semenova MG .  Emulsifying properties of covalent protein\u2014dextran hybrids .  _Colloids and Surfaces_.  1992 ;64 (3-4) : 299 \u2013 310 .\n\nDickinson E ,  Semenova MG ,  Antipova AS ,  Pelan EG .  Effect of high-methoxy pectin on properties of casein-stabilized emulsions .  _Food Hydrocolloids_.  1998 ;12 (4) : 425 \u2013 432 .\n\nDonato L ,  Garnier C ,  Novales B ,  Doublier JL .  Gelation of globular protein in presence of low methoxyl pectin: Effect of Na+ and\/or Ca2+ ions on rheology and microstructure of the systems .  _Food Hydrocolloids_.  2005 ;19 (3) : 549 \u2013 556 .\n\nDoublier JL ,  Garnier C ,  Renard D ,  Sanchez C .  Protein-polysaccharide interactions .  _Current Opinion in Colloid and Interface Science_.  2000 ;5 (3-4) : 202 \u2013 214 .\n\nDumay E ,  Laligant A ,  Zasypkin D ,  Cheftel JC .  Pressure- and heat-induced gelation of mixed \u03b2-lactoglobulin\/polysaccharide solutions: scanning electron microscopy of gels .  _Food Hydrocolloids_.  1999 ;13 (4) : 339 \u2013 351 .\n\nDunlap CA ,  C\u00f4t\u00e9 GL .  \u03b2-lactoglobulin\u2013dextran conjugates: Effect of polysaccharide size on emulsion stability .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (2) : 419 \u2013 423 .\n\nElmer C ,  Karaca AC ,  Low NH ,  Nickerson MT .  Complex coacervation in pea protein isolate-chitosan mixtures .  _Food Research International_.  2011 ;44 (5) : 1441 \u2013 1446 .\n\nEnnis MP ,  Mulvihill DM .  Milk proteins .  In:  Phillips GO ,  Williams PA ,  eds.  _Handbook of hydrocolloids_ .  Cambridge, UK :  Woodhead Publishing Limited ;  2000 : 189 \u2013 217 .\n\nErcelebi EA ,  Ibanoglu E .  Influence of hydrocolloids on phase separation and emulsion properties of whey protein isolate .  _Journal of Food Engineering_.  2007 ;80 (2) : 454 \u2013 459 .\n\nEttelaie R ,  Akinshina A ,  Maurer S .  Mixed protein-polysaccharide interfacial layers: Effect of polysaccharide charge distribution .  _Soft Matter_.  2012 ;8 (29) : 7582 \u2013 7597 .\n\nEuston SR ,  Finnigan SR ,  Hirst RL .  Kinetics of droplet aggregation in heated whey protein-stabilized emulsions: Effect of polysaccharides .  _Food Hydrocolloids_.  2002 ;16 (5) : 499 \u2013 505 .\n\nFaergemand M ,  Otte J ,  Qvist KB .  Cross-linking of whey proteins by enzymatic oxidation .  _Journal of Agricultural and Food Chemistry_.  1998 ;46 (4) : 1326 \u2013 1333 .\n\nFang Y ,  Dalgleish DG .  The conformation of \u03b1-lactalbumin as a function of pH, heat treatment and adsorption at hydrophobic surfaces studied by FTIR .  _Food Hydrocolloids_.  1998 ;12 (2) : 121 \u2013 126 .\n\nFlanagan J ,  Singh H .  Conjugation of sodium caseinate and gum arabic catalyzed by transglutaminase .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 (19) : 7305 \u2013 7310 .\n\nGalazka VB ,  Ledward DA ,  Sumner IG ,  Dickinson E .  Influence of high pressure on bovine serum albumin and its complex with dextran sulfate .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 (9) : 3465 \u2013 3471 .\n\nGalazka VB ,  Smith D ,  Ledward DA ,  Dickinson E .  Complexes of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment .  _Food Chemistry_.  1999 ;64 (3) : 303 \u2013 310 .\n\nGalazka VB ,  Sumner IG ,  Ledward DA .  Changes in protein\u2013protein and protein\u2013polysaccharide interactions induced by high pressure .  _Food Chemistry_.  1996 ;57 (3) : 393 \u2013 398 .\n\nGao JY ,  Dubin PL .  Binding of proteins to copolymers of varying hydrophobicity .  _Biopolymers_.  1999 ;49 (2) : 185 \u2013 193 .\n\nGarti N ,  Slavin Y ,  Aserin A .  Portulaca oleracea gum and casein interactions and emulsion stability .  _Food Hydrocolloids_.  1999 ;13 (2) : 127 \u2013 138 .\n\nGirard M ,  Sanchez C ,  Laneuville SI ,  Turgeon SL ,  Gauthier SF .  Associative phase separation of \u03b2-lactoglobulin\/pectin solutions: A kinetic study by small angle static light scattering .  _Colloids and Surfaces B: Biointerfaces_.  2004 ;35 (1) : 15 \u2013 22 .\n\nGirard M ,  Turgeon SL ,  Gauthier SF .  Interbiopolymer complexing between \u03b2-lactoglobulin and low- and high-methylated pectin measured by potentiometric titration and ultrafiltration .  _Food Hydrocolloids_.  2002 ;16 (6) : 585 \u2013 591 .\n\nGirard M ,  Turgeon SL ,  Gauthier SF .  Thermodynamic parameters of \u03b2-lactoglobulin-pectin complexes assessed by isothermal titration calorimetry .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 (15) : 4450 \u2013 4455 .\n\nGirard M ,  Turgeon SL ,  Gauthier SF .  Quantification of the interactions between \u03b2-lactoglobulin and pectin through capillary electrophoresis analysis .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 (20) : 6043 \u2013 6049 .\n\nGrinberg VY ,  Tolstoguzov VB .  Thermodynamic compatibility of gelatin and some \u03b2D-glucans in aqueous media .  _Carbohydrate Research_.  1972 ;25 : 313 \u2013 320 .\n\nGrinberg VY ,  Tolstoguzov VB .  Thermodynamic incompatibility of proteins and polysaccharides in solutions .  _Food Hydrocolloids_.  1997 ;11 : 145 \u2013 158 .\n\nGu YS ,  Decker EA ,  McClements DJ .  Influence of pH and carrageenan type on properties of \u03b2-lactoglobulin stabilized oil-in-water emulsions .  _Food Hydrocolloids_.  2005 ;19 (1) : 83 \u2013 91 .\n\nGu YS ,  Regnier L ,  McClements DJ .  Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by \u03b2-lactoglobulin-\u03b9-carrageenan membranes .  _Journal of Colloid and Interface Science_.  2005 ;286 (2) : 551 \u2013 558 .\n\nGuido S ,  Simeone M ,  Alfani A .  Interfacial tension of aqueous mixtures of Na-caseinate and Na-alginate by drop deformation in shear flow .  _Carbohydrate Polymers_.  2002 ;48 (2) : 143 \u2013 152 .\n\nGuzey D ,  Kim HJ ,  McClements DJ .  Factors influencing the production of o\/w emulsions stabilized by [beta]-lactoglobulin-pectin membranes .  _Food Hydrocolloids_.  2004 ;18 (6) : 967 \u2013 975 .\n\nGuzey D ,  McClements DJ .  Characterization of \u03b2-lactoglobulin-chitosan interactions in aqueous solutions: A calorimetry, light scattering, electrophoretic mobility and solubility study .  _Food Hydrocolloids_.  2006 ;20 (1) : 124 \u2013 131 .\n\nG\u00fczey D ,  McClements DJ .  Influence of environmental stresses on O\/W emulsions stabilized by \u03b2-lactoglobulin\u2013pectin and \u03b2-lactoglobulin\u2013pectin\u2013chitosan membranes produced by the electrostatic layer-by-layer deposition technique .  _Food Biophysics_.  2006 ;1 (1) : 30 \u2013 40 .\n\nHansen PMT ,  Hidalgo J ,  Gould IA .  Reclamation of whey protein with carboxymethylcellulose .  _Journal of Dairy Science_.  1974 ;54 (6) : 830 \u2013 834 .\n\nHarnsilawat T ,  Pongsawatmanit R ,  McClements DJ .  Characterization of [beta]-lactoglobulin-sodium alginate interactions in aqueous solutions: A calorimetry, light scattering, electrophoretic mobility and solubility study .  _Food Hydrocolloids_.  2006 ;20 (5) : 577 \u2013 585 .\n\nHattori M ,  Nagasawa K ,  Amenati A ,  Kaminogawa S ,  Takahashi K .  Functional changes in \u03b2-lactoglobulin by conjugation with carboxymethyl dextran .  _Journal of Agricultural and Food Chemistry_.  1994 ;42 (10) : 2120 \u2013 2125 .\n\nHattori M ,  Yang W ,  Takahashi K .  Functional changes of carboxymethyl potato starch by conjugation with whey proteins .  _Journal of Agricultural and Food Chemistry_.  1995 ;43 (8) : 2007 \u2013 2011 .\n\nHemar Y ,  Tamehana M ,  Munro PA ,  Singh H .  Influence of xanthan gum on the formation and stability of sodium caseinate oil-in-water emulsions .  _Food Hydrocolloids_.  2001 ;15 (4-6) : 513 \u2013 519 .\n\nHemar Y ,  Tamehana M ,  Munro PA ,  Singh H .  Viscosity, microstructure and phase behavior of aqueous mixtures of commercial milk protein products and xanthan gum .  _Food Hydrocolloids_.  2001 ;15 (4-6) : 565 \u2013 574 .\n\nHidalgo J ,  Hansen PMT .  Interactions between food stabilizers and \u03b2-lactoglobulin .  _Journal of Agricultural and Food Chemistry_.  1969 ;17 (5) : 1089 \u2013 1092 .\n\nHolt C ,  Sawyer L .  Primary and predicted secondary structures of the caseins in relation to their biological functions .  _Protein Engineering_.  1988 ;2 (4) : 251 \u2013 259 .\n\nHong Y-H ,  McClements D .  Modulation of pH sensitivity of surface charge and aggregation stability of protein-coated lipid droplets by chitosan addition .  _Food Biophysics_.  2007 ;2 (1) : 46 \u2013 55 .\n\nImeson AP .  On the nature of interactions betwen some anionic polysaccharides and proteins .  _Journal of the Science of Food and Agriculture_.  1977 ;28 : 661 \u2013 667 .\n\nKasran M ,  Cui SW ,  Goff HD .  Emulsifying properties of soy whey protein isolate-fenugreek gum conjugates in oil-in-water emulsion model system .  _Food Hydrocolloids_.  2013 ;30 (2) : 691 \u2013 697 .\n\nKato A ,  Mifuru R ,  Matsudomi N ,  Kobayashi K .  Functional casein\u2013polysaccharide conjugates prepared by controlled dry heating .  _Bioscience Biotechnology and Biochemistry_.  1992 ;56 : 567 \u2013 571 .\n\nKazmiersi M ,  Wicker L ,  Corredig M .  Interactions of \u03b2-lactoglobulin and high-methoxyl pectins in acidified systems .  _Journal of Food Science_.  2003 ;68 (5) : 1673 \u2013 1679 .\n\nKim HJ ,  Decker EA ,  Julian McClements D .  Preparation of multiple emulsions based on thermodynamic incompatibility of heat-denatured whey protein and pectin solutions .  _Food Hydrocolloids_.  2006 ;20 (5) : 586 \u2013 595 .\n\nKinsella JE .  Milk proteins: Physical and functional properties .  _Critical Reviews in Food Science and Nutrition_.  1984 ;21 (3) : 197 \u2013 262 . \nLaneuville SI ,  Paquin P ,  Turgeon SL .  Effect of preparation conditions on the characteristics of whey protein\u2014xanthan gum complexes .  _Food Hydrocolloids_.  2000 ;14 (4) : 305 \u2013 314 .\n\nLangendorff V ,  Cuvelier G ,  Launay B ,  Michon C ,  Parker A ,  De Kruif CG .  Casein micelle\/iota carrageenan interactions in milk: Influence of temperature .  _Food Hydrocolloids_.  1999 ;13 (3) : 211 \u2013 218 .\n\nLangendorff V ,  Cuvelier G ,  Launay B ,  Parker A .  Gelation and flocculation of casein-micelle\u2013carrageenan mixtures .  _Food Hydrocolloids_.  1997 ;11 (1) : 35 \u2013 40 .\n\nLangendorff V ,  Cuvelier G ,  Michon C ,  Launay B ,  Parker A ,  De kruif CG .  Effects of carrageenan type on the behaviour of carrageenan\/milk mixtures .  _Food Hydrocolloids_.  2000 ;14 (4) : 273 \u2013 280 .\n\nLangton M ,  Hermansson AM .  Fine-stranded and particulate gels of beta-lactoglobulin and whey-protein at varying pH .  _Food Hydrocolloids_.  1992 ;5 (6) : 523 \u2013 539 .\n\nLaplante S ,  Turgeon SL ,  Paquin P .  Effect of pH, ionic strength, and composition on emulsion stabilising properties of chitosan in a model system containing whey protein isolate .  _Food Hydrocolloids_.  2005 ;19 (4) : 721 \u2013 729 .\n\nLi J ,  Ould Eleya MM ,  Gunasekaran S .  Gelation of whey protein and xanthan mixture: Effect of heating rate on rheological properties .  _Food Hydrocolloids_.  2006 ;20 (5) : 678 \u2013 686 .\n\nLiu LY ,  Zhao QZ ,  Liu TX ,  Long Z ,  Kong J ,  Zhao MM .  Sodium caseinate\/xanthan gum interactions in aqueous solution: Effect on protein adsorption at the oil-water interface .  _Food Hydrocolloids_.  2012 ;27 (2) : 339 \u2013 346 .\n\nLucey JA ,  Srinivasan M ,  Singh H ,  Munro PA .  Characterization of commercial and experimental sodium caseinates by multiangle laser light scattering and size-exclusion chromatography .  _Journal of Agricultural and Food Chemistry_.  2000 ;48 (5) : 1610 \u2013 1616 .\n\nLundin L ,  Williams MAK ,  Foster TJ .  Chapter 3: Phase separation in foods .  In:  McKenna BM ,  ed.  _Texture in foods. Volume 1: Semi-solid foods_ .  Cambridge :  Woodhead Publishing Ltd ;  2003 : 63 \u2013 85 .\n\nMa S ,  Wang ZH .  Pulsed electric field-assisted modification of pectin from sugar beet pulp .  _Carbohydrate Polymers_.  2013 ;92 (2) : 1700 \u2013 1704 .\n\nMackie AR ,  Mingins J ,  Dann R .  Preliminary studies of \u03b2-lactoglobulin adsorbed on polystyrene latex .  In:  Dickinson E ,  Walstra P ,  eds.  _Food colloids and polymers: Stability and mechanical properties_ .  Cambridge, UK :  The Royal Society of Chemistry ;  1993 : 96 \u2013 112 .\n\nMarkman G ,  Livney YD .  Maillard-conjugate based core-shell co-assemblies for nanoencapsulation of hydrophobic nutraceuticals in clear beverages .  _Food and Function_.  2012 ;3 (3) : 262 \u2013 270 .\n\nMaroziene A ,  de Kruif CG .  Interaction of pectin and casein micelles .  _Food Hydrocolloids_.  2000 ;14 (4) : 391 \u2013 394 .\n\nMartinez KD ,  Baeza RI ,  Millan F ,  Pilosof AMR .  Effect of limited hydrolysis of sunflower protein on the interactions with polysaccharides in foams .  _Food Hydrocolloids_.  2005 ;19 (3) : 361 \u2013 369 .\n\nMatia-Merino L ,  Lau K ,  Dickinson E .  Effects of low-methoxyl amidated pectin and ionic calcium on rheology and microstructure of acid-induced sodium caseinate gels .  _Food Hydrocolloids_.  2004 ;18 (2) : 271 \u2013 281 .\n\nMcClements DJ .  _Food emulsions in practice. Food Emulsions: Principles, practices and techniques_ .  2nd ed. Boca Raton, FL :  CRC Press ;  2005 : pp. 515-544 .\n\nMession JL ,  Assifaoui A ,  Lafarge C ,  Saurel R ,  Cayot P .  Protein aggregation induced by phase separation in a pea proteins-sodium alginate-water ternary system .  _Food Hydrocolloids_.  2012 ;28 (2) : 333 \u2013 343 .\n\nMiquelim JN ,  Lannes SCS ,  Mezzenga R .  pH Influence on the stability of foams with protein-polysaccharide complexes at their interfaces .  _Food Hydrocolloids_.  2010 ;24 (4) : 398 \u2013 405 .\n\nMishra S ,  Mann B ,  Joshi VK .  Functional improvement of whey protein concentrate on interaction with pectin .  _Food Hydrocolloids_.  2001 ;15 (1) : 9 \u2013 15 .\n\nMleko S ,  Li-Chan ECY ,  Pikus S .  Interactions of \u03b9-carrageenan with whey proteins in gels formed at different pH .  _Food Research International_.  1997 ;30 (6) : 427 \u2013 433 .\n\nMoreau L ,  Kim H-J ,  Decker EA ,  McClements DJ .  Production and characterization of oil-in-water emulsions containing droplets stabilized by \u03b2-lactoglobulin-pectin membranes .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 (22) : 6612 \u2013 6617 .\n\nMorr CV ,  Ha EYW .  Whey protein concentrates and isolates: Processing and functional properties .  _Critical Reviews in Food Science and Nutrition_.  1993 ;33 (6) : 431 \u2013 476 .\n\nMorris GA ,  Sims IM ,  Robertson AJ ,  Furneaux RH .  Investigation into the physical and chemical properties of sodium caseinate-maltodextrin glyco-conjugates .  _Food Hydrocolloids_.  2004 ;18 (6) : 1007 \u2013 1014 .\n\nMorris VJ ,  Multicomponent gels .  Philips GO ,  Wedlock DJ ,  Williams PA ,  eds.  _Gums and stabilisers for the food industry_ ,  3 .  London :  Elsevier ;  1986 : 87 \u2013 99 .\n\nMun S ,  Decker E ,  Park Y ,  Weiss J ,  McClements DJ .  Influence of interfacial composition on in vitro digestibility of emulsified lipids: Potential mechanism for chitosan's ability to inhibit fat digestion .  _Food Biophysics_.  2006 ;1 (1) : 21 \u2013 29 .\n\nNakamura S ,  Kato A ,  Kobayashi K .  New antimicrobial characteristics of lysozyme-dextran conjugate .  _Journal of Agricultural and Food Chemistry_.  1991 ;39 (4) : 647 \u2013 650 .\n\nNakamura S ,  Kobayashi K ,  Kato A .  Role of positive charge of lysozyme in the excellent emulsifying properties of Maillard-type lysozyme\u2013polysaccharide conjugate .  _Journal of Agricultural and Food Chemistry_.  1994 ;42 (12) : 2688 \u2013 2691 .\n\nNeirynck N ,  Van der Meeren P ,  Bayarri Gorbe S ,  Dierckx S ,  Dewettinck K .  Improved emulsion stabilizing properties of whey protein isolate by conjugation with pectins .  _Food Hydrocolloids_.  2004 ;18 (6) : 949 \u2013 957 .\n\nNeiser S ,  Draget KI ,  Smidsrod O .  Gel formation in heat-treated bovine serum albumin-sodium alginate systems .  _Food Hydrocolloids_.  1998 ;12 (2) : 127 \u2013 132 .\n\nNorton IT ,  Frith WJ .  Microstructure design in mixed biopolymer composites .  _Food Hydrocolloids_.  2001 ;15 (4\u20136) : 543 \u2013 553 .\n\nO'Regan J ,  Mulvihill DM .  Sodium caseinate-maltodextrin conjugate stabilized double emulsions: Encapsulation and stability .  _Food Research International_.  2010 ;43 (1) : 224 \u2013 231 .\n\nOudgenoeg G ,  Hilhorst R ,  Piersma SR ,  Boeriu CG ,  Gruppen H ,  Hessing M ,  Laane C .  Peroxidase- mediated cross-linking of a tyrosine-containing peptide with ferulic acid .  _Journal of Agricultural and Food Chemistry_.  2001 ;49 (5) : 2503 \u2013 2510 .\n\nOuld Eleya MM ,  Turgeon SL .  Rheology of \u03b9-carrageenan and \u03b2-lactoglobulin mixed gels .  _Food Hydrocolloids_.  2000 ;14 (1) : 29 \u2013 40 .\n\nPanteloglou AG ,  Bell AE ,  Ma F .  Effect of high-hydrostatic pressure and pH on the rheological properties of gum arabic .  _Food Chemistry_.  2010 ;122 (4) : 972 \u2013 979 .\n\nParaskevopoulou A ,  Tsioga E ,  Biliaderis CG ,  Kiosseoglou V .  Acid-induced gelation of aqueous WPI-CMC solutions: Effect on orange oil aroma compounds retention .  _Food Hydrocolloids_.  2013 ;30 (1) : 368 \u2013 374 .\n\nPereyra R ,  Schmidt KA ,  Wicker L .  Interaction and stabilization of acidified casein dispersions with low and high methoxyl pectins .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 (9) : 3448 \u2013 3451 .\n\nPiculell L ,  Lindman B .  Association and segregation in aqueous polymer\/polymer, polymer\/surfactant and surfactant\/surfactant mixtures: Similarities and differences .  _Advances in Colloid and Interface Science_.  1992 ;41 : 149 \u2013 178 .\n\nPolyakov VI ,  Grinberg VY ,  Tolstoguzov VB .  Application of phase-volume-ratio method for determining the phase diagram of water-casein-soybean globulins system .  _Polymer Bulletin_.  1980 ;2 (11) : 757 \u2013 760 .\n\nPolyakov VI ,  Grinberg VY ,  Tolstoguzov VB .  Thermodynamic incompatibility of proteins .  _Food Hydrocolloids_.  1997 ;11 : 171 \u2013 180 .\n\nPongsawatmanit R ,  Harnsilawat T ,  McClements DJ .  Influence of alginate, pH and ultrasound treatment on palm oil-in-water emulsions stabilized by \u03b2-lactoglobulin .  _Colloids and Surfaces A: Physicochemical and Engineering Aspects_.  2006 ;287 (1\u20133) : 59 \u2013 67 .\n\nRao MA .  Rheological behavior of food gel systems .  In:  Rao MA ,  ed.  _Rheology of fluid and semisolid foods: Principles and applications_ .  Gaithersburg, Maryland :  Aspen Publishers ;  1999 : 357 .\n\nRu QM ,  Wang YW ,  Lee J ,  Ding YT ,  Huang QR .  Turbidity and rheological properties of bovine serum albumin\/pectin coacervates: Effect of salt concentration and initial protein\/polysaccharide ratio .  _Carbohydrate Polymers_.  2012 ;88 (3) : 838 \u2013 846 .\n\nSamant SK ,  Singhal RS ,  Kulkarni PR ,  Rege DV .  Protein-polysaccharide interactions: A new approach in food formulation .  _International Journal of Food Science and Technology_.  1993 ;28 : 547 \u2013 562 .\n\nSanchez C ,  Mekhloufi G ,  Renard D .  Complex coacervation between beta-lactoglobulin and acacia gum: A nucleation and growth mechanism .  _Journal of Colloid and Interface Science_.  2006 ;299 (2) : 867 \u2013 873 .\n\nSanchez C ,  Mekhloufi G ,  Schmitt C ,  Renard D ,  Robert P ,  Lehr C-M ,  Hardy J .  Self-assembly of \u03b2-lactoglobulin and acacia gum in aqueous solvent: Structure and phase-ordering kinetics .  _Langmuir_.  2002 ;18 (26) : 10323 \u2013 10333 .\n\nSanchez C ,  Renard D .  Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates .  _International Journal of Pharmaceutics_.  2002 ;242 (1\u20132) : 319 \u2013 324 .\n\nScaman C ,  Nakai S ,  Aminlari M .  Effect of pH, temperature and sodium bisulfite or cysteine on the level of Maillard-based conjugation of lysozyme with dextran, galactomannan and mannan .  _Food Chemistry_.  2006 ;99 (2) : 368 \u2013 380 .\n\nSchmitt C ,  Sanchez C ,  Desobry-Banon S ,  Hardy J .  Structure and technofunctional properties of protein-polysaccharide complexes: A Review .  _Critical Reviews in Food Science and Nutrition_.  1998 ;38 (8) : 689 \u2013 753 .\n\nSchmitt C ,  Sanchez C ,  Despond S ,  Renard D ,  Thomas F ,  Hardy J .  Effect of protein aggregates on the complex coacervation between \u03b2-lactoglobulin and acacia gum at pH 4 .  _2. Food Hydrocolloids_.  2000 ;14 (4) : 403 \u2013 413 .\n\nSchmitt C ,  Sanchez C ,  Lamprecht A ,  Renard D ,  Lehr C-M ,  de Kruif CG ,  Hardy J .  Study of \u03b2-lactoglobulin\/acacia gum complex coacervation by diffusing-wave spectroscopy and confocal scanning laser microscopy .  _Colloids and Surfaces B: Biointerfaces_.  2001 ;20 (3) : 267 \u2013 280 .\n\nSchmitt C ,  Sanchez C ,  Thomas F ,  Hardy J .  Complex coacervation between \u03b2-lactoglobulin and acacia gum in aqueous medium .  _Food Hydrocolloids_.  1999 ;13 (6) : 483 \u2013 496 .\n\nSchorsch C ,  Jones MG ,  Norton IT .  Thermodynamic incompatibility and microstructure of milk protein\/locust bean gum\/sucrose systems .  _Food Hydrocolloids_.  1999 ;13 (2) : 89 \u2013 99 .\n\nShepherd R ,  Robertson A ,  Ofman D .  Dairy glycoconjugate emulsifiers: casein-maltodextrins .  _Food Hydrocolloids_.  2000 ;14 (4) : 281 \u2013 286 .\n\nSherony DF ,  Kintner RC .  Van der Waals forces in a three-phase system .  _American Institute of Chemical Engineers_.  1971 ;17 (2) : 291 \u2013 294 .\n\nSimeone M ,  Mole F ,  Guido S .  Measurement of average drop size in aqueous mixtures of Na-alginate and Na-caseinate by linear oscillatory tests .  _Food Hydrocolloids_.  2002 ;16 (5) : 449 \u2013 459 .\n\nSingh H ,  Tamehana M ,  Hemar Y ,  Munro PA .  Interfacial compositions, microstuctures and properties of oil-in-water emulsions formed with mixtures of milk proteins and \u03b9-carrageenan: 1. Sodium caseinate .  _Food Hydrocolloids_.  2003 ;17 (4) : 539 \u2013 548 .\n\nSingh V ,  Kumar P ,  Sanghi R .  Use of microwave irradiation in the grafting modification of the polysaccharides\u2014A review .  _Progress in Polymer Science_.  2012 ;37 (2) : 340 \u2013 364 .\n\nSnoeren THM .  Electrostatic interaction between \u03b9-carrageenan and kappa-casein .  _Milchwissenchaft_.  1975 ;30 : 393 \u2013 395 .\n\nSong Y ,  Babiker EE ,  Usui M ,  Saito A ,  Kato A .  Emulsifying properties and bactericidal action of chitosan-lysozyme conjugates .  _Food Research International_.  2002 ;35 (5) : 459 \u2013 466 .\n\nSouza CJF ,  Rojas EEG ,  Melo NR ,  Gaspar A ,  Lins JFC .  Complex coacervates obtained from interaction egg yolk lipoprotein and polysaccharides .  _Food Hydrocolloids_.  2013 ;30 (1) : 375 \u2013 381 .\n\nStading M ,  Langton M ,  Hermansson AM .  Microstructure and rheological behavior of particulate beta-lactoglobulin gels .  _Food Hydrocolloids_.  1993 ;7 (3) : 195 \u2013 212 .\n\nStainsby G .  Proteinaceous gelling systems and their complexes with polysaccharides .  _Food Chemistry_.  1980 ;6 (1) : 3 \u2013 14 .\n\nSuchkov VV ,  Grinberg VY ,  Muschiolik G ,  Schmandke H ,  Tolstoguzov VB .  Mechanical and functional-properties of anisotropic gel fibers obtained from the 2-phase system of water casein sodium alginate .  _Nahrung-Food_.  1988 ;32 (7) : 661 \u2013 668 .\n\nSuchkov VV ,  Grinberg VY ,  Tolstogusov VB .  Steady-state viscosity of the liquid two-phase disperse system water-casein-sodium alginate .  _Carbohydrate Polymers_.  1981 ;1 (1) : 39 \u2013 53 .\n\nSurh J ,  Decker EA ,  McClements DJ .  Influence of pH and pectin type on properties and stability of sodium-caseinate stabilized oil-in-water emulsions .  _Food Hydrocolloids_.  2006 ;20 (5) : 607 \u2013 618 .\n\nSyrbe A ,  Bauer WJ ,  Klostermeyer H .  Polymer science concepts in dairy systems\u2014An overview of milk protein and food hydrocolloid interaction .  _International Dairy Journal_.  1998 ;8 (3) : 179 \u2013 193 .\n\nTaravel MN ,  Domard A .  Collagen and its interaction with chitosan: II. Influence of the physicochemical characteristics of collagen .  _Biomaterials_.  1995 ;16 (11) : 865 \u2013 871 .\n\nTavares C ,  Lopes da Silva JA .  Rheology of galactomannan-whey protein mixed systems .  _International Dairy Journal_.  2003 ;13 (8) : 699 \u2013 706 .\n\nTavares C ,  Monteiro SR ,  Moreno N ,  Lopes da Silva JA .  Does the branching degree of galactomannans influence their effect on whey protein gelation? .  _Colloids and Surfaces a-Physicochemical and Engineering Aspects_.  2005 ;270 : 213 \u2013 219 .\n\nThaiudom S ,  Goff HD .  Effect of \u03b9-carrageenan on milk protein polysaccharide mixtures .  _International Dairy Journal_.  2003 ;13 (9) : 763 \u2013 771 .\n\nTiebackx FWZ .  Gleichzeitige ausflockung zweier kolloide .  _Zeitschrift f \u00fcr Chemie und Industrie der Kolloide_.  1911 ;8 : 198 \u2013 201 .\n\nTolstogusov VB .  Functional properties of protein-polysaccharide mixtures .  In:  Mitchell JR ,  Ledwards DA ,  eds.  _Functional properties of macromolecules_ .  London :  Elsevier Applied Science Publishers ;  1986 : 385 \u2013 415 .\n\nTolstoguzov V .  Thermodynamic aspects of dough formation and functionality .  _Food Hydrocolloids_.  1997 ;11 (2) : 181 \u2013 193 .\n\nTolstoguzov V .  Some thermodynamic considerations in food formulation .  _Food Hydrocolloids_.  2003 ;17 (1) : 1 \u2013 23 .\n\nTolstoguzov V .  Phase behavior in mixed polysaccharide systems .  In:  Alistair MS ,  Glyn OP ,  Peter AW ,  eds.  _Food polysaccharides and their applications_ .  Boca Raton, FL :  CRC Press ;  2006 : 590 \u2013 620 .\n\nTolstoguzov VB .  Some physico-chemical aspects of protein processing into foodstuffs .  _Food Hydrocolloids_.  1988 ;2 : 339 \u2013 370 .\n\nTolstoguzov VB .  Functional properties of food proteins and role of protein\u2013polysaccharide interaction .  _Food Hydrocolloids_.  1991 ;4 (6) : 429 \u2013 468 .\n\nTolstoguzov VB .  Protein-polysaccharide interactions .  In:  Damodaran S ,  Paraf A ,  eds.  _Food proteins and their applications_ .  New York :  Marcel Dekker ;  1997 : 171 \u2013 198 .\n\nTolstoguzov VB .  Thermodynamic aspects of biopolymer functionality in biological systems, foods, and beverages .  _Critical Reviews in Biotechnology_.  2002 ;22 (2) : 89 \u2013 174 .\n\nTolstoguzov VB ,  Grinberg VY ,  Gurov AN .  Some physicochemical approaches to the problem of protein texturization .  _Journal of Agricultural and Food Chemistry_.  1985 ;33 : 151 \u2013 159 .\n\nTran T ,  Rousseau D .  Stabilization of acidic soy protein-based dispersions and emulsions by soy soluble polysaccharides .  _Food Hydrocolloids_.  2013 ;30 (1) : 382 \u2013 392 .\n\nTuinier R ,  De Kruif CG .  Phase behavior of casein micelles\/exocellular polysaccharide mixtures: Experiment and theory .  _Journal of Chemical Physics_.  1999 ;110 (18) : 9296 \u2013 9304 .\n\nTuinier R ,  Dhont JKG ,  De Kruif CG .  Depletion-induced phase separation of aggregated whey protein colloids by an exocellular polysaccharide .  _Langmuir_.  2000 ;16 (4) : 1497 \u2013 1507 .\n\nTuinier R ,  ten Grotenhuis E ,  Holt C ,  Timmins PA ,  de Kruif CG .  Depletion interaction of casein micelles and an exocellular polysaccharide .  _Physical Review E_.  1999 ;60 (1) : 848 \u2013 856 .\n\nTurgeon SL ,  Beaulieu M .  Improvement and modification of whey protein gel texture using polysaccharides .  _Food Hydrocolloids_.  2001 ;15 (4\u20136) : 583 \u2013 591 .\n\nTurgeon SL ,  Beaulieu M ,  Schmitt C ,  Sanchez C .  Protein-polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects .  _Current Opinion in Colloid and Interface Science_.  2003 ;8 (4\u20135) : 401 \u2013 414 .\n\nvan Gruijthuijsen K ,  Herle V ,  Tuinier R ,  Schurtenberger P ,  Stradner A .  Origin of suppressed demixing in casein\/xanthan mixtures .  _Soft Matter_.  2012 ;8 (5) : 1547 \u2013 1555 .\n\nWang Q ,  Qvist KB .  Investigation of the composite system of \u03b2-lactoglobulin and pectin in aqueous solutions .  _Food Research International_.  2000 ;33 (8) : 683 \u2013 690 .\n\nWang S ,  van Dijk JAPP ,  Odijk T ,  Smit JAM .  Depletion-induced demixing in aqueous protein-polysaccharide solutions .  _Biomacromolecules_.  2001 ;2 (4) : 1080 \u2013 1088 .\n\nWeinbreck F ,  de Vries R ,  Schrooyen P ,  de Kruif CG .  Complex coacervation of whey proteins and gum arabic .  _Biomacromolecules_.  2003 ;4 (2) : 293 \u2013 303 .\n\nWeinbreck F ,  Nieuwenhuijse H ,  Robijn GW ,  De Kruif CG .  Complexation of whey proteins with carrageenan .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 (11) : 3550 \u2013 3555 .\n\nWeinbreck F ,  Nieuwenhuijse H ,  Robjin GW ,  De Kruif CG .  Complex formation of whey proteins-exocellular polysaccharide EPS B40 .  _Langmuir_.  2003 ;19 (22) : 9404 \u2013 9410 .\n\nWeinbreck F ,  Rollema HS ,  Tromp RH ,  deKruif CG .  Diffusivity of whey protein and gum arabic in their coacervates .  _Langmuir_.  2004 ;20 (15) : 6389 \u2013 6395 .\n\nWeinbreck F ,  Tromph RH ,  De Kruif CG .  Composition and structure of whey protein\/gum arabic coacervates .  _Biomacromolecules_.  2004 ;5 (4) : 1437 \u2013 1445 .\n\nWeinbreck F ,  Wientjes RHW .  Rheological properties of whey protein\/gum arabic coacervates .  _Journal of Rheology_.  2004 ;48 (6) : 1215 \u2013 1228 .\n\nWong DWS ,  Camirand WM ,  Pavlath AE .  Structures and functionalities of milk proteins .  _Critical Reviews in Food Science and Nutrition_.  1996 ;36 (8) : 807 \u2013 844 .\n\nWong SS ,  Sun Y ,  Ngiam ZJ ,  Kasapis S ,  Huang D .  Polysaccharide ultrasonication-beyond depolymerization .  _Gums and stabilizers for the food industry_.  2010 ;15 : 77 \u2013 83 .\n\nXu DX ,  Wang XY ,  Jiang JP ,  Yuan F ,  Gao YX .  Impact of whey protein\u2014Beet pectin conjugation on the physicochemical stability of beta-carotene emulsions .  _Food Hydrocolloids_.  2012 ;28 (2) : 258 \u2013 266 .\n\nYadav MP ,  Parris N ,  Johnston DB ,  Onwulata CI ,  Hicks KB .  Corn fiber gum and milk protein conjugates with improved emulsion stability .  _Carbohydrate Polymers_.  2010 ;81 (2) : 476 \u2013 483 .\n\nYe A ,  Hemar Y ,  Singh H .  Influence of polysaccharides on the rate of coalescence in oil-in-water emulsions formed with highly hydrolyzed whey proteins .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 (17) : 5491 \u2013 5498 .\n\nYe A ,  Singh H .  Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: Influence of polysaccharides .  _Food Hydrocolloids_.  2006 ;20 (2\u20133) : 269 \u2013 276 .\n\nYe AQ ,  Flanagan J ,  Singh H .  Formation of stable nanoparticles via electrostatic complexation between sodium caseinate and gum arabic .  _Biopolymers_.  2006 ;82 (2) : 121 \u2013 133 .\n\nYe A ,  Edwards PJB ,  Gilliland J ,  Jameson GB ,  Singh H .  Temperature-dependent complexation between sodium caseinate and gum arabic .  _Food Hydrocolloids_.  2012 ;26 (1) : 82 \u2013 88 .\n\nYin BR ,  Deng W ,  Xu KK ,  Huang LW ,  Yao P .  Stable nano-sized emulsions produced from soy protein and soy polysaccharide complexes .  _Journal of Colloid and Interface Science_.  2012 ;380 : 51 \u2013 59 .\n\nZasypkin DV ,  Dumay E ,  Cheftel JC .  Pressure- and heat-induced gelation of mixed \u03b2-lactoglobulin\/xanthan solutions .  _Food Hydrocolloids_.  1996 ;10 (2) : 203 \u2013 211 .\n\nZhang JB ,  Wu NN ,  Yang XQ ,  He XT ,  Wang LJ .  Improvement of emulsifying properties of Maillard reaction products from beta-conglycinin and dextran using controlled enzymatic hydrolysis .  _Food Hydrocolloids_.  2012 ;28 (2) : 301 \u2013 312 . \nChapter 14\n\n# Interactions between Milk Proteins and Micronutrients\n\nTh\u00e9r\u00e8se Considine*\n\nJohn Flanagan**\n\nSimon M. Loveday***\n\n* Fonterra Research and Development Centre, Palmerston North, New Zealand  \n** Previously: Riddet Institute, Massey University, Palmerston North, New Zealand; Currently: Naturex S.A., Avignon, France  \n*** Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nMilk proteins can interact with micronutrients through a variety of mechanisms, with hydrophobic interactions being of particular importance. This chapter focuses on the interactions of milk proteins with a range of micronutrients, including vitamins, fatty acids, sugars, and minerals. Milk proteins can potentially be used as micronutrient carriers in foods, thereby increasing the nutritional benefit of milk and milk-based products.\n\nIt is widely known that the processing of milk proteins via heat or high pressure can result in the modification of protein structure, resulting in altered interactions between proteins and micronutrients. Interestingly, the presence of some micronutrients can retard the denaturation of some milk proteins. The addition of specific micronutrients may therefore be used as a processing tool to prevent denaturation of milk proteins under physical conditions that normally result in denaturation.\n\n## Keywords\n\nMilk proteins\n\n\u03b2-lactoglobulin\n\n\u03b1-lactalbumin\n\ncasein\n\nmicronutrient\n\nvitamin\n\nmineral\n\nfatty acid\n\nsugar\n\nsurfactant\n\nfood processing\n\nmicrostructures\n\nbiopolymers\n\nphase separation\n\ndepletion flocculation\n\nthermodynamic incompatibility\n\nco-solubility\n\ncoacervates\n\nemulsion\n\nMaillard\n\ninterface\n\nOutline\n\nIntroduction 421\n\nInteractions between native milk proteins and micronutrients 422\n\nVitamin A 422\n\nVitamin C 424\n\nVitamin D 424\n\nOther Vitamins 426\n\nMinerals 426\n\nFatty Acids 427\n\nSurfactants 430\n\nSugars and Polyols 431\n\nFlavors 433\n\nOther Micronutrients 434\n\nInteractions between process-modified milk proteins and micronutrients 435\n\nProtein Denaturation by Thermal and Pressure Treatments and Effect of Micronutrients 436\n\nProcessing Treatments Involving Ligands 437\n\nProcessing Treatments Involving Sugars or Polyols 438\n\nConclusions 441\n\n## Introduction\n\nThe existence of a three-dimensional, folded protein structure is dependent on several forces. These include hydrogen bonding, hydrophobic interactions, van der Waals forces, and electrostatic interactions. Some amino acid residues exhibit a hydrophobic character, and electrostatic forces are based on interactions between charged residues. Thus, the conformation of a protein is dependent on the presence of particular amino acids and the variation of residues within the primary structure. Although proteins may be in the native state, interactions through hydrophobic, electrostatic, van der Waals, and other forces can occur at exposed regions on the protein's surface or in cavities and pockets. It is through these mechanisms that interactions between milk proteins and various micronutrients such as vitamins, fatty acids, minerals, and surfactants can occur.\n\nProtein structures can be readily destabilized from their native state by relatively minor changes in the environmental conditions. Variations in pH, temperature, and pressure, for example, can all induce structural transitions in proteins. In some cases, the objective of processing is to induce changes in protein structure, for example, the heating of whey proteins to form gels. In other cases, however, changes in environmental conditions can elicit changes in protein structure that result in undesirable functional properties (e.g., loss of solubility or biological activity).\n\nIn addition to pH-, temperature- and pressure-induced changes in protein structure, the presence of micronutrients can affect how the protein structure reacts to variations in pH, temperature or pressure. By interacting with specific sites within the protein's three-dimensional structure, micronutrients can render a protein more, or less, susceptible to denaturation.\n\n## Interactions Between native Milk Proteins and Micronutrients\n\nMicronutrients such as vitamins, sugars, fatty acids, and minerals may interact with milk proteins through a variety of mechanisms. The main mechanism is through hydrophobic interaction, and the majority of studies of interactions between milk proteins and micronutrients focus on globular whey proteins, which have hydrophobic cavities and extensive secondary and tertiary structures. By contrast, the interactions between caseins and micronutrients are based mostly on electrostatic driving forces. Interactions between milk proteins and micronutrients can have appreciable effects on the physical and chemical properties of proteins during processing, and these effects are discussed in the next section.\n\n### Vitamin A\n\nVitamin A refers to a group of molecules structurally related to retinol, which consists of a \u03b2-ionone ring and an isoprenoid 'tail' containing four conjugated double bonds and a terminal hydroxyl group. These 'retinoids' may have carboxylic acid, aldehyde, or esterified fatty acid terminal groups, but the all-trans conjugated double-bond structure is needed for biological activity (Weiser and Somorjai, 1992).\n\nCis-trans isomerization, which leads to loss of biological activity, is catalyzed by heat, transition metal ions, free radicals, and light, especially at wavelengths below 500 nm (Loveday and Singh, 2008). In foods, retinoids are often dissolved in the fat matrix, where they are protected from the oxidizing action of atmospheric oxygen by vitamin E and other antioxidants (Ball, 1988). They may also be dispersed in various colloidal carrier systems (Loveday and Singh, 2008).\n\nMost lipocalin molecules such as \u03b2-lactoglobulin (\u03b2-LG) have clear biological roles as ligand carriers. Papiz et al. (1986) observed that the structure of \u03b2-LG was remarkably similar to the structure of retinol-binding protein (RBP). Nonetheless, no definite biological function has been attributed to \u03b2-LG (Creamer et al., 2011). One high-affinity retinoid binding site has been identified on \u03b2-LG, located inside the calyx, and there is some evidence for additional low-affinity binding on the outside of the molecule (Qin et al., 1998; Wu et al., 1999). The structural changes induced in different environments have been determined by x-ray crystallography (Qin et al., 1998). Further information about \u03b2-LG structure and binding can be found in Chapter 7.\n\nDifferent researchers have used a variety of methods to determine binding constants for the \u03b2-LG retinoid complexation, thus making comparison between studies difficult. For example, Muresan et al. (2001) compared fluorimetry with equilibrium dialysis and found that fluorimetry yielded higher binding affinities than equilibrium analysis. The pH, the genetic variant, and the source of the protein all contribute to the discrepancies in the literature. The range of retinoid binding constants reported for \u03b2-LG and the factors affecting their measurement are discussed in detail by Kontopidis et al. (2004).\n\nFree retinol is a rather unstable compound, especially in an aqueous environment, but its stability is greatly improved when bound to an RBP (Futterman and Heller, 1972). However, little endogenous retinol is found bound to \u03b2-LG when it is first purified, and the ligand most closely associated with the protein is palmitate (P\u00e9rez et al., 1989).\n\nIn 1972, Futterman and Heller used fluorescence measurements to deduce that bovine \u03b2-LG, like RBP, forms water-soluble complexes with retinol. Complexation with \u03b2-LG can make vitamin A more resistant to heat and UV light (Loveday and Singh, 2008). The \u03b2-LG-retinoid complex is slightly more resistant to tryptic hydrolysis than uncomplexed \u03b2-LG (Shimoyamada et al., 1996).\n\nDufour et al. (1991) monitored the binding of retinol, retinyl acetate, retinoic acid, and \u03b2-carotene to native, esterified, or alkylated \u03b2-LG by the quenching of tryptophan fluorescence. The retinoids were bound to native or modified \u03b2-LG in a 1:1 molar ratio with apparent dissociation constants in the range of 10\u20138 M, whereas the molar ratio was 1:2 (ligand:protein) for \u03b2-carotene. Chemical modification of \u03b2-LG by methods such as methylation, ethylation (Dufour and Haertle, 1990), or alkylation (Dufour et al., 1991) has been shown to enhance the binding affinity for retinol by opening up a second binding site. It may therefore be assumed that the partial change of the \u03b2-LG secondary structure produced by these treatments does not destroy the structure of the retinol-binding pocket. Similarly, moderate amounts of surfactants do not appear to affect the retinol binding site of \u03b2-LG (Sahihi et al., 2012; Taheri-Kafrani et al., 2008).\n\nVery few studies have been carried out with ligand binding to \u03b1-lactalbumin (\u03b1-LA), in comparison with the vast range of studies with \u03b2-LG. However, there is the potential for ligands to bind to \u03b1-LA. Puyol et al. (1991) studied the binding of retinol and palmitic acid in a whey protein mixture. From this study, \u03b1-LA was shown to bind retinol more strongly than \u03b2-LG, but a much lower percentage of palmitic acid was bound to \u03b1-LA in comparison to \u03b2-LG.\n\nFutterman and Heller (1972) showed that, as with \u03b2-LG, bovine serum albumin (BSA) forms a strong fluorescent water-soluble complex with retinol. They also postulated that, although no detectable retinol is bound to BSA in vivo, the possibility exists that this protein could serve as an auxiliary carrier if excess free retinol were introduced into the circulation. BSA can inhibit photo-catalyzed oxidation of retinol, but not retinoic acid (Shimoyamada et al., 1996). BSA is sometimes used in cell culture to reduce nonspecific binding of retinoids to plastic surfaces, but binding of retinoids to BSA can reduce their bioavailability (Klaassen et al., 1999).\n\nRaica et al. (1959) reported that retinol can also be bound to casein, and Mohan et al. (2013) found that a significant proportion of the vitamin A in commercial skim milk was bound to unmodified casein micelles. The insolubilization of the casein micelles in the acid casein and rennet casein forms promotes nutritional activity in retinol, which is not observed for the milk proteins in the native state (Adrian et al., 1984). Reassembled casein micelles have been shown to afford thermal protection to \u03b2-carotene (S\u00e1iz-Abajo et al., 2013).\n\n### Vitamin C\n\nFew studies have explored the interactions between vitamin C and milk proteins. A single binding site on BSA for ascorbic acid was recorded by Tukamoto et al. (1974). Oelrichs et al. (1984) investigated the interactions between ascorbate and BSA. They suggested an intrinsic association constant of 2600 M\u22121 at 20 \u00b0C. Dai-Dong et al. (1990) observed an increased stability of ascorbic acid in the presence of \u03b2-LG compared with that in pure water, and vitamin C was more thermoresistant when heated in the presence of \u03b2-LG. In contrast to these studies, Puyol et al. (1994) reported the lack of interaction of ascorbic acid with \u03b2-LG or indeed any of the other whey proteins. Puyol et al. (1994) suggested that the discrepancy between their work and that of Dai-Dong et al. (1990) may have been related to the methods used. Monitoring the reducing ability of ascorbic acid may not provide sufficient allowance for the effects of ascorbate losses through autoxidation. Puyol et al. (1994) also suggested that the antioxidant effect of reductive thiols in \u03b2-LG and serum albumin may have a protective effect.\n\n### Vitamin D\n\nThe affinity of \u03b2-LG for vitamin D2 is about 10-fold greater than that for vitamin A and other retinoids (Cho et al., 1994; Dufour and Haertle, 1990; Wang et al., 1997b). For vitamin D2, 2 mol\/mol of protein is bound, but only 1 mol of the various retinoids binds to \u03b2-LG (Cho et al., 1994; Dufour and Haertle, 1990; Wang et al., 1997a). Ergosterol and vitamin D3 will bind to \u03b2-LG at 2 mol\/mol, and the binding affinity appears to be significantly greater than for vitamin D2. Wang et al. (1997b) showed that both vitamin D and cholesterol can bind to \u03b2-LG. However, they reported that only one molecule of vitamin D or cholesterol would bind in the calyx, and so they suggested that the other molecule could be bound to an external site, as postulated by Monaco et al. (1987) and later confirmed using x-ray crystallography (Yang et al., 2008). Figure 14.1 illustrates the sites at which vitamin D molecules will bind to \u03b2-LG, as identified crystallographically. The external site is located in a hydrophobic cleft between the \u03b2-I strand and an \u03b1-helix, where hydrophobic ligands are stabilized by interactions with hydrophobic residues near the C-terminus (residues 136\u2013149) (Yang et al., 2008).\n\nFigure 14.1 Structure of \u03b2-LG (gray) complexed with two molecules of vitamin D3 (white). Source: Drawn using PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schr\u00f6dinger, LLC) and PDB entry 2GJ5.\n\nThe binding of retinal, vitamin D2, and retinyl palmitate by \u03b2-LG was studied by Wang et al. (1999). Analysis of competitive binding experiments with palmitate indicated that retinal and palmitate did not compete for the same site; however, vitamin D2 appeared to displace palmitate at higher concentrations. Retinoids and vitamin D2 were bound more tightly than palmitate.\n\nFogolari et al. (2000) demonstrated the importance of pH when binding ligands to \u03b2-LG, in some cases due to the effect of pH on monomer\u2013dimer\u2013oligomer equilibria (Mercadante et al., 2012). Forrest et al. (2005) reported on the interactions of vitamin D3 with \u03b2-LG A under a range of environmental conditions (i.e., pH and ionic strength). These results showed that binding depended greatly on the solution conditions. For example, at low pH, 2.5 (I = 0.15 M), the EF loop (gate) is closed, and thus vitamin D3 was probably weakly bound in the external hydrophobic surface. Upon lowering the ionic strength to 0.08 M, binding increased. It has been suggested that lowering the salt concentration allowed more surface binding (Aymard et al., 1996). A dissociation constant of 0.02\u20130.29 \u03bcM was reported for \u03b2-LG A, with apparent mole ratios of vitamin D3 bound per mole of \u03b2-LG A ranging from 0.51 to 2.04 (Forrest et al., 2005).\n\nForrest et al. (2005) also studied the binding of vitamin D3 to \u03b2-casein and reported on interactions under a range of environmental conditions. The binding constants of vitamin D3 to \u03b2-casein were dependent on pH and ionic strength. In agreement with the study of Lietaer et al. (1991), an increase in binding as a function of ionic strength was apparent at pH 6.6. This was attributed to enhanced hydrophobic interactions, creating more surface area for binding (Lietaer et al., 1991). Increased binding was associated with a weaker affinity, compared with lower ionic strength where binding was stronger. Although stronger interactions at low ionic strength were attributed to fewer protein interactions, Forrest et al. (2005) could not identify a reason for decreased binding at pH 8. A dissociation constant of 0.06\u20130.26 \u03bcM was reported for \u03b2-casein, with apparent mole ratios of vitamin D3 bound per mole of \u03b2-casein ranging from 1.16 to 2.05. Forrest et al. (2005) suggested that the rheomorphic nature of \u03b2-casein allowed the hydrophobic area to bind strongly with vitamin D3, in the most thermodynamically stable conformation. The hydrophobic interactions were aligned with the perturbation of phenylalanine and the quenching of tryptophan, both of which are located in the hydrophobic core.\n\nHaham et al. (2012) recently reported that encapsulating vitamin D in reassembled casein micelles improves its stability during thermal treatment or prolonged storage, while maintaining bioavailability. The concept of delivering hydrophobic compounds in casein micelles is claimed in a patent from this group (Livney and Dalgleish, 2007). The potential of \u03b2-LG as a carrier for vitamin D3 in fortified foods has recently been explored (Diarrassouba et al., 2013).\n\n### Other Vitamins\n\nMilk also contains an array of vitamin-binding proteins, including vitamin-B12-binding protein, folate-binding protein (FBP), vitamin-D-binding protein, and riboflavin-binding protein. These proteins occur at low concentrations but may play a significant role in the uptake of vitamins from the diet (Salter and Mowlem, 1983). FBPs are specifically involved in the uptake of folate in the intestine. They also reduce the availability of folate to bacteria in the gut and hence may have antibacterial properties (Ford, 1974). Raw bovine milk contains a riboflavin-binding protein (Kanno et al., 1991), and riboflavin bound to this milk protein has similar antioxidant activities to riboflavin bound to egg white riboflavin-binding protein (Toyosaki and Mineshita, 1988). This area, including the binding of trace elements, has been reviewed in detail by Vegarud et al. (2000). Subsequent to that review, Nixon et al. (2004) investigated the source of the cooperativity between FBP and folate, and their results suggested stoichiometric interactions.\n\n### Minerals\n\nThe abilities of certain milk proteins, in particular the caseins, to bind calcium are well known. The extent of binding of the caseins is directly related to the number of phosphoserine residues and thus follows the order \u03b1S2\\- > \u03b1S1\\- > \u03b2\\- > \u03ba-casein (see Chapter 6). Increased binding of calcium to the caseins results in reduced negative charges on the casein molecule, producing diminished electrostatic repulsion and consequently inducing precipitation.\n\nCaseins with high numbers of phosphoserine residues, such as \u03b1S1-casein B, \u03b1S1-casein C and the \u03b1S2-caseins, are insoluble in Ca2+ concentrations above about 4 mM (Singh and Flanagan, 2005). However, \u03b2-casein is soluble at high concentrations of Ca2+ (0.4 M) at temperatures below 18 \u00b0C, but very insoluble above 18 \u00b0C, even in the presence of low concentrations of Ca2+ (4 mM). \u03ba-Casein, with only one phosphoserine, binds little calcium and remains soluble in Ca2+ at all concentrations. Although \u03ba-casein does not bind calcium to any great extent, its ability to stabilize \u03b1S1-, \u03b1S2-, and \u03b2-caseins against precipitation by Ca2+ is well known and plays a large part in the stabilization of the casein micelle. This is discussed in more detail in Chapter 6.\n\nSugiarto et al. (2009) tested whether sodium caseinate and\/or whey protein isolate (WPI) could bind and solubilize iron (ferrous sulfate) for food fortification. Caseinate had more binding sites than WPI, and Fe was bound more strongly to caseinate, but caseinate was increasingly precipitated at >4 mM Fe. Caseinate-iron complexes with 2 mM Fe remained soluble as the pH was decreased from 7 to 5.5, where the solubility of WPI-iron complexes decreased with decreasing pH. Chelation of iron with milk proteins mitigated iron-catalyzed oxidation in emulsions, although some contribution from antioxidant amino acid side chains was also postulated (Sugiarto et al., 2010). It was recently discovered that prior depletion of calcium from milk proteins dramatically improves Fe binding, allowing much higher levels of Fe to be stabilized for food fortification (Das et al., 2013).\n\nIntact casein will bind zinc and calcium, but tryptic hydrolysates of \u03b1S1-, \u03b1S2-, \u03b2-, and \u03ba-caseins also display mineral-binding properties. Termed caseinophosphopeptides (CPPs), these peptides can bind and solubilize high concentrations of calcium because of their highly polar acidic domain. Calcium-binding CPPs can have an anticariogenic effect in that they inhibit caries lesions through recalcification of the dental enamel (FitzGerald, 1998). This effect has been exploited in CPP-fortified chewing gums (Recaldent and Trident brands) using ingredients developed by CSIRO Australia. CPPs have also been reported to improve the intestinal absorption of zinc, as studied using an isolated perfused rat intestinal loop system (Peres et al., 1998). The amount of iron bound to CPPs produced with the enzyme alcalase depends on the degree of hydrolysis as well as the temperature and pH during the binding reaction (Wang et al., 2011). Binding of iron to CPPs reduces iron-induced peroxidation in Caco-2 cells, suggesting that CPPs could help mitigate against the unintended side effects of iron fortification (Kibangou et al., 2008). Enzymatic hydrolysis of \u03b2-LG dramatically increases its iron-binding capacity, which may be due to improved contact between iron and aromatic amino acids (Zhou et al., 2012).\n\nLactoferrin has the ability to bind iron very strongly. In vivo, the ferric III form of iron is bound to lactoferrin (Anderson et al., 1989). Considerable interest has been expressed in supplementing bovine-milk-based infant formulas with lactoferrin, as bovine milk contains much lower levels of lactoferrin than human milk and lactoferrin, isolated from human milk, can bind two moles of iron per mole of protein (Bezwoda and Mansoor, 1986). Nagasko et al. (1993) reported that lactoferrin can bind iron at sites other than its chelate-binding sites, probably on the surface of the molecule. The thermal stability of lactoferrin-iron complexes is enhanced by soluble soybean polysaccharide, which was apparently due to enhanced electrostatic repulsion (Ueno et al., 2012). Other studies involving the interactions of minerals\/ions and milk proteins are listed in Table 14.1.\n\nTable 14.1\n\nInteraction of Milk Proteins and Minerals\n\nMilk protein | Mineral | Reference  \n---|---|---  \n\u03b2-LG A  \n\u03b2-LG A and B  \nCaseins and \u03b2-LG  \n\u03b1-LA | Chromium  \nLead  \nMercury  \nCopper | Divsalar et al. (2006)  \nDivsalar et al. (2005)  \nMata et al. (1997)  \nPermyakov et al. (1988)\n\n### Fatty Acids\n\nMost of the fatty acids present in milk are found as triglycerides, which form the fat globule. P\u00e9rez et al. (1992) proposed that ruminant \u03b2-LG, because of its activity to bind fatty acids, might play a role in the activity of pregastric lipases. P\u00e9rez et al. (1989) demonstrated that two types of lipids, namely, free fatty acids and triglycerides, bound to \u03b2-LG. The total amount of fatty acids extracted from \u03b2-LG was 0.71 mol per mol of monomer protein. The predominant saturated fatty acids found were palmitic (31\u201335%) and myristic (14\u201317%) acids, which when combined account for 66\u201375% of the total fatty acids bound to \u03b2-LG. The remaining fatty acids extracted from \u03b2-LG were unsaturated (<31% of the total fatty acids) and were mainly oleic (22\u201323%) and palmitoleic (4\u20135%) acids. Although \u03b2-LG is often associated with fatty acids in milk at physiological pH, Frapin et al. (1993) showed that \u03b2-LG isolated at acid pH contained minimal bound fatty acids, and delipidating had almost no effect on fatty acid binding affinities.\n\nAs with retinol, there also seems to be controversy regarding the binding location of fatty acids. Narayan and Berliner (1998) suggested that fatty acids bind at the 'external site' of \u03b2-LG. However, this conflicts with earlier studies by Puyol et al. (1991), which suggested competitive binding, and by Creamer (1995), which suggested an internal location as the primary binding site for fatty acids. Since then, several studies have shown ligands to bind in the internal cavity. Qin et al. (1998), using x-ray crystallography, showed 12-bromododecanoic acid binding inside the calyx, and Wu et al. (1999) revealed that palmitate binds in the central cavity (Fig. 14.2) in a manner similar to the binding of retinol to the related lipocalin, serum RBP. Ragona et al. (2000) provided further evidence for cavity binding of \u03b2-LG and palmitic acid, as did Zsila et al. (2002) using circular dichroism (CD) spectroscopy, electronic absorption spectroscopy, and electrospray ionization mass spectrometry (ESI-MS) with cis-parinaric acid.\n\nFigure 14.2 Diagram of the three-dimensional structure of \u03b2-LG that shows the relative positions of the five Cys residues, Lys60, Lys69, and the bound palmitate (Wu et al., 1999). The helix and the strands that constitute Sheets 1 and 2 are also labeled. Source: The diagram was drawn from the PDB file 1GXA using RASMOL Ver 2.6. Reproduced with the permission of Considine et al. (2005). Copyright 2005 Journal of Agricultural and Food Chemistry, American Chemical Society.\n\nKonuma et al. (2007) examined palmitic acid binding to a dimeric \u03b2-LG mutant A34C using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. Their results suggested a 1:1 binding stoichiometry. They indicated that the protein conformation should be complementary, at least in part, to the ligand's structure if tight binding (dissociation constant of <10\u20137 M) is to occur. They further highlighted the role of the flexible loops above the barrel in ligand binding. Konuma et al. (2007) hypothesized that the barrel's entrance accommodates a variety of ligands, because of its plasticity, whereas the bottom of the cavity shows rigid and somewhat selective binding.\n\nThus, it has been established that \u03b2-LG strongly binds one mole of long-chain fatty acids (myristic, palmitic, stearic acid, etc.) per mole of monomeric protein (Dufour et al., 1994; Frapin et al., 1993). The relative binding strength for saturated fatty acids from C8 to C20 has been measured with a variety of methods, including isothermal titration calorimetry, fluorescence spectroscopy, mass spectrometry, and equilibrium partition analysis (Loch et al., 2012). There is remarkable agreement on both the order and the relative magnitude of association constants, and data clearly show increasing affinity with increasing chain length: C8 \u2248 C10 < C12 < C14 < C16. Binding with arachidonic acid (C20) is weaker than with palmitic acid (C16), and there is disagreement for stearic acid (C18), which is thought to bind with a similar strength to palmitic acid or more strongly (Loch et al., 2012). Frapin et al. (1993) reported binding constants for several monounsaturated fatty acids, and Bello et al. (2011) measured the binding of lauric acid (C10) and SDS to \u03b2-LG genetic variants A and B.\n\nFatty acid binding to \u03b2-LG is sensitive to changes in pH. Changes in binding constants are observed over the pH range 5.5\u20138.5 (P\u00e9rez and Calvo, 1995). This may be due to the electrostatic interactions; for example, as the pH increases, \u03b2-LG becomes negatively charged, thus making it less electrostatically inviting for a negatively charged fatty acid. The two lysine residues at the opening of \u03b2-LG's ligand-binding cavity, Lys60 and Lys69, are likely to play a significant role in ligand affinity. The inability of porcine \u03b2-LG to bind fatty acids may be due to the substitution of Lys69 by glutamate, as suggested by Frapin et al. (1993) and P\u00e9rez et al. (1993). Creamer (1995) also hypothesized that lysine was involved in the binding process, whereby at neutral pH the carboxylate group of the fatty acid salt bridged to the positively charged \u025b-amino group. Loch et al. (2012) reported x-ray crystallography data suggesting that fatty acid head groups could form hydrogen bonds with Glu62 and Lys69.\n\nPuyol et al. (1991) studied the competition between the binding of retinol and free fatty acids to \u03b2-LG. They observed that, when the ratio between the concentrations of the total fatty acids (as palmitic acid) and retinol is similar to that found in milk, the fatty acids compete with retinol for binding to \u03b2-LG. Using intrinsic fluorescence studies, Frapin et al. (1993) and Dufour et al. (1994) suggested that an external, independent fatty-acid-binding site on the \u03b2-LG\u2013retinol complex was in the groove between the \u03b1-helix and the \u03b2-sheets of the protein. Narayan and Berliner (1997) supported simultaneous binding of retinoids and fatty acids to \u03b2-LG. However, binding is more difficult to determine when several ligands are present.\n\nThe organic-anion-binding sites of BSA are composed of two parts: a pocket lined with nonpolar amino acid chains and a cationic group located at or near the surface of the pocket (Swaney and Klotz, 1970). Most of the information available on the mechanism of BSA binding has been obtained using organic dyes, anionic detergents, and fluorinated or spin-labeled derivatives. Free fatty acid binding involves hydrophobic interactions with the hydrocarbon chain and electrostatic interactions with the carboxylate anion of BSA (Spector et al., 1969). Andersson et al. (1971) suggested that the fatty-acid-binding sites are located in clefts between the globular regions of the albumin polypeptide. One tryptophan is located deep inside the globular structure, whereas the other is superficially located where it is fairly accessible to solvent (Futterman and Heller, 1972). Several of the strong fatty-acid-binding sites are located within 10 \u00c5 of the buried tryptophan residue (Spector, 1975). Spector et al. (1969) reported that palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate and much more tightly than laurate. When a long-chain hydrocarbon did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexane), they were bound to a limited extent.\n\nAccording to P\u00e9rez et al. (1989), the amount of fatty acids found bound to BSA in milk was 4.8 mol per mol, and the predominant acids were oleic, palmitic, and stearic acids. Although the number of high-affinity binding sites and the values of apparent association constants for fatty acids to \u03b2-LG are lower than those for BSA (Anel et al., 1989), the molar concentration of \u03b2-LG in milk is much higher than that of BSA (about 30 times higher). Therefore, \u03b2-LG is considered to be the main fatty-acid-binding protein in ruminant whey (P\u00e9rez et al., 1989; ), whereas \u03b1-LA binds little or no palmitic acid (Barbana et al., 2006).\n\nBarbana et al. (2006) reported that bovine holo-\u03b1-LA neither contains bound fatty acids in vivo nor has the ability to bind them in vitro. Cawthern et al. (1997) observed the lack of binding of stearic acid with bovine holo-\u03b1-LA, using the fluorescent indicator acrylodated intestinal fatty-acid-binding (ADIFAB) protein. However, these results were in contrast to their spin resonance and intrinsic protein fluorescence results (Cawthern et al., 1997), which showed that stearic acid was bound to holo-\u03b1-LA with a dissociation constant of 10\u2013100 \u00d7 10\u22126 M. On the other hand, interactions of apo-\u03b1-LA with fatty acids have been reported by Barbana et al. (2006). Bovine apo-\u03b1-LA displayed apparent affinity binding constants of 4.6 \u00d7 106 and 5.4 \u00d7 105 M\u22121 for oleic acid and palmitic acid, respectively, using partition equilibrium, and fluorescence spectroscopy showed a binding constant of 3.3 \u00d7 106 M\u22121 for oleic acid. The small fluorescence changes observed for palmitic acid made it difficult to obtain a binding constant.\n\nComplexes of \u03b1-LA with oleic acid have strong cytotoxic effects on cancer cells, and the complex has been named HAMLET\/BAMLET (Human\/Bovine Alpha-lactalbumin Made LEthal to Tumor cells) (Brinkmann et al., 2011). Similar cytotoxic activity has recently been demonstrated with \u03b2-LG-oleate complexes (Li\u0161kov\u00e1 et al., 2011).\n\n### Surfactants\n\nSome interesting amphiphilic surfactants have been widely used to study ligand binding to \u03b2-LG, including cationic, anionic, and non-ionic surfactants. These ligands provide useful information regarding the structure of the molecule and the way in which it unfolds in response to chemical and physical stimuli. At low concentration, ionic surfactants are thought to bind in the hydrophobic calyx of \u03b2-LG, whereas they unfold the protein at higher concentration (Hansted et al., 2011).\n\nSodium dodecyl sulfate (SDS) is an anionic surfactant, and it binds strongly to a small number of sites on \u03b2-LG at low SDS concentration (Jones and Wilkinson, 1976; Lamiot et al., 1994). Creamer (1995) demonstrated that SDS had a profound effect on the equilibrium unfolding of bovine \u03b2-LG by maintaining \u03b2-LG in the native confirmation, despite high concentrations of urea. Busti et al. (2005) examined the interaction of alkyl sulfonates (AS) with \u03b2-LG and demonstrated one binding site per molecule. The efficiency of AS stabilizing native \u03b2-LG was related to the length of the hydrocarbon tail: Longer AS generally stabilized \u03b2-LG better against urea denaturation (Busti et al., 2005). AS also retarded thermal denaturation of \u03b2-LG to an extent that depended on the length of the alkyl group\u2014longer tails were again more effective (Busti et al., 2006). This mirrors the link between alkyl group length and binding affinity that occurs with fatty acids (see above).\n\nWaninge et al. (1998) showed a substantial increase in unfolding temperature of \u03b2-LG\u2013SDS complex at a molar ratio of 1:1. In contrast, a decrease in the unfolding temperature was observed with the addition of a cationic surfactant dodecyl trimethyl ammonium chloride (DTAC) at a similar ratio. Whereas DTAC was easily removed by dialysis, it was impossible to remove SDS by this method. Viseu et al. (2007) showed that DTAC disrupted the tertiary structure of \u03b2-LG, but also drove an increase in the amount of \u03b1-helical secondary structure, in contrast to total disruption of structure caused by guanidine-HCl.\n\nLu et al. (2006) showed that the anionic surfactant sodium perfluorooctanoate was a strong denaturant of \u03b2-LG. However, the denaturing ability of sodium perfluorooctanoate could be tempered with cationic surfactants, such as alkyl trimethyl ammonium bromide. Maulik et al. (1996) observed the binding of cetyl trimethyl ammonium bromide with \u03b2-LG and reported a two-stage interaction by first-order kinetics. Tetradecyl trimethyl ammonium bromide (TTAB) was also shown to interact with \u03b1-LA and cause protein unfolding below TTAB concentrations of 2 mM (Housaindokht et al., 2001).\n\nThe interactions of non-ionic sucrose esters with the casein micelle (Fontecha and Swaisgood, 1995) and \u03b2-casein (Clark et al., 1992) have also been studied. Creamer (1980) examined the effect of SDS on the \u03b2-casein self-association. The results indicated that SDS binds on an external site of \u03b2-casein, such that the hydrophobic tail of SDS becomes involved in the casein self-association. This is supported by the lack of displacement of 8-anilino-1-naphthalene sulfonate (ANS) by SDS. It was also postulated that SDS binds to sites in or on the protein such that the amino acid residues involved in the self-association reaction can interact more favorably with one another. At low concentrations, SDS is thought to bind to a limited number of sites. Despite the increase in the negative charge of the protein when low concentrations of SDS are added, the normal monomer\u2013polymer equilibrium moves predominantly to the polymer in solution, whereas at high concentrations of SDS, only protein monomers are present.\n\n### Sugars and Polyols\n\nSugars and polyols belong to a family of small hydrophilic molecules that stabilize proteins, and can be referred to as osmolytes, cosolvents, compatible solutes, or cosolutes (the last named will be used here). The effect of sugars and polyols on the unfolding and denaturation of proteins is generally attributed to preferential exclusion of these solutes from the protein surface, or, equivalently, 'preferential hydration' of the protein. Two main elements contribute to this effect (McClements, 2002; Timasheff, 1998), as illustrated in Figure 14.3. First, nonspecific steric exclusion arises from the fact that sugars and polyols are larger than water molecules. This effect is common across a wide range of cosolutes and depends more on the size of cosolute molecules than on their chemical nature (Ebel et al., 2000; R\u00f6sgen, 2007). Second, hydroxyl-rich polyols and sugars have a strong affinity for hydrogen-bonded water molecule networks and a strong phobia of protein nonpolar groups, so they are drawn to the aqueous environment in preference to the protein surface. This leads to the strengthening of hydrophobic interactions in cosolute solutions (Kamiyama et al., 1999; Timasheff, 1998).\n\nFigure 14.3 Protein\u2013cosolvent\u2013solvent interactions as a result of (a) steric interaction or (b) differential interaction effects. In (b), the exclusion of the cosolvent from the region surrounding the protein is clearly shown. Source: Taken from McClements (2002).\n\nA recent study comparing the effects of trehalose, maltose, and sucrose on the structure of water found that trehalose binds to a larger number of water molecules than do maltose or sucrose, thus affecting the structure of water to a greater extent (Lerbret et al., 2005). The effect of cosolutes on the surface tension at the protein\u2013solvent interface may also contribute to preferential exclusion, but these effects are very complex and somewhat controversial (Chanasattru et al., 2007c; Kaushik and Bhat, 1998; Lin and Timasheff, 1996).\n\nWater is able to get into the layer immediately adjacent to the protein, but cosolutes are sterically excluded; thus, a concentration gradient of the cosolvent molecules between the inner layer and the outer solution arises. This is a thermodynamically unfavorable situation because of the free energy that is required to maintain this concentration gradient. Subsequent movement of water molecules from the area surrounding the protein to outer parts leads to a dehydration of the protein molecule. This dehydration can result in tighter folding of the protein molecules.\n\nUnfolding of a compact protein structure increases the area of contact between the protein and the solvent, and reveals nonpolar groups that were buried in the interior in the native protein. For these reasons, preferential exclusion is greater in the denatured state than in the native state, and it follows that denaturation is accompanied by a net increase in the degree of preferential exclusion (i.e., a net increase in cosolute activity in the aqueous phase). This adds to the energetic cost of unfolding, so denaturation is inhibited (Timasheff, 1998).\n\nAn alternative explanation of the effect of sugars on the unfolding and denaturation of proteins has been put forward by Semenova et al. (2002), who proposed a direct hydrogen bonding between sugars and proteins, which results in additional hydration. However, this hypothesis does not fully explain the exclusion of sugars from the protein domain (Hammou et al., 1998; Timasheff, 2002).\n\nHigher concentrations of polyols and sugars increase viscosity substantially, possibly inhibiting diffusion-limited reactions such as protein aggregation (Kulmyrzaev et al., 2000). However, the more compact protein conformation induced by preferential exclusion may diffuse faster, overcoming viscosity effects (Rodr\u00edguez Ni\u00f1o and Rodr\u00edguez Patino, 2002). The stabilizing effect of polyols increases with more hydroxyl groups on the polyols (Politi and Harries, 2010; Romero et al., 2007; Tiwari and Bhat, 2006), and with lower pH (Singh et al., 2011) and lower temperature (Xie and Timasheff, 1997b).\n\nThe Timasheff research group has been dominant in research into the interactions of proteins and sugars, or cosolvents as they describe them (Timasheff, 1993). Xie and Timasheff (1997a) reported the exclusion of trehalose from the domain of ribonuclease A at low temperatures. However, at 52 \u00b0C there was preferential binding of trehalose in both the native and unfolded state (pH 5.5 and 2.8, respectively). Binding was stronger in the native state than the unfolded state, meaning that denaturation in the presence of trehalose was still accompanied by a net increase in preferential exclusion (or in other words a net decrease in binding), and therefore denaturation was inhibited. The same group conducted a lot of earlier research showing the exclusion of water from the domains of a range of globular proteins, in the presence of sucrose (Lee and Timasheff, 1981), lactose and glucose (Arakawa and Timasheff, 1982). In all cases, they argued that the exclusion of sugars from the protein domain made unfolding of the protein less thermodynamically favorable.\n\nThe Timasheff group has measured the preferential exclusion of various cosolutes from BSA and \u03b2-LG in a number of studies (Timasheff, 1998). In a rare study involving sugars and casein, Mora-Gutierrez and Farrell (2000) also proposed preferential exclusion of sugar molecules from the casein domain, resulting in preferential hydration of the caseins. The ability of sugars to alter the heat- and pressure-induced denaturation of milk proteins is discussed later in this chapter.\n\n### Flavors\n\nThe interaction of milk proteins and volatile flavor has been reviewed in detail by K\u00fchn et al. (2006), and the reader should refer to this review for more in-depth discussion of protein\u2013flavor interactions. However, this section covers the area briefly. A number of flavor compounds are known to bind to milk proteins. Despite this knowledge, there are large discrepancies in the binding data because of the use of different methodologies, which appears to be a common feature of determining binding constants.\n\n\u03b2-LG is known to interact with a variety of flavor compounds, including ionones (Jouenne and Crouzet, 2000; Jung and Ebeler, 2003), lactones (Guth and Fritzler, 2004; Sostmann and Guichard, 1998), alkanes (Mohammadzadeh et al., 1969), aldehydes (van Ruth et al., 2002), esters, and ketones (Guichard and Langourieux, 2000; Jouenne and Crouzet, 2000). In contrast, very few studies have explored flavor binding to \u03b1-LA, with an exception being the binding of aldehydes and methyl ketones (Franzen and Kinsella, 1974), and 2-nonanone and 2-nonanal (Jasinski and Kilara, 1985). BSA has been shown to bind alkanes (Mohammadzadeh et al., 1969), and studied interactions of 2-nonanone and BSA. Jasinski and Kilara (1985) compared the binding of 2-nonanone and nonanal to BSA.\n\nThe binding of flavors to caseins or sodium caseinate has also received some attention, including the binding of diacetyl (Reineccius and Coulter, 1969), vanillin (McNeill and Schmidt, 1993), \u03b2-ionone, n-hexanol, ethylhexanoate, and isomyl acetate (Voilley et al., 1991) to sodium caseinate.\n\nThe most extensively studied flavor compound is 2-nonanone. Thus the binding strengths of this flavor to the whey proteins can be compared. Although different authors have reported different affinity constants, the binding strength consistently follows the trend BSA >\u03b2-LG >\u03b1-LA. Table 14.2 illustrates the interactions between 2-nonanone and various milk proteins (K\u00fchn et al., 2006).\n\nTable 14.2\n\nBinding Data for the Interactions between 2-Nonanone and Milk Proteins (25 \u00b0C): n, Number of Binding Sites per Monomer; K, Intrinsic Binding Constant\n\nProtein | n | K (M\u20131) | Method | Reference  \n---|---|---|---|---  \nWPCa | 61 | 1,920,000 | Equilibrium dialysis | Jasinski & Kilara (1985)  \n| 0.2 | 53,000,000 | Fluorescence spectroscopy | Liu et al. (2005)  \nWPIb | 1 | 2059 | Headspace SPMEc | Zhu (2003)  \nsodium caseinate | 0.3 | 1858 | Headspace SPME | Zhu (2003)  \n\u03b2-LG | 1 | 2439 | Equilibrium dialysis | O'Neill & Kinsella (1987)  \n| 0.2  \n0.5 | 6250 (\u226440 ppm)  \n1667 (\u226545 ppm) | Static headspace analysis | Charles et al. (1996)  \n| 14 | 122 | Equilibrium dialysis | Jasinski & Kilara (1985)  \n\u03b1-LA | 33 | 11 | Equilibrium dialysis | Jasinski & Kilara (1985)  \nBSA | 5\u20136 | 1800 | Liquid\u2013liquid partitioning | Damodaran & Kinsella (1980)  \n| 15 | 14,100 | Equilibrium dialysis | Jasinski & Kilara (1985)  \n| 7 | 833 | PFG-NMRd spectroscopy | Jung et al. (2002)\n\na WPC: whey protein concentrate\n\nb WPI: whey protein isolate\n\nc SPME: solid phase microextraction\n\nd PFG-NMR: pulsed-field gradient NMR\n\nSource: Reproduced with the permission of K\u00fchn et al. (2006); copyright 2006 Journal of Food Science, Institute of Food Technologists.\n\n### Other Micronutrients\n\nSome of the milk proteins, most particularly the whey proteins, have been used as model proteins in studies involving a range of other micronutrients. The interaction of small heat-shock proteins, such as alpha-crystallin, prevents the precipitation of \u03b1-LA when in the molten globule state (Lindner et al., 1997). This finding was confirmed by Sreelakshmi and Sharma (2001), who found that the active site of alpha-cystallin by itself can maintain a significantly denatured and unfolded protein in soluble form. Zhang et al. (2005) reported on the chaperone-like activity of \u03b2\\- and \u03b1-caseins. \u03b2-Casein was able to suppress the thermal and chemical aggregation of insulin, lysozyme, and catalase. A similar chaperone-like effect is seen with \u03b2-LG, \u03b1-LA, and BSA (Kehoe and Foegeding, 2011).\n\nThe use of milk proteins as chaperones for drugs has also been studied. The interaction of chlorpromazine with \u03b2-LG and \u03b1s-casein affected the proteins in different ways. Far UV CD studies revealed that chlorpromazine increased the secondary structure of \u03b2-LG, whereas the structure of casein became further disordered (Bhattacharyya and Das, 2001). Divsalar et al. (2006) also reported on the interaction between genetic variants of \u03b2-LG and an anticancer component.\n\nA number of recent studies that examined the interactions between milk proteins and various bioactive compounds are listed in Table 14.3. Most studies were carried out with a view to using milk proteins as carrier molecules or particles for protecting and\/or delivering bioactives. This potential application was examined in several recent reviews (Abd El-Salam and El-Shibiny, 2012; Elzoghby et al., 2011; Livney, 2010). The number of studies on the binding of polyphenols by milk proteins is notable.\n\nTable 14.3\n\nRecent Studies of Milk Protein Interactions with Miscellaneous Biologically Active Compounds\n\nProtein | Active agent | Notes | Reference  \n---|---|---|---  \nPolyphenolic compounds  \n\u03b2-LG | Epigallocatechin | Binding study | Wu et al. (2011)  \n\u03b2-LG | Epigallocatechin-3-gallate | Binding study | Wu et al. (2013)  \n\u03b2-LG | Tea polyphenols | Binding study | Kanakis et al. (2011)  \nCasein | Quercitin | Chitosan-casein nanoparticles | Ha et al. (2013)  \nCasein | Polymethoxyflavones | Raman spectroscopy study of binding | He et al. (2013)  \nVarious milk proteins | Flavonoids | \u03b2-casein showed strongest interactions | Bohin et al. (2012)  \nVarious milk proteins | Polyphenols | Protein sequence influence on noncovalent binding | Nagy et al. (2012)  \nVarious milk proteins | Green tea catechins | Effect of milk proteins on bioaccessibility | Xie et al. (2013)  \nVarious bioactive compounds  \n---  \nCasein micelles | Curcumin | Bioactive component of turmeric | Benzaria et al. (2013)  \nRahimi et al. (2012)  \n\u03b2-LG | Curcumin | Binding and encapsulation study | Sneharani et al. (2010)  \n\u03b1\\- and \u03b2-caseins | Folic acid |  | Bourassa and Tajmir-Riahi (2012)  \nVarious milk proteins | Norbixin | Cheese coloring agent | Zhang and Zhong (2013a, b)  \nReassembled casein micelles | n-3 Polyunsaturated fatty acids |  | Zimet et al. (2011)  \n\u03b2-LG | Piperine (pepper alkaloid) | Binding study | Zsila et al. (2005)  \nPharmaceutical compounds  \n---  \n\u03b2-LG A | Bioactive peptides | Antihypertensive peptide | Roufik et al. (2006)  \n\u03b2-LG | Doxorubicin | Antibiotic | Agudelo et al. (2012)  \nCasein | Alfuzosin | Prostate cancer drug in genipin-cross-linked casein nanoparticles | Elzoghby et al. (2013)  \n\u03b2-casein nanoparticles | Paclitaxel, vinblastine, mitoxantrone | Antitumor drugs | Shapira et al. (2010)  \nLactoferrin | Gambogic acid | Antitumor compound | Zhang et al. (2013)\n\n## Interactions between process-modified milk proteins and micronutrients\n\nHeat has been used extensively in food processing for centuries and is a widely applied treatment in food production, primarily for the control of microbial populations. Fields of application include pasteurization under mild temperatures and sterilization under higher temperatures. However, heating may also affect texture as well as taste development and may result in flavor and color changes. The latter effects are often described as disadvantages of heat treatment. Changes in the organoleptic properties are generally a result of structural changes occurring within the constituents of the food, namely, the proteins, polysaccharides, or fats.\n\nAnother technology that is similar in its control of the microbial population of food products is high-pressure treatment. Foods are preserved with minor changes in texture, flavor, or color, in contrast to heat processing, and high pressure can be considered a cold preservation technology. High pressure is a long-used technique in Japan and has also become increasingly popular worldwide. However, high-pressure treatment may cause some conformational and structural changes to the individual constituents of the food, possibly resulting in altered functional and organoleptic properties.\n\nHeat and high-pressure treatment may both cause the denaturation of globular whey proteins such as \u03b2-LG; although there may be differences in the mechanisms behind the denaturation process, the general process appears to be similar. These processes are examined in detail in Chapter 9.\n\nThe denaturation of whey proteins during the heat treatment of milk, the interactions of the denatured whey proteins with other milk components, and the effect of these reactions on the physical and functional properties of milk products have been extensively reported and reviewed in great detail (O'Connell and Fox, 2003; Singh and Havea, 2003). Studies have shown that heat-induced aggregation and gelation occur along detailed pathways and are influenced by the types of proteins and forces present (disulfide bonding and hydrophobic interactions) (Abbasi and Dickinson, 2002; Havea et al., 2001; Schokker et al., 1999). The use of heat to induce self-assembly and co-assembly of milk proteins into micro\/nanoparticles is discussed in Loveday et al. (2012).\n\nThe effect of high pressure on whole milk and individual constituents has become a subject of much recent activity, particularly regarding the effect of pressure treatment on the physical and functional properties of milk products (Anema, 2010), and the pressure-induced changes to individual proteins (Anema, 2012). Interested readers are referred to the reviews of Huppertz et al. (2006) and Considine et al. (2007). The mechanistic effects of high-pressure processing and several other novel processing technologies were reviewed in the context of the industrial potential of these technologies in yogurt manufacture (Loveday et al., 2013). The use of high pressure in other dairy systems, such as whey or casein gels, has also been reviewed (Devi et al., 2013).\n\n### Protein Denaturation by Thermal and Pressure Treatments and Effect of Micronutrients\n\nThe caseins have not been suitable candidates for observing changes in protein denaturation due to their lack of defined secondary and tertiary structure. In contrast, the whey proteins have been studied widely as model globular proteins because of their well-defined secondary and tertiary structures, as outlined above.\n\nInteractions between whey proteins and other species induced by either heat treatment or pressure treatment may be divided into two separate classes: covalent interactions and noncovalent interactions. The most important covalent interaction involving whey proteins upon storage is their reaction with reducing sugars via the Maillard reaction to form discolored protein powders, which also have reduced solubilities and diminished nutritional properties. Noncovalent interactions can also occur; these too may lead to a loss of protein solubility after association of the proteins with polysaccharides, and these noncovalent interactions are primarily driven by reversible electrostatic interactions.\n\nIn the present work, the effects of noninteracting species on the unfolding and structural transitions of whey proteins are of specific interest. The marked increase in the thermal and conformational stability of globular proteins in aqueous media in the presence of sugars is well known and has been extensively studied.\n\n### Processing Treatments Involving Ligands\n\nSeveral studies have shown that ligands can retard the heat- or pressure-denaturation of \u03b2-LG, and the type of ligand has an impact on this process. For example, during the heat denaturation of \u03b2-LG, both SDS and palmitate stabilized the native structure of \u03b2-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation up to approximately 70 \u00b0C, whereas both retinol and ANS provided very little stabilization (Considine et al., 2005). When a similar range of ligands were used during pressure denaturation, a similar effect was noted; that is, higher pressures were required to cause unfolding of \u03b2-LG when a ligand was present (Considine et al., 2005). These studies and the comparison study of heat and pressure using myristate and conjugated linoleic acid as ligands showed that \u03b2-LG unfolds slightly differently with respect to the type of treatment (Fig. 14.4) (Considine et al., 2007).\n\nFigure 14.4 Proposed three-stage model of the pressure denaturation of \u03b2-LG B, and \u03b2-LG B with added ANS, retinol, or SDS. Source: Reproduced with the permission of Considine et al. (2005). Copyright 2005 Journal of Agricultural and Food Chemistry.\n\nBarbiroli et al. (2011) have shown that endogenous ligands bound to \u03b2-LG (mostly palmitic and stearic acid) stabilize the tertiary structure against denaturation by urea or heat. They reported evidence that the binding of palmitic acid in the calyx enhanced the thermal stability of both the calyx region and the helix held against the outside of the beta barrel (the helix conceals the free thiol at Cys 121). They believed that fatty acid binding in the calyx made the whole structure 'tighter,' and inhibited the movement of the helix region and exposure of Cys 121, which is crucially involved in disulfide-bonded aggregation. In related work with synthetic ligands, Busti et al. (2006) reported that alkyl sulfonates (AS) with a chain length >10 increased the denaturation temperature of \u03b2-LG at pH 6.8 by up to 13 \u00b0C.\n\nHansted et al. (2011) conducted a detailed investigation of how surfactants affect thermally-induced unfolding and aggregation of \u03b2-LG, using homologous a series of cationic (alky trimethyl ammonium chlorides, xTAC), anionic (sodium alkyl sulfates, SxS), and non-ionic (alkyl maltopyranosides, xM) surfactants. SxS inhibited thermal unfolding and aggregation at concentrations well below the critical micelle concentration (CMC), indicating that surfactant monomers were responsible for the effect. xM also inhibited aggregation, though only above the CMC, and smaller xM promoted unfolding at such concentrations. xTAC strongly promoted aggregation at sub-CMC concentrations. The findings highlight the effect of surfactant charge on aggregation at pH 6.5: anionic SxS and non-ionic xM reduced aggregation, whereas cationic xTAC promoted aggregation. The authors also showed how the concentration of surfactants strongly modifies their effects, and they postulated surface interactions between \u03b2-LG and micelles of non-ionic or cationic surfactants (Hansted et al., 2011).\n\nCelej et al. (2005) compared the effects of the binding of two ANS derivatives, namely, 1,8-ANS and 2,6-ANS, on BSA thermostability. They reported that 1,8-ANS had a stronger effect on BSA thermal stability and that the binding parameters of the two ANS derivatives were quite different. This was thought to indicate that stereochemistry is an important factor in determining protein\u2013ligand interactions. Thus electrostatic interactions should also be considered along with hydrophobic interactions. The authors emphasized the importance of free ligand concentration rather than the ligand:protein mole ratio when determining protein stability.\n\nAs discussed earlier, the binding of retinol to casein is through hydrophobic interactions (Poiffait and Adrian, 1991). \u03b2-Casein is the most hydrophobic casein and has a highly charged N-terminal domain, containing an anionic phosphoserine cluster, that is clearly distinct from a very hydrophobic C-terminal domain (Swaisgood, 2003). Little work has been done on the ability of the caseins to bind retinol, although Poiffait and Adrian (1991) reported that casein plays an important role in stabilizing retinol over time or during heat treatment. However, information in this area is limited.\n\n### Processing Treatments Involving Sugars or Polyols\n\nThe effect of up to 70% w\/w glycerol or sorbitol on the properties and functionality of \u03b2-LG was examined in several studies by Chanasattru and co-workers. Sorbitol strongly increased the thermal denaturation temperature of \u03b2-LG at pH 7, whereas glycerol had a very minor effect (Chanasattru et al., 2007b). This translated to much stronger gels with glycerol when 10% \u03b2-LG solutions were heated to 90 \u00b0C for 70 min. Both polyols increased the complex modulus (G*) relative to controls, which was attributed to the strengthening of protein\u2013protein interactions, but the inhibitory effect of sorbitol on denaturation was thought to explain the low G* with this polyol. Later studies noted that glycerol decreases the surface tension at hexadecane\/water interfaces, whereas sorbitol slightly increases it (Chanasattru et al., 2007c). The authors proposed that glycerol could interact with nonpolar regions on the surface of proteins in a way that counterbalanced steric exclusion effects, leading to small net effects on denaturation temperature (Chanasattru et al., 2008).\n\nThis group also studied the effects of polyols in \u03b2-LG-stabilized emulsions (Chanasattru et al., 2007a). Glycerol and sorbitol improved emulsion stability against salt-induced flocculation to an approximately equal extent on a % w\/w basis. This effect was attributed partly to increased viscosity (especially for sorbitol) but also a predicted reduction in attractive van der Waals and hydrophobic interactions large enough to overcome a slight weakening of electrostatic repulsion. Similar studies on \u03b2-LG- and casein-stabilized emulsions were discussed by Dickinson (2010).\n\nThe effect of small mono- and polyhydroxy alcohols on \u03b2-LG thermal stability at pH 5.5 was examined in more detail by Romero et al. (2007), using a homologous series of 4-carbon alcohols with 1 to 4 hydroxyl groups. All alcohols destabilized \u03b2-LG, but to an extent that decreased as the number of hydroxyl groups increased. The authors proposed that 1-butanol was hydrophobic enough to interact with nonpolar regions on the surface of \u03b2-LG, whereas more hydroxylated (and therefore more hydrophilic) alcohols interacted preferentially with water instead of protein, and thereby had a less destabilizing effect. This theory aligns well with the proposal from Chanasattru et al. (2008) that glycerol (1,2,3-propanetriol) interacts with nonpolar regions on the surface of protein.\n\nBoye and Alli (2000) reported on the thermal denaturation of 1:1 mixtures of \u03b1-LA and \u03b2-LG in the presence of a range of sugars, using differential scanning calorimetry (DSC). Sugars protected against heat-induced denaturation, and the protection offered (i.e., size of the increase in the thermal transition temperature of \u03b2-LG) was in the order galactose = glucose > fructose = lactose > sucrose > sugar-free control. No significant effects of sugar were observed with apo-\u03b1-LA. Interestingly, an earlier study by the same authors solely on \u03b1-LA found an increase in the thermal transition temperature of both the apo and holo forms of \u03b1-LA when either 50% sucrose or 50% glucose was added (Boye et al., 1997). This increase was fully reversible in the holo form, but only partly reversible in the apo form. The thermal transition temperature of \u03b2-LG was found to be increased in the presence of sucrose, lactose, and glucose at 10\u201350% (Boye, Ismail, et al., 1996).\n\nJou and Harper (1996) found an increase in the DSC thermal transition temperature of whey protein concentrates following the addition of sugars, and the protection offered by the sugars was in the order maltose > trehalose > sucrose. Lactose was also found to provide some protection against heat-induced denaturation. Dierckx and Huyghebaert (2002) followed the heat-induced gelation of a WPI solution using DSC and small-amplitude oscillatory rheometry. They found that by adding increasing concentrations of sucrose or sorbitol, both the thermal transition temperature of the protein denaturation process and the gelation temperature were increased, with a linear relationship existing between the transition and gelation temperatures. They suggested that because of the differences in the gelation mechanisms observed at different pH values, sucrose and sorbitol affected protein\u2013protein interactions in gels through enhancement of hydrophobic interactions.\n\nKulmyrzaev et al. (2000) had previously conducted a study on the effect of sucrose on the thermal denaturation, gelation, and emulsion stabilization of WPI. They also observed increases in the thermal transition temperatures on the addition of increasing concentrations of sucrose, as well as improved gel formation and enhanced emulsification flocculation. They postulated that sucrose played different roles in a pre-denatured (improved heat stability) and a post-denatured (enhanced protein\u2013protein interactions) whey protein solution system.\n\nIn a study on the effects of different lactose concentrations (within a naturally occurring range) on the formation of whey protein microparticles, Spiegel (1999) put forward a two-stage process in the aggregation of whey proteins: Up to approximately 85 \u00b0C, the aggregation of whey proteins is limited by the slow unfolding of the individual proteins; above 100 \u00b0C aggregation is the rate-limiting step, as the rate of unfolding is high. Lactose (at 500 mM) was also found to increase the temperature of the denaturation of WPI at pH 9.0 by approximately 3 \u00b0C. However, the authors realized the effect that the Maillard reaction was having on these systems, a factor that some reports seem to ignore.\n\nBaier and McClements (2001) found that increased concentrations of sucrose (up to 40%) could increase the thermal stability of BSA. These systems had a higher gelation temperature and produced gels with a lower complex shear modulus. Similar effects were found in a subsequent study (Baier and McClements, 2003). A further study by the same group (Baier et al., 2004) showed that 40% glycerol increased the temperature of gelation of BSA, but no change in the temperature of denaturation of BSA with increasing concentration of glycerol was detected.\n\nSome early DSC work (Dumay et al., 1994) showed that the presence of 5% sucrose was enough to reduce the extent of \u03b2-LG unfolding by 22% following high-pressure treatment at 450 MPa for 15 min. In a later study, Dumay et al. (1998) found that adding sucrose to \u03b2-LG solutions prior to pressure-induced gelation resulted in gels with decreased pore size and strand thickness. They attributed this to a reduction in the number of protein\u2013protein interactions occurring under the influence of pressure.\n\nKeenan et al. (2001) reported that low concentrations of sucrose aided in the pressure-induced gel formation of a range of milk-protein-containing systems, but that gel formation was reduced at higher sucrose concentrations. In another group of studies, the pressure- induced gelation properties of skim milk powder were found to be improved by adding low concentrations of sucrose, glucose, or fructose, whereas high (45\u201350%) sugar concentrations inhibited gel formation (Abbasi and Dickinson, 2001).\n\nBoye et al. (1996) described how lactose, sucrose, and glucose increased the temperature of denaturation of BSA, with 50% glucose having a greater stabilizing effect than 50% sucrose. Wendorf et al. (2004) studied the ability of different proteins (ribonuclease A, BSA, and egg white lysozyme) to adsorb to a liquid\u2013solid interface in the presence of a range of sugars. They found that the ability of sugars to reduce protein adsorption followed the trend trisaccharides > disaccharides > 6-carbon polyols > monosaccharides, and this was explained by the stabilization of the protein in the native state in solution.\n\nOther studies have also shown the beneficial effects of sugars in protecting against denaturation induced by freeze drying, spray drying, and chemicals. At low temperatures, high concentrations of sugars cause a substantial increase in solution viscosities and can thus affect protein denaturation. Tang and Pikal (2005) reported that, by negating the thermal stabilizing effects of sucrose by adding denaturants, the increased stability of \u03b2-LG in the freeze drying process could be directly attributed to a viscosity effect. Murray and Liang (1999) explored the addition of sucrose, trehalose, lactose, and lactitol to whey protein concentrate solutions prior to spray drying and found that the foaming properties of the spray-dried powders were dramatically decreased when sugars were absent. Trehalose was particularly successful in retaining the original foaming properties of both whey protein concentrate and \u03b2-LG, but did not perform as well in spray-dried BSA powders (Murray and Liang, 1999).\n\n## Conclusions\n\nThe interaction of milk proteins with various micronutrients is primarily governed by the physicochemical properties of the proteins. The whey proteins, with extensive secondary and tertiary structures and significant hydrophobicity (albeit largely shielded in the native form), tend toward hydrophobic interactions with ligands. Preferential exclusion effects govern the interaction of sugars and polyols with proteins, thus affecting their denaturing properties in the presence of pressure or heat. Electrostatic interactions drive the association of minerals and proteins.\n\nIn the food industry, an increasing emphasis is being placed on foods that will have a physiologically functional benefit, in addition to the nutritional benefit of the food. This emphasis is being driven by consumers who are becoming increasingly more health aware and health responsible. The challenge for the food scientist is now to deliver the required physiologically functional activities into the final food product, while retaining product quality and shelf life. Knowledge of the interactions of these micronutrients with milk proteins, a major component in many food products, is necessary to achieve this aim. Relevant examples of this concept are detailed in patents concerning the delivery of micronutrients in complexes with \u03b2-LG (Swaisgood, 2001) or casein micelles (Livney and Dalgleish, 2007).\n\nWhile the concept of using milk proteins as nutrient carriers has been explored in a range of protein\u2013nutrient combinations, there is relatively little knowledge about how nutrient interactions can be used to manipulate the functionality of proteins during processing. Binding of ligands to certain whey proteins increases their resistance to thermal denaturation, and noninteracting solutes like sugars can also stabilize proteins against heat processing. Mineral binding to caseins affects their solubility, which has obvious implications for beverage products. A stronger knowledge of protein\u2013micronutrient interactions will enable the use of milk proteins as nutrient carriers and allow the use of micronutrients as processing aids; perhaps both objectives could even be achieved simultaneously.\n\n# References\n\nAbbasi S ,  Dickinson E .  Influence of sugars on high-pressure induced gelation of skim milk dispersions .  _Food Hydrocolloids_.  2001 ;15 : 315 \u2013 319 .\n\nAbbasi S ,  Dickinson E .  Influence of high-pressure treatment on gelation of skim milk powder + low methoxyl pectin dispersions .  _High Pressure Res._ 2002 ;22 : 643 \u2013 647 .\n\nAbd El-Salam MH ,  El-Shibiny S .  Formation and potential uses of milk proteins as nano delivery vehicles for nutraceuticals: A review .  _Int. J. Dairy Technol._ 2012 ;65 : 13 \u2013 21 .\n\nAdrian J ,  Frangne R ,  Rabache M .  Effect of milk-proteins on retinol efficiency .  _Sci. Aliments_.  1984 ;4 : 305 \u2013 308 .\n\nAgudelo D ,  Beauregard M ,  B\u00e9rub\u00e9 G ,  Tajmir-Riahi HA .  Antibiotic doxorubicin and its derivative bind milk \u03b2-lactoglobulin .  _J. Photochem. Photobiol._ 2012 ;B117 : 185 \u2013 192 .\n\nAnderson BF ,  Baker HM ,  Norris GE ,  Rice DW ,  Baker EN .  Structure of human lactoferrin: Crystallographic structure analysis and refinement at 2.8 \u00c5 resolution .  _J. Mol. Biol._ 1989 ;209 : 711 \u2013 734 .\n\nAndersson LO ,  Brandt J ,  Johansson S .  The use of trinitrobenzenesulfonic acid in studies on the binding of fatty acid anions to bovine serum albumin .  _Arch. Biochem. Biophys._ 1971 ;146 : 428 \u2013 440 .\n\nAnel A ,  Calvo M ,  Naval J ,  Iturralde M ,  Alava MA ,  Pineiro A .  Interaction of rat \u03b1-fetoprotein and albumin with polyunsaturated and other fatty acids: Determination of apparent association constants .  _FEBS Lett._ 1989 ;250 : 22 \u2013 24 .\n\nAnema SG .  Instability of pressure-treated reconstituted skim milk to acidification .  _Food Biophysics_.  2010 ;5 : 321 \u2013 329 .\n\nAnema SG .  Pressure-induced denaturation of \u03b2-lactoglobulin in skim milk: Effect of milk concentration .  _J. Agric. Food Chem._ 2012 ;60 : 6565 \u2013 6570 .\n\nArakawa T ,  Timasheff SN .  Stabilization of protein structure by sugars .  _Biochemistry_.  1982 ;21 : 6536 \u2013 6544 .\n\nAymard P ,  Durand D ,  Nicolai T .  The effect of temperature and ionic strength on the dimerization of \u03b2-lactoglobulin .  _Int. J. Biol. Macromol._ 1996 ;19 : 213 \u2013 221 .\n\nBaier S ,  McClements DJ .  Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions .  _J. Agric. Food Chem._ 2001 ;49 : 2600 \u2013 2608 .\n\nBaier SK ,  Decker EA ,  McClements DJ .  Impact of glycerol on thermostability and heat-induced gelation of bovine serum albumin .  _Food Hydrocolloids_.  2004 ;18 : 91 \u2013 100 .\n\nBaier SK ,  McClements DJ .  Combined influence of NaCl and sucrose on heat-induced gelation of bovine serum albumin .  _J. Agric. Food Chem._ 2003 ;51 : 8107 \u2013 8112 .\n\nBall GFM .  _Fat-soluble vitamin assays in food analysis, a comprehensive review_ .  London :  Elsevier Applied Science ;  1988 .\n\nBarbana C ,  P\u00e9rez MD ,  S\u00e1nchez L ,  Dalgalarrondo M ,  Chobert JM ,  Haertl\u00e9 T ,  Calvo M .  Interaction of bovine \u03b1-lactalbumin with fatty acids as determined by partition equilibrium and fluorescence spectroscopy .  _Int. Dairy J._ 2006 ;16 : 18 \u2013 25 .\n\nBarbiroli A ,  Bonomi F ,  Ferranti P ,  Fessas D ,  Nasi A ,  Rasmussen P ,  Iametti S .  Bound fatty acids modulate the sensitivity of bovine \u03b2-lactoglobulin to chemical and physical denaturation .  _J. Agric. Food Chem._ 2011 ;59 : 5729 \u2013 5737 .\n\nBello M ,  Portillo-T\u00e9llez MDC ,  Garc\u00eda-Hern\u00e1ndez E .  Energetics of ligand recognition and self-association of bovine \u03b2-lactoglobulin: Differences between variants A and B .  _Biochemistry_.  2011 ;50 : 151 \u2013 161 .\n\nBenzaria A ,  Maresca M ,  Taieb N ,  Dumay E .  Interaction of curcumin with phosphocasein micelles processed or not by dynamic high-pressure .  _Food Chem._ 2013 ;138 : 2327 \u2013 2337 .\n\nBezwoda WR ,  Mansoor N .  Isolation and characterization of lactoferrin separated from human whey by adsorption chromatography using Cibacron Blue F3G-A linked affinity adsorbent .  _Clin. Chim. Acta_.  1986 ;157 : 89 \u2013 93 .\n\nBhattacharyya J ,  Das KP .  Interactions of chlorpromazine with milk proteins .  _Mol. Cell. Biochem._ 2001 ;221 : 11 \u2013 15 .\n\nBohin MC ,  Vincken JP ,  Van Der Hijden HTWM ,  Gruppen H .  Efficacy of food proteins as carriers for flavonoids .  _J. Agric. Food Chem._ 2012 ;60 : 4136 \u2013 4143 .\n\nBourassa P ,  Tajmir-Riahi HA .  Locating the binding sites of folic acid with milk \u03b1\\- and \u03b2-caseins .  _J. Phys. Chem. B_.  2012 ;116 : 513 \u2013 519 .\n\nBoye JI ,  Alli I .  Thermal denaturation of mixtures of \u03b1-lactalbumin and \u03b2-lactoglobulin: A differential scanning calorimetric study .  _Food Res. Int._ 2000 ;33 : 673 \u2013 682 .\n\nBoye JI ,  Alli I ,  Ismail AA .  Interactions involved in the gelation of bovine serum albumin .  _J. Agric. Food Chem._ 1996 ;44 : 996 \u2013 1004 .\n\nBoye JI ,  Alli I ,  Ismail AA .  Use of differential scanning calorimetry and infrared spectroscopy in the study of thermal and structural stability of \u03b1-lactalbumin .  _J. Agric. Food Chem._ 1997 ;45 : 1116 \u2013 1125 .\n\nBoye JI ,  Ismail AA ,  Alli I .  Effects of physicochemical factors on the secondary structure of \u03b2-lactoglobulin .  _J. Dairy Res._ 1996 ;63 : 97 \u2013 109 .\n\nBrinkmann CR ,  Thiel S ,  Larsen MK ,  Petersen TE ,  Jensenius JC ,  Heegaard CW .  Preparation and comparison of cytotoxic complexes formed between oleic acid and either bovine or human \u03b1-lactalbumin .  _J. Dairy Sci._ 2011 ;94 : 2159 \u2013 2170 .\n\nBusti P ,  Gatti CA ,  Delorenzi NJ .  Binding of alkylsulfonate ligands to bovine \u03b2-lactoglobulin: Effects on protein thermal unfolding .  _Food Res. Int._ 2006 ;39 : 503 \u2013 509 .\n\nBusti P ,  Scarpeci S ,  Gatti CA ,  Delorenzi NJ .  Binding of alkylsulfonate ligands to bovine \u03b2-lactoglobulin: Effects on protein denaturation by urea .  _Food Hydrocolloids_.  2005 ;19 : 249 \u2013 255 .\n\nCawthern KM ,  Narayan M ,  Chaudhuri D ,  Permyakov EA ,  Berliner LJ .  Interactions of \u03b1-lactalbumin with fatty acids and spin label analogs .  _J. Biol. Chem._ 1997 ;272 : 30812 \u2013 30816 .\n\nCelej MS ,  Dassie SA ,  Freire E ,  Bianconi ML ,  Fidelio GD .  Lingand-induced thermostability in proteins:Thermodynamic analysis of ANS-albumin interaction .  _Biochim. Biophys. Acta_.  2005 ;1750 : 122 \u2013 133 .\n\nChanasattru W ,  Decker EA ,  McClements DJ .  Inhibition of droplet flocculation in globular-protein stabilized oil-in-water emulsions by polyols .  _Food Res. Int._ 2007 ;40 : 1161 \u2013 1169 .\n\nChanasattru W ,  Decker EA ,  McClements DJ .  Modulation of thermal stability and heat-induced gelation of \u03b2-lactoglobulin by high glycerol and sorbitol levels .  _Food Chem._ 2007 ;103 : 512 \u2013 520 .\n\nChanasattru W ,  Decker EA ,  McClements DJ .  Physicochemical basis for cosolvent modulation of \u03b2\\- lactoglobulin functionality: Interfacial tension study .  _Food Res. Int._ 2007 ;40 : 1098 \u2013 1105 .\n\nChanasattru W ,  Decker EA ,  McClements DJ .  Impact of cosolvents (polyols) on globular protein functionality: Ultrasonic velocity, density, surface tension and solubility study .  _Food Hydrocolloids_.  2008 ;22 : 1475 \u2013 1484 .\n\nCharles M ,  Bernal B ,  Guichard E .  Interaction of \u03b2-lactoglobulin with flavour compounds .  In:  Taylor AJ ,  Mottram DS ,  eds.  _Flavour science: Recent developments_ .  Cambridge, UK :  Royal Society of Chemistry ;  1996 : 433 \u2013 436 .\n\nCho YJ ,  Batt CA ,  Sawyer L .  Probing the retinol-binding site of bovine \u03b2-lactoglobulin .  _J. Biol. Chem._ 1994 ;269 : 11102 \u2013 11107 .\n\nClark DC ,  Wilde PJ ,  Wilson DR ,  Wustneck R .  The interaction of sucrose esters with \u03b2-lactoglobulin and \u03b2-casein from bovine-milk .  _Food Hydrocolloids_.  1992 ;6 : 173 \u2013 186 .\n\nConsidine T ,  Patel HA ,  Anema SG ,  Singh H ,  Creamer LK .  Interactions of milk proteins during heat and high hydrostatic pressure treatments\u2014a review .  _Innov. Food Sci. Emerging Technol._ 2007 ;8 : 1 \u2013 23 .\n\nConsidine T ,  Patel HA ,  Singh H ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _J. Agric. Food Chem._ 2005 ;53 : 3197 \u2013 3205 .\n\nConsidine T ,  Patel HA ,  Singh H ,  Creamer LK .  Influence of binding conjugated linoleic acid and myristic acid on the heat- and high-pressure-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _Food Chem._ 2007 ;102 : 1270 \u2013 1280 .\n\nConsidine T ,  Singh H ,  Patel HA ,  Creamer LK .  Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of \u03b2-lactoglobulin B .  _J. Agric. Food Chem._ 2005 ;53 : 8010 \u2013 8018 .\n\nCreamer L .  A study of the effect of sodium dodecyl sulfate on bovine \u03b2-casein self-association .  _Archives Biochemistry and Biophysics_.  1980 ;199 : 172 \u2013 178 .\n\nCreamer LK .  Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine \u03b2-lactoglobulin .  _Biochemistry_.  1995 ;34 : 7170 \u2013 7176 .\n\nCreamer LK ,  Loveday SM ,  Sawyer L .  _Milk proteins | \u03b2-Lactoglobulin, Encyclopedia of Dairy Sciences_ .  2nd ed. Oxford :  Elsevier ;  2011 .\n\nDai-Dong JX ,  Novak G ,  Hardy J .  Stabilization of vitamin C by \u03b2-lactoglobulin during heat treatment .  _Sci. Aliments_.  1990 ;10 : 393 \u2013 401 .\n\nDamodaran S ,  Kinsella JE .  Flavor protein interactions. Binding of carbonyls to bovine serum albumin\u2014thermodynamic and conformational effects .  _J. Agric. Food Chem._ 1980 ;28 : 567 \u2013 571 .\n\nDamodaran S ,  Kinsella JE .  The effect of neutral salts on the stability of macromolecules \u2013 a new approach using a protein-ligand binding system .  _J. Biol. Chem._ 1981 ;256 : 3394 \u2013 3398 .\n\nDas, S., Ellis, A., Mittal, V.A., Singh, H., Ye, A., 2013. Micronutrient fortification process and its uses, PCT Application No. PCT\/NZ2013\/000109.\n\nDevi AF ,  Buckow R ,  Hemar Y ,  Kasapis S .  Structuring dairy systems through high pressure processing .  _J. Food Eng._ 2013 ;114 : 106 \u2013 122 .\n\nDiarrassouba F ,  Remondetto G ,  Liang L ,  Garrait G ,  Beyssac E ,  Subirade M .  Effects of gastrointestinal pH conditions on the stability of the \u03b2-lactoglobulin\/vitamin D3 complex and on the solubility of vitamin D3 .  _Food Res. Int. 52, 515-521_.  2013 ; .\n\nDickinson E .  Flocculation of protein-stabilized oil-in-water emulsions .  _Colloids Surf., B_.  2010 ;81 : 130 \u2013 140 .\n\nDierckx S ,  Huyghebaert A .  Effects of sucrose and sorbitol on the gel formation of a whey protein isolate .  _Food Hydrocolloids_.  2002 ;16 : 489 \u2013 497 .\n\nDivsalar A ,  Saboury AA .  Comparative structural and conformational studies on two forms of \u03b2-lactoglobulin (A and B) upon interaction with lead ion .  _FEBS J._ 2005 ;272 : 340 .\n\nDivsalar A ,  Saboury AA ,  Mansoori-Torshizi H ,  Hemmatinejad B .  Comparative and structural analysis of the interaction between \u03b2-lactoglobulin type A and B with a new anticancer component (2,2\u2032-bipyridin n-hexyl dithiocarbamato Pd(II) nitrate) .  _Bull. Korean Chem. Soc._ 2006 ;27 : 1801 \u2013 1808 .\n\nDivsalar A ,  Saboury AA ,  Moosavi-Movahedi AA .  A study on the interaction between \u03b2-lactoglobulin a and a new antitumor reagent (2,2-bipyridinglycinato Pd(II) chloride) .  _FEBS J._ 2006 ;273 : 333 .\n\nDufour E ,  Haertle T .  Alcohol induced changes of \u03b2-lactoglobulin-retinol-binding stoichiometry .  _Protein Eng._ 1990 ;4 : 185 \u2013 190 .\n\nDufour E ,  Hoa GHB ,  Haertle T .  High-pressure effects on \u03b2-lactoglobulin interactions with ligands studied by fluorescence .  _Biochim. Biophys. Acta_.  1994 ;1206 : 166 \u2013 172 .\n\nDufour E ,  Haertle T .  Binding of retinoids and \u03b2-carotene to \u03b2-lactoglobulin. Influence of protein modifications .  _Biochim. Biophys. Acta_.  1991 ;1079 : 316 \u2013 320 .\n\nDumay EM ,  Kalichevsky MT ,  Cheftel JC .  High-pressure unfolding and aggregation of \u03b2-lactoglobulin and the baroprotective effects of sucrose .  _J. Agric. Food Chem._ 1994 ;42 : 1861 \u2013 1868 .\n\nDumay EM ,  Kalichevsky MT ,  Cheftel JC .  Characteristics of pressure-induced gels of \u03b2-lactoglobulin at various times after pressure release .  _Food. Sci. Technol. \u2014LWT_.  1998 ;31 : 10 \u2013 19 .\n\nEbel C ,  Eisenberg H ,  Ghirlando R .  Probing protein-sugar interactions .  _Biophys. J._ 2000 ;78 : 385 \u2013 393 .\n\nElzoghby AO ,  Abo El-Fotoh WS ,  Elgindy NA .  Casein-based formulations as promising controlled release drug delivery systems .  _J. Control. Release_.  2011 ;153 : 206 \u2013 216 .\n\nElzoghby AO ,  Samy WM ,  Elgindy NA .  Novel spray-dried genipin-crosslinked casein nanoparticles for prolonged release of alfuzosin hydrochloride .  _Pharm. Res._ 2013 ;30 : 512 \u2013 522 .\n\nFitzGerald RJ .  Potential uses of caseinophosphopeptides .  _Int. Dairy J._ 1998 ;8 : 451 \u2013 457 .\n\nFogolari F ,  Ragona L ,  Licciardi S ,  Romagnoli S ,  Michelutti R ,  Ugolini R ,  Molinari H .  Electrostatic properties of bovine \u03b2-lactoglobulin .  _Proteins: Struct., Funct., Genet._ 2000 ;39 : 317 \u2013 330 .\n\nFontecha J ,  Swaisgood HE .  Interaction of sucrose ester with casein micelles as characterized by size-exclusion chromatography .  _J. Dairy Sci._ 1995 ;78 : 2660 \u2013 2665 .\n\nFord JE .  Some observations on possible nutritional significance of vitamin-B12-binding and folate-binding proteins in milk .  _Br. J. Nutr._ 1974 ;31 : 243 \u2013 257 .\n\nForrest SA ,  Yada RY ,  Rousseau D .  Interactions of vitamin D3 with bovine \u03b2-lactoglobulin a and \u03b2-casein .  _J. Agric. Food Chem._ 2005 ;53 : 8003 \u2013 8009 .\n\nFranzen KL ,  Kinsella JE .  Parameters affecting binding of volatile flavor compounds in model food systems 1 .  _Proteins. J. Agric. Food Chem._ 1974 ;22 : 675 \u2013 678 .\n\nFrapin D ,  Dufour E ,  Haertle T .  Probing the fatty acid binding site of \u03b2-lactoglobulins .  _J. Protein Chem._ 1993 ;12 : 443 \u2013 449 .\n\nFutterman S ,  Heller J .  The enhancement of fluorescence and the decreased susceptibility to enzymatic oxidation of retinol complexed with bovine serum albumin, \u03b2-lactoglobulin, and the retinol-binding protein of human plasma .  _J. Biol. Chem._ 1972 ;247 : 5168 \u2013 5172 .\n\nGuichard E ,  Langourieux S .  Interactions between \u03b2-lactoglobulin and flavour compounds .  _Food Chem._ 2000 ;71 : 301 \u2013 308 .\n\nGuth H ,  Fritzler R .  Binding studies and computer-aided modelling of macromolecule\/odorant interactions .  _Chemical Biodiversity_.  2004 ;1 : 2001 \u2013 2023 .\n\nHa HK ,  Kim JW ,  Lee MR ,  Lee WJ .  Formation and characterization of quercetin-loaded chitosan oligosaccharide\/\u03b2-lactoglobulin nanoparticle .  _Food Res. Int._ 2013 ;52 : 82 \u2013 90 .\n\nHaham M ,  Ish-Shalom S ,  Nodelman M ,  Duek I ,  Segal E ,  Kustanovich M ,  Livney YD .  Stability and bioavailability of vitamin D nanoencapsulated in casein micelles .  _Food Funct._ 2012 ;3 : 737 \u2013 744 .\n\nHammou HO ,  Plaza del Pino IM ,  Sanchez-Ruiz JM .  Hydration changes upon protein unfolding: Cosolvent effect analysis .  _New J. Chem._ 1998 ;22 : 1453 \u2013 1461 .\n\nHansted JG ,  Wejse PL ,  Bertelsen H ,  Otzen DE .  Effect of protein-surfactant interactions on aggregation of \u03b2-lactoglobulin .  _Biochim. Biophys. Acta, Proteins Proteomics_.  2011 ;1814 : 713 \u2013 723 .\n\nHavea P ,  Singh H ,  Creamer LK .  Characterization of heat-induced aggregates of \u03b2-lactoglobulin, \u03b1-lactalbumin and bovine serum albumin in a whey protein concentrate environment .  _J. Dairy Res._ 2001 ;68 : 483 \u2013 497 .\n\nHe L ,  Zheng J ,  Labuza TP ,  Xiao H .  A surface enhanced Raman spectroscopic study of interactions between casein and polymethoxyflavones .  _J. Raman Spectrosc_.  2013 ; : 531 \u2013 535 .\n\nHousaindokht MR ,  Chamani J ,  Saboury AA ,  Moosavi-Movahedi AA ,  Bahrololoom M .  Three binding sets analysis of \u03b1-lactalbumin by interaction of tetradecyl trimethyl ammonium bromide .  _Bull. Korean Chem. Soc._ 2001 ;22 : 145 \u2013 148 .\n\nHuppertz T ,  Fox PF ,  de Kruif KG ,  Kelly AL .  High pressure-induced changes in bovine milk proteins: A review .  _Biochim. Biophys. Acta, Proteins Proteomics_.  2006 ;1764 : 593 \u2013 598 .\n\nJasinski E ,  Kilara A .  Flavor binding by whey proteins .  _Milchwissenschaft_.  1985 ;40 : 596 \u2013 599 .\n\nJones MN ,  Wilkinson A .  The interaction between \u03b2-lactoglobulin and sodium n-dodecylsulfate .  _Biochem J._ 1976 ;153 : 713 \u2013 718 .\n\nJou KD ,  Harper WJ .  Effect of di-saccharides on the thermal properties of whey proteins determined by differential scanning calorimetry (DSC) .  _Milchwissenschaft_.  1996 ;51 : 509 \u2013 511 . \nJouenne E ,  Crouzet J .  Determination of apparent binding constants for aroma compounds with \u03b2-lactoglobulin by dynamic coupled column liquid chromatography .  _J. Agric. Food Chem._ 2000 ;48 : 5396 \u2013 5400 .\n\nJung DM ,  de Ropp JS ,  Ebeler SE .  Application of pulse field gradient NMR techniques for investigating binding of flavor compounds to macromolecules .  _J. Agric. Food Chem._ 2002 ;50 : 4262 \u2013 4269 .\n\nJung DM ,  Ebeler SE .  Investigation of binding behaviour of \u03b1\\- and \u03b2-ionones to \u03b2-lactoglobulin at different pH values using a diffusion-based NOE pumping technique .  _J. Agric. Food Chem._ 2003 ;51 : 1988 \u2013 1993 .\n\nKamiyama T ,  Sadahide Y ,  Nogusa Y ,  Gekko K .  Polyol-induced molten globule of cytochrome C: An evidence for stabilization by hydrophobic interaction .  _Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol._ 1999 ;1434 : 44 \u2013 57 .\n\nKanakis CD ,  Hasni I ,  Bourassa P ,  Tarantilis PA ,  Polissiou MG ,  Tajmir-Riahi HA .  Milk \u03b2-lactoglobulin complexes with tea polyphenols .  _Food Chem._ 2011 ;127 : 1046 \u2013 1055 .\n\nKanno C ,  Kanehara N ,  Shirafuji K ,  Tanji R ,  Imai T .  Binding form of vitamin-B2 in bovine-milk\u2014its concentration, distribution and binding linkage .  _J. Nutr. Sci. Vitaminol._ 1991 ;37 : 15 \u2013 27 .\n\nKaushik JK ,  Bhat R .  Thermal stability of proteins in aqueous polyol solutions: Role of the surface tension of water in the stabilizing effect of polyols .  _J. Phys. Chem. B_.  1998 ;102 : 7058 \u2013 7066 .\n\nKeenan RD ,  Young DJ ,  Tier CM ,  Jones AD ,  Underdown J .  Mechanism of pressure-induced gelation of milk .  _J. Agric. Food Chem._ 2001 ;49 : 3394 \u2013 3402 .\n\nKehoe JJ ,  Foegeding EA .  Interaction between beta-casein and whey proteins as a function of pH and salt concentration .  _J. Agric. Food Chem._ 2011 ;59 : 349 \u2013 355 .\n\nKibangou I ,  Bouhallab S ,  Bureau F ,  Allouche S ,  Thouvenin G ,  Bougl\u00e9 D .  Caseinophosphopeptide-bound iron: Protective effect against gut peroxidation .  _Ann. Nutr. Metab._ 2008 ;52 : 177 \u2013 180 .\n\nKlaassen I ,  Brakenhoff RH ,  Smeets SJ ,  Snow GB ,  Braakhuis BJM .  Considerations for in vitro retinoid experiments: Importance of protein interaction .  _Biochim. Biophys. Acta, Gen. Subj._ 1999 ;1427 : 265 \u2013 275 .\n\nKontopidis G ,  Holt C ,  Sawyer L .  Invited review: \u03b2-lactoglobulin: Binding properties, structure, and function .  _J. Dairy Sci._ 2004 ;87 : 785 \u2013 796 .\n\nKonuma T ,  Sakurai M ,  Goto Y .  Promiscuous binding of ligands by \u03b2-lactoglobulin involves hydrophobic interactions and plasticity .  _J. Mol. Biol._ 2007 ;368 : 209 \u2013 218 .\n\nK\u00fchn J ,  Considine T ,  Singh H .  Interactions of milk proteins and volatile compounds: Implications in the development of protein foods .  _J. Food Sci._ 2006 ;71 : R72 \u2013 R82 .\n\nKulmyrzaev A ,  Bryant C ,  McClements DJ .  Influence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins .  _J. Agric. Food Chem._ 2000 ;48 : 1593 \u2013 1597 .\n\nLamiot E ,  Dufour E ,  Haertle T .  Insect sex pheromone binding by bovine \u03b2-lactoglobulin .  _J. Agric. Food Chem._ 1994 ;42 : 695 \u2013 699 .\n\nLee JC ,  Timasheff SN .  The stabilization of proteins by sucrose .  _J. Biol. Chem._ 1981 ;256 : 7193 \u2013 7201 .\n\nLerbret A ,  Bordat P ,  Affouard F ,  Descamps M ,  Migliardo F .  How homogeneous are the trehalose, maltose, and sucrose water solutions? An insight from molecular dynamics simulations .  _J. Phys. Chem. B_.  2005 ;109 : 11046 \u2013 11057 .\n\nLietaer E ,  Poiffait A ,  Adrian J .  Studies on the interaction between casein and vitamin A .  _Food. Sci. Technol. \u2014LWT_.  1991 ;24 : 39 \u2013 45 .\n\nLin TY ,  Timasheff SN .  On the role of surface tension in the stabilization of globular proteins .  _Protein Sci._ 1996 ;5 : 372 \u2013 381 .\n\nLindner RA ,  Kapur A ,  Carver JA .  The interaction of the molecular chaperone, alpha-crystallin, with molten globule states of bovine-lactalbumin .  _J. Biol. Chem._ 1997 ;272 : 27722 \u2013 27729 .\n\nLi\u0161kov\u00e1 K ,  Auty MAE ,  Chaurin V ,  Min S ,  Mok KH ,  O'Brien N ,  Kelly AL ,  Brodkorb A .  Cytotoxic complexes of sodium oleate with \u03b2-lactoglobulin .  _Eur. J. Lipid Sci. Technol._ 2011 ;113 : 1207 \u2013 1218 .\n\nLiu X ,  Powers JR ,  Swanson BG ,  Hill HH ,  Clark S .  Modification of whey protein concentrate hydrophobicity by high hydrostatic pressure .  _Innov. Food Sci. Emerging Technol._ 2005 ;6 : 310 \u2013 317 .\n\nLivney YD .  Milk proteins as vehicles for bioactives .  _Curr. Opin. Colloid Interface Sci._ 2010 ;15 : 73 \u2013 83 .\n\nLivney, Y.D., and Dalgleish, D.G. 2007. Casein micelles for nanoencapsulation of hydrophobic compounds. Patent application WO2007122613 A1.\n\nLoch JI ,  Polit A ,  Bonarek P ,  Olszewska D ,  Kurpiewska K ,  Dziedzicka-Wasylewska M ,  Lewiski K .  Structural and thermodynamic studies of binding saturated fatty acids to bovine \u03b2-lactoglobulin .  _Int. J. Bio. Macromol._ 2012 ;50 : 1095 \u2013 1102 .\n\nLoveday SM ,  Rao MA ,  Singh H .  Food protein nanoparticles: Formation, properties and applications .  In:  Bhandari B ,  Roos YH ,  eds.  _Food materials science and engineering_ .  Oxford :  Blackwell Publishing ;  2012 : 263 \u2013 294 .\n\nLoveday SM ,  Sarkar A ,  Singh H .  Innovative yoghurts: Novel processing technologies for improving acid milk gel texture .  _Trends Food Sci. Tech._ 2013 ;33 : 5 \u2013 20 .\n\nLoveday SM ,  Singh H .  Recent advances in technologies for vitamin A protection in foods .  _Trends Food Sci. Tech._ 2008 ;19 .\n\nLu R-C ,  Cao A-N ,  Lai L-H ,  Xiao J-X .  Effect of cationic surfactants on the interaction between sodium perfluorooctanoate and \u03b2-lactoglobulin .  _J. Colloid Interface Sci._ 2006 ;293 : 61 \u2013 68 .\n\nMata L ,  Sanchez L ,  Calvo M .  Interaction of mercury with human and bovine milk proteins .  _Biosci. Biotechol. Biochem._ 1997 ;61 : 1641 \u2013 1645 .\n\nMaulik S ,  Moulik DSP ,  Chattoraj DK .  Biopolymer\u2014surfactant interaction: 4 kinetics of binding of cetyltrimethyl ammonium bromide with gelatin, hemoglobin, \u03b2-lactoglobulin and lysozyme .  _J. Biomol. Struct. Dyn._ 1996 ;13 : 771 \u2013 780 .\n\nMcClements DJ .  Modulation of globular protein functionality by weakly interacting cosolvents .  _Crit. Rev. Food Sci. Nutr._ 2002 ;42 : 417 \u2013 471 .\n\nMcNeill VL ,  Schmidt KA .  Vanillin interaction with milk protein isolates in sweetened drinks .  _J. Food Sci._ 1993 ;58 : 1142 \u2013 1144 : 1147 .\n\nMercadante D ,  Melton LD ,  Norris GE ,  Loo TS ,  Williams MAK ,  Dobson RCJ ,  Jameson GB .  Bovine \u03b2-lactoglobulin is dimeric under imitative physiological conditions: Dissociation equilibrium and rate constants over the pH range of 2.5\u20137.5 .  _Biophys. J._ 2012 ;103 : 303 \u2013 312 .\n\nMohammadzadeh KA ,  Smith GM ,  Feeney RE .  Hydrophobic binding of hydrocarbons by proteins II. Relationship of protein structure .  _Biochim. Biophys. Acta_.  1969 ;194 : 256 \u2013 264 .\n\nMohan MS ,  Jurat-Fuentes JL ,  Harte F .  Binding of vitamin A by casein micelles in commercial skim milk .  _J. Dairy Sci._ 2013 ;96 : 790 \u2013 798 .\n\nMonaco HL ,  Zanotti G ,  Spadon P ,  Bolognesi M ,  Sawyer L ,  Eliopoulos EE .  Crystal structure of the trigonal form of bovine \u03b2-lactoglobulin and of its complex with retinol at 2.5 \u00c5 resolution .  _J. Mol. Biol._ 1987 ;197 : 695 \u2013 706 .\n\nMora-Gutierrez A ,  Farrell HM .  Sugar\u2013casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: A case of preferential interactions .  _J. Agric. Food Chem._ 2000 ;48 : 3245 \u2013 3255 .\n\nMuresan S ,  van der Bent A ,  de Wolf FA .  Interaction of \u03b2-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: Nonlinear quenching effects related to protein\u2013protein association .  _J. Agric. Food Chem._ 2001 ;49 : 2609 \u2013 2618 .\n\nMurray BS ,  Liang HJ .  Enhancement of the foaming properties of protein dried in the presence of trehalose .  _J. Agric. Food Chem._ 1999 ;47 : 4984 \u2013 4991 .\n\nNagasako Y ,  Saito H ,  Tamura Y ,  Shimamura S ,  Tomita M .  Iron-binding properties of bovine lactoferrin in iron-rich solution .  _J. Dairy Sci._ 1993 ;76 : 1876 \u2013 1881 .\n\nNagy K ,  Courtet-Compondu MC ,  Williamson G ,  Rezzi S ,  Kussmann M ,  Rytz A .  Non-covalent binding of proteins to polyphenols correlates with their amino acid sequence .  _Food Chem._ 2012 ;132 : 1333 \u2013 1339 .\n\nNarayan M ,  Berliner LJ .  Fatty acids and retinoids bind independently and simultaneously to \u03b2-lactoglobulin .  _Biochemistry_.  1997 ;36 : 1906 \u2013 1911 .\n\nNarayan M ,  Berliner LJ .  Mapping fatty acid binding to \u03b2-lactoglobulin: Ligand binding is restricted by modification of cys 121 .  _Protein Sci._ 1998 ;7 : 150 \u2013 157 .\n\nNixon PF ,  Jones M ,  Winzor DJ .  Quantitative description of the interaction between folate and the folate-binding protein from cow's milk .  _Biochem J._ 2004 ;382 : 215 \u2013 221 .\n\nO'Connell JE ,  Fox PF .  Heat-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry \u2014Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 879 \u2013 945 .\n\nO'Neill TE ,  Kinsella JE .  Binding of alkanone favors to \u03b2-lactoglobulin-effects of conformational and chemical modification .  _J. Agric. Food Chem._ 1987 ;35 : 770 \u2013 774 .\n\nOelrichs BA ,  Kratzing CC ,  Kelly JD ,  Winzor DJ .  The binding of ascorbate to bovine serum-albumin .  _Int. J. Vitam. Nutr. Res._ 1984 ;54 : 61 \u2013 64 .\n\nPapiz MZ ,  Sawyer L ,  Eliopoulos EE ,  North ACT ,  Findlay JBC ,  Sivaprasadarao R ,  Jones TA ,  Newcomer ME ,  Kraulis PJ .  The structure of \u03b2-lactoglobulin and its similarity to plasma retinol-binding protein .  _Nature_.  1986 ;324 : 383 \u2013 385 .\n\nPeres JM ,  Bouhallab S ,  Petit C ,  Bureau F ,  Maubois JL ,  Arhan P ,  Bougle D .  Improvement of zinc intestinal absorption and reduction of zinc\/iron interaction using metal bound to the caseinophosphopeptide 1-25 of \u03b2-casein .  _Reprod., Nutr., Dev._ 1998 ;38 : 465 \u2013 472 .\n\nP\u00e9rez MD ,  Calvo M .  Interaction of \u03b2-lactoglobulin with retinol and fatty acids and its role as a possible biological function for this protein: A review .  _J. Dairy Sci._ 1995 ;78 : 978 \u2013 988 .\n\nP\u00e9rez MD ,  Diaz de Villegas C ,  Sanchez L ,  Aranda P ,  Ena JM ,  Calvo M .  Interaction of fatty acids with \u03b2-lactoglobulin and albumin from ruminant milk .  _J. Biochem._ 1989 ;106 : 1094 \u2013 1097 .\n\nP\u00e9rez MD ,  Puyol P ,  Ena JM ,  Calvo M .  Comparison of the ability to bind lipids of \u03b2-lactoglobulin and serum-albumin of milk from ruminant and non-ruminant species .  _J. Dairy Res._ 1993 ;60 : 55 \u2013 63 .\n\nP\u00e9rez MD ,  Sanchez L ,  Aranda P ,  Ena JM ,  Calvo M .  Synthesis and evolution of concentration of \u03b2-lactoglobulin and \u03b1-lactalbumin from cow and sheep colostrums and milk throughout early lactation .  _Cell. Mol. Biol._ 1990 ;36 : 205 \u2013 212 .\n\nP\u00e9rez MD ,  Sanchez L ,  Aranda P ,  Ena JM ,  Oria R ,  Calvo M .  Effect of \u03b2-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk .  _Biochim. Biophys. Acta_.  1992 ;1123 : 151 \u2013 155 .\n\nPermyakov EA ,  Morozova LA ,  Kahnichenko LP ,  Derezhkov VY .  Interaction of \u03b1-lactalbumin with Cu2+ .  _Biophys. Chem._ 1988 ;32 : 37 \u2013 42 .\n\nPoiffait A ,  Adrian J .  Interaction between casein and vitamin A during food processing .  _Adv. Exp. Med. Biol._ 1991 ;289 : 61 \u2013 73 .\n\nPoliti R ,  Harries D .  Enthalpically driven peptide stabilization by protective osmolytes .  _Chem. Comm._ 2010 ;46 : 6449 \u2013 6451 .\n\nPuyol P ,  Perez MD ,  Mata L ,  Calvo M .  Study on interaction between \u03b2-lactoglobulin and other bovine whey proteins with ascorbic acid .  _Milchwissenschaft_.  1994 ;49 : 25 \u2013 27 .\n\nPuyol P ,  Perez MD ,  Peiro JM ,  Calvo M .  Interaction of bovine \u03b2-lactoglobulin and other bovine and human whey proteins with retinol and fatty acids .  _Agric. Biol. Chem._ 1991 ;55 : 2515 \u2013 2520 .\n\nQin BY ,  Creamer LK ,  Baker EN ,  Jameson GB .  12-bromododecanoic acid binds inside the calyx of bovine \u03b2-lactoglobulin .  _FEBS Lett._ 1998 ;438 : 272 \u2013 278 .\n\nRagona L ,  Fogolari F ,  Zetta L ,  Perez DM ,  Puyol P ,  De Kruif K ,  Lohr F ,  Ruterjans H ,  Molinari H .  Bovine \u03b2-lactoglobulin: Interaction studies with palmitic acid .  _Protein Sci._ 2000 ;9 : 1347 \u2013 1356 .\n\nRahimi Yazdi S ,  Corredig M .  Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study .  _Food Chem._ 2012 ;132 : 1143 \u2013 1149 .\n\nRaica Jr N ,  Vavich MG ,  Kemmerer AR .  The effects of several milk components and similar compounds on the utilization of carotene by the rat .  _Arch. Biochem. Biophys._ 1959 ;83 : 376 \u2013 380 .\n\nReineccius GA ,  Coulter ST .  Flavour rentention during drying .  _J. Dairy Sci._ 1969 ;52 : 1219 \u2013 1223 .\n\nRodr\u00edguez Ni\u00f1o MR ,  Rodr\u00edguez Patino JM .  Effect of the aqueous phase composition on the adsorption of bovine serum albumin to the air\u2013water interface .  _Ind. Eng. Chem. Res._ 2002 ;41 : 1489 \u2013 1495 .\n\nRomero CM ,  Lozano JM ,  Sancho J ,  Giraldo GI .  Thermal stability of \u03b2-lactoglobulin in the presence of aqueous solution of alcohols and polyols .  _Int. J. Bio. Macromol._ 2007 ;40 : 423 \u2013 428 .\n\nR\u00f6sgen J .  Molecular basis of osmolyte effects on protein and metabolites .  _Methods Enzymol._ 2007 ;428 : 459 \u2013 486 .\n\nRoufik S ,  Gauthier SF ,  Leng X ,  Turgeon SL .  Thermodynamics of binding interactions between bovine \u03b2-lactoglobulin a and the antihypertensive peptide \u03b2-lg f142-148 .  _Biomacromolecules_.  2006 ;7 : 419 \u2013 426 .\n\nSahihi M ,  Ghayeb Y ,  Bordbar A-K .  Fluorescence spectroscopic study on interaction of retinol with \u03b2-lactoglobulin in the presence of cetylpyridinium chloride .  _Spectroscopy_.  2012 ;27 : 27 \u2013 34 .\n\nS\u00e1iz-Abajo MJ ,  Gonz\u00e1lez-Ferrero C ,  Moreno-Ruiz A ,  Romo-Hualde A ,  Gonz\u00e1lez-Navarro CJ .  Thermal protection of \u03b2-carotene in re-assembled casein micelles during different processing technologies applied in food industry .  _Food Chem._ 2013 ;138 : 1581 \u2013 1587 .\n\nSalter DN ,  Mowlem A .  Neonatal role of milk folate-binding protein\u2014studies on the course of digestion of goats milk folate binder in the 6-d-old kid .  _Br. J. Nutr._ 1983 ;50 : 589 \u2013 596 .\n\nSchokker EP ,  Singh H ,  Pinder DN ,  Norris GE ,  Creamer LK .  Characterization of intermediates formed during heat-induced aggregation of \u03b2-lactoglobulin ab at neutral pH .  _Int. Dairy J._ 1999 ;9 : 791 \u2013 800 .\n\nSemenova MG ,  Antipova AS ,  Belyakova LE .  Food protein interactions in sugar solutions .  _Curr. Opin. Colloid Interface Sci._ 2002 ;7 : 438 \u2013 444 .\n\nShapira A ,  Assaraf YG ,  Epstein D ,  Livney YD .  \u03b2-casein nanoparticles as an oral delivery system for chemotherapeutic drugs: Impact of drug structure and properties on co-assembly .  _Pharm. Res._ 2010 ;27 : 2175 \u2013 2186 .\n\nShimoyamada M ,  Yoshimura H ,  Tomida K ,  Watanabe K .  Stabilities of bovine \u03b2-lactoglobulin\/retinol or retinoic acid complexes against tryptic hydrolysis, heating and light-induced oxidation .  _Lebensm. -Wiss. Technol._ 1996 ;29 : 763 \u2013 766 .\n\nSingh H ,  Flanagan J .  Milk proteins .  In:  Hui YH ,  ed.  _Handbook of Food Science, Technology and Engineering_ .  New York :  CRC Press ;  2005 : 1 \u2013 23 .\n\nSingh H ,  Havea P .  Thermal denaturation, aggregation and gelation of whey proteins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry \u2014Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 1261 \u2013 1287 .\n\nSingh LR ,  Poddar NK ,  Dar TA ,  Kumar R ,  Ahmad F .  Protein and DNA destabilization by osmolytes: The other side of the coin .  _Life Sci._ 2011 ;88 : 117 \u2013 125 .\n\nSneharani AH ,  Karakkat JV ,  Singh SA ,  Rao AGA .  Interaction of curcumin with \u03b2-lactoglobulin; stability, spectroscopic analysis, and molecular modeling of the complex .  _J. Agric. Food Chem._ 2010 ;58 : 11130 \u2013 11139 .\n\nSostmann K ,  Guichard E .  Immobilized \u03b2-lactoglobulin on a HPLC-column: A rapid way to determine protein-flavour interactions .  _Food Chem._ 1998 ;62 : 509 \u2013 513 .\n\nSpector AA .  Fatty acid binding to plasma albumin .  _J. Lipid Res._ 1975 ;16 : 165 \u2013 179 .\n\nSpector AA ,  John K ,  Fletcher JE .  Binding of long-chain fatty acids to bovine serum albumin .  _J. Lipid Res._ 1969 ;10 : 56 \u2013 67 .\n\nSpiegel T .  Whey protein aggregation under shear conditions\u2014effects of lactose and heating temperature on aggregate size and structure .  _Int. J. Food Sci. Technol._ 1999 ;34 : 523 \u2013 531 .\n\nSreelakshmi Y ,  Sharma KK .  Interaction of \u03b1-lactalbumin with mini-\u03b1a-crystallin .  _J. Protein Chem._ 2001 ;20 : 123 \u2013 130 .\n\nSugiarto M ,  Ye A ,  Singh H .  Characterisation of binding of iron to sodium caseinate and whey protein isolate .  _Food Chem._ 2009 ;114 : 1007 \u2013 1013 .\n\nSugiarto M ,  Ye A ,  Taylor MW ,  Singh H .  Milk protein-iron complexes: Inhibition of lipid oxidation in an emulsion .  _Dairy Sci. Technol._ 2010 ;90 : 87 \u2013 98 .\n\nSwaisgood HE .  Protein ingredient for carrying lipophilic nutrients .  _United States Patent 6_.  2001 ;290 : 274 .\n\nSwaisgood HE .  Chemistry of the caseins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry \u2014Proteins_ .  New York :  Kluwer Academic\/Plenum Publishers ;  2003 : 139 \u2013 201 .\n\nSwaney JB ,  Klotz IM .  Amino acid sequence adjoining the lone tryptophan of human serum albumin. A binding site of the protein .  _Biochem J._ 1970 ;9 : 2570 \u2013 2574 .\n\nTaheri-Kafrani A ,  Bordbar A-K ,  Mousavi SH-A ,  Haertle T .  \u03b2-lactoglobulin structure and retinol binding changes in presence of anionic and neutral detergents .  _J. Agric. Food Chem._ 2008 ;56 : 7528 \u2013 7534 .\n\nTang X ,  Pikal MJ .  Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying .  _Pharm. Res._ 2005 ;22 : 1176 \u2013 1185 .\n\nTimasheff SN .  The control of protein stability and association by weak-interactions with water - how do solvents affect these processes .  _Annu. Rev. Biophys. Biomol. Struct._ 1993 ;22 : 67 \u2013 97 .\n\nTimasheff SN .  Control of protein stability and reactions by weakly interacting cosolvents: The simplicity of the complicated .  _Adv. Protein Chem._ 1998 ;51 : 355 \u2013 432 .\n\nTimasheff SN .  Protein hydration, thermodynamic binding, and preferential hydration .  _Biochemistry_.  2002 ;41 : 13473 \u2013 13482 .\n\nTiwari A ,  Bhat R .  Stabilization of yeast hexokinase A by polyol osmolytes: Correlation with the physicochemical properties of aqueous solutions .  _Biophys. Chem._ 2006 ;124 : 90 \u2013 99 .\n\nToyosaki T ,  Mineshita T .  Antioxidant effects of protein-bound riboflavin and free riboflavin .  _J. Food Sci._ 1988 ;53 : 1851 \u2013 1853 .\n\nTukamoto T ,  Ozeki S ,  Hattori F ,  Ishida T .  Drug interactions. 1. Binding of ascorbic-acid and fatty-acid ascorbyl esters to bovine serum-albumin .  _Chem. Pharm. Bull._ 1974 ;22 : 385 \u2013 389 .\n\nUeno HM ,  Ueda N ,  Morita M ,  Kakehi Y ,  Kobayashi T .  Thermal stability of the iron-lactoferrin complex in aqueous solution is improved by soluble soybean polysaccharide .  _Food Biophysics_.  2012 ;7 : 183 \u2013 189 .\n\nvan Ruth SM ,  de Vries G ,  Geary M ,  Giannouli P .  Influence of composition and structure of oil-in-water emulsions on retention of aroma compounds .  _J. Sci. Food Agric._ 2002 ;82 : 1028 \u2013 1035 .\n\nVegarud GE ,  Langsrud T ,  Svenning C .  Mineral-binding milk proteins and peptides; occurrence, biochemical and technological characteristics .  _Br. J. Nutr._ 2000 ;84 (Suppl 1) : S91 \u2013 98 .\n\nViseu MI ,  Melo EP ,  Carvalho TI ,  Correia RF ,  Costa SMB .  Unfolding kinetics of \u03b2-lactoglobulin induced by surfactant and denaturant: A stopped-flow\/fluorescence study .  _Biophys. J._ 2007 ;93 : 3601 \u2013 3612 .\n\nVoilley A ,  Beghin V ,  Charpentier C ,  Peyron D .  Interaction between aroma substances and macromolecules in model wine .  _Lebensm. -Wiss. Technol._ 1991 ;24 : 469 \u2013 472 .\n\nWang Q ,  Allen JC ,  Swaisgood HE .  Binding of lipophilic nutrients to \u03b2-lactoglobulin prepared by bioselective adsorption .  _J. Dairy Sci._ 1999 ;82 : 257 \u2013 264 .\n\nWang QW ,  Allen JC ,  Swaisgood HE .  Binding of retinoids to \u03b2-lactoglobulin isolated by bioselective adsorption .  _J. Dairy Sci._ 1997 ;80 : 1047 \u2013 1053 .\n\nWang QW ,  Allen JC ,  Swaisgood HE .  Binding of vitamin D and cholesterol to \u03b2-lactoglobulin .  _J. Dairy Sci._ 1997 ;82 : 257 \u2013 264 .\n\nWang X ,  Li M ,  Li M ,  Mao X ,  Zhou J ,  Ren F .  Preparation and characteristics of yak casein hydrolysate-iron complex .  _Int. J. Food Sci. Technol._ 2011 ;46 : 1705 \u2013 1710 .\n\nWaninge R ,  Paulsson M ,  Nylander T ,  Ninham B ,  Sellers P .  Binding of sodium dodecyl sulfate and dodecyl trimethyl ammonium chloride to \u03b2-lactoglobulin: A calorimetric study .  _Int. Dairy J._ 1998 ;8 : 141 \u2013 148 .\n\nWeiser H ,  Somorjai G .  Bioactivity of cis and dicis isomers of vitamin A esters .  _Int. J. Vitam. Nutr. Res._ 1992 ;62 : 201 \u2013 208 .\n\nWendorf JR ,  Radke CJ ,  Blanch HW .  Reduced protein adsorption at solid interfaces by sugar excipients .  _Biotechnol. Bioeng._ 2004 ;87 : 565 \u2013 573 .\n\nWu SY ,  Perez MD ,  Puyol P ,  Sawyer L .  \u03b2-Lactoglobulin binds palmitate within its central cavity .  _J. Biol. Chem._ 1999 ;274 : 170 \u2013 174 .\n\nWu X ,  Dey R ,  Wu H ,  Liu Z ,  He Q ,  Zeng X .  Studies on the interaction of -epigallocatechin-3-gallate from green tea with bovine \u03b2-lactoglobulin by spectroscopic methods and docking .  _Int. J. Dairy Technol._ 2013 ;66 : 7 \u2013 13 .\n\nWu X ,  Wu H ,  Liu M ,  Liu Z ,  Xu H ,  Lai F .  Analysis of binding interaction between (-)-epigallocatechin (EGC) and \u03b2-lactoglobulin by multi-spectroscopic method .  _Spectrochim. Acta, Part A_.  2011 ;82 : 164 \u2013 168 .\n\nXie G ,  Timasheff SN .  The thermodynamic mechanism of protein stabilization by trehalose .  _Biophys. Chem._ 1997 ;64 : 25 \u2013 43 .\n\nXie GF ,  Timasheff SN .  Mechanism of the stabilization of ribonuclease A by sorbitol: Preferential hydration is greater for the denatured than for the native protein .  _Protein Sci._ 1997 ;6 : 211 \u2013 221 .\n\nXie Y ,  Kosinska A ,  Xu H ,  Andlauer W .  Milk enhances intestinal absorption of green tea catechins in in vitro digestion\/Caco-2 cells model .  _Food Res. Int._ 2013 ; : 793 \u2013 800 .\n\nYang MC ,  Guan HH ,  Liu MY ,  Lin YH ,  Yang JM ,  Chen WL ,  Chen CJ ,  Mao SJT .  Crystal structure of a secondary vitamin D3 binding site of milk \u03b2-lactoglobulin .  _Proteins: Struct., Funct., Genet._ 2008 ;71 : 1197 \u2013 1210 .\n\nZhang X ,  Fu X ,  Zhang H ,  Liu C ,  Jiao W ,  Chang Z .  Chaperone-like activity of \u03b2-casein .  _Int. J. Biochem. Cell Biol._ 2005 ;37 : 1232 \u2013 1240 .\n\nZhang Y ,  Zhong Q .  Encapsulation of bixin in sodium caseinate to deliver the colorant in transparent dispersions .  _Food Hydrocolloids_.  2013 ;33 : 1 \u2013 9 .\n\nZhang Y ,  Zhong Q .  Probing the binding between norbixin and dairy proteins by spectroscopy methods .  _Food Chem._ 2013 ;139 : 611 \u2013 616 .\n\nZhang ZH ,  Wang XP ,  Ayman WY ,  Munyendo WL ,  Lv HX ,  Zhou JP .  Studies on lactoferrin nanoparticles of gambogic acid for oral delivery .  _Drug Delivery_.  2013 ;20 : 86 \u2013 93 .\n\nZhou J ,  Wang X ,  Ai T ,  Cheng X ,  Guo HY ,  Teng GX ,  Mao XY .  Preparation and characterization of \u03b2-lactoglobulin hydrolysate-iron complexes .  _J. Dairy Sci._ 2012 ;95 : 4230 \u2013 4236 .\n\nZhu XQ .  _Interactions between flavour compounds and milk proteins_ .  Palmerston North, New Zealand :  Institute of Food, Nutrition and Human Health. Massey University ;  2003 .\n\nZimet P ,  Rosenberg D ,  Livney YD .  Re-assembled casein micelles and casein nanoparticles as nano-vehicles for \u03c9-3 polyunsaturated fatty acids .  _Food Hydrocolloids_.  2011 ;25 : 1270 \u2013 1276 .\n\nZsila F ,  Hazai E ,  Sawyer L .  Binding of the pepper alkaloid piperine to bovine \u03b2-lactoglobulin: Circular dichroism spectroscopy and molecular modeling study .  _J. Agric. Food Chem._ 2005 ;53 : 10179 \u2013 10185 .\n\nZsila F ,  Imre T ,  Szabo BZ ,  Simonyi M .  Induced chirality upon binding of cis-parinaric acid to bovine \u03b2-lactoglobulin: Spectroscopic characterization of the complex .  _FEBS Lett._ 2002 ;520 : 81 \u2013 87 . \nChapter 15\n\n# Model Food Systems and Protein Functionality\n\nW. James Harper Department of Food Science and Technology, The Ohio State University, Ohio, USA\n\n## Abstract\n\nFabricated foods generally comprise a mixture of components made up of lipids, proteins, simple and complex carbohydrates, emulsifiers, and salts, which are capable of interacting with each other and modifying the final characteristics of the food. Often, processing utilized in the manufacture of the food also modifies these interactions. Model food systems were first developed because of the disparity between laboratory functional tests for proteins and the functionality in the food.\n\nModel food systems today find utility for the functionality of many other food components, including starches, gums, and emulsifiers, as well as areas of continued interest (lipid oxidation, Maillard reaction, etc.). Therefore, model food systems provide a means of determining how the ingredients and the process alter the characteristics of the final product, as well as evaluating the sensitivity of the characteristics of the food to the different ingredients and processing steps. Model food systems are based on the formulation and processing of real foods, using laboratory and pilot plant facilities. Generally, ingredients that do not have a main effect on the final characteristics of the product are eliminated. One potential limitation is the use of processing equipment that does not scale up to commercial production.\n\nThe utilization of carefully selected statistical designs is essential to unravel the multiple interactions that do occur and to optimize food formulation and processing.\n\nA major limitation of model food systems is that they do not provide any information as to the mechanisms by which the ingredients and the process control the final characteristics of the product. Thus they have application to only the food under investigation. They do have a major role in food product development.\n\n## Keywords\n\nModel food systems, protein functionality, food characteristics, sodium caseinate, pasting characteristics, potato starch, milk proteins, statistical design\n\nOutline\n\nIntroduction 451\n\nProtein functionality in foods 453\n\nRole of interactions in determining food characteristics 453\n\nA Case Study 455\n\nEffect of Sodium Caseinate on the Pasting Characteristics of Different Starches 455\n\nEffect of Milk Proteins on the Pasting Characteristics of Potato Starch, with Emphasis on Peak Viscosity 455\n\nProcessing effects 459\n\nUses of model food systems 461\n\nInitial Steps to Developing Model Food Systems 461\n\nStatistical Design 461\n\nApplications of model food systems 462\n\nBakery Products 463\n\nDairy Analogues 464\n\nCoffee Whiteners 464\n\nWhipped Toppings 465\n\nSalad Dressings 465\n\nMeat Products 466\n\nUse of model food systems for other food components 467\n\nLimitations 467\n\nConclusions 467\n\n## Introduction\n\nThe utilization of proteins in food for nutritional and functional purposes goes back many centuries, but the relationship between structure and function has been given close attention only during the past 30\u201340 years (Owusu-Apenten, 2004). Numerous studies and many reviews have contributed to gaining an understanding of precisely how proteins act in a complex food system and how their structure and function are altered by the other ingredients in the food, its intrinsic properties, and its processing. These include Anfinsen (1972), Kinsella (1982), Nakai (1983), Mulvihill and Fox (1987), Mangino et al. (1994), Zayas (1996), Li-Chan (2004), Luyten et al. (2004), Owusu-Apenten (2004), Turgeon et al. (2007), Ghousch et al. (2008), Foegeding and Davis (2011), Singh (2011), and Dickinson (2012).\n\nThere are two broad ways of gaining knowledge of the structure and function of protein systems: (1) study of pure proteins in simple systems and (2) study of commercial proteins in the food systems in which they are used. These methods are entirely different (Luyten et al., 2004; Owusu-Apenten, 2004) and provide quite different information. Functionality tests can be very useful in obtaining reproducible functional properties, even though such tests cannot be used to predict the final characteristics in a real food system (de Wit, 1984; 1989; Harper, 1984; Owusu-Apenten, 2004). Some differences include the following.\n\n\u2022 In pure structure\/function studies, pure proteins are generally used and are used at concentrations much lower than those used in food systems (Owusu-Apenten, 2004).\n\n\u2022 In food systems, proteins are seldom pure and may actually involve complex mixtures of proteins from a given food source (such as milk proteins, egg proteins, and soy proteins) or proteins from multiple food sources (i.e., meat, soy, milk, and gluten) or proteins that have been selectively denatured to provide the desired functionality (Mangino et al., 1994).\n\n\u2022 In structure\/function studies, care is taken to avoid interactions with other components and to avoid modifying the secondary and tertiary structure during the experiments. The proteins are fully hydrated (Kinsella, 1982; Owusu-Apenten, 2004).\n\n\u2022 In food systems, the proteins are constantly exposed to other ingredients, which can modify the structure and hence function, as well as being modified by processes that often include pH, heat, and shear (Lee et al., 1992; Kilara, 1994). Competition for water can also modify functionality, as can changes in intrinsic properties (Zayas, 1996).\n\n\u2022 In structure\/function studies, outcome is generally measured for a specific and single response (Owusu-Apenten, 2004).\n\n\u2022 In food systems, the ingredients can influence product functionality at different points in the process, or functionality can be expressed in more than one outcome with respect to the characteristics of the food (de Wit, 1984; 1989; Harper, 1984).\n\nUnquestionably, proteins and other hydrocolloids are important and are required to give the food desirable characteristics. However, our knowledge remains incomplete today because we still cannot fully predict the characteristics of a formulated food on the basis of our knowledge of the structure and function of pure proteins or hydrocolloids under strictly controlled conditions (Kinsella, 1982; de Wit, 1984; 1989; Harper, 1984; Owusu-Apenten, 2004; Zayas, 1996).\n\nTesting for functionality using simplified systems has been reviewed in depth by numerous investigators, including Kinsella (1982), Kilara (1984), Modler and Jones (1987), Mulvihill and Fox (1987), Patel and Fry (1987), Hall (1996), Zayas (1996), and Owusu-Apenten (2004). The continued need to develop standardized testing for protein solubility, viscosity, water absorption, gelation, emulsification, and foaming properties has been emphasized by Mulvihill and Fox (1987), Patel and Fry (1987), German and Phillips (1994), Kilara (1994), Hall (1996), Zayas (1996), and Luyten et al. (2004).\n\n## Protein functionality in foods\n\nProteins used in foods include plant proteins (soy, wheat, rice, corn, and other plant sources), milk proteins (caseins, caseinates, whey proteins, and milk protein concentrates\u2014both caseins and whey proteins), egg proteins (egg white and egg yolk proteins), meat proteins, and fish proteins. Each type of protein exhibits different functional properties and has application in different types of food products (Inglett and Inglett, 1992; Kinsella, 1982; Lee et al., 1992; Kilara, 1994; Mangino et al., 1994; Owusu-Apenten, 2004).\n\nThe major functionalities of food proteins include solubility, emulsification, gelation and foaming, water binding, and heat stability. As shown in Table 15.1, different types of foods have different functional requirements and may require multiple functionalities.\n\nTable 15.1\n\nMultiple Functionalities in Selected Food Products\n\nFood Type | Multiple Functionalities  \n---|---  \nBeverages | Solubility, heat stability, pH stability, color  \nBaked goods | Emulsification, foaming, gelation  \nDairy analogues | Gelation, foaming, emulsification  \nEgg substitutes | Foaming, gelation  \nMeat emulsions | Emulsification, foaming, gelation, adhesion\/cohesion  \nSoups and sauces | Viscosity, emulsification, water adsorption  \nInfant formulae | Emulsification, heat stability  \nToppings | Foaming, emulsification  \nFrozen desserts | Foaming, gelation, emulsification\n\nSource: Adapted from Kinsella (1982), de Wit (1984), Kilara (1994) and Owusu-Apenten (2004).\n\nFactors that may modify the protein during processing, and hence its effect on the product characteristics, include heat, shear, salts, and other hydrocolloids (de Wit, 1984; Mangino et al., 1987; Yada, 2004).\n\n## Role of interactions in determining food characteristics\n\nInteractions between ingredients and modifications caused by processing are the primary reasons why the functionality of proteins and other colloids cannot be predicted in food systems (de Wit, 1984; 1989; Harper, 1984; Zayas, 1996; Owusu-Apenten, 2004; Yada, 2004).\n\nThe following diagram provides an overview of the potential interactions that can occur in a food product (adapted from Harper, 1984):\n\nEssentially, almost everything can modify the functionality of everything else. Salts are somewhat unique in that they do not in themselves affect product characteristics, but can act on proteins, surfactants, polysaccharides, and, to some extent, polar lipids to modify the functionality of each in the food system. The nature and extent of these interactions will be modified by pH, ionic strength, ingredient concentrations, and process-induced modifications. Some examples include the following.\n\n\u2022 Surfactants and proteins can interact competitively at the surface of an oil to modify the characteristics of the emulsion, such as stability, size distribution, and light scattering. The extent to which a given component will dominate the characteristics will depend on the relative concentrations (Fig. 15.1) and chemical natures of the surfactant and protein.\n\nFigure 15.1 Effect of emulsifier (mono- and diglycerides) on protein and phase separation of an oil-in-water emulsion (coffee whitener). Adapted from Harper (1984).\n\n\u2022 Starch is frequently used to provide texture in food products. However, the viscosity during processing and the final viscosity can be greatly altered by interactions with other components. The data in Figure 15.2 show that interactions that are observed with two-component systems do not always predict the effect of three- and four-ingredient interactions. In addition, 'who sees who first' can further modify other interactions and change product characteristics.\n\nFigure 15.2 Effect of components singly and in combination on the peak viscosity of potato starch (starch (S)): S + X = starch + xanthan gum; S + M = starch + mono- and diglycerides; S + F = starch + fructose; S + X + M = starch + xanthan gum + mono- and diglycerides; S + X + F = starch + xanthan gum + fructose; S + M + F = starch + mono- and diglycerides + fructose; S + X + M + F = starch + xanthan gum + mono- and diglycerides + fructose. Adapted from Harper (2006).\n\nRecent reviews of the use of simple model systems to obtain a better understanding of protein food ingredient interactions and the mechanisms involved include Dickinson (2011; 2012), Foegeding and Davis (2011), and Singh (2011).\n\n### A Case Study\n\nTo further expand on protein interactions, the following presents a case study of the effect of various milk proteins on the modification of the pasting characteristics of potato starch, including the effect of different types of milk proteins and of differences in the concentrations of protein and\/or starch.\n\nThe texture of formulated foods is strongly modified by interactions. This section gives results of a simplified model system that provides a better understanding of food formulations that involve protein\/food component interactions. The information provides an understanding of how milk proteins can modify the pasting properties of potato starch as investigated by Harper and Illingworth (unpublished data); Harper and Hemar (unpublished data); Doublier et al. (2001); and Bertolini et al. (2005). Starch is important in a number of food products in which the texture can be modified by protein\/starch interactions to change the textural properties of the food.\n\n#### Effect of Sodium Caseinate on the Pasting Characteristics of Different Starches\n\nThe Rapid Visco Analyzer (RVA) was used to evaluate the pasting properties of starch. Figure 15.3 shows a typical starch pasting curve for potato starch. Generally, an 8% starch paste was utilized, and the various characteristics are shown as a function of time. The two most important characteristics are peak height and final viscosity. Peak height shows the maximum viscosity during manufacture, and the final viscosity gives a measure of the final texture.\n\nFigure 15.3 Diagram of the steps in starch pasting, including time to initiate gelation. Peak viscosity, trough, final viscosity, breakdown, and setback\n\nThe addition of sodium caseinate to six different starches (corn, rice, wheat, potato, cassava, and waxy maize) influenced the peak (pasting) temperature, the time to reach peak viscosity, the peak viscosity, and the viscosity after cooling. Sodium caseinate had different effects on these parameters for the different starches.\n\nThe percent change in peak viscosity of the six sodium caseinate\/starch mixtures is shown in Figure 15.4. All starches, except potato starch, showed an increase in peak viscosity, whereas potato starch showed a dramatic decrease in peak viscosity. There was no statistically significant change in the final viscosity.\n\nFigure 15.4 Peak viscosity during pasting of six different starches\n\nThe differences in the pasting characteristics will be important with respect to both processing and the characteristics of different food products using starch and caseinate. Based on the results of the marked difference between the pasting characteristics of potato starch and those for all the other starches, attention was given only to potato starch for further studies.\n\n#### Effect of Milk Proteins on the Pasting Characteristics of Potato Starch, with Emphasis on Peak Viscosity\n\nProteins and polysaccharides, including starch, are frequently used together in food systems to import specific attributes to the final product. Hardacre et al., (2004) showed that sodium caseinate decreased the peak viscosity of potato starch at a starch to protein ratio of 1000:1. Different milk proteins were used to determine their effect on the pasting characteristics of potato starch. These included individual milk casein fractions, sodium caseinate with different concentrations of protein and starch, sodium caseinate, milk protein concentrate, and milk protein isolate.\n\n##### Individual Casein Fractions\n\nCasein was fractionated into \u03b1s\\- (\u03b1s1 \\+ \u03b1s2) and \u03b2-caseins, and these were in turn converted to their sodium and calcium salts. The peak viscosity, final viscosity, and pasting time of starch, starch + 1% calcium caseinate + starch, 1% calcium \u03b1s-caseinate starch + 1% calcium \u03b2-caseinate are shown in Table 15.2 and Figure 15.5 using standard starch concentrations (8%).\n\nTable 15.2\n\nRVA Results Showing the Effect of the Calcium Salts of Caseinate Fractions on the Gelation and Pasting of Potato Starch\n\n| Viscosity (cP) | Pasting time (s)  \n---|---|---  \n| Peak | Final |   \nStarch | 7315 | 2952 | 196.2  \nStarch + 1% calcium \u03b1s-caseinate | 4487 | 2975 | 232.2  \nStarch + 1% calcium \u00df-caseinate | 3806 | 2834 | 288  \nStarch + 1% calcium caseinate | 3467 | 2543 | 336\n\nFigure 15.5 Effect of caseinate fractions and sodium casein on the peak viscosity of potato starch.\n\nPasting time increased in the order of starch < starch + calcium caseinate < starch + \u03b2-caseinate < starch + sodium caseinate. As the starch concentration decreased, there was a loss in final viscosity, especially at a 2% starch concentration. This finding would indicate a definite lack in texture for food products using 2% or less of starch.\n\n##### Effect of the Concentration of Sodium Caseinate on the Pasting Characteristics of Potato Starch\n\nThe effect of concentration of sodium caseinate, from 0.1% to 2.5%, on the pasting characteristics of potato starch was evaluated. Data for time to reach the maximum viscosity, peak viscosity, breakdown, setback, and final viscosity are shown in Table 15.3. The time to reach maximum viscosity increased. Peak viscosity and setback decreased with concentration, where there was no significant change in setback or final viscosity. The data suggest that the addition of casein would put less stress on processing, through a decrease in peak viscosity, without a significant effect on the final viscosity\n\nTable 15.3\n\nRVA Results for the Effect of Casein Fractions on the Gelation and Pasting of Potato Starch\n\n| Viscosity (cP) | Pasting time (s)  \n---|---|---  \n| Peak | Final |   \nStarch | 7361 | 2968 | 199.8  \nStarch + 2.5% \u03b1s-casein | 5885 | 3106 | 228.0  \nStarch + 2.5% \u03b2-casein | 4359 | 2712 | 228.0  \nStarch + 2.5% sodium caseinate | 2113 | 2671 | 343.8  \nStarch + 5% \u03b1s-casein | 5676 | 3329 | 220.2  \nStarch + 5% \u03b2-casein | 4727 | 2947 | 211.8\n\n##### Effect of the Concentration of Starch and Milk Proteins on Potato Starch Pasting Characteristics\n\nThe concentration of starch for determining the effects of starch on pasting characteristics generally has been 6\u20138%. However, the use level in most food applications ranges from 2% to 4%. Therefore, from a practical viewpoint, effects on starch pasting characteristics in food use concentration would be useful.\n\nThe effect of protein concentration and starch concentration on the loss of peak viscosity, final viscosity, and phase separation on storage was determined for sodium caseinate, milk protein concentrate, and milk protein isolate. All of the different proteins gave similar results, and only the effect of milk protein isolate is presented in this investigation, for illustrative purposes.\n\nThe loss in peak viscosity increased as the starch content decreased, and the protein content increased as illustrated in Figure 15.6. The loss of peak viscosity increased to some degree as the starch content increased. The effect of protein concentration was very significant and linear, from 0.02% to 1%. However, once the protein concentration reached 1%, the loss was the same for higher protein concentration increases, ranging from 80% to nearly 100%.\n\nFigure 15.6 Effect of protein and starch concentration of peak viscosity of potato starch\n\nUnder standard test conditions with the RVA (8% starch), there was little to no loss in final viscosity with the addition of milk protein. However, as the starch content was decreased below 6%, the loss in final viscosity markedly increased, as shown in Figure 15.7. This would limit the advantage of using potato starch in foods containing milk protein, especially at starch concentrations below 4%.\n\nFigure 15.7 Effect of MPI concentrations on loss of final viscosity as a function of starch concentration.\n\n## Processing effects\n\nThe functionality of commercial food proteins and other hydrocolloids can be modified both during their production and during the processing of the food product itself. An overview of the conversion of a raw protein source to a functional food ingredient and the subsequent further processing during food manufacture is outlined in Figure 15.8.\n\nFigure 15.8 Steps in the manufacture of food proteins and the subsequent processes during food manufacture. From Owusu-Apenten (2004).\n\nDuring the production of commercial food proteins for use as food ingredients, the proteins may be exposed to a wide range of processing steps that can include thermal processes (pasteurization, sterilization), shear (pumping, mixing, homogenization), pressure (high-pressure processing, retorting), concentration (membrane processing, evaporation, drying), and precipitation (heat, acid, salts, solvents). Each of these steps will modify the functional properties of the protein and thus will affect the final characteristics of the food (Kinsella, 1982; de Wit, 1984; 1989; Harper, 1984; Dybing and Smith, 1991; Kilara, 1994; Zayas, 1996; Owusu-Apenten, 2004). Such processes can alter functionality in food through a number of different modifications of the protein, including changes in sulfydryl interactions, modification of secondary and quaternary structure, and shifts in the hydrophilic\/lipophilic balance (Kinsella, 1982). Subsequent processing during use of the protein as a functional ingredient in food will bring further changes in the system, especially those occurring in the presence of other interacting ingredients. Such changes in the characteristics of the food generally cannot be predicted; thus, there is a need for use of model food systems as an intermediate step in product development (Owusu-Apenten, 2004).\n\n## Uses of model food systems\n\nModel food systems can be used in a variety of ways (de Wit, 1984; Harper, 1984; Owusu-Apenten, 2004), including the following.\n\n\u2022 Determining the relative significance of the main effects of ingredients.\n\n\u2022 Studying factors in food that affect chemical and physical changes (Maillard reaction, lipid oxidation, etc.).\n\n\u2022 Evaluating the sensitivity of the food to alterations in formulation and processing.\n\n\u2022 Defining ingredient interactions.\n\n\u2022 Optimizing the formulation for robustness.\n\n\u2022 Determining critical steps in the processing of the product.\n\n\u2022 Determining interrelationships between ingredients and the process.\n\n\u2022 Tailor-making ingredients for a specific food application.\n\n\u2022 Evaluating and minimizing the sensitivity of product attributes to the formulation and the process.\n\nOwusu-Apenten (2004) stated that the advantages of model food systems over standard functionality tests included: (a) their ease of use, (b) the lack of a need for specialized equipment and methodologies, (c) the ability to aid in product optimization, and (d) the ability to test for multiple factors and interactions with respect to formulation and processing.\n\n### Initial Steps to Developing Model Food Systems\n\nThe approach to developing a model food system is the same, whether the ingredient being investigated is a protein, lipid, emulsifier, starch, or gum.\n\nThe development of a model food system begins by reviewing as many formulations as can be found and selecting those that are common to all formulations at a concentration that is at the central point of the various formulas (Harper, 1984). Next, a small-scale process for making the products is developed using processing steps and conditions as close to the commercial process as possible. When more than four or five ingredients are involved, it is often necessary to do a screening experiment to eliminate ingredients that do not have a main effect on important characteristics.\n\nEach different food will have different characteristics, which may include taste, color, and texture, that can be modified by the formulation and the process. Key attributes and methods for their evaluation need to be selected. Generally, the evaluation methods are different from those that are used in research (Owusu-Apenten, 2004).\n\n### Statistical Design\n\nStatistical design is an essential component of model food systems because of the information it provides on ingredient and processing interactions (Dziezak, 1990; Earle et al., 2001; Hanrahan and Lu, 2006). Most fabricated food products have from 5 to 25 variables when both the ingredients and the processing steps are taken into consideration. This makes full factorial designs, which would exceed several hundred experiments, an impractical choice. Thus fractional factorial screening designs are generally required. For most food products, the experimental design is a stepwise process, starting with screening experiments to minimize the variables that do not have major effects on the characteristics of the products. One of the most common screening designs is the Plackett-Burman, which can be used with up to 36 variables (Mullen and Ennis, 1985; Hanrahan and Lu, 2006). The screening experiments allow determination of the main effects that can be used in further fractional factorial designs; these designs will provide a better understanding of ingredient and process interactions and will generate response surfaces that give an understanding of the interactions (Hanrahan and Lu, 2006).\n\nIn developing a fractional factorial experimental design in model food systems, it is necessary to know: (a) the critical factors associated with the ingredients and the process, (b) the region of interest where the factor levels influencing the product characteristics are known, (c) that the factors vary continuously throughout the experimental range tested, (d) that a mathematical function relates the variable factors to the measured response, and (e) that the response defined by the function is a smooth curve.\n\nNumerous studies have used statistical design and response surface methodology to determine the effect of interactions on product characteristics and to optimize specific characteristics in a food (Dziezak, 1990).\n\nIn developing an experimental design, consultation with a statistician familiar with the factors that affect the outcomes of the specific design is needed to avoid common pitfalls. Among these pitfalls are the following: (a) incorrectly defined or specified critical factors, (b) too narrow or too broad a range of factors selected, so that the optimum cannot be defined, (c) lack of the use of good statistical practices, (d) too large a variation in the range of the factors utilized, introducing bias and error, (e) over-reliance on computer-generated results, and (f) failure to ensure that the results make good sense.\n\n## Applications of model food systems\n\nInitially, model food systems were applied to milk proteins to gain a better understanding of what was required to get desired characteristics in complex food products that could not be predicted from standard functionality tests. de Wit (1998) has stated: \"Information obtained from functional characterization tests in model systems is more suitable to explain retroactively protein behaviors in complex food systems than to predict functionality.\" What has been learned using milk proteins in model food systems has been shown to be equally applicable to other food proteins. In addition to understanding the protein being used, there is a need to know the functionality of other ingredients in the food, the probability of how they will interact and modify the function of the food protein, and the use of statistical design to gain the full potential of the model system approach.\n\nStudies of model food systems, used to assess their performance in foods, have included a large number of different types of foods, as shown in Table 15.4. These include bakery products, dairy products, dairy analogues, meat products, sauces and dressings, fermented foods, wine, and infant formulas.\n\nTable 15.4\n\nModel Food Systems Used to Assess Functionality in foodsa\n\nFirst-generation model foodsa | Additional examplesb  \n---|---  \nCakes (angel food, chocolate, yellow, pound) | Low-fat spreads  \nMeringues | Beer batters  \nBread | Beef patties  \nCoffee whitener | Gravies  \nHam, restructured meats | Meat emulsions  \nInfant formula | Cream  \nSalad dressing | Milk  \nSausage | Cheese  \nStarch pudding | Processed cheese  \nWhipped topping | Soups and sauces  \nIce cream | Surimi  \n| Wine  \n| Yogurt\n\na Adapted from Harper (1984) and de Wit (1984).\n\nb Adapted from Owusu-Apenten (2004).\n\nThe examples of the model food systems used to illustrate applications in this chapter are primarily from the first generation category. They include bakery products, dairy analogues, meat products, salad dressings, and sauces.\n\n### Bakery Products\n\nBread represents a system in which the methods of evaluation of ingredients have been standardized and covered by AACC-approved methods (AACC methods 10-9, 10-10 and 10-11). Details of the procedures and evaluation techniques have been given by various investigators (Lindblom, 1977; Pomeranz et al., 1984; Ranhortra et al., 1992; Fenn et al., 1994; Cauvain and Young, 2006). In general, the substitution or addition of other proteins (milk, whey proteins, etc.) leads to a loss of loaf volume (Harper et al., 1980; de Wit, 1984). Harper and Zadow (1984) found that heat treatments that prevented loss of loaf volume in bread made with milk powder were ineffective in preventing loss of loaf volume in bread made with whey protein concentrates.\n\nModel food systems have been used widely in cake systems including: pound cake (Lee, 1999), Madeira cake (de Wit, 1984), white cake (Harper et al., 1980), and angel food cake (Kissell and Bean, 1978).\n\nOf these, angel food cake has received the most attention (Lowe et al., 1969; DeVilbiss et al., 1974; Cunningham, 1976; Regenstein et al., 1978; Johnson and Zabik, 1981a,b; Ball and Winn, 1982; Froning et al., 1987; Froning, 1988; Martinez et al., 1995). The primary protein evaluated has been egg white, for which cake height and texture can be related to the individual egg white proteins (Johnson and Zabik, 1981a,b; Ball and Winn, 1982). Attempts to replace egg white with whey proteins have never been completely successful (DeVilbiss et al., 1974; Harper et al., 1980). Arunepanlop et al. (1996) were able to replace 25\u201350% of the egg white with whey protein and could achieve greater replacement by the addition of xanthan gum. Cake volume is essentially the same as it is with egg white, but the cakes collapse upon baking. This emphasizes the requirement for both foaming and gelation (Owusu-Apenten, 2004). This is due in part to the lower gelation temperature for foams made with egg white (Pernell et al., 2002) and in part to the shear-induced denaturation of egg white with mixing (DeVilbiss et al., 1974).\n\nOther proteins evaluated for angel food cake include blood plasma protein (Kahn et al., 1979; Raeker and Johnson, 1995) and dried beef plasma (Duxbury, 1988). Factors that should be considered in developing a model food system for bakery products are outlined briefly in Table 15.5.\n\nTable 15.5\n\nFactors Affecting Functionality of Protein in Bakery Products\n\nProduct type | Functional requirement of protein | Ingredient modifying functionality | Processing factors affecting functionality  \n---|---|---|---  \nBread | Dough formation, water binding, gelation, elasticity of dough | Protein source, polar lipids, oxidizing and reducing agents, other proteins with sulfhydryl groups | Mixing, method of bread making (sponge dough versus mechanical development)  \nCakes | Fat binding, foaming, gelation | Protein type and concentration, gums, fat, sugar concentration | Mixing speed and time, pre-emulsification\n\n### Dairy Analogues\n\nDairy analogues include coffee whiteners, whipped toppings, and processed cheese products.\n\n#### Coffee Whiteners\n\nCoffee whiteners, first developed in the 1950s, generally are protein-stabilized oil-in water emulsions, with vegetable oil as the dispersed phase. A model system developed by Harper and Raman (1979) and Harper et al. (1980) utilized caseinate, soy bean oil, carbohydrate, phosphate, emulsifier, and a gum (xanthan gum or carrageenan). The role of the ingredients has been reviewed by Knightly (1969) and Patel et al. (1992), and the process has been reviewed by Owusu-Apenten (2004). This is summarized in Table 15.6.\n\nTable 15.6\n\nFactors Affecting Functionality of Protein in Coffee Whiteners\n\nProduct type | Functional requirement of protein | Ingredient modifying functionality | Processing factors affecting functionality  \n---|---|---|---  \nCoffee whiteners | Emulsification, stability to the pH and temperature of coffee | Emulsifiers, gums, phosphate, calcium | Homogenization, pasteurization time and temperature, temperature and pH of coffee\n\nPatented processes include using milk protein retentate (Kosikowski and Jimenez- Florez 1987), reformed casein micelles (McKenna et al., 1992), phosphate-modified milk protein (Melachouris et al., 1994), and soy proteins (Melmychyn, 1973).\n\nAlternative proteins that have been suggested to replace caseinate include milk protein concentrate (Euston and Hirst, 2000), whey protein (Hlavacek et al., 1970; Gruetzmacher and Bradley, 1991; Euston and Hirst, 2000), wheat protein (Golde and Schmidt, 2005; Patil et al., 2006), soy protein (Hlavacek et al., 1970; Golde and Schmidt, 2005), peanut protein (Malundo et al., 1992), and cottonseed protein (Choi et al., 1982).\n\nCoffee whiteners are evaluated to ensure that they provide an emulsion with a small particle size to maximize whiteness, minimize astringency of the coffee by binding with the coffee tannins, maintain stability in hot coffee under acidic conditions, minimize feathering in the presence of hard water salts, and readily disperse in the coffee (Pearce and Harper, 1982; Tran and Einerson, 1987; Kneifel et al., 1992; Kelly et al., 1999).\n\nGolde and Schmidt (2005) compared coffee whiteners made from sodium caseinate, soy protein isolate, and wheat protein isolate, and found that they gave similar whiteness (L*) to the coffee. However, the liquid coffee whiteners made with wheat protein tended to separate upon storage, whereas the whiteners made with soy protein isolate tended to show feathering.\n\n#### Whipped Toppings\n\nMost commercial whipped toppings contain sodium caseinate as the protein of choice (Knightly, 1968). Other proteins used for whipped toppings include whey protein concentrate (Peltonen-Shalaby and Mangino, 1986; Liao and Mangino, 1987) and soy protein isolates (Kolar et al., 1979; Lah et al., 1980; Chow et al., 1988; Abdullah et al., 1993; Shurtleff et al, 1994).\n\nWhipped toppings are high-fat, foamed emulsions with about 40% total solids, and model food systems generally also contain sugars, gums, and small-molecular-weight emulsifiers (Knightly, 1968; Harper et al., 1980). A brief summary of key factors that affect the functionality of protein in whipped toppings is given in Table 15.7. The model system differs from whipping or foaming tests with respect to both compositions and much lower fat content (Owusu-Apenten, 2004). Min and Thomas (1977) found that calcium addition to a 15%-fat-containing whipped topping stabilized with sodium caseinate gave improved stability to the system. Peltonen-Shalaby and Mangino (1986) showed that pasteurization also improved the overrun of the topping. Liao and Mangino (1987) used whey proteins to make a model whipped topping and found a positive correlation between exposed hydrophobicity and overrun. Other factors that affect overrun and stability include the hardness of the fat, the type and percentage of emulsifier, and the equipment used for mixing (Harper, 1984).\n\nTable 15.7\n\nFactors Affecting Functionality of Protein in Whipped Toppings\n\nProduct type | Functional requirement of protein | Ingredient modifying functionality | Processing factors affecting functionality  \n---|---|---|---  \nWhipped toppings | Emulsification, whipping to \u2248200% overrun in the presence of high fat and high solids | Emulsifiers, gums, phosphate, calcium | Homogenization, pasteurization time, equipment used to produce the whipped product\n\n### Salad Dressings\n\nSalad dressings are high-fat emulsions, frequently stabilized by high shear in the presence of egg yolk as the primary emulsifier (Parker et al., 1995). Mayonnaise, a spoonable dressing, contains 75% oil by definition. Subsequently, starch pastes were used to make a spoonable dressing with about 40% oil. Today, the most common dressings are pourable, with a wide range of oil contents and are stabilized primarily by xanthan gum (Franco et al., 1995).\n\nModel food systems have been used to gain a better understanding of both ingredients (Smith, 1977; Paredes et al., 1988) and processing (Parker et al., 1995).\n\nSmith (1977), using a central composite statistical design, found that the coefficients of the regression analysis were larger for the interaction terms than for the main effect terms in pourable salad dressing with 40% oil and containing egg, vinegar, xanthan gum, and mustard powder. The order of addition was also found to be important to the viscous properties of the pourable salad dressing.\n\n### Meat Products\n\nModel meat products including beef, pork, lamb, poultry, or fish, have been utilized for recombined meats (ham, steaks, etc.) and meat macroemulsions that include bologna, sausages, liver sausages, frankfurters, and meat loaves.\n\nNon-meat proteins have been injected into beef and ham, together with water, followed by tumbling to maintain nutritionally equivalent protein levels, increase yield, and improve texture (Zayas, 1996; Yada, 2004; Szerman et al., 2007). Szerman et al. (2007) found whey protein isolates to be superior to vegetable proteins on the basis of flavor.\n\nMeat emulsions generally have size distributions between 0.1 and 50 \u03bcm, and many investigators suggest that they are three-dimensional gel networks with entrapped fat (Regenstein, 1989; Krishnan and Sharma, 1990; Xiong et al., 1992; Correia and Mittal, 1993; Barbut, 1995). However, most reviewers continue to classify them as meat emulsions (Gordon, 1969; Webb, 1974; Owusu-Apenten, 2004).\n\nThe factors that affect the functionality of proteins in these products include meat extraction temperature, emulsification temperature, shear during emulsification, fat melting point, pH, ionic strength, ratios of ingredients, salt, soluble protein concentration, and type of salt (salt, phosphates, citrates, etc.).\n\nAchievement of functionality has been determined by a number of different methods, including:\n\n\u2022 emulsification capacity (EC) (Swift et al., 1961; Swift and Sulzbacher, 1963)\n\n\u2022 emulsion activity (EA) (Acton and Saffle, 1972)\n\n\u2022 emulsion stability (ES) (Carpenter and Saffle, 1964; Townsend et al., 1968; Marshall et al., 1975)\n\nAlthough the tests for EC and ES for comminuted meat products are widely used, there does not appear to be much collaborative testing of the different methods (Owusu-Apenten, 2004). The type of protein affects the EC of meat emulsions, with T isolated muscle proteins giving different EC values. In general, the EC was in the order of myosin > actomycin > actin for different types of meat (Tsai et al., 1972; Galluzzo and Regenstein, 1978; Li-Chan et al., 1984). Substitution of meat protein by other protein in meat emulsions, as measured by large deformation rheological testing, showed that\n\n\u2022 gluten, soy protein isolate, or egg white increased the yield after the cooking of meat emulsions (Randall et al., 1976)\n\n\u2022 corn germ protein at 2% substitution reduced the shear force and reduced cooking losses (Mittal and Usborne, 1985)\n\n\u2022 partial substitution of meat with sodium caseinate, soy protein isolate, whey protein concentrate, or wheat germ protein all increased cook yield, increased protein level, and decreased fat in frankfurters, without affecting quality (Atughonu et al., 1988)\n\n\u2022 addition of bovine blood plasma to meat emulsion products improved emulsion stability and yield, and contents of protein, phenylalanine, and valine (Marquez et al., 1997)\n\n## Use of model food systems for other food components\n\nIn addition to evaluating the performance of proteins in food systems, a wide range of other applications have been utilized. During the past several years, more than 200 papers have been published on other uses of model food systems. A full review of such uses is outside the scope of this chapter. However, selected applications from studies over the past several years are cited both to present a basis for understanding the scope of the use of model food systems in the food industry and to provide a starting point for obtaining more detailed information.\n\nApplications include:\n\n\u2022 factors affecting flavor release in foods (Bylaite et al., 2005; Heinemann et al., 2005; Conde-Petit et al., 2006; Nongonierma et al., 2006; Seuvre et al., 2007)\n\n\u2022 factors affecting D values in food (Rodriguez et al., 2006)\n\n\u2022 lipid oxidation (Jaswir et al., 2004; Sakanaka and Tachibana, 2006; Wijeratne et al., 2006)\n\n\u2022 water migration in foods (Guignon et al., 2005; Doona and Moo, 2007)\n\n\u2022 Maillard reaction investigations (Severini, et al., 2003; Miao and Roos, 2005; Acevedo, 2006; Casal, 2006)\n\n\u2022 effects of high-pressure processing of food (Severini et al., 2003; Sila et al., 2007)\n\n## Limitations\n\nModel food systems can tell you 'what,' but they cannot tell you the mechanism(s) by which the effects occur. Frequently, the results with a model system cannot be scaled up to full commercial practice because of differences in equipment and processes. However, they do provide insight into directions to take to overcome scale-up problems. Generally, the results are valid only within the parameters that have been established. Optimization of a food system can sometimes be outside the limits of either the processing equipment or the functionality of a specific ingredient.\n\n## Conclusions\n\nHistorically, model food systems were used first to improve the functionality of milk proteins in food systems. Currently, there are very few publications on the use of milk proteins for these purposes, although it is known that a number of dairy food companies use model food systems in their product development programs. Today, the publications concerning model food systems have a much broader usage, with attention being given to a better understanding of how complex food systems affect such factors as oxidation, Maillard reactions, and shelf life.\n\nModel food systems can be a valuable tool in product development with respect to developments of both formulations and manufacturing processes and have a role in the development of ingredients for new foods.\n\nModel food systems do not provide information on why interactions occur, but they can provide insights into which interactions need basic study to provide a more robust product.\n\nIn the future, model food systems can be expected to continue to provide a better understanding of how interactions modify the functionality of proteins in complex food systems and to give insight into how to use this information to interface with studies on the basis of protein structure\/function.\n\n# References\n\nAbdullah A ,  Resurreccion AVA ,  Beuchat LR .  Formulation and evaluation of a peanut milk based whipped topping using response surface methodology .  _Lebensmittel-Wissenschaft und -Technologie_.  1993 ;26 : 167 \u2013 170 .\n\nAcevedo N ,  Schebor C ,  Buera MP .  Water solids interactions: Maxtric, structural properties and the rate of non-enzymatic browning .  _Journal of Food Engineering_.  2006 ;77 (4) : 1108 \u2013 1115 .\n\nActon JC ,  Saffle RL .  Emulsifying capacity of muscle protein: phase volumes at emulsion collapse .  _Journal of Food Science_.  1972 ;37 : 904 \u2013 906 .\n\nAnfinsen CB .  _Studies on the principles that govern the folding of protein chains. Nobel Lecture_ .  Nobel Foundation ;  1972 .\n\nArunepanlop B ,  Morr CV ,  Karleskind D ,  Laye I .  Partial replacement of egg white proteins with whey proteins in angel food cakes .  _Journal of Food Science_.  1996 ;61 : 1085 \u2013 1093 .\n\nAtughonu A-G ,  Zayas J-F ,  Herald T-J ,  Harbers L-H .  Thermo-rheology, quality characteristics, and microstructure of frankfurters prepared with selected plant and milk additives .  _Journal of Food Quality_.  1988 ;21 : 223 \u2013 238 .\n\nBall Jr HR ,  Winn SE .  Acylation of egg white proteins with acetic anhydride and succinic anhydride .  _Poultry Science_.  1982 ;61 : 1041 \u2013 1046 .\n\nBarbut S .  Importance of fat emulsification and protein matrix characteristics in meat batter stability .  _Journal of Muscle Foods_.  1995 ;6 : 161 \u2013 177 .\n\nBertolini AC ,  Creamer L ,  Eppink M ,  Boland M .  Some rheological properties of sodium-caseinate starch gels .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (2248\u20132254) .\n\nBylaite E ,  Adler-Nisse J ,  Meyer AS .  Effect of xanthan on flavor release from thickened food model systems .  _Journal of Agricultural and Food Chemistry_.  2005 ; 3577 \u2013 3583 .\n\nCauvain S ,  Young L .  _Baked Products_ .  Oxford :  Blackwell Publishing Ltd ;  2006 .\n\nCarpenter JA ,  Saffle RL .  A simple method of estimating the emulsifying capacity of various sausage meats .  _Journal of Food Science_.  1964 ;29 : 774 \u2013 781 .\n\nCasal E ,  Ramirez P ,  Ibanez E ,  Corzo N ,  Olano A .  Effect of supercritical carbon dioxide treatment on the Maillard reaction in model food systems .  _Food Chemistry_.  2006 ;97 (2) : 272 \u2013 276 .\n\nChoi YR ,  Lucas EW ,  Rhee KC .  Formulation of non-dairy coffee whiteners with cottonseed protein isolates .  _Journal of the American Oil Chemists ' Society_.  1982 ;59 : 564 \u2013 567 .\n\nChow ETS ,  Wei LS ,  DeVor RE ,  Steinberg MP .  Performance of ingredients in a soybean whipped topping: a response surface analysis .  _Journal of Food Science_.  1988 ;53 : 1761 \u2013 1765 .\n\nConde-Petit B ,  Escher F ,  Nuessli J .  Structural features of starch-flavor complexation in food model systems .  _Trends in Food Science and Technology_.  2006 ;17 (5) : 227 \u2013 235 .\n\nCorreia LR ,  Mittal GS .  Selection of fillers based on zp values of meat emulsion properties during smokehouse cooking .  _International Journal of Food Science and Technology_.  1993 ;28 (5) : 443 \u2013 451 .\n\nCunningham FE .  Properties of egg white foam drainage .  _Poultry Science_.  1976 ;55 : 738 \u2013 743 .\n\nde Wit JN .  Functional properties of whey proteins in food systems .  _Netherlands Milk and Dairy Journal_.  1984 ;38 : 71 \u2013 89 .\n\nde Wit JN ,  The use of whey protein products .  Fox PF ,  ed.  _Developments in Dairy Chemistry_ ,  Volume 3 .  London :  Elsevier Science Publishers ;  1989 : 323 \u2013 345 .\n\nde Wit JN .  Nutrition and functional characteristics of whey protein in foods .  _Journal of Dairy Science_.  1998 ;81 : 597 \u2013 608 .\n\nDeVilbiss ED ,  Holsinger VH ,  Posati LP ,  Pallansch MJ .  Properties of whey protein concentrates .  _Food Technology_.  1974 ;28 (3) : 40 \u2013 42 : 46, 48 .\n\nDickinson E .  Mixed biopolymers at interfaces: Competitive absorption and multilayer structures .  _Food Hydrocolloids_.  2011 ;25 : 1966 \u2013 1983 .\n\nDickinson E .  Emulsion gels: The structuring of soft gels with protein- stabilized oil droplets .  _Food Hydrocolloids_.  2012 ;28 (1) : 224 \u2013 241 .\n\nDoona CJ ,  Moo YB .  Molecular mobility in model dough systems studied by time-domain nuclear magnetic resonance spectroscopy .  _Journal of Cereal Science_.  2007 ;45 (3) : 257 \u2013 262 .\n\nDoublier J-L ,  Garnier C ,  Renard D ,  Sanchez C .  Protein-polysaccharide interactions .  _Current Opinion in Colloid and Interface Science_.  2001 ;5 (3\/4) : 202 \u2013 214 .\n\nDuxbury DD .  Powdered beef plasma replaces eggs in cakes .  _Food Processing_.  1988 ;49 (2) : 73 \u2013 74 .\n\nDybing ST ,  Smith DE .  Relation of chemistry and processing procedures to whey protein functionality .  _Cultured Dairy Products Journal_.  1991 ;26 (1) : 4 \u2013 12 .\n\nDziezak JD .  Taking the gamble out of food product development .  _Food Technology_.  1990 ;44 (6) : 109 \u2013 117 .\n\nEarle MD ,  Earle RL ,  Anderson A .  _Food Product Development_ .  Cambridge :  Woodhead Publishing ;  2001 .\n\nEuston SR ,  Hirst RL .  The emulsifying properties of commercial milk protein products in simple oil-in-water emulsions and in a model system .  _Journal of Food Science_.  2000 ;65 : 934 \u2013 940 .\n\nFenn D ,  Lukow OM ,  Bushuk W ,  Depauw RM .  Milling and baking quality of 1BL\/1RS translocation wheats. I. Effects of genotype and environment .  _Cereal Chemistry_.  1994 ;71 : 189 \u2013 195 .\n\nFoegeding EA ,  Davis JP .  Food protein functionality: A comprehensive approach .  _Food Hydrocolloids_.  2011 ;25 : 1853 \u2013 1864 .\n\nFranco JM ,  Guerrero A ,  Gallegos C .  Rheology and processing of salad dressing emulsions .  _Rheologica Acta_.  1995 ;34 : 513 \u2013 524 .\n\nFroning GW ,  Nutritional and functional properties of egg proteins .  Hudson BJF ,  ed.  _Developments in Food Proteins_ ,  Volume 6 .  London :  Elsevier Applied Science Publishers ;  1988 : 1 \u2013 34 .\n\nFroning GW ,  Wehling RL ,  Ball HR ,  Hill RM .  Effect of ultrafiltration and reverse osmosis on the composition and functional properties of egg white .  _Poultry Science_.  1987 ;66 : 1168 \u2013 1173 .\n\nGalluzzo SJ ,  Regenstein JM .  Role of chicken breast muscle proteins in meat emulsion formation: myosin, actin and synthetic actomyosin .  _Journal of Food Science_.  1978 ;43 (6) : 1761 \u2013 1765 .\n\nGerman JB ,  Phillips L ,  Protein interactions in foams: Protein-gas interactions .  Hettiarachchy NS ,  Ziegler GR ,  eds.  _Protein Functionality in Food Systems_ New York :  Marcel Dekker ;  1994 : 181 \u2013 208 .\n\nGolde AE ,  Schmidt KA .  Quality of coffee creamers as a function of protein sources .  _Journal of Food Quality_.  2005 ;28 (1) : 46 \u2013 61 .\n\nGordon A .  Emulsification in comminuted meat systems. II .  _Food Processing Industry_.  1969 ;38 (459) : 50 \u2013 52 : 54 .\n\nGhoush MA ,  Samhouri M ,  Al-Holy M ,  Hera T .  Formulation and fuzzy modeling of emulsion stability and viscosity of a gum\u2013protein emulsifier in a model mayonnaise system .  _Journal of Food Engineering_.  2008 ;84 (2) : 348 \u2013 357 .\n\nGruetzmacher TJ ,  Bradley Jr RL .  Acid whey as a replacement for sodium caseinate in spray-dried coffee whiteners .  _Journal of Dairy Science_.  1991 ;74 : 2838 \u2013 2849 .\n\nGuignon B ,  Otero L ,  Molina-Garcia AD ,  Sang PD .  Liquid water-ice phase diagrams under high pressure: Sodium chloride and suggested model food systems .  _Biotechnology Progress_.  2005 ;21 : 439 \u2013 445 .\n\nHall GM ,  ed.  _Methods of Testing Protein Functionality_ .  London :  Blackie Academic and Professional ;  1996 .\n\nHanrahan G ,  Lu K .  Application of factorial and response surface methodology in modern experimental design and optimization .  _Critical Reviews in Analytical Chemistry_.  2006 ;36 : 141 \u2013 151 .\n\nHardacre A ,  Clark SN ,  Harper J ,  Hemar Y ,  Illingworth D ,  Boland M .  Variation in the rheological properties of potato starches and interaction with casein and phosphorus. Report 2 .  _Crop and Foods Research Report_.  2004 ; .\n\nHarper WJ .  Model food system approaches for evaluating whey protein functionality .  _Journal of Dairy Science_.  1984 ;67 : 2745 \u2013 2756 .\n\nHarper WJ ,  Raman RV .  Protein interactions in whey protein concentrates and functionality in model food systems .  _New Zealand Journal of Dairy Science and Technology_.  1979 ;14 : 198 \u2013 199 .\n\nHarper WJ ,  Zadow G .  Heat-induced changes in whey protein concentrates as related to bread manufacture .  _New Zealand Journal of Dairy Science and Technology_.  1984 ;19 : 229 \u2013 237 .\n\nHarper WJ ,  Peltonen R ,  Hayes J .  Model food systems yield clearer utility evaluations of whey protein .  _Food Product Development_.  1980 ;14 (10) : 52 : 54, 56 .\n\nHeinemann C ,  Zinsli M ,  Renggli A ,  Escher F ,  Conde-Petit B .  Influence of amylose-flavor complexation on build-up and breakdown of starch structures in aqueous food model systems .  _Lebensmittel-Wissenschaft und -Technologie_.  2005 ;38 : 885 \u2013 894 .\n\nHlavacek RG ,  Robe K ,  Stinson WS ,  Belshaw F .  Reduce fat at 1\/3 in coffee whiteners, emulsifiers\u2014stabilizer also improves flavor .  _Food Processing_.  1970 ;31 (5) : 18 .\n\nInglett MJ ,  Inglett GE .  _Food Products Formulary, Volume 4, Fabricated Foods_ .  Westport, CT :  AVI Publishing Co ;  1982 .\n\nJaswir I ,  Hassan TH ,  Said MZM .  Effect of Malaysian plant extracts in preventing peroxidation reactions in model food oil systems .  _Journal of Oleo Science_.  2004 ;53 : 525 \u2013 529 .\n\nJohnson TM ,  Zabik ME .  Response surface methodology for analysis of protein interactions in angel cake food system .  _Journal of Food Science_.  1981 ;46 : 1226 \u2013 1230 .\n\nJohnson TM ,  Zabik ME .  Egg albumen protein interactions in an angel food system .  _Journal of Food Science_.  1981 ;46 : 1231 \u2013 1236 .\n\nKahn MN ,  Rooney LW ,  Dill CW .  Baking properties of plasma protein isolate .  _Journal of Food Science_.  1979 ;44 : 274 \u2013 276 .\n\nKelly PM ,  Oldfield DJ ,  O'Kennedy BT .  The thermostability of spray dried coffee whiteners .  _International Journal of Dairy Technology_.  1999 ;52 (3) : 107 \u2013 113 .\n\nKilara A .  Standardization of methodology for evaluating whey proteins .  _Journal of Dairy Science_.  1984 ;67 : 2734 \u2013 2744 .\n\nKilara A .  Whey protein functionality .  In:  Hettiarachchy NS ,  Ziegler GR ,  eds.  _Protein Functionality in Food Systems_ .  New York :  Marcel Dekker ;  1994 : 325 \u2013 355 .\n\nKinsella JN .  Relationship between structure and functional properties of food proteins .  In:  Fox PF ,  Condon JJ ,  eds.  _Food Proteins_ .  New York :  Applied Science Publishers ;  1982 : 51 \u2013 103 .\n\nKissell LT ,  Bean MM .  AACC technical committee report: Development of a method for angel food cake .  _Cereal Foods World_.  1978 ;23 (3) : 136 \u2013 142 .\n\nKneifel W ,  Ulbeth F ,  Shaffer E .  Evaluation of coffee whitening ability of dairy products and coffee whiteners by means of reflectance colorimetry .  _Milchwissenschaft_.  1992 ;47 : 567 \u2013 569 .\n\nKnightly WH .  The role of ingredients in the formulation of whipped toppings .  _Food Technology_.  1968 ;22 (6) : 73 \u2013 74 : 77\u201378, 81, 85\u201386 .\n\nKnightly WH .  The role of ingredients in the formulation of coffee whiteners .  _Food Technology_.  1969 ;32 (2) : 37 \u2013 48 .\n\nKolar CW ,  Cho IC ,  Watrous WL .  Vegetable protein applications in yogurt, coffee creamers and whip toppings .  _Journal of the American Oil Chemists ' Society_.  1979 ;56 : 389 \u2013 391 .\n\nKosikowski, F. and Jimenez-Flores, R. (1987) Low-fat dairy coffee whitener. United States Patent US4689245.\n\nKrishnan KR ,  Sharma N .  Studies on emulsion-type buffalo meat sausages incorporating skeletal and offal meat with different levels of pork fat .  _Meat Science_.  1990 ;28 (1) : 51 \u2013 60 .\n\nLah CL ,  Cheryan M ,  DeVor RE .  A response surface methodology approach to the optimization of whipping properties of an ultrafiltered soy product .  _Journal of Food Science_.  1980 ;45 : 1720 \u2013 1726 : 1731 .\n\nLee KM .  _Functionality of 34% whey protein concentrate (WPC) and its application in selected model food systems_ .  Columbus, Ohio :  PhD Dissertation. The Ohio State University ;  1999 .\n\nLee SY ,  Morr CV ,  Ha EYW .  Structural and functional properties of caseinate and whey protein isolate as affected by temperature and pH .  _Journal of Food Science_.  1992 ;57 : 1210 \u2013 1213 .\n\nLi-Chan E ,  Nakai S ,  Wood DF .  Hydrophobicity and solubility of meat proteins and their relationship to emulsifying properties .  _Journal of Food Science_.  1984 ;49 (2) : 345 \u2013 350 .\n\nLi-Chan ECY .  Properties of proteins in food systems: an introduction .  In:  Yada RY ,  ed.  _Proteins in Food Processing_ .  Cambridge :  Woodhead Publishing ;  2004 : 2 \u2013 29 .\n\nLiao SY ,  Mangino ME .  Characterization of the composition, physicochemical and functional properties of acid whey protein concentrates .  _Journal of Food Science_.  1987 ;52 : 1033 \u2013 1037 .\n\nLindblom M .  Bread baking properties of yeast protein concentrates .  _Lebensmittel-Wissenschaft und -Technologie_.  1977 ;10 : 341 \u2013 345 .\n\nLowe E ,  Durkee EL ,  Mersen RL ,  Ijichi K ,  Cimino SL .  Egg white concentrated by reverse osmosis .  _Food Technology_.  1969 ;23 (6) : 753 : 757-759, 760-762 .\n\nLuyten H ,  Vereijken J ,  Buecking M .  Using proteins as additives in foods: an introduction .  In:  Yada RY ,  ed.  _Proteins in Food Processing_ .  Cambridge :  Woodhead Publishing ;  2004 : 421 \u2013 437 .\n\nMalundo TMM ,  Resurrecci\u00f3n AVA ,  Koehler PE .  Sensory quality and performance of spray-dried coffee whitener from peanuts .  _Journal of Food Science_.  1992 ;57 : 222 \u2013 225 : 226 .\n\nMangino ME ,  Liao YY ,  Harper NJ ,  Morr CV ,  Zadow JG .  Effects of heat processing on the functionality of whey protein concentrates .  _Journal of Food Science_.  1987 ;52 : 1522 \u2013 1524 .\n\nMangino ME ,  Hettiarachchy NS ,  Ziegler GR .  _Protein Functionality in Food Systems_ .  New York :  Marcel Dekker ;  1994 .\n\nMarshall WH ,  Dutson TR ,  Carpenter ZL ,  Smith GC .  A simple method for emulsion end-point determinations .  _Journal of Food Science_.  1975 ;40 : 896 \u2013 897 .\n\nMarquez E ,  de Barboza MY ,  Izquierdo P ,  Torres G .  Studies on the incorporation of bovine plasma in emulsion type of meat product .  _Journal of Food Science and Technology, India_.  1997 ;34 (4) : 337 \u2013 339 .\n\nMartinez RM ,  Dawson PL ,  Ball Jr HR ,  Swartzel KR ,  Winn SE ,  Giesbrecht FG .  The effects of ultrapasteurization with and without homogenization on the chemical, physical, and functional properties of aseptically packaged liquid whole egg .  _Poultry Science_.  1995 ;74 (4) : 742 \u2013 752 .\n\nMcKenna, R.J., Keller, D.J. and Andersen, D.L. (1992) Process for preparing an alternative protein source for coffee whiteners and other products. United States Patent US5128156, US616909.\n\nMelachouris, N., Moffitt, K.R., Rasilowicz, C.E. and Tonner, G.F. (1994) Powdered coffee whitener containing reformed casein micelles. United States Patent US5318793, US937574.\n\nMiao S ,  Roos YH .  Nonezymatic browning kinetics in low-moisture food system as affected by matrix composition and crystallization .  _Journal of Food Science_.  2005 ;70 (2) : 69 \u2013 77 .\n\nMelmychyn, P. (1973) Acelated protein for coffee whitener formulations. United States Patent US3764711.\n\nMin DBS ,  Thomas EL .  A study of physical properties of dairy whipped topping mixtures .  _Journal of Food Science_.  1977 ;42 : 221 \u2013 224 .\n\nMittal GS ,  Usborne WR .  Meat emulsion extenders .  _Food Technology_.  1985 ;39 (4) : 121 \u2013 130 .\n\nModler H ,  Jones JD .  Selected processes to improve the functionality of dairy ingredients .  _Food Technology_.  1987 ;41 (10) : 114 \u2013 117 : 129 .\n\nMullen K ,  Ennis D .  Fractional factorials in food product development .  _Food Technology_.  1985 ;39 (5) : 90 : 92, 94, 97\u201398, 100, 102\u2013103 .\n\nMulvihill DM ,  Fox PF .  Assessment of the functional properties of milk protein products .  _Bulletin of the International Dairy Federation_.  1987 ;209 : 3 \u2013 11 .\n\nNakai S .  Structure-function relationships of food proteins: With an emphasis on the importance of protein hydrophobicity .  _Journal of Agricultural and Food Chemistry_.  1983 ;31 : 876 \u2013 883 .\n\nNongonierma AB ,  Springett M ,  Le Quere J-L ,  Cavot P ,  Voilley A .  Flavor release at gastric\/matrix interfaces of stirred yogurt models .  _International Dairy Journal_.  2006 ;16 : 102 \u2013 110 .\n\nOwusu-Apenten RK .  Testing protein functionality .  In:  Yada RY ,  ed.  _Proteins in Food Processing_ .  Cambridge :  Woodhead Publishing ;  2004 : 217 \u2013 244 .\n\nParedes MDC ,  Rao MA ,  Bourne MC .  Rheological characterization of salad dressings. 1. Steady shear, thixotropy and effect of temperature .  _Journal of Texture Studies_.  1988 ;19 : 247 \u2013 258 .\n\nParker A ,  Gunning PA ,  Ng K ,  Robins MM .  How does xanthan stabilize salad dressing .  _Food Hydrocolloids_.  1995 ;9 : 333 \u2013 342 .\n\nPatel PD ,  Fry JC ,  The search for standardized methods for assessing protein functionality .  Hudson BJF ,  ed.  _Developments in Food Proteins_ ,  Volume 5 .  London :  Elsevier Applied Science ;  1987 : 299 \u2013 333 .\n\nPatel JR ,  Dave RI ,  Joshi NS ,  Thakar PN .  Coffee whiteners .  _Journal of Dairy Science Association_.  1992 ;44 : 18 \u2013 25 .\n\nPatil SK ,  Baczynsk ,  i M ,  McCurry T .  Wheat protein isolates\u2014alternative to sodium caseinate .  _Cereal Foods World_.  2006 ;51 (5) : 279 \u2013 281 .\n\nPearce RJ ,  Harper WJ .  A method for the quantitative evaluation of emulsion stability .  _Journal of Food Science_.  1982 ;47 : 680 \u2013 681 .\n\nPeltonen-Shalaby R ,  Mangino ME .  Compositional factors that affect the emulsifying and foaming properties of whey protein concentrate .  _Journal of Food Science_.  1986 ;51 : 91 \u2013 95 .\n\nPernell CW ,  Luck PJ ,  Foegeding EA ,  Daubert CR .  Heat-induced changes in angel food cakes containing egg-white protein or whey protein isolate .  _Journal of Food Science_.  2002 ;67 : 2945 \u2013 2951 .\n\nPomeranz Y ,  Bolling H ,  Zwingelberg H .  Wheat hardness and baking properties of wheat flours .  _Journal of Cereal Science_.  1984 ;2 (3) : 137 \u2013 143 .\n\nRaeker MO ,  Johnson LA .  Cake-baking (high-ratio white layer) properties of egg white bovine blood plasma, and their protein fractions .  _Cereal Chemistry_.  1995 ;72 : 299 \u2013 303 .\n\nRandall CJ ,  Raymond DP ,  Voisey PW .  Effects of various animal and vegetable materials on replacing the beef component in a meat emulsion system .  _Canadian Institute of Food Science and Technology Journal_.  1976 ;9 (4) : 216 \u2013 221 .\n\nRanhortra GS ,  Gelroth JA ,  Eisenbraun GJ .  Gluten index and bread-making quality of commercial dry glutens .  _Cereal Foods World_.  1992 ;37 (3) : 261 \u2013 263 .\n\nRegenstein JM ,  Grunden LP ,  Baker PC .  Proteolytic enzymes and the functionality of chicken egg albumin .  _Journal of Food Science_.  1978 ;43 : 279 \u2013 280 .\n\nRegenstein JM .  _Are comminuted meat products emulsions or a gel matrix? Proceedings of Food Processing and Preservation Meeting_ .  Cornell University ;  1989 .\n\nRodriguez O ,  Castell-Perez ME ,  Moreira RG .  Effect of sugar and storage temperature on the survival and recovery of irradiated Escherichia coli K-12 MG 1655 .  _Food Science and Technology_.  2007 ;40 (4) : 690 \u2013 696 .\n\nSakanaka S ,  Tachibana Y .  Active oxygen scavenging activity of egg-yolk protein hydrolysates and their effects on lipid oxidation in beef and tuna homogenates .  _Food Chemistry_.  2006 ;95 : 243 \u2013 249 .\n\nSeuvre A-M ,  Philippe E ,  Rochard S ,  Voilley A .  Kinetic study of the release of aroma compounds in different model food systems .  _Food Research International_.  2007 ;40 (4) : 480 \u2013 492 .\n\nSeverini C ,  Baiano A ,  Rovere P ,  Dall'Aglio G .  Effect of high pressure on olive oil oxidation and the Maillard reaction in model and food systems .  _Italian Food and Beverage Technology_.  2003 ;31 : 5 \u2013 10 .\n\nShurtleff W ,  Aoyagi A .  _Non-dairy Whip Toppings (With or Without Soy Protein) \u2014 Bibliography and Source Book: 1944 to 1994_ .  Lafayette, California :  Soy Foods Centre ;  1994 .\n\nSila DN ,  Smout C ,  Satara Y ,  Vu-Truong L ,  Hendricloc M .  Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity .  _Journal of Food Engineering_.  2007 ;78 (3) : 755 \u2013 764 .\n\nSingh H .  Aspects of milk-protein stabilized emulsions .  _Food Hydrocolloids_.  2011 ;24 : 1938 \u2013 1944 .\n\nSmith RA .  _Emulsion formation and stability in salad dressings. M.S. Thesis_ .  Columbus, Ohio :  The Ohio State University ;  1997 .\n\nSong M ,  Roos YH .  Isothermal study of non-enzymatic browning kinetics in spray-dried systems at different relative vapor pressure environments .  _Innovative Food Science and Emerging Technologies_.  2006 ;7 (3) : 182 \u2013 194 .\n\nStanley DW ,  Goff HD ,  Smith AK .  Texture-structure relationships in foamed dairy emulsions .  _Food Research International_.  1996 ;29 (1) : 1 \u2013 3 .\n\nSwift CE ,  Sulzbacher WL .  Comminuted meat emulsions: Factors affecting meat proteins as emulsion stabilizers .  _Food Technology_.  1963 ;17 : 224 \u2013 226 .\n\nSwift CE ,  Lockett C ,  Fryar AS .  Comminuted meat emulsions. The capacity of meats for emulsifying fat .  _Food Technology_.  1961 ;15 (11) : 468 \u2013 473 .\n\nSzerman N ,  Gonzalez CB ,  Sancho AM ,  Grigioni G ,  Carduza F ,  Vaudagna SR .  Effect of whey protein concentrate and sodium chloride addition plus tumbling procedures on technological parameters, physical properties and visual appearance of sous vide cooked beef .  _Meat Science_.  2007 ;76 : 463 \u2013 473 .\n\nTownsend WE ,  Witnauer LP ,  Riloff JA ,  Swift CE .  Comminuted meat emulsions: Differential thermal analysis of fat transitions .  _Food Technology_.  1968 ;22 : 319 \u2013 323 .\n\nTran KH ,  Einerson MA .  A rapid method for the evaluation of emulsion stability of non-dairy creamers .  _Journal of Food Science_.  1987 ;52 : 1109 \u2013 1110 .\n\nTsai R ,  Cassens RG ,  Briskey EJ .  The emulsifying properties of purified muscle proteins .  _Journal of Food Science_.  1972 ;37 : 286 \u2013 288 .\n\nTurgeon SL ,  Schmitt C ,  Sanchez C .  Protein\u2013polysaccharide complexes and coacervates .  _Current Opinion in Colloid and Interface Science_.  2007 ;12 (4\u20135) : 166 \u2013 178 .\n\nWebb, N.B. (1974) Emulsion technology. Proceedings of the Meat Industry Research Conference. 1-15.\n\nWijeratne SSK ,  Amarowicz R ,  Shahidi F .  Antioxidant activity of almonds and their by-products in food model systems .  _Journal of the American Oil Chemists ' Society_.  2006 ;83 : 223 \u2013 230 .\n\nXiong YL ,  Blanchard SP ,  Means WJ .  Properties of broiler myofibril gels containing emulsified lipids .  _Poultry Science_.  1992 ;71 (9) : 1548 \u2013 1555 .\n\nYada RY .  _Proteins in Food Processing_ .  Cambridge :  Woodhead Publishing ;  2004 .\n\nZayas JF .  _Functionality of Proteins in Food_ .  Berlin :  Springer-Verlag ;  1996 . \nChapter 16\n\n# Sensory Properties of Dairy Proteins\n\nM.A. Drake\n\nR.E. Miracle\n\nJ.M. Wright Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, North Carolina, USA\n\n## Abstract\n\nProduction and applications of dairy proteins are increasing globally. The dairy protein category is a wide one, encompassing milk proteins and whey proteins, as well as many subcategories of these products such as caseins, hydrolysates, and serum or native whey proteins. Although functionality and nutrition continue to be key aspects of these products, flavor is a critical parameter that should not be overlooked. An array of sensory analysis techniques can be applied to measure flavor intensities and flavor variability and to determine flavor sources when applied in conjunction with analytical chemistry. This chapter addresses the current status and ongoing research on the sensory properties of dairy proteins.\n\n## Keywords\n\nSensory properties\n\ndairy proteins\n\nmilk proteins\n\nwhey proteins\n\nflavor performance\n\nflavor properties\n\nflavor variability\n\nOutline\n\nIntroduction 473\n\nSensory analysis 474\n\nWhey proteins 474\n\nMilk proteins 485\n\nCaseins and hydrolysates 488\n\nFlavor binding 489\n\nConclusions 489\n\nAcknowledgment 490\n\n## Introduction\n\nDairy proteins are valuable dried ingredients with a host of functional and nutritional properties (Foegeding et al., 2002; Miller, 2005; O'Connell & Flynn, 2007). Within the category of dairy proteins, there are a variety of ingredients, including whey proteins and milk proteins of various protein contents. Dried caseins and caseinates as well as serum proteins ('native' whey proteins or whey proteins separated from milk prior to the cheesemaking process) are also in this category. Dairy proteins (primarily dried) are used in an increasingly wide array of ingredient applications for functionality, but with the current consumer focus on health and nutrition, these ingredients are also used widely to enhance nutrition.\n\nMilk proteins play a crucial role in the flavor of all dairy foods. As part of the sensory experience, proteins provide mouthfeel, viscosity, and structure for dairy foods. Amino acids and peptides can elicit basic tastes but can also serve as the starting substrates for numerous volatile aroma-active compounds. Proteolysis and the subsequently released amino acids and peptides are the sources as well as the substrates for many desirable and undesirable flavors in cheeses and other fermented dairy products (Singh et al. 2003; 2005; Carunchia Whetstine et al., 2005a; Drake et al., 2007). Heat processing influences the flavor potential of proteins via denaturation and the release of sulfuric compounds and the typical eggy aroma of scalded milk. Denaturation can also make proteins more susceptible to breakdown and thus influences flavor and flavor development in this manner as well. Theoretically, pure undegraded protein should be flavorless. However, dairy proteins as food ingredients are not 100% protein. Fat, ash, carbohydrate, and other components are present in various amounts and also clearly influence the final flavor and flavor stability of dairy proteins.\n\nAs with all foods, flavor plays a large role in product acceptance and success. Dried ingredients certainly affect the quality of the final product (Caudle et al., 2005), and the sensory properties of these valuable ingredients should not be overlooked. Dried dairy proteins should ideally be bland or mild and dairy-like in flavor. Recent research has demonstrated that dairy proteins are not flavorless and display a wide array of flavor variability. Understanding and documenting the flavor of these proteins is the key to strategic research and marketing. This chapter addresses and reviews current research on the sensory properties of dairy proteins.\n\n## Sensory analysis\n\nSensory analysis is a scientific discipline that encompasses the depth and breadth of all properties of a food that are perceived by the human senses (Drake, 2007). As such, sensory properties are crucial for product success. Dairy foods continue to enjoy a positive flavor perception by the consumer (Drake & Gerard, 2003; Russell et al., 2006) and this competitive edge should ideally be maximized. This means that a complete understanding of flavor, flavor variability, sources of flavors, and consumer perception is mandatory.\n\nA wide array of sensory tests is available to objectively or subjectively measure the sensory properties of foods. These tests and their specific application to dairy products are covered in several textbooks and review articles (Lawless & Heymann, 1999; Singh et al., 2003; Drake, 2004; 2007; Meilgaard et al., 2007). Two basic categories exist: analytical tests and affective tests. Several types of tests exist within each category, and selection of the specific and appropriate test is dependent on the specific objective in mind. Analytical sensory tests are a group of sensory tests that are objective in nature and use trained or screened panelists. Some examples include descriptive analysis, discrimination tests, and threshold tests. Affective tests are subjective tests and comprise tests that use consumers in qualitative or quantitative measurements.\n\n## Whey proteins\n\nWhey proteins are recovered from membrane processing and concentration of the liquid whey stream resulting from cheesemaking. Thus, one source of flavor and flavor variability of whey proteins is the flavor of the liquid whey source. The flavor of liquid whey varies, not surprisingly, with the type of cheese (Tomaino et al., 2004; Gallardo Escamilla et al., 2005). The flavor (sensory perception and volatile components) of fresh liquid whey from thermophilic starters (pasta filata cheeses) differs from that from mesophilic starter cultures (Cheddar cheese) (Liaw et al., 2011). The flavor of whey from direct acid-set curd will deviate further (Table 16.1) (Campbell et al., 2011a,b). The addition of enzymes such as lipases will increase the free fatty acid content of the whey, and this will also influence flavor in the form of rancid, waxy, and\/or animal flavors, depending on the lipase and the milk source.\n\nTable 16.1\n\nFlavor Profiles of Fluid Whey Obtained from Mozzarella Cheese, Cheddar Cheese, or Acid Casein Manufacturea\n\nAttributeb | Cheddar | Mozzarella | Acid casein  \n---|---|---|---  \nHeated milkc | 41a | 20c | 27b  \nCaramelized milk | 21a | 4b | 4b  \nNatural yogurt | 4c | 29a | 5c  \nStale | 9b | 9b | 22a  \nRancid | 16c | 24b | 38a  \nOaty | 8a | 1b | 7a  \nDirty | 2b | 7b | 35a  \nAcid | 5b | 39a | 36a  \nSweet | 37a | 8b | 12b  \nBitter | 2b | 14a | 7ab  \nSalty | 6c | 12b | 18a\n\na Adapted from Gallardo Escamilla et al. (2005).\n\nb Attributes were scored on a scale from 0 to 100.\n\nc Means in a row followed by different letters are different (p < 0.05).\n\nWithin a single type of cheese, the flavor of the whey will vary depending on starter culture rotation and\/or other variables in the cheesemaking process. Carunchia Whetstine et al. (2003) documented tremendous variability in flavor and volatile compound profiles within and between two Cheddar cheese facilities with starter culture rotation. These results were further confirmed by Karagul-Yuceer et al. (2003a). Free fatty acid profiles and proteolysis were also distinct. Tomaino et al. (2004) documented that cold storage of liquid pasteurized whey increased lipid oxidation products and resulted in cardboard flavors. They speculated that lactic starter culture enzymes accelerated these storage-induced changes because the concentrations of lipid oxidation products were higher in fermented whey than in whey from direct acid coagulation. Further, differences in lipid oxidation products were observed in fresh whey from three different single mesophilic starter strains. Campbell et al. (2011a, b) confirmed the role of the starter culture in lipid oxidation as well as differences in oxidative contributions between strains of starter cultures. Liaw et al. (2011) also documented consistent differences in flavor initially and following storage between mesophilic starter cultures and thermophilic starter cultures, concurrent with distinct volatile compound profiles. Clearly, the initial raw product stream in whey protein processing displays tremendous flavor, flavor variability, and flavor precursors.\n\nIt is not unexpected then that finished dried protein concentrates and isolates also display flavor variability. Liquid whey is subjected to a host of processing techniques to concentrate and separate the whey protein. Pasteurization, membrane filtration, concentration, and spray drying are all steps that can induce the formation of flavor compounds. Although there is a general process of whey protein production, each facility is distinct, with facility-specific storage parameters and\/or time\/temperature profiles that further contribute variability to the finished product. In the case of whey protein from colored Cheddar cheese, a bleaching process with hydrogen or benzoyl peroxide is also involved. The process of oxidizing the whey stream to decolorize it will result in a host of possible flavors and flavor precursors. Recent published work has highlighted the impact of these specific processing steps (cheesemaking procedure, liquid storage, dry storage, bleaching, solids) on final whey protein flavor (Whitson et al., 2011; Wright et al., 2009; Croissant et al., 2009; Evans et al. 2009; 2010; Jervis et al., 2012; Campbell et al., 2013).\n\nRecently, application of defined sensory analysis in combination with instrumental volatile analysis has shed light on the sources of many dairy flavors and will ultimately aid in identifying methods to control flavor. This approach recently led to the identification of a method to enhance the nutty flavor in Cheddar cheese (Avsar et al., 2004; Carunchia Whetstine et al., 2006) and the specific identification of a cabbage off-flavor in whey protein isolate (Wright et al., 2006). The reader is referred to three recent reviews on the application of these techniques to control the flavor in dairy products (Singh et al., 2003; Drake, 2004; Drake et al., 2006). A host of defined flavors in whey proteins have been documented (Drake et al., 2003; Carunchia Whetstine et al., 2005b; Drake, 2006; Russell et al., 2006; Wright et al., 2006) (Table 16.2), and volatile compound flavor variability has also been documented (Morr & Ha, 1991; Mills, 1993; Quach et al., 1999; Carunchia Whetstine et al., 2005b; Wright et al., 2006). The many processing variables listed above undoubtedly contribute to these differences in flavor among fresh products. Figure 16.1 demonstrates the flavor variability in fresh (<1 month old) product collected from different manufacturers. Products 4, 5, 6, 8, 9, and 10 were manufactured from Mozzarella or white Cheddar cheese whey, whereas products 1, 2, 3, and 7 were manufactured from colored Cheddar cheese whey. These differences in flavor and volatile compounds also suggest that some flavors and volatile components are specifically formed from whey protein manufacturing bleaching processes. Certainly this area of research should be investigated in ongoing efforts to minimize whey protein flavor. The flavor intensities of many whey proteins are comparable with those of soy proteins (Russell et al., 2006); this is a crucial issue for competitive global marketing.\n\nTable 16.2\n\nFlavors Reported in Whey Proteins [Whey Protein Concentrate (WPC80) and Whey Protein Isolate (WPI)] by Sensory Analysisa\n\nTerm | Definition | Reference | Example\/Preparation  \n---|---|---|---  \nOverall aroma intensity | The overall orthonasal aroma impact |  | Evaluated as the lid is removed from the cupped sample  \nFlavors, tastes, feeling factors (evaluated in the mouth) |  |  |   \nSweet aromatic | Sweet aroma associated with dairy products |  | Vanilla cake mix or 20 ppm vanillin in milk  \nCooked\/milky | Aromatics associated with | cooked milk | Heating skim milk to 85 \u00b0C for 30 min  \nDoughy\/fatty\/  \npasta | Aromatics associated with canned biscuit dough and cooked pasta | (Z)-4-heptenal | 1 ppm (Z)-4-heptenal in water from canned biscuit dough, or cooked pasta water  \nFatty\/frying oil | Aromatics associated with old frying oil and lipid oxidation products | 2,4-decadienal | Old (stored) vegetable oil  \nMetallic\/meat serum | Aromatics associated with metals or with juices of raw or rare beef | Aromatics of fresh raw beef steak or ground beef or juices from seared beef steak |   \nCucumber | Aromatics associated with freshly sliced cucumber | (E)-2-nonenal | 1 ppm (E)-2-nonenal or freshly sliced cucumbers  \nBrothy | Aromatics associated with broth or boiled potatoes | Methional | 1 ppm methional in water or boiled potatoes  \nCabbage | Aromatics associated with medium-chain fatty acids and soaps | Dimethyl trisulfide | Boiled fresh cut cabbage, 10 ppb dimethyl trisulfide on filter paper in sniff jar  \nCardboard\/ wet paper | Aromatics associated with cardboard | Cardboard paper | Brown cardboard or brown paper bag cut into strips and soaked in water  \nAnimal\/wet dog | Aromatics associated with wet dog hair | Knox gelatin | One bag of gelatin (28 g) dissolved in two cups of distilled water  \nPasta water | Aromatics associated with water after pasta has been boiled in it |  | Pasta boiled in water for 30 min  \nSoapy | Aromatics associated with soap | Lauric acid | 1 ppm lauric acid or shaved bar soap  \nBitter | Basic taste associated with bitterness | Caffeine | Caffeine, 0.5% in waterb  \nAstringency | Drying tongue sensation | Alum | Alum, 1% in waterb\n\na Adapted from Drake et al. (2003), Karagul-Yuceer et al. (2003a), Carunchia Whetstine et al. (2005b), and Wright et al. (2006).\n\nb From Meilgaard et al. (2007).\n\nFigure 16.1 Principal component biplot of descriptive sensory analysis of WPC80. The WPC80s are represented by numbers. Underlined samples were evaluated by consumers.\n\nStorage of whey proteins is another source of flavor variability. The purported shelf life of whey protein concentrate (WPC80) and whey protein isolate (WPI) varies from 12 to 24 months, depending on the supplier. Volatile compound work on WPC80 subjected to accelerated storage conditions has demonstrated key volatile component changes with storage (Javidipour & Qian, 2007). Wright et al. (2009) went on to apply sensory and volatile compound analysis to demonstrate changes in flavor with storage time and instantization. Products are often agglomerated to enhance their functional properties. The agglomeration process (rewet or single pass) can include addition of lecithin to further increase wettability (instantization). Both of these processes (agglomeration and agglomeration with lecithin) may impact flavor and decrease shelf life. Figure 16.2 demonstrates the flavor changes with storage time of nonagglomerated, agglomerated, and instantized (agglomerated with lecithin) WPC80 from a single supplier, as documented by a trained sensory panel. The results suggest that agglomeration, especially agglomeration with lecithin, affects the storage stability of WPC80, with more rapid development of fatty, cucumber, and lipid oxidation types of off-flavors.\n\nFigure 16.2 Flavor changes during storage for 12 months at 21 \u00b0C of nonagglomerated, agglomerated, and instantized (agglomerated with lecithin) WPC80s. C, control nonagglomerated product; A, steam-agglomerated product; AL, product agglomerated with lecithin.\n\nSamples of agglomerated and nonagglomerated WPI and WPC80 were collected from suppliers whose products were previously noted to develop a cucumber off-flavor. The samples were stored at 21 \u00b0C and were monitored by descriptive sensory analysis (rehydrated to 10% solids w\/w) and by instrumental volatile analysis [headspace solid phase microextraction gas chromatography\u2013olfactometry (HS-SPME GC\u2013O) with gas chromatography\u2013mass spectrometry (GC\u2013MS)]. Samples for volatile analysis were prepared as previously described (Wright et al., 2006). Briefly, 20 g of each reconstituted whey protein, a stirring bar, and 1 g of NaCl were placed in a 40 mL amber glass SPME vial and sealed air tight with a Teflon\u2122-sided silicon septum (PTFE\/silicon) and a plastic cap (Supelco, Bellefonte, Pennsylvania, USA). Samples were heated to 40 \u00b0C and stirred for 30 min before the SPME fiber (three phase: 2 cm \u2013 50\/30 \u03bcm DVB\/Carboxed\u2122\/PDMS Stable Flex, Supelco) was exposed in the headspace at a depth of 3.8 cm for an additional 30 min prior to injection on to the gas chromatograph. The fiber was desorbed at 250 \u00b0C for 5 min in the injection port fitted with an SPME inlet at a depth of 7.6 cm. GC\u2013O analysis was performed using an HP 5890 series II gas chromatograph equipped with a flame ionization detector (FID), a sniffing port, and a splitless injector. For GC\u2013MS, samples were prepared analogously prior to injection on to the GC\u2013MS system by a CTC Analytics CombiPal Autosampler (Zwingen, Switzerland). The fiber was desorbed at 250 \u00b0C for 5 min in the injection port fitted with an SPME inlet at a depth of 50 mm. GC\u2013MS analysis was performed using an Agilent 6890N gas chromatograph with a 5973 inert MSD with a DB-5ms (20 m \u00d7 0.25 mm internal diameter x 0.25 \u03bcm film thickness) column. Each sample was analyzed in triplicate.\n\nThe sensory profiles confirm that agglomerated samples, particularly those with lecithin, are more prone to cucumber off-flavor (Figs. 16.3 and 16.4). A number of compounds with cucumber or fatty aromas were recorded by GC\u2013O in samples with and without cucumber flavor (Table 16.3). Unfortunately, these compounds were at or below MS detection limits by the extraction technique used. The aroma of a compound when it is isolated is not necessarily the same aroma or flavor that compound elicits when it is in a food matrix, which means that sensory analysis of the compound in the food matrix is recommended (Drake & Civille, 2003). The character of an aroma can also change with compound concentration (Drake & Civille, 2003). However, when suspect compounds were placed into WPC80 without cucumber flavor within their reported threshold range and were presented to trained panelists (n = 8), many of them elicited cucumber flavors (Table 16.4), suggesting that one or a combination of these compounds are responsible for this off-flavor that develops during the storage of whey proteins. In agreement with the previous example (Fig. 16.2), these compounds are also lipid oxidation compounds, again indicating that lipid oxidation is a major source of off-flavor development in these protein products.\n\nFigure 16.3 Flavor changes during storage for 18 months at 21 \u00b0C of nonagglomerated, agglomerated, and instantized (agglomerated with lecithin) WPC80s. L, lecithinated; C, control; S, steam agglomerated. Number indicates months of storage.\n\nFigure 16.4 Flavor changes during storage for 18 months at 21 \u00b0C of nonagglomerated, agglomerated, and instantized (agglomerated with lecithin) WPIs. L, lecithinated agglomerated; C, control. Number indicates months of storage.\n\nTable 16.3\n\nAroma-active 'Green' Compounds Identified by HS-SPME GC\u2013O from Stored Agglomerated and Nonagglomerated WPC80 and WPI\n\n|  | Post-peak intensityb |  |   \n---|---|---|---|---  \nCompound | Odor a | C8 | C10 | C12 | C14 | S8 | S10 | S12 | S14 | L8 | L10 | L12 | L14 | RI c | Method of ID d\n\nWPC80\n\nHexanal | Grassy\/green | 2.50 | 2.50 | 2.50 | 3.00 | 3.25 | 2.50 | 2.50 | 3.00 | 2.50 | 2.75 | 1.50 | 3.25 | 806 | RI, odor, MSe  \nE-2-nonenal | Carpet\/green | NDf | 2.00 | 1.50 | 2.25 | 3.00 | 2.00 | 3.00 | ND | ND | 2.75 | 2.00 | 3.00 | 1163 | RI, odor  \nE,Z-2,6-nonadienal | Cucumber\/herb | ND | 3.00 | 2.00 | ND | 3.50 | ND | 3.00 | ND | 2.00 | 3.00 | ND | 3.00 | 1168 | RI, odor  \nIsobutyl-methoxy-pyrazine | Green pepper | 3.00 | ND | ND | ND | 3.00 | ND | ND | ND | 3.00 | 2.50 | ND | ND | 1185 | RI, odor  \nE,Z-2,4-nonadienal | Carpet\/green | ND | ND | ND | ND | ND | ND | ND | 2.25 | 1.75 | ND | ND | ND | 1189 | RI, odor  \n6-Decenal | Cucumber\/rosy | 2.50 | ND | 2.50 | ND | 2.00 | ND | 1.50 | ND | ND | 2.00 | 1.50 | ND | 1205 | RI, odor\n\nWPI\n\n---  \n|  | Post-peak intensityb |  |   \n---|---|---|---|---  \nCompound | Odor a | C8 | C10 | C12 | C14 | L8 | L10 | L12 | L14 | RI c | Method of ID d  \nMethyl-2-butenol | Green\/hay | NDf | ND | ND | ND | 1.50 | ND | ND | ND | 777 | RI, odor  \nHexanal | Grassy\/green | ND | 2.25 | 1 | 2 | 2.00 | 2.00 | 1.5 | 2.5 | 806 | RI, odor, MSe  \nE-2-nonenal | Carpet\/green | ND | ND | ND | ND | ND | 1.75 | ND | ND | 1163 | RI, odor  \nE,Z-2,6-nonadienal | Cucumber\/herb | ND | ND | ND | ND | 1.75 | 1.75 | ND | 3.0 | 1168 | RI, odor  \nIsobutyl-methoxy-pyrazine | Green pepper | 3.25 | 2.50 | ND | ND | 5.00 | ND | ND | ND | 1185 | RI, odor  \nE,Z-2,4-nonadienal | Carpet\/green | ND | ND | ND | ND | 2.00 | ND | ND | ND | 1189 | RI, odor  \n6-Decenal | Cucumber\/rosy | 2.75 | ND | ND | ND | 3.50 | 1.50 | ND | ND | 1205 | RI, odor\n\nNote: C refers to nonagglomerated product, S to steam agglomerated product, and L to product agglomerated with added lecithin. The number following the treatment letter designation indicates storage time at 21\u00b0C in months.\n\na Odor description at the gas chromatograph sniffing port.\n\nb Mean post-peak intensities as determined by two experienced sniffers at the gas chromatograph sniffing port (van Ruth, 2001).\n\nc Retention index was calculated from GC\u2013O data on a DB-5 column.\n\nd Compounds were identified by comparison with authentic standards on the following criteria: retention index (RI) on a DB-5 column, odor property at the gas chromatograph sniffing port, and mass spectra in the electron impact mode. Positive identifications indicate that mass spectral data were compared with authentic standards.\n\ne MS = mass spectra.\n\nf ND = not detected.\n\nTable 16.4\n\nSensory Analysis of Model Whey Protein Systems for Cucumber Flavor\n\nCompound | Concentration in WPC80 Model (ppb) | Aromaa | Reported threshold (ppb)  \n---|---|---|---  \nE-2-nonenal | 0.4 | New carpet\/cucumber | 0.4  \nE-2-nonenal | 4.0 | Cucumber | -  \nE,Z-2,6-nonadienal | 0.14 | Cucumber | 0.14  \nE,Z-2,6-nonadienal | 1.4 | Cucumber\/fatty | -  \nE,Z-2,4-nonadienal | 0.158 | Fatty\/cucumber | Not reported  \nE,Z-2,4-nonadienal | 1.58 | Fatty\/cucumber | -  \nHexanal | 73.3 | Sweet chemical-like | 50  \n2-Pentyl furan | 7.97 | Cucumber\/fatty (very faint) | 6  \n2-Pentyl furan | 79.7 | Hay\/licorice | -  \n2-Ethyl 1 hexanol | 300 | Chemical\/cleaning agent | 300\n\nNote: WPC80 without discernible cucumber flavor was used as the base. WPC80 was rehydrated to 10% solids (w\/w) and then spiked with suspect compounds at their reported threshold concentration.\n\na Aroma as perceived by eight trained sensory panelists who were also experienced GC\u2013O sniffers. Panelists were provided with the list of descriptors used for GC\u2013O and were asked to select one or two descriptors that best described the aroma.\n\nRecent work has suggested that native whey proteins might provide a product with the functional and nutritional benefits of whey proteins with superior flavor properties (Evans et al. 2009; 2010; Campbell et al., 2013). Native whey proteins are simply whey proteins that are removed from fluid milk prior to the initiation of cheesemaking. In fact, serum or whey proteins can be removed from fluid milk, and the cheesemaking procedure can subsequently be initiated as normal with few or no effects on cheese yield. As the native whey proteins have not been subjected to the normal cheesemaking and whey protein processing procedures, their flavor profiles are remarkably bland and nearly free of flavor (Fig. 16.5). Significant economic challenges face the industrial scale-up of these products but, at a minimum, research with these products may reveal key information on minimizing the flavor of traditional whey proteins.\n\nFigure 16.5 Sensory profiles of rehydrated WPC34 from Cheddar whey and rehydrated native WPC34 (serum proteins).\n\nDairy products and, by association, dairy ingredients continue to enjoy a positive flavor image with consumers (Drake & Gerard, 2003; Russell et al., 2006). However, many consumers are unaware that whey proteins are dairy proteins; this is a sensory issue because consumer perception influences consumer liking. Drake (2006) found that consumers were generally less sure of their responses when asked to comment on the properties of specific protein types. The U.S. consumer was generally less informed about whey proteins and was more confident and aware of soy protein than were New Zealand consumers (Jones et al., 2007). In a follow-up study (Childs et al., 2007), focus groups with U.S. consumers confirmed that most U.S. consumers were unaware that whey proteins were dairy or milk proteins. Consumer education is a current challenge to the dairy protein industry.\n\nOne other issue pertinent to whey proteins and consumer acceptance is whether the flavor variability documented by trained panelists is detected by consumers and\/or whether it affects the quality of the finished product. Intuitively, the freshest and highest quality ingredients make the best finished product. However, research also indicates that consumers can discern differences in whey protein flavors and that these flavors carry through into ingredient applications (Drake, 2006). Figure 16.6 demonstrates this concept with protein beverages manufactured from different fresh WPC80s. The nature of the off-flavor and the ingredient application will also influence flavor carry-through (Drake, 2006). Some ingredient applications will be more tolerant than others of variability in the flavor of the ingredients. Childs et al. (2007) recently demonstrated flavor and texture\/mouthfeel differences between meal replacement beverages and bars made with whey proteins, soy proteins, or mixtures of whey and soy proteins. The ingredient applications were made using standard formulas to allow direct comparison of the influence of the different proteins. Trained panelists documented discernible flavor carry-through of whey and soy proteins in vanilla meal replacement beverages (Fig. 16.6). In contrast, no differences in flavor between bars made with whey or soy proteins were noted, although several differences in bar texture were impacted by the protein type (Figs. 16.7 and 16.8). Wright et al. (2009) and Evans et al. (2009; 2010); conducted consumer acceptance testing with whey proteins that were documented as distinct when profiled in 10% solution by trained panelists. For consumer testing, proteins were incorporated into clear acidic beverages. Consumers documented differences in flavor liking among beverages that differed only in protein source, confirming that (off) flavors from whey proteins carry through into ingredient applications.\n\nFigure 16.6 Trained panel flavor and mouthfeel profiles of vanilla meal replacement shakes made with whey protein, soy protein, or a mixture of whey protein and soy protein. * Indicates significant attributes (p <0.05). Adapted from Childs et al. (2007).\n\nFigure 16.7 Trained panel flavor profiles of peanut butter-flavored meal replacement bars made with whey protein, soy protein, or a mixture of whey protein and soy protein. No attribute differences were noted (p > 0.05). Adapted from Childs et al. (2007).\n\nFigure 16.8 Trained panel texture profiles of peanut butter-flavored meal replacement bars made with whey protein, soy protein, or a mixture of whey protein and soy protein. * Indicates significant attributes (p <0.05). Adapted from Childs et al. (2007).\n\n## Milk proteins\n\nMilk protein concentrates (MPCs) and isolates (MPIs) represent a newer category of dried dairy ingredients that are rapidly gaining in popularity. These products are manufactured by concentrating milk proteins (whey proteins and caseins) from fluid milk by membrane processing, followed by spray drying. Recent work in the primary author's laboratory has addressed the sensory properties of milk proteins across increasing protein concentration. MPCs with lower protein content (56, 70% protein dry weight) are characterized by fluid milk types of flavors: cooked\/milky, sweet aromatic, sweet taste, and cereal (Fig. 16.9). As the protein content is increased, the flavor profiles change, and MPC77, MPC80, and MPI are characterized by tortilla, brothy, cardboard, and animal flavors as well as higher astringency.\n\nFigure 16.9 Principal component biplot of trained panel flavor profiles of rehydrated MPC and MPI. Number indicates protein content.\n\nChanges in flavor with increasing protein content were also observed when the sensory properties of lower protein WPCs were compared with those of WPC80 and WPI. Increases in whey protein content also resulted in decreases in sweet aromatic and milky flavors. These changes in flavor are probably directly linked to changes in composition and different concentrations of resulting volatile components. A comparison of aroma-active volatile components isolated from WPC80\/WPI and whey powder revealed few differences (Mahajan et al., 2004; Carunchia Whetstine et al., 2005b). Differences in flavor are probably due to differences in the relative abundance of specific compounds. Similarly, volatile compound changes are evident in MPCs and MPIs as the protein content is increased. MPCs with higher final protein content have lower sulfur compound response as well as lower aldehyde levels when analyzed by HS-SPME techniques (Table 16.5). Changes appear to be due to changes in relative abundance rather than the evolution of new compounds.\n\nTable 16.5\n\nMean Relative Concentration (ppb) of Selected Volatile Components Extracted from the Headspace of Rehydrated (10% solids w\/w) Domestic and International MPCs with Various Protein Contents\n\nMPC Sample |  | Dimethyl sulfide | Propanal, 2-methyl- | Furan, 2-methyl- | Butanal, 3-methyl- | Butanoic acid, methyl ester | Hexanal | 2-Heptanone | Heptanal | Hexanoic acid, methyl ester | Pentanoic acid, 1-methyl ethyl ester | Benzaldehyde | Furan, 2-pentyl- | Octanal | 2-Nonanone | Nonanal | Octanoic acid, methyl ester | Decanal  \n---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---  \n| Protein (%)  \n1 | 56 | 0.66  \n(0.18) | 2.33  \n(0.06) | 0.22  \n(0.03) | 0.07  \n(0.10) | 0.22  \n(0.03) | 2.40  \n(0.14) | 0.33  \n(0.07) | 0.44  \n(0.13) | 0.15  \n(0.01) | 0.50  \n(0.07) | 0.32  \n(0.05) | 0.56  \n(0.02) | 0.17  \n(0.02) | 0.18  \n(0.02) | 1.12  \n(0.22) | 0.56  \n(0.05) | 0.10  \n(0.02)  \n2 | 56 | 0.45  \n(0.04) | 2.73  \n(0.42) | 0.21  \n(0.02) | 0.35  \n(0.03) | 0.43  \n(0.01) | 7.44  \n(0.13) | 0.70  \n(0.01) | 1.72  \n(0.11) | 1.11  \n(0.18) | 0.46  \n(0.01) | 1.49  \n(0.10) | 1.86  \n(0.01) | 0.36  \n(0.00) | 0.33  \n(0.01) | 1.43  \n(0.04) | 0.76  \n(0.01) | 0.13  \n(0.02)  \n3 | 56 | 0.53  \n(0.00) | 2.70  \n(0.69) | 0.15  \n(0.00) | 0.20  \n(0.02) | 0.26  \n(0.01) | 3.12  \n(0.06) | 0.74  \n(0.03) | 0.49  \n(0.02) | 0.06  \n(0.01) | 0.49  \n(0.02) | 0.61  \n(0.24) | 0.71  \n(0.00) | 0.18  \n(0.01) | 0.30  \n(0.02) | 1.00  \n(0.04) | 0.08  \n(0.00) | 0.19  \n(0.03)  \n4 | 56 | 0.26  \n(0.02) | 3.72  \n(0.09) | 0.11  \n(0.00) | 0.11  \n(0.01) | 0.18  \n(0.02) | 3.96  \n(0.08) | 0.59  \n(0.00) | 0.59  \n(0.11) | 0.07  \n(0.02) | 0.53  \n(0.01) | 0.77  \n(0.05) | 0.80  \n(0.01) | 0.26  \n(0.01) | 0.24  \n(0.02) | 1.07  \n(0.03) | 0.09  \n(0.01) | 0.18  \n(0.02)  \n5 | 70 | 0.23  \n(0.01) | 2.56  \n(0.44) | 0.11  \n(0.01) | 0.00  \n(0.00) | 0.24  \n(0.02) | 2.25  \n(0.04) | 0.29  \n(0.04) | 0.40  \n(0.04) | 0.04  \n(0.01) | 0.45  \n(0.02) | 0.95  \n(0.01) | 0.43  \n(0.01) | 0.15  \n(0.00) | 0.17  \n(0.00) | 1.29  \n(0.02) | 0.17  \n(0.01) | 0.10  \n(0.01)  \n6 | 70 | 0.39  \n(0.05) | 2.50  \n(0.16) | 0.08  \n(0.01) | 0.00  \n(0.00) | 0.31  \n(0.05) | 0.27  \n(0.04) | 0.36  \n(0.01) | 0.05  \n(0.01) | 0.12  \n(0.03) | 0.52  \n(0.01) | 0.26  \n(0.01) | 0.41  \n(0.03) | 0.08  \n(0.00) | 0.30  \n(0.01) | 0.18  \n(0.02) | 0.38  \n(0.01) | 0.16  \n(0.01)  \n7 | 70 | 0.18  \n(0.02) | 2.60  \n(0.44) | 0.09  \n(0.01) | 0.14  \n(0.06) | 0.29  \n(0.07) | 3.27  \n(0.42) | 0.39  \n(0.07) | 0.44  \n(0.04) | 0.30  \n(0.06) | 0.53  \n(0.07) | 1.03  \n(0.12) | 0.94  \n(0.06) | 0.20  \n(0.02) | 0.22  \n(0.02) | 1.37  \n(0.20) | 1.04  \n(0.03) | 0.15  \n(0.01)  \n8 | 70 | 0.34  \n(0.07) | 4.13  \n(0.45) | 0.06  \n(0.02) | 0.07  \n(0.01) | 0.30  \n(0.13) | 2.34  \n(0.31) | 0.20  \n(0.01) | 0.58  \n(0.19) | 0.15  \n(0.01) | 0.52  \n(0.00) | 0.23  \n(0.00) | 0.20  \n(0.03) | 0.16  \n(0.06) | 0.16  \n(0.02) | 1.28  \n(0.21) | 0.20  \n(0.01) | 0.18  \n(0.02)  \n9 | 70 | 0.00  \n(0.00) | 2.69  \n(0.04) | 0.29  \n(0.01) | 0.14  \n(0.01) | 0.28  \n(0.08) | 0.94  \n(0.03) | 0.15  \n(0.00) | 0.94  \n(0.05) | 0.03  \n(0.00) | 0.58  \n(0.02) | 1.61  \n(0.13) | 0.09  \n(0.03) | 0.18  \n(0.05) | 0.10  \n(0.03) | 0.80  \n(0.01) | 0.01  \n(0.01) | 0.07  \n(0.01)  \n10 | 70 | 0.11  \n(0.02) | 3.08  \n(0.40) | 0.04  \n(0.01) | 0.08  \n(0.01) | 0.84  \n(0.24) | 0.82  \n(0.21) | 0.39  \n(0.04) | 0.21  \n(0.02) | 1.21  \n(0.04) | 0.52  \n(0.01) | 0.19  \n(0.02) | 0.12  \n(0.04) | 0.06  \n(0.00) | 0.16  \n(0.01) | 0.17  \n(0.01) | 1.41  \n(0.06) | 0.06  \n(0.00)  \n11 | 80 | 0.01  \n(0.02) | 1.62  \n(1.00) | 0.01  \n(0.01) | 0.17  \n(0.09) | 0.15  \n(0.14) | 2.97  \n(0.29) | 0.72  \n(0.04) | 0.46  \n(0.08) | 0.04  \n(0.00) | 0.44  \n(0.05) | 2.08  \n(0.20) | 1.75  \n(0.08) | 0.17  \n(0.03) | 0.75  \n(0.04) | 0.92  \n(0.08) | 0.10  \n(0.00) | 0.24  \n(0.03)  \n12 | 80 | 0.00  \n(0.00) | 4.20  \n(0.30) | 0.02  \n(0.01) | 0.03  \n(0.05) | 0.19  \n(0.00) | 1.92  \n(0.19) | 0.30  \n(0.02) | 0.26  \n(0.13) | 0.03  \n(0.00) | 0.45  \n(0.04) | 1.83  \n(0.13) | 0.34  \n(0.04) | 0.11  \n(0.05) | 0.12  \n(0.01) | 0.33  \n(0.05) | 0.00  \n(0.00) | 0.13  \n(0.02)  \n13 | 80 | 0.00  \n(0.00) | 0.00  \n(0.00) | 0.03  \n(0.04) | 0.04  \n(0.06) | 0.14  \n(0.20) | 0.49  \n(0.69) | 0.82  \n(0.74) | 0.09  \n(0.13) | 0.19  \n(0.27) | 0.26  \n(0.37) | 0.00  \n(0.00) | 0.00  \n(0.00) | 0.00  \n(0.00) | 0.00  \n(0.00) | 0.00  \n(0.00) | 1.69  \n(1.61) | 2.39  \n(3.28)  \n14 | 80 | 0.06  \n(0.00) | 2.35  \n(0.04) | 0.02  \n(0.02) | 0.00  \n(0.00) | 0.20  \n(0.00) | 0.47  \n(0.00) | 0.21  \n(0.04) | 0.32  \n(0.05) | 0.03  \n(0.00) | 0.49  \n(0.06) | 0.46  \n(0.04) | 0.03  \n(0.01) | 0.04  \n(0.02) | 0.09  \n(0.00) | 0.32  \n(0.05) | 0.08  \n(0.02) | 0.08  \n(0.00)  \n56 | 0.48 | 2.87 | 0.17 | 0.18 | 0.27 | 4.23 | 0.59 | 0.81 | 0.35 | 0.50 | 0.80 | 0.98 | 0.24 | 0.26 | 1.16 | 0.37 | 0.15  \n70 | 0.21 | 2.93 | 0.11 | 0.07 | 0.38 | 1.65 | 0.30 | 0.44 | 0.31 | 0.52 | 0.71 | 0.37 | 0.14 | 0.19 | 0.85 | 0.54 | 0.12  \n80 | 0.02 | 2.04 | 0.02 | 0.06 | 0.17 | 1.46 | 0.51 | 0.28 | 0.07 | 0.41 | 1.09 | 0.53 | 0.08 | 0.24 | 0.39 | 0.47 | 0.71\n\nNote: All concentrations given as mean relative concentration and (standard deviation)\n\nNote: MPC volatile compound mean relative concentration (ppb); all concentrations given as mean relative concentration\n\n## Caseins and hydrolysates\n\nCaseins represent the primary protein constituent of milk; whey or serum proteins are the other fraction. Just as whey proteins comprise a large group of functional ingredients, so do caseins and caseinates. Caseins are traditionally produced by acid or rennet precipitation of casein, followed by spray or roller drying. Caseinate or soluble casein is produced when casein curd (usually acid precipitated) is treated with alkali at pH 6\u20137 and fully dissolved prior to spray drying (O'Connell & Flynn, 2007). Potassium, sodium, and calcium are commonly used counter-ions. Caseins display a unique set of functional properties, including solubility and heat stability, and are thus used for a host of ingredient applications. Caseins have relied on functionality for their success because a host of relatively intense and unpleasant flavors, including sulfur, animal, tortilla, musty, cardboard, burnt feathers, glue, and bitter taste, have been associated with them (Ramshaw & Dunstone, 1969; Walker & Manning, 1976; Drake et al., 2003; Karagul-Yuceer et al., 2003b) (Fig. 16.10). Micellar casein can be manufactured by membrane fractionation and spray drying of fluid milk and may represent a blander option. The net result is a more mildly flavored product that still displays some of the previously reported flavors.\n\nFigure 16.10 Trained panel flavor profiles of rehydrated caseins [10% solids (w\/w)]. Caseins 1 and 2 are rennet caseins, caseins 3 and 5 are acid caseins, and casein 4 is a sodium caseinate.\n\nDairy protein hydrolysates are another promising category of protein-derived ingredients with valuable functional and nutritional properties (Nnanna & Wu, 2007). Hydrolysis improves digestibility, and hydrolysates are used widely in infant formulas. Recent research has demonstrated that peptides with specific bioactive properties can also be generated, and certainly an array of functional properties such as solubility and heat stability can be altered via hydrolysis. Whey protein hydrolysates are commonly added to meal replacement bars to inhibit bar hardening with storage time (Childs et al., 2007). These products can be manufactured from casein, whey protein, or milk protein by enzymatic hydrolysis and are classified based on their degree of protein hydrolysis (molecular weight). Flavor is a significant challenge to increased usage of these products, particularly in beverages. Brothy and free fatty acid flavors and bitter tastes are distinct (Fig. 16.11), and intensities can vary with the degree of hydrolysis, the specific processing steps, the enzyme used, and the protein source (Leksrisompong et al., 2010). Because of their intense aromas and flavors, these products should be rehydrated to a lower solids concentration prior to sensory analysis (e.g., 5% w\/w compared with 10% w\/w for all other dairy proteins).\n\nFigure 16.11 Trained panel flavor profiles of commercial rehydrated whey protein hydrolysates (5% solids w\/w). pH 1 has a higher degree of hydrolysis than pH 2\u2013pH 6. pH 2\u2013pH 6 represent different enzymatic digestions.\n\n## Flavor binding\n\nAlthough somewhat beyond the scope of this chapter, it is important to note that, in addition to displaying and contributing flavors, dairy proteins can interact and bind with desirable flavors in foods and influence flavor in this manner as well. An excellent review on this subject has been published (Kuhn et al., 2006). Most of the research in this arena has been conducted with instrumental analysis (e.g., headspace analysis and calculation of binding constants), and very little research to relate these results directly back to sensory perception has been attempted. Future research should address this issue.\n\n## Conclusions\n\nApplications for dairy proteins continue to increase, and flavor will remain a crucial aspect. An abundance of research on the functional properties of dairy proteins exists, but there is still a relative dearth of information on the flavor of dairy proteins. Flavor sources, flavor formation during processing, and flavor carry-through and stability in ingredient applications are key areas for future research.\n\nThe flavor of dairy proteins and their flavor performance in ingredient applications will ultimately influence their widespread usage and competitiveness with other protein sources. Published research has only recently begun to reflect and emphasize the importance of this issue. The positive flavor image of dairy foods, combined with the numerous functional and nutritional benefits of dairy proteins, provides a powerful marketing juggernaut for these products, but specific flavor properties and the flavor variability of these proteins should not be overlooked in ongoing research.\n\n## Acknowledgment\n\nThe authors gratefully acknowledge Dairy Management, Inc. and the California Dairy Research Foundation for providing financial support.\n\n# References\n\nAvsar YK ,  Karagul-Yuceer Y ,  Drake MA ,  Singh T ,  Yoon Y ,  Cadwallader KR .  Characterization of nutty flavor in Cheddar cheese .  _Journal of Dairy Science_.  2004 ;87 : 1999 \u2013 2010 .\n\nCampbell RE ,  Miracle RE ,  Drake MA .  The effect of starter culture and annatto on the flavor and functionality of whey protein concentrate .  _Journal of Dairy Science_.  2011 ;94 : 1185 \u2013 1193 .\n\nCampbell RE ,  Miracle RE ,  Gerard PD ,  Drake MA .  Effects of starter culture and storage on the flavor of liquid whey .  _Journal of Food Science_.  2011 ;76 : S354 \u2013 361 .\n\nCampbell RE ,  Adams S ,  Drake MA ,  Barbano DM .  Effect of bleaching permeate from microfiltered skim milk on 80% serum protein concentrate .  _Journal of Dairy Science_.  2013 ;96 : 1387 \u2013 1400 .\n\nCarunchia Whetstine ME ,  Parker JD ,  Drake MA ,  Larick DK .  Determining flavor and flavor variability in commercially produced liquid Cheddar whey .  _Journal of Dairy Science_.  2003 ;86 : 439 \u2013 448 .\n\nCarunchia Whetstine ME ,  Cadwallader KR ,  Drake MA .  Characterization of aroma compounds responsible for the rosy\/floral flavor in Cheddar cheese .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 3126 \u2013 3132 .\n\nCarunchia Whetstine ME ,  Croissant AE ,  Drake MA .  Characterization of WPC80 and WPI flavor .  _Journal of Dairy Science_.  2005 ;88 : 3826 \u2013 3829 .\n\nCarunchia Whetstine ME ,  Drake MA ,  Broadbent JR ,  McMahon DJ .  Enhanced nutty flavor formation in Cheddar cheese made with a \"malty\" Lactococcus lactis adjunct culture .  _Journal of Dairy Science_.  2006 ;89 : 3277 \u2013 3284 .\n\nCaudle AD ,  Yoon Y ,  Drake MA .  Influence of flavor variability in skim milk powder on consumer acceptability of ingredient applications .  _Journal of Food Science_.  2005 ;70 : S427 \u2013 S431 .\n\nChilds JL ,  Yates MD ,  Drake MA .  Sensory properties of meal replacement bars and beverages made from whey or soy proteins .  _Journal of Food Science_.  2007 ;72 : S425 \u2013 S434 .\n\nCroissant AE ,  Kang EJ ,  Campbell RE ,  Bastian E ,  Drake MA .  Impact of bleaching agent on the flavor and flavor chemistry of whey proteins .  _Journal of Dairy Science_.  2009 ;92 : 5917 \u2013 5927 .\n\nDrake MA .  Defining dairy flavors .  _Journal of Dairy Science_.  2004 ;87 : 777 \u2013 784 .\n\nDrake MA .  Flavor and flavor carry-through of whey proteins in beverages .  _The Wonders of Whey... Catch the Power. Proceedings of the 4th International Whey Conference_ .  Elmhurst, IL :  American Dairy Products Institute ;  2006 : 292 \u2013 300 .\n\nDrake MA .  Sensory analysis of dairy foods .  _Journal of Dairy Science_.  2007 ;90 : 4925 \u2013 4937 .\n\nDrake MA ,  Civille GV .  Flavor lexicons .  _Comprehensive Reviews in Food Science and Food Safety_.  2003 ;2 : 33 \u2013 40 .\n\nDrake MA ,  Gerard PD .  Consumer attitudes and acceptability of soy-fortified yogurts .  _Journal of Food Science_.  2003 ;68 : 1118 \u2013 1122 .\n\nDrake MA ,  Karagul-Yuceer Y ,  Cadwallader KR ,  Civille GV ,  Tong PS .  Determination of the sensory attributes of dried milk powders and dairy ingredients .  _Journal of Sensory Studies_.  2003 ;18 : 199 \u2013 216 .\n\nDrake MA ,  Miracle RE ,  Caudle AD ,  Cadwallader KR .  Relating sensory and instrumental analyses .  In:  Marsili R ,  ed.  _Sensory-directed Flavor Analysis_ .  Boca Raton, FL :  CRC Press, Taylor and Francis ;  2006 : 23 \u2013 55 .\n\nDrake SL ,  Carunchia Whetstine ME ,  Drake MA ,  Courtney P ,  Fligner K ,  Jenkins J ,  Pruitt C .  Sources of umami taste in Cheddar and Swiss cheeses .  _Journal of Food Science_.  2007 ;72 : S360 \u2013 S366 .\n\nEvans J ,  Zulewska J ,  Newbold M ,  Drake MA ,  Barbano DM .  Comparison of composition, sensory and volatile components of 34% whey protein and milk serum protein concentrates .  _Journal of Dairy Science_.  2009 ;92 : 4773 \u2013 4791 .\n\nEvans JP ,  Zulewska J ,  Newbold M ,  Drake MA ,  Barbano DM .  Comparison of composition and sensory properties of 80% whey protein and milk serum protein concentrates .  _Journal of Dairy Science_.  2010 ;93 : 1824 \u2013 1843 .\n\nFoegeding EA ,  Davis JP ,  Doucet D ,  McGuffey M .  Advances in modifying and understanding whey protein functionally .  _Trends in Food Science and Technology_.  2002 ;13 : 151 \u2013 159 .\n\nGallardo Escamilla FJ ,  Kelly AL ,  Delahunty CM .  Sensory characteristics and related volatile flavor compound profiles of different types of whey .  _Journal of Dairy Science_.  2005 ;88 : 2689 \u2013 2699 .\n\nJavidipour I ,  Qian M .  Volatile component change in whey protein concentrate during storage investigated by headspace solid-phase microextraction gas chromatography .  _Dairy Science and Technology_.  2008 ;88 : 95 \u2013 104 .\n\nJervis SM ,  Campbell RE ,  Wojciechowski K ,  Drake MA ,  Barbano DM .  Impact of bleaching whey on sensory and functional properties of 80% whey protein concentrate .  _Journal of Dairy Science_.  2012 ;95 : 2828 \u2013 2862 .\n\nJones VS ,  Drake MA ,  Harding R ,  Kuhn-Sherlock B .  Consumer perception of soy and dairy products: A cross-cultural study .  _Journal of Sensory Studies_.  2008 ;23 : 65 \u2013 79 .\n\nKaragul-Yuceer Y ,  Drake MA ,  Cadwallader KR .  Aroma active components of liquid Cheddar whey .  _Journal of Food Science_.  2003 ;68 : 1215 \u2013 1219 .\n\nKaragul-Yuceer Y ,  Vlahovich KN ,  Drake MA ,  Cadwallader KR .  Characteristic aroma components of rennet casein .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 6797 \u2013 6801 .\n\nKuhn J ,  Considine T ,  Singh H .  Interactions of milk proteins and volatile flavor compounds: Implications in the development of protein foods .  _Journal of Food Science_.  2006 ;71 : R72 \u2013 R82 .\n\nLawless HT ,  Heymann H .  _Sensory Evaluation of Food: Principles and Practices_ .  1st ed. New York :  Chapman and Hall ;  1999 .\n\nLeksrisompong PP ,  Miracle RE ,  Drake MA .  Characterization of flavor of whey protein hydrolysates .  _Journal of Agricultural Food Chemistry_.  2010 ;58 : 6318 \u2013 6327 .\n\nLeksrisompong PP ,  Gerard PD ,  Lopetcharat K ,  Drake MA .  Bitter taste inhibiting agents for whey protein hydrolysate and whey protein hydrolysate beverages .  _Journal of Food Science_.  2012 ;77 : S282 \u2013 S287 .\n\nLiaw I ,  Miracle RE ,  Jervis SM ,  Listiyani M ,  Drake MA .  Comparison of the flavor chemistry and flavor stability of Mozzarella and Cheddar whey .  _Journal of Food Science_.  2011 ;76 : C1188 \u2013 C1194 .\n\nMahajan SS ,  Goddik L ,  Qian MC .  Aroma compounds in sweet whey powder .  _Journal of Dairy Science_.  2004 ;87 : 4057 \u2013 4063 .\n\nMeilgaard MM ,  Civille GV ,  Carr T .  _Sensory Evaluation Techniques_ .  4th ed. New York :  CRC Press ;  2007 .\n\nMiller G .  Healthy growth ahead for wellness drinks .  _Food Technology_.  2005 ;59 : 21 \u2013 26 .\n\nMills OE .  Flavour of whey protein concentrate .  _Food Flavours, Ingredients, and Composition. Proceedings of the 7th International Flavour Conference_ .  New York :  Elsevier ;  1993 : 139 \u2013 149 .\n\nMorr CV ,  Ha EYW .  Off-flavors in whey protein concentrates: A literature review .  _International Dairy Journal_.  1991 ;1 : 1 \u2013 11 .\n\nNnanna IA ,  Wu C .  Dairy protein hydrolysates .  In:  Hui YH ,  ed.  _Handbook of Food Products Manufacturing_ .  Hoboken, NJ :  John Wiley and Sons ;  2007 : 537 \u2013 556 .\n\nO'Connell JE ,  Flynn C .  The manufacture and applications of casein-derived ingredients .  In:  Hui YH ,  ed.  _Handbook of Food Products Manufacturing_ .  Hoboken, NJ :  John Wiley and Sons ;  2007 : 557 \u2013 591 .\n\nQuach ML ,  Chen XG ,  Stevenson RL .  Headspace samplings of whey protein concentrate solutions using solid phase microextraction .  _Food Research International_.  1999 ;31 : 371 \u2013 379 .\n\nRamshaw EH ,  Dunstone EA .  Volatile compounds associated with the off-flavor in stored casein .  _Journal of Dairy Research_.  1969 ;36 : 215 \u2013 223 .\n\nRussell TA ,  Drake MA ,  Gerard PD .  Sensory properties of whey and soy proteins .  _Journal of Food Science_.  2006 ;71 : S447 \u2013 S455 .\n\nSingh T ,  Drake MA ,  Cadwallader KR .  Flavor of Cheddar cheese: A chemical and sensory perspective .  _Comprehensive Reviews in Food Science and Food Safety_.  2003 ;2 : 139 \u2013 162 .\n\nSingh TK ,  Young ND ,  Drake MA ,  Cadwallader KR .  Production and sensory characterization of a bitter peptide from \u03b2 casein .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 1185 \u2013 1189 .\n\nTomaino RM ,  Turner LG ,  Larick DL .  The effect of Lactococcus lactic starter cultures on the oxidative stability of liquid whey .  _Journal of Dairy Science_.  2004 ;87 : 300 \u2013 307 .\n\nvan Ruth S .  Methods for gas chromatography-olfactometry: A review .  _Biomolecular Engineering_.  2001 ;17 : 121 \u2013 128 .\n\nWalker NJ ,  Manning DJ .  Components of the musty off-flavor of stored dried lactic casein .  _New Zealand Journal of Dairy Science and Technology_.  1976 ;11 : 1 \u2013 9 .\n\nWhitson ME ,  Miracle RE ,  Bastian E ,  Drake MA .  Effect of liquid retentate storage on flavor of spray dried whey protein concentrate and isolate .  _Journal of Dairy Science_.  2011 ;94 : 3747 \u2013 3760 .\n\nWright JM ,  Carunchia Whetstine ME ,  Miracle RE ,  Drake MA .  Characterization of a cabbage off-flavor in whey protein isolate .  _Journal of Food Science_.  2006 ;71 : C91 \u2013 C96 .\n\nWright BJ ,  Zevchak SE ,  Wright JM ,  Drake MA .  Impact of agglomeration on flavor and flavor stability of whey proteins .  _Journal of Food Science_.  2009 ;74 : S17 \u2013 S29 . \nChapter 17\n\n# Milk Protein Gels\n\nJohn A. Lucey Department of Food Science, University of Wisconsin-Madison, USA\n\n## Abstract\n\nThe formation and properties of the main types of milk protein gels are described, that is, casein gels made with rennet or acid, heat-induced whey protein gels, and gels made by a combination of approaches. The impact of various factors on these gelation properties is discussed. Recent key advances are highlighted, including the use of high pressure, exopolysaccharides, and transglutaminase cross-linking of proteins, and new insights are given into the ubiquitous use of thermal processing to alter the texture of these gels.\n\n## Keywords\n\nmilk protein gels\n\ncasein gels\n\nheat-induced whey protein gels\n\nrennet-induced gels\n\nacid-induced milk gels\n\nmixed gels, casein micelles\n\nOutline\n\nIntroduction 494\n\nRennet-induced gels 494\n\nPrimary Phase of Rennet Coagulation 494\n\nSecondary Phase of Rennet Coagulation 495\n\nMonitoring Gelation 497\n\nRheological Properties of Rennet-induced Milk Gels 497\n\nSyneresis of Rennet-induced Milk Gels 498\n\nFactors Influencing the Texture of Rennet-induced Gels 500\n\nMilk Heat Treatment 501\n\nEnzymatic Cross-linking of Caseins 501\n\nHigh Hydrostatic Pressure 502\n\nStarch Addition 502\n\nAcid-induced milk gels 502\n\nImpact of Acid on Casein Micelles 502\n\nSome Factors Influencing the Texture of Yogurt Gels 503\n\nHomogenization and Fat Globule Surface Material 504\n\nHigh Hydrostatic Pressure 504\n\nEnzymatic Modification of Proteins 504\n\nHeat Treatment 505\n\nIncubation Temperature 508\n\nProduction of Exopolysaccharides 508\n\nWhey protein gels 509\n\nThermal Denaturation of Whey Proteins 510\n\nTypes and Properties of Whey Protein Gels 512\n\nOther Factors Influencing Properties of Whey Protein Gels 513\n\nCold Gelation of Whey Proteins 514\n\nEnzymatic Modification of Whey Protein for Gelation Purposes 515\n\nMixed Gels Made with Rennet and Acid 515\n\nConclusions 516\n\nAcknowledgment 516\n\n## Introduction\n\nGelation of the proteins in milk is the basis for the manufacture of cheese and fermented milk products. Various approaches can be used to destabilize the milk proteins, including heat (whey proteins), use of rennet enzyme (caseins), and acidification (caseins and denatured whey proteins). Combinations of these approaches can also be used to form dairy products, for example, the use of a low concentration of rennet in cottage cheese (or quarg), which is primarily a cultured product. Yogurt is a cultured product in which caseins and denatured whey proteins are responsible for the gelation properties. Milk protein gels are irreversible, in contrast to many polysaccharide gels that are thermoreversible. Milk gels are often classified as particle gels, although it is now recognized that they are not simple particle gels, as the internal structure of the casein particle plays an important role in their rheological properties (Horne 2001; ). The properties of milk protein gels have been reviewed (Green, 1980; de Kruif et al., 1995; Lucey 2002; van Vliet et al., 2004). The casein particles in rennet gels undergo rearrangement, fusion, and syneresis in the process of forming cheese curd. Thus they are inherently dynamic in nature, and the rearrangement processes involved have been studied (e.g., see the review by Dejmek & Walstra, 2004).\n\n## Rennet-induced gels\n\nCoagulation of milk by rennet probably occurred initially by accident, as warm milk was stored in sacks made from the stomachs of ruminant animals that contained some residual proteinase enzymes. Crude extracts, prepared from the fourth stomach of young calves (called rennets, which are a type of aspartic proteinase), have been used for cheesemaking for thousands of years. Pepsin is the predominant proteinase in adult mammals. Naturally produced calf chymosin (EC 3.4.23.4) may contain up to six molecular species, which have slight differences in their amino acid residues (Crabbe, 2004). Chymosin has been cloned into several genetically modified organisms to produce fermentation-derived chymosin, which is widely used in many countries around the world (Crabbe, 2004). Recently, Kappeler et al. (2006) expressed the gene for camel (Camelus dromedarius) chymosin in Aspergillus niger and produced camel chymosin by fermentation. The rennet coagulation of milk has been reviewed (Dalgleish 1987; ; Hyslop, 2003; Horne & Banks, 2004).\n\n### Primary Phase of Rennet Coagulation\n\nThe basic building blocks of rennet-induced gels are the casein micelles. Both \u03b1s\\- (\u03b1s1\\- and \u03b1s2-) and \u03b2-caseins are sensitive to precipitation by the Ca2+ in milk and are protected by association with \u03ba-casein, which is one reason for the formation of micelles. \u03ba-Casein molecules are thought to have a predominantly surface position on micelles (although some \u03ba-casein is also present in the interior of the micelle), where the hydrophilic C-terminal apparently acts as a 'hairy' layer providing steric stabilization and a barrier against association with other micelles (Walstra, 1990).\n\nThe two stages of the rennet coagulation of milk are shown in Figure 17.1. In the primary phase of rennet coagulation, the C-terminal part (residues 106\u2013169) of the \u03ba-casein molecule is hydrolyzed, and this hydrophilic peptide diffuses away from the micelle (called para-casein) into the serum phase. This macropeptide is called caseinomacropeptide (CMP) or, if it is highly glycosylated, glycomacropeptide (GMP). Most microbial coagulants, including those derived from Rhizomucor miehei, hydrolyze the same Phe105\u2013Met106 bond as chymosin; however, Cryphonectria parasitica hydrolyzes the Ser104\u2013Phe105 bond (Dr\u00f8hse & Foltmann, 1989). The proteolysis of other proteins in milk by chymosin occurs at a much slower rate (Crabbe, 2004).\n\nFigure 17.1 The two stages of the rennet coagulation of milk.\n\nThe enzymatic reaction in milk appears to obey first-order kinetics. The proteolysis of \u03ba-casein is usually described by standard Michaelis\u2013Menten kinetics, although Hyslop (2003) questioned whether this was truly appropriate. It should be noted that the primary phase and the secondary phase of clotting overlap as the aggregation begins before the enzymatic reaction is complete.\n\n### Secondary Phase of Rennet Coagulation\n\nThe stability of the casein micelles of milk is attributed to their net negative charge and to steric repulsion by the flexible macropeptide region of \u03ba-casein (the so-called hairs that extend out into the solution), calcium-induced interactions between protein molecules, hydrogen bonding, and electrostatic and hydrophobic interactions. The release of the CMP (or GMP), which diffuses away from the micelles, leads to a decrease in the zeta potential, by \u22485\u20137 mV (\u224850%), which reduces electrostatic repulsion between rennet-altered micelles. Removal of the 'hairs' results in a decrease in the hydrodynamic diameter by \u22485 nm and a loss of steric stabilization, and causes a slight minimum in the viscosity during the initial lag phase of renneting.\n\nVarious attempts have been made to model the aggregation reaction (see the review by Horne & Banks, 2004). The nature of the attractive forces during the aggregation of casein micelles is still not completely clear, although calcium bridges, van der Waals forces, and hydrophobic interactions may be involved. Destabilized micelles will aggregate only in the presence of free Ca2+. Rennet acts on casein at temperatures as low as 0 \u00b0C, but milk does not clot at temperatures below 15 \u00b0C, whereas aggregation is very rapid at high temperature (e.g., 55 \u00b0C).\n\nWhen milk is clotted under normal conditions of pH and protein content, the viscosity does not increase until the enzymatic phase is mostly complete, that is, at >60% of the (visual) rennet coagulation time. Coagulation does not occur until the enzymatic phase is at least \u224887% complete. Sandra et al. (2007) studied the rennet gelation process using diffusing wave spectroscopy, which allowed gelation to be monitored without the need for dilution. Sandra et al. (2007) suggested that partially renneted casein micelles do not begin to approach one another until the extent of breakdown of the \u03ba-casein hairs has reached about 70%; above this point, they interact increasingly strongly with an increase in the extent of proteolysis. This interaction initially restricts the diffusive motion of the particles rather than causing true aggregation. Only after more extensive removal of the protective \u03ba-casein hairs does true aggregation occur, with the appearance of a space-filling gel (as defined by rheology terms, such as having a loss tangent value <1). A micrograph of a rennet-induced skim milk gel is shown in Figure 17.2.\n\nFigure 17.2 A confocal laser scanning micrograph of a rennet gel made from skim milk. Protein is white; dark areas are water. Scale bar = 10 \u03bcm.\n\nSrinivasan and Lucey (2002) studied the impact of plasmin enzyme on the rennet coagulation of skim milk. They found that hydrolysis of \u03b1s\\- and \u03b2-caseins (as plasmin hardly degrades \u03ba-casein) accelerated the rennet coagulation time. Srinivasan and Lucey (2002) hypothesized that plasmin could have degraded non-\u03ba-casein 'hairs' present on the surface of micelles and that this could have reduced the repulsive barrier to aggregation of rennet-altered micelles such that aggregation could take place at a lower degree of \u03ba-casein hydrolysis. At low temperatures \u03b2-caseins protrude from the micelle surface due to a weakening of intramicellar hydrophobic interactions, and these proteins provide a substantial barrier to the aggregation of renneted micelles. This phenomenon could be involved in the slow rennet coagulation of milk at low temperatures (Walstra, 1990). There is also a reduction in the amount of calcium ions bound to caseins with a decrease in temperature (Walstra, 1990).\n\nCompletely hydrolyzed micelles initially form small linear chains, and these continue to aggregate to form clumps, clusters and eventually a system-spanning network that has a fractal-like appearance.\n\nLittle aggregation occurs at low temperatures (e.g., <15 \u00b0C), which is usually taken as an indication of the importance of hydrophobic interactions. It is more likely that, with decreasing temperature, the activation free energy for flocculation increases, presumably because of the presence of \u03b2-casein chains on the outside of the micelle (Walstra, 1993). There is an increase in the strength of rennet gels at lower temperatures (where hydrophobic interactions are weak), reflecting swelling of casein particles, which results in an increase in the contact area between aggregated particles and strands.\n\n### Monitoring Gelation\n\nThere have been several recent reviews of techniques to monitor milk gelation (Lucey, 2002; O'Callaghan et al., 2002; Klandar et al. 2007). The interest in monitoring gelation comes from the cheesemaker's desire to know the 'optimum' time to initiate cutting, as well as from the researcher's desire to better understand this complex process.\n\nTwo promising techniques for the study of milk gels are diffusing wave spectroscopy and ultrasonic spectroscopy (Alexander & Dalgleish, 2004; Dalgleish et al., 2006). These techniques could be used to complement existing approaches. For example, Wang et al. (2007) used both ultrasonic and (traditional) rheological methods to investigate the effects of milk pretreatment at ultra-high temperatures on the rennet gelation of a whey-protein-free casein solution. Wang et al. (2007) found that the ultrasonic velocity was able to measure the enzymatic hydrolysis and aggregation process, but was not as sensitive in detecting gel formation. In contrast, the oscillatory rheological method was not able to detect the enzymatic hydrolysis reaction, but was very suitable for characterizing the formation of a gel network.\n\n### Rheological Properties of Rennet-induced Milk Gels\n\nRennet-induced gels are viscoelastic and their rheological properties can be characterized using dynamic low-amplitude oscillatory rheology, which determines both the viscous component and the elastic component. These measurements should be performed in the linear viscoelastic range, where the deformation (strain) is proportional to the applied stress. Often, for rennet and acid gels, that means trying to operate at \u22643% strain, which can be difficult during the early stage of gel formation for many (of the popular) controlled stress rheometers (because of the very low torque resulting on the measuring geometry of the rheometer from such a weak gel). Some new techniques\/software can be used to reduce this problem in commercial rheometers (e.g., Lauger et al., 2002).\n\nParameters that can be determined include the elastic or storage modulus (G'), which is a measure of the energy stored per oscillation cycle; the viscous or loss modulus (G''), which is a measure of the energy dissipated as heat per cycle; and the loss tangent (tan \u03b4), which is the ratio of the viscous properties to the elastic properties (loss tangent = G''\/G'). The loss tangent is related to the relaxation of bonds in the gel during deformation and is a useful parameter.\n\nDuring gelation, there is a lag period before a measurable storage modulus value is obtained (this depends on the sensitivity of the rheometer to measure events close to the gelation point). The loss tangent decreases from \u226b1 to <1 at the gelation point and then attains a relatively constant value (about 0.35 for rennet gels). The dynamic moduli initially increase relatively rapidly and then, after a period of several hours, tend to plateau. In commercial practice, rennet-induced gels are cut once they have attained a certain firmness (usually assessed subjectively by the cheesemaker) or, more commonly, at a fixed time after rennet addition. The increase in the moduli after gelation probably reflects ongoing fusion of micelles, which results in an increase in the contact area between aggregated particles, and possibly the incorporation of additional particles into the gel network. Some micelles that have incomplete hydrolysis of their \u03ba-casein hairs could be trapped within the space-filling network at the point of network formation and they might later become attached to the matrix once their \u03ba-casein hairs get completely hydrolyzed. Mellema et al. (2002) reported that their analysis suggested that nearly all casein was incorporated in the rennet gel, at least very soon after network formation. Mellema et al. (2002) also considered that changes in the storage modulus and microstructure during aging could be explained in terms of (various types of) rearrangements of the gel network at various length scales.\n\nTypical plateau values for the storage modulus of rennet-induced gels (made from unconcentrated milk) range from 100 to 200 Pa. Both moduli have lower values at low frequencies, reflecting relaxation of more bonds when the time scale of the applied stress is longer. The loss tangent at low frequencies is an important indicator of rearrangements as this is approximately the same timescale as that over which rearrangement processes related to syneresis in rennet gels are estimated to occur (van Vliet et al., 1991).\n\nThe development of the complex or shear modulus as a function of time after rennet addition can be re-plotted against a reduced time t\/t g, where t g is the gelation time. Various individual renneted milk curves can be normalized against its complex or shear modulus value at a low multiple (two or three) of the t g. These various curves then collapse into a single or master curve because of the scaling behavior of the dynamics of the gel formation process (Horne 1995; ).\n\nVarious mathematical, empirical, and kinetic models have been applied to predict the development of gel firmness or shear moduli; their effectiveness in performing this function has been reviewed by Horne and Banks (2004).\n\n### Syneresis of Rennet-induced Milk Gels\n\nThe syneresis of rennet-induced gels has been reviewed (Walstra et al., 1985; Pearse & MacKinlay, 1989; Walstra 1993 van Vliet & Walstra, 1994; Dejmek & Walstra, 2004). Rennet-induced gels remain stable for several hours if left undisturbed. They rapidly synerese if disturbed by cutting or by wetting the gel surface. A rennet-induced gel may lose up to two-thirds of its volume (as whey) under quiescent conditions and more than 90% if external pressure is applied (Dejmek & Walstra, 2004). Cheesemaking can be viewed as a dehydration process, and syneresis is the crucial method by which most of the moisture is lost from curd particles. As syneresis is the main method that cheesemakers have to control cheese moisture, it is also the process that is most often manipulated; it also helps to facilitate differentiation between cheese varieties. Most of the water in milk gels is not chemically bound to proteins but rather is physically entrapped in the network structure (van Vliet & Walstra, 1994).\n\nBecause of the complexity of the syneresis process, researchers have often used thin gel slabs to monitor one-dimensional shrinkage (e.g., van Dijk & Walstra, 1986). The one-dimensional syneresis of rennet gels is related to the flow of liquid (whey) through the network (because liquid flows out of the gel concomitantly with gel shrinkage) and is governed by the equation of Darcy:\n\nv = B \u03b7 p x\n\nwhere v is the superficial flow velocity of the syneresing liquid, B is the permeability coefficient, \u03b7 is the viscosity of the liquid, p is the pressure acting on the liquid, and x is the distance over which the liquid must flow.\n\nIt is believed (e.g., Walstra, 1993) an internal (endogenous) pressure or driving force within rennet gels is responsible for the shrinkage of the gel once the initial gel is disturbed (presumably this overcomes the yield stress of the system). It has not been possible to measure this small endogenous pressure experimentally. Endogenous syneresis pressure (i.e., the pressure within the rennet gel causing the syneresis) is not constant. It increases initially as a function of time after renneting but decreases at longer times, presumably because of the fusion of para-casein micelles and a reduction in permeability of the contracting network. In practice, syneresis in curd particles occurs in three dimensions simultaneously and is much harder to study than the one-dimensional model.\n\nIn rennet-induced milk gels, the mechanism responsible for the gels' strong tendency to exhibit syneresis is related to the (extensive) rearrangements of the casein network that occur after gel formation. As acid-induced gels undergo much less rearrangement, they synerese less. The rearrangement process is accelerated, and is more extensive, at high temperatures and lower pH values (\u22655.1) (the loss tangent is also higher under these conditions). Aging of rennet-induced gels results in a coarsening (sometimes called 'microsyneresis') of the gel (i.e., as a result of more rearrangements), and an increase in the permeability and the fractal dimensionality takes place.\n\nRearrangements of casein particles into a more compact structure would increase the number of bonds and hence would decrease the total free energy of the system (Walstra, 1993). However, the casein particles are already part of the gel network, which must be deformed or broken locally to form new junctions. Breakage of the bonds in the strands requires a sufficiently low-yield stress if it is to be exceeded. In cheesemaking, conditions such as cutting, stirring, acid production, and the increase in the cooking temperature all encourage syneresis and the rearrangement processes that facilitate syneresis of the gel network. If the strands become too thick (e.g., because of a very high casein concentration), syneresis hardly occurs.\n\nOne-dimensional syneresis of rennet-induced skim milk gels was studied in gels with different thicknesses and at pH values of 6.4 and 6.0 using a laser displacement sensor (Lodaite et al., 2000). Syneresis was much faster at the lower pH, and the initial syneresis rate increased linearly with slab thickness.\n\nSeveral (mostly empirical) techniques have been used to estimate the syneresis of rennet gels, including shrinkage of gel slabs, determining the volume of whey expelled as a function of time, the dry matter content or density of curd particles, and low-resolution nuclear magnetic resonance (NMR) (Dejmek & Walstra, 2004). A recent development has been a light backscatter sensor, with a large field of view relative to curd size, for continuous online monitoring of coagulation and syneresis to help cheesemakers improve their control over the moisture content of the curd (Fagan et al., 2006).\n\n### Factors Influencing the Texture of Rennet-induced Gels\n\nMany factors influence the milk-clotting process and the consistency of rennet gels, including pH, temperature, casein content, ionic strength, enzyme concentration, calcium content, presence of homogenized fat globules, concentration of denatured whey proteins, and casein hydrolysis by proteinases such as plasmin. These factors have been reviewed many times (Dalgleish, 1987; 1993 Green & Grandison, 1993; Lomholt & Qvist, 1999; Hyslop, 2003; Horne & Banks, 2004) because of the importance of rennet gels for the cheese industry.\n\nThe effects of pH (5.19\u20136.21) and NaCl concentration (0, 1.75, and 3.5%) on the rheological and microstructural properties of rennet-induced casein gels made from ultrafiltered skim milk (19.8%, w\/w casein) were recently investigated (Karlsson et al., 2007a). Low pH and high NaCl concentration reduced the rate of development of the gel elasticity after coagulation. Strain at fracture and stress at fracture 48 h after coagulation showed maximum and minimum values at pH 5.8 and 5.29, respectively. The microstructure examined with confocal laser scanning microscopy was unaffected by the changes in pH and the concentrations of NaCl, probably because of the very high-volume fraction of caseins in this type of gel (Karlsson et al., 2007a). Rennet-induced coagulation of ultrafiltered skim milk (19.8%, w\/w casein) at pH 5.8 was studied and compared with coagulation of unconcentrated skim milk of the same pH (Karlsson et al., 2007b). At the same rennet concentration, coagulation occurred at a slower rate in ultrafiltered skim milk but started at a lower degree of \u03ba-casein hydrolysis, compared with the unconcentrated skim milk. Confocal laser scanning micrographs revealed that, during storage for up to 60 days (at 13 \u00b0C), the microstructure and the size of the protein strands of the ultrafiltered gel hardly changed, probably because of the high zero shear viscosity of the concentrated system (Karlsson et al., 2007b).\n\nPlant coagulants obtained from the flowers of Cynara sp. have been used to make rennet gels and cheeses (Esteves et al., 2001; ; ). These coagulants are less sensitive to changes in gelation temperature, they cause more casein rearrangements during gelation, and they have higher values for the storage modulus (at least during the initial stages of gelation), compared with gels made with chymosin (probably because of greater proteolysis of the caseins).\n\nThe properties of fermentation-produced camel chymosin have been compared with those of calf chymosin. Camel chymosin has a 70% higher clotting activity per mol on bovine milk than calf chymosin, but only 20% of its general proteolytic activity and, hence, has a seven-fold higher ratio of clotting to general proteolytic activity (Kappeler et al., 2006).\n\nChoi et al. (2007) demonstrated that the concentration of colloidal calcium phosphate (CCP) associated with the casein micelles had an important influence on the properties of rennet gels. Removal of some CCP from milk prior to gelation using calcium-chelating agents lowered the storage modulus of rennet gels because of the reduction in the amount of CCP cross-linking in the casein micelles. Reduction in the CCP content prior to rennet gelation resulted in gels with higher loss tangent values, indicating greater bond mobility. Choi et al. (2007) also studied the impact of preacidification of milk prior to gelation. They found that gels made at pH 6.4 had higher storage modulus values than gels made at pH 6.7, probably because of the reduction in electrostatic repulsion, whereas the CCP content only slightly decreased at this pH value. The storage modulus values were highest at pH 6.4 and decreased with decreasing pH from 6.4 to 5.4 because of the reduction in CCP cross-linking within the casein micelles (Choi et al., 2007).\n\n### Milk Heat Treatment\n\nIt is well known that severe heat treatment of milk at temperatures sufficiently high to denature the whey proteins results in an increased rennet coagulation time as well as weaker gels (Lucey, 1995). There are some reports that the interaction of denatured whey proteins with the \u03ba-casein inhibits the primary phase of rennet action on \u03ba-casein (to some extent). For example, Reddy and Kinsella (1990) reported that very high heat treatments decreased the initial velocity (V i) and GMP release. However, most studies have concluded that the secondary phase of the coagulation process is the step that is mainly inhibited by the presence of denatured whey proteins on the micelle surface. These denatured whey proteins probably sterically interfere with the (normal) aggregation of rennet-altered micelles (Lucey, 1995). Vasbinder et al. (2003) concluded that whey protein denaturation had only a small effect on rennet activity and that the release of GMP (or the formation of para-\u03ba-casein) was similar in heated and unheated milks. Anema et al. (2007) adjusted the pH of the milk prior to heat treatment, which allowed them to manipulate the distribution of denatured whey proteins and \u03ba-casein between the serum and micellar phases. They reported that the retardation in rennet gelation as a result of heat treatment was observed regardless of whether the denatured whey proteins were associated with the casein micelles or in the serum phase.\n\n### Enzymatic Cross-linking of Caseins\n\nTransglutaminase (TGase; EC 2.3.2.13) catalyzes covalent intermolecular protein cross-linking through an acyl-transfer reaction, between the \u03b3-carboxyamide group of a peptide-bound glutamine residue (acyl donor) and the primary amino group of an amine (acyl acceptor). The application of TGase in various types of dairy products has been reviewed (Jaros et al., 2006). In a system where caseins and whey proteins are available as substrates for TGase, such as milk, the caseins are preferentially cross-linked over native whey proteins (Han & Damodaran, 1996).\n\nLorenzen (2000) incubated preheated milk with TGase for various incubation times prior to rennet addition, and found that increasing TGase incubation times, as well as an increasing intensity of preheat treatment of the milk, resulted in increasing coagulation times up to the point of a complete absence of coagulation. Lorenzen (2000) attributed the reduced rennetability of preheated milk to a 'surface sealing' of the casein micelles with cross-linked \u03b2-lactoglobulin, leading to a steric inhibition of the release of the macropeptide from the surface of the casein micelle. O'Sullivan et al. (2002b) also attributed the loss of rennetability to the impact of TGase cross-linking on the primary enzymatic phase, that is, reduced rate of hydrolysis of \u03ba-casein. Huppertz and de Kruif (2007) criticized the analytical method used by O'Sullivan et al. (2002b) to study the hydrolysis reaction because they suggested that this method detects only the products of hydrolysis of non\u2013cross-linked milk; hydrolysis products of cross-linked \u03ba-casein would not be adequately detected because the macropeptide remains attached to the micelle. Huppertz and de Kruif (2007) suggested instead that TGase treatment affects mainly the secondary stage of rennet-induced coagulation. They suggested that this inhibition was due to the progressive cross-linking of the \u03ba-casein located on the surface of the casein micelles, which provided additional steric hindrance to the aggregation of renneted micelles.\n\n### High Hydrostatic Pressure\n\nHigh hydrostatic pressure influences various properties of milk, including a reduction in the size of the casein micelles, denaturation of \u03b2-lactoglobulin, and a reduction in the CCP content. Huppertz et al. (2005) studied the impact of milk heat treatment (90 \u00b0C for 10 min) and subsequent application of high-pressure treatment at pressures from 0 to 600 MPa. Heated unpressurized milk or heated milk treated for 0 min (immediate release of pressure) at 100 MPa was not coagulable by rennet. However, heated milk treated at 250\u2013600 MPa for 0\u201330 min had a rennet coagulation time equal to, or lower than, that of unheated unpressurized milk; the coagulation time decreased with increasing pressure and treatment time. The strength of the rennet-induced coagulum from heated milk treated at 250\u2013600 MPa for 30 min or 400 or 600 MPa for 0 min was considerably greater than that of the rennet-induced coagulum from unheated unpressurized milk. There was also an increase in the yield of cheese curd by \u224815%. Other studies on applying a combination of pressures and heat treatment for rennet gels have also been reported (Al-Nabulsi et al., 2012). The impact of high pressure and heat treatment on milk proteins has been reviewed (Considine et al., 2007).\n\n### Starch Addition\n\nStarch is sometimes added in cheesemaking in order to bind water and increase cheese yield. It is not a permitted ingredient in many cheese types, but where permitted they are used to replace more expensive milk proteins. Waxy corn or rice starch appear to be mostly retained in rennet curds, whereas modified tapioca starch appears to interfere with the rennet gelation process, producing very weak rennet gels (Brown et al., 2012). Losses of starch into the cheese whey would be a significant concern for whey producers due to greatly increased viscosity and increased likelihood of fouling during membrane filtration.\n\n## Acid-induced milk gels\n\n### Impact of Acid on Casein Micelles\n\nIn cultured products, such as yogurt, as the pH of milk is reduced, CCP is dissolved, and the internal casein micelle structure is altered because of the loss of CCP. Little casein dissociation occurs at the high temperatures (>40 \u00b0C) commonly used for yogurt manufacture. Aggregation of casein occurs as the isoelectric point (pH \u22484.6) is approached (Horne, 1999). Native casein micelles (in milk of normal pH) are stabilized by their negative charge and steric repulsion (Lucey & Singh, 2003). Lucey (2003) distinguished three (arbitrary) pH regions in the acidification of milk from pH 6.7 to 4.6. (a) pH from 6.7 to \u22486.0. The decrease in pH causes a reduction in the net negative charge on the casein micelles, thereby reducing electrostatic repulsion. As only a relatively small amount of CCP is dissolved above pH 6.0, the structural features of the micelles are relatively unchanged. (b) pH from \u22486.0 to \u22485.0. The decrease in pH causes a reduction in the net negative charge on the casein micelles, thereby reducing electrostatic repulsion. As the \u03ba-casein 'hairs' on the micelle surface are charged, these charged 'hairs' may shrink\/collapse as the pH decreases. The net result is a decrease in both electrostatic repulsion and steric stabilization. The CCP within the casein micelles is dissolved completely by pH \u22485.0. (c) pH \u22645.0. The net negative charge on the casein micelles declines with the approach of the isoelectric point, and there are increased +\/\u2013 charge interactions (and van der Waals forces). The reduction in electrostatic repulsion allows increased hydrophobic interactions (Horne, 1998; 2001). In unheated milk gels where acidification is the only coagulation method, gelation occurs at around pH 4.9; if acidification is performed at very high temperatures, a higher gelation pH is observed.\n\nOn acidification, casein particles aggregate as a result of (mainly) charge neutralization. With acidification, the overall charge density of the colloidal system changes and together with it the van der Waals attraction\/electrostatic repulsion balance (Mezzenga & Fischer, 2013). Acidification eventually leads to the formation of chains and clusters that are linked together to form a three-dimensional network (Kal\u00e1b et al., 1983). Acid casein gels can be formed from sodium caseinate (this ingredient is sometimes used as a yogurt stabilizer). Another approach to acid gel formation involves direct acidification of milk at a low temperature and subsequent warming. Glucono-\u03b4-lactone (GDL) is also used to acidify milk, but these acid-induced gels have different rheological and structural properties from gels produced by bacterial cultures (Lucey et al., 1998a; Renan et al., 2008).\n\nHydrophobic interactions are unlikely to play a direct role in the strength of acid gels as the storage modulus of acid gels increases with decreasing measurement temperature (Lucey, 2003). Cooling gels result in an increase in the storage modulus, probably as a result of the swelling of casein particles (caused by the weaker hydrophobic interactions) and an increase in the contact area between particles (Lucey, 2003). With increasing ionic strength, the charged groups on casein are screened, thereby weakening interactions between casein particles.\n\nMilk has been reversibly acidified by means of carbonation, injecting pressurized CO2 as the acidifying agent, in order to reduce the pH (usually done at low temperature). Neutralization is obtained by pressure release followed by degassing under vacuum. Upon CO2 treatment, the zeta potential and the size of the casein micelles were restored, although the total amount of CCP was not restored (Raouche et al., 2007). The rheological properties of acid gels (made using GDL) from CO2-treated milk were similar to those of acid gels from untreated milk (Raouche et al., 2007).\n\n### Some Factors Influencing the Texture of Yogurt Gels\n\nIt is well established that the way in which the milk is handled or prepared, including the processing conditions used in yogurt manufacture, greatly influences the gel texture, strength, and stability (Lucey & Singh, 1998; Walstra, 1998; Tamime and Robinson, 1999; Jaros & Rohm, 2003a, b). These factors include (a) fortification level and material(s) used in the mix, (b) stabilizer type and usage levels, (c) fat content and homogenization conditions, (d) milk heat treatment conditions, (e) starter culture (type, rate of acid development, and production of exopolysaccharides), (f) incubation temperature (which influences growth of starter cultures, gel aggregation, and bond strength), (g) pH at the breaking of the gel (stirred) and\/or the start of cooling (set), (h) cooling conditions (when cooling is started, rate of cooling), and (i) postmanufacture handling of the product, for example, vibration and temperature fluctuations (i.e., if the product is not maintained at \u22485 \u00b0C).\n\n### Homogenization and Fat Globule Surface Material\n\nThe fat globules in milk are surrounded by membrane proteins, and, unless homogenized, fat acts as an inert filler in milk gels. Cho et al. (1999) prepared fat globules with different surface materials and studied the effects of these surface materials on the rheological properties of acid milk gels. Gels containing fat globules stabilized by noninteracting materials ('structure breaker'), i.e., Tween and unheated whey protein concentrate (WPC) had low storage moduli compared with interacting surface materials ('structure promoter') (skim milk powder, sodium caseinate, and heated WPC).\n\nMilk for the manufacture of yogurt is normally homogenized (15\u201320 MPa) in order to increase the yogurt's consistency and to decrease whey separation during storage (Tamime & Robinson, 1999). High-pressure homogenization has a similar principle to conventional homogenization but works at significantly higher pressures (up to 400 MPa). Milk given a high-pressure (>200 MPa) treatment gave firmer yogurt gels than heat treated milk (90 \u00b0C for 90 s) and traditionally homogenized at 15 MPa (Serra et al., 2007). Presumably this effect reflects a combination of the creation of very small fat globules, whey protein denaturation, and possible modification to the CCP content (Huppertz & de Kruif, 2006; L\u00f3pez-Fandi\u00f1o, 2006). The combination of high-pressure homogenization and heat treatments has been explored to improve the properties of acid gels (Hernandez & Harte, 2008).\n\n### High Hydrostatic Pressure\n\nHigh hydrostatic-pressure treatment of milk enhances the mechanical properties of yogurt gels (Needs et al., 2000). The storage moduli of gels made from high-pressure-treated milk were considerably higher than those of gels made from heat-treated milk (85 \u00b0C for 20 min), although the yield stress and the yield strain were lower in the pressure-treated gel (Needs et al., 2000). The combined use of high thermal treatment and high hydrostatic pressure results in extensive whey protein denaturation and casein micelle disruption, respectively (Harte et al., 2003). The net effect of the combined high hydrostatic pressure and thermal treatments was an improvement in the yield stress of the yogurt and a reduction in syneresis (Harte et al., 2003). High-pressure treatment up to 600 MPa (for 20 min) improved the viscosity of stirred yogurt, which had similar rheological properties to yogurt made from milk heated at 90 \u00b0C for 30 min (Knudsen et al., 2006). The pH of milk at pressure treatment also influences acid gelation (Anema, 2010), with higher pH values producing firmer gels as a result of greater whey protein denaturation.\n\n### Enzymatic Modification of Proteins\n\nAcid-induced gelation of TGase-cross-linked casein resulted in increased gel firmness, lower permeability, finer protein networks, and improved whey drainage (Faergemand & Qvist, 1997; F\u00e6rgemand et al., 1999; Schorsch et al., 2000). Lauber et al. (2000) reported that even a very small amount of casein cross-linking, due to the action of TGase, is capable of inducing significant changes in yogurt texture (i.e., a large increase in gel strength). TGase treatment may reduce protein rearrangements during the gelation phase, thereby lessening the likelihood of (unwanted) whey separation (Ercili-Cura et al., 2013). A slightly slower acidification rate by the starter culture was observed in yogurts made from TGase-treated milk; possibly there was a reduction in availability of the low-molecular-weight peptides required by Streptococcus thermophilus as a result of the cross-linking reaction (Faergemand et al., 1999; Ozer et al. 2007). Cross-linking of caseins restored the sensory texture profile of a lower protein yogurt to be comparable with that of a higher protein yogurt, suggesting that TGase could be used instead of some of the milk solids currently used in yogurt fortification (Faergemand et al., 1999). Excessive protein cross-linking increased the gel firmness, but the yogurt became grittier than the control samples (Faergemand et al., 1999). TGase is capable of cross-linking caseins even under high pressure (Lauber et al., 2001). When TGase treatment was performed during high hydrostatic-pressure treatment, a markedly higher final storage modulus was observed in acid milk gels compared with gels with only pressure treatment or when a separate TGase treatment was performed (Anema et al., 2005). Anema et al. (2005) proposed that there is an increase in cross-linking of the whey proteins and an increase in cross-linking between the whey proteins and casein when TGase treatment is performed under high pressure. It appears that treatment of goat's milk with TGase is less effective than cow's milk, resulting in weaker acid gels (Ardelean et al., 2013).\n\n### Heat Treatment\n\nAcid gels formed from unheated milk are very weak, at least partly because the interparticle contact area is still dominated by the presence of the \u03ba-casein hairs (GMP), which have collapsed but are still present (Li & Dalgleish, 2006). The \u03ba-casein hairs are rich in hydroxylated amino acids, some of which are glycosylated, and also acidic and basic residues. Thus, the interface between the aggregating particles will tend to be highly hydrated, and attractive interactions will be partly offset by the hydrophilic tendency of the \u03ba-casein hairs (Li & Dalgleish, 2006).\n\nThere has been considerable recent research on the topic of how whey proteins influence yogurt texture. Native whey proteins in unheated milk are inert fillers in yogurt (Lucey et al., 1999). Added whey proteins alter yogurt gelation and texture as long as the mix is given a sufficiently high-heat treatment to denature the whey proteins and cause them to associate with the casein micelles (Lucey et al., 1999). Commercially, WPC is often used to increase the solids content of yogurt and to give improved viscosity and lower whey drainage. High-heat treatment causes considerable whey protein denaturation (e.g., 85 \u00b0C for 15 min results in >80% \u03b2-lactoglobulin denaturation). As a result, \u03b2-lactoglobulin becomes mostly attached to the \u03ba-casein of the casein micelles or forms soluble complexes (with serum casein), depending on the heating conditions (i.e., pH) (Lucey et al., 1998b).\n\nDenatured whey proteins (DWPs) attached to the surface of casein micelles during heating (i.e., bound DWP) are a critical factor in the increased stiffness of yogurt gels made from heated milk. DWPs cause micelles to aggregate at higher pH because of the higher isoelectric pH (\u22485.3) of the main whey protein, \u03b2-lactoglobulin, than that of caseins (Lucey et al., 1997; Guyomarc'h et al., 2003; Morand et al. 2012). An alternative view is that the DWPs associated with the micelles alter the hydrophobic interactions between heated micelles, which facilitates gelation at higher pH values (although there is greater electrostatic repulsion at higher pH) (Jean et al., 2006). More cross-linking of gels by bound DWP increases the gel strength. Soluble DWPs are not able to increase the gel stiffness of milk in which there are no bound DWP present. That is, the micelle surface does not contain any 'bound' DWP and can be created experimentally (Lucey et al., 1998b) (Fig. 17.3).\n\nFigure 17.3 Storage modulus as a function of time during the formation of acid-induced milk gels made from heated milk ( ), heated milk containing bound DWP (\u25cb), heated milk containing soluble DWP (\u25be), and unheated milk (\u25b5). Heat treatment was at 80 \u00b0C for 30 min, and acidification was at 30 \u00b0C with 1.3% GDL. Reproduced, with the permission of Cambridge University Press, from Lucey et al., 1998b.\n\nIn industrial practice, heating milk always creates some bound DWP, which allows soluble DWP to become attached to the micelles and to contribute to the gel strength. The pH at heating influences the association of DWP with casein micelles. At pH 6.5, most DWPs are associated with micelles (e.g., >70% for milk heated at 90 \u00b0C for 30 min). At higher pH (e.g., 7.0), fewer DWPs are associated with micelles as more \u03ba-casein dissociates from the micelles to interact with \u03b2-lactoglobulin during heating. The gel strength of acid gels made from milk heated at high pH is higher than that of acid gels made from milk heated at the natural pH of milk (Lucey et al., 1998b; Anema et al., 2004); this may not be valid for situations in which there is a lot of added whey protein. At high pH values, there is an increase in the concentration of CCP (additional cross-linking) in milk (McCann & Pyne, 1960), which could potentially increase the stiffness of acid gels made from high-pH milk. Increasing the pH of heat treatment of the milk from 6.5 to 7.0 should also alter protein unfolding and disulfide bond formation, involving \u03b2-lactoglobulin, as the pK value of its free thiol group is 9.35 (Kella & Kinsella, 1988a).\n\nThe creation of additional covalent disulfide bonds that involve whey protein and caseins should increase the strength of the yogurt gel. Regardless of the pH of the milk at heating, DWPs (i.e., those designated as 'soluble' and 'bound' at the pH of heat treatment) are insoluble at low pH and should associate with casein at the pH values involved in yogurt fermentation. As the pH decreases during fermentation, virtually all the residual soluble complexes become attached to caseins via the bound DWP. The rate of acidification and the gelation temperature may also influence how these complexes associate with the caseins during acidification. The extent of denaturation of the whey proteins is often determined by their loss of solubility at pH 4.6 (de Wit, 1981), so that all the DWP should precipitate as the pH approaches 4.6.\n\nThe addition of WPC to milk that was then given a high-heat treatment resulted in an increase in the pH of gelation, an increase in gel stiffness, and a reduction in fracture strain compared with gels made from heated milk without added WPC (Lucey et al., 1999). If WPC was added to heated milk and this mixture was not given any further heat treatment, the acid gels formed after acidification were weaker than those made from heated milk without WPC. This suggests that any added whey proteins must be denatured in order to reinforce the network, even when DWPs are already present in the milk. Schorsch et al. (2001) examined the effect of heating whey proteins in the presence or absence of casein micelles on the subsequent acid gelation properties of milk. The acid-induced gelation occurred at a higher pH (around pH 6.0) and in a shorter time when the whey proteins (concentration of 1 g whey protein\/kg) were denatured separately from the casein micelles than when the whey proteins were heated in the presence of the casein micelles. However, the gels formed were very weak, probably because of the formation of a weak network in which whey proteins entrapped caseins.\n\nVarious studies have shown some conflicting results about the relative importance of the soluble and bound DWP fractions to the texture of acid milk gels (Lucey et al., 1998b; Guyomarc'h et al., 2003; Anema et al. 2004). Differences in the proportions of soluble and bound DWP fractions in these studies could have contributed to these conflicting results. Guyomarc'h et al. (2003) had only a small proportion (10\u201315%) of \u03b2-lactoglobulin in the bound DWP fraction, whereas Lucey et al. (1998b) had around 80%. Guyomarc'h et al. (2003) suggested that differences in the quantitative amounts of aggregates (and the total amount of DWP) present in the systems, independently of whether or not they were soluble, could be the reason for some of the conflicting results reported by the different groups.\n\nIn gels made from heated milk, because of the high gelation pH, the gel goes through a period of solubilization of the CCP that is present within casein particles that are already part of the gel network (this event is responsible for the maximum in the loss tangent during gelation) (Lucey et al., 1997). This process loosens the interactions between caseins in the gel network, and the higher bond mobility in yogurt gels during this period has been associated with whey separation (Lucey, 2001). The rheological changes during the acid-induced gelation (with GDL) of unheated and heated milk at 30 \u00b0C are shown in Figure 17.4. Note the much shorter gelation time, the large increase in the storage modulus, and the maximum in the loss tangent (as indicated by the hatched region between the two arrows, region A) in the heated milk sample. As the low gelation pH (4.8) of the unheated milk gel occurs after most or all of the CCP is already solubilized, there is no maximum in the loss tangent in this type of gel. When acid-induced gelation of heated milk occurs rapidly at high temperature, a plateau in the storage modulus, which corresponds to the region where there is a maximum in the loss tangent, can be observed (Horne, 2001).\n\nFigure 17.4 Storage modulus (solid lines) and loss tangent (dashed lines) of acid gels made from heated milk ( ) and unheated milk (\u25cb). Heat treatment was at 80 \u00b0C for 30 min, and acidification was at 30 \u00b0C with 1.3% GDL. The area marked by the letter A indicates the region in which the loss tangent increases after gelation because of solubilization of CCP in casein particles that are already part of the gel network.\n\nBikker et al. (2000) reported that the addition of \u03b2-lactoglobulin variant B or variant C to the milk prior to heating and acidification caused a larger increase in the storage modulus of acid gels than the addition of \u03b2-lactoglobulin variant A.\n\nSoluble whey protein polymers have been used as ingredients for yogurt applications (Britten & Giroux, 2001). The use of whey protein polymers to standardize the protein content of milk increased the yogurt viscosity to about twice that obtained using skim milk powder at the same protein concentration. The water-holding capacity of yogurt standardized with whey protein polymers was considerably higher than that of yogurt standardized with skim milk powder (Britten & Giroux, 2001).\n\n### Incubation Temperature\n\nAlthough 42 \u00b0C is a commonly used fermentation temperature for yogurt, the use of slightly lower incubation temperatures (e.g., 40 \u00b0C) leads to slightly longer gelation times, but firmer and more viscous gels that are less prone to whey syneresis are formed (Lee & Lucey, 2004). At a lower incubation temperature, there is an increase in the size of the casein particles because of a reduction in hydrophobic interactions, which, in turn, leads to an increased contact area between the casein particles (Lee & Lucey, 2004); a similar trend occurs when the gels are cooled. A high incubation temperature also makes the gel network more prone to rearrangements (more flexible) during gelation, and these changes can lead to greater whey separation (Lucey, 2001; Mellema et al. 2002). Peng et al. (2010) investigated the effect of altering temperature immediately after gel formation. Cooling after gelation resulted in an increase in gel stiffness and greater inter-cluster strand formation, whereas heating of gels may promote intra-cluster fusion and breakage of strands between clusters.\n\n### Production of Exopolysaccharides\n\nSome yogurt starter cultures produce exopolysaccharides (EPS) during the fermentation process. They can be viewed as a naturally produced thickener. This EPS can be produced as a capsular layer around the bacterial cell or excreted into the medium to produce an effect sometimes called 'ropy' or 'stringy' (Hassan, 2008). Popular examples of ropy yogurt are Viili and L\u00e5ngfil from Scandinavia. Capsular EPS has little impact on yogurt gelation or texture. Ropy EPS can be either charged or uncharged. It is possible that charged EPS may associate electrostatically with the caseins, depending on the pH of the milk, whereas uncharged EPS may influence gelation via a depletion flocculation-type mechanism (Girard & Schaffer-Lequart, 2007). The molar mass, concentration of EPS, and the exact period during fermentation (before, during, or after gelation) when EPS is produced may all play a critical role in determining the impact of EPS on yogurt gels.\n\n## Whey protein gels\n\nWhey is usually obtained as a by-product of cheesemaking (although recent developments in membrane technology mean that, in future, whey will come not necessarily from a cheese vat but as native whey directly from milk prior to cheesemaking). Its composition, however, depends on the cheesemaking conditions; for example, acid whey derived from cottage cheese has different mineral (ash), lactic acid, and pH values from whey derived from rennet-coagulated cheeses such as Cheddar (Table 17.1). Whey products are widely used as food ingredients because of their excellent functional and nutritional properties. Various types of whey products are made commercially, ranging from dried whey to WPC (WPC has protein contents ranging from \u224835 to 80%) to whey protein isolate (WPI) (protein contents \u226590%) (Table 17.2). Membrane filtration, that is, ultrafiltration (UF) and diafiltration (DF), is used to concentrate the protein fraction before spray drying into WPC. Two different approaches are used to produce WPI: (a) membrane filtration (microfiltration, UF and DF) and (b) ion-exchange chromatography coupled with UF\/DF. These two approaches result in WPI with different protein profiles (Table 17.3; Wang & Lucey, 2003). Many serum proteins take part in heat-induced gelation, whereas GMP and proteose peptones do not (Walstra et al., 2005).\n\nTable 17.1\n\nComposition of Rennet and Acid Wheys\n\n| Average composition  \n---|---  \n| Rennet whey | Acid whey  \nTotal whey protein (g\/L) | 6.7 | 5.8  \n\u03b2-lactoglobulin (g\/L) | 3.5 | 3.5  \n\u03b1-lactalbumin (g\/L) | 1.3 | 1.3  \nSerum albumin (g\/L) | 0.1 | 0.1  \nImmunoglobulins (g\/L) | 0.4 | 0.4  \nProteose peptones (g\/L) | 0.2 | 0.2  \nGlycomacropeptide (g\/L) | 1.0 | \u2014  \nLactose (g\/L) | 5.0 | 4.4  \nLipid (g\/L) | 0.6 | 0.1  \nAsh (g\/L) | 0.5 | 0.6  \nNa (mg\/100 g) |  35 | 40  \nK (mg\/100 g) |  109 | 133  \nCa (mg\/100 g) |  22 | 86  \nMg (mg\/100 g) |  6 | 9  \nP (mg\/100 g) |  42 | 63\n\n(Adapted from Oakenfull et al., 1997)\n\nTable 17.2\n\nTypical Composition of Some Whey Powders (approximate, wet, or as-is basis)\n\nWhey ingredient | Moisture (%) | Fat (%) | Protein (%) | Lactose (%) | Ash (%)  \n---|---|---|---|---|---  \nSweet whey | 3\u20135 | 1.1\u20131.5 | 11\u201314.5 | 75 | 8\u201310  \nAcid whey | 3.5 | 0.5\u20131.5 | 11\u201313.5 | 70 | 10\u201312  \nWPC35 | 3\u20134.5 | 3\u20134.5 | 34\u201336 | 48\u201352 | 6.5\u20138  \nWPC80 | 3.5-4.5 | 6\u20138 | 80\u201382 | 4\u20138 | 3\u20134  \nWPI | 4\u20135 | <1.0 | 90\u201392 | <1.0 | 2.5\u20133.5\n\nTable 17.3\n\nApproximate Protein Composition of Whey Protein Isolates Made by Different Technologies\n\nProtein type | Membrane filtration | Ion-exchange chromatography  \n---|---|---  \n\u03b2-lactoglobulin | 48\u201355% | 60\u201373%  \n\u03b1-lactalbumin | 15\u201322% | 12\u201325%  \nBovine serum albumin and Immunoglobulins | 4\u20137% | 6\u201316%  \nGlycomacropeptide | 17\u201326% | 0.2\u20131.4%\n\n(Data from several sources, including Wang and Lucey, 2003)\n\nWhey proteins are globular proteins, and heating induces denaturation and aggregation. Gelation depends on many factors, especially pH. At sufficiently high protein levels (usually \u22656%, except for purified individual whey proteins), gelation occurs during heating or cooling. The formation and the properties of whey protein gels are influenced by many factors (Table 17.4). There have been several reviews of the aggregation\/gelation of globular proteins (Oakenfull, 1987; Clark 1992; ; ; Doi, 1993; Oakenfull et al., 1997; Gosal & Ross-Murphy, 2000). The thermal denaturation and gelation of whey proteins have been reviewed (Mulvihill & Kinsella, 1987; Mangino, 1992; Aguilera, 1995; Singh & Havea, 2003; Foegeding, 2006; Mezzenga & Fischer, 2013).\n\nTable 17.4\n\nFactors that Influence the Heat-induced Gelation Properties of Whey Proteins\n\npH\n\nProtein content\n\nIonic strength\n\nRate and temperature\/time of heating\n\nTypes and ratios of the serum proteins\n\nConcentration of divalent ions (e.g., Ca2+)\n\nConcentration of sugars\n\nConcentration of lipids, including phospholipids\n\nUpon denaturation, whey proteins unfold and expose their hydrophobic residues, which were initially buried within the folded structure to minimize contact with water. The protein molecules will then tend to aggregate, thereby minimizing the contact of the hydrophobic residues toward water (Mezzenga & Fischer, 2013).\n\n### Thermal Denaturation of Whey Proteins\n\nMany studies on the denaturation of whey proteins have been conducted (see the review by Mulvihill & Donovan, 1987), especially \u03b2-lactoglobulin, as this is the major whey protein and its behavior dominates the gelation behavior of whey protein products. Denaturation has been used to describe both the loss of native structure (conformational change) and the loss of solubility (e.g., at pH values close to the isoelectric point). At around neutral pH values, denaturation becomes irreversible above about 65 \u00b0C (Holt & Sawyer, 2003); with a decrease in the pH, the denaturation temperature increases (Kella & Kinsella, 1988b). Disulfide bond formation is favored as the pH is increased toward the pK value of the thiol group on \u03b2-lactoglobulin (9.35; Kella & Kinsella, 1988b).\n\nDenaturation, that is, conformational change, can be reversible and, for whey proteins, the cause of irreversibility is often the formation of new covalent (mostly disulfide) bonds. Various mechanisms for the thermal denaturation\/aggregation of \u03b2-lactoglobulin (at neutral pH) have been proposed, in which the basic steps are: (a) the dissociation of the dimer into monomers and a conformational change leading to the exposure of Cys121, which initiates sulfydryl\u2013disulfide interchange reactions, (b) an endothermic transition to a molten globule state, and (c) the unfolding of the protein and a second, high-temperature endothermic transition (Holt & Sawyer, 2003). The reactive monomers formed during the denaturation process initially form dimers and trimers via the thiol\u2013disulfide exchange reaction, and the conversion of dimer to trimer is considered to be the rate-limiting step in the aggregation process (Prabakaran & Damodaran, 1997). Patel et al. (2006) proposed that the following reactions occur when milk is heated at \u224885 \u00b0C. The major whey proteins (\u03b2-lactoglobulin and \u03b1-lactalbumin) alter their structures and the free cysteine (CysH121) of \u03b2-lactoglobulin initially reacts reversibly with the adjacent Cys106\u2013Cys119 disulfide bond to give a free CysH119, which, in turn, reacts with the Cys66\u2013Cys160 disulfide bond of the same or another \u03b2-lactoglobulin molecule to give a free CysH160. CysH160 is mobile and free to move because it is so close to the C-terminus of the molecule. Thus, it reacts with disulfide bonds in other proteins, allowing a chain reaction with other \u03b2-lactoglobulin or casein molecules to occur (Patel et al., 2006).\n\nA possible model of these reactions during the denaturation and aggregation of \u03b2-lactoglobulin is shown in Figure 17.5. In the presence of different types of whey proteins, various heteropolymers (e.g., \u03b2-lactoglobulin\u2013\u03b1-lactalbumin or \u03b2-lactoglobulin\u2013bovine serum albumin) are formed during heating (Havea et al., 2001).\n\nFigure 17.5 Model for the aggregation and formation of heat-induced \u03b2-lactoglobulin gels.\n\nDuring the heating of \u03b2-lactoglobulin, the loss of native structure occurs via both disulfide-linked aggregate formation and noncovalently linked aggregates (e.g., hydrophobic interactions) (McSwiney et al., 1994). When \u03b2-lactoglobulin was heated at 75 \u00b0C, gelation was not observed until most of the protein had aggregated (McSwiney et al., 1994). Pure \u03b1-lactalbumin is very heat stable (because it does not have a free thiol group), although it does undergo a reversible transition at 64 \u00b0C (Ruegg et al., 1977). In the presence of \u03b2-lactoglobulin, it undergoes irreversible aggregation via the thiol\u2013disulfide exchange reaction as well as other types of interactions (Elfagm & Wheelock, 1978).\n\nDuring the heating of \u03b2-lactoglobulin, most of the helical conformation is lost by about 65 \u00b0C; with increasing temperature there is progressive loss of \u03b2-sheet structure (Qi et al., 1997). However, in \u03b2-lactoglobulin, a considerable amount of secondary structure, particularly \u03b2-sheet, still remains intact even at 90 \u00b0C (Bhattacharjee et al., 2005). Aggregation of globular proteins starts when heat causes some unfolding of the molecule, which exposes reactive groups or sites (e.g., hydrophobic regions) that favor intermolecular interactions (Foegeding, 2006). If \u03b2-lactoglobulin is denatured at low temperatures (e.g., around 60 \u00b0C), the molecules partially unfold into the state of a molten globule, while higher denaturation temperatures trigger a complete unfolding and rapid aggregation (Mezzenga & Fischer, 2013).\n\nGupta et al. (1999), using Monte Carlo computer simulations, indicated that protein-like molecules need to only partially unfold before they are susceptible to aggregation. Aggregation ultimately results in gelation if the protein concentration and other gelling conditions are favorable. This aggregation process is governed by a balance between attractive hydrophobic and repulsive electrostatic interactions.\n\nFractal aggregation theory has been applied to the aggregation and formation of whey protein gels (Vreeker et al., 1992; Ikeda et al., 1999; Mezzenga & Fischer, 2013). Euston (2004) argued that theories of fractal aggregation are not necessarily a good representation of protein gel structure as they treat the aggregating protein as a rigid particle and ignore any structural changes that occur in the protein during denaturation and aggregation. This criticism could be particularly important for the gelation of globular proteins, such as \u03b2-lactoglobulin.\n\nA gel is formed when the extent of aggregation exceeds a critical level for the formation of a self-supporting network that is able to entrap the solvent.\n\n### Types and Properties of Whey Protein Gels\n\nDifferent types of gel networks can be formed by globular proteins, such as whey proteins. The network structure in a heat-induced globular protein gel is strongly dependent on the balance between attractive and repulsive forces among (partially) denatured protein molecules during the aggregation process. As whey proteins have isoelectric points (pI) in the vicinity of pH 5, they are negatively charged at neutral pH values. In whey protein solutions, the ionic strength is important as it regulates the amount of ions available for the screening of charged groups on the proteins. At neutral pH values and under low ionic strengths, there is intermolecular repulsion. Aggregation of denatured proteins proceeds via hydrophobic sites, and this leads to the formation of fine-stranded gels (with a transparent or translucent appearance and strands that are often 10\u201320 nm in thickness) (Stading & Hermansson, 1991).\n\nIntermolecular repulsion can be reduced by increasing the ionic strength or by adjusting the pH to be closer to the isoelectric point of the whey proteins (\u22485). Under gelation conditions of high ionic strength or pH values close to 5, whey proteins form opaque or particulate or turbid gels. The particles\/clusters in this type of gel are in the micron-size range. This type of gel structure has a poorer water-holding capacity than fine-stranded gels (Bottcher & Foegeding, 1994). Particulate gels break down rapidly during mastication to yield a homogeneous distribution of small particles, whereas fine-stranded gels break down into large, inhomogeneous particles with irregular shapes (Foegeding, 2006). A fine-stranded gel formed at neutral pH is rubbery and deformable to a large strain, with a small fracture stress (Stading & Hermansson, 1991). At acidic pH values, intermolecular disulfide bonding is unlikely to occur, and the fine-stranded networks formed at very low pH values (e.g., 3) are brittle. Particulate gels normally fracture at a small strain, but the stress required to reach the fracture strain is relatively large (Stading & Hermansson, 1991; Bottcher & Foegeding, 1994; Foegeding et al., 1995). After heat-induced gelation, cooling results in strengthening of the network because of hydrogen bond formation.\n\nHeat-induced \u03b2-lactoglobulin gels exhibit the characteristics of a 'strong' gel. That is, they have a low-frequency dependence on the storage modulus (the linear slope, n, of the plot of log frequency versus log storage modulus is <0.06) (Stading & Hermansson, 1990). The slope n is slightly higher for particulate gels than for fine-stranded gels (Stading & Hermansson, 1990).\n\nAt pH values around 2 and low ionic strengths, whey protein gels that have some similarities in structure to \u03b2-amyloid fibrils are formed (Gosal et al., 2004; Bolder et al. 2006). Fibrils are usually rigid, nonbranching and filamentous structures, around 8 nm (or larger) in width (for \u03b2-lactoglobulin) and often more than 1 \u03bcm long, that arise from linear aggregation of partly unfolded proteins (Gosal et al., 2004). \u03b1-lactalbumin and bovine serum albumin can also form fibrils during heating at pH 2 (Goers et al., 2002; Veerman et al. 2003). Extensive hydrolysis occurs at low pH, and aggregation occurs through \u03b2-strands and \u03b2-sheets creating fibrils (Mezzenga & Fischer, 2013).\n\n### Other Factors Influencing Properties of Whey Protein Gels\n\npH and ionic strength greatly impact the type of gel formed and its properties. The strength of whey protein gels increases with protein content. The minimum protein content needed for gelation depends on whether an individual whey protein (e.g., \u03b2-lactoglobulin) or a commercial mixture (e.g., WPC) is used, as well as the gelation conditions (e.g., pH, heat treatment, ionic strength). Pure solutions of \u03b2-lactoglobulin can form a self-supporting gel at 5% protein content when tested at pH 8.0 and a heat treatment of 90 \u00b0C for 15 min (100 mM Tris-HCl buffer) (Matsudomi et al., 1991).\n\nThe protein profile is important for whey gelation. For example, higher gelling whey products can be made by increasing the proportion of \u03b2-lactoglobulin and decreasing the proportion of GMP. As \u03b1-lactalbumin is a poorer gelling protein than \u03b2-lactoglobulin, increasing the proportion of \u03b2-lactoglobulin to \u03b1-lactalbumin also increases the gelation properties of whey products (Hines & Foegeding, 1993). Commercial whey products with a higher ratio of \u03b2-lactoglobulin to \u03b1-lactalbumin are available (e.g., WPI made by ion-exchange chromatography compared with WPI made by membrane filtration, or acid whey WPC; both have little or no GMP).\n\nSalts have a major effect on the type of whey protein gel formed as a result of heat treatment and its mechanical\/sensory properties. It is generally recognized that the addition of NaCl or CaCl2 to dialyzed samples of WPC or WPI results in an increase in gel strength. Above a level of 10\u201320 mM CaCl2 and 100\u2013200 mM NaCl, the gel firmness starts to decrease (Schmidt et al., 1979; Kuhn & Foegeding, 1991). Excessive calcium has been speculated to cause rapid protein aggregation, which limits protein unfolding and network formation (Mangino, 1992). Caussin et al. (2003) reported that the addition of calcium to whey proteins resulted in the formation of very large protein aggregates during heating. Most commercially available WPC products probably have calcium contents that are greater than that required for optimal gel strength (Mangino, 1992).\n\nThere is considerable variability in the thermal aggregation behavior of commercial whey products; some of these differences could be removed by dialysis of these samples to a common ionic strength (McPhail & Holt, 1999). The concentration of divalent cations is higher in WPC made from cheese whey than in WPC made from acid whey. These cations are not easily removed by dialysis, suggesting some binding with the whey proteins (Havea et al., 2002). Although acid whey starts with a higher calcium content than cheese whey (Table 17.1), it is presumably easier to remove these salts in the manufacture of acid whey WPC than in the manufacture of cheese whey WPC. Acid whey WPC is known as a superior heat-gelling product compared with cheese whey WPC (Veith & Reynolds, 2004). These differences could be due to the absence of GMP and the low calcium concentration in acid whey WPC. It has been reported that polyphosphates have been added to WPC to improve the gelling properties (Veith & Reynolds, 2004). There are various possible processing approaches to reduce the calcium\/mineral content of cheese whey WPC (e.g., electrodialysis, addition of chelating agents, low-pH UF\/DF) in order to improve its gelling properties.\n\nThe gelling time is also dependent on temperature, with the time required for gelation decreasing with increasing temperature, although, at very high temperatures, gelation may occur only during the subsequent cooling stage (Hillier & Cheeseman, 1979). Many reports show that, when all other factors are kept constant, the gel strength increases with increasing temperature (presumably reflecting greater unfolding and reactivity of the proteins) (Mulvihill & Kinsella, 1987). The presence of lipids and lactose impairs the gelation of whey proteins (Mulvihill & Kinsella, 1987). Sugars, such as lactose, are known to protect the protein against loss of solubility during heat treatment and increase the thermal denaturation temperature of whey proteins (de Wit, 1981; Jou & Harper, 1996). Possibly, lipids might interfere with the hydrophobic interactions that play a role in the aggregation of partly unfolded whey proteins during heat treatment.\n\n### Cold Gelation of Whey Proteins\n\nGels can also be produced using a two-step process that involves heat treatment at low ionic strength and\/or far from the isoelectric point, followed by an increase in ionic strength and\/or an adjustment in pH (Barbut & Foegeding, 1993; Britten & Giroux, 2001). These gels are labeled cold-set gels, as the initial heat treatment produces a polymerized solution, with gelation occurring during the subsequent cold-set conditions through screening of the repulsive forces. To obtain gels via the cold-set gelation method, it is necessary to prepare a heat-denatured solution, with a protein concentration below the critical gelation concentration. Gelation can then be induced at low temperatures by the addition of mono- or polyvalent cations (e.g., Ca2+).\n\nBritten and Giroux (2001) acidified whey protein polymers to pH 4.6 with GDL and formed opaque particulate gels. The storage modulus and the firmness of the gels were affected by the conditions used to prepare the protein polymers.\n\n### Enzymatic Modification of Whey Protein for Gelation Purposes\n\nExtensive hydrolysis of whey protein using proteinases results in gelation mainly via hydrophobic interactions, with hydrogen bonding and electrostatic interactions also playing a minor role (Otte et al., 1996; Doucet et al. 2003;).\n\nThe casein fractions in milk are more susceptible to TGase cross-linking than the globular whey protein fractions (Jaros et al., 2006). Some unfolding of \u03b2-lactoglobulin improves the extent of cross-linking with TGase (Faergemand et al., 1997; O'Sullivan et al. 2002a). Cold-set whey protein gels at low pH have been cross-linked with the TGase enzyme under either low pH or alkaline conditions (Eissa et al., 2004; Eissa & Khan, 2005). One approach involved two steps; first, cross-linking whey proteins with TGase at pH 8 and 50 \u00b0C; and second, cold-set acidifying the resulting solution using GDL (Eissa et al., 2004). During the first step, the whey proteins undergo enzyme-catalyzed \u025b-(\u03b3-glutamyl) lysine bond formation with a substantial increase in viscosity. Enzyme-cross-linked gels had significantly higher yield\/fracture stress and strain than cold-set gels prepared without TGase enzyme or conventional heat-set gels. In addition, the elastic modulus of the enzyme-catalyzed gel was higher than that of its nonenzyme-treated counterpart.\n\n### Mixed Gels Made with Rennet and Acid\n\nMilk coagulation can be induced by the combined action of acid and enzyme (i.e., mixed gels). The study of mixed milk coagulation has received very little attention when compared with rennet- or acid-induced coagulation (Roefs et al., 1990; Lucey et al. 2000; 2001; Tranchant et al., 2001). Cottage cheese is generally manufactured by acid coagulation of pasteurized skim milk, and a small concentration of rennet is sometimes added after the starter has been allowed to develop some acidity (i.e., at pH around 5.5) (Castillo et al., 2006). The use of rennet in combination with acid development initiates gelation at a high pH, and the gel can undergo a 'weakening' stage (as indicated by a decrease\/plateau of the storage modulus, a decrease in the light backscatter ratio, or an increase in the loss tangent). This weakening is more pronounced with unheated milk gels and where there have been very high levels of \u03ba-casein hydrolysis prior to acidification (Li & Dalgleish, 2006). This 'weakening' stage is related to rearrangements caused by CCP demineralization of the casein particles in the gel network because this CCP solubilization occurs after gelation (gelation is initiated at a high pH in mixed gels) (Lucey et al., 2000). The final storage modulus of mixed gels can be considerably higher than that of acid gels made without rennet. Mixed gels made from heated milk formed firmer gels, as they were cross-linked by denatured whey proteins and underwent fewer large-scale rearrangements (Lucey et al., 2000).\n\nThe rheological and microstructural properties of mixed gels are complex, and these properties can be adjusted by varying the rennet level or the acidification rate (Tranchant et al., 2001). The use of low rennet levels during the fermentation of milk resulted in a coarser acid gel network and higher syneresis (Aichinger et al., 2003). Micelle fusion was faster in gels with added rennet because of the removal of the \u03ba-casein hairs (Aichinger et al., 2003). Gastaldi et al. (2003) studied the acid-induced gelation of milk samples in which chymosin was used to vary the degree of \u03ba-casein hydrolysis prior to acidification (further chymosin activity during acidification was blocked using an inhibitor). The gelation pH increased and the gelation time decreased with an increasing degree of \u03ba-casein hydrolysis. Gels with much higher storage moduli were formed as a result of partial \u03ba-casein hydrolysis prior to gelation, although the loss tangent and the serum-holding capacity were lower (Gastaldi et al., 2003). Presumably, partial \u03ba-casein hydrolysis prior to acid gelation facilitated greater rearrangements\/fusion of casein, which was responsible for the increase in the storage modulus but also increased the serum separation (Lucey et al., 2001).\n\n## Conclusions\n\nThe formation and the physical properties of milk protein gels have been the subject of intense study because of the great economic impact of these gels on dairy products such as cheese, yogurt, and heat-set whey gels. There is a growing recognition that the internal structure of casein micelles plays an important role in the structural properties of rennet, acid, and mixed gels. These gels are dynamic in nature and undergo rearrangements. Technologists have recently studied the impact of high pressure and enzymatic cross-linking of these proteins to modify their functionality. The interaction between DWP and caseins has received a lot of attention; this interaction has been used to alter the texture of acid gels, although there is disagreement about the exact mechanism(s) involved. DWP polymers have been used for making cold-set gels, and they have interesting possible applications in various milk gels\/products. Fine-stranded whey proteins made at very low pH values have been shown to be similar in structure to amyloid fibrils. From an industrial viewpoint, these fine-stranded fibril types of gels might have some useful applications because they gel at low-protein concentrations.\n\n## Acknowledgment\n\nThis material is based on work supported by the National Institute of Food and Agriculture, United States Department of Agriculture, under project WIS01650.\n\n# References\n\nAichinger P-A ,  Michel M ,  Servais C ,  Dillmann M-L ,  Rouvet M ,  D'Amico N ,  Zink R ,  Klostermeyer H ,  Horne DS .  Fermentation of a skim milk concentrate with Streptococcus thermophilus and chymosin: structure, viscoelasticity and syneresis of gels .  _Colloids and Surfaces B: Biointerfaces_.  2003 ;31 : 243 \u2013 255 .\n\nAguilera JM .  Gelation of whey proteins .  _Food Technology_.  1995 ;49 : 83 \u2013 89 .\n\nAl-Nabulsi A ,  Shaker R ,  Osailli T ,  Clark S ,  Harte F ,  Barboas-Canovas G .  Impact of high hydrostatic pressure and heat treatments on milk gel properties .  _International Journal of Food Properties_.  2012 ;15 : 613 \u2013 627 .\n\nAlexander M ,  Dalgleish DG .  Application of transmission diffusive wave spectroscopy to the study of gelation of milk by acidification and rennet .  _Colloids and Surfaces B: Biointerfaces_.  2004 ;38 : 83 \u2013 90 .\n\nAnema SG .  Effect of pH at pressure treatment on the acid gelation of skim milk .  _Innovative Food Science and Emerging Technologies_.  2010 ;11 : 265 \u2013 273 .\n\nAnema SG ,  Lee SK ,  Lowe EK ,  Klostermeyer H .  Rheological properties of acid gels prepared from heated pH-adjusted skim milk .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 337 \u2013 343 .\n\nAnema SG ,  Lauber S ,  Lee SK ,  Henle T ,  Klostermeyer H .  Rheological properties of acid gels prepared from pressure- and transglutaminase-treated skim milk .  _Food Hydrocolloids_.  2005 ;19 : 879 \u2013 887 .\n\nAnema SG ,  Lee SK ,  Klostermeyer H .  Effect of pH at heat treatment on the hydrolysis of \u03ba-casein and the gelation of skim milk by chymosin .  _Lebensmittel-Wissenschaft und -Technologie_.  2007 ;40 : 99 \u2013 106 .\n\nArdelean AI ,  Jaros D ,  Rohm H .  Influence of microbial transglutaminase cross-linking on gelation kinetics and texture of acid gels made from whole goats and cows milk .  _Dairy Science and Technology_.  2013 ;93 : 63 \u2013 71 .\n\nBarbut S ,  Foegeding EA .  Calcium-induced gelation of preheated whey protein isolate .  _Journal of Food Science_.  1993 ;58 : 867 \u2013 871 .\n\nBhattacharjee C ,  Saha S ,  Biswas A ,  Kundu M ,  Ghosh L ,  Das KP .  Structural changes of \u03b2-lactoglobulin during thermal unfolding and refolding\u2014an FT-IR and circular dichroism study .  _Protein Journal_.  2005 ;24 : 27 \u2013 35 .\n\nBikker JF ,  Anema SG ,  Li Y ,  Hill JP .  Rheological properties of acid gels prepared from heated milk fortified with whey protein mixture containing the A, B, and C variants of \u03b2-lactoglobulin .  _International Dairy Journal_.  2000 ;10 : 723 \u2013 732 .\n\nBolder SG ,  Hendrickx H ,  Sagis LMC ,  van der Linden E .  Fibril assemblies in aqueous whey protein mixtures .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 4229 \u2013 4234 .\n\nBottcher SR ,  Foegeding EA .  Whey protein gels\u2014fracture stress and strain and related microstructural properties .  _Food Hydrocolloids_.  1994 ;8 : 113 \u2013 123 .\n\nBritten M ,  Giroux HJ .  Acid-induced gelation of whey protein polymers: effects of pH and calcium concentration during polymerization .  _Food Hydrocolloids_.  2001 ;15 : 609 \u2013 617 .\n\nBrown KM ,  McManus WR ,  McMahon DJ .  Starch addition in renneted milk gels: partitioning between curd and whey and effect on curd syneresis and gel microstructure .  _Journal of Dairy Science_.  2012 ;95 : 6871 \u2013 6881 .\n\nCastillo M ,  Lucey JA ,  Payne FA .  The effect of temperature and inoculum concentration on rheological and light scatter properties of milk coagulated by a combination of bacterial fermentation and chymosin. Cottage cheese-type gels .  _International Dairy Journal_.  2006 ;16 : 131 \u2013 146 .\n\nCaussin F ,  Famelart MH ,  Maubois J-L ,  Bouhallab S .  Mineral modulation of thermal aggregation and gelation of whey proteins: From \u03b2-lactoglobulin model system to whey protein isolate .  _Lait_.  2003 ;83 : 1 \u2013 12 .\n\nCho YH ,  Lucey JA ,  Singh H .  Rheological properties of acid milk gels as affected by the nature of the fat globule surface material and heat treatment of milk .  _International Dairy Journal_.  1999 ;9 : 537 \u2013 545 .\n\nChoi J ,  Horne DS ,  Lucey JA .  Effect of insoluble calcium concentration on rennet coagulation properties of milk .  _Journal of Dairy Science_.  2007 ;90 : 2612 \u2013 2623 .\n\nClark AH .  Gels and gelling .  In:  Schwartzberg HG ,  Hartel RW ,  eds.  _Physical Chemistry of Foods_ .  New York :  Marcel Dekker ;  1992 : 263 \u2013 305 .\n\nClark AH .  Biopolymer gels .  _Current Opinion in Colloid and Interface Science_.  1996 ;1 : 712 \u2013 717 .\n\nClark AH .  Gelation of globular proteins .  In:  Hill SE ,  Ledward DA ,  Mitchell JR ,  eds.  _Functional Properties of Food Macromolecules_ .  2nd ed. Gaithersburg, MD :  Aspen Publishers ;  1998 : 77 \u2013 142 .\n\nConsidine T ,  Patel HA ,  Anema SG ,  Singh H ,  Creamer LK .  Interactions of milk proteins during heat and high hydrostatic pressure treatments\u2014a review .  _Innovative Food Science and Emerging Technologies_.  2007 ;8 : 1 \u2013 23 .\n\nCrabbe MJC .  Rennet: general and molecular aspects .  In:  Fox PF ,  McSweeney PLH ,  Cogan TM ,  Guinee TP ,  eds.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  3rd ed. London :  Elsevier ;  2004 : 19 \u2013 43 .\n\nDalgleish DG .  The enzymatic coagulation of milk .  In:  Fox PF ,  ed.  _Cheese: Chemistry, Physics and Microbiology_ .  London :  Elsevier Applied Science ;  1987 : 63 \u2013 95 .\n\nDalgleish DG .  The enzymatic coagulation of milk .  In:  Fox PF ,  ed.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  2nd ed. London :  Chapman & Hall ;  1993 : 69 \u2013 100 .\n\nDalgleish DG ,  Alexander M ,  Corredig M .  Studies of the acid gelation of milk using ultrasonic spectroscopy and diffusing wave spectroscopy .  _Food Hydrocolloids_.  2006 ;18 : 747 \u2013 755 .\n\nde Kruif KG ,  Hoffman MAM ,  van Marle ME ,  van Mil PJJM ,  Roefs SPFM ,  Verheul M ,  Zoon N .  Gelation of proteins from milk .  _Faraday Discussions_.  1995 ;101 : 185 \u2013 200 .\n\nde Wit JN .  Structure and functional behaviour of whey proteins .  _Netherlands Milk and Dairy Journal_.  1981 ;35 : 47 \u2013 64 .\n\nDejmek P ,  Walstra P .  The syneresis of rennet-coagulated curd .  In:  Fox PF ,  McSweeney PLH ,  Cogan TM ,  Guinee TG ,  eds.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  3rd ed. London :  Elsevier ;  2004 : 71 \u2013 103 .\n\nDoi E .  Gels and gelling of globular proteins .  _Trends in Food Science and Technology_.  1993 ;4 : 1 \u2013 5 .\n\nDoucet D ,  Gauthier SF ,  Otter DE ,  Foegeding EA .  Enzyme-induced gelation of extensively hydrolyzed whey proteins by alcalase: Comparison with the plastein reaction and characterization of interactions .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 6036 \u2013 6042 .\n\nDr\u00f8hse HB ,  Foltmann B .  Specificity of milk-clotting enzymes towards bovine \u03ba-casein .  _Biochimica et Biophysica Acta_.  1989 ;995 : 221 \u2013 224 .\n\nEissa AS ,  Khan SA .  Acid-induced gelation of enzymatically modified, preheated whey proteins .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 5010 \u2013 5017 .\n\nEissa AS ,  Bisram S ,  Khan SA .  Polymerization and gelation of whey protein isolates at low pH using transglutaminase enzyme .  _Journal of Agricultural and Food Chemistry_.  2004 ;52 : 4456 \u2013 4464 .\n\nElfagm AA ,  Wheelock JV .  Interaction of bovine \u03b1-lactalbumin and \u03b2-lactoglobulin during heating .  _Journal of Dairy Science_.  1978 ;61 : 28 \u2013 32 .\n\nErcili-Cura D ,  Lille M ,  Legland D ,  Gaucel S ,  Poutanen K ,  Partanen R ,  Lantto R .  Structural mechanisms leading to improved water retention in acid gels by use of transglutaminase .  _Food Hydrocolloids_.  2013 ;30 : 419 \u2013 427 .\n\nEsteves CLC ,  Lucey JA ,  Pires EMV .  Mathematical modelling of the formation of rennet-induced gels by plant coagulants and chymosin .  _Journal of Dairy Research_.  2001 ;68 : 499 \u2013 510 .\n\nEsteves CLC ,  Lucey JA ,  Pires EMV .  Rheological properties of milk gels made using coagulants of plant origin and chymosin .  _International Dairy Journal_.  2002 ;12 : 427 \u2013 434 .\n\nEsteves CLC ,  Lucey JA ,  Hyslop DB ,  Pires EMV .  Effect of gelation temperature on the properties of skim milk gels made from plant coagulants and chymosin .  _International Dairy Journal_.  2003 ;13 : 877 \u2013 885 .\n\nEuston SR .  Computer simulation of proteins: Adsorption, gelation and self-association .  _Current Opinion in Colloid and Interface Science_.  2004 ;9 : 321 \u2013 327 .\n\nFaergemand M ,  Qvist KB .  Transglutaminase: Effect on rheological properties, microstructure and permeability of set style acid skim milk gel .  _Food Hydrocolloids_.  1997 ;11 : 287 \u2013 292 .\n\nFaergemand M ,  Otte J ,  Qvist KB .  Enzymatic crosslinking of whey proteins by a Ca2+ independent microbial transglutaminase from Streptomyces lydicus .  _Food Hydrocolloids_.  1997 ;11 : 19 \u2013 25 .\n\nFaergemand M ,  S\u00f8rensen MV ,  J\u00f8rgensen U ,  Budolfsen G ,  Qvist KB .  Transglutaminase: Effect on instrumental and sensory texture of set style yoghurt .  _Milchwissenschaft_.  1999 ;54 : 563 \u2013 566 .\n\nFagan CC ,  Leedy M ,  Castillo M ,  Payne FA ,  O'Donnell CP ,  O'Callaghan DJ .  Development of a light scatter sensor technology for on-line monitoring of milk coagulation and whey separation .  _Journal of Food Engineering_.  2006 ;83 : 61 \u2013 67 .\n\nFoegeding EA .  Food biophysics of protein gels: A challenge of nano and macroscopic proportions .  _Food Biophysics_.  2006 ;1 : 41 \u2013 50 .\n\nFoegeding EA ,  Bowland EI ,  Hardin CC .  Factors that determine the fracture properties and microstructure of globular protein gels .  _Food Hydrocolloids_.  1995 ;9 : 237 \u2013 249 .\n\nGastaldi E ,  Trial N ,  Guillaume C ,  Bourret E ,  Gontard N ,  Cuq JL .  Effect of controlled \u03ba-casein hydrolysis on rheological properties of acid milk gels .  _Journal of Dairy Science_.  2003 ;86 : 704 \u2013 711 .\n\nGirard M ,  Schaffer-Lequart C .  Gelation of skim milk containing anionic exopolysaccharides and recovery of texture after shearing .  _Food Hydrocolloids_.  2007 ;21 : 1031 \u2013 1040 .\n\nGoers J ,  Permyakov SE ,  Permyakov EA ,  Uversky VN ,  Fink AL .  Conformational prerequisites for alpha-lactalbumin fibrillation .  _Biochemistry_.  2002 ;41 : 12546 \u2013 12551 .\n\nGosal WS ,  Ross-Murphy SB .  Globular protein gelation .  _Current Opinion in Colloid and Interface Science_.  2000 ;5 : 188 \u2013 194 .\n\nGosal WS ,  Clark AH ,  Ross-Murphy SB .  Fibrillar \u03b2-lactoglobulin gels: Part 1. Fibril formation and structure .  _Biomacromolecules_.  2004 ;5 : 2408 \u2013 2419 .\n\nGreen ML .  The formation and structure of milk protein gels .  _Food Chemistry_.  1980 ;6 : 41 \u2013 49 .\n\nGreen ML ,  Grandison AS .  Secondary (non-enzymatic) phase of rennet coagulation and post-coagulation phenomena .  In:  Fox PF ,  ed.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  2nd ed Gaithersburg, MD :  Aspen Publishers ;  1993 : 101 \u2013 140 .\n\nGupta P ,  Hall CK ,  Voegler A .  Computer simulation of the competition between protein folding and aggregation .  _Fluid Phase Equilibria_.  1999 ; : 158 \u2013 160 : 87-93 .\n\nGuyomarc'h F ,  Queguiner C ,  Law AJR ,  Horne DS ,  Dalgleish DG .  Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels .  _Journal of Agricultural and Food Chemistry_.  2003 ;51 : 7743 \u2013 7750 .\n\nHan X-Q ,  Damodaran S .  Thermodynamic compatability of substrate proteins affects their cross-linking by transglutaminase .  _Journal of Agricultural and Food Chemistry_.  1996 ;44 : 1211 \u2013 1217 .\n\nHarte F ,  Luedecke L ,  Swanson B ,  Barbosas-C\u00e1novas GV .  Low-fat set yogurt made from milk subjected to combinations of high hydrostatic pressure and thermal processing .  _Journal of Dairy Science_.  2003 ;86 : 1074 \u2013 1082 .\n\nHassan AN .  Possibilities and challenges of exopolysaccharide-producing lactic cultures in dairy foods .  _Journal of Dairy Science_.  2008 ;91 : 1282 \u2013 1298 .\n\nHavea P ,  Singh H ,  Creamer LK .  Characterization of heat-induced aggregates of \u03b2-lactoglobulin, \u03b1-lactalbumin and bovine serum albumin in a whey protein concentrate environment .  _Journal of Dairy Research_.  2001 ;68 : 483 \u2013 497 .\n\nHavea P ,  Singh H ,  Creamer LK .  Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition .  _Journal of Agricultural and Food Chemistry_.  2002 ;50 : 4674 \u2013 4681 .\n\nHernandez A ,  Harte FM .  Manufacture of acid gels from skim milk using high-pressure homogenization .  _Journal of Dairy Science_.  2008 ;91 : 3761 \u2013 3767 .\n\nHillier RM ,  Cheeseman GC .  Effect of proteose-peptone on the heat gelation of whey protein isolate .  _Journal of Dairy Research_.  1979 ;46 : 113 \u2013 120 .\n\nHines ME ,  Foegeding EA .  Interactions of \u03b1-lactalbumin and bovine serum albumin with \u03b2-lactoglobulin in thermally induced gelation .  _Journal of Agricultural and Food Chemistry_.  1993 ;41 : 341 \u2013 346 .\n\nHolt C ,  Sawyer L .  The principal bovine whey protein \u03b2-lactoglobul: a structure: function analysis .  In:  Aalbersberg WY ,  Hamer RJ ,  Jasperse P ,  de Jong HHJ ,  de Kruif CG ,  Walstra P ,  de Wolf FA ,  eds.  _Industrial Proteins in Perspective, Progress in Biotechnology 23_ .  Amsterdam :  Elsevier Science ;  2003 : 44 \u2013 49 .\n\nHorne DS .  Scaling behaviour of shear moduli during the formation of rennet milk gels .  In:  Dickinson E ,  Lorient D ,  eds.  _Food Macromolecules and Colloids_ .  Cambridge :  Royal Society of Chemistry ;  1995 : 456 \u2013 461 .\n\nHorne DS .  Aspects of scaling behaviour in the kinetics of particle formation .  _Journal de Chimie Physique et de Physico-Chimie Biologique_.  1996 ;93 : 977 \u2013 986 .\n\nHorne DS .  Casein interactions: Casting light on the black boxes, the structure in dairy products .  _International Dairy Journal_.  1998 ;8 : 171 \u2013 177 .\n\nHorne DS .  Formation and structure of acidified milk gels .  _International Dairy Journal_.  1999 ;9 : 261 \u2013 268 .\n\nHorne DS .  Factors influencing acid-induced gelation of skim milk .  In:  Dickinson E ,  Miller R ,  eds.  _Food Hydrocolloids: Fundamentals of Formulation_ .  Cambridge :  Royal Society of Chemistry ;  2001 : 345 \u2013 351 .\n\nHorne DS .  Casein micelles as hard spheres: Limitations of the model in acidified milk gels .  _Colloids and Surfaces A: Physicochemical Engineering Aspects_.  2003 ;213 : 255 \u2013 263 .\n\nHorne DS ,  Banks JM .  Rennet-induced coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  Cogan T ,  Guinee T ,  eds.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  3rd ed. London :  Elsevier ;  2004 : 47 \u2013 70 .\n\nHuppertz T ,  de Kruif CG .  Disruption and reassociation of casein micelles under high pressure: Influence of milk serum composition and casein micelle concentration .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 5903 \u2013 5909 .\n\nHuppertz T ,  de Kruif CG .  Rennet-induced coagulation of enzymatically cross-linked casein micelles .  _International Dairy Journal_.  2007 ;17 : 442 \u2013 447 .\n\nHuppertz T ,  Hinz K ,  Zobrist MR ,  Uniacke T ,  Kelly AL ,  Fox PF .  Effects of high pressure treatment on the rennet coagulation and cheese-making properties of heated milk .  _Innovative Food Science and Emerging Technologies_.  2005 ;6 : 279 \u2013 285 .\n\nHyslop DB .  Enzymatic coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Proteins_ .  2nd ed. Gaithersburg, MD :  Aspen Publishers ;  2003 : 839 \u2013 878 .\n\nIkeda S ,  Foegeding EA ,  Hagiwara T .  Rheological study of the fractal nature of the protein gel structure .  _Langmuir_.  1999 ;15 : 8584 \u2013 8589 .\n\nJaros D ,  Rohm H .  The rheology and textural properties of yoghurt .  In:  McKenna BM ,  ed.  _Texture in Food, Volume 1_ .  Woodhead Publishing :  Cambridge ;  2003 : 321 \u2013 349 .\n\nJaros D ,  Rohm H .  Controlling the texture of fermented dairy products: The case of yoghurt .  In:  Smit G ,  ed.  _Dairy Processing: Improving Quality_ .  Cambridge :  Woodhead Publishing ;  2003 : 155 \u2013 184 .\n\nJaros D ,  Partschefeld C ,  Henle T ,  Rohm H .  Transglutaminase in dairy products: Chemistry, physics, applications .  _Journal of Texture Studies_.  2006 ;37 : 113 \u2013 155 .\n\nJean K ,  Renan M ,  Famelart MH ,  Guyomarc'h F .  Structure and surface properties of the serum heat-induced protein aggregates isolated from heated skim milk .  _International Dairy Journal_.  2006 ;16 : 303 \u2013 315 .\n\nJou KD ,  Harper WJ .  Effect of di-saccharides on the thermal properties of whey proteins determined by differential scanning calorimetry (DSC) .  _Milchwissenschaft_.  1996 ;51 : 509 \u2013 512 .\n\nKal\u00e1b M ,  Allan-Wojtas P ,  Phipps-Todd BE .  Development of microstructure in set-style nonfat yoghurt\u2014a review .  _Food Microstructure_.  1983 ;2 : 51 \u2013 66 .\n\nKappeler SR ,  van den Brink HJ ,  Rahbek-Nielsen H ,  Farah Z ,  Puhan Z ,  Hansen EB ,  Johansen E .  Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk .  _Biochemical and Biophysical Research Communications_.  2006 ;342 : 647 \u2013 654 .\n\nKarlsson AO ,  Ipsen R ,  Ard\u00f6 Y .  Influence of pH and NaCl on rheological properties of rennet-induced casein gels made from UF concentrated skim milk .  _International Dairy Journal_.  2007 ;17 : 1053 \u2013 1062 .\n\nKarlsson AO ,  Ipsen R ,  Ard\u00f6 Y .  Rheological properties and microstructure during rennet induced coagulation of UF concentrated skim milk .  _International Dairy Journal_.  2007 ;17 : 674 \u2013 682 .\n\nKella NK ,  Kinsella JE .  Structural stability of \u03b2-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study .  _International Journal of Peptide and Protein Research_.  1988 ;32 : 396 \u2013 405 .\n\nKella NK ,  Kinsella JE .  Enhanced thermodynamic stability of \u03b2-lactoglobulin at low pH. A possible mechanism .  _Biochemical Journal_.  1988 ;255 : 113 \u2013 118 .\n\nKlandar AH ,  Lagaude A ,  Chevalier-Lucia D .  Assessment of the rennet coagulation of skim milk: A comparison of methods .  _International Dairy Journal_.  2007 ;17 : 1151 \u2013 1160 .\n\nKnudsen JC ,  Karlsson AO ,  Ipsen R ,  Skibsted LH .  Rheology of stirred acidified skim milk gels with different particle interactions .  _Colloids and Surfaces A: Physicochemical and Engineering Aspects_.  2006 ;274 : 56 \u2013 61 .\n\nKuhn PR ,  Foegeding EA .  Mineral salt effects on whey protein gelation .  _Journal of Agricultural and Food Chemistry_.  1991 ;39 : 1013 \u2013 1016 .\n\nLauber S ,  Henle T ,  Klostermeyer H .  Relationship between the crosslinking of caseins by transglutaminase and the gel strength of yoghurt .  _European Food Research and Technology Journal_.  2000 ;210 : 305 \u2013 309 .\n\nLauber S ,  Noack I ,  Klostermeyer H ,  Henle T .  Stability of microbial transglutaminase to high pressure treatment .  _European Food Research and Technology Journal_.  2001 ;213 : 273 \u2013 276 .\n\nLauger J ,  Wollny K ,  Huck S .  Direct strain oscillation: A new oscillatory method enabling measurements at very small shear stresses and strains .  _Rheologica Acta_.  2002 ;41 : 356 \u2013 361 .\n\nLee WJ ,  Lucey JA .  Structure and physical properties of yogurt gels: Effect of inoculation rate and incubation temperature .  _Journal of Dairy Science_.  2004 ;87 : 3153 \u2013 3164 .\n\nLi J ,  Dalgleish DG .  Controlled proteolysis and the properties of milk gels .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 4687 \u2013 4695 .\n\nLodaite K ,  \u00d6stergren K ,  Paulsson M ,  Dejmek P .  One-dimensional syneresis of rennet-induced gels .  _International Dairy Journal_.  2000 ;10 : 829 \u2013 834 .\n\nLomholt SB ,  Qvist KB .  The formation of cheese curd .  In:  Law BA ,  ed.  _Technology of Cheesemaking_ .  Sheffield :  Sheffield Academic Press ;  1999 : 66 \u2013 98 .\n\nL\u00f3pez-Fandi\u00f1o R .  High pressure-induced changes in milk proteins and possible applications in dairy technology .  _International Dairy Journal_.  2006 ;16 : 1119 \u2013 1131 .\n\nLorenzen PC .  Renneting properties of transglutaminase-treated milk .  _Milchwissenschaft_.  2000 ;55 : 433 \u2013 437 .\n\nLucey JA .  Effect of heat treatment on the rennet coagulability of milk .  In:  Fox PF ,  ed.  _Heat-induced Changes in Milk_ .  2nd ed. Brussels :  International Dairy Federation ;  1995 : 171 \u2013 187 : IDF Special Issue 9501 .\n\nLucey JA .  The relationship between rheological parameters and whey separation in milk gels .  _Food Hydrocolloids_.  2001 ;15 : 603 \u2013 608 .\n\nLucey JA .  Formation and physical properties of milk protein gels .  _Journal of Dairy Science_.  2002 ;85 : 281 \u2013 294 .\n\nLucey JA .  Formation, structure, properties and rheology of acid-coagulated milk gels .  In:  Fox PF ,  McSweeney PLH ,  Cogan TM ,  Guinee TP ,  eds.  _In Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  3rd ed. London :  Elsevier ;  2003 : 105 \u2013 122 .\n\nLucey JA ,  Singh H .  Formation and physical properties of acid milk gels: A review .  _Food Research International_.  1998 ;30 : 529 \u2013 542 .\n\nLucey JA ,  Singh H .  Acid coagulation of milk .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Proteins_ .  2nd ed. Gaithersburg, MD :  Aspen Publishers ;  2003 : 1001 \u2013 1026 .\n\nLucey JA ,  Teo CT ,  Munro PA ,  Singh H .  Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk .  _Journal of Dairy Research_.  1997 ;64 : 591 \u2013 600 .\n\nLucey JA ,  Tamehana M ,  Singh H ,  Munro PA .  A comparison of the formation, rheological properties and microstructure of acid skim milk gels made with a bacterial culture or glucono-\u03b4-lactone .  _Food Research International_.  1998 ;31 : 147 \u2013 155 .\n\nLucey JA ,  Tamehana M ,  Singh H ,  Munro PA .  Effect of interactions between denatured whey proteins and casein micelles on the formation and rheological properties of acid skim milk gels .  _Journal of Dairy Research_.  1998 ;65 : 555 \u2013 567 .\n\nLucey JA ,  Munro PA ,  Singh H .  Effects of heat treatment and whey protein addition on the rheological properties and structure of acid skim milk gels .  _International Dairy Journal_.  1999 ;9 : 275 \u2013 279 .\n\nLucey JA ,  Tamehana M ,  Singh H ,  Munro PA .  Rheological properties of milk gels formed by a combination of rennet and glucono-\u03b4-lactone .  _Journal of Dairy Research_.  2000 ;67 : 415 \u2013 427 .\n\nLucey JA ,  Tamehana M ,  Singh H ,  Munro PA .  Effect of heat treatment on the physical properties of milk gels made with both rennet and acid .  _International Dairy Journal_.  2001 ;11 : 559 \u2013 565 .\n\nMangino ME .  Gelation of whey-protein concentrates .  _Food Technology_.  1992 ;46 : 114 \u2013 117 .\n\nMatsudomi N ,  Rector D ,  Kinsella JE .  Gelation of bovine serum albumin and \u03b2-lactoglobulin; effects of pH, salts and thiol reagents .  _Food Chemistry_.  1991 ;40 : 55 \u2013 69 . \nMcCann TCA ,  Pyne GT .  The colloidal phosphate of milk. III. Nature of its association with casein .  _Journal of Dairy Research_.  1960 ;27 : 403 \u2013 417 .\n\nMcPhail D ,  Holt C .  Effect of anions on the denaturation and aggregation of \u03b2-lactoglobulin as measured by differential scanning microcalorimetry .  _International Journal of Food Science and Technology_.  1999 ;34 : 477 \u2013 481 .\n\nMcSwiney M ,  Singh H ,  Campanella OH .  Thermal aggregation and gelation of bovine \u03b2-lactoglobulin .  _Food Hydrocolloids_.  1994 ;8 : 441 \u2013 453 .\n\nMellema M ,  Walstra P ,  van Opheusden JHJ ,  van Vliet T .  Effects of structural rearrangements on the rheology of rennet-induced casein particle gels .  _Advances in Colloid and Interface Science_.  2002 ;98 : 25 \u2013 50 .\n\nMezzenga R ,  Fischer P .  The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions .  _Reports on Progress in Physics_.  2013 ; : 76 .\n\nMorand M ,  Guyomarc'h F ,  Legland D ,  Famelart M-H .  Changing the isoelectric point of the heat-induced whey protein complexes affects the acid gelation of skim milk .  _International Dairy Journal_.  2012 ;23 : 9 \u2013 17 .\n\nMulvihill DM ,  Donovan M .  Whey proteins and their thermal denaturation. A review .  _Irish Journal of Food Science and Technology_.  1987 ;11 : 43 \u2013 77 .\n\nMulvihill DM ,  Kinsella JE .  Gelation characteristics of whey proteins and \u03b2-lactoglobulin .  _Food Technology_.  1987 ;41 : 102 \u2013 111 .\n\nNeeds EC ,  Capellas M ,  Bland AP ,  Manoj P ,  Macdougal D ,  Paul G .  Comparison of heat and pressure treatments of skim milk, fortified with whey protein concentrate, for set yogurt preparation: Effects on milk proteins and gel structure .  _Journal of Dairy Research_.  2000 ;67 : 329 \u2013 348 .\n\nO'Callaghan DJ ,  O'Donnell CP ,  Payne FA .  Review of systems for monitoring curd setting during cheesemaking .  _International Journal of Dairy Technology_.  2002 ;55 : 65 \u2013 74 .\n\nO'Sullivan MM ,  Kelly AL ,  Fox PF .  Effect of transglutaminase on the heat stability of milk: A possible mechanism .  _Journal of Dairy Science_.  2002 ;85 : 1 \u2013 7 .\n\nO'Sullivan MM ,  Kelly AL ,  Fox PF .  Influence of transglutaminase treatment on some physico-chemical properties of milk .  _Journal of Dairy Research_.  2002 ;69 : 433 \u2013 442 .\n\nOakenfull D .  Gelling agents .  _CRC Critical Reviews in Food Science and Nutrition_.  1987 ;26 : 1 \u2013 25 .\n\nOakenfull D ,  Pearce J ,  Burley RW .  Protein gelation .  In:  Damodaran S ,  Paraf A ,  eds.  _Food Proteins and their Applications_ .  New York :  Marcel Dekker ;  1997 : 111 \u2013 142 .\n\nOtte J ,  Ju ZY ,  Faergemand M ,  Lomholt SB ,  Qvist KB .  Protease-induced aggregation and gelation of whey proteins .  _Journal of Food Science_.  1996 ;61 : 911 \u2013 915 : 923 .\n\nOzer B ,  Kirmaci HA ,  Oztekin S ,  Hayalogluc A ,  Atamer M .  Incorporation of microbial transglutaminase into non-fat yogurt production .  _International Dairy Journal_.  2007 ;17 : 199 \u2013 207 .\n\nPatel HA ,  Singh H ,  Anema SG ,  Creamer LK .  Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk .  _Journal of Agricultural and Food Chemistry_.  2006 ;54 : 3409 \u2013 3420 .\n\nPearse MJ ,  MacKinlay AG .  Biochemical aspects of syneresis: A review .  _Journal of Dairy Science_.  1989 ;72 : 1401 \u2013 1407 .\n\nPeng Y ,  Horne DS ,  Lucey JA .  Physical properties of acid milk gels prepared at 37C up to gelation but at different incubation temperatures for the remainder of fermentation .  _Journal of Dairy Science_.  2010 ;93 : 1910 \u2013 1917 .\n\nPrabakaran S ,  Damodaran S .  Thermal unfolding of \u03b2-lactoglobulin: Characterization of initial unfolding events responsible for heat-induced aggregation .  _Journal of Agricultural and Food Chemistry_.  1997 ;45 : 4303 \u2013 4308 .\n\nQi XL ,  Holt C ,  McNulty D ,  Clarke DT ,  Brownlow S ,  Jones GR .  Effect of temperature on the secondary structure of \u03b2-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis .  _Biochemical Journal_.  1997 ;324 : 341 \u2013 346 .\n\nRaouche S ,  Dobenesque M ,  Bot A ,  Lagaude A ,  Cuq JL ,  Marchesseau S .  Stability of casein micelle subjected to reversible CO2 acidification: impact of holding time and chilled storage .  _International Dairy Journal_.  2007 ;17 : 873 \u2013 880 .\n\nReddy IM ,  Kinsella JE .  Interaction of \u03b2-lactoglobulin with \u03ba-casein in micelles as assessed by chymosin hydrolysis: Effect of temperature, heating time, \u03b2-lactoglobulin concentration, and pH .  _Journal of Agricultural and Food Chemistry_.  1990 ;38 : 50 \u2013 58 .\n\nRenan M ,  Arnoult-Delest V ,  Paquet D ,  Brule G ,  Famelart M-H .  Changes in the rheological properties of stirred acid milk gels as induced by the acidification procedure .  _Dairy Science and Technology_.  2008 ;88 : 341 \u2013 353 .\n\nRoefs SPFM ,  van Vliet T ,  van den Bijgaart HJCM ,  de Groot-Mostert AEA ,  Walstra P .  Structure of casein gels made by combined acidification and rennet action .  _Netherlands Milk and Dairy Journal_.  1990 ;44 : 158 \u2013 188 .\n\nRuegg M ,  Moor U ,  Blanc B .  A calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate .  _Journal of Dairy Research_.  1977 ;44 : 509 \u2013 520 .\n\nSandra S ,  Alexander M ,  Dalgleish DG .  The rennet coagulation mechanism of skim milk as observed by transmission diffusing wave spectroscopy .  _Journal of Colloid and Interface Science_.  2007 ;308 : 364 \u2013 373 .\n\nSchmidt RH ,  Illingworth BL ,  Deng JC ,  Cornell JA .  Multiple regression and response surface analysis of the effects of calcium chloride and cysteine on heat-induced whey protein gelation .  _Journal of Agricultural and Food Chemistry_.  1979 ;27 : 529 \u2013 532 .\n\nSchorsch C ,  Carrie H ,  Norton IT .  Cross-linking casein micelles by a microbial transglutaminase: Influence of cross-links in acid-induced gelation .  _International Dairy Journal_.  2000 ;10 : 529 \u2013 539 .\n\nSchorsch C ,  Wilkins DK ,  Jones MG ,  Norton IT .  Gelation of casein-whey mixtures: effects of heating whey proteins alone or in the presence of casein micelles .  _Journal of Dairy Research_.  2001 ;68 : 471 \u2013 481 .\n\nSerra M ,  Trujillo AJ ,  Quevedo JM ,  Guamis B ,  Ferragut V .  Acid coagulation properties and suitability for yogurt production of cows' milk treated by high-pressure homogenization .  _International Dairy Journal_.  2007 ;17 : 782 \u2013 790 .\n\nSingh H ,  Havea P .  Thermal denaturation, aggregation and gelation of whey proteins .  In:  Fox PF ,  McSweeney PLH ,  eds.  _Advanced Dairy Chemistry, Volume 2, Proteins_ .  2nd ed. Gaithersburg, MD :  Aspen Publishers ;  2003 : 1261 \u2013 1287 .\n\nSrinivasan M ,  Lucey JA .  Effects of added plasmin on the formation and rheological properties of rennet-induced skim milk gels .  _Journal of Dairy Science_.  2002 ;85 : 1070 \u2013 1078 .\n\nStading M ,  Hermansson A-M .  Viscoelastic behaviour of \u03b2-lactoglobulin gel structures .  _Food Hydrocolloids_.  1990 ;4 : 121 \u2013 135 .\n\nStading M ,  Hermansson A-M .  Large deformation properties of \u03b2-lactoglobulin gel structures .  _Food Hydrocolloids_.  1991 ;5 : 339 \u2013 352 .\n\nTamime AY ,  Robinson RK .  _Yoghurt\u2014Science and Technology_ .  Cambridge :  Woodhead Publishers ;  1999 .\n\nTranchant CC ,  Dalgleish DG ,  Hill AR .  Different coagulation behaviour of bacteriologically acidified and renneted milk: The importance of fine-tuning acid production and rennet action .  _International Dairy Journal_.  2001 ;11 : 483 \u2013 494 .\n\nvan Dijk HJM ,  Walstra P .  Syneresis of curd. 2. One-dimensional syneresis of rennet curd in constant conditions .  _Netherlands Milk and Dairy Journal_.  1986 ;40 : 3 \u2013 30 .\n\nvan Vliet T ,  Walstra P .  Water in casein gels: How to get it out or keep it in .  _Journal of Food Engineering_.  1994 ;22 : 75 \u2013 88 .\n\nvan Vliet T ,  van Dijk HJM ,  Zoon P ,  Walstra P .  Relation between syneresis and rheological properties of particle gels .  _Colloid and Polymer Science_.  1991 ;269 : 620 \u2013 627 .\n\nvan Vliet T ,  Lakemond CMM ,  Visschers RW .  Rheology and structure of milk protein gels .  _Current Opinion in Colloid and Interface Science_.  2004 ;9 : 298 \u2013 304 .\n\nVasbinder AJ ,  Rollema HS ,  de Kruif CG .  Impaired rennetability of heated milk; study of enzymatic hydrolysis and gelation kinetics .  _Journal of Dairy Science_.  2003 ;86 : 1548 \u2013 1555 .\n\nVeerman C ,  Sagis LMC ,  Heck J ,  van der Linden E .  Mesostructure of fibrillar bovine serum albumin gels .  _International Journal of Biological Macromolecules_.  2003 ;31 : 139 \u2013 146 .\n\nVeith PD ,  Reynolds EC .  Production of a high gel strength whey protein concentrate from cheese whey .  _Journal of Dairy Science_.  2004 ;87 : 831 \u2013 840 .\n\nVreeker R ,  Hoekstra LL ,  den Boer DC ,  Agterof WGM .  Fractal aggregation of whey proteins .  _Food Hydrocolloids_.  1992 ;6 : 423 \u2013 435 .\n\nWalstra P .  On the stability of casein micelles .  _Journal of Dairy Science_.  1990 ;73 : 1965 \u2013 1979 .\n\nWalstra P .  The syneresis of curd .  In:  Fox PF ,  ed.  _Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects_ .  2nd ed. London :  Chapman & Hall ;  1993 : 141 \u2013 191 .\n\nWalstra P ,  Relationship between structure and texture of cultured milk products .  _Texture of Fermented Milk Products and Dairy Desserts_ Brussels :  International Dairy Federation ;  1998 : 9 \u2013 15 : IDF Special Issue 9802 .\n\nWalstra P ,  van Dijk HJM ,  Geurts TJ .  The syneresis of curd. 1. General considerations and literature review .  _Netherlands Milk and Dairy Journal_.  1985 ;39 : 209 \u2013 246 .\n\nWalstra P ,  Wouters JTM ,  Geurts TJ .  _Dairy Science and Technology_ .  2nd ed. New York :  CRC Press ;  2005 .\n\nWang Q ,  Bulca S ,  Kulozik U .  A comparison of low-intensity ultrasound and oscillating rheology to assess the renneting properties of casein solutions after UHT heat pre-treatment .  _International Dairy Journal_.  2007 ;17 : 50 \u2013 58 .\n\nWang T ,  Lucey JA .  Use of multi-angle laser light scattering and size-exclusion chromatography to characterize the molecular weight and types of aggregates present in commercial whey protein products .  _Journal of Dairy Science_.  2003 ;86 : 3090 \u2013 3101 . \nChapter 18\n\n# Milk Proteins\u2014A Cornucopia for Developing Functional Foods\n\nPaul J. Moughan Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nMilk proteins have a central role to play in the development of functional foods, foods that have targeted physiological effects in the body over and above the normal effects of food nutrients. Milk proteins contain high amounts of bioavailable amino acids, making them ideal ingredients for the manufacture of nutritionals, foods designed for specific nutritional purposes. Certain amino acids have specific physiological roles (e.g., tryptophan as a precursor of serotonin or leucine in the regulation of muscle metabolism), and some isolated milk proteins have particularly high concentrations of these amino acids, allowing foods to be developed to target physiological end points. Milk proteins, especially whey protein and glycomacropeptide, have an application in inducing satiety in humans, and the relatively low yield of adenosine triphosphate (ATP) per unit amino acid in comparison with glucose or fatty acids means that milk proteins are ideal ingredients for weight-loss foods. Finally, milk proteins are known to be a rich source of bioactive peptides, released in the gut naturally during digestion. These peptides have a plethora of physiological effects including effects locally at the gut level. The multiple nutritional and physiological properties of milk proteins and peptides in the context of functional foods are discussed.\n\n## Keywords\n\nMilk\n\nproteins\n\namino acids\n\nbioactive peptides\n\nfunctional foods\n\nOutline\n\nIntroduction 525\n\nFunctional foods 526\n\nMilk proteins as a source of amino acids\u2014specialized nutritionals 528\n\nMilk proteins as a source of amino acids\u2014specific physiological roles 531\n\nMilk proteins as a source of amino acids\u2014role in providing calories and in promoting satiety 533\n\nMilk proteins as a source of bioactive peptides 534\n\nConclusions 537\n\n## Introduction\n\nOver the last century, scientists have gradually come to better understand the complexity of foods. In addition to delivering nutrients, satisfying hunger, and providing pleasure, food components are now known to have a role in directly influencing physiological processes in the body. In particular, certain food components may assist in maintaining or promoting health and well-being and preventing the development of disease. These components may be non-nutrients (e.g., the antioxidant effect of polyphenols or the blood cholesterol-lowering effect of plant sterols) or nutrients (e.g., the effects of short-chain volatile fatty acids in modulating gut development and function). In fact, the multiple effects of some chemical compounds released from foods during digestion, traditionally considered to have a sole role in nourishment, call into question the very definition of 'a nutrient.'\n\nIt is also now widely appreciated that body growth and maintenance processes are a subtle interaction between nutrient supply and genetically regulated metabolism. The assimilation and metabolism of food compounds is subject to genetic and epigenetic control, which will vary among individuals and diverse populations, leading to important gene\u2013nutrient interactions. In turn, nutrients and food non-nutrients also greatly influence gene expression, with effects once again varying among individuals, and the complexity and the subtlety of nutrigenomics and nutrigenetics are really only beginning to be understood (Ferguson, 2011). Over the next 50 years, it is expected that great advances will be made in understanding how food influences gene expression, how genetic regulation influences the assimilation and utilization of nutrients, and how the individual's genome explains differences among individuals in their physiological and nutritional response to different foods under different conditions. Such understanding will pave the path toward personalized nutrition and personalized health foods and dietary\/exercise regimens.\n\nMilk is an excellent example of a food having both nutritional and non-nutritional physiological roles in the human diet. Milk and, in particular, milk proteins not only supply the body with amino acids necessary for the maintenance and growth of body protein, but during food manufacture and\/or food digestion give rise to a myriad of protein fragments and large and small peptides that have distinct biological functions (Ward and German, 2004). Certain amino acids (e.g., leucine, tryptophan) released during digestion have regulatory functions or act as precursors for the synthesis of key nonprotein metabolites. Such compounds are a rich source of bioactive components for the development of functional foods. Although these compounds are undoubtedly of major significance to the suckled infant, whereby milk should be regarded as a biological fluid specifically designed by nature for optimal growth and development, they are also probably of importance in the adult diet, where milk has long been an important constituent.\n\n## Functional foods\n\nA functional food has been defined by Diplock et al. (1999, p. 1) as follows:\n\nA food can be regarded as functional if it has beneficial effects on target functions in the body beyond nutritional effects in a way that is relevant to health and well-being and\/or the reduction of disease.\n\nIn a sense, and following this definition strictly, most if not all foods could be described as 'functional.' Perhaps, then, the definition needs to be expanded to include the notion that the 'beneficial effect on target functions' occurs at a meaningful level. Minor beneficial effects on target functions, which would be consequent upon the ingestion of many mainstream foods, may be relevant to health, well-being, and disease prevention in the context of a long-term balanced diet, but such foods are not widely regarded as functional foods.\n\nUnder the latter definition of a functional food, foods may be functional naturally (e.g., oily fish containing high amounts of omega 3 fatty acids) or may be rendered functional, usually by adding a bioactive component or by removing some component that is inhibiting bioactivity. Foods may also be enriched with a given bioactive component or components, through conventional animal or plant breeding practices, by genetic engineering or by manipulation of the feeding and nutrition of the plant or animal (Moughan, 2011).\n\nFunctional foods is a rapidly growing sector of the international food industry, with development spurred by a number of technical, social, and economic drivers. First, there is a high degree of awareness of the link between diet and health, which has been established largely through well-publicized epidemiological studies. This knowledge has been expanded by the more cogent 'proof of cause and effect' from human intervention studies. Well-educated consumers, aware of the importance of diet to health, are demanding healthy and functional foods and are prepared to pay a price premium. Escalating health care costs, a major concern to governments, encourage 'disease prevention rather than cure' community health strategies. Completion of the human genome project with the subsequent rapid accumulation of knowledge concerning single and multiple gene effects (heralding presymptomatic testing for disposition to particular conditions) is being coupled with a better understanding of intergenerational nutritional effects (Barker hypothesis) on predisposition to chronic disease states. This is likely to lead in the near future to personalized nutrition strategies and to further demand for specific functional foods tailored to the requirements of the individual (mass customization).\n\nIf the functional foods industry is to achieve its full potential, it will be critical that regulatory bodies have a clear, consistent, and rigorous approach to safety, labeling, and health claims issues, and that food manufacturers contract reputable science providers to independently establish 'proof of concept' around their products (Roberfroid, 2000). There will also need to be a considerable investment in research and development to clearly establish cause-and-effect relationships between food compounds and targeted physiological end points and recognized disease risk factors. Consumer confidence can quickly be eroded by conflicting messages received from the scientific community. This underlines the critical role of regulatory authorities in ensuring that sufficient credible information is available and is correctly analyzed using meta-analysis techniques in order to make sustainable health claims, both qualified and approved claims. The burden of proof for generic health claims and for claims on the efficacy of specific food products needs to be considerable, such that consumers can have high levels of confidence.\n\nFoods undoubtedly have a major role to play in preventing disease and ensuring health and vitality. However, if functional foods are to achieve their potential as part of an overall lifestyle stratagy toward healthfulness, then consumers must be guided by the highest quality information and distilled findings that have a strong likelihood of remaining substantiated over time. The food industry, science providers, and government bodies all have a responsibility to ensure that the functional foods movement is led by ethical and informed decision making. Although the Institute of Food Technologists' (IFT, USA) Expert Report on Functional Foods (2005) recognized the enormous potential for functional foods, it stated: \"But that is not to say that IFT believes that all foods on the market for which claims are being made are being properly represented based on science and proper regulatory policies. IFT does not support some claims on foods marketed today because they are not supported by today's science.\" Clearly there is a challenge! The IFT Expert Panel goes on to recommend basing structure\/function health claims on broad-based scientific criteria that address the underlying link between health and nutrition and meet the need for sound scientific substantiation supporting the structure\/function effect. The panel discusses principles around ensuring the safety of functional foods and has introduced the concept of GRAE (generally recognized as efficacious), analogous to GRAS (generally recognized as safe), to encourage public confidence in the labeling of functional foods.\n\nThe dairy industry is in an excellent position to take advantage of the trend toward more healthy diets and more healthy and functional foods. Because milk proteins are readily available sources of amino acids and give rise to many bioactive compounds, they have a central place in the development of both specialized nutritionals and functional foods.\n\n## Milk proteins as a source of amino acids\u2014specialized nutritionals\n\nMilk and milk proteins have long been regarded as a rich source of readily digestible and nutritionally available amino acids. However, early in vivo determinations of protein digestibility were based on fecal measurement, which is now known to be flawed. This is due to the significant degree of colonic microbial metabolism now known to occur, and the fact that the resulting amino acids are at best absorbed from the large intestine to only a very limited extent (Moughan, 2003). The preferred accurate method for determining amino acid digestibility is to determine unabsorbed amino acids at the end of the small bowel (terminal ileum). This can be achieved in humans using naso-intestinal intubation or through the cooperation of ileostomates. Alternatively, animal models can be used, with both rat and pig ileal digestibility assays being suitable (Moughan et al., 1994). Where a protein has undergone structural alteration due to processing or storage (especially at high temperatures), conventional digestibility measures are inappropriate for some amino acids, in particular the often first-limiting amino acid, lysine. A new lysine bioavailability assay has been developed that can be usefully applied to damaged proteins, based on the collection of ileal digesta and application of the guanidination reaction (Moughan, 2003). When digestibility determinations are based on the sampling of ileal digesta, it is important to recognize that digesta contain copious quantities of endogenous (of body origin) protein in addition to undigested protein. This endogenous protein component needs to be taken into account to yield 'true' rather than 'apparent' estimates of digestibility (Moughan et al., 1998; Moughan and Rutherfurd, 2012).\n\nThere are very few published data on true ileal protein digestibility as determined using human subjects. A comprehensive set of studies in humans demonstrated a high true ileal digestibility of protein in milk of around 95% (Gaudichon et al. 1994; 1995; 1996; Mah\u00e9 et al., 1995; 1996). Comparable values, using the same methodology, for soya and pea proteins were 91 and 89%, respectively. Sandstrom et al. (1986) gave soya- and meat-based diets to ileostomates and reported ileal digestibility coefficients for total nitrogen in the range 80\u201385%. The naso-intestinal intubation method with normal volunteers has also been used to determine digestibility coefficients for individual amino acids (Gaudichon et al., 2002). For cow's milk, true ileal digestibility ranged from 92% for glycine to 99% for tyrosine, whereas for soya bean, digestibility ranged from 89% for threonine to 97% for tyrosine. In our own laboratory, true ileal amino acid digestibility determined using ileostomates ranged from 98% for aspartate to 100% for cysteine in sodium caseinate; from 93% for threonine to 99% for cysteine in whey protein concentrate; from 95% for glycine to 99% for arginine in soya protein isolate; and from 91% for cysteine to 100% for arginine in soya protein concentrate (Moughan et al., 2005a). There are more comprehensive data on the true ileal digestibility of amino acids in various milk proteins, which have been obtained using animal models for digestion in humans (Rutherfurd and Moughan, 1997). Table 18.1 shows ileal digestibility data obtained using the laboratory rat for selected amino acids in a range of protein sources. These data confirm the very high digestibility of milk-derived proteins to the end of the ileum in simple-stomached mammals. The dietary essential amino acids are virtually completely digested.\n\nTable 18.1\n\nMean True Ileal Digestibility of Selected Amino Acids in a Range of Soya and Dairy Products\n\nAmino acid | Soya protein concentrate | Soya protein isolate | Lactic casein | Sodium caseinate | Whey protein concentrate | \u03b1-Lactalbumin | Milk protein concentrate  \n---|---|---|---|---|---|---|---  \nLysine | 97.3 | 98.5 | 98.8 | 98.0 | 98.2 | 94.7 | 98.3  \nMethionine | 95.3 | 100.0 | 100.0 | 99.6 | 100.0 | 99.2 | 100.0  \nCysteine | 86.9 | 95.3 | 99.2 | 93.0 | 99.6 | 96.1 | 97.8  \nIsoleucine | 96.4 | 96.8 | 94.8 | 90.6 | 98.1 | 95.4 | 94.9  \nLeucine | 95.7 | 95.3 | 99.1 | 97.6 | 99.1 | 96.1 | 98.9\n\nAdapted from Rutherfurd and Moughan (1997), with the permission of the publisher.\n\nAlmost all dairy proteins have been subjected to some type of processing during their manufacture. Given that milk products often contain the reducing sugar lactose, they are susceptible to damage to the amino acid lysine. A specific assay designed to allow an accurate determination of lysine bioavailability in processed foods (Moughan and Rutherfurd, 1996) has recently been applied to a range of commercially available milk protein-based products (Table 18.2). Once again, this underscores the high bioavailability of milk proteins and the limited amount of lysine damage incurred by proteins with modern controlled dairy processing. In contrast, when the same bioassay was applied to grain-based processed foods often containing high amounts of sucrose, including cereals for children, a substantial degree of lysine damage was found (Table 18.3) (Rutherfurd et al., 2006). Milk and milk-based products have an important role in complementing cereal foods and in supplying available lysine.\n\nTable 18.2\n\nTrue Ileal Reactive Lysine Digestibility (Bioavailability, %) and Digestible Total and Reactive Lysine Contents (g\/kg air-dry) for 12 Dairy Protein Sources\n\n| | Digestible lysine  \n---|---|---  \nProduct | Bioavailability | Total | Reactivea  \nWhole milk protein | 98.3 | 26.2 | 24.0  \nInfant formula A | 91.0 | 8.3 | 8.6  \nInfant formula B | 92.3 | 9.1 | 9.2  \nInfant formula C | 93.1 | 11.1 | 11.7  \nWhey protein concentrate | 98.5 | 79.9 | 77.5  \nUHT milk | 100.0 | 31.7 | 31.4  \nEvaporated milk | 96.7 | 23.4 | 20.5  \nWeight-gain formula | 99.0 | 24.4 | 24.1  \nSports formula | 98.0 | 20.4 | 19.1  \nElderly formula | 97.1 | 11.7 | 11.8  \nHydrolyzed lactose milk powder | 98.6 | 27.2 | 25.1  \nHigh-protein supplement | 99.9 | 14.3 | 14.3\n\na Bioavailable lysine; minimal difference between total lysine and reactive lysine denotes minimal Maillard damage.\n\nAdapted from Rutherfurd and Moughan (2005), with the permission of the publisher.\n\nTable 18.3\n\nTrue Ileal Digestible Total and Reactive Lysine Contents (g\/kg air-dry) in Selected Cereal-based Foods\n\n| Digestible lysine  \n---|---  \nCereal product | Total | Reactivea  \nWheat-based (shredded) | 1.3 | 0.8  \nCorn-based (flaked) | 0.4 | 0.2  \nRice-based (puffed) | 1.1 | 0.6  \nMixed cereal (rolled) | 3.2 | 1.9\n\na Bioavailable lysine; a difference between total lysine and reactive lysine denotes Maillard damage.\n\nAdapted from Rutherfurd et al. (2006), with the permission of the publisher.\n\nFigure 18.1 highlights the substantial differences that exist in the amounts of digestible amino acids supplied by plant proteins (e.g., soya) and milk proteins (e.g., \u03b1-lactalbumin). The often first-limiting amino acids (lysine and methionine plus cysteine) are found in much higher concentrations in the milk proteins. This makes them excellent sources of amino acids and very important dietary constituents in order to achieve a balanced dietary protein intake. Recently, an international consensus view has formed that the traditional protein digestibility corrected AA score (PDCAAS) method for describing dietary protein quality for humans should be replaced by a method based on true ileal amino acid digestibility and availability and not involving truncation of the score for individual food ingredients (DIAAS; digestible indispensable amino acid score). Calculated DIAAS values for whey protein isolate, milk protein concentrate, and whey protein concentrate are 1.25, 1.31, and 1.10, and values for soya protein isolate and pea protein concentrate are 0.99 and 0.93, further showing the significant complementary value of milk proteins.\n\nFigure 18.1 Digestible (true ileal) amino acid contents of a plant protein and a milk protein. Adapted from Rutherfurd and Moughan, 1997, with the permission of the publisher.\n\nBecause of their relatively high levels of nutritionally important amino acids, milk proteins are utilized efficiently by humans when given as a sole protein source. Tom\u00e9 and Bos (2000) reported net postprandial protein utilization values of 80 and 72% for milk protein and soya protein, respectively, measured over 8 h after the ingestion of standard meals by healthy human subjects.\n\nGiven the high bioavailability of amino acids in milk proteins and their abundant supply, it is hardly surprising that milk proteins are commonly used for the manufacture of so-called nutritionals\u2014foods designed for a specific nutritional purpose (e.g., formulas for infants, the elderly, and athletes).\n\nIn the future, with increasing human population growth and greater pressure on food supplies, milk proteins will likely play an ever more important role as protein 'balancers' in plant-based processed foods.\n\n## Milk proteins as a source of amino acids\u2014specific physiological roles\n\nAmino acids may have physiological roles that are unrelated to their direct involvement in protein synthesis. These include their roles as neurotransmitters (e.g., glutamate, aspartate, and glycine) and as precursors for the synthesis of other molecules involved in neuromuscular function (e.g., creatine and taurine) and in host defenses (e.g., glutathione and nitric oxide). Tryptophan is a precursor for the synthesis of serotonin, potentially impacting mood control (van de Poll et al., 2006) and appetite regulation (Fernstrom and Wurtman, 1972; Fernstrom and Fernstrom, 1995). The nitrogen-rich amino acid arginine leads to the production of nitric oxide (Wu and Morris, 1998), which is considered to have a significant role in cell signaling and the control of endothelial tone. Depending on its site of release, nitric oxide has several functions, including stimulation of the pituitary gland, vasodilation, neurotransmission and immune modulation. Another example of an amino acid with a specific metabolic role is the branched-chain amino acid leucine, which has a unique role in the regulation of muscle protein synthesis (Kimball and Jefferson, 2001; McNurlan, 2012). Interestingly, leucine stimulates protein synthesis directly in skeletal muscle but not in liver. The other branched-chain amino acids, isoleucine and valine, are less effective in stimulating muscle protein synthesis. Leucine supplementation has been shown to stimulate recovery of muscle protein synthesis during food restriction and after endurance exercise (Gautsch et al., 1998; Anthony et al., 2000). It has also been suggested that leucine has a potential regulatory role in glycemic control (Layman 2002; 2003).\n\nIt has been known for many years that glutamine, glutamate, and aspartate are preferred oxidative fuels for the gut, a highly metabolic organ. Consequently, many studies with humans and animals have been undertaken to investigate the effects on intestinal mucosal integrity, glutathione synthesis, and immune function, especially using dietary glutamine. This has led to debate as to whether glutamine should be regarded as a 'conditionally essential' dietary amino acid (Grimble, 1993). In the traumatized patient, dietary glutamine may be needed to maintain immune responsiveness and for maintenance of the mucosal barrier against bacterial action and endotoxins.\n\nThere are also amino acids with specific physiological functions that are not found in proteins (i.e., nonprotein amino acids). The classic example is taurine (\u03b2-aminoethanesulfonic acid), which is synthesized by the body from cysteine or methionine and is essential for the production of conjugated bile salts (taurocholic acid). Taurine is found in milk, normally in the free form. It is recognized that cow's milk has low concentrations of taurine relative to human milk, and so taurine is now often added to cow's milk-based infant formulas.\n\nThe above are examples of specific physiological functions of amino acids; there are many others. It is anticipated that over the next decade our understanding of the physiological roles of individual amino acids will increase, leading to opportunities to develop functional foods containing higher or lower amounts of certain amino acids. Summaries of current evidence for proven functional effects (clinical benefits) in humans consequent upon dietary supplementation with specific amino acids have been provided by van de Poll et al. (2006) and Jonker et al. (2012). Arginine has been widely used in supplemental nutrition for surgical patients and patients with burns, to modify the inflammatory response, to enhance organ perfusion, and to stimulate wound healing. However, the benefits of arginine supplementation are not uniformly proven and accepted. There is some evidence that taurine supplementation improves retinal development in premature babies receiving parenteral nutrition. Glutamine is one of the more extensively studied amino acids and has been used in the preparation of medical foods. There is evidence that glutamine supplementation may reduce infectious morbidity and the length of hospital stay in surgical patients. Phenylalanine-free preparations have application in phenylketonuria.\n\nEtzel (2004) highlighted an opportunity for the dairy industry, whereby a number of refined high-quality proteins are produced and marketed. The diverse amino acid compositions of these proteins can be exploited. The mixtures of proteins in milk and whey may be fractionated to give isolated proteins (\u03b1-lactalbumin, \u03b2-lactoglobulin, immunoglobulins, bovine serum albumin, the caseins, lactoferrin, lactoperoxidase, and the peptide glycomacropeptide that is cleaved from \u03ba-casein by chymosin) and blends of proteins with unique amino acid patterns. Etzel (2004) compared the amino acid compositions of several milk proteins with the composition of a theoretical 'average' protein calculated from the frequency of occurrence of each amino acid in 207 unrelated proteins of known sequence. Table 18.4 provides an abridged version of the Etzel (2004) dataset. Some interesting comparisons can be made. First, glycomacropeptide is completely devoid of cysteine, histidine, phenylalanine, tyrosine, and tryptophan. Cysteine content is relatively high in \u03b1-lactalbumin, whereas glutamine has a relatively high concentration in \u03b2-lactoglobulin, and glutamic acid is found at a high concentration in three of the dairy products. Leucine content is some twofold higher in \u03b2-lactoglobulin compared with the 'average' protein, and the threonine content of glycomacropeptide is extraordinarily high. As a group, the branched-chain amino acids in milk proteins are higher in concentration than the 'average' protein. It is clear from this type of comparison that milk-based food products with amino acid compositions targeting particular physiological end points can be developed.\n\nTable 18.4\n\nThe Amounts (% air-dry) of Selected Amino Acids in Various Milk Proteins and in an 'Average' Protein\n\nAmino acid | \u03b2-Lactoglobulin | \u03b1-Lactalbumin | Glycomacropeptide | Whey protein isolate | 'Average' proteina  \n---|---|---|---|---|---  \nCysteine | 2.8 | 5.8 | 0 | 1.7 | 2.6  \nGlutamine | 6.3 | 4.5 | 3.8 | 3.4 | 4.6  \nGlutamic acid | 11.3 | 7.3 | 15.5 | 15.4 | 7.3  \nHistidine | 1.5 | 2.9 | 0 | 1.7 | 2.6  \nIsoleucine | 6.2 | 6.4 | 11.9 | 4.7 | 4.8  \nLeucine | 13.6 | 10.4 | 1.7 | 11.8 | 7.8  \nValine | 5.4 | 4.2 | 8.9 | 4.7 | 6.2  \nPhenylalanine | 3.2 | 4.2 | 0 | 3.0 | 4.7  \nTryptophan | 2.0 | 5.3 | 0 | 1.3 | 1.9  \nTyrosine | 3.6 | 4.6 | 0 | 3.4 | 5.2  \nThreonine | 4.4 | 5.0 | 16.7 | 4.6 | 5.5\n\na Based on amino acid compositions of 207 unrelated sequenced proteins.\n\nAdapted from Etzel (2004), with the permission of the publisher.\n\n## Milk proteins as a source of amino acids\u2014role in providing calories and in promoting satiety\n\nIn addition to their role as a substrate for body protein synthesis and for the synthesis of various nonprotein nitrogenous compounds, amino acids may also be used as a source of dietary energy, and the interaction between dietary protein and energy has long been understood.\n\nIt is often overlooked, however, that different dietary macronutrients give rise, biologically, to quite different amounts of free energy (adenosine triphosphate\u2014ATP) per unit gross energy (bomb calorimeter). It is argued that 'a calorie is a calorie' regardless of the macronutrient giving rise to the energy. This is true, but what often fails to be appreciated is that the numbers of calories in a food deemed to be derived from the respective macronutrients are usually estimates, not absolute measures. Conversion factors such as the Atwater factors are applied to determine the amounts of macronutrients in a food, and 'available' energy is estimated. The point is that the conversion factors are not completely accurate, and thus neither are the estimated calories. This point has particular relevance in the case of amino acids.\n\nAtwater factors attempt to take into account the loss of energy due to incomplete absorption of the amino acid during digestion and the loss of energy in excreted urinary metabolites post catabolism. However, the net yield of ATP during the catabolism of the amino acid and the ATP cost of synthesizing urea are not accounted for. The capture of net energy as ATP for an amino acid is less efficient than for other nutrients such as glucose and fatty acids, with an accompanying higher dietary-induced thermogenesis compared with glucose and fatty acids. This has important implications for designing weight-loss diets. Foods containing high amounts of protein will provide less 'available energy' (i.e., ATP) per unit dry matter or gross energy, compared with foods high in available carbohydrate and\/or fat.\n\nDairy proteins are a highly versatile source of amino acids for the design of weight-loss foods, and more care should be taken in describing the calorific values, especially for functional foods designed for weight loss. Protein has a further advantage for the formulation of weight-loss foods, as it is now widely accepted that it is a satiating nutrient and is more effective than carbohydrate and fat in suppressing voluntary food intake independent of its calorific value. The role of dietary protein in the regulation of food intake and body weight in humans, and underlying mechanisms, has been the subject of recent reviews (Anderson and Moore, 2004; Westerterp-Plantenga and Lejeune, 2005). There is strong evidence that the protein content of a food is a determinant of short-term satiety and of how much food is subsequently eaten. The role of protein in the regulation of long-term food intake and body weight is less clear because of a paucity of relevant experimental observations.\n\nThe role of protein in regulating body weight, in comparison with other macronutrients, is considered to consist of several often-related but different aspects: satiety, thermogenesis, metabolic energy efficiency, and body composition. First, the highly satiating effect of protein has been observed both postprandially and postabsorptively. Second, and also as discussed, high-protein diets are associated with a high dietary-induced thermogenesis, which could be related to the satiety effect of proteins. Third, high-protein diets help maintain or increase fat-free body mass, and the maintenance of a higher lean mass is costly energetically (i.e., a higher resting energy expenditure), leading to a lower associated metabolic efficiency of energy utilization.\n\nOf particular interest to the dairy industry is the observation that protein source per se may be a factor influencing short-term satiety in humans. Whey protein has been identified as potentially more effective in promoting satiety (Vandewater and Vickers, 1996; Portman et al., 2000; Hall et al., 2003). A basis for differences in satiety related to source of protein may be found in amino acid composition (e.g., a high leucine content stimulating protein synthesis and altering body energetics), in bioactive peptides released from the protein during digestion (refer to the following section), in different kinetics of protein digestion, and, in the case of whey, in the presence of glycomacropeptide.\n\n## Milk proteins as a source of bioactive peptides\n\nMilk is known to contain proteins (e.g., lactoferrin, lactoperoxidase, immunoglobulins) and free peptides having specific non-nutritional physiological functions. These compounds are undoubtedly important in the case of the human infant and may also have a functional role in adults. Of potentially greater significance, however, are the many small (from 3 to 20 amino acids) bioactive peptides encrypted in food protein amino acid sequences and released during digestion. Bioactive peptides are specific protein fragments that influence body function. These peptides are inactive within the sequence of the parent protein and can be released during proteolysis or fermentation. Bioactive peptides may act as physiological modulators both locally in the gut and systemically. Most, if not all, proteins appear to contain bioactive sequences, although the majority of research to date has been conducted with milk proteins.\n\nAn opioid activity of peptides derived from partial enzymatic digestions of milk proteins and wheat gluten was reported in the literature as early as 1979 (Brantl et al., 1979; Zioudrou et al., 1979). Since then, a considerable body of research has been undertaken, and many different bioactive amino acid sequences have been discovered and physiological functions have been defined. The potential for bioactive peptides in the development of functional foods is great. It is now appreciated that bioactive peptides have a wide range of physiological effects, some of which are listed in Table 18.5. Specific bioactive peptides and protein hydrolysates can now be produced commercially, allowing for dietary supplementation and protein fortification. Casein-derived peptides are already in commercial use as food supplements (e.g., phosphopeptides) and as pharmaceuticals (Meisel, 1997).\n\nTable 18.5\n\nReported Effects of Natural Food-derived Peptides\n\nModulation of gastrointestinal motility\n\nStimulation of secretory processes\n\nMineral binding\n\nAntibacterial properties\n\nImmunomodulation\n\nAntithrombotic activity\n\nInhibition of angiotensin-converting enzyme (ACE) in the control of hypertension\n\nAnalgesic (pain relief) and other neuroactive effects\n\nThe remainder of this section focuses on the first two functions listed in Table 18.5 (i.e., gut function), as an example of the potential of food-derived peptides as natural bioactive peptides. Several studies have described the involvement of bioactive peptides (exorphins) in regulating the stomach-emptying rate, gastrointestinal motility, and gut secretory activity in mammals (Rutherfurd-Markwick and Moughan, 2005). A role for bioactive peptides in influencing gut function is not unexpected, as the effects may be mediated both directly and hormonally, involving receptor sites in the gut without the need for absorption and systemic uptake of the peptide.\n\nOur own quantitative studies within the Riddet Institute at Massey University highlight the potential importance of the net effect of food-derived peptides on overall gut metabolism. The gut is a highly metabolic organ, with changes in the rate of organ metabolism having significant implications for total body energetics, protein dynamics, and amino acid and other nutrient requirements. In our series of studies, terminal ileal digesta amino acid or nitrogen flow was determined as an indicator of overall protein dynamics in the upper digestive tract consequent upon the ingestion of a meal. This is reflective of the various cellular and dietary controls on the protein secretion and amino acid reabsorption processes. Endogenous amino acid flows (the net result of secretion and reabsorption) at the end of the ileum were determined following the provision of semisynthetic corn starch-based diets, differing in the source of dietary nitrogen (protein-free, synthetic amino acids, protein, hydrolyzed protein). A range of methods to determine endogenous (of body origin) as opposed to exogenous (diet origin) nitrogen were applied (Moughan et al., 1998).\n\nTable 18.6 gives results for endogenous lysine (a marker for total protein) flow at the end of the small bowel from a representative study from our series of experiments using the pig as a generalized mammalian model. The results clearly demonstrate that when amino acids were present in the gut (either directly from the hydrolyzed casein or after being released from the digestion of dietary zein, a corn-derived protein), endogenous protein loss at the end of the small bowel was significantly enhanced. The peptides led to an enhanced secretion of protein into the gut lumen and\/or a reduced reabsorption of endogenous amino acids. In any case, the loss of extra protein into the colon, whereupon the amino acids are not salvageable, represents a considerable loss of amino acids and is associated with a high metabolic energy cost. Further work has demonstrated that the quite dramatic effect (an almost 60% increase in endogenous flow for the hydrolyzed casein) of dietary peptides is dose dependent (Hodgkinson et al., 2000; Hodgkinson and Moughan, 2006).\n\nTable 18.6\n\nEndogenous Lysine Loss at the Terminal Ileum of the Growing Pig Given Protein-free, Synthetic Amino Acid, Hydrolyzed Casein- or Zein-based Diets\n\n| Diet  \n---|---  \n| PF | SAAa | EHCb | Znc | Significance  \nLysine loss (mg\/kg dry matter intake) | 252 | 284 | 448 | 389 | P < 0.05\n\nPF, protein-free; SAA, synthetic amino acid; EHC, hydrolyzed casein; Zn, zein.\n\na Devoid of lysine, with intravenous lysine infusion.\n\nb Digesta were centrifuged and ultrafiltered (10,000 Da molecular weight cut off).\n\nc Naturally devoid of lysine, with intravenous lysine infusion.\n\nAdapted from Butts et al. (1993), with the permission of the publisher.\n\nThese results, combined with those of several other similar studies, provide compelling evidence for a significant influence of dietary peptides on gut protein dynamics and overall metabolism. Little is known about how these effects are mediated or how the magnitude of the effect is influenced by the source of dietary protein. Claustre et al. (2002) have recently shown that casein and lactalbumin hydrolysates (but not egg or meat hydrolysate) greatly stimulate mucin secretion in rat jejunum. The casein hydrolysate-mediated effect was blocked by the administration of naloxone (an opioid antagonist), and \u03b2-casomorphin-7, an opioid peptide released from \u03b2-casein during digestion, also induced mucin secretion. The peptide effect was also inhibited by naloxone. It may be that the effects are more pronounced with milk proteins.\n\nThe cooperation of ileostomates has allowed our results obtained from animal studies to be confirmed in humans (Moughan et al., 2005b; refer to Table 18.7).\n\nTable 18.7\n\nEndogenous Ileal Lysine and Total Nitrogen Losses (\u03bcg\/g dry matter intake) in Adult Humans Receiving a Protein-free or Hydrolyzed Casein-based Diet\n\n| Diet  \n---|---  \n| PF | EHCa | Significance  \nLysine | 383 | 614 | P < 0.01  \nTotal nitrogen | 2061 | 4233 | P < 0.001\n\nPF, protein-free; EHC, hydrolyzed casein.\n\na Digesta were centrifuged and ultrafiltered (10,000 Da molecular weight cutoff).\n\nAdapted from Moughan et al. (2005b), with the permission of the publisher.\n\nBioactive peptides (and it would seem particularly those arising from the digestion of milk proteins) have been shown to have multiple physiological effects often at modest dietary intakes. As more is understood about these effects, it will become possible to develop novel functional foods. The potential to develop protein hydrolysates, peptide fractions, and commercially synthesized peptides, to target physiological end points associated with gut motility, digestion, energetics and satiety, is particularly promising.\n\n## Conclusions\n\nIn this chapter, a case has been made for the central place of dairy proteins in the development of functional foods and specialized nutritionals. Dairy proteins are a source of highly bioavailable amino acids and offer a diverse range of amino acid patterns and specific amino acid concentration ratios. Certain amino acids found in milk proteins at high concentration have direct physiological as well as nutritional functions in humans. Milk proteins may have antimicrobial and immunomodulatory functions, and also induce and maintain satiety. Additionally, amino acids have a relatively high dietary thermogenesis; thus dairy proteins are ideal for the formulation of specialized weight-loss foods. Almost every week, new information is reported concerning bioactive peptides, which are released in the gut during the natural digestion of milk proteins. Milk is indeed a veritable cornucopia for developing functional foods.\n\n# References\n\nAnderson GH ,  Moore SE .  Dietary proteins in the regulation of food intake and body weight in humans .  _J. Nutr_.  2004 ;134 : 974S \u2013 979S .\n\nAnthony JC ,  Yoshizawa F ,  Gautsch-Anthony T ,  Vary TC ,  Jefferson LS ,  Kimball SR .  Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway .  _J. Nutr_.  2000 ;130 : 2413 \u2013 2419 .\n\nBrantl VH ,  Teschemacher H ,  Blasig J ,  Henschen A ,  Lottspeich F .  Novel opioid peptides derived from casein (-casomorphins). I. Isolation from bovine casein peptone .  _Hoppe-Seyler's Z. Physiol. Chem_.  1979 ;360 : 1211 .\n\nButts CA ,  Moughan PJ ,  Smith WC ,  Carr DH .  Endogenous lysine and other amino acid flows at the terminal ileum of the growing pig (20 kg body weight): The effect of protein-free, synthetic amino acid, peptide and protein alimentation .  _J. Sci. Food Agric_.  1993 ;61 : 31 \u2013 40 .\n\nClaustre J ,  Toumi F ,  Trompette A ,  Jourdan G ,  Guignard H ,  Chayvialle JA ,  Plaisanci\u00e9 P .  Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum .  _Am. J. Physiol. Gastrointest. Liver Physiol_.  2002 ;283 : G521 \u2013 G528 .\n\nDiplock AT ,  Aggett PJ ,  Ashwell M ,  Bornet F ,  Fern EB ,  Roberfroid MB .  Scientific concepts for functional foods in Europe. Consensus document .  _Br. J. Nutr_.  1999 ;81 : S1 \u2013 S27 .\n\nEtzel MR .  Manufacture and use of dairy protein fractions .  _J. Nutr_.  2004 ;134 : 996S \u2013 1002S .\n\nFerguson LR .  Nutrigenomics .  In:  Moughan PJ ,  McCool P ,  eds.  _Floreat Scientia_ .  New Zealand :  Wairau Press ;  2011 : 223 \u2013 227 .\n\nFernstrom MH ,  Fernstrom JD .  Brain tryptophan concentrations and serotonin synthesis remain responsive to food consumption after the ingestion of sequential meals .  _Am. J. Clin. Nutr_.  1995 ;61 : 312 \u2013 319 .\n\nFernstrom JD ,  Wurtman RJ .  Brain serotonin content: physiological regulation by plasma neutral amino acids .  _Science_.  1972 ;178 : 414 \u2013 416 .\n\nGaudichon C ,  Roos N ,  Mah\u00e9 S ,  Sick H ,  Bouley C ,  Tom\u00e9 D .  Gastric emptying regulates the kinetics of nitrogen absorption from 15N-labelled milk and 15N-labelled yogurt in miniature pigs .  _J. Nutr_.  1994 ;124 : 1970 \u2013 1977 .\n\nGaudichon C ,  Mah\u00e9 S ,  Roos N ,  Benamouzig R ,  Luengo C ,  Huneau JF ,  Sick H ,  Bouley C ,  Rautureau J ,  Tom\u00e9 D .  Exogenous and endogenous nitrogen flow rates and level of protein hydrolysis in the human jejunum after [15N]-labeled milk and [15N]-labeled yogurt ingestion .  _Br. J. Nutr_.  1995 ;74 : 251 \u2013 260 .\n\nGaudichon C ,  Mah\u00e9 S ,  Luengo C ,  Laurent C ,  Meaugeais M ,  Krempf M ,  Tom\u00e9 D .  A 15N-leucine-dilution method to measure endogenous contribution to luminal nitrogen in the human upper jejunum .  _Eur. J. Clin. Nutr_.  1996 ;50 : 261 \u2013 268 .\n\nGaudichon C ,  Bos C ,  Morens C ,  Petzke KJ ,  Mariotti F ,  Everwand J ,  Benamouzig R ,  Dar\u00e9 S ,  Tom\u00e9 D ,  Metges CC .  Ileal losses of nitrogen and amino acids in humans and their importance to the assessment of amino acid requirements .  _Gastroenterology_.  2002 ;123 : 50 \u2013 59 .\n\nGautsch TA ,  Anthony JC ,  Kimball SR ,  Paul GL ,  Layman DK ,  Jefferson LS .  Availability of eIF-4E regulates skeletal muscle protein synthesis during recovery from exercise .  _Am. J. Physiol_.  1998 ;274 : C406 \u2013 C414 .\n\nGrimble GK .  Essential and conditionally-essential nutrients in clinical nutrition .  _Nutr. Res. Rev_.  1993 ;6 : 97 \u2013 119 .\n\nHall WL ,  Millward DJ ,  Long SJ ,  Morgan LM .  Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite .  _Br. J. Nutr_.  2003 ;89 : 239 \u2013 248 .\n\nHodgkinson SM ,  Moughan PJ .  An effect of dietary protein content on endogenous ileal lysine flow in the growing rat .  _J. Sci. Food Agric_.  2006 ;87 : 233 \u2013 238 .\n\nHodgkinson SM ,  Moughan PJ ,  Reynolds GW ,  James KAC .  The effect of dietary peptide concentration on endogenous ileal amino acid loss in the growing pig .  _Br. J. Nutr_.  2000 ;83 : 421 \u2013 430 .\n\nInstitute of Food Technologists. 2005. Expert Report onFunctional Foods. Available at: www.ift.org.\n\nJonker R ,  Engelen MPKJ ,  Deutz NEP .  Role of specific dietary amino acids in clinical conditions .  _Br. J. Nutr_.  2012 ;108 : S139 \u2013 S148 .\n\nKimball SR ,  Jefferson LS .  Regulation of protein synthesis by branched-chain amino acids .  _Current Opinion in Clinical Nutrition and Metabolic Care_.  2001 ;4 : 39 \u2013 43 .\n\nLayman DK .  Role of leucine in protein metabolism during exercise and recovery .  _Canadian Journal of Applied Physiology_.  2002 ;27 : 592 \u2013 608 .\n\nLayman DK .  The role of leucine in weight loss diets and glucose homeostasis .  _J. Nutr_.  2003 ;133 : 261S \u2013 267S .\n\nMah\u00e9 S ,  Benamouzig R ,  Gaudichon C ,  Huneau JF ,  De Cruz I ,  Tom\u00e9 D .  Nitrogen movements in the upper jejunum lumen in humans fed low amounts of caseins or \u03b2-lactoglobulin .  _Gastroenterology and Clinical Biology_.  1995 ;19 : 20 \u2013 26 .\n\nMah\u00e9 S ,  Roos N ,  Benamouzig R ,  Davin L ,  Luengo C ,  Gagnon L ,  Gausser\u00e8s N ,  Rautureau J ,  Tom\u00e9 D .  Gastrojejunal kinetics and the digestion of [15N]\u03b2-lactoglobulin and casein in humans: The influence of the nature and quantity of the protein .  _Am. J. Clin. Nutr_.  1996 ;63 : 546 \u2013 552 .\n\nMcNurlan M .  New perspectives in the control of body protein metabolism .  _Br. J. Nutr_.  2012 ;108 : S94 \u2013 S104 .\n\nMeisel H .  Biochemical properties of regulatory peptides derived from milk proteins .  _Biopolymers_.  1997 ;43 : 119 \u2013 128 .\n\nMoughan PJ .  Amino acid availability: Aspects of chemical analysis and bioassay methodology .  _Nutr. Res. Rev_.  2003 ;16 : 127 \u2013 141 .\n\nMoughan PJ .  Functional foods .  In:  Moughan PJ ,  McCool P ,  eds.  _Floreat Scientia_ .  New Zealand :  Wairau Press ;  2011 : 218 \u2013 221 .\n\nMoughan PJ ,  Rutherfurd SM .  A new method for determining digestible reactive lysine in foods .  _J. Agric. Food Chem_.  1996 ;44 : 2202 \u2013 2209 .\n\nMoughan PJ ,  Rutherfurd SM .  Gut luminal endogenous protein: Implications for the determination of ileal amino acid digestibility in humans .  _Br. J. Nutr_.  2012 ;108 : S258 \u2013 S263 .\n\nMoughan PJ ,  Cranwell PD ,  Darragh AJ ,  Rowan AM ,  The domestic pig as a model for studying digestion in humans .  Souffrant WB ,  Hagemeister H ,  eds.  _Digestive Physiology in the Pig_ ,  Volume II .  Dummerstorf, Germany :  Forschungsinstitut fur die Biologie Landwirtschaftlicher Nutztiere (FBN) ;  1994 : 389 \u2013 396 .\n\nMoughan PJ ,  Souffrant WB ,  Hodgkinson SM .  Physiological approaches to determining gut endogenous amino acid flows in the mammal .  _Arch. Anim. Nutr_.  1998 ;51 : 237 \u2013 252 .\n\nMoughan PJ ,  Butts CA ,  van Wijk H ,  Rowan AM ,  Reynolds GW .  An acute ileal amino acid digestibility assay is a valid procedure for use in human ileostomates .  _J. Nutr_.  2005 ;135 : 404 \u2013 409 .\n\nMoughan PJ ,  Butts CA ,  Rowan AM ,  Deglaire A .  Dietary peptides increase endogenous amino acid losses from the gut in adults .  _Am. J. Clin. Nutr_.  2005 ;81 : 1359 \u2013 1365 .\n\nPortman R ,  Bakal A ,  Peikin SR .  Ingestion of a premeal beverage designed to release CCK reduces hunger and energy consumption in overweight females .  _Obes. Res_.  2000 ;8 (Suppl 1) : PB59 (Abstract) .\n\nRoberfroid MB .  Concepts and strategy of functional food science: The European perspective .  _Am. J. Clin. Nutr_.  2000 ;71 (Suppl) : 1660S \u2013 1664S .\n\nRutherfurd SM ,  Moughan PJ .  The digestible amino acid composition of several milk proteins: Application of a new bioassay .  _J. Dairy Sci_.  1997 ;81 : 909 \u2013 917 .\n\nRutherfurd SM ,  Moughan PJ .  Digestible reactive lysine in selected milk-based products .  _J. Dairy Sci_.  2005 ;88 : 40 \u2013 48 .\n\nRutherfurd-Markwick KJ ,  Moughan PJ .  Bioactive peptides derived from food .  _J. AOAC Int_.  2005 ;88 : 955 \u2013 966 .\n\nRutherfurd SM ,  Torbatinejad NM ,  Moughan PJ .  Available (ileal digestible reactive) lysine in selected cereal-based food products .  _J. Agric. Food Chem_.  2006 ;54 : 9453 \u2013 9457 .\n\nSandstrom B ,  Andersson H ,  Kivisto B ,  Sandberg AS .  Apparent small intestinal absorption of nitrogen and minerals from soy and meat-protein based diets .  _A study on ileostomy subjects. J. Nutr_.  1986 ;116 : 2209 \u2013 2218 .\n\nTom\u00e9 D ,  Bos C .  Dietary protein and nitrogen utilization .  _J. Nutr_.  2000 ;130 : 1868S \u2013 1873S .\n\nvan de Poll MCG ,  Luiking YC ,  Dejong CHC ,  Soeters PB .  Amino acids\u2014specific functions .  In:  Caballero B ,  Allen L ,  Prentice A ,  eds.  _Encyclopedia of Human Nutrition_ .  2nd ed. Amsterdam :  Elsevier ;  2006 : 92 \u2013 100 .\n\nVandewater K ,  Vickers Z .  Higher-protein foods produce greater sensory-specific satiety .  _Physiol. Behav_.  1996 ;59 : 579 \u2013 583 .\n\nWard RE ,  German JB .  Understanding milk's bioactive components: A goal for the genomics toolbox .  _J. Nutr_.  2004 ;134 : 962S \u2013 967S .\n\nWesterterp-Plantenga MS ,  Lejeune MPGM .  Protein intake and body-weight regulation .  _Appetite_.  2005 ;45 : 187 \u2013 190 .\n\nWu G ,  Morris SM .  Arginine metabolism: Nitric oxide and beyond .  _Biochem. J_.  1998 ;336 : 1 \u2013 17 .\n\nZioudrou C ,  Streaty RA ,  Klee WA .  Opioid peptides derived from food proteins, the exorphins .  _J. Biol. Chem_.  1979 ;254 : 2446 \u2013 2449 . \nChapter 19\n\n# Milk Proteins and Human Health\n\nRobin A. McGregor*,**\n\nSally D. Poppitt*,**,***\n\n* Human Nutrition Unit, University of Auckland, Auckland, New Zealand  \n** Institute for Innovation in Biotechnology, School of Biological Sciences, University of Auckland, Auckland, New Zealand  \n*** Riddet Institute, Palmerston North, New Zealand\n\n## Abstract\n\nCow's milk proteins and related bioactive peptides are purported to have a wide range of effects on human health across the life span. Casein and whey protein have both been proposed to play a role in the prevention of chronic age-related conditions such as adverse metabolic health and type 2 diabetes, muscle wasting and sarcopenia, atherosclerosis, hypertension and cardiovascular disease risk, as well as bone health and osteoporosis. Epidemiological studies have shown regular dairy consumption to be associated with decreased prevalence of cardiometabolic risk, an outcome of improved metabolic health, while intervention studies have shown milk proteins to promote postprandial insulin secretion and glycemic control, and under some conditions improve postprandial lipemia and hypertension. The branched-chain amino acids of dairy protein also promote muscle anabolism, important for the maintenance of muscle mass and mobility during aging and, in our current epidemic of obesity, for the maintenance of lean body mass during energy restriction and weight loss. Dietary protein is known to suppress satiety and food intake, and if consumed from a dairy source may support preferential loss of fat rather than lean mass during weight loss. Milk proteins may also be beneficial for bone health during aging. In addition, milk proteins are important for maternal and infant nutrition, with protein playing a major role in growth and development during early life. While public health recommendations for breastfeeding underpin nutrition in young infants, commercial infant formulas are required for mothers unable to breastfeed long term, and formulas containing cow's milk protein aim to optimize infant health. In this chapter we review the evidence for the potential health benefits of milk proteins, based on human clinical trials, with a particular focus on metabolic health.\n\n## Keywords\n\nMilk protein\n\nwhey protein\n\ncasein\n\nmetabolic health\n\ncardiovascular\n\nskeletal muscle\n\nbone\n\ninfant\n\nOutline\n\nIntroduction 541\n\nMilk proteins, metabolic health, and type 2 diabetes 542\n\nMilk proteins, obesity, and weight control 543\n\nMilk Proteins, Muscle Wasting, and Sarcopenia 545\n\nMilk Proteins and Heart Health 547\n\nAtherosclerosis 547\n\nBlood Pressure and Vascular Reactivity 547\n\nMilk proteins and bone health 548\n\nMilk Proteins and Infant Health 549\n\nConclusions 550\n\n## Introduction\n\nMilk provides a rich source of proteins that are not found in any other food source and that have a range of putative positive health outcomes in both adults and children. Epidemiology has pointed to a positive association between cow's milk and human health, the underpinning mechanisms of which are as yet not well understood (Elwood et al., 2008). Humans consume milk primarily from cows, but around the world communities consume milk from many other animals, including buffalo, yaks, goats, sheep, reindeer, and camels. Cow's milk protein consists of approximately 80% (w\/w) casein and 20% (w\/w) whey protein, both of which may elicit beneficial health outcomes, and which are the source of a wide range of peptides with potential bioactive properties. The processing of milk protein, which may be an important factor in digestion and absorption kinetics, may also have physiological effects and in turn potential health benefits (Morr and Ha, 1993; Dalgleish and Corredig, 2012).\n\nIn clinical studies, whey protein concentrate (WPC), micellar casein, or sodium caseinate have most commonly been investigated, but trials have also used milk protein concentrate (MPC), calcium caseinate, casein hydrolysate, whey hydrolysates, and whey protein isolate (WPI) as sources of cow's milk protein. The production of these products is described elsewhere in this volume (see in particular Chapters 2 and ).\n\nThe impact of milk proteins and their associated peptides on health outcomes across the life span is a growing area of research interest. Whey protein in particular has been a focus due to its properties of rapid absorption, serum AA profile, and insulinotropic effects among other properties of interest. In this chapter, we review the evidence (primarily from human clinical trials) that milk proteins may improve or prevent a range of age-related chronic health conditions, particularly those associated with metabolic health, including metabolic syndrome, type 2 diabetes (T2DM), atherosclerosis, and hypertension, as well as the role that they may play in the control of body weight and maintenance of lean body and\/or skeletal muscle mass during aging and weight loss. We also review recent evidence in support of bone health and maternal and infant nutrition.\n\n## Milk proteins, metabolic health, and type 2 diabetes\n\nOver the past two decades poor metabolic health has emerged as a major public health concern. It has been linked to a more sedentary lifestyle and poor diet, and is a forerunner of T2DM and adverse cardiovascular health. Metabolic syndrome has been defined to represent a cluster of commonly measured metabolic markers of adverse health, including abdominal obesity, hyperglycemia, dyslipidemia, and hypertension (Grundy et al., 2005; Alberti et al., 2009). These provide intermediary biomarkers of poor health that can be targeted for improvement. Individuals with metabolic syndrome are at higher risk of developing T2DM and also cardiovascular disease (CVD) (Alberti et al., 2009), and younger, lean individuals typically have a better metabolic profile than older, overweight men and women. This gradual impairment is commonly accompanied by physiological changes such as an excess accumulation of lipids in adipose tissue and subsequent overspill into liver, skeletal muscle, and other tissues. Adipose tissue provides a major lipid storage depot; however, excessive expansion of abdominal and possibly other adipose stores and consequent lipid overspill and infiltration into other tissues is linked to release of proinflammatory mediators (Despres and Lemieux, 2006). Blunting of CHO, fat and protein metabolism, insulin resistance, and impairment in endothelial function are other common consequences. Metabolically active lean body mass is important for good metabolic health, not least as a mediator for glucose regulation among many other roles. For example, skeletal muscle accounts for up to 75% of whole-body glucose uptake. In combination, progressive loss of skeletal muscle and excess accumulation of abdominal fat (recently termed 'sarcobesity' (Parr et al., 2013)) can severely impair metabolic health.\n\nEvidence from large epidemiological studies has shown that the consumption of dairy products may be associated with a lower risk of metabolic disorders and CVD (Elwood et al. 2008; Rice et al., 2011), which in turn has been attributed to milk protein and in particular to the whey protein component. Insulin is sensitive to both the composition and concentration of plasma AAs, and both whey protein and casein ingestion stimulate increased insulin secretion (Nilsson et al. 2004; 2007;). Ingestion of whey protein leads to more rapid secretion of insulin than micellar casein (Boirie et al., 1997), a consequence of its rapid absorption kinetics, and hence underpins better glucose control in metabolically impaired individuals (Pal and Radavelli-Bagatini, 2012). Evidence predominantly generated from single-meal postprandial studies shows that milk proteins may increase tissue glucose uptake and suppress post-meal blood glucose fluctuations (Claessens et al., 2008; ; Pal and Ellis, 2010a; Akhavan et al., 2010).\n\nThese effects have also been shown in patients with T2DM. In a postprandial study of T2DM patients, the addition of 18 g whey protein into a breakfast or lunch meal caused a marked increase in plasma insulin concentrations compared to an isoenergetic substitution of nondairy protein and carbohydrate (CHO). The higher insulin levels were associated with a greater suppression of postprandial blood glucose levels (Frid et al., 2005). Casein also has effects on blood glucose. In a group of overweight individuals with T2DM, casein hydrolysate (0.3 g\/kg) with leucine (0.1 g\/kg) decreased plasma glucose levels over 24 hours when consumed after breakfast, lunch, and dinner (Manders et al., 2006). Notably, however, the casein supplement had been enriched in this study with the branched-chain amino acid (BCAA) leucine. Conversely, ingestion of 40 g casein hydrolysate at breakfast, lunch, and dinner caused little improvement in 24-hour blood glucose levels in a group of patients with long-standing T2DM. The discrepancy in outcome has been attributed to impairment of the insulin-secreting pancreatic beta-cell in long-standing T2DM patients, impairing the response to the milk protein (Manders et al., 2009). This remains unconfirmed, with some preliminary evidence that free AAs may in fact reactivate the insulin secretory mechanism in this metabolically impaired T2DM patient group (van Loon et al., 2003). Obesity and metabolic syndrome are often accompanied by the development of nonalcoholic fatty liver disease (NAFLD), where excessive lipids accumulate in the liver, eventually leading to fibrosis and cirrhosis. Experimental evidence from rodents has shown whey protein to decrease lipid accumulation in the liver (Hamad et al., 2011), but there are as yet no clinical studies.\n\nThere have been few long-term clinical studies of milk proteins on hyperglycemia. In overweight and obese individuals with increased metabolic risk through impaired glucose tolerance (IGT), raised plasma triglyceride (TG) levels, low HDL-cholesterol levels, and\/or abdominal obesity, daily ingestion of a high-dose WPI supplement (54 g\/day) for 12 weeks successfully decreased fasting blood insulin levels and insulin resistance but unexpectedly did not in turn decrease fasting glucose concentrations (Pal et al., 2010a). Clearly, more long-term studies of insulin and glucose control are needed.\n\n## Milk proteins, obesity, and weight control\n\nThere is growing evidence that high-protein diets may be efficacious for weight loss and\/or longer-term weight-loss maintenance (Larsen et al., 2010), but the issue as to whether different protein sources may be more or less successful is not well understood. The primary mechanisms that underpin high-protein diets or protein-based supplements for weight loss are enhanced appetite control and suppression of food intake (Poppitt et al., 1998; Anderson and Moore, 2004; Paddon-Jones et al., 2008; Dove et al., 2009). Sparing of skeletal muscle in favor of adipose loss during weight reduction through use of a higher-protein (>20% of energy as protein), energy-restricted diet may also be important. This is of particular relevance to dairy protein, where the high BCAA content drives muscle protein synthesis, which will contribute to the maintenance of metabolically active lean body mass. High-protein diets suppressing hunger and food intake have been reported in many studies, although the majority of data have been from shorter-term, postprandial interventions. Gradually, evidence for longer-term suppression and weight loss is growing (Clifton et al., 2008; Larsen et al., 2010). Milk proteins are commonly used in meal replacement drinks, which provide a convenient way to undertake a low-energy, high-protein diet. Some preliminary data show that milk proteins may promote greater satiety than other protein sources, but it is not well substantiated (Anderson et al., 2004; Veldhorst et al., 2009; Baer et al., 2011). Whether whey protein or casein may have differential effects is also not well understood. In a recent study, ingestion of an isoenergetic bolus of skimmed milk containing both casein and whey protein was reported to decrease energy intake more than protein alone (Lorenzen et al., 2012). In contrast, other studies have reported whey protein to be more anorectic than casein (Hall et al., 2003), with \u03b1-lactalbumin proposed as the whey protein fraction responsible for the greatest suppression of intake in a study comparing whey, whey protein plus glycomacropeptide (GMP), casein, and soy (Veldhorst et al., 2009). In a recent study on overweight women, there was no difference in energy intake between four whey protein fractions comprising WPC, colostrum WPC, GMP, and \u03b2-lactoglobulin, despite promising rodent studies, although \u03b2-lactoglobulin induced greater fullness (Poppitt et al., 2013). GMP has long been purported to differentially suppress appetite, but there is little evidence to support this hypothesis (Veldhorst et al., 2009; Keogh et al., 2010).\n\nTo date, there have been few long-term studies investigating the role of milk proteins in weight loss, in the absence of other lifestyle interventions. A 6-month study of overweight and obese individuals reported that 56 g\/day WPC resulted in significant loss of body weight, fat mass, and waist circumference compared to a CHO control (Baer et al., 2011), while a shorter 12-week study found no effect of 54 g\/day of WPI (Pal et al., 2010a). Interestingly, fasting blood lipids and insulin levels improved in this study, supporting the hypothesis that whey protein may improve metabolic health even in the absence of weight loss (Pal et al., 2010a). Energy-restricted diets are a common way to successfully decrease body weight, at least in the short term, but characteristic of this is loss of both fat mass and lean mass. Loss of lean mass results in concomitant reduction in basal metabolic requirements, and a return to habitual dietary habits may then result in rapid weight regain. Middle-aged or older individuals with the age-related sarcopenia and obesity known as 'sarcobesity' (Parr et al., 2013) who lose weight through traditional energy-restricted diets may be even more susceptible to rapid weight regain due to already low muscle mass. Ingestion of whey protein as part of an energy-restricted diet has been proposed as a strategy to decrease fat mass while preserving lean mass. There is some data to support this proposal. In a study of obese individuals undertaking a severely energy-restricted diet of \u223c2 MJ\/day for 12 weeks, supplementation twice daily with a milk protein successfully led to greater weight loss, fat loss, and maintenance of lean muscle mass (Frestedt et al., 2008). Another similar study supplementing with a high-protein meal replacement comprising whey, soy, and free AAs led to greater loss of body fat compared to a low-protein meal replacement (Treyzon et al., 2008). Milk proteins may also be beneficial for maintaining a lower body weight after energy restriction and fat loss. In a study of whey protein and casein supplementation, there was significantly better weight loss maintenance over a 12-week period compared with a CHO control (Claessens et al., 2009a).\n\nWhether whey protein or casein supplements may be more effective for preservation of lean muscle mass during periods of weight loss induced by an energy-restricted diet was addressed in a recent trial (Adechian et al., 2012) where obese individuals underwent a 6-week energy-restricted diet with whey protein or casein supplementation. While all individuals showed weight loss after 6 weeks, there were no differential effects of whey protein or casein for weight loss, fat loss, or preservation of lean muscle mass. Intriguingly, assessment of whole body protein synthesis and whole body protein breakdown showed casein to cause greater inhibition of protein breakdown, while whey protein increased protein synthesis (Adechian et al., 2012). The implications are that casein supplementation may be optimal for preservation of skeletal muscle mass during energy restriction. Notably, however, no long-term trials have as yet compared milk proteins with other protein sources for long-term weight loss and maintenance.\n\n### Milk Proteins, Muscle Wasting, and Sarcopenia\n\nAdvancing age and a sedentary lifestyle is associated with a gradual decline in skeletal muscle mass, function, and strength, which in the extreme form is termed sarcopenia or muscle wasting. Loss of skeletal muscle mass or function of the muscle has major implications for quality of life since activities of daily living such as walking upstairs become difficult or are no longer possible. At the extreme end, patients with chronic or end-stage diseases, including cancer, heart failure, AIDS, and chronic obstructive lung disease and sepsis, are also often susceptible to muscle wasting (Tan and Fearon, 2008; Fearon et al., 2011).\n\nIn addition to these mobility issues, skeletal muscle also has a significant impact on metabolic health. As one of the major organs responsible for insulin-stimulated glucose uptake, loss of skeletal muscle mass is often associated with insulin resistance (Evans, 2010). In addition to the insulin stimulatory effect of milk protein, it is the high level of BCAAs in whey protein and casein that prevents loss of lean body mass through increased skeletal muscle protein synthesis and\/or decrease in breakdown (Adechian et al., 2012). There is evidence that the anabolic effect of milk protein is decreased during aging, which has been termed anabolic resistance (Volpi et al., 2000). Whether this is an anabolic resistance to dietary intake of BCAAs or simply a reflection of underutilization of the major muscle groups in older people as exercise levels decline is a matter of considerable debate. Whether milk proteins can prevent the development of anabolic resistance or overcome established anabolic resistance in older individuals is of great interest.\n\nBoth circulating insulin and AAs are important for the activation of muscle protein synthesis. Whey protein and casein are high-quality proteins based on both the protein digestibility corrected AA score (PDCAAS) (Boye et al., 2012) and the recently developed digestible indispensable AA score (DIAAS) (FAO, 2013), both of which take into account human AA requirements and protein digestibility. However, whey protein contains a greater amount of the BCAAs leucine, isoleucine, and valine than does casein. Of the BCAAs, leucine is thought to be the most potent activator of protein synthesis (Katsanos et al., 2006; van Loon, 2012), although a recent study has shown that high levels of nonleucine BCAAs can induce equivalent protein synthesis when given with a whey protein supplement (Churchward-Venne et al., 2012). Casein in turn contains several essential amino acids (EAAs), including histine, methionine, and phenylalanine in a greater amount than whey protein, and also contains a greater amount of the non-EAAs arginine, glutamic acid, proline, serine, and tyrosine (Hall et al., 2003). The EAAs have been demonstrated to be primarily responsible for the activation of muscle protein synthesis (Volpi et al., 2003), although they are not necessarily efficacious for inhibition of protein breakdown. As noted above, studies have demonstrated greater whole body protein synthesis following ingestion of whey protein versus casein, while protein catabolism was greater following casein supplementation (Boirie et al., 1997; Adechian et al., 2012). The faster digestion rate of whey protein compared with casein, which due to its micellar structure tends to clot in the stomach, has been commonly believed to lead to more rapid delivery of AAs into plasma following whey protein, and longer, more sustained delivery following casein ingestion. There is certainly a body of data showing greater muscle protein accretion over the 6 hours following whey protein supplementation compared to casein or casein hydrolysate (Pennings et al., 2011).\n\nConversely, a recent study of intrinsically labeled whey protein and casein, co-ingested as milk, showed the absorption and retention of AAs from whey protein and casein to be similar after 2 hours, with AAs derived from casein showing greater absorption and retention rates beyond 3 hours (Soop et al., 2012). Some discrepancies in outcome might be due to the differential effects of aging on the response to these two dairy proteins. It may be worthwhile to increase the typically low whey protein content of milk, which then theoretically would provide both an early (whey) and sustained (casein) stimulation of muscle protein synthesis and an inhibition of muscle protein breakdown (Reitelseder et al., 2011). However, it remains to be seen whether milk protein-enhanced muscle protein synthesis is effective for prevention of muscle wasting and promotion of muscle protein accretion in healthy aging or elderly populations, or in patients with chronic end-stage disease. Additional evidence is also required to confirm that this in turn results in functional improvements in strength and\/or mobility.\n\nRegular resistance-type exercise transiently activates muscle protein synthesis and over time can lead to increases in skeletal muscle mass. Milk protein supplements are popular among recreational gym users seeking to increase muscle mass, but there is also research interest in whether milk protein ingestion in conjunction with resistance exercise may be beneficial for individuals with sarcopenia. Whey protein and casein ingestion after resistance exercise have been shown to both cause comparable increases in net protein balance (Tipton et al., 2004) and myofibrillar protein synthesis rate (Reitelseder et al., 2011), while other data show that whey protein ingestion causes greater increase in the muscle protein synthesis rate during the early 3-h period (Tang et al., 2009). Longer-term studies to date have produced mixed findings. In obese postmenopausal women, WPI in combination with an energy-restricted diet plus exercise over 6 months led to 4% greater weight loss than in the control group, and notably greater loss of subcutaneous adipose tissue and greater increase in leg muscle mass (Mojtahedi et al., 2011). Conversely, in elderly men who undertook resistance training for 12 weeks, protein supplementation did not improve muscle hypertrophy (Verdijk et al., 2009). Although the effects on muscle anabolism and catabolism are clear, the evidence underpinning clinically significant gains in lean body remain equivocal (Cermak et al., 2013).\n\n### Milk Proteins and Heart Health\n\n#### Atherosclerosis\n\nAtherosclerosis is a common cause of myocardial infarction, stroke, and peripheral vascular disease. It represents the progressive damage to the vascular endothelium due to build-up of lipids, immune cell infiltration, and plaque formation, leading to impaired endothelial function and reduced blood flow. It has been suggested that milk protein may improve adverse circulating lipid levels, one of the primary metabolic risk factors for CVD. Most of these studies have focused on the effects on postprandial lipemia, based on the premise that rapid clearance of blood lipids after a meal decreases arterial exposure to these circulating lipids. One study in overweight postmenopausal women given a high-fat meal found WPI and sodium caseinate to both decrease circulating TG and TG:ApoB48 ratio compared with glucose ().\n\nAnother study of obesity compared the effect of different milk protein fractions, including WPI, whey protein hydrolysate, \u03b1-lactalbumin, and GMP, on postprandial lipemia after a high-fat meal, but found no significant differences (Holmer-Jensen et al., 2012). Whey protein has also been shown to suppress postprandial circulating TG, free fatty acids (FFA), and chylomicron-rich lipoprotein appearance in diabetic patients following a high-fat meal, compared to controls of casein, fish, or plant protein sources (Mortensen et al., 2009). Casein alone, on the other hand, failed to improve postprandial TG in T2DM patients (Brader et al., 2010). There have been few long-term clinical studies. A 3-month trial of a fermented whey product in individuals with metabolic syndrome found some improvements in metabolic markers, although confounding effects on body weight make this trial difficult to interpret (Gouni-Berthold et al., 2012).\n\n#### Blood Pressure and Vascular Reactivity\n\nObservational and clinical studies have shown that the consumption of dairy products is associated with decreased risk of hypertension (Soedamah-Muthu et al., 2012). Much work has focused on identifying and isolating the bioactive peptides that may be responsible. The discovery that milk-derived peptides inhibit angiotensin converting enzyme (ACE) activity, and hence alter vasoconstriction, vasodilation, and blood pressure (BP) in vitro, led to a plethora of animal and subsequently human trials. Despite promising evidence that lactokinins or caseinkinins can reduce BP in spontaneously hypertensive animals, these findings are yet to be confirmed.\n\nThe most well-studied milk protein-derived peptides are isoleucine-proline-proline (IPP) and valine-proline-proline (VPP), which are generated by the fermentation of milk (Saito, 2008; Boelsma and Kloek, 2009). IPP and VPP have been shown to be weak ACE inhibitors based on in vitro experiments (FitzGerald and Meisel, 2000). Several meta-analyses of clinical trials of IPP and VPP on BP have been published which suggest these milk-derived peptides may have antihypertensive effects in humans (Xu et al., 2008; Turpeinen et al., 2013; Cicero et al., 2013). For example, a recent meta-analysis of 19 clinical trials of a daily dose of <10 mg milk tripeptides on BP over the last 15 years reported that overall systolic BP was decreased \u20134.0 mmHg, and diastolic BP was decreased \u20131.9 mmHg (Turpeinen et al., 2013). Previous meta-analysis reports also reported that VPP and IPP supplementation resulted in significant decreases in systolic and diastolic BP, although suppression was greater in hypertensive individuals (Xu et al., 2008).\n\nNot all studies have been positive, however. A European trial of VPP and IPP supplementation in a workplace environment found no effect on BP (Engberink et al., 2008). In the few human trials that assessed changes in ACE activity, neither VPP nor IPP appears to result in detectable inhibition of in vivo ACE activity. Hence, the mechanism behind the BP lowering effects observed in many studies is not yet clear. A recent report from the European Food Safety Authority (EFSA), which assesses the scientific basis for health claims of nutraceutical food products, concluded there was currently insufficient evidence to substantiate the claim that consumption of the milk-derived peptides IPP or VPP help maintain normal BP (European Food Safety Authority, 2012). Efforts are ongoing to identify further milk-derived peptides with potential to improve cardiovascular health.\n\nThere is evidence that supplementation with intact milk proteins may also potentially exert beneficial effects on endothelial function and BP control. An acute trial showed that ingestion of 5 g whey protein extract increased brachial artery flow mediated dilation in older, overweight individuals (Ballard et al., 2012). This was not associated with an inhibition of ACE activity. Hence regulation of vascular reactivity\/endothelial function takes place by some other as yet unknown mechanism. In overweight individuals, whey protein hydrolysate or WPC (28 g\/day) decreased systolic and diastolic BP compared to baseline, but only in subjects with hypertension at the start of the trial, and was again not associated with any detectable changes in ACE activity. A more recent study found 12-week supplementation with WPI or sodium caseinate (54 g\/day) decreased BP compared to glucose supplementation (Pal and Ellis, 2010b). In summary, human intervention studies suggest that milk peptides or milk protein supplementation may improve hypertension, but this is primarily in individuals with increased risk. Moreover, the mechanism underlying these improvements is not well understood.\n\n## Milk proteins and bone health\n\nBone remodeling occurs across the life span in response to environmental changes such as diet, physical activity, and external loading. Nutrition during childhood and adolescence is one of the major factors that influences the development of bone mass and strength, alongside physical activity, endocrine status, and exposure to risk factors (Caroli et al., 2011). Calcium, protein, and vitamin D are essential nutrients for bone development and bone health maintenance (Pampaloni et al., 2011), and dairy foods such as milk and cheese are a rich source of both Ca and protein, arguably providing an optimal source of essential nutrients for bone health (Caroli et al., 2011). Aging is associated with decreased bone mineral density (BMD), which leads to increased susceptibility to fracture in the elderly.\n\nAlthough Ca derived from cow's milk has long been shown to ameliorate bone loss (Tang et al., 2007) and provides a more conservative and arguably safer route by which to increase dietary Ca than through supplementation with Ca salts per se (Reid et al., 2006; Bolland et al. 2008; 2010;), recent studies show that cow's milk protein may influence bone remodeling (Tsuji-Naito and Jack, 2012). In these studies, whey proteins have been shown to promote growth of bone cells through a number of mechanisms, including stimulation of osteoblast differentiation, activation of intracellular signaling molecules, increased alkaline phosphatase activity, and mineralization (Tsuji-Naito and Jack, 2012). It has long been known that milk protein supplementation increases serum insulin-like growth factor 1 (IGF-1). In turn it has been hypothesized that this may be a mechanism for protection of BMD. Supplementation with MPC over a period of 6 months successfully increased serum IGF-1 in elderly individuals with recent osteoporotic hip fracture, decreased mean rehabilitation ward stay to 33 days compared with 54 days in control patients, and 6 months after the intervention was completed continued to show attenuation of proximal femur BMD loss (Sch\u00fcrch et al., 1998). Conversely, in a longer 2-year study of elderly women, while a high-protein whey drink increased IGF-1 levels when measured after 1 and 2 years of supplementation, there were no effects on BMD outcomes (Zhu et al., 2011). Whether whey protein may enhance the effects of multinutrient supplementation for improving recovery from fractures in the elderly is of interest, but is yet to be established.\n\nThe effects of overweight, obesity, and weight loss have long been of concern to the field of bone remodeling and fracture risk. Energy-restricted diets for weight loss have long been reported to increase the risk of bone loss, possibly due to inadequate energy and nutrients for maintenance of BMD (Villareal et al., 2006). Interestingly, there is some evidence that bone health may be preserved when the diet is rich in dairy products (Josse et al., 2012). Which component of dairy may be responsible for this protective effect, however, is not clear.\n\n### Milk Proteins and Infant Health\n\nAppropriate early nutrition is essential for the growth and development of infants. The World Health Organization and other health bodies recommend breastfeeding infants up to 6 months of age and continuing breastfeeding with complementary foods up to 2 years of age. However, commercial infant formulas (IF) have been widely developed to provide appropriate nutrition should breastfeeding not be possible. Cow's milk proteins are often used as the main protein source in IF, the composition of which is governed by strict regulatory guidelines. Milk protein, in particular whey-modified protein, is used as a primary protein source. Cow's milk requires modification in order for it to be the basis of IF since it contains two to three times the level of total protein compared with human breast milk, and has a different protein composition. Whether high-protein IFs may exceed infant requirements is widely debated, with discussion focused on later development of obesity and associated noncommunicable diseases (NCDs) (Michaelsen et al., 2012), although systematic reviews examining associations between early feeding and later-life obesity or BMI are not conclusive (Owen et al., 2005a,b).\n\nCertainly there may be differences in infant body composition driven by feeding practices (Gale et al., 2012). In light of these issues, IFs with low protein levels close to that of human milk have been developed, and these lower protein content IFs remain an area of great interest and a current 'hot topic.' Differences in protein composition between human and cow's milk include the whey protein:casein ratio and the level of free AAs, which are high in human milk. Mature human milk contains \u223c60% whey protein, of which \u03b1-lactalbumin is the major component, and \u223c40% casein. It also has high levels of EAAs. Conversely, the ratio of whey protein:casein in cow's milk is 20:80, and it contains a major whey protein \u03b2-lactoglobulin that is absent in human breast milk. The primary objective in protein-modified IFs has been to achieve a low-protein composition with sufficient indispensable and conditionally indispensable AAs but with decreased nonessential AAs. Adequate protein nutrition in infants given low-protein IF (as demonstrated by comparable growth curves to infants fed standard IF) has been demonstrated in formulas enriched with components such as \u03b1-lactalbumin (Lien et al., 2004).\n\nIn low-birth-weight (LBW) infants, dairy protein provides a useful way to increase intake in order to improve growth and development. A systematic review of IF feeding showed that 3\u20134 g\/kg body weight per day of protein may successfully accelerate weight gain (Premji et al., 2006). Again there has been debate as to whether this accelerated growth may increase susceptibility to weight and adipose gain in later life, with data showing that it may be associated with school age obesity (Koletzko et al., 2009). Studies are ongoing to establish whether the benefits of increased protein intake in infancy may be offset in later adolescence and adulthood, or whether IF-fed infants have better long-term health in adult life. Outcomes remain equivocal, with prospective studies showing both IF and breast milk to be more beneficial in areas such as bone health in later adult life (Fewtrell et al., 2009; M\u00f8lgaard et al., 2011; Piril\u00e4 et al., 2011).\n\nAn area of protein composition that has been reviewed in detail is immune function. Breast milk contains a range of fractions, including immunoglobulins, lactoferrins, antibodies, macrophages, neutrophils, lymphocytes, and cytokines among others, which may aid immune development (Field, 2005). Because of the importance of this fact, IF commonly also contains factors that may help support immunity. In addition, the use of hydrolyzed proteins for suppression of allergy is of great interest. Hydrolyzed whey protein in IF has been shown to decrease the risk of atopic dermatitis, an inflammatory, chronic form of childhood eczema, after birth and up to 3 years of age. This beneficial effect has been attributed to the presence of immune components such as \u03b2-lactoglobulin, GMP, and lactoferrin (Osborn and Sinn, 2006). Also of possible relevance with respect to allergy is recent evidence from a probiotic supplementation study. Having initially supplemented mothers during pregnancy with two different probiotic strains, these were then given to their infants using a range of delivery formats, including cow's milk IF. This study showed Lactobacillus rhamnosus to decrease the risk of childhood eczema (Wickens et al., 2012). Whether the mother or the infant is the critical window for supplementation, and whether there may be synergies between specific strains of probiotics and the protein component of dairy, is not known.\n\n## Conclusions\n\nThere is an evolving body of evidence showing that milk proteins or milk-derived peptides may have significant functional properties for the prevention and\/or treatment of important chronic health conditions, although much of the science is as yet at an early stage. Clearly, there remains considerable scope for optimizing the processing of whey protein or casein in order to maximize positive health outcomes. The role that dairy protein may play in the prevention of adverse metabolic health and T2DM, atherosclerosis, hypertension and CVD risk, muscle wasting and sarcopenia, as well as osteoporosis, is gradually being elucidated, but research to identify specific protein components, optimum protein dose, synergies with other nutrients, and also the underpinning mechanisms of these positive effects on health are needed. Bovine milk proteins are also an important component of infant formulas and provide an essential alternative when breastfeeding cannot be achieved or maintained. However, further clinical intervention studies, particularly long-term well-controlled studies, are required to build the evidence base necessary to fully support the development of functional foods based on bovine milk proteins for the maintenance and improvement of human health.\n\n##### Disclosures\n\nSDP holds the Fonterra Chair in Human Nutrition at the University of Auckland. RAM receives financial support from the New Zealand Primary Growth Partnership (PGP) program, funded by Fonterra Co-operative group and the New Zealand Ministry for Primary Industries (MPI).\n\n# References\n\nAdechian S ,  Balage M ,  Remond D ,  Mign\u00e9 C ,  Quignard-Boulang\u00e9 A ,  Marset-Baglieri A ,  Rousset S ,  Boirie Y ,  Gaudichon C ,  Dardevet D ,  Mosoni L .  Protein feeding pattern, casein feeding, or milk-soluble protein feeding did not change the evolution of body composition during a short-term weight loss program .  _Am. J. Physiol. Endocrinol. Metab_.  2012 ;303 : E973 \u2013 982 .\n\nAkhavan T ,  Luhovyy BL ,  Brown PH ,  Cho CE ,  Anderson GH .  Effect of premeal consumption of whey protein and its hydrolysate on food intake and postmeal glycemia and insulin responses in young adults .  _Am. J. Clin. Nutr_.  2010 ;91 : 966 \u2013 975 .\n\nAlberti KGMM ,  Eckel RH ,  Grundy SM ,  Zimmet PZ ,  Cleeman JI ,  Donato KA ,  Fruchart J-C ,  James WPT ,  Loria CM ,  Smith Jr SC .  Harmonizing the metabolic syndrome: A joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity .  _Circulation_.  2009 ;120 : 1640 \u2013 1645 .\n\nAnderson GH ,  Moore SE .  Dietary proteins in the regulation of food intake and body weight in humans .  _J. Nutr_.  2004 ;134 : 974S \u2013 979S .\n\nAnderson GH ,  Tecimer SN ,  Shah D ,  Zafar TA .  Protein source, quantity, and time of consumption determine the effect of proteins on short-term food intake in young men .  _J. Nutr_.  2004 ;134 : 3011 \u2013 3015 .\n\nBaer DJ ,  Stote KS ,  Paul DR ,  Harris GK ,  Rumpler WV ,  Clevidence BA .  Whey protein but not soy protein supplementation alters body weight and composition in free-living overweight and obese adults .  _J. Nutr_.  2011 ;141 : 1489 \u2013 1494 .\n\nBallard KD ,  Kupchak BR ,  Volk BM ,  Mah E ,  Shkreta A ,  Liptak C ,  Ptolemy AS ,  Kellogg MS ,  Bruno RS ,  Seip RL ,  Maresh CM ,  Kraemer WJ ,  Volek JS .  Acute effects of ingestion of a novel whey-derived extract on vascular endothelial function in overweight, middle-aged men and women .  _Br. J. Nutr_.  2012 ; 1 \u2013 12 .\n\nBoelsma E ,  Kloek J .  Lactotripeptides and antihypertensive effects: A critical review .  _Br. J. Nutr_.  2009 ;101 : 776 \u2013 786 .\n\nBoirie Y ,  Dangin M ,  Gachon P ,  Vasson MP ,  Maubois JL ,  Beaufr\u00e8re B .  Slow and fast dietary proteins differently modulate postprandial protein accretion .  _Proc. Natl. Acad. Sci. U.S.A_.  1997 ;94 : 14930 \u2013 14935 .\n\nBolland MJ ,  Avenell A ,  Baron JA ,  Grey A ,  MacLennan GS ,  Gamble GD ,  Reid IR .  Effect of calcium supplements on risk of myocardial infarction and cardiovascular events: Meta-analysis .  _BMJ_.  2010 ;341 : c3691 .\n\nBolland MJ ,  Barber PA ,  Doughty RN ,  Mason B ,  Horne A ,  Ames R ,  Gamble GD ,  Grey A ,  Reid IR .  Vascular events in healthy older women receiving calcium supplementation: randomised controlled trial .  _BMJ_.  2008 ;336 : 262 \u2013 266 .\n\nBoye J ,  Wijesinha-Bettoni R ,  Burlingame B .  Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method .  _Br. J. Nutr_.  2012 ;108 : S183 \u2013 211 .\n\nBrader L ,  Holm L ,  Mortensen L ,  Thomsen C ,  Astrup A ,  Holst JJ ,  De Vrese M ,  Schrezenmeir J ,  Hermansen K .  Acute effects of casein on postprandial lipemia and incretin responses in type 2 diabetic subjects .  _Nutr. Metab. Cardiovasc. Dis_.  2010 ;20 : 101 \u2013 109 .\n\nCaroli A ,  Poli A ,  Ricotta D ,  Banfi G ,  Cocchi D .  Invited review: Dairy intake and bone health: A viewpoint from the state of the art .  _J. Dairy Sci_.  2011 ;94 : 5249 \u2013 5262 .\n\nCermak NM ,  De Groot LC ,  Van Loon LJC .  Perspective: Protein supplementation during prolonged resistance type exercise training augments skeletal muscle mass and strength gains .  _J. Am. Med. Dir. Assoc_.  2013 ;14 : 71 \u2013 72 .\n\nChurchward-Venne TA ,  Burd NA ,  Mitchell CJ ,  West DW ,  Philp A ,  Marcotte GR ,  Baker SK ,  Baar K ,  Phillips SM .  Supplementation of a suboptimal protein dose with leucine or essential amino acids: Effects on myofibrillar protein synthesis at rest and following resistance exercise in men .  _J Physiol_.  2012 ;590 : 2751 \u2013 2765 .\n\nCicero AFG ,  Aubin F ,  Azais-Braesco V ,  Borghi C .  Do the lactotripeptides isoleucine-proline-proline and valine-proline-proline reduce systolic blood pressure in European subjects? A meta-analysis of randomized controlled trials .  _Am. J. Hypertens_.  2013 ;26 : 442 \u2013 449 .\n\nClaessens M ,  Van Baak MA ,  Monsheimer S ,  Saris WHM .  The effect of a low-fat, high-protein or high-carbohydrate ad libitum diet on weight loss maintenance and metabolic risk factors .  _Int. J. Obes_.  2009 ;33 : 296 \u2013 304 .\n\nClaessens M ,  Calame W ,  Siemensma AD ,  Van Baak MA ,  Saris WHM .  The effect of different protein hydrolysate\/carbohydrate mixtures on postprandial glucagon and insulin responses in healthy subjects .  _Eur. J. Clin. Nutr_.  2009 ;63 : 48 \u2013 56 .\n\nClaessens M ,  Saris WHM ,  Van Baak MA .  Glucagon and insulin responses after ingestion of different amounts of intact and hydrolysed proteins .  _Br. J. Nutr_.  2008 ;100 : 61 \u2013 69 .\n\nClifton PM ,  Keogh JB ,  Noakes M .  Long-term effects of a high-protein weight-loss diet .  _Am. J. Clin. Nutr_.  2008 ;87 : 23 \u2013 29 .\n\nDalgleish DG ,  Corredig M .  The structure of the casein micelle of milk and its changes during processing .  _Annu. Rev. Food. Sci. Technol_.  2012 ;3 : 449 \u2013 467 .\n\nDespr\u00e9s JP ,  Lemieux I .  Abdominal obesity and metabolic syndrome .  _Nature_.  2006 ;444 : 881 \u2013 887 .\n\nDove ER ,  Hodgson JM ,  Puddey IB ,  Beilin LJ ,  Lee YP ,  Mori TA .  Skim milk compared with a fruit drink acutely reduces appetite and energy intake in overweight men and women .  _Am. J. Clin. Nutr_.  2009 ;90 : 1 \u2013 6 .\n\nElwood PC ,  Givens DI ,  Beswick AD ,  Fehily AM ,  Pickering JE ,  Gallacher J .  The survival advantage of milk and dairy consumption: An overview of evidence from cohort studies of vascular diseases, diabetes and cancer .  _J. Am. Coll. Nutr_.  2008 ;27 : S723 \u2013 S734 .\n\nEngberink MF ,  Schouten EG ,  Kok FJ ,  Van Mierlo LAJ ,  Brouwer IA ,  Geleijnse JM .  Lactotripeptides show no effect on human blood pressure: Results from a double-blind randomized controlled trial .  _Hypertension_.  2008 ;51 : 399 \u2013 405 .\n\nEuropean Food Safety Authority .  Scientific opinion on the substantiation of health claims related to isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) and maintenance of normal blood pressure .  _EFSA. J_.  2012 ;10 : 2715 .\n\nEvans WJ .  Skeletal muscle loss: Cachexia, sarcopenia, and inactivity .  _Am. J. Clin. Nutr_.  2010 ;91 : 1123S \u2013 1127S .\n\nFearon K ,  Strasser F ,  Anker SD ,  Bosaeus I ,  Bruera E ,  Fainsinger RL ,  Jatoi A ,  Loprinzi C ,  MacDonald N ,  Mantovani G ,  Davis M ,  Muscaritoli M ,  Ottery F ,  Radbruch L ,  Ravasco P ,  Walsh D ,  Wilcock A ,  Kaasa S ,  Baracos VE .  Definition and classification of cancer cachexia: An international consensus .  _Lancet. Oncol_.  2011 ;12 : 489 \u2013 495 .\n\nFewtrell MS ,  Williams JE ,  Singhal A ,  Murgatroyd PR ,  Fuller N ,  Lucas A .  Early diet and peak bone mass: 20 year follow-up of a randomized trial of early diet in infants born preterm .  _Bone_.  2009 ;45 : 142 \u2013 149 .\n\nField CJ .  The immunological components of human milk and their effect on immune development in infants .  _J. Nutr_.  2005 ;135 : 1 \u2013 4 .\n\nFitzGerald RJ ,  Meisel H .  Milk protein-derived peptide inhibitors of angiotensin-I-converting enzyme .  _Br. J. Nutr_.  2000 ;84 : S33 \u2013 37 .\n\nFood and Agriculture Organization (FAO), 2013. Dietary protein quality evaluation in human nutrition. Report of an FAO expert consultation. http:\/\/www.fao.org\/ag\/humannutrition\/35978-02317b979a686a 57aa4593304ffc17f06.pdf.\n\nFrestedt JL ,  Zenk JL ,  Kuskowski MA ,  Ward LS ,  Bastian ED .  A whey-protein supplement increases fat loss and spares lean muscle in obese subjects: A randomized human clinical study .  _Nutr. Metab_.  2008 ;5 : 8 .\n\nFrid AH ,  Nilsson M ,  Holst JJ ,  Bj\u00f6rck IME .  Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects .  _Am. J. Clin. Nutr_.  2005 ;82 : 69 \u2013 75 .\n\nGale C ,  Logan KM ,  Santhakumaran S ,  Parkinson JRC ,  Hyde MJ ,  Modi N .  Effect of breastfeeding compared with formula feeding on infant body composition: a systematic review and meta-analysis .  _Am. J. Clin. Nutr_.  2012 ;95 : 656 \u2013 669 .\n\nGouni-Berthold I ,  Schulte DM ,  Krone W ,  Lapointe J-F ,  Lemieux P ,  Predel H-G ,  Berthold HK .  The whey fermentation product malleable protein matrix decreases TAG concentrations in patients with the metabolic syndrome: A randomised placebo-controlled trial .  _Br. J. Nutr_.  2012 ;107 : 1694 \u2013 1706 .\n\nGrundy SM ,  Cleeman JI ,  Daniels SR ,  Donato KA ,  Eckel RH ,  Franklin BA ,  Gordon DJ ,  Krauss RM ,  Savage PJ ,  Smith Jr SC ,  Spertus JA ,  Costa F .  Diagnosis and management of the metabolic syndrome: An American Heart Association\/National Heart, Lung, and Blood Institute Scientific Statement .  _Circulation_.  2005 ;112 : 2735 \u2013 2752 .\n\nHall WL ,  Millward DJ ,  Long SJ ,  Morgan LM .  Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite .  _Br. J. Nutr_.  2003 ;89 : 239 \u2013 248 .\n\nHamad EM ,  Taha SH ,  Abou Dawood A-GI ,  Sitohy MZ ,  Abdel-Hamid M .  Protective effect of whey proteins against nonalcoholic fatty liver in rats .  _Lipids Health Dis_.  2011 ;10 : 57 .\n\nHolmer-Jensen J ,  Hartvigsen ML ,  Mortensen LS ,  Astrup A ,  De Vrese M ,  Holst JJ ,  Thomsen C ,  Hermansen K .  Acute differential effects of milk-derived dietary proteins on postprandial lipaemia in obese non-diabetic subjects .  _Eur. J. Clin. Nutr_.  2012 ;66 : 32 \u2013 38 .\n\nJosse AR ,  Atkinson SA ,  Tarnopolsky MA ,  Phillips SM .  Diets higher in dairy foods and dietary protein support bone health during diet- and exercise-induced weight loss in overweight and obese premenopausal women .  _J. Clin. Endocrinol. Metab_.  2012 ;97 : 251 \u2013 260 .\n\nKatsanos CS ,  Kobayashi H ,  Sheffield-Moore M ,  Aarsland A ,  Wolfe RR .  A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly .  _Am. J. Physiol. Endocrinol. Metab_.  2006 ;291 : E381 \u2013 387 .\n\nKeogh JB ,  Woonton BW ,  Taylor CM ,  Janakievski F ,  Desilva K ,  Clifton PM .  Effect of glycomacropeptide fractions on cholecystokinin and food intake .  _Br. J. Nutr_.  2010 ;104 : 286 \u2013 290 .\n\nKoletzko B ,  Von Kries R ,  Monasterolo RC ,  Sub\u00edas JE ,  Scaglioni S ,  Giovannini M ,  Beyer J ,  Demmelmair H ,  Anton B ,  Gruszfeld D ,  Dobrzanska A ,  Sengier A ,  Langhendries J-P ,  Cachera M-FR ,  Grote V .  Infant feeding and later obesity risk .  _Adv. Exp. Med. Biol_.  2009 ;646 : 15 \u2013 29 .\n\nLarsen TM ,  Dalskov S-M ,  Van Baak M ,  Jebb SA ,  Papadaki A ,  Pfeiffer AFH ,  Martinez JA ,  Handjieva-Darlenska T ,  Kune\u0161ov\u00e1 M ,  Pihlsg\u00e5rd M ,  Stender S ,  Holst C ,  Saris WHM ,  Astrup A .  Diets with high or low protein content and glycemic index for weight-loss maintenance .  _N. Engl. J. Med_.  2010 ;363 : 2102 \u2013 2113 .\n\nLien EL ,  Davis AM ,  Euler AR ,  Multicenter Study Group .  Growth and safety in term infants fed reduced-protein formula with added bovine alpha-lactalbumin .  _J Pediatr Gastroenterol Nutr_.  2004 ;38 : 170 \u2013 176 .\n\nLorenzen J ,  Frederiksen R ,  Hoppe C ,  Hvid R ,  Astrup A .  The effect of milk proteins on appetite regulation and diet-induced thermogenesis .  _Eur. J. Clin. Nutr_.  2012 ;66 : 622 \u2013 627 .\n\nManders RJF ,  Praet SFE ,  Meex RCR ,  Koopman R ,  De Roos AL ,  Wagenmakers AJM ,  Saris WHM ,  Van Loon LJC .  Protein hydrolysate\/leucine co-ingestion reduces the prevalence of hyperglycemia in type 2 diabetic patients .  _Diabetes Care_.  2006 ;29 : 2721 \u2013 2722 .\n\nManders RJF ,  Praet SFE ,  Vikstr\u00f6m MH ,  Saris WHM ,  Van Loon LJC .  Protein hydrolysate co-ingestion does not modulate 24 h glycemic control in long-standing type 2 diabetes patients .  _Eur. J. Clin. Nutr_.  2009 ;63 : 121 \u2013 126 .\n\nMichaelsen KF ,  Larnkj\u00e6r A ,  M\u00f8lgaard C .  Amount and quality of dietary proteins during the first two years of life in relation to NCD risk in adulthood .  _Nutr. Metab. Cardiovasc. Dis_.  2012 ;22 : 781 \u2013 786 .\n\nMojtahedi MC ,  Thorpe MP ,  Karampinos DC ,  Johnson CL ,  Layman DK ,  Georgiadis JG ,  Evans EM .  The effects of a higher protein intake during energy restriction on changes in body composition and physical function in older women .  _J. Gerontol. A Biol. Sci. Med. Sci_.  2011 ;66 : 1218 \u2013 1225 .\n\nM\u00f8lgaard C ,  Larnkj\u00e6r A ,  Mark AB ,  Michaelsen KF .  Are early growth and nutrition related to bone health in adolescence? The Copenhagen Cohort Study of infant nutrition and growth .  _Am. J. Clin. Nutr_.  2011 ;94 : 1865S \u2013 1869S .\n\nMorr CV ,  Ha EY .  Whey protein concentrates and isolates: Processing and functional properties .  _Crit. Rev. Food. Sci. Nutr_.  1993 ;33 : 431 \u2013 476 .\n\nMortensen LS ,  Hartvigsen ML ,  Brader LJ ,  Astrup A ,  Schrezenmeir J ,  Holst JJ ,  Thomsen C ,  Hermansen K .  Differential effects of protein quality on postprandial lipemia in response to a fat-rich meal in type 2 diabetes: comparison of whey, casein, gluten, and cod protein .  _Am. J. Clin. Nutr_.  2009 ;90 : 41 \u2013 48 .\n\nNilsson M ,  Holst JJ ,  Bj\u00f6rck IM .  Metabolic effects of amino acid mixtures and whey protein in healthy subjects: Studies using glucose-equivalent drinks .  _Am. J. Clin. Nutr_.  2007 ;85 : 996 \u2013 1004 .\n\nNilsson M ,  Stenberg M ,  Frid AH ,  Holst JJ ,  Bj\u00f6rck IME .  Glycemia and insulinemia in healthy subjects after lactose-equivalent meals of milk and other food proteins: The role of plasma amino acids and incretins .  _Am. J. Clin. Nutr_.  2004 ;80 : 1246 \u2013 1253 .\n\nOsborn, D.A., and Sinn, J., 2006. Formulas containing hydrolysed protein for prevention of allergy and food intolerance in infants. Cochrane. Database. Syst. Rev. CD003664.\n\nOwen CG ,  Martin RM ,  Whincup PH ,  Davey-Smith G ,  Gillman MW ,  Cook DG .  The effect of breastfeeding on mean body mass index throughout life: A quantitative review of published and unpublished observational evidence .  _Am. J. Clin. Nutr_.  2005 ;82 : 1298 \u2013 1307 .\n\nOwen CG ,  Martin RM ,  Whincup PH ,  Smith GD ,  Cook DG .  Effect of infant feeding on the risk of obesity across the life course: A quantitative review of published evidence .  _Pediatrics_.  2005 ;115 : 1367 \u2013 1377 .\n\nPaddon-Jones D ,  Westman E ,  Mattes RD ,  Wolfe RR ,  Astrup A ,  Westerterp-Plantenga M .  Protein, weight management, and satiety .  _Am. J. Clin. Nutr_.  2008 ;87 : 1558S \u2013 1561S .\n\nPal S ,  Ellis V .  The acute effects of four protein meals on insulin, glucose, appetite and energy intake in lean men .  _Br. J. Nutr_.  2010 ;104 : 1241 \u2013 1248 .\n\nPal S ,  Ellis V .  The chronic effects of whey proteins on blood pressure, vascular function, and inflammatory markers in overweight individuals .  _Obesity_.  2010 ;18 : 1354 \u2013 1359 .\n\nPal S ,  Ellis V ,  Dhaliwal S .  Effects of whey protein isolate on body composition, lipids, insulin and glucose in overweight and obese individuals .  _Br. J. Nutr_.  2010 ;104 : 716 \u2013 723 .\n\nPal S ,  Ellis V ,  Ho S .  Acute effects of whey protein isolate on cardiovascular risk factors in overweight, post-menopausal women .  _Atherosclerosis_.  2010 ;212 : 339 \u2013 344 .\n\nPal S ,  Radavelli-Bagatini S .  The effects of whey protein on cardiometabolic risk factors .  _Obes. Rev_.  2013 ; .\n\nPampaloni B ,  Bartolini E ,  Brandi ML .  Parmigiano Reggiano cheese and bone health .  _Clin. Cases. Miner. Bone. Metab_.  2011 ;8 : 33 \u2013 36 .\n\nParr EB ,  Coffey VG ,  Hawley JA .  Sarcobesity: A metabolic conundrum .  _Maturitas_.  2013 ;74 : 109 \u2013 113 .\n\nPennings B ,  Boirie Y ,  Senden JM ,  Gijsen AP ,  Kuipers H ,  van Loon LJ .  Whey protein stimulates postprandial muscle protein accretion more effectively than do casein and casein hydrolysate in older men .  _Am. J. Clin. Nutr_.  2011 ;93 : 997 \u2013 1005 .\n\nPiril\u00e4 S ,  Taskinen M ,  Viljakainen H ,  Kajosaari M ,  Turanlahti M ,  Saarinen-Pihkala UM ,  M\u00e4kitie O .  Infant milk feeding influences adult bone health: A prospective study from birth to 32 years .  _PLoS. ONE_.  2011 ;6 : e19068 .\n\nPoppitt S ,  Strik C ,  McArdle B ,  McGill A ,  Hall R .  Evidence of enhanced serum amino acid profile but not appetite suppression by dietary glycomacropeptide (GMP): A comparison of dairy whey proteins .  _J. Am. Coll. Nutr_.  2013 ; .\n\nPoppitt SD ,  McCormack D ,  Buffenstein R .  Short-term effects of macronutrient preloads on appetite and energy intake in lean women .  _Physiol. Behav_.  1998 ;64 : 279 \u2013 285 .\n\nPremji, S.S., Fenton, T.R., Sauve, R.S., 2006. Higher versus lower protein intake in formula-fed low birth weight infants. Cochrane Database Syst. Rev. 25 (1), CD003959.\n\nReid IR ,  Mason B ,  Horne A ,  Ames R ,  Reid HE ,  Bava U ,  Bolland MJ ,  Gamble GD .  Randomized controlled trial of calcium in healthy older women .  _Am. J. Med_.  2006 ;119 : 777 \u2013 785 .\n\nReitelseder S ,  Agergaard J ,  Doessing S ,  Helmark IC ,  Lund P ,  Kristensen NB ,  Frystyk J ,  Flyvbjerg A ,  Schjerling P ,  van Hall G ,  Kjaer M ,  Holm Lars .  Whey and casein labeled with l-[1-13C]leucine and muscle protein synthesis: Effect of resistance exercise and protein ingestion .  _Am. J. Physiol. Endocrinol. Metab_.  2011 ;300 : E231 \u2013 E242 .\n\nRice BH ,  Cifelli CJ ,  Pikosky MA ,  Miller GD .  Dairy components and risk factors for cardiometabolic syndrome: Recent evidence and opportunities for future research .  _Adv. Nutr_.  2011 ;2 : 396 \u2013 407 .\n\nSaito T .  Antihypertensive peptides derived from bovine casein and whey proteins .  _Adv. Exp. Med. Biol_.  2008 ;606 : 295 \u2013 317 .\n\nSch\u00fcrch MA ,  Rizzoli R ,  Slosman D ,  Vadas L ,  Vergnaud P ,  Bonjour JP .  Protein supplements increase serum insulin-like growth factor-I levels and attenuate proximal femur bone loss in patients with recent hip fracture. A randomized, double-blind, placebo-controlled trial .  _Ann. Intern. Med_.  1998 ;128 : 801 \u2013 809 .\n\nSheikholeslami Vatani D ,  Ahmadi Kani Golzar F .  Changes in antioxidant status and cardiovascular risk factors of overweight young men after six weeks supplementation of whey protein isolate and resistance training .  _Appetite_.  2012 ;59 : 673 \u2013 678 .\n\nSoedamah-Muthu SS ,  Verberne LD ,  Ding EL ,  Engberink MF ,  Geleijnse JM .  Dairy consumption and incidence of hypertension: A dose-response meta-analysis of prospective cohort studies .  _Hypertension_.  2012 ;60 : 1131 \u2013 1137 .\n\nSoop M ,  Nehra V ,  Henderson GC ,  Boirie Y ,  Ford GC ,  Nair KS .  Coingestion of whey protein and casein in a mixed meal: Demonstration of a more sustained anabolic effect of casein .  _Am. J. Physiol. Endocrinol. Metab_.  2012 ;303 : E152 \u2013 E162 .\n\nTan BHL ,  Fearon KCH .  Cachexia: Prevalence and impact in medicine .  _Curr. Opin. Clin. Nutr. Metab. Care_.  2008 ;11 : 400 \u2013 407 .\n\nTang BMP ,  Eslick GD ,  Nowson C ,  Smith C ,  Bensoussan A .  Use of calcium or calcium in combination with vitamin D supplementation to prevent fractures and bone loss in people aged 50 years and older: A meta-analysis .  _Lancet_.  2007 ;370 : 657 \u2013 666 .\n\nTang JE ,  Moore DR ,  Kujbida GW ,  Tarnopolsky MA ,  Phillips SM .  Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men .  _J. Appl. Physiol_.  2009 ;107 : 987 \u2013 992 .\n\nTipton KD ,  Elliott TA ,  Cree MG ,  Wolf SE ,  Sanford AP ,  Wolfe RR .  Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise .  _Med. Sci. Sports. Exerc_.  2004 ;36 : 2073 \u2013 2081 .\n\nTreyzon L ,  Chen S ,  Hong K ,  Yan E ,  Carpenter CL ,  Thames G ,  Bowerman S ,  Wang H-J ,  Elashoff R ,  Li Z .  A controlled trial of protein enrichment of meal replacements for weight reduction with retention of lean body mass .  _Nutr. J_.  2008 ;7 : 23 .\n\nTsuji-Naito K ,  Jack RW .  Concentrated bovine milk whey active proteins facilitate osteogenesis through activation of the JNK-ATF4 pathway .  _Biosci. Biotechnol. Biochem_.  2012 ;76 : 1150 \u2013 1154 .\n\nTurpeinen AM ,  J\u00e4rvenp\u00e4 \u00e4 S ,  Kautiainen H ,  Korpela R ,  Vapaatalo H .  Antihypertensive effects of bioactive tripeptides\u2014a random effects meta-analysis .  _Ann. Med_.  2013 ;45 : 51 \u2013 56 .\n\nvan Loon LJC .  Leucine as a pharmaconutrient in health and disease .  _Curr. Opin. Clin. Nutr. Metab. Care_.  2012 ;15 : 71 \u2013 77 .\n\nvan Loon LJC ,  Kruijshoop M ,  Menheere PPCA ,  Wagenmakers AJ ,  Saris WHM ,  Keizer HA .  Amino acid ingestion strongly enhances insulin secretion in patients with long-term type 2 diabetes .  _Diabetes Care_.  2003 ;26 : 625 \u2013 630 .\n\nVeldhorst MAB ,  Nieuwenhuizen AG ,  Hochstenbach-Waelen A ,  Van Vught AJAH ,  Westerterp KR ,  Engelen MPKJ ,  Brummer R-JM ,  Deutz NEP ,  Westerterp-Plantenga MS .  Dose-dependent satiating effect of whey relative to casein or soy .  _Physiol. Behav_.  2009 ;96 : 675 \u2013 682 .\n\nVeldhorst MAB ,  Nieuwenhuizen AG ,  Hochstenbach-Waelen A ,  Westerterp KR ,  Engelen MPKJ ,  Brummer R-JM ,  Deutz NEP ,  Westerterp-Plantenga MS .  A breakfast with alpha-lactalbumin, gelatin, or gelatin + TRP lowers energy intake at lunch compared with a breakfast with casein, soy, whey, or whey-GMP .  _Clin. Nutr_.  2009 ;28 : 147 \u2013 155 .\n\nVerdijk LB ,  Jonkers RAM ,  Gleeson BG ,  Beelen M ,  Meijer K ,  Savelberg HHCM ,  Wodzig WKWH ,  Dendale P ,  Van Loon LJC .  Protein supplementation before and after exercise does not further augment skeletal muscle hypertrophy after resistance training in elderly men .  _Am. J. Clin. Nutr_.  2009 ;89 : 608 \u2013 616 .\n\nVillareal DT ,  Fontana L ,  Weiss EP ,  Racette SB ,  Steger-May K ,  Schechtman KB ,  Klein S ,  Holloszy JO .  Bone mineral density response to caloric restriction-induced weight loss or exercise-induced weight loss: A randomized controlled trial .  _Arch. Intern. Med_.  2006 ;166 : 2502 \u2013 2510 .\n\nVolpi E ,  Kobayashi H ,  Sheffield-Moore M ,  Mittendorfer B ,  Wolfe RR .  Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults .  _Am. J. Clin. Nutr_.  2003 ;78 : 250 \u2013 258 .\n\nVolpi E ,  Mittendorfer B ,  Rasmussen BB ,  Wolfe RR .  The response of muscle protein anabolism to combined hyperaminoacidemia and glucose-induced hyperinsulinemia is impaired in the elderly .  _J. Clin. Endocrinol. Metab_.  2000 ;85 : 4481 \u2013 4490 .\n\nWickens K ,  Black P ,  Stanley TV ,  Mitchell E ,  Barthow C ,  Fitzharris P ,  Purdie G ,  Crane J .  A protective effect of Lactobacillus rhamnosus HN001 against eczema in the first 2 years of life persists to age 4 years .  _Clin. Exp. Allergy_.  2012 ;42 : 1071 \u2013 1079 .\n\nXu J-Y ,  Qin L-Q ,  Wang P-Y ,  Li W ,  Chang C .  Effect of milk tripeptides on blood pressure: A meta-analysis of randomized controlled trials .  _Nutrition_.  2008 ;24 : 933 \u2013 940 .\n\nZhu K ,  Meng X ,  Kerr DA ,  Devine A ,  Solah V ,  Binns CW ,  Prince RL .  The effects of a two-year randomized, controlled trial of whey protein supplementation on bone structure, IGF-1, and urinary calcium excretion in older postmenopausal women .  _J. Bone Miner. Res_.  2011 ;26 : 2298 \u2013 2306 . \nChapter 20\n\n# Milk Proteins: Digestion and Absorption in the Gastrointestinal Tract\n\nDidier Dupont*\n\nDaniel Tome**\n\n* UMR 1253 INRA, Rennes, France  \n** UMR 914 INRA, Paris, France\n\n## Abstract\n\nMilk proteins are present in numerous foods and are key players in the human diet. They are known to be highly digestible and excellent sources of indispensable amino acids. Milk proteins can be divided into two families: the caseins, which are proteins with a loose structure that are part of a supramolecular structure called the micelle; and the whey proteins, which exhibit a compact and well-characterized three-dimensional structure. These opposite structures give them opposite behaviors when entering the gastrointestinal tract. Whereas caseins are rapidly hydrolyzed by digestive enzymes, whey proteins show great resistance to hydrolysis by pepsin in the stomach. However, caseins can coagulate and be retained longer in the stomach, and for this reason have been referred to as 'slow proteins,' whereas whey proteins remain soluble and are rapidly transferred into the small intestine. Processing can significantly affect protein digestion and absorption through phenomena such as aggregation, denaturation, coagulation, and hydrolysis.\n\n## Keywords\n\nProtein\n\namino acids\n\nmilk\n\ndigestion\n\nabsorption\n\nhydrolysis\n\naggregation\n\ncoagulation\n\nprocessing\n\nOutline\n\nIntroduction 557\n\nDigestion of milk proteins 558\n\nMilk protein hydrolysis in the intestinal lumen 559\n\nCaseins 559\n\nWhey Proteins 559\n\nPeptides released during digestion 561\n\nImpact of processing on milk protein digestion and absorption 562\n\nHeat Treatment of Milk 562\n\nHomogenization of Milk 563\n\nPhysicochemical Modifications of Proteins 563\n\nCoagulation of Milk 563\n\nConclusions 566\n\n## Introduction\n\nDairy products are an important part of the diet in the industrialized world, especially in northern Europe and North America. In these regions, milk products contribute around 30% of the total dietary protein supply and represent about 65% of the intake of animal protein.\n\nThe protein content of cow's milk ranges from 32 to 35 g\/L. There are two major types of milk protein: the caseins (80%), which are represented by four distinct proteins (\u03b1s1, \u03b1s2, \u03b2, and \u03ba) and the whey proteins (20%), which are represented by proteins such as \u03b2-lactoglobulin and \u03b1-lactalbumin (see Chapter 2 for more details). These two families of proteins are opposite in terms of structure. Caseins exhibit a loose, highly flexible structure and are part of a supramolecular structure called the micelle, whereas whey proteins have a globular, well-defined three-dimensional (3D) structure. These structural differences between the two families markedly affect the behavior of these proteins in the gastrointestinal tract, particularly their susceptibility to hydrolysis by the digestive enzymes.\n\nThe nutritive value of milk protein is generally assessed via two components: nitrogen (indicative of total protein level, a marker of protein quantity) and essential amino acids (a marker of protein quality). The most common method for measuring protein retention is via nitrogen retention. In terms of protein quality, the nutritive value is related to the amino acid content and the bioavailability of these amino acids. The content of indispensable amino acids, that is, those that cannot be synthesized in the body and consequently must be supplied through the diet, is of particular concern.\n\n## Digestion of milk proteins\n\nIn the evaluation of the nutritive value of dietary proteins, nitrogen and individual amino acid digestibility, ileal and fecal digestibility, and apparent and true digestibility should be considered (Fuller & Tom\u00e9, 2005).\n\nThe true digestibility of milk protein, as measured in the ileum, averages 95%, which corresponds to one of the highest digestibilities for dietary proteins (Table 20.1) (AFSSA, 2007). The ileal digestibility of caseins has been estimated to be around 93% in pigs and 94% in humans; that of whey proteins appears to be even higher (97\u221298%) (Gilani & Sepehr, 2003; Lacroix et al., 2006; Rutherfurd & Moughan, 2003) but has never been precisely assessed in humans. Measurement of the true digestibility values of dietary nitrogen and amino acids in healthy human volunteers after the ingestion of milk indicated that ileal digestibility values for the individual amino acids ranged from 92% for serine to 99% for tyrosine, with an average amino acid digestibility of 95.3% (Gaudichon et al., 2002), that is, the same value as for nitrogen digestibility.\n\nTable 20.1\n\nFecal versus Ileal Digestibility (%) of Milk Proteins in Humans\n\n| Fecal | Ileal  \n---|---|---  \nProtein | True | Apparent | True | References  \nMilk protein | 96.6 | 91 | 95 | Bos et al., 2003; Gaudichon et al., 2002; Mah\u00e9 et al., 1994b  \nFermented milk | \u2014 | 90 | \u2014 |  Mah\u00e9 et al., 1994a  \nCasein | \u2014 | \u2014 | 94.1 |  Deglaire et al., 2009\n\nIn the context of digestion, although caseins exhibit a structure that makes them highly sensitive to hydrolysis by digestive enzymes, they are considered to be 'slow proteins' (Boirie et al., 1997) because they cause a slow postprandial release of amino acids in the plasma. This contrasts with whey proteins, which rapidly give rise to an intense peak of amino acids in the plasma. This property of caseins has been attributed to their ability to form a coagulum in the stomach through the joint action of acidic secretions and digestive enzymes. This coagulum of caseins is retained in the stomach longer than the whey proteins, which remain soluble and pass rapidly through the stomach and into the small intestine. These differences in gastric emptying lead to differences in the rate at which dietary amino acids enter the bloodstream (Lacroix et al., 2006; Mah\u00e9 et al., 1996). The longer retention of caseins in the stomach leads to a lower level, but a longer persistence, of amino acids in the plasma than is observed for amino acids from whey proteins (see Fig. 20.1). The type of protein can also specifically influence post-meal aminoacidemia. The chemical composition of whey protein is characterized by high leucine and isoleucine contents, and its ingestion is followed by a peripheral plasma elevation of these amino acids, which are known to be poorly oxidized in the liver. Similarly, the higher plasma proline concentration observed after the ingestion of casein is due to the higher proline content of this fraction (Lacroix et al., 2006).\n\nFigure 20.1 Mean (\u00b1 standard deviation) changes from baseline in serum total amino acid (AA), indispensable AA (IAA), branched-chain AA (BCAA), and dispensable AA (DAA) concentrations in subjects after the ingestion of total milk protein (TMP; n = 8), micellar casein (MC; n = 8), and milk soluble protein isolate (MSPI; n = 7). A significant effect of time (P = 0.0001) and a significant meal-by-time interaction (P = 0.01) were observed for all variables, as tested on the crude values using a mixed-model analysis of variance (ANOVA) with time as a repeated measure.*, **, *** Significantly different from baseline: *P = 0.05, **P = 0.01, ***P = 0.005. Lacroix et al., 2006.\n\n## Milk protein hydrolysis in the intestinal lumen\n\n### Caseins\n\nCaseins are extensively degraded during the gastric phase of digestion, an observation that is consistent with the fact that pepsin has a preference for mobile, loosely structured polypeptides (Dupont et al., 2010a). All in vitro studies on purified proteins have clearly demonstrated that caseins are hydrolyzed within the first minutes of pepsin hydrolysis, in adults as well as in infants (Fig. 20.2A).\n\nFigure 20.2 Evolution of residual immunoreactivity of \u03b2-lactoglobulin (A) and \u03b2-casein (B) during in vitro gastric (left) and duodenal (right) digestion using an infant model ( ) and an adult model ( ), as determined by inhibition enzyme-linked immunosorbent assay (data are the results of three independent determinations made in duplicate).\n\nMore recently, an animal trial conducted on mini-pigs fed skim milk and yogurt also showed a rapid and extensive hydrolysis of caseins during the first minutes of digestion, with intact caseins being detected for only 20 min after the meal intake (Barb\u00e9 et al., 2013). Similarly, in piglets fed milk-based infant formulas, caseins were shown to be more rapidly hydrolyzed than whey proteins, with only 23% of intact caseins being present in the stomach 30 min after ingestion (Bouzerzour et al., 2012).\n\nA recent in vivo study on human volunteers fed caseins showed extensive release in the jejunum of medium-sized peptides (750\u22121050 kDa) during the first 6 h after the meal intake. Most of the identified peptides originated from the two major caseins, that is, \u03b2-casein (61%) and \u03b1s1-casein (25%), and most contained two or more proline residues; the largest contained seven proline residues out of the 26 residues in its sequence (Boutrou et al., 2013). This finding is in agreement with the generally reported resistance of proline-containing peptides to gastric and pancreatic digestive enzymes (Agudelo et al., 2004; Vanhoof et al., 1995) and to epithelial proteinases (Bauchart et al., 2007).\n\n### Whey Proteins\n\nIn contrast to caseins, whey proteins, because of their globular structure, are known to be extremely resistant to proteolysis. This is particularly the case for \u03b2-lactoglobulin, which is not affected during gastric digestion, being virtually unaltered after 60 min of simulated digestion (Fig. 20.2B) (Dupont et al., 2010a; Mandalari et al., 2009b; Schmidt et al., 1995). It has also been shown that the molecular interaction of \u03b2-lactoglobulin with phosphatidylcholine from the gastric mucosa protects the protein from duodenal digestion by trypsin and chymotrypsin (Mandalari, 2009a). However, \u03b2-lactoglobulin has been found to be more sensitive to pepsinolysis when located at the interface of a lipid droplet than when in solution because of drastic conformational changes (Macierzanka et al., 2009).\n\nConflicting results have been published for the second major bovine whey protein, that is, \u03b1-lactalbumin. Whereas some researchers have found that \u03b1-lactalbumin is even more resistant to simulated digestion than \u03b2-lactoglobulin (Inglingstad et al., 2010a), others have found that \u03b1-lactalbumin is susceptible to hydrolysis in solution (Nik et al., 2010). In contrast to \u03b2-lactoglobulin, \u03b1-lactalbumin appears to be more resistant to digestion when located at the oil\u2212water interface than when in solution. The same protective effect of phospholipids from the gastric mucosa on the susceptibility of \u03b1-lactalbumin to hydrolysis by pancreatic enzymes has been described (Moreno et al., 2005).\n\nIn contrast to \u03b1-lactalbumin and \u03b2-lactoglobulin, lactoferrin has been shown to be extensively degraded during simulated gastric digestion (Furlund et al., 2013). Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cleavage sites were located on the surface and mainly on the nonglycosylated half of lactoferrin.\n\n## Peptides released during digestion\n\nThe hydrolysis of milk proteins in the gastrointestinal tract will result in the production of myriad peptides (Jahan-Mihan et al., 2011), some of which have been shown to exert biological activities such as antihypertensive (Martinez-Maqueda et al., 2012), antiatherogenic (Ricci-Cabello et al., 2012), antimicrobial, and immunomodulatory activities (Agyei & Danquah, 2012). Mass spectrometry is the best tool for tracking peptides released during digestion, and the concept of nutritional peptidomics has recently been proposed (Panchaud et al., 2012).\n\nTo date, milk peptides have been identified by submitting food to simulated in vitro digestion (Dupont et al., 2010b; Kopf-Bolanz et al., 2012; Picariello et al., 2010). In the in vitro situation, identifying and quantifying the peptides in digested samples is rather easy because most of the proteins in the samples originate from the food itself. However, it is still questionable whether mimicking digestion with in vitro models perfectly reflects the physiological reality. Only a few in vivo studies have been conducted; detection of dietary peptides is made more difficult by the presence of endogenous proteins secreted in the different compartments of the gut. In a pioneer work, milk caseinomacropeptide, that is, \u03ba-casein (f106\u2212169), was detected in the jejunum of humans fed15N-labeled casein, whey protein, and yogurt (Ledoux et al., 1999). Similarly, caseinophosphopeptides were identified in the effluent collected from milk-fed humans (Meisel et al., 2003). More recently, Bauchart et al. (2007) identified 26 dietary peptides in the duodenum and jejunum of pigs fed beef meat or cooked trout fillets; the peptides were short (<2000 Da) and particularly rich in proline residues. In all these studies, only a limited number of targeted peptides were followed, and the food peptidome found in the lumen of the small intestine was not extensively characterized. In 2013, Boutrou et al. established the peptidome of jejunal effluents collected from milk protein-fed humans. Totals of 356 and 146 peptides were detected and sequenced in the jejunum following casein and whey protein ingestion, respectively. However, the subjects were fed pure protein fractions, and the possible effect of the food structure was not investigated. Indeed, the structure of the chyme, as affected by food structure, could limit or modify the accessibility of digestive enzymes to some cleavage sites.\n\nIn recent work, the impact of the structure of the dairy matrix on the number and nature of milk peptides released in the duodenum was investigated using cannulated mini-pigs fed dairy liquid or acid or rennet dairy gels of identical composition. The formation of peptides in vivo was followed by tandem mass spectrometry over a postprandial period of 5 h after ingestion of the dairy matrices by the mini-pigs. The effect of the meal structure was investigated at two levels: the microstructure, as modified by thermal treatment, and the macrostructure, as modified by milk gelation. More than 16,000 peptides were sequenced and unambiguously identified. The results obtained showed that the structure of the dairy products had only little influence on the location of the cleavage sites on the protein sequences (Barb\u00e9 et al., submitted). However, the structure markedly impacted the number of peptides identified, especially for the rennet dairy gels; about three times fewer peptides were detected than for the other matrices. This effect was attributed to greater extents of dilution by digestive secretions associated with longer gastric retentions for the rennet gels. Potential bioactive peptides were also produced over time, and their identification has increased our knowledge of peptides present in the lumen in vivo. Our results indicate that the structure of dairy matrices markedly affects the kinetics of milk protein digestion in vivo, more than the mechanism of proteolysis itself.\n\n## Impact of processing on milk protein digestion and absorption\n\nMilk proteins are introduced into the human diet as processed milk products. It is therefore critical to determine the impact of the major processing technologies on milk protein digestion.\n\n### Heat Treatment of Milk\n\nOne of the most common processes applied to milk is heat treatment to ensure product safety. Because of the structural differences already mentioned, heat treatment affects caseins and whey proteins quite differently. Heat treatment highly modifies the 3D structure of whey proteins, resulting in an 'opening' of the globular structure and making whey proteins more sensitive to the action of digestive enzymes, as demonstrated for \u03b2-lactoglobulin (Barb\u00e9 et al., 2013) and \u03b1-lactalbumin (Inglingstad et al., 2010b). In contrast, caseins, with their loose and highly flexible structure, are not strongly modified by heat treatment. Heat treatment at high temperature results in an increased resistance of the caseins to simulated digestion (Almaas et al., 2006; Dupont et al., 2010b), which has been attributed to the formation of thermally induced aggregates between caseins and between caseins and whey proteins.\n\n### Homogenization of Milk\n\nHomogenization of milk results in the disruption of the milk fat globule membranes. Lipids are present as smaller droplets that are stabilized by milk proteins covering the oil\u2212water interface. \u03b2-Lactoglobulin and \u03b2-casein have been shown to be more susceptible to pepsinolysis when they are adsorbed to an oil\u2212water interface than when they are in solution (Macierzanka et al., 2009; Sarkar et al., 2009). This has been attributed to the unfolding of the proteins at the droplet surface, which improves their accessibility to pepsin. It has been found that the rate of gastric digestion of \u03b2-casein is twice as fast as when adsorbed to the oil\u2212water interface than when in solution. In the small intestine, proteins are displaced from the interface by bile salts (Sarkar et al., 2010), making triglycerides more accessible to the pancreatic lipase.\n\n### Physicochemical Modifications of Proteins\n\nMilk protein modification with cross-linking enzymes such as transglutaminase (TG) has been used extensively to change the functionality of proteins and thereby to improve the textural quality and stability of protein-based food products. In dairy products, TG-induced cross-linking can increase the firmness and water-holding capacity of acid-induced gels in products with low solids and fat contents or to improve the stability of emulsions and foams. The effect of the TG-induced cross-linking of sodium caseinate on postprandial metabolic and appetite responses was recently investigated in 13 healthy individuals (Juvonen et al., 2012). The results indicated that enzymatically cross-linked sodium caseinate and native sodium caseinate had comparable metabolic responses in a liquid matrix, suggesting similar digestion and absorption rates and first pass metabolism despite the structural modification of the cross-linked sodium caseinate.\n\nThe hydrolysis of milk proteins has been widely used to reduce their allergenicity properties in infant nutrition. However, hydrolysis could also be considered to be a 'pre-digestion' of proteins, facilitating their digestion and absorption in the gastrointestinal tract. This was confirmed in a study on 10 elderly subjects who received either intact or hydrolyzed caseins (Koopman et al., 2009). The plasma amino acid concentrations increased extensively (25\u221250%) after ingestion of the hydrolyzed casein, compared with the intact casein (P < 0.01).\n\n### Coagulation of Milk\n\nMilk coagulation (liquid\/gel\/solid transition) is used extensively in the dairy industry, especially for yogurt and cheese manufacture, even though the mechanisms of milk clotting for these two types of product are quite different. Studies on the digestion of real dairy matrices (yogurt, cheese) are scarce, compared with studies on purified fractions of casein or whey protein. Gaudichon et al. (1994) showed, using mini-pigs, that the half gastric emptying time of the liquid phase was not different between milk and yogurt. However, the intestinal deliveries of both the liquid phase and the nitrogenous fraction of the chyme were more delayed in pigs fed yogurt than in pigs fed milk (Fig. 20.3). The kinetics of exogenous nitrogen delivery into the intestine were correlated with those of exogenous nitrogen absorption. These results suggest that milk proteins are rapidly absorbed after they reach the intestine and that gastric emptying is a major factor controlling the kinetics of milk nitrogen absorption.\n\nFigure 20.3 Remaining fraction of exogenous nitrogen in the stomach of mini-pigs after the ingestion of 500 mL of milk or 500 g of yogurt. The ingested milk and yogurt contained 17 and 18 g of nitrogen, respectively. Values are means \u00b1 standard error of the mean (SEM) for three or four pigs. No significant differences were found by ANOVA, P < 0.05.\n\nRychen et al. (2002) examined the postprandial portal absorption of15N in the growing pig after the ingestion of milk, yogurt, and heat-treated yogurt. Although the total portal absorption was similar between the three products, yogurt nitrogen was absorbed more slowly than milk nitrogen, with significant differences being observed after 30, 60, and 180 min. Heat-treated yogurt showed similar behavior to milk; it was hypothesized that heat treatment of the gel was responsible for destroying the natural body and viscosity of yogurt. These effects were therefore attributed to different emptying rates between milk, yogurt, and heat-treated yogurt.\n\nMore recently, a determination of the kinetics of milk protein digestion and amino acid absorption after the ingestion of liquid or gelled (acid and rennet gels) dairy matrices by six mini-pigs showed that the gelation of milk slowed down the outflow of the meal from the stomach, and the subsequent absorption of amino acids, and decreased their bioavailability in peripheral blood (Fig. 20.4) (Barb\u00e9 et al., 2013). The nature of the matrix seemed to affect the release of the gastrointestinal hormones involved in appetite regulation, with the gel matrices appearing to be potentially more satiating. It was also shown that two gels with the same composition and similar rheological and structural properties, but differing in their mode of coagulation (acidification\/renneting), exhibited different behaviors during digestion. Indeed, ingestion of the rennet gel resulted in lower levels of both proteins in the duodenum and lower levels of amino acids in the plasma, compared with ingestion of the acid gel. This was probably due to the formation of a coagulum with high stiffness after ingestion of the rennet gel, under the simultaneous action of the stomach acidity and the rennet, leading to a very long retention of the rennet matrix in the stomach (Barb\u00e9 et al., 2013). The plasma cholecystokinin and ghrelin concentrations suggested a potentially more satiating effect of the rennet gel than the acid gel.\n\nFigure 20.4 Plasma leucine concentration (\u03bcmol\/h) in mini-pigs over a 7-h period after the ingestion of liquid (L) and gel (G) matrices, from unheated (R) and heated (H) milk products. Values are means \u00b1 SEM calculated for four mini-pigs (n = 4). The data were analyzed using a mixed-model ANOVA. The time effect was significant (P < 0.001), and the lines at the bottom of the figure indicate a significant difference (P < 0.05) from baseline for each curve. The time-by-matrix interaction was significant (P < 0.001), and, at a given time, differences between matrices are indicated by different letters a and b (P < 0.05).\n\nStudies performed on cheese digestion are scarce. A recent study compared the kinetics of the matrix degradation of different cheeses in a gastrointestinal environment (Lamothe et al., 2012). The relationship between the physical characteristics of the cheeses (rheological properties, microstructure) and their digestion patterns was also studied. Rheological measurements and compositional and microstructural analyses were performed on mild cheddar, aged cheddar, light cheddar, and mozzarella cheeses. Mozzarella cheese showed the highest rate of matrix degradation. Aged cheddar cheese showed rapid degradation during the gastric phase, but was more resistant to the duodenal environment. Light cheddar showed the opposite behavior, being highly resistant to the gastric environment; however, it underwent extensive degradation at the end of the duodenal phase. The extent of matrix degradation for mild cheddar was similar to that for mozzarella in the gastric phase but was much lower than that for the other cheeses in the duodenal phase. The results suggest that degradation of the cheeses was driven mainly by their physical characteristics.\n\nThe production of parmigiano reggiano cheese is closely related to the nutritional quality of the final product; in particular, the high digestibility of its proteins is claimed to be proportional to the ripening stage of the cheese. The effect of cheese aging on the kinetics of protein digestion was recently investigated. Two different kinds of parmigiano reggiano, young (aged 15 months) and old (aged 30 months), were separately digested using an in vitro system that simulated digestive processes in the mouth, stomach, and small intestine (Bordoni et al., 2011). The results indicated that the digestion of cheeses with different aging times, although starting from different initial compositions, concluded in similar ways, in terms of free amino acids and small organic compounds, but evolved with different kinetics of hydrolysis and peptide formation, discriminating the young cheese from the old cheese.\n\n## Conclusions\n\nThe digestion and absorption of milk proteins have been extensively studied, and the mechanisms involved are well described. However, many of the studies have been performed either in vitro or with purified protein fractions, and more work is needed to better understand the disintegration of real dairy products in the human gastrointestinal tract. Nevertheless, it appears that casein and whey protein exhibit different behaviors in the gastrointestinal tract because of differences in their structure and physicochemical properties, and that processing has a significant impact on the kinetics of protein digestion by modifying the residence time of the products in the stomach. In the context of the nutritional properties of food, it appears that the micro- and macrostructures of a meal, resulting from technological processes used in the food industry, markedly affect the different steps of milk protein digestion. Thus, the design of food matrices at the technological level is of particular interest in the control of the delivery of nutrients, especially for specific subpopulations, such as the elderly or overweight people.\n\n# References\n\nAFSSA, 2007. Protein intake: Consumption, quality, requirements and recommendations. In: AFSSA., (Ed.), Maisons-Alfort. France.\n\nAgudelo RA ,  Gauthier SF ,  Pouliot Y ,  Marin J ,  Savoie L .  Kinetics of peptide fraction release during in vitro digestion of casein .  _Journal of the Science of Food and Agriculture_.  2004 ;84 (4) : 325 \u2013 332 .\n\nAgyei D ,  Danquah MK .  Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activities .  _Trends in Food Science & Technology_.  2012 ;23 (2) : 62 \u2013 69 .\n\nAlmaas H ,  Cases AL ,  Devold TG ,  Holm H ,  Langsrud T ,  Aabakken L ,  Aadnoey T ,  Vegarud GE .  In vitro digestion of bovine and caprine milk by human gastric and duodenal enzymes .  _International Dairy Journal_.  2006 ;16 (9) : 961 \u2013 968 .\n\nBarb\u00e9 F ,  M\u00e9nard O ,  Le Gouar Y ,  Buffi\u00e8re C ,  Famelart M-H ,  Laroche B ,  Le Feunteun S ,  Dupont D ,  R\u00e9mond D .  The heat treatment and the gelation are strong determinants of the kinetics of milk proteins digestion and of the peripheral availability of amino acids .  _Food Chemistry_.  2013 ;136 : 1203 \u2013 1212 .\n\nBauchart C ,  Morzel M ,  Chambon C ,  Mirand PP ,  Reynes C ,  Buffiere C ,  Remond D .  Peptides reproducibly released by in vivo digestion of beef meat and trout flesh in pigs .  _British Journal of Nutrition_.  2007 ;98 (6) : 1187 \u2013 1195 .\n\nBoirie Y ,  Dangin M ,  Gachon P ,  Vasson MP ,  Maubois JL ,  Beaufrere B .  Slow and fast dietary proteins differently modulate postprandial protein accretion .  _Proceedings of the National Academy of Sciences of the United States of America_.  1997 ;94 (26) : 14930 \u2013 14935 .\n\nBordoni A ,  Picone G ,  Babini E ,  Vignali M ,  Danesi F ,  Valli V ,  Di Nunzio M ,  Laghi L ,  Capozzi F .  NMR comparison of in vitro digestion of Parmigiano Reggiano cheese aged 15 and 30 months .  _Magnetic Resonance in Chemistry_.  2011 ;49 : S61 \u2013 S70 .\n\nBoutrou R ,  Gaudichon C ,  Dupont D ,  Jardin J ,  Airinei G ,  Marsset-Baglieri A ,  Benamouzig R ,  Tome D ,  Leonil J .  Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans .  _American Journal of Clinical Nutrition_.  2013 ;97 (6) : 1314 \u2013 1323 .\n\nBouzerzour K ,  Morgan F ,  Cuinet I ,  Bonhomme C ,  Jardin J ,  Le Huerou-Luron I ,  Dupont D .  In vivo digestion of infant formula in piglets: Protein digestion kinetics and release of bioactive peptides .  _British Journal of Nutrition_.  2012 ;108 (12) : 1 \u2013 10 .\n\nDeglaire A ,  Bos C ,  Tome D ,  Moughan PJ .  Ileal digestibility of dietary protein in the growing pig and adult human .  _British Journal of Nutrition_.  2009 ;102 (12) : 1752 \u2013 1759 .\n\nDupont D ,  Mandalari G ,  Molle D ,  Jardin J ,  Leonil J ,  Faulks RM ,  Wickham MSJ ,  Mills ENC ,  Mackie AR .  Comparative resistance of food proteins to adult and infant in vitro digestion models .  _Molecular Nutrition and Food Research_.  2010 ;54 (6) : 767 \u2013 780 .\n\nDupont D ,  Mandalari G ,  Molle D ,  Jardin J ,  Rolet-Repecaud O ,  Duboz G ,  Leonil J ,  Mills CEN ,  Mackie AR .  Food processing increases casein resistance to simulated infant digestion .  _Molecular Nutrition and Food Research_.  2010 ;54 (11) : 1677 \u2013 1689 .\n\nFuller MF ,  Tome D .  In vivo determination of amino acid bioavailability in humans and model animals .  _Journal of AOAC International_.  2005 ;88 (3) : 923 \u2013 934 .\n\nFurlund CB ,  Ulleberg EK ,  Devold TG ,  Flengsrud R ,  Jacobsen M ,  Sekse C ,  Holm H ,  Vegarud GE .  Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes .  _Journal of Dairy Science_.  2013 ;96 (1) : 75 \u2013 88 .\n\nGaudichon C ,  Bos C ,  Morens C ,  Petzke KJ ,  Mariotti F ,  Everwand J ,  Benamouzig R ,  Dare S ,  Tome D ,  Metges CC .  Ileal losses of nitrogen and amino acids in humans and their importance to the assessment of amino acid requirements .  _Gastroenterology_.  2002 ;123 (1) : 50 \u2013 59 .\n\nGaudichon C ,  Roos N ,  Mahe S ,  Sick H ,  Bouley C ,  Tome D .  Gastric-emptying regulates the kinetics of nitrogen absorption from N-15-labeled milk and N-15-labeled yogurt in miniature pigs .  _Journal of Nutrition_.  1994 ;124 (10) : 1970 \u2013 1977 .\n\nGilani GS ,  Sepehr E .  Protein digestibility and quality in products containing antinutritional factors are adversely affected by old age in rats .  _Journal of Nutrition_.  2003 ;133 (1) : 220 \u2013 225 .\n\nInglingstad RA ,  Devold TG ,  Eriksen EK ,  Holm H ,  Jacobsen M ,  Liland KH ,  Rukke EO ,  Vegarud GE .  Comparison of the digestion of caseins and whey proteins in equine, bovine, caprine and human milks by human gastrointestinal enzymes .  _Dairy Science and Technology_.  2010 ;90 (5) : 549 \u2013 563 .\n\nInglingstad RA ,  Devold TG ,  Eriksen EK ,  Holm H ,  Jacobsen M ,  Liland KH ,  Rukke EO ,  Vegarud GE .  Comparison of the digestion of caseins and whey proteins in equine, bovine, caprine and human milks by human gastrointestinal enzymes .  _Dairy Science and Technology_.  2010 ;90 (5) : 549 \u2013 563 .\n\nJahan-Mihan A ,  Luhovyy BL ,  El Khoury D ,  Anderson GH .  Dietary proteins as determinants of metabolic and physiologic functions of the gastrointestinal tract .  _Nutrients_.  2011 ;3 (5) : 574 \u2013 603 .\n\nJuvonen KR ,  Lille ME ,  Laaksonen DE ,  Mykkanen HM ,  Niskanen LK ,  Herzig KH ,  Poutanen KS ,  Karhunen LJ .  Crosslinking with transglutaminase does not change metabolic effects of sodium caseinate in model beverage in healthy young individuals .  _Nutrition Journal_.  2012 ;11, 35 .\n\nKoopman R ,  Crombach N ,  Gijsen AP ,  Walrand S ,  Fauquant J ,  Kies AK ,  Lemosquet S ,  Saris WHM ,  Boirie Y ,  Van Loon LJC .  Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein .  _American Journal of Clinical Nutrition_.  2009 ;90 (1) : 106 \u2013 115 .\n\nKopf-Bolanz KA ,  Schwander F ,  Gijs M ,  Vergeres G ,  Portmann R ,  Egger L .  Validation of an in vitro digestive system for studying macronutrient decomposition in humans .  _Journal of Nutrition_.  2012 ;142 (2) : 245 \u2013 250 .\n\nLacroix M ,  Bos C ,  Lonil J ,  Airinei G ,  Luengo C ,  Dar S ,  Benamouzig R ,  Fouillet H ,  Fauquant J ,  Tom D ,  Gaudichon C .  Compared with casein or total milk protein, digestion of milk soluble proteins is too rapid to sustain the anabolic postprandial amino acid requirement .  _American Journal of Clinical Nutrition_.  2006 ;84 (5) : 1070 \u2013 1079 .\n\nLamothe S ,  Corbeil MM ,  Turgeon SL ,  Britten M .  Influence of cheese matrix on lipid digestion in a simulated gastro-intestinal environment .  _Food and Function_.  2012 ;3 (7) : 724 \u2013 731 .\n\nLedoux N ,  Mahe S ,  Dubarry M ,  Bourras M ,  Benamouzig R ,  Tome D .  Intraluminal immunoreactive caseinomacropeptide after milk protein ingestion in humans .  _Nahrung-Food_.  1999 ;43 (3) : 196 \u2013 200 .\n\nMacierzanka A ,  Sancho AI ,  Mills ENC ,  Rigby NM ,  Mackie AR .  Emulsification alters simulated gastrointestinal proteolysis of beta-casein and \u03b2-lactoglobulin .  _Soft Matter_.  2009 ;5 (3) : 538 \u2013 550 .\n\nMahe S ,  Gaudichon C ,  Roos N ,  Benamouzig R ,  Luengo C ,  Bouley C ,  Tome D .  N-15-labeled milk and yogurt digestion and absorption in the human jejunum .  _FASEB Journal_.  1994 ;8 (5) : A714 \u2013 A1714 .\n\nMahe S ,  Roos N ,  Benamouzig R ,  Sick H ,  Baglieri A ,  Huneau JF ,  Tome D .  True exogenous and endogenous nitrogen fractions in the human jejunum after ingestion of small amounts of N-15-labeled casein .  _Journal of Nutrition_.  1994 ;124 (4) : 548 \u2013 555 .\n\nMahe S ,  Roos N ,  Benamouzig R ,  Davin L ,  Luengo C ,  Gagnon L ,  Gausserges N ,  Rautureau J ,  Tome D .  Gastrojejunal kinetics and the digestion of N-15 \u03b2-lactoglobulin and casein in humans: The influence of the nature and quantity of the protein .  _American Journal of Clinical Nutrition_.  1996 ;63 (4) : 546 \u2013 552 .\n\nMandalari G ,  Mackie AM ,  Rigby NM ,  Wickham MS ,  Mills E .  Physiological phosphatidylcholine protects bovine \u03b2-lactoglobulin from simulated gastrointestinal proteolysis .  _Molecular Nutrition and Food Research_.  2009 ;53 : S131 \u2013 S139 .\n\nMandalari G ,  Mackie AM ,  Rigby NM ,  Wickham MSJ ,  Mills ENC .  Physiological phosphatidylcholine protects bovine \u03b2-lactoglobulin from simulated gastrointestinal proteolysis .  _Molecular Nutrition and Food Research_.  2009 ;53 : 131 \u2013 139 .\n\nMartinez-Maqueda D ,  Miralles B ,  Recio I ,  Hernandez-Ledesma B .  Antihypertensive peptides from food proteins: a review .  _Food & Function_.  2012 ;3 (4) : 350 \u2013 361 .\n\nMeisel H ,  Bernard H ,  Fairweather-Tait S ,  FitzGerald RJ ,  Hartmann R ,  Lane CN ,  McDonagh D ,  Teucher B ,  Wal JM .  Detection of caseinophosphopeptides in the distal ileostomy fluid of human subjects .  _British Journal of Nutrition_.  2003 ;89 (3) : 351 \u2013 358 .\n\nMoreno FJ ,  Mackie AR ,  Mills ENC .  Phospholipid interactions protect the milk allergen \u03b1-lactalbumin from proteolysis during in vitro digestion .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 (25) : 9810 \u2013 9816 .\n\nNik AM ,  Wright AJ ,  Corredig M .  Surface adsorption alters the susceptibility of whey proteins to pepsin-digestion .  _Journal of Colloid and Interface Science_.  2010 ;344 (2) : 372 \u2013 381 .\n\nPanchaud A ,  Affolter M ,  Kussmann M .  Mass spectrometry for nutritional peptidomics: How to analyze food bioactives and their health effects .  _Journal of Proteomics_.  2012 ;75 (12) : 3546 \u2013 3559 .\n\nPicariello G ,  Ferranti P ,  Fierro O ,  Mamone G ,  Caira S ,  Di Luccia A ,  Monica S ,  Addeo F .  Peptides surviving the simulated gastrointestinal digestion of milk proteins: Biological and toxicological implications .  _Journal of Chromatography B \u2013 Analytical Technologies in the Biomedical and Life Sciences_.  2010 ;878 (3-4) : 295 \u2013 308 .\n\nRicci-Cabello I ,  Herrera MO ,  Artacho R .  Possible role of milk-derived bioactive peptides in the treatment and prevention of metabolic syndrome .  _Nutrition Reviews_.  2012 ;70 (4) : 241 \u2013 255 .\n\nRutherfurd SM ,  Moughan PJ .  The rat as a model animal for the growing pig in determining ileal amino acid digestibility in soya and milk proteins .  _Journal of Animal Physiology and Animal Nutrition_.  2003 ;87 (7\u20138) : 292 \u2013 300 .\n\nRychen G ,  Mpassi D ,  Jurjanz S ,  Mertes M ,  Lenoir-Wijnkoop I ,  Antoine JM ,  Laurent F .  N-15 as a marker to assess portal absorption of nitrogen from milk, yogurt and heat-treated yogurt in the growing pig .  _Journal of Dairy Research_.  2002 ;69 (1) : 95 \u2013 101 .\n\nSarkar A ,  Goh KKT ,  Singh RP ,  Singh H .  Behaviour of an oil-in-water emulsion stabilized by \u03b2-lactoglobulin in an in vitro gastric model .  _Food Hydrocolloids_.  2009 ;23 (6) : 1563 \u2013 1569 .\n\nSarkar A ,  Horne DS ,  Singh H .  Interactions of milk protein-stabilized oil-in-water emulsions with bile salts in a simulated upper intestinal model .  _Food Hydrocolloids_.  2010 ;24 (2\u20133) : 24 .\n\nSchmidt DG ,  Meijer R ,  Slangen CJ ,  Vanberesteijn ECH .  Raising the pH of the pepsin-catalyzed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolysates .  _Clinical and Experimental Allergy_.  1995 ;25 (10) : 1007 \u2013 1017 .\n\nVanhoof G ,  Goossens F ,  Demeester I ,  Hendriks D ,  Scharpe S .  Proline motifs in peptides and their biological processing .  _FASEB Journal_.  1995 ;9 (9) : 736 \u2013 744 . \nChapter 21\n\n# Milk Proteins: The Future\n\nMike J. Boland*\n\n* Riddet Institute, Massey University, Palmerston North, New Zealand\n\n## Abstract\n\nThis final chapter contemplates future trends and their likely impact on the production and use of milk proteins. First, we consider global issues, including energy consumption, the global water economy, and specific issues for dairy relating to greenhouse gases. We then review current and emerging trends in consumer demands and how they might impact the market for milk proteins. Important factors are expected to be food safety and traceability, as well as an increasing concern for the effect of food on health and an increasing importance of personalized nutrition. Finally, we consider some emerging technologies and how they might affect the future of milk protein production and processing.\n\n## Keywords\n\nGlobal issues for food\n\nmilk and energy\n\ndairy methane production\n\nfood safety and traceability\n\nallergies to milk\n\nA2 milk\n\nbioactive peptides\n\ngenetic modification\n\nOutline\n\nIntroduction 571\n\nGlobal issues for food 571\n\nCompetition for Land Use 572\n\nMilk and Energy 572\n\nMilk and the Water Economy 573\n\nImplications of Dairy Methane Production 574\n\nConsumer demands and trends for food and ingredients 575\n\nFood Safety and Traceability 575\n\nFood and Health: Nutrigenomics and Personalized Nutrition 576\n\nNew technologies and their possible effect on milk protein ingredients and products 578\n\nGenetic Modification 578\n\nNovel Processing 579\n\nNew Analytical Methods 580\n\nMaterials Science and Nanotechnology 580\n\nConclusions 581\n\n## Introduction\n\nAs a wrap-up of our journey from expression to food, this chapter takes a look at the possible future of food, especially as it relates to milk proteins. Global macroenvironmental factors are considered first, and then we examine consumer demands and trends, and the likely impact of new technologies.\n\n## Global issues for food\n\nThe demand for high-quality protein for nutrition, especially for dairy protein, has been discussed in the first chapter of this volume. The Food and Agriculture Organization (FAO) has predicted a demand for dairy production by 2050 of 843 million tons\u2014more than double that of today. In order to achieve this figure sustainably, the industry will need to address a range of resource constraints. Global resource issues expected to have a major impact on future food production, including production of milk proteins, are:\n\n\u2022 competition for land use;\n\n\u2022 increasing cost of energy\u2014primarily because of the greenhouse gas implications of energy use, but also because of the rising cost of energy production;\n\n\u2022 the water economy; and\n\n\u2022 methane emissions from cows and the effect on global warming.\n\nThe position of animal production in a constrained world has been discussed in Chapter 1 of this volume and is covered in more depth by Steinfeld et al. (2006), FAO (2009), and Godfray et al. (2010).\n\n### Competition for Land Use\n\nAn important consideration related to competition for land use is the opportunity cost incurred in land used for milk production, including land used for growing crops to feed cows. If cows are fed using arable crops, the equation is simple: The opportunity cost is the value of the crop that has been fed to cows that could otherwise have been used to feed humans. In the case of pasture grazing, the consideration changes to one of the value of competing uses for land (e.g., Godfray et al., 2010). In some cases, when the land is unsuitable for arable farming or horticulture, for example, because the terrain is not suitable, the opportunity cost is low. In cases where the land is suitable for cropping, a comparison has to be made on the value of the crop in relation to the value of the equivalent milk production from that land. This comes down to the purpose of production. If the purpose is simply production of energy, a cereal crop will always come out best. The global need for protein nutrition was addressed in Chapter 1. If the purpose of farming is to produce protein, calculations based on product yields and composition suggest that under the right circumstances, the yield of bioavailable protein per hectare per annum can be at least as high for milk as it is for a cereal crop such as wheat once both are converted into a food format. Pastoral farming can be relatively efficient, partly because of the perennial nature and low maintenance costs of pasture and partly because the cow is doing the work of harvesting as well as re-fertilizing the pasture.\n\n### Milk and Energy\n\nMilk is energetically very expensive. Milk is an animal product: To produce it requires that the cow eat vegetable material that has already been produced in a nutritional format\u2014although not one necessarily edible by humans. However, milk is one of the most efficiently produced of the animal-produced foods, largely because the animal itself is not consumed. It has been estimated that production of 50 kg of milk protein in the United States requires 7 x 106 kcal of feed energy (i.e., 585 kJ\/kg), an energy efficiency of 30:1 (Pimentel and Pimentel, 1979). In contrast, the total energy input per kilogram for production of corn or soy protein in the United States calculates out to 58 kJ\/kg (calculated from data in Pimentel and Pimentel, 1979), one-tenth of the energy. These figures do not take into account the uptake of direct solar energy through photosynthesis as the crops grow, or the opportunity cost in energy for other use of the land that grows these products. It is generally accepted that for grain-fed beef, about 10 kg of grain is required to produce 1 kg of meat. Included in this calculation is the requirement to replace the animal and a loss of around 50% of carcass mass either as waste or by-products. It has been estimated that the energy efficiency for milk is 4:1 and the protein efficiency is 4.75:1 (Council for Agricultural Science and Technology, quoted in Fairlie, 2010). More recent calculated values of the efficiency of conversion of grain and forage into animal protein have been provided by Pimentel (2006), and selected values are given in Table 20.1. A significant difference between the inputs for grass-fed and grain-fed beef production is evident, and presumably a similar difference can be expected for dairy production, although the conversion factor presented is a hybrid.\n\nTable 20.1\n\nEnergy Efficiency of Conversion of Animal Feed to Animal Protein\n\nLivestock | Grain  \n(kg) | Forage  \n(kg) | Energy input \/ energy protein  \n---|---|---|---  \nLamb | 21 | 30 | 57:1  \nBeef cattle | 13 | 30 | 40:1  \nGrass-fed beef cattle | \\-- | 200 | 20:1  \nSwine | 5.9 | \\-- | 14:1  \nDairy (milk protein) | 0.7 | 1 | 14:1\n\nData are from Pimentel (2006).\n\nEnergy sensitivity in some markets, particularly in Europe, has resulted in the use of 'food miles'\u2014an inappropriately named descriptor of the carbon footprint (i.e., the energy cost) expended in producing, distributing, and consuming foods. The methods used in calculating carbon footprints have varied, sometimes leading to inappropriate comparisons. Calculations can be expected to become more accurate and more meaningful in the future as the methodology becomes standardized, but they are also open to misuse as nontariff barriers in some jurisdictions. Most food products are shipped by sea, and the greenhouse gas component of shipping is small compared with production costs, even when food is shipped long distances. For example, in shipping from New Zealand to the UK, the contribution of CO2 from the shipping was estimated at 125 kg CO2\/ton milk solids out of a total of 1422 kg CO2\/ton for the life-cycle footprint, which in turn compared favorably with the equivalent figure of 2921 kg CO2\/ton milk solids for the locally produced equivalent in the UK (Saunders et al., 2006). It is becoming increasingly recognized that sea freight is a minor part of the carbon footprint of a food product, typically 5\u201310%, and that low carbon footprints through more efficient production systems can more than offset this.\n\n### Milk and the Water Economy\n\nIncreasingly, international attention is being paid to the 'water economy' as water becomes a limiting resource in many regions due to the effects of population growth and of climate change. The amount of 'virtual water' in a product is the amount of water required to produce it throughout the production chain. The amount of virtual water in a range of products is given in Table 20.2.\n\nTable 20.2\n\nVirtual Water Content of Dairy and Related Products\n\nProduct | Virtual water  \n(m3\/ton) | Reference  \n---|---|---  \nMilk | 990 |  Hoekstra and Chapagain (2007)  \nMilk powder | 4,602 |  Hoekstra and Chapagain (2007)  \nMilk protein powders | 18,400 | Calculated from above  \nSoybeans | 1,789 |  Hoekstra and Chapagain (2007)  \nSoy protein | 5,400 | Calculated from above\n\nMost of the virtual water in these products arises from on-farm activities, with processing water a minor component. Hence, protein product values have been calculated here by simply adjusting for the amount of protein in the parent product, without adjusting for processing water or credit for the water value of any co-products. The key point is that, as with energy, the cost of water for producing milk-origin products is several-fold higher than for producing similar plant-origin products. This means that only countries that are water-rich can sustainably produce animal-based products for export. This fact will impact in future as water distribution changes with climate change, but also threatens production in some parts of the world where existing water use is unsustainable, such as parts of Australia where water offtake has led to saline ingress into soils (Anderies et al., 2006).\n\nThe use of water for dairy production must be considered in relation to the availability of water and competition for that water. In countries such as New Zealand and Brazil, there is an abundance of fresh water in many regions, and water not used for dairying is unlikely to be used for another more beneficial purpose; thus there is no real opportunity cost. As water availability becomes limiting in different parts of the world, due to the effects of population growth and climate change, the locus of dairy production may shift to water-rich regions. An interesting development is the emergence of 'seawater greenhouse' farming based on using seawater as the primary source of water, with various methods of production of fresh water to support plant growth. This method is already in operation in a number of countries for horticulture. Whether this practice will extend significantly to dairy production remains to be seen.\n\n### Implications of Dairy Methane Production\n\nMethane merits special mention as a greenhouse gas because emissions from cows contribute substantially to the global greenhouse gas load as a by-product of rumen digestion. Methane is recognized as a greenhouse gas and is rated as having a global warming potential 21 times that of the equivalent amount of carbon dioxide, based on a 100-year time scale. It has been estimated that, of all the greenhouse gases, methane is second in effect only to carbon dioxide and is responsible for around 10\u201315% of the present greenhouse gas effect in the atmosphere. Globally, ruminant livestock produce about 28% of methane emissions from human-related activities. A single adult cow is a relatively minor contributor, emitting only 80\u2013110 kg of methane, but, with about 100 million cattle in the United States alone and 1.2 billion large ruminants in the world, ruminants are one of the largest sources of methane. In the United States, cattle emit about 5.5 million tons of methane per annum into the atmosphere, accounting for 20% of U.S. methane emissions, with dairy cattle producing around one-quarter of the total (see www.epa.gov\/methane\/rlep\/faq.html). Although the greenhouse gas effect of methane is a matter of concern, to put it in perspective, it has been estimated that the methane emissions from the entire dairy herd in the United States in 2007 were 112 billion kg CO2 equivalent, less than half of the emissions calculated for the buffalo (American bison) herd in 1860 (228 billion kg CO2 equivalent) (Capper, 2011). It is further recognized that increasing efficiencies in farming have a substantial effect in reducing the carbon footprint of milk. In the United States, it has been estimated that the carbon footprint of milk was reduced by 63% between 1944 and 2007 (Capper et al., 2009). In New Zealand, a strong negative correlation with greenhouse gas emissions has been shown for both kg milk solids per hectare per year and kg milk solids per cow, indicating that more efficient production systems have a lower footprint in terms of their product (Judge et al., 2010).\n\nMost governments recognize the need to limit greenhouse gases, and international negotiations following the Kyoto Protocol were expected to impose penalties on greenhouse gas producers (carbon taxes). To date, governments have shied away from imposing carbon taxes on pastoral farming, and it remains to be seen whether this tax will ever be levied.\n\nNevertheless, methane generation represents both a source of pollution and a waste of energy, and research efforts to specifically target the removal of methanogenic organisms from the rumen are important for the future economic viability of the industry. Because methanogens are believed to have an important role in managing the hydrogen concentration in the rumen, it may be necessary to find or create a microorganism that can transfer hydrogen into a product other than methane.\n\n## Consumer demands and trends for food and ingredients\n\n### Food Safety and Traceability\n\nThroughout the world, awareness of foodborne disease has risen in response to the high-level publicity that such outbreaks receive. The toll exacted in human and economic terms is considerable. Notable dairy outbreaks in recent years include Salmonella in ice cream (United States, 1994: 224,000 cases of illness) and staphylococcal enterotoxin in milk (Japan, 2000: 15,000 cases). Contaminated soft cheeses and raw milk are often in the news. Most dairy products, processed to modern standards of hygiene, have an excellent safety record, but consumers are demanding increased surveillance and control of all foods, including dairy. The contamination of animal feed with dioxin in Belgium in 1999 highlighted the importance consumers place on the absence of toxic chemicals in their food. The deliberate adulteration of infant formula with melamine in China in 2008 resulted in the illnesses and deaths of infants. There will be no lessening in the demands on food producers to control risks and deliver assurances of safety. The increased costs from providing this assurance through effective process control will become the norm for dairy businesses in the future.\n\nIn recent times, increasing attention has been paid to traceability, so that any food safety issue can be quickly traced to its origin and other food from the same batch can be quickly quarantined. Traceability can also be important because of consumers' desire for products that are sustainably produced or have other connotations of quality (such as organically produced products). Traceability is usually managed through the labeling and tracking of products through manufacture and distribution, usually by means of labels on the packaging. This is usually well handled by most food manufacturers and distributors. There have, however, been attempts at 'false-flagging' products in the past, and this will no doubt continue.\n\nFor products containing milk proteins, it is often possible to obtain an internal check on the origin of the product: Dairy herds in different countries and regions tend to have a rather unique mixture of breeds and genetics. This is reflected in the distribution of polymorphisms of the proteins, which can be relatively simply analyzed using gel electrophoresis and\/or mass spectroscopy. Additional information about processing can be gained from mass spectroscopic analysis of postproduction changes in the chemistry of milk proteins (see Chapter 11).\n\nA recent concern has arisen because of deliberate adulteration of milk used for infant formula in China with melamine, leading to the deaths of babies in the summer of 2008 (Tyan et al., 2009). Melamine has a high nitrogen content (66% w\/w) and is relatively inexpensive, and when milk is paid for on the basis of nitrogen content as a proxy for protein content, melamine can be a cheap way to fraudulently boost the apparent protein content. An unfortunate side effect of the addition of melamine can be the presence of cyanuric acid, a common contaminant and hydrolysis product of melamine. Although neither melamine nor cyanuric acid on its own is particularly toxic, the combination of the two can result in the formation of melamine cyanurate crystals, which are very insoluble and tend to form in the kidneys. This can lead to kidney failure. Protein measurements using Fourier transform infrared spectroscopy (FTIR), now a common standard method for routine milk testing in factories, can detect low levels of melamine in dairy products (Balabin and Smirnoff, 2011), and melamine itself is relatively easy to test for in other ways (Tyan et al., 2009).\n\n### Food and Health: Nutrigenomics and Personalized Nutrition\n\nConsumers are being increasingly sensitized to the effects of diet on health (and on personal appearance). The success of diet clinics attests to this trend. The occurrence of (and attention being paid to) current high levels of obesity in affluent societies is spurring interest in diet at all levels of society, from individual to government. Food products on supermarket shelves are increasingly differentiated by the presence of (omega-3 fats, antioxidants) or absence of (fat-free, gluten-free) components believed to affect health.\n\nIndividuals can now obtain information about their own genetic profile, with respect to known genetic polymorphisms related to health and metabolism. Although early-entrant mail-order gene-sequencing companies such as Sciona and Genelex failed, it is now possible for anyone to have their DNA genotyped for under 300US$ (23andMe DNA Spit Kit). The combination of the availability of individual genetic data on an unprecedented scale with a detailed understanding of nutrition has led to the field of 'nutrigenomics', the study of the relationship between a person's genetic make up and their nutritional needs. While early studies in nutrigenomics suggested quick wins, with simple gene differences (single-nucleotide polymorphisms, or SNPs) indicating specific dietary effects, the situation has been found to be much more complex. The focus is now on understanding 'nutritional phenotypes,' which take into account not only SNPs, but other genetic differences such as copy number variants of genes (CNVs), as well as differences in expression of those genes that can be caused by a range of factors, including epigenetic effects, health status, and medications (van Ommen et al., 2010).\n\n'Personalized nutrition' is a nutritional response to differences between individuals\u2014whether from a nutrigenomics input or through other identified needs and preferences\u2014and attempts to balance an individual's diet to specific individual (nutritional phenotype) and situational needs. Nutrition today is not just about balance of macro- and micronutrients: A plethora of functional (bioactive) food components are also known to affect health in ways that extend far beyond the simple supply of nutrients. They can also be modifiers of nutrient uptake and usage, thus modifying the effect of nutritional balance as seen by the body's metabolism. The kinetics of nutrient uptake are just as important as overall absolute uptakes of nutrients. In the case of carbohydrates, this has translated into the 'glycemic index'\u2014an indicator of the rapidity of glucose uptake and thus the effect of a food on insulin production in the body. In the case of proteins, 'fast' and 'slow' proteins have been identified that exit the stomach either rapidly or slowly following ingestion (Boirie et al., 1997), and these may have significant effects on hormone levels and satiety. Personalized nutrition attempts to take this into account, to provide optimal customized nutrition for the individual.\n\nIn sophisticated markets today, there is increasing acceptance of the idea that nutrition has a profound effect on health and wellness; as individuals become more aware of their specific nutritional needs, the demand for personalized nutrition is set to increase.\n\nThe impact of all this on milk proteins has to date been minimal. However, three aspects are notable.\n\n\u2022 Allergies to milk. Such allergies, particularly in infants, have been attributed to \u03b2-lactoglobulin in cow's milk (although recent work has cast some doubt on this theory\u2014see Brix et al., 2003). This protein is not produced in human milk and is the dominant whey protein in bovine milk (see Chapter 2). Whey proteins are important nutritionally, as they are a valuable source of essential amino acids. \u03b2-Lactoglobulin is a particularly important source of branched-chain amino acids. So-called hypoallergenic products are therefore produced by hydrolyzing milk proteins, more particularly whey proteins, so that fragments are sufficiently small to be nonallergenic.\n\n\u2022 A2 milk: Some literature suggests a weak correlation between consumption of milk containing the \u03b2-casein A1 variant and some diseases, notably type 1 diabetes (Elliott at al., 1997) and ischemic heart disease (McLachlan, 2001). Further studies on diabetes have proved to be inconclusive (Beales et al., 2004), and the heart disease data do not stand up to scrutiny. Furthermore, other epidemiological data show that the A2 hypothesis does not hold up (Truswell, 2005). Nevertheless, a New Zealand-based company, the A2 Corporation, markets a niche milk product from cows that do not carry the A1 gene. The milk is sold mostly in Australia, where the company is now very careful not to make claims about any specific health benefits after having been prosecuted and fined $15,000 in Queensland in 2004 for making such claims. The product has since then been launched in New Zealand, where it has limited availability, and more recently in the United Kingdom. A report on A2 milk has been released by the European Food Safety Authority (De Noni et al., 2009), which concludes: \"Based on this review, EFSA concluded that a cause and effect relationship is not established between the dietary intake of BCM7, related peptides or their possible protein precursors and non-communicable diseases. Consequently, a formal EFSA risk assessment is not recommended.\"\n\n\u2022 Bioactive peptides: There is increasing evidence that some milk proteins, and more particularly peptides, have physiological functionality. Effects on cardiovascular health, immune modulation, health of bones and teeth, and anticancer effects have all been reported (see Chapters 18  ,). The validity of these effects and the efficacy of functional foods based on them remain to be fully proven, but in time may lead to new functional foods based on milk proteins and their products.\n\n## New technologies and their possible effect on milk protein ingredients and products\n\nA range of new technologies have the potential to affect dairy production and processing in the near future. They include gene technologies that could lead to new, different milk proteins, new kinds of processing that can produce novel milk protein materials and products containing them, and new analytical techniques that have the potential to improve processing and place ever more stringent requirements on product quality.\n\n### Genetic Modification\n\nMilk proteins have been genetically modified and expressed in nonbovine animals (e.g., Bleck et al., 1998) and in cows (Brophy et al., 2003). However, it seems unlikely that transgenic modification of milk proteins for functional or nutritional purposes will occur widely in the foreseeable future. There are several reasons for this (Creamer et al., 2002).\n\n\u2022 Consumer acceptance of genetically modified (GM) foods is still variable throughout the world, with most countries now having strict labeling requirements. Because milk is a liquid product handled in large volumes during processing, maintaining batch identity and keeping GM milk separate are more problematic than is the case with discrete products, although recent efforts with organic milk have proven that this separation is possible.\n\n\u2022 Milk is an animal product that is strongly targeted at the health of babies and young people. This area has been identified in consumer surveys as very sensitive (compared, for example, with the acceptability of GM fruit and vegetables), and milk will probably be one of the last foods for which genetic modification is acceptable to most consumers.\n\n\u2022 The cost of producing herds of GM cows will be very high, and developing herds will be very slow unless expensive cloning and embryo transfer methods are used. This is not justified by a small premium for improved nutrition or functionality arising from genetic modification.\n\n\u2022 More importantly, a switch to genetic modification will severely limit genetic gain because the gene pool will be restricted to the genetics of the donor animals for the original GM parents. This segregation from the global bovine gene pool will prevent, or severely limit, participation in the ongoing genetic improvement of the species, which is currently occurring at about 2% per annum.\n\nNotwithstanding these points, if a strong nutriceutical or pharmaceutical component were to be identified, enhanced expression through genetic modification would not be out of the question. However, much-touted 'gene-pharming' in dairy animals has not yet been notably successful commercially.\n\nThe announcement of development of a female calf that does not produce \u03b2-lactoglobulin in its milk is of great interest (Jabed et al., 2012). Because \u03b2-lactoglobulin does not occur in human milk, it is often regarded as an undesirable component in milk for infant formula, and it has been implicated in some milk allergies (see above). Hydrolysis of whey protein has been an approach to overcome this problem, but this approach incurs a processing cost and raises the osmolarity of the resulting formula, which is considered undesirable. Although the composition of milk from this experimental animal has been shown to be quite unusual, with no detectable \u03b2-lactoglobulin and elevated casein levels, this composition must be viewed with caution, as it was artificially produced from a female calf (induced by hormones) rather than from natural lactation. It remains to be seen if this animal is able to breed. The fact that this construct removes a protein from milk, rather than adding a foreign protein to it, may lead to a product that meets with less consumer resistance.\n\nIn contrast to milk proteins, competitive plant-origin proteins are well advanced in improvement, using genetic modification. GM soybeans are now predominant in world soybean crops, covering 69 million hectares in 2009, which makes up 77% of worldwide soybean production (www.gmo-compass.org\/eng\/agri_biotechnology\/gmo_planting\/342.genetically_modified_soybean_global_area_under_cultivation.html). Soybeans can be genetically modified to remove undesirable proteins such as trypsin inhibitor, soy hemagglutinin, and allergens (e.g., Friedman et al., 1991), while at the same time soy proteins can be modified to provide a more favorable nutritional balance of essential amino acids (Mandal and Mandal, 2000). The more efficient production of soy proteins in terms of energy and water, coupled with these improvements from genetic modification, means that soy proteins will increasingly outcompete dairy proteins as generic nutritional and functional food ingredients. Having said that, we can expect increasing demand for high-quality protein, so there is likely to be a continuing strong market pull for all forms of food protein.\n\n### Novel Processing\n\nHigh-pressure processing was originally developed in the late 19th century (Hite, 1899) but did not find application in food processing until the 1990s, when new materials enabled the development of production-scale processing equipment. High-pressure processing has been used commercially as an alternative method for preservation, particularly for acidic foods (Dunne and Kluter, 2001).\n\nWhen milk is subjected to high pressure, the casein micelle undergoes dramatic nonreversible changes, leading to a smaller micelle that is less opaque (Chapter 6). It has also been reported that high-pressure processing can alter the functionality of whey proteins (Patel et al., 2005). Whether these novel modified proteins will find application in new foods remains to be seen.\n\nA range of other 'nonthermal' processes for the preservation of food have been developed. These include pulsed electric field, irradiation, and cold plasma. Although some of these innovations have been successful in the processing of acidic foods and solid foods (in the case of cold plasma), their commercial application to dairy products is not an immediate prospect.\n\n### New Analytical Methods\n\nRecent years have seen a range of new and improved analytical methods that have potential to improve process control and tighten product specification. Particularly important are methods that can control product safety (especially microbiology), as well as nutritional and functional properties.\n\nOne weakness in product safety in the past has been the need to grow samples on Petri dishes to test for the presence of undesirable microbial species. This process is time consuming and laborious, and can identify issues with process and product only well after they have occurred. A range of novel microbial detection methods is emerging that may allow at-line detection of microbiological problems, or, conversely, provide early assurance of food safety. For example, use of flow cytometry was able to reduce times for measuring bacterial numbers from the 3 days required for the traditional plate count to 2 h (Flint et al., 2006). This advance could be particularly important for proteins manufactured using ultrafiltration, such as whey protein concentrates and milk protein concentrates, because the ultrafiltration step co-concentrates any microbial contaminants that may be present.\n\nModern electrospray mass spectrometric analysis has enhanced our ability to understand and control processing effects that can alter the nutritional value of milk proteins, particularly the loss of bioavailable lysine due to processing and storage effects (see Chapter 11). Similarly, once the relationship between functionality and protein chemistry is well understood, the same techniques will allow better management of functional properties. Novel inline and at-line methods are becoming possible through a range of techniques, including nuclear magnetic resonance, Fourier transform infrared spectroscopy, selected ion flow tube mass spectrometry, and surface plasmon resonance analysis. These methods can, in principle, measure attributes such as water activity inside packaging and can be used to predict the flavor characteristics of cheeses at maturation (for example, Langford et al., 2012).\n\n### Materials Science and Nanotechnology\n\nFood structure is important at all dimensional scales for the sensory properties of food (including texture, mouthfeel, and flavor release) and may have an important effect on nutrient release and bioavailability (Parada and Aguilera, 2007). Increasingly, attention is being paid to materials science approaches to understanding and potentially managing these effects. An example is the physics of soft materials being applied to food (Mezzenga et al., 2005; Ubbink et al., 2008).\n\nThe potential application of nanotechnology and nanoscience to food can be expected to become an important area. Much of the higher dimensional structure of food is a consequence of nanostructures. It is unlikely that nano robotics will be applied to food in the foreseeable future: Regardless of the considerable technical challenges, public acceptance can be expected to be a major barrier. Notwithstanding this attitude, nanotechnology is having a considerable impact on food science, in part through the use of new improved instrumentation that is becoming available to support nanotechnology research (Foegeding, 2006; Weiss et al., 2006). One of the important features of nanotechnology is the occurrence of self-assembling molecular superstructures (nanostructures). It turns out that foods naturally contain many such systems, examples being the actin-myosin complex in the muscle fibres of meat, starch granules in plant foods, and the casein micelle in milk. Whey proteins have been shown to form self-assembling systems under a variety of conditions\u2014as whey protein (a mixture), as \u03b2-lactoglobulin, and as \u03b1-lactalbumin. Early publications on these (Bolder et al., 2006; Graveland-Bikker and de Kruif, 2006) have been followed by a wide range of published work, recently reviewed by Loveday et al. (2012) and Jones and Mezzenga (2012), that shows how to control the generation and properties of such structures and their physical functional properties.\n\n## Conclusions\n\nThis brief chapter has provided a glimpse of some of the global issues and new technologies that may influence the future development and use of dairy protein products. Global trends such as rising energy costs, scarcity of water, and effect of greenhouse gases will increase the cost of production of milk proteins and will restrict land areas where they can be sustainably produced, especially to areas that are water-rich. Milk proteins are relatively expensive nutritional and functional food ingredients, and although they are nutritionally superior to plant origin competitors, they are likely to be increasingly restricted to niche applications as less expensive plant-based alternatives become more widely available.\n\n# References\n\nAnderies JM ,  Ryan P ,  Walker BH .  Loss of resilience, crisis, and institutional change: lessons from an intensive agricultural system in Southeastern Australia .  _Ecosystems_.  2006 ;9 : 865 \u2013 878 .\n\nBalabin RM ,  Smirnov SV .  Melamine detection by mid- and near-infrared (MIR\/NIR) spectroscopy: A quick and sensitive method for dairy products analysis including liquid milk, infant formula, and milk powder .  _Talanta_.  2011 ;85 : 562 \u2013 568 .\n\nBeales P ,  Elliott R ,  Floh\u00e9 S ,  Hill J ,  Kolb H ,  Pozzilli P ,  Wang G-S ,  Wasmuth H ,  Scott F .  A multi-centre, blinded international trial of the effect of A1 and A2 \u00df-casein variants on diabetes incidence in two rodent models of spontaneous Type I diabetes .  _Diabetologia_.  2004 ;45 : 1240 \u2013 1246 .\n\nBleck GT ,  White BR ,  Miller DJ ,  Wheeler MB .  Production of bovine alpha-lactalbumin in the milk of transgenic pigs .  _Journal of Animal Science_.  1998 ;76 : 3072 \u2013 3078 .\n\nBoirie Y ,  Dangin M ,  Gachon P ,  Vasson MP ,  Maubois JL ,  Beaufrere B .  Slow and fast dietary proteins differently modulate postprandial accretion .  _Proceedings of the National Academy of Science (USA)_.  1997 ;94 : 14930 \u2013 14935 .\n\nBolder SG ,  Hendrickx H ,  Sagis LMG ,  van der Linden E .  Fibril assemblies in aqueous whey protein mixtures .  _Journal of Agricultural Food Chemistry_.  2006 ;54 : 4229 \u2013 4234 .\n\nBrix S ,  Bovetto L ,  Fritsch\u00e9 R ,  Barkholt V ,  Fr\u00f8kiaer H .  Immunostimulatory potential of \u03b2-lactoglobulin preparations: effects caused by endotoxin contamination .  _Journal of Allergy and Clinical Immunology_.  2003 ;112 : 1216 \u2013 1222 .\n\nBrophy B ,  Smolenski G ,  Wheeler T ,  Wells D ,  L'Huillier P ,  Laible G .  Cloned transgenic cattle produce milk with higher \u03b2-lactoglobulin and \u03ba-casein .  _Nature Biotechnology_.  2003 ;21 : 157 \u2013 162 .\n\nCapper JL .  Replacing rose-tinted spectacles with a high-powered microscope: The historical versus modern carbon footprint of animal agriculture .  _Animal Frontiers_.  2011 ;1 : 26 \u2013 32 .\n\nCapper JL ,  Cady RA ,  Bauman DE .  The environmental impact of dairy production: 1944 compared with 2007 .  _Journal of Animal Science_.  2009 ;87 : 2160 \u2013 2167 .\n\nCreamer LK ,  Pearce LE ,  Hill JP ,  Boland MJ .  Milk and dairy products in the 21st century .  _Journal of Agricultural and Food Chemistry_.  2002 ;50 : 7187 \u2013 7193 .\n\nDe Noni I ,  FitzGerald RJ ,  Korhonen HJT ,  Le Roux Y ,  Livesey CT ,  Thorsdottir I ,  Tom\u00e9 D ,  Witkamp R .  Scientific Report of EFSA prepared by a DATEX Working Group on the potential health impact of (-casomorphins and related peptides .  _EFSA Scientific Report (2009)_.  2009 ;231 : 1 \u2013 107 : Available at: www.efsa.europa.eu\/en\/efsajournal\/pub\/231r.htm .\n\nDunne CP ,  Kluter RA .  Emerging non-thermal processing technologies: criteria for success .  _Australian Journal of Dairy Technology_.  2001 ;56 : 109 \u2013 112 .\n\nElliott RB ,  Wasmuth HE ,  Bibby NJ ,  Hill JP .  The role of beta-casein variants in the induction of insulin-dependent diabetes in the non-obese diabetic mouse and humans .  _Milk Protein Polymorphism_ .  Brussels, Belgium :  International Dairy Federation ;  1997 : 445 \u2013 453 .\n\nFairlie S .  _Meat: A benign extravagance_ .  White River Junction :  Chelsea Green Publishing ;  2010 .\n\nFAO .  _The State of Food and Agriculture: Livestock in the Balance_ .  Rome :  Food and Agriculture Organization ;  2009 .\n\nFlint S ,  Drocourt J-L ,  Walker K ,  Stevenson B ,  Dwyer M ,  Clarke I ,  McGill D .  A rapid, two-hour method for the enumeration of total viable bacteria in samples from commercial milk powder and whey protein concentrate powder manufacturing plants .  _International Dairy Journal_.  2006 ;16 : 379 \u2013 384 .\n\nFoegeding EA .  Food biophysics of protein gels: A challenge of nano and macroscopic proportions .  _Food Biophysics_.  2006 ;1 : 41 \u2013 50 .\n\nFriedman M ,  Brandon DL ,  Bates AH ,  Hymowitz T .  Comparison of a commercial soybean cultivar and an isoline lacking the Kunitz trypsin inhibitor: Composition, nutritional value, and effects of heating .  _Journal of Agricultural and Food Chemistry_.  1991 ;39 : 327 \u2013 335 .\n\nGodfray HCJ ,  Beddington JR ,  Cruste IR ,  Haddad L ,  Lawrence D ,  Muir JF ,  Pretty J ,  Robinson S ,  Thomas SM ,  Toulmin C .  Food security: The challenge of feeding 9 billion people .  _Science_.  2010 ;327 : 812 \u2013 818 .\n\nGraveleand-Bikker JF ,  de Kruif CG .  Unique milk protein based nanotubes: Food and nanotechnology meet .  _Trends in Food Science and Technology_.  2006 ;17 : 196 \u2013 203 .\n\nHite BH .  The effect of pressure in the preservation of milk .  _Bulletin of the West Virginia University Agricultural Experiment Station_.  1899 ;58 : 15 .\n\nHoekstra AY ,  Chapagain AK .  Water footprints of nations: Water use by people as a function of their consumption pattern .  _Water Resource Management_.  2007 ;21 : 35 \u2013 48 .\n\nJabed A ,  Wagner S ,  McCracken J ,  Wells DN ,  Laible G .  Targeted microRNA expression in dairy cattle directs production of \u03b2-lactoglobulin-free, high-casein milk .  _Proceedings of the National Academy of Science (USA)_.  2012 ; : doi\/10.1073\/pnas.1210057109 .\n\nJones OG ,  Mezzenga R .  Inhibiting, promoting, and preserving stability of functional protein fibrils .  _Soft Matter_.  2012 ;8 : 876 \u2013 895 .\n\nJudge A ,  Ledgard S ,  Smeaton D ,  Boyes M .  _Greenhouse gas emissions from Rotorua dairy farms_ .  Hamilton, New Zealand :  Report to MAF. AgResearch ;  2010 .\n\nLangford VS ,  Reed CJ ,  Milligan DB ,  McEwan MJ ,  Barringer SA ,  Harper WJ .  Headspace analysis of Italian and New Zealand Parmesan cheeses .  _Journal of Food Science_.  2012 ;77 : C719 \u2013 C726 .\n\nLoveday SM ,  Su J ,  Rao MA ,  Anema SG ,  Singh H .  Whey protein nanofibrils: The environment \u2212 morphology \u2212 functionality relationship in lyophilization, rehydration, and seeding .  _Journal of Agricultural and Food Chemistry_.  2012 ;60 : 5229 \u2013 5236 .\n\nMandal S ,  Mandal RK .  Seed storage proteins and approaches for improvement of their nutritional quality by genetic engineering .  _Current Science_.  2000 ;79 : 576 \u2013 589 .\n\nMcLachlan CN .  Beta-casein A1, ischaemic heart disease mortality, and other illnesses .  _Medical Hypotheses_.  2001 ;56 : 262 \u2013 272 .\n\nMezzenga R ,  Schurtenberger P ,  Burbridge A ,  Michel M .  Understanding foods as soft materials .  _Nature Materials_.  2005 ;4 : 729 \u2013 740 .\n\nParada J ,  Aguilera JM .  Food microstructure affects the bioavailability of several nutrients .  _Journal of Food Science_.  2007 ;72 : R21 \u2013 R32 .\n\nPatel HS ,  Singh H ,  Havea P ,  Considine T ,  Creamer LK .  Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions .  _Journal of Agricultural and Food Chemistry_.  2005 ;53 : 9590 \u2013 9601 .\n\nPimentel D .  Impacts of organic farming on the efficiency of energy use in agriculture .  _An Organic Center State of Science Review_.  2006 ; : 2013 : www.organiccenter.org\/science.pest.php?action=view&report_id=59 downloaded May 2013 .\n\nPimentel D ,  Pimentel M .  _Food, energy, and society_ .  New York :  Wiley ;  1979 .\n\nSaunders C ,  Barber A ,  Taylor G .  _Food Miles\u2014 Comparative Energy\/Emissions Performance of New Zealand's Agriculture Industry_ .  Lincoln, New Zealand :  Research Report No. 285. Lincoln University ;  2006 .\n\nSteinfeld H ,  Gerber P ,  Wassenaar T ,  Castel V ,  Rosales M ,  de Haan C .  _Livestock's long shadow: Environmental issues and options_ .  Rome :  Food and Agriculture Organization ;  2006 .\n\nTyan Y-C ,  Yang M-H ,  Jong S-B ,  Wang C-K ,  Shiea J .  Melamine contamination .  _Analytical and Bioanalytical Chemistry_.  2009 ;395 : 729 \u2013 735 .\n\nTruswell AS .  The A2 milk case: A critical review .  _European Journal of Clinical Nutrition_.  2005 ;59 : 623 \u2013 631 .\n\nUbbink J ,  Burbidge A ,  Mezzenga R .  Food structure and functionality: A soft matter perspective .  _Soft Matter_.  2008 ;4 : 1569 \u2013 1581 .\n\nvan Ommen B ,  Bouwman J ,  Dragsted LO ,  Drevon CA ,  Elliott R ,  de Groot P ,  Kaput J ,  Mathers JC ,  Muller M ,  Pepping F ,  Saito J ,  Scalbert A ,  Radonjic M ,  Rocca-Sera P ,  Travis A ,  Woperis S ,  Evelo CT .  Challenges of molecular nutrition research 6: the nutritional phenotype database to store, share and evaluate nutritional systems biology studies .  _Genes and Nutrition_.  2010 ;5 : 189 \u2013 203 .\n\nWeiss J ,  Takhistov P ,  McClements DJ .  Functional materials in food nanotechnology .  _Journal of Food Science_.  2006 ;71 : R107 \u2013 R116 . \n\n# Index\n\nA\n\nA2 milk, see A2 protein\n\nA2 protein, ,\n\nAllergenic effects, ,\n\nAlpha-cystallin,\n\nAcid gels\n\nbonds, types of, 305\u2013306\n\ncarbonation,\n\n\u03ba-casein, , ,\n\nCCP, ,\n\ncross-linking,\n\ndirect acidification,\n\nenzymatic modification of proteins,\n\nfat globule surface material,\n\nglucono-\u03b4-lactone (GDL), , , ,\n\nhomogenization,\n\nhydrolysis\n\n\u03ba-casein,\n\nwhey proteins,\n\nmicelles, effect of acidification, , 502\u2013503\n\nmicelle-whey protein association, , 302\u2013304\n\nmolecular interactions,\n\nnon sedimentable protein, , ,\n\npH, 192\u2013193, , , 305\u2013306, 502\u2013503,\n\npressure,\n\nprocessing variables, , ,\n\nprotein concentration, , , , 305\u2013306, ,\n\nrheological properties, 192\u2013193, , , ,\n\nsoluble aggregates and acid gel properties, , , 304\u2013305\n\nstorage modulus, , ,\n\nstructure, , , , , ,\n\nsyneresis, , ,\n\ntemperature\n\nacidification temperature,\n\nincubation temperature, ,\n\npH, , , 298\u2013299, 305\u2013306,\n\npressure,\n\npre-treatment, , , , 505\u2013508\n\nwhey protein denaturation, 296\u2013311, 504\u2013508\n\ntexture, 192\u2013193, , , ,\n\ntransglutaminase (TGase),\n\ntrisodium citrate (TSC),\n\nwhey protein denaturation, , 505\u2013508\n\nin the presence of micelles, , 297\u2013307\n\nseparate denaturation to micelles, 310\u2013311\n\nzeta potential,\n\nAcid precipitation, , 274\u2013275, ,\n\nAlkysulfonate (AL),\n\nAlveolus, see Mammary gland\n\nAmino acids\n\ncalorie provision, 533\u2013534\n\ncaseins,\n\ncomparison of proteins,\n\ncysteine, , , ,\n\ndietary essential,\n\ncountries deficient, identification of, 9\u201310\n\ndietary availability of,\n\nfood items, contents of,\n\nformation of lysinoalanine,\n\nlysine, see Lysine\n\nflavor,\n\nlanthionine, , ,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin variants,\n\nlysine, 352\u2013353, , , , , ,\n\nmethionine, , , ,\n\nnutrition, 528\u2013531\n\nphysiological roles, 531\u2013532\n\nproline,\n\nsatiety, 533\u2013534\n\ntaurine, , ,\n\ntryptophan, , , , 439\u2013440\n\nAngiogenins,\n\nAnimal protein sources,\n\ngrowing global demand, 8\u20139\n\nBennett's Law,\n\ncarbon footprint,\n\ngreen revolution,\n\nAtherosclerosis,\n\ncommon cause of,\n\nmilk protein fractions,\n\npostmenopausal women, overweight,\n\nwhey protein\n\nfunctions,\n\nvs. casein,\n\nB\n\nBakery products, 463\u2013464\n\ninclusion of milk proteins,\n\nBAMLET, ,\n\nBiologically active molecules, see Functional components; See also Functional foods Peptides\n\nBiopolymers, see Polysaccharide-protein systems\n\nBlood serum albumin, see Serum albumin\n\nBlood pressure, 547\u2013548\n\ndairy products consumption,\n\nintact milk proteins supplementation,\n\nlowering effects,\n\nmilk-derived peptides\n\nantihypertensive effects,\n\nACE inhibitory activity,\n\nisoleucine-proline-proline (IPP), ,\n\nmeta analysis,\n\nvaline-proline-proline (VPP), ,\n\nwhey protein hydrolysate,\n\nBone health, 548\u2013550\n\nbone mineral density (BMD),\n\nbone remodeling,\n\ncow's milk,\n\nCa derived from,\n\nenergy-restricted diets, weight loss,\n\nessential nutrients,\n\ninfant, see Infant health\n\ninsulin-like growth factor 1 (IGF-1),\n\nnutrition\n\nadolescence,\n\nchildhood,\n\nwhey protein,\n\nfunction,\n\nBonds, see Molecular interactions\n\nBovine classification, 22\u201323\n\nBovine serum albumin, see Serum albumin\n\nBradykinin, ,\n\nC\n\nCalcium-binding proteins, , , , , , , ,\n\nCalcium phosphate\n\nequilibria, 180\u2013181\n\nforms in milk,\n\ngeneral, 53\u201356,\n\nmicelles, role in, 55\u201356, , , , 270\u2013273\n\npH, effect on, , , 502\u2013503,\n\npressure, effect on, ,\n\nrennet gels,\n\nCape fur seal\n\napoptosis, protection from, 99\u2013103\n\ncDNA library,\n\nchanges in milk composition,\n\ncomparison with other seals, ,\n\nFIL protein,\n\ngene expression, 102\u2013103\n\ninvolution, 102\u2013103\n\n\u03b1-lactalbumin,\n\nlactation cycle,\n\nprotein content of milk,\n\nCarbohydrates, see Lactose; See also Oligosaccharides\n\nCarbon footprint,\n\nCarrageenans\n\nCasein\n\navailable amine,\n\ncalcium binding,\n\ncomparison with whey proteins,\n\nflavors, interactions with,\n\nfunctionality, 171\u2013173,\n\ngenes, ,\n\nglycosylation, , 150\u2013151\n\nheterogeneity, , See also \u03ba-casein\n\ncasein phosphoproteins,\n\nexperimental modifications, 130\u2013132\n\nfunction, 125\u2013127\n\nmicelle, see Casein micelle\n\nprecipitation,\n\nprotein characterization,\n\nvariants\n\nbovine \u03b1-Lg,\n\nbovine \u03b1s2-casein,\n\nbovine \u03b1s1-casein, B variant,\n\nbovine \u03b2-casein,\n\nbovine \u03b2-Lg,\n\nbovine \u03ba-casein,\n\n\u03ba-casein genes,\n\ngenetic,\n\n\u03b2-Lg genes,\n\nhistory of, 37\u201338\n\nhydrolysis, , , , , , ,\n\nintestinal lumen,\n\nin vitro studies,\n\nin vivo study, human,\n\nhydrophobicity, ,\n\ninterspecies comparison,\n\nisoelectric point, ,\n\nisopeptide bonds, , ,\n\nmanufacture, 37\u201338, ,\n\nmetal binding,\n\nmicroheterogeneity,\n\nmolecular interactions\n\ndisulfide bonds, , , , , , ,\n\nelectrostatic repulsion, , , , , , , ,\n\nhydrophobic bonds, , , , , 174\u2013175, , 182\u2013184, , , , , , , , ,\n\nproperties, , 43\u201345\n\nrehydration of powders, 337\u2013338\n\nretinol, interactions with, , ,\n\nresidual immunoreactivity, evolution of \u03b2-caesin, digestion of,\n\nsensory characteristics,\n\nstorage and processing changes, , , ,\n\nstructure, ,\n\nsugars, interactions with,\n\ntransglutaminase (TGase), , ,\n\nvariants, proportions of,\n\nvitamin D, interactions with, , see also \u03b1-casein; \u03b2-casein; Casein micelles; \u03b3-casein; \u03ba-casein\n\n\u03b1-Casein\n\naggregation, ,\n\ncalcium binding,\n\ncalcium sensitivity, , ,\n\n\u03ba-casein, interactions with, ,\n\nchaperone-like activity,\n\ndissociation,\n\ndisulfide bonding, ,\n\nemulsions, , , , ,\n\ngenes, ,\n\nhydrolysis, ,\n\nhydrophobicity,\n\nhydrophobic bonds, , , 173\u2013174, 178\u2013179\n\ninterspecies comparison, ,\n\nisolation, ,\n\n\u03b2-lactoglobulin, interactions with, , , ,\n\nlocation in the micelle,\n\nminerals, interactions with, ,\n\nphosphorylation, , , ,\n\npolymorphism,\n\npost-translational modification, ,\n\nproportion in milk,\n\nself-association, , 178\u2013179\n\nstability,\n\nstructure, , ,\n\nvariants, , , , ,\n\nwhey proteins, interactions with,\n\n\u03b2-Casein\n\nA2 protein, ,\n\ncalcium sensitivity, ,\n\nCape fur seal,\n\nchaperone-like activity,\n\ndissociation, , ,\n\nmolecular interactions\n\ndisulfide bonding,\n\nhydrophobic bonds, , , , 173\u2013174, , ,\n\nemulsions, 171\u2013173, , , , ,\n\ngenes, , ,\n\nhydrolysis,\n\nhydrophobicity, ,\n\ninteractions with \u03ba-casein,\n\ninteractions with minerals, , ,\n\ninteractions with surfactants,\n\ninteractions with vitamin D,\n\ninterspecies comparison, , ,\n\nisolation,\n\nphosphorylation, , , ,\n\npost-translational modifications, ,\n\nproportion in milk,\n\nself-association, 173\u2013175, 178\u2013179\n\nstability,\n\nstructure, ,\n\ntemperature changes, ,\n\nvariants, , , ,\n\n\u03b3-Casein\n\nhydrolysis,\n\nisolation,\n\n\u03ba-Casein\n\nacid gels, , ,\n\naggregation,\n\ndissociation\n\nchanges in physical properties of milk,\n\npH, effect of, 282\u2013285,\n\nstabilization of other caseins,\n\ntemperature, effect of, , ,\n\ndisulfide bonding, , , 152\u2013153\n\nemulsions, , ,\n\ngenes, , , ,\n\nglycosylation, , , , , , 150\u2013151, 155\u2013160\n\nheterogeneity\n\nenvironmental influences, ,\n\nhydrolysis, effect on,\n\nlactation phase,\n\nmicelle stability, effect on, ,\n\nbiological significance,\n\npolymorphism,\n\nrennet clotting time (RCT), effect on,\n\nvariants, ,\n\nhydrolysis, , , ,\n\nglycosylation, effect of, 157\u2013158\n\nkinetics,\n\nrennet, , , , ,\n\ninteractions with other whey proteins, ,\n\ninterspecies comparison, , ,\n\nisolation, ,\n\n\u03b2-lactoglobulin, interactions with, , , , 279\u2013293, 505\u2013507\n\ncomplexes, formation of,\n\npH, effect of, 282\u2013285\n\nspecific disulfide bonds, 290\u2013293\n\nstabilizing caseins against calcium ions,\n\nlocation in micelle, , , , , , , 270\u2013273,\n\nmacropeptides, , , , , ,\n\nminerals, interaction with, , ,\n\nmolecular interactions\n\ndisulfide bonds, , , , 152\u2013153, 182\u2013183, , 280\u2013281, 290\u2013293, 305\u2013306\n\nsteric effects, , , , , , , ,\n\nNeuAc, , , ,\n\nphosphorylation, , , ,\n\npost-translational modification,\n\npolymorphism, , ,\n\nproportion in milk,\n\nstability\n\nethanol, , ,\n\npH, 282\u2013285, ,\n\nthermal treatment, , , ,\n\nstructure, ,\n\nvariants, , , , ,\n\nCasein micelles, 53\u201358\n\nacid gels, 192\u2013193\n\naggregation, 495\u2013497, 497\u2013498\n\nbiological purpose, ,\n\ncalcium phosphate, , , 55\u201356, , , 180\u2013186, , , , 270\u2013273\n\ncasein aggregation\n\n\u03b1-casein aggregation, ,\n\n\u03b2-casein self association, 173\u2013174, , 178\u2013179\n\nforces involved, , , 182\u2013183\n\nionic concentration, effect of, 174\u2013175,\n\nmicellization, 173\u2013174\n\ntemperature, effect of,\n\n\u03b1-casein, location in the micelle,\n\n\u03ba-casein\n\ndissociation, , 282\u2013285, ,\n\ndistribution, ,\n\nglycosylation,\n\nhydrolysis, , , ,\n\nlocation in the micelle, , , , , , , 270\u2013273,\n\ncentrifugation, , ,\n\ncomposition\n\ngeneral, ,\n\npH, effect of, , , 184\u2013186, ,\n\nsize fractionation, effect of,\n\ntemperature, effect of, , , , , , ,\n\nconcentration, effect of, 186\u2013188\n\nenzymes, interactions with,\n\ninterspecies comparison,\n\nisoelectric point,\n\nisolation,\n\nmetal binding,\n\npara-\u03ba-casein, , , , , ,\n\npolysaccharides, interactions with\n\n\u03b9-carrageenan,\n\ngalactomannans,\n\nlocust bean gum,\n\npectin,\n\npost-translational modification,\n\nproperties, , 186\u2013188\n\nrennet gels, 189\u2013190, 494\u2013497\n\nrheology, 186\u2013188\n\nself-assembly,\n\nsize, , , , , 191\u2013192, , , ,\n\nsurfactants,\n\nstability\n\nalcohols, , , , , , 191\u2013192,\n\ncalcium,\n\n\u03ba-casein glycosylation,\n\ncompaction, ,\n\ncooling, , , , ,\n\nheating, , , , 282\u2013285\n\npH changes, , , , , , , 191\u2013192, , 282\u2013285, , , 502\u2013503\n\npressure, ,\n\nproteinases, , , ,\n\nsequestrants, , ,\n\ntrifluoroethanol (TFE),\n\nurea, , ,\n\nstructural models, dual-binding\n\nacid gels, 192\u2013193\n\nassembly, 178\u2013179, 182\u2013183\n\ncalcium phosphate equilibria, 180\u2013181\n\nethanol, 191\u2013192\n\nmicellar interactions, 184\u2013186, 186\u2013188\n\nmineral addition, ,\n\npH, , 184\u2013187, 190\u2013192\n\nrennet gels, 186\u2013190\n\nsequestrants, effect of,\n\nstructure, 182\u2013183,\n\ntemperature, effect of, ,\n\ntrifluoroethanol (TFE),\n\nurea, effect of,\n\nstructural models, general, 54\u201356, , , 270\u2013273\n\nsubmicelles, 55\u201356,\n\nsurface structure, , , , , ,\n\nSXE peptide,\n\nwhey proteins, interactions with\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , , 280\u2013293, 305\u2013306, , see also \u03ba-casein; hydrolysis\n\nCaseinomacropeptide (CMP), see Macropeptide\n\nCaseinate\n\nacid gel stabilization, ,\n\nemulsions\n\ncalcium caseinate, , , ,\n\nsodium caseinate, , , 368\u2013369, , ,\n\nflavors, interactions with,\n\nfunctionality,\n\nhydrophobic bonding,\n\nlanthionine,\n\nmanufacture, , , ,\n\nnon-dairy food applications\n\ncoffee whitener,\n\nmeat products, 466\u2013467\n\nwhipped toppings,\n\npolysaccharides, interactions with\n\ngum Arabic, ,\n\npectin, ,\n\nxanthan gum,\n\nproperties, ,\n\nstability, ,\n\nstorage and processing changes, , ,\n\nCaseinophosphopeptides (CPPs),\n\nCCP, see Calcium phosphate\n\nCeruloplasmin,\n\nChaperone-like activity,\n\nChemical denaturants\n\n\u03b1-lactalbumin,\n\nLactoferrin,\n\n\u03b2-lactoglobulin, 218\u2013219\n\nserum albumin,\n\nsugars, effect of,\n\nChitosan\n\nChymosin, , , , , , , , , , see also Rennet\n\nCircular dichroism (CD), , , 216\u2013218, ,\n\nComplex coacervation, see Polysaccharide-protein systems, mixing behaviors\n\nCoagulation, 563\u2013566\n\ncheese,\n\naging effect,\n\ncheddar vs. mozzarella,\n\ndigestion studies,\n\nParmigiano Reggiano, production of,\n\nrheological measurements,\n\ndairy industry, use in,\n\ngel matrices,\n\nkinetics of\n\namino acids absorption,\n\nmilk protein digestion,\n\nmini-pigs stomach,\n\nexogenous nitrogen, remaining fraction of,\n\nplasma leucine concentration,\n\n15N, postprandial portal absorption of,\n\nreal dairy matrices, digestion of,\n\nrennet\n\ngel,\n\nmatrix,\n\nyogurt,\n\ndigestion studies,\n\nheat treated,\n\nColloidal calcium phosphate (CCP), see Calcium phosphates\n\nCommercial milk protein products, , ,\n\nConfocal laser scanning microscope (CLSM), ,\n\nCo-solubility, see Polysaccharide-protein systems, mixing behaviors\n\nCosts of milk production\n\nenergy,\n\nmethane, 574\u2013575\n\nwater, 573\u2013574\n\nCryoglobulin,\n\nCysteine, , , , , , , ,\n\nCysteine residues, , ,\n\nD\n\nDairy food, global\n\ncheese,\n\nexporters, major,\n\nproduction,\n\nconsumption of,\n\nfood consumption patterns, global,\n\nimportance of,\n\nimporters, major,\n\nIndia's dairy industry,\n\nmajor dairy exports, volume of,\n\nprotein consumption, global,\n\nprotein demand, global,\n\nspecialty foods,\n\nworld milk production,\n\nregional distribution, pattern of,\n\nworld protein trade,\n\nDairy product analogues\n\ncoffee whitener,\n\nwhipping products,\n\nDehydration, see Spray drying\n\nDenaturation\n\ndefinition, , see also beta-lactoglobulin\n\nDepletion interactions, see Polysaccharide-protein systems, mixing behaviors\n\nDifferential scanning calorimetry (DSC), , , , , ,\n\nDiffusing wave spectroscopy, ,\n\nDigestion, milk protein\n\namino acids digestibility,\n\ncaseins\n\ncoagulum,\n\nretention time,\n\nslow proteins,\n\nvs. whey protein,\n\nileal digestibility\n\ncaseins,\n\nvs. fecal digestibility,\n\nwhey,\n\npeptides released during digestion,\n\ntrue digestibility of,\n\nDried proteins, see Drying by desorption; See also Protein powders; Spray drying\n\nDrying by desorption\n\ncaseinates, ,\n\ncitrate addition,\n\ndrying slopes,\n\nion addition,\n\nprinciples,\n\nWPC, , see also Spray drying\n\nE\n\nEchidna (Tachyglossus aculeatus)\n\ndevelopment stages,\n\nextant monotreme,\n\nhabitats,\n\nmaternal care, phases of,\n\ngestation,\n\nincubation,\n\nlactation,\n\nweaning,\n\nmonotreme milk\n\ncomponents,\n\ncomposition,\n\nEdman sequencing,\n\nElderly, see also Sarcopenia; Atherosclerosis; Blood pressure\n\nessential amino acids,\n\nanabolic resistance,\n\nhigh-quality protein,\n\nleucine, , see also Leucine\n\nresponsible for,\n\nsupplements,\n\nwhey protein, see Whey protein\n\nprotein nutritional needs of, 12\u201313\n\naging process, characterized by,\n\nhigh-quality protein intake, daily,\n\nmuscle protein synthesis,\n\nprotein dietary allowance, recommended,\n\nsarcopenia, see Sarcopenia\n\nElectron microscopy, , 55\u201356, , 176\u2013178, , , , , , , ,\n\nElectrophoresis, 41\u201342, , ,\n\n2-dimensional (2-DE), , , , , , , ,\n\nnative-PAGE,\n\nSDS-PAGE, , , , ,\n\nElectrospray ionization mass spectrometry,\n\nEmulsifying characteristics of milk constituents, , ,\n\nEmulsions\n\ncasein emulsions\n\ncalcium caseinate, , , ,\n\n\u03b1-casein, , , , ,\n\n\u03b2-casein, 171\u2013173, , , , ,\n\n\u03ba-casein, , ,\n\nformation, ,\n\nsodium caseinate, , , 368\u2013369, , ,\n\nwhey emulsion, stabilization of,\n\ncharacteristics,\n\ncomparison of milk proteins,\n\ncompetitive adsorption, ,\n\ndroplet size, , ,\n\neffect of calcium addition, ,\n\neffect of polysaccharide addition,\n\nformation drivers,\n\nformation process, ,\n\ngel, emulsion,\n\nheat-induced changes, 370\u2013371\n\nhomogenization, ,\n\nhydrolysate-stabilised\n\nbenefits of hydrolysis,\n\ncauses of instability,\n\neffect of polysaccharide addition,\n\nwhey hydrolysates, 373\u2013374\n\ninstability\n\nbridging flocculation, ,\n\ncalcium-induced instability, 373\u2013374\n\ncoalescence, ,\n\ncreaming, , , ,\n\ndefinition,\n\ndepletion flocculation, , 368\u2013369, 371\u2013373\n\nlactoferrin-based emulsions, 374\u2013375\n\nmeat emulsions, 466\u2013467\n\nMPC emulsions, , , ,\n\nmultilayered emulsions,\n\nprocessing of proteins,\n\nprotein load, ,\n\nstability\n\ndefinition,\n\nheat, 370\u2013371\n\nions, , , , , 373\u2013374\n\npH, , ,\n\npressure,\n\nprotein concentration, ,\n\nprotein-oil ratios, 363\u2013364, ,\n\nsynergistic effects,\n\ntypes of instability,\n\nstructure,\n\nsurface activity, ,\n\nsurface tension,\n\nwhey protein emulsions\n\ncasein addition,\n\nformation, , ,\n\nhydrolysates, 373\u2013374\n\nion addition, , 373\u2013374\n\n\u03b1-lactalbumin, , , ,\n\n\u03b2-lactoglobulin, , , , , , ,\n\nmixed whey proteins, , , ,\n\nEnergy to produce milk,\n\nEnzymes, , , ,\n\nEvolutionary changes in proteins, ,\n\nExpressed sequence tag (EST), , ,\n\nExtreme adaptation to lactation, see Cape fur seal; See also Tammar wallaby\n\nF\n\nFAO, see Food and agriculture organization\n\nFood and agriculture organization, , see also Hunger\n\nfood insecurity, definition of,\n\nprotein quality evaluation in human nutrition,\n\nFatty acids\n\nconjugated linoleic acid (CLA), ,\n\nflavors,\n\nproperties,\n\nproteins, interactions with\n\nbinding sites, , , 428\u2013429,\n\ncompetitive binding, ,\n\n\u03b1-lactalbumin, , ,\n\n\u03b2-lactoglobulin, , , , , , , 428\u2013429\n\npH, effect of,\n\nserum albumin, , , , 429\u2013430\n\nstructure, 32\u201334\n\nsynthesis, ,\n\ntriglycerides, distribution in,\n\nFeedback inhibitor of lactation (FIL) protein, see Cape fur seal, Tammar wallaby\n\nFerroxidase, see Ceruloplasmin\n\nFIL, see Cape fur seal; See also Tammar wallaby\n\nFlavor, see Sensory characteristics\n\nFlavor binding, ,\n\nFlocculation, see Polysaccharide-protein systems, mixing behaviors\n\nFluorescence spectroscopy, , , , , ,\n\nFolate-binding proteins (FBPs),\n\nFolate\n\nlactation, regulation of, 119\u2013120\n\nlactation, requirements in,\n\nprotein synthesis, role in,\n\nreceptors,\n\nsupplementation during lactation,\n\nFood miles,\n\nFood safety, 575\u2013576,\n\nFood traceability, 575\u2013576\n\nFourier transform infrared (FTIR) spectroscopy, , , 274\u2013275\n\nFourier transform of Raman spectra,\n\nFreeze drying, protective effect of sugars, 440\u2013441\n\nFunctional characteristics\n\nACE inhibitors, 128\u2013129,\n\nangiogenesis,\n\nantimicrobial, , , , , , , , ,\n\nanticancer, , , , , ,\n\nanticariogenic, ,\n\nanti-hypertensive, 128\u2013129\n\nanti-inflammatory,\n\nantioxidant, , ,\n\nantiviral, , ,\n\nblood coagulation,\n\nbone-cell activity, ,\n\ncopper delivery,\n\ngastrointestinal motility, 535\u2013536\n\ngrowth promotion,\n\nimmunity, , , , , , see also Immunoglobulins\n\nintestinal absorption,\n\nmuscle contraction,\n\nlipase stimulation,\n\nsatiety, 533\u2013534\n\nsecretory processes,\n\nthermogenesis,\n\nvitamin-binding,\n\nFunctional components\n\namino acids\n\narginine, , ,\n\nglutamine,\n\nleucine, , , ,\n\ntaurine, , ,\n\ntryptophan, , ,\n\nbioactive peptides, , , 128\u2013129, 534\u2013536\n\ncaseinophosphopeptides (CPPs),\n\nexorphins,\n\nlactoferricin, ,\n\nmacropeptides, , ,\n\ndiscovery of,\n\nwhole proteins\n\nangiogenins,\n\nenzymes,\n\nglycoproteins, 50\u201351\n\nimmunoglobulins,\n\nkininogens,\n\n\u03b1-lactalbumin,\n\nlactoferrin, , , ,\n\nosteopontin (OPN), 50\u201351\n\nwhey proteins, , 49\u201351\n\nFunctional foods\n\nbackground, , , ,\n\nconsumer considerations,\n\ndefinition,\n\ndrivers, , 128\u2013129,\n\nexamples\n\n\"Anadis\" tablets to prevent diarrhoea,\n\nAnti-hypertensive dairy products, 128\u2013129\n\nPossible future product,\n\n\"Recaldent\" chewing gum to repair tooth enamel,\n\nmanipulation of milk composition, 130\u2013132\n\nregulatory considerations, 526\u2013527, see also Functional compounds; Nutrigenomics; Personalized nutrition\n\nFunctionality of protein systems, see Structure-function relationships\n\nFurosine, ,\n\nG\n\nGalactomannans\n\n\u03b2-Galactosidase,\n\nGels, see Acid-induced gels; See also Mixed gels; Polysaccharide-protein systems; Rennet-induced gels\n\nGene expression, see Cape fur seal; See also Post-translational modifications; Tammar wallaby\n\nGenetic modification\n\nbarriers to uptake, ,\n\neffects on milk characteristics, 130\u2013132\n\nmodification of mammals, ,\n\nmodification of other organisms, , ,\n\ntechniques for manipulating milk proteins,\n\nGenetic polymorphism, , , , ,\n\nCharacteristics influenced, ,\n\nhuman gene polymorphisms,\n\ntypes of milk protein polymorphism, , see also Variants\n\nGenomics\n\nbovine\n\ngenome sequencing project, ,\n\nDNA sequence,\n\nfunctional genomics, 117\u2013118\n\ngenome map,\n\npolymorphisms, ,\n\nCape fur seal, 102\u2013103\n\ncDNA library,\n\ndefinition, 114\u2013115\n\nExpressed sequence tag (EST), , ,\n\nFIL protein,\n\nfolate metabolism and milk production, 119\u2013120\n\ngene expression during lactation cycle, , , ,\n\n\u03b1-lactalbumin, , ,\n\n\u03b2-lactoglobulin, , , ,\n\nMammalian Genome Project,\n\nTammar wallaby\n\ngrowth regulation,\n\ngene expression, 102\u2013103\n\nwhey acidic protein (WAP), , , 90\u201393,\n\nGlobal hunger index\n\nglobal and regional trends of,\n\nhunger indicators,\n\nhunger scenarios, different,\n\nGlucono-\u03b4-lactone (GDL), , , , ,\n\nGlutathione peroxidase,\n\nGlycomacropeptide (GMP), see Macropeptide\n\nGlycoproteins, , , , , ,\n\nGlycosylation, , , , , , , 150\u2013151, 155\u2013160\n\nGrowth factors, ,\n\nGuar gum\n\nGum Arabic\n\nH\n\nHAMLET, ,\n\nHeterogeneity, see Casein; See also \u03ba-casein\n\nHigh pressure processing (HPP), see Pressure treatment\n\nHormones, , , ,\n\nHunger\n\ndefinition of,\n\nGHI, see Global hunger index\n\nprotein, importance of, 5\u20139, see also Protein bioavailability; Protein composition\n\nFAOSTAT database,\n\nprotein daily intake for adults, recommended,\n\nprotein intake, country population,\n\nworld protein supply,\n\nreduction targets,\n\ngoal 1, millennium development goals,\n\nworld food summit target,\n\nworld hunger and undernutrition status,\n\nundernourished people,\n\nundernourishment by region,\n\nHydrolysates\n\nbioactive hydrolysates\n\ncasein, ,\n\n\u03b1-lactalbumin,\n\nfunctional properties\n\nemulsification properties, 373\u2013374\n\nprevention of bar hardening,\n\nnutritional benefits, ,\n\nsensory characteristics, ,\n\nHydrolysis\n\n\u03b1-casein, ,\n\n\u03b2-casein,\n\n\u03ba-casein, , , , , , , , , ,\n\nchymosin, , , , , , , , ,\n\nlactoferrin, ,\n\nplasmin, , ,\n\nrennet, , 39\u201340, , 46\u201347, , , , , , 189\u2013190, ,\n\nHyper-immune milk, ,\n\nHypoallergenic milk products, ,\n\nI\n\nIFPRI, see International food policy research institute\n\nImmunoglobulins\n\nbioactive potential, ,\n\nconcentration in milk, ,\n\nhyper-immunization,\n\nImmunoglobulin G (IgG)\n\nbiological purpose,\n\nstability, , ,\n\nstructure,\n\ninterspecies variation, ,\n\nstability, ,\n\nvariants,\n\nInfant health\n\nbreastfeeding,\n\nbreast milk,\n\nfractionation,\n\ncommercial infant formulas (IF)\n\nhigh-protein content,\n\nhydrolyzed whey protein,\n\nlow-protein levels,\n\nobesity,\n\nprotein-modified,\n\nwhey-modified protein,\n\ncow's milk proteins,\n\nhuman's milk\n\nvs. cow's milk protein composition,\n\nlow-birth-weight (LBW) infants,\n\nInternational food policy research institute,\n\nIsopeptide bonds, , ,\n\nK\n\nKininogens,\n\nL\n\n\u03b1-Lactalbumin\n\nalpha-crystallin, interactions with,\n\napoptosis, protection against,\n\nBAMLET, , , ,\n\nbiological purpose, ,\n\ncasein micelles\/\u03ba-casein, interactions with, 279\u2013281,\n\nemulsions,\n\nfatty acids, interactions with, , ,\n\nflavors, interactions with,\n\nforms, ,\n\ngels, ,\n\ngenes, ,\n\nglycosylation,\n\nHAMLET,\n\nheat-shock proteins, interactions with,\n\nhydrolysates,\n\ninterspecies variation, , 47\u201348,\n\ninvolution, role in,\n\nisolation, ,\n\nlactoferrin, interactions with,\n\n\u03b2-lactoglobulin, interactions with, , , , , , , , , ,\n\nlactose synthesis,\n\nligand binding, ,\n\nlysozyme, similarity to, , , ,\n\nminerals, interactions with, ,\n\nmineral binding\n\nbinding site, ,\n\ngeneral, ,\n\nstability, effect on, ,\n\nmolecular interactions\n\ndisulfide bonding, , , , ,\n\nelectrostatic repulsion,\n\noverview, 47\u201348\n\nphospholipids, interactions with,\n\nproteolysis,\n\nproportion in milk, ,\n\nretinol, interactions with,\n\nserum albumin, interactions with, ,\n\nstability\n\nbound calcium, , , ,\n\nchemical denaturants,\n\ndenaturation kinetics, 276\u2013279\n\nions, , ,\n\nnon-protein soluble components,\n\npH, , ,\n\npressure, , 250\u2013253, ,\n\nprotein concentration,\n\nsugars, 438\u2013440\n\ntemperature, , , , 276\u2013279,\n\nstructure\n\nbovine, 220\u2013222\n\ngeneral, 47\u201348, ,\n\nnon-bovine,\n\nrecombinant,\n\nsurfactants, interactions with,\n\nsynthesis,\n\nvariants,\n\nLactase, see beta-galactosidase\n\nLactation\n\nbovine, ,\n\nCape fur seal, , , 96\u201397\n\neffect of folate supplementation,\n\ngene expression, , 102\u2013103,\n\ninterspecies comparison, 24\u201325, , , 90\u201397\n\noverview, ,\n\nTammar wallaby, , , , ,\n\nLactoferricin, ,\n\nLactoferrin\n\napplications,\n\nbioactivity, , 126\u2013127,\n\ncalcium-binding,\n\nconcentration in milk, ,\n\nemulsification properties, 374\u2013375\n\nhydrolysis, ,\n\ninteractions with other milk proteins\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulm, , 374\u2013375\n\nserum albumin,\n\ninterspecies comparison,\n\niron-binding, , , ,\n\nisolation,\n\nnon-bovine,\n\npH, ,\n\npI,\n\nstability, , ,\n\nstructure, ,\n\n\u03b2-Lactoglobulin\n\nallergenic effects, ,\n\nanalogy to WAP,\n\nbioactivity,\n\nbiological purpose, , ,\n\n\u03b1-casein, interactions with,\n\n\u03ba-casein, interactions with, , , , 279\u2013293, ,\n\nconcentration in milk,\n\ncysteine,\n\ndynamics,\n\ndisulfide bonding, , , , , 216\u2013217, , , , , , 290\u2013293,\n\nemulsions, , , , , ,\n\nfatty acids, interactions with, , , , , , 428\u2013429\n\nbinding site,\n\nflavors, interactions with, ,\n\ngels, ,\n\ngenes, ,\n\ninterspecies variation, , , , 121\u2013122, , ,\n\nisoelectric point,\n\nisoelectric pH,\n\nisolation, ,\n\n\u03b1-lactalbumin, interactions with, , , , , , , , ,\n\nlactoferrin, interactions with, , 374\u2013375\n\nlactose, interactions with,\n\nligand binding, , , , , , 210\u2013213, , ,\n\nbinding sites,\n\ncompetitive binding between retinol and fatty acids,\n\nlipase stimulation,\n\noverview, 46\u201347\n\nphospholipids, interactions with,\n\npolymorphism, , see also Variants\n\npolysaccharides, interactions with,\n\nrennet gels, ,\n\nretinol-binding protein, similarities to, ,\n\nSDS, interactions with, , 430\u2013431\n\nserum albumin interactions with, , , , ,\n\nstability\n\nchemical denaturants, 218\u2013219\n\ndenaturation process, 213\u2013216,\n\ndenaturation kinetics, 276\u2013279\n\nions, ,\n\nligand binding,\n\nnon-protein soluble component, concentration,\n\npH, , 213\u2013216, 218\u2013219,\n\npressure, 216\u2013217, , , , , ,\n\npressure and pH,\n\npressure and temperature, ,\n\nprotein concentration, 213\u2013216,\n\nproteolysis, , ,\n\nsugars, protective effect of, 438\u2013440\n\ntemperature, , 213\u2013216, , 276\u2013279, , 438\u2013440, ,\n\nstructure\n\nbovine, 203\u2013205\n\nchemical denaturants, effect on, 218\u2013219\n\ngeneral, , ,\n\nnon-bovine, , , ,\n\npH dependence, , , 203\u2013205, ,\n\nsugars, protective effect of\n\npressure,\n\nthermal, 438\u2013440\n\nsurfactants, interactions with, 430\u2013431\n\ntransglutaminase (TGas), interactions with,\n\nvariants, , , , ,\n\ndenaturation process,\n\ndifferences between, ,\n\ndistinguishing between variants,\n\ndynamics,\n\nNMR-friendlyAla34Cys mutant, ,\n\nother mutants,\n\npressure, 216\u2013217,\n\nSDS binding,\n\nstability, ,\n\nstructure,\n\nvitamins, interactions with\n\nbinding sites,\n\nvitamin A, , , , , 422\u2013424,\n\nvitamin C,\n\nLactollin, see beta-Microglobulin\n\nLactoperoxide,\n\nLactose, 26\u201328\n\napplications,\n\nbiological purpose,\n\nconcentration, , 28\u201329,\n\ncrystallization, , ,\n\ngenetic engineering to modify concentration, 28\u201329\n\ninterspecies comparison, , ,\n\nintolerance, ,\n\nKoesler Number,\n\n\u03b1-lactalbumin involvement, ,\n\n\u03b2-lactoglobulin, interactions with,\n\nmanufacture, ,\n\nosmotic pressure,\n\npreferential hydration theory,\n\nproperties, 27\u201328\n\nprotection against protein degradation, 439\u2013440\n\nproteolysis,\n\nstructure,\n\nsynthesis, ,\n\nwhey protein denaturation, stabilizing effect on, , see also Lactulosyl lysine; Maillard browning\n\nLactose synthetase,\n\nLactulosyl lysine\n\nbioavailability, 352\u2013353\n\ndetection,\n\nformation kinetics, 345\u2013349\n\nstorage trial, 348\u2013349\n\n\u03b2-lactoglobulin, , , , , 210\u2013213, , ,\n\ncasein,\n\nLeucine\n\nanabolic stimuli, muscle,\n\nimportance of,\n\nas signaling molecule,\n\nLight scattering techniques, , , , , ,\n\nLipids\n\nCLA, ,\n\nclasses,\n\nconcentration,\n\ndegradation, , 35\u201337\n\nform in milk, ,\n\ninterspecies comparison, , 32\u201334,\n\nphospholipid binding,\n\nprotein binding,\n\nPUFA,\n\nsynthesis, , , see also Fatty acids\n\nLipocalin,\n\nLipoprotein lipase (LPL), , , ,\n\nLocust bean gum (LBG)\n\nLysine, , 352\u2013353, , , , , ,\n\nbioavailablity of,\n\ndairy protein,\n\ndeficient countries,\n\nMaillard reaction,\n\nreaction conditions,\n\nmeat, best source of,\n\nLysoalanine, , ,\n\nLysozyme, ,\n\n\u03b1-lactalbumin, similarity to,\n\nM\n\nMaillard reactions, , , 345\u2013349, , , , , ,\n\nMacropeptide, , ,\n\nbioactivity, , ,\n\nformation, , , ,\n\ntypes of macropeptide,\n\nzeta potential, effect on,\n\nMammals\n\nclassification of, 22\u201323\n\nevolution of, 21\u201322, see also Mammary gland\n\nMammary gland\n\nevolution,\n\ninterspecies comparison, 24\u201325\n\nstructure,\n\nhormones,\n\nManipulation of bovine proteins genes, 130\u2013132\n\nMass spectrometry (MS), , , , 291\u2013292\n\nMastitis,\n\nMetabolic health, 542\u2013543\n\nbranched-chain amino acid (BCAA) leucine,\n\ncardiovascular disease (CVD),\n\ndairy products consumption,\n\nepidemiological studies,\n\nhyperglycemia,\n\nimpaired glucose tolerance (IGT),\n\nmilk proteins, long-term clinical studies,\n\ninsulin,\n\nmetabolically active lean body mass,\n\nimportance of,\n\nmetabolic syndrome\n\ndefinition,\n\nphysiological changes,\n\nproinflammatory mediators,\n\nT2DM, see Type 2 diabetes\n\nMeat products, inclusion of milk proteins, 466\u2013467\n\nMetal-binding proteins, see Mineral-binding proteins\n\nMethane production, 574\u2013575\n\nMicrobiological techniques,\n\n\u03b2-Microglobulin,\n\nMicronutrients,\n\nMicroRNAs (miRNAs)\n\nbovine milk,\n\nexogenous,\n\nfunction of,\n\nmammary gland development,\n\nmilk bioactives,\n\nmilk exosomes,\n\nphysical properties,\n\nsignificance,\n\nstage-specific expression,\n\nMilk\n\nbiological purpose, , , , , ,\n\ncommercial products,\n\ncomposition\n\nchanges during lactation, ,\n\ngeneral, , , , , , ,\n\nmanipulation of composition,\n\nratio of components, , see also Lactose; Lipids; Minerals and specific protein names\n\ncosts of production\n\nenergy, 572\u2013573\n\nmethane, 574\u2013575\n\nwater, 573\u2013574\n\nglobal production, ,\n\nheat treatment,\n\neffect on caseins,\n\nwhey protein, modified,\n\nhomogenization,\n\nmilk fat globule membranes, disruption of, , see also Milk fat globule membranes\n\npepsinolysis,\n\nphysicochemical modifications\n\nallergenicity, to reduce,\n\npre-digestion of proteins,\n\nprotein-based food products, stability of,\n\ntransglutaminase (TG), cross-linking enzyme,\n\nsecretion, see Lactation\n\nsynthesis, 24\u201325, see also Bovine; Lactation; Mammary gland; Non-bovine species\n\nMilk fat globule membrane (MFGM)\n\ndamage to,\n\nformation,\n\nglycolipids,\n\ninterspecies comparison, 35\u201337\n\nproperties,\n\nprotein components, , , ,\n\nstructure, ,\n\nMilk protein concentrates\n\ncommercial products,\n\nemulsions, , , ,\n\nfunctionality,\n\ninteractions with xanthan gum,\n\nmanufacture,\n\nnon-dairy food applications\n\nbakery products, 463\u2013464\n\ncoffee whitener,\n\nproduction, global,\n\nsensory characteristics, 483\u2013484\n\nstorage changes\n\navailable amine,\n\nlactulosyl lysine, 345\u2013349, 352\u2013353\n\nMaillard browning, 345\u2013349\n\nMinerals\n\nconcentration in milk, , 28\u201329,\n\ninteractions with proteins,\n\nmodification of product functionality, , ,\n\noverview,\n\ntypes of minerals in milk, , see also Casein micelles; Colloidal calcium phosphate; Mastitis\n\nMineral-binding proteins, , , , see also Casein; Casein micelle; Ceruloplasmin; Glutathione peroxidase; Lactoferrin; Transferrin\n\nMixed gels,\n\nModel food systems\n\nadvantages,\n\napplications,\n\ndevelopment\n\ninitial stages,\n\nstatistical design, 461\u2013462\n\nexample models\n\nbakery products, 463\u2013464\n\ncoffee whitener,\n\nmeat products, 466\u2013467\n\nwhipped toppings,\n\nlimitations,\n\nMolecular interactions\n\ndisulfide bonds\n\ncaseins, , , , , , ,\n\n\u03ba-casein, , , , 152\u2013153, 182\u2013183, , 280\u2013281, 290\u2013293, 305\u2013306\n\nimmunoglobulins,\n\n\u03b1-lactalbumin, , , , ,\n\n\u03b2-lactoglobulin, , , , , 216\u2013217, , , , , , , 290\u2013293, 305\u2013306, , 510\u2013512,\n\n\u03b2-lactoglobulin and \u03ba-casein, , , , , , 280\u2013281, 290\u2013293, 305\u2013306\n\nmixed milk proteins, , ,\n\nserum albumin, , , , , ,\n\nWAP,\n\nelectrostatic repulsion\n\ncaseins, , , , , , , ,\n\nemulsions, , ,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , ,\n\nmicelles, , , , , , , , , 191\u2013192, ,\n\nprotein-polysaccharide mixtures, , , ,\n\nwhey proteins and other species, , ,\n\ngeneral covalent bonds, , , , , , , ,\n\nhydrogen bonds\n\ngels, ,\n\n\u03b2-lactoglobulin, , ,\n\nmicelle structure, , ,\n\nprotein-polysaccharide mixtures,\n\nsugars,\n\nhydrophobic bonds\n\n\u03b1-casein, , , 173\u2013174, 178\u2013179\n\n\u03b2-casein, , , , 173\u2013174, , ,\n\ncaseins, , , , , 174\u2013175, , 182\u2013184, , , , , , , , ,\n\n\u03b2-lactoglobulin, , , , , , , , , 424\u2013426, , , ,\n\nmicelle structure, , , 182\u2013187, 189\u2013192, ,\n\nprotein-polysaccharide mixtures,\n\nwhey proteins, , , , , , , ,\n\nsteric effects\n\n\u03ba-casein, , , , , , ,\n\nemulsions, , , ,\n\nprotein-polysaccharide mixtures, , ,\n\nsugars,\n\nwhey proteins,\n\nvan der Waals' bonds, , , ,\n\nMucins, 35\u201337\n\nN\n\nNanotechnology,\n\nNational dairy development board,\n\nNDDB, see National dairy development board\n\nNative-PAGE, see Electrophoresis Non-bovine species\n\n\u03b1-caseins, ,\n\n\u03b2-caseins, ,\n\n\u03ba-caseins, , ,\n\nclassification of, 21\u201322\n\ncryoglobulin,\n\nenzymes,\n\ngenome,\n\nimmunoglobulins, ,\n\n\u03b1-lactalbumin, 47\u201348,\n\n\u03b2-lactoglobulin, , 121\u2013122, , ,\n\nlactose, , ,\n\nlipid classes, , 32\u201334, ,\n\nmilk fat globule membrane, 35\u201337\n\noligosaccharide levels, ,\n\nprotein comparison, 56\u201358\n\nwhey acidic protein (WAP), , , 90\u201393, , see also Cape fur seal; Mammary gland; Tammar wallaby\n\nNon-protein nitrogen,\n\nNuclear magnetic resonance (NMR)\n\ngels,\n\n\u03b2-lactoglobulin, 205\u2013208, , , 216\u2013218, , ,\n\npowders, ,\n\nserum albumin,\n\nNutraceuticals, see Functional foods\n\nNutrigenomics, 576\u2013578\n\nNutrigenetics, 576\u2013578\n\nNutritional characteristics\n\namino acids, 528\u2013534\n\ncalorie provision, 533\u2013534\n\nmanufacture of nutritional products, , see also Functional foods Nutrigenomics\n\nO\n\nObesity and weight control, 543\u2013548\n\nadipose loss,\n\nbranched-chain amino acids (BCAA),\n\nmuscle protein synthesis,\n\nenergy-restricted diets,\n\nwhey protein,\n\nheart health, see Atherosclerosis; See also Blood pressure\n\nhigh-protein diets,\n\nsuppressing hunger,\n\nhigh-protein meal replacement,\n\nmilk protein role,\n\nlong-term studies,\n\nlower body weight,\n\nmuscle wasting, see Sarcopenia\n\nsarcobesity,\n\nskimmed milk,\n\nisoenergetic bolus, ingestion of,\n\nwhey protein vs. casein,\n\nsupplements\n\ncasein,\n\nwhey protein,\n\nOligosaccharides\n\nbiological purpose, , ,\n\nconcentrations,\n\ninterspecies comparison, ,\n\nosmotic pressure,\n\nstructure,\n\nsynthesis,\n\nOsmotic pressure, ,\n\nOsteopontin (OPN),\n\nP\n\nparacasein, ,\n\nPectin, see Polysaccharide-protein systems\n\nPeptides, , , 128\u2013129, , see also Functional components\n\nPeptides released, digestion\n\nbioactive peptides,\n\nbiological activities,\n\n\u03ba-casein,\n\ndetection of,\n\ncaseinophosphopeptides,\n\nidentification of,\n\nin vitro digestion of food,\n\nin vivo peptide formation,\n\nin duodenum,\n\nin jejenum,\n\nmeal structure, effect of,\n\nmilk proteins hydrolysis,\n\nPepsin, , , , see also Chymosin; Rennet\n\nPersonalised nutrition, 576\u2013578\n\nPhospholipid binding,\n\nPhosphorylation, , ,\n\nPlasmin, , ,\n\nPolymorphism, , , , , , ,\n\nPolysaccharide-protein systems\n\ncasein micelles\n\n\u03b9-carrageenan,\n\ngalactomannans,\n\nlocust bean gum,\n\npectin,\n\ncaseinate\n\ngum Arabic, ,\n\npectin, ,\n\nxanthan gum,\n\nemulsions,\n\ngel formation,\n\ngelling phase-separated systems, 404\u2013406,\n\ninteracting mixtures, 406\u2013409\n\n\u03b2-lactoglobulin\n\n\u03ba-carrageenan,\n\nchitosan,\n\npectin,\n\nmixing behaviors\n\nco-solubility, 388\u2013389,\n\ncomplex coacervation, , , ,\n\ndepletion flocculation, , , , ,\n\ndepletion interaction, , , , ,\n\nthermodynamic incompatibility, , , ,\n\nmolecular interactions\n\nattractive, 393\u2013394\n\ncovalent,\n\nrepulsive, 392\u2013393\n\nMPC and xanthan gum,\n\nnon-gelling phase-separated systems, , 407\u2013408\n\nnon-interacting mixtures, 402\u2013406\n\nphase diagrams, 390\u2013391\n\nrheological measurements, ,\n\ntransglutaminase cross-linking,\n\nwhey proteins, mixed\n\nEPS,\n\ngalactomannans,\n\nguar gum,\n\ngum Arabic,\n\nxanthan gum, ,\n\nPost-translational modifications (PTMs)\n\nanalytical techniques,\n\n\u03b1-casein, ,\n\n\u03b2-casein, ,\n\n\u03ba-casein, , 150\u2013151\n\ndisulfide bonding, 152\u2013153\n\nglycosylation, , 150\u2013151, 155\u2013156\n\nimportance of,\n\noccurrence, ,\n\nphosphorylation, , , ,\n\nPressure treatment\n\nacid gels,\n\nbond disruption,\n\ncommercially pressure-treated products,\n\ncomparison with thermal, , , ,\n\ndenaturation of proteins caseins,\n\ncasein-whey interactions, , ,\n\neffect of processing variables,\n\ngeneral, 247\u2013248\n\nimmunoglobulins, ,\n\n\u03b1-lactalbumin, 250\u2013251,\n\nlactoferrin, ,\n\n\u03b2-lactoglobulin, , 248\u2013250, , ,\n\nmicelles, ,\n\nmilk systems, 256\u2013261\n\nserum albumin, ,\n\nwhey proteins, mixtures of, ,\n\nwhey protein products, commercial, 252\u2013253\n\nemulsions,\n\ngelation of whey proteins\n\ncomparison with thermal gels,\n\neffect of processing variables, ,\n\nformation, ,\n\n\u03b2-lactoglobulin, ,\n\nproperties,\n\nsugars,\n\nstorage,\n\nWPI,\n\nimmunoglobulins, ,\n\n\u03b1-lactalbumin, , 250\u2013253, ,\n\nlactoferrin,\n\n\u03b2-lactoglobulin\n\nconcentration,\n\ndenaturation, , 248\u2013250, , , ,\n\nenzymatic cleavage,\n\ngels,\n\ninteractions with \u03b1-casein,\n\ninteractions with \u03ba-casein,\n\ninteractions with whey proteins,\n\npH,\n\npresence of ligands, ,\n\npressure level, , , 248\u2013250,\n\npre-denatured state,\n\nprocessing variables,\n\nstructural changes, 216\u2013217, 248\u2013250\n\ntemperature, ,\n\nvariants,\n\nLe Chatelier-Braun's principle,\n\nmilk systems\n\nwhey protein denaturation,\n\nwhey proteins and casein micelles, 258\u2013261,\n\npreservation,\n\nprocess development, ,\n\npurpose, , , ,\n\nserum albumin, , , ,\n\nstructural modification, , , , ,\n\nanalytical techniques, 244\u2013247\n\ntemperature of pressure treatment,\n\nwhey protein products, commercial, 252\u2013253,\n\nProtein bioavailability\n\nanimal-derived proteins,\n\ncommon food proteins\n\ntrue protein digestibility,\n\nfecal digestibility,\n\nplant-derived proteins,\n\nprotein breakdown, gastrointestinal tract,\n\nprotein supply, adequate,\n\nProtein composition\n\ndietary essential amino acids,\n\nlinear chains amino acids, composed of,\n\nProtein powders\n\nagglomeration, 476\u2013479\n\napplications, 295\u2013296,\n\ncommercial products, 321\u2013323,\n\ndenaturation during spray drying, , , , ,\n\nfunctionality, effect of denaturation, 295\u2013296\n\nglobal production,\n\nhistory of drying proteins,\n\nphysical properties,\n\nrehydration\n\ncaseins, 337\u2013338\n\nions, effect of,\n\ngranulation,\n\nstages of water transfer, ,\n\ntechniques for monitoring rehydration, 335\u2013337\n\nwhey powders,\n\nsensory characteristics\n\ncasein and hydrolysates,\n\nflavor binding, ,\n\nmixed proteins, 483\u2013484\n\nwhey proteins, 474\u2013482\n\nstorage changes\n\ncysteine, , ,\n\nflavor changes, , 476\u2013479\n\nisopeptide bonds, , , ,\n\nlanthionine, , ,\n\nlysine, 352\u2013353, ,\n\nMaillard compounds, 345\u2013349, ,\n\nmethionine, , , ,\n\nnutritional consequences, , 352\u2013355\n\ntryptophan, , , see also Drying by desorption Spray drying; WPNI\n\nProteins, see \u03b1-casein; See also \u03b2-casein; \u03ba-casein; Immunoglobulins; \u03b1-lactalbumin; Lacto-ferrin; \u03b2-lactoglobulin; Serum albumin\n\nProtein quality, need for\n\naging,\n\nassociated with,\n\nsarcopenia,\n\nchallenges,\n\nelderly population, , see also Elderly\n\ndemographic trends,\n\npopulation percentage in 2050,\n\nglobal demographic trends,\n\nProteolysis, , , , , , , , , , , see also chymosin; rennet\n\nProteomics, , ,\n\nProteose peptones, , , \nR\n\nRecombinant DNA technology, ,\n\nRegulatory issues\n\ncarbon footprint,\n\nfunctional foods, 526\u2013527,\n\nfood safety, 575\u2013576\n\nfood traceability, 575\u2013576\n\ngenetic modification, 578\u2013579\n\nmethane production, 574\u2013575\n\nRennet, , 39\u201340, , 46\u201347, , , , , , 189\u2013190, , see also chymosin; rennet gels\n\nRennet clotting time (RCT),\n\nRennet gels\n\naggregation of micelles, , 495\u2013497\n\ncalcium phosphate, ,\n\ncross-linking of caseins,\n\nformation\n\n\u03ba-casein hydrolysis, , 494\u2013497, ,\n\nions, effect of,\n\nmodeling aggregation,\n\npH, effect of, ,\n\nprimary phase, 494\u2013495\n\nrheological changes, 497\u2013498\n\nsecondary phase, 495\u2013497\n\ntemperature, effect of, , ,\n\nzeta potential, changes in,\n\nhistory,\n\nions, ,\n\nmonitoring gelation,\n\npH, 189\u2013190, , ,\n\nplant coagulants,\n\npre-heat treatment, 505\u2013508\n\npressure,\n\nprotein concentration,\n\nrennet concentration, ,\n\nrennets, types of,\n\nrheological changes during formation, 497\u2013498\n\nrheological properties, 189\u2013190, 497\u2013499\n\nrheological parameters,\n\nrheological techniques,\n\nsequestrants, 189\u2013190\n\nstorage,\n\nstorage modulus, , ,\n\nstructure, , 497\u2013499,\n\nsyneresis\n\ncasein concentration,\n\nendogenous pressure,\n\nfree energy,\n\nfunctional purpose,\n\nmechanism,\n\nmicrosyneresis,\n\nmodeling,\n\nmonitoring,\n\npH,\n\ntemperature,\n\ntemperature, 499\u2013501\n\ntexture, ,\n\ntransglutaminase (TGase),\n\nwhey protein denaturation, ,\n\nRetinol-binding protein, , ,\n\nRetinol-protein interactions, see Vitamin A-protein interactions\n\nRheology, see Rennet gels\n\nS\n\nS100A19 proteins\n\nidentification of,\n\npeptide sequence,\n\nS100A19 gene,\n\ndifferential\n\nexpression,\n\nregulation,\n\nexpression analysis\n\nwallaby lactation cycle,\n\nwallaby stomach development,\n\nfunctions,\n\nvariants\n\nS100A8,\n\nS100A9,\n\nS100A12,\n\nSalts, see Minerals\n\nSarcopenia\n\naging effects,\n\ncasein,\n\nbranched-chain amino acid (BCAA),\n\ndigestion rate,\n\nessential amino acids (EAAs),\n\ncauses of,\n\nend-stage diseases,\n\nmilk protein,\n\nanabolic effect,\n\nmuscle protein\n\nbreakdown, inhibition of,\n\nsynthesis,\n\nactivation of,\n\nessential amino acids (EAAs),\n\npreventive strategies,\n\ndiet,\n\nexercise,\n\nresistance-type exercise, regular,\n\nlong-term studies,\n\nmilk protein supplements,\n\nskeletal muscle\n\ninsulin resistance,\n\nloss of function,\n\nloss of mass,\n\nmetabolic health, impact on,\n\nwhey protein,\n\nbranched-chain amino acid (BCAA),\n\ndigestion rate,\n\nSatiety, 533\u2013534\n\nSelf-assembly of protein structures, ,\n\nSensory analysis,\n\nSensory characteristics\n\ncasein,\n\nflavor binding, ,\n\nhydrolysates, ,\n\nmixed protein powders, 483\u2013484\n\nrennet gels,\n\ntexture, , 192\u2013193, , , ,\n\nwhey proteins, 474\u2013482\n\nagglomeration, effect of, 476\u2013479\n\nconsumer issues, 480\u2013482\n\nenzymes, effect of, ,\n\nflavor control,\n\nnative whey proteins,\n\nprocess variations, effect of,\n\nstorage, effect of, 474\u2013479\n\nwhey type, effect of,\n\nwhole milk,\n\nSerum albumin\n\nbiological purpose, ,\n\n\u03ba-casein, interactions with,\n\nconcentration in milk,\n\nfatty acids, interactions with, ,\n\nflavors, interactions with,\n\n\u03b1-lactalbumin, interactions with,\n\nlactoferrin, interactions with,\n\n\u03b2-lactoglobulin, interactions with, , ,\n\nminerals, interactions with,\n\ninterspecies comparison, ,\n\nstability\n\nchemical denaturants,\n\nligand binding, effect of, ,\n\nions,\n\npressure, , , ,\n\npressure and temperature,\n\nsugars, ,\n\ntemperature, , , ,\n\nstructure\n\nbovine,\n\ngeneral,\n\nnon-bovine,\n\nvitamins, interactions with, ,\n\nSmall-angle X-ray scattering (SAXS), 176\u2013178, ,\n\nSodium dodecyl sulfate (SDS)\n\n\u03b2-lactoglobulin\n\nbinding, , , 430\u2013431\n\nstabilizing in presence of urea,\n\ncasein micelle dissociation, ,\n\nSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), see Electrophoresis\n\nSoft materials science,\n\nSpray drying\n\nagglomeration,\n\ncaseins, ,\n\nCompact Drier Instantization (CDI),\n\ndenaturation, , , , ,\n\nindustrial implications,\n\nions,\n\noptimization,\n\nprinciples\n\ncomponents, 325\u2013326\n\nkinetics,\n\nsingle-stage system,\n\nthree-stage system,\n\ntwo-stage system,\n\nsugars, protective effects,\n\nwater transfer\n\ncaseins,\n\nwhey proteins, ,\n\nStorage\n\nbar hardening,\n\nchanges during storage\n\ncysteine, , ,\n\nisopeptide bonds, , , ,\n\nlanthionine, , ,\n\nlysine, 352\u2013353, ,\n\nMaillard compounds, 345\u2013349, ,\n\nmethionine, , , ,\n\ntryptophan, ,\n\nflavor changes, , 476\u2013479\n\nnutritional consequences, , 352\u2013355\n\npowder stability,\n\nStructure, techniques for assessing\n\ncircular dichroism (CD), , , , 216\u2013218, ,\n\nconfocal laser scanning microscope (CLSM), ,\n\ndifferential scanning calorimetry (DSC), , , , , ,\n\ndiffusing wave spectroscopy, ,\n\nelectron microscopy, , 55\u201356, , 176\u2013178, , , , , , , ,\n\nelectrospray ionization mass spectrometry,\n\nFourier transform infrared (FTIR)\n\nspectroscopy, , , 274\u2013275\n\nFourier transform of Raman spectra,\n\nfluorescence spectroscopy, , , , , ,\n\nlight scattering techniques, , , , , ,\n\nmass spectrometry (MS), , , , 291\u2013292\n\nnuclear magnetic resonance (NMR) gels,\n\n\u03b2-lactoglobulin, 205\u2013208, , , 216\u2013218, , ,\n\npowders, 336\u2013338\n\nserum albumin,\n\nsmall-angle X-ray scattering (SAXS), 176\u2013178, ,\n\nneutron small angle scattering,\n\nultrasonic spectroscopy,\n\nUV differential absorption, ,\n\nX-ray crystallography\n\nCaseins,\n\nIgG,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , , , , ,\n\nserum albumin,\n\nStructure of proteins, see \u03b1-casein; See also \u03b2-casein; \u03ba-casein; immunoglobulins; \u03b1-lactalbumin; lactoferrin; \u03b2-lactoglobulin\n\nStructure-function relationships\n\nhistory, 451\u2013452\n\nfunctionality, modification of\n\ningredients, , 453\u2013458\n\nprocessing, 359\u2013361, 459\u2013460\n\nsimple model systems vs complex real\n\nsystems, 451\u2013452\n\nprediction of function, 451\u2013453, ,\n\ntypes of functionality, ,\n\nSugar-protein interactions\n\ncasein,\n\ncomparison of different sugars, , , 439\u2013440,\n\ndenaturation, protective effect chemical denaturants,\n\nfoaming,\n\nfreeze drying, 440\u2013441\n\noverview, 431\u2013433\n\npressure, ,\n\npromotion,\n\nthermal treatments, 436\u2013440\n\ndirect bonding, ,\n\n\u03b1-lactalbumin, 438\u2013440\n\n\u03b2-lactoglobulin, 438\u2013441\n\nMaillard reactions,\n\nserum albumin,\n\nsteric exclusion effect, 431\u2013433\n\nthermodynamics,\n\nSurfactant-protein interactions, 430\u2013431,\n\nSyneresis, see Acid gel; See also Rennet gel\n\nT\n\nTammar wallaby, ,\n\nasynchronous lactation,\n\ncDNA library,\n\nchanges in milk composition, , 84\u201385\n\nchanges in gene expression,\n\ncomparative milk genomics, 119\u2013120\n\neffect of milk composition on growth of young,\n\nexpressed sequence tag (EST),\n\nFIL protein,\n\ngenomics,\n\ngrowth regulation, 23\u201324,\n\nlactation\n\npattern, ,\n\nstrategy,\n\noligosaccharide levels,\n\nwhey acidic protein (WAP), , , 90\u201393, see also Mammary gland\n\nThermal treatments\n\n\u03b1-casein and whey protein complexes,\n\n\u03ba-casein glycosylation,\n\nchanges in milk due to heat\n\ncaseins, , , , , ,\n\nmicelles, , , , , , 282\u2013285\n\nminor proteins, , ,\n\nmixed proteins, , , , 279\u2013289,\n\nnon-protein components, , ,\n\nwhey proteins, , , , 276\u2013277, 291\u2013292\n\nwhole milk, ,\n\nurea,\n\ncomparison with pressure treatments, , , ,\n\ndenaturation, effect on milk product functionality\n\nacid gel strength, heating whey proteins and caseins together, 303\u2013306\n\nacid gel strength, heating whey proteins and caseins separately, 310\u2013311\n\nhigh pH, heating at,\n\nlow pH, heating at,\n\nmicelle-bound \u03ba-casein, 304\u2013306\n\nnon-sedimentable \u03ba-casein, 304\u2013306\n\nyoghurt properties,\n\ndenaturation, whey proteins\n\nchanges in functional properties of milk products, 295\u2013311\n\ninteractions with \u03ba-casein in milk systems, 280\u2013289\n\ninteractions with \u03ba-casein in model systems, 279\u2013280\n\nkinetics of denaturation, 276\u2013279\n\nmeasurement of denaturation, 274\u2013275,\n\nwhey protein nitrogen index (WPNI), 274\u2013275, ,\n\ndirect heating,\n\nindirect heating,\n\nion binding, protective effect,\n\nkinetics of denaturation, 276\u2013277\n\n\u03b1-lactalbumin and \u03ba-casein complexes, ,\n\n\u03b2-lactoglobulin\n\ninteractions with \u03ba-casein in model systems, 279\u2013280\n\ninteractions with \u03ba-casein in milk systems, 280\u2013289, , 291\u2013292\n\ndenaturation, 213\u2013216, , , ,\n\nthermal stability,\n\nlactoferrin,\n\nmicelles, interactions with,\n\npH, effect on\n\ndissociation of \u03b1-casein,\n\ndissociation of \u03b2-casein,\n\ndissociation of \u03ba-casein, 282\u2013285,\n\nhigh temperature stability,\n\n\u03b2-lactoglobulin,\n\nmoderate temperature stability,\n\nnon-sedimentable protein, , ,\n\nsensory changes, , ,\n\nsugars, 436\u2013440\n\ntemperatures,\n\ntypes of thermal treatments,\n\npurpose, ,\n\nThermodynamic incompatibility, see Polysaccharide-protein systems, mixing behaviors\n\nTransferrin,\n\nTransgenic animals, ,\n\nTransgenic plants,\n\nTransglutaminase, (TGase), , , ,\n\nType 2 diabetes (T2DM)\n\ncasein supplement,\n\ninsulin, high levels of,\n\nmetabolically impaired,\n\noverweight,\n\npostprandial study,\n\nU\n\nUltrasonic spectroscopy,\n\nV\n\nVariants\n\n\u03b1-casein, , , , ,\n\n\u03b2-casein, , , ,\n\n\u03ba-casein, , , , ,\n\nexisting variation in genes,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , , , , , , see also Genetic polymorphism\n\nVegetable protein sources\n\nproduction efficiencies,\n\nvegetarianism,\n\nmerits of,\n\nVitamin-binding proteins, , 424\u2013426\n\nVitamin A-protein interactions\n\ncaseins,\n\ncompetitive binding, ,\n\ndifferent forms of vitamin A,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , 422\u2013424\n\nretinol-binding protein, ,\n\nserum albumin,\n\nstabilization of retinol,\n\nVitamin B2-protein interactions,\n\nVitamin C-protein interactions\n\n\u03b2-lactoglobulin,\n\nserum albumin,\n\nVitamin D-protein interactions\n\n\u03b2-casein,\n\n\u03b2-lactoglobulin, 424\u2013426\n\nVitamins, other,\n\nVitamins present in milk, ,\n\nW\n\nWAP four-disulfide core domain protein 2\n\nantibacterial acitivity, 89\u201391\n\ndomain structure,\n\nduring lactation,\n\nstructure,\n\nin tammar wallaby,\n\nWFDC2 gene,\n\nepididymis, identified in,\n\nexpression pattern,\n\nexpression profile, ,\n\nmammary gland, tammar wallaby,\n\nWater\n\ncomponent of milk,\n\nsyneresis, loss through,\n\nwater activity in powders, , , , ,\n\nwater economy, 573\u2013574\n\nwater transfer in powders, , 330\u2013332, , , 337\u2013338\n\nWhey four-disulfide core\n\nWAP, see Whey acidic protein\n\nWFDC2, see WAP four-disulfide core domain protein 2\n\nWhey proteins\n\nbioactivity, ,\n\n\u03ba-casein, interactions with, ,\n\ncommercial products\n\nagglomerated products,\n\napplications,\n\nmanufacture, , , ,\n\nshelf-life,\n\ntypes of whey products,\n\ncomparison with caseins,\n\nconsumer acceptance,\n\nconcentration in milk,\n\ndenaturation\n\nassessment, 274\u2013275\n\ndefinition,\n\nfunctionality, effect on, 295\u2013311\n\ninteractions with \u03ba-casein in milk systems, 280\u2013289, 505\u2013507\n\ninteractions with \u03ba-casein in model systems, 276\u2013280\n\nsugars, protective effect of, 438\u2013440\n\ntemperature, , , , 510\u2013512\n\nemulsions\n\ncasein addition,\n\nformation, , , ,\n\nhydrolysates, 373\u2013374\n\nion addition, ,\n\n\u03b1-lactalbumin, , ,\n\n\u03b2-lactoglobulin, , , , , , ,\n\nmixed whey proteins, , ,\n\ngenetic modification,\n\nhistory of,\n\nhydrolysates,\n\nhydrolysis, intestinal lumen\n\n\u03b1-lactalbumin,\n\nvs. \u03b2-lactalbumin,\n\nlactoferrin,\n\nmultiple sequence analysis,\n\npepsinolysis, sensitivity to,\n\nproteolysis, resistant to,\n\nfractions, , ,\n\nfunctionality,\n\ngels\n\ncold gelation, 514\u2013515\n\nenzymatic modification,\n\nformation variables,\n\nionic strength, 513\u2013514\n\nnon-protein compounds, effect of,\n\npH, , 512\u2013513\n\npolysaccharide gels,\n\nproperties of gels, 512\u2013513\n\nprotein concentration,\n\nstructure, 512\u2013513\n\ntemperature,\n\nthermal denaturation, 510\u2013512\n\ntypes of whey protein gels, 512\u2013513,\n\nisoelectric points,\n\nintact,\n\ninterspecies comparison, 56\u201357\n\n\u03b2-lactoglobulin and \u03ba-casein interactions, , , 279\u2013293, ,\n\nmineral binding,\n\nmuscle protein anabolism, elderly\n\nbeneficial effects,\n\nnative whey proteins, , ,\n\nnon-dairy food applications\n\nbakery products, 463\u2013464\n\ncoffee whitener,\n\nmeat products, 466\u2013467\n\nwhipped toppings,\n\npolysaccharides, interactions with\n\nEPS,\n\ngalactomannans,\n\nguar gum,\n\ngum Arabic,\n\nxanthan gum, ,\n\npreparation, ,\n\npressure treatments of commercial products, 252\u2013253\n\npressure-induced gels, 253\u2013256\n\nproperties,\n\nproteolysis,\n\nrehydration,\n\nself-assembly,\n\nsensory characteristics\n\nagglomeration, effect of, 476\u2013479\n\nconsumer issues,\n\nenzymes, effect of,\n\nflavor control,\n\nnative whey proteins,\n\nprocess variations, effect of,\n\nstorage, effect of, 474\u2013480\n\nwhey type, effect of,\n\nstorage changes\n\n\u03b2-lactoglobulin-lactose interactions,\n\nlactulosyl lysine, 346\u2013349, 352\u2013353\n\nMaillard reactions, 346\u2013349, ,\n\nstructure,\n\nsugars, interactions with, , 438\u2013440,\n\ntransglutaminase (TGase),\n\nyoghurt, roles in, , see also \u03b1-lactalbumin; \u03b2-lactoglobulin; immunoglobulins\n\nWhey acidic protein (WAP), , , , 90\u201393,\n\nWhey protein nitrogen index (WPNI), 274\u2013275, ,\n\nX\n\nXanthan gum\n\nXanthine oxidoreductase (XOR),\n\nX-ray crystallography\n\nCaseins,\n\nIgG,\n\n\u03b1-lactalbumin,\n\n\u03b2-lactoglobulin, , , 212\u2013213, , ,\n\nserum albumin,\n\nY\n\nYoghurt\n\nwhey protein denaturation, effect of,\n\nwhey proteins, role of, 505\u2013507,\n\nZ\n\nZeta potential, , , \n\n# Food Science and Technology: International Series\n\nAmerine, M.A., Pangborn, R.M., and Roessler, E.B., 1965. Principles of Sensory Evaluation of Food.\n\nGlicksman, M., 1970. Gum Technology in the Food Industry.\n\nJoslyn, M.A., 1970. Methods in Food Analysis, Second Ed.\n\nStumbo, C. R., 1973. Thermobacteriology in Food Processing, Second Ed.\n\nAltschul, A.M. (Ed.), New Protein Foods: Volume 1, Technology, Part A\u20141974. Volume 2, Technology, Part B\u20141976. Volume 3, Animal Protein Supplies, Part A\u20141978. Volume 4, Animal Protein Supplies, Part B\u20141981. Volume 5, Seed Storage Proteins\u20141985.\n\nGoldblith, S.A., Rey, L., and Rothmayr, W.W., 1975. Freeze Drying and Advanced Food Technology.\n\nBender, A.E., 1975. Food Processing and Nutrition.\n\nTroller, J.A., and Christian, J.H.B., 1978. Water Activity and Food.\n\nOsborne, D.R., and Voogt, P., 1978. The Analysis of Nutrients in Foods.\n\nLoncin, M., and Merson, R.L., 1979. Food Engineering: Principles and Selected Applications.\n\nVaughan, J. G. (Ed.), 1979. Food Microscopy.\n\nPollock, J. R. A. (Ed.), Brewing Science, Volume 1\u20141979. Volume 2\u20141980. Volume 3\u20141987.\n\nBauernfeind, J. C. (Ed.), 1981. Carotenoids as Colorants and Vitamin A Precursors: Technological and Nutritional Applications.\n\nMarkakis, P. (Ed.), 1982. Anthocyanins as Food Colors.\n\nStewart, G.G., and Amerine, M.A. (Eds.), 1982. Introduction to Food Science and Technology, Second Ed.\n\nIglesias, H.A., and Chirife, J., 1982. Handbook of Food Isotherms: Water Sorption Parameters for Food and Food Components.\n\nDennis, C. (Ed.), 1983. Post-Harvest Pathology of Fruits and Vegetables.\n\nBarnes, P.J. (Ed.), 1983. Lipids in Cereal Technology.\n\nPimentel, D., and Hall, C.W. (Eds.), 1984. Food and Energy Resources.\n\nRegenstein, J.M., and Regenstein, C.E., 1984. Food Protein Chemistry: An Introduction for Food Scientists.\n\nGacula Jr. M.C., and Singh, J., 1984. Statistical Methods in Food and Consumer Research.\n\nClydesdale, F.M., and Wiemer, K.L. (Eds.), 1985. Iron Fortification of Foods.\n\nDecareau, R.V., 1985. Microwaves in the Food Processing Industry.\n\nHerschdoerfer, S.M. (Ed.), Quality Control in the Food Industry, second edition. Volume 1\u20141985. Volume 2\u20141985. Volume 3\u20141986. Volume 4\u20141987.\n\nUrbain, W.M., 1986. Food Irradiation.\n\nBechtel, P.J., 1986. Muscle as Food.\n\nChan, H.W.-S., 1986. Autoxidation of Unsaturated Lipids.\n\nCunningham, F.E., and Cox, N.A. (Eds.), 1987. Microbiology of Poultry Meat Products.\n\nMcCorkle Jr. C.O., 1987. Economics of Food Processing in the United States.\n\nJaptiani, J., Chan Jr., H.T., and Sakai, W.S., 1987. Tropical Fruit Processing.\n\nSolms, J., Booth, D.A., Dangborn, R.M., and Raunhardt, O., 1987. Food Acceptance and Nutrition.\n\nMacrae, R., 1988. HPLC in Food Analysis, Second Ed.\n\nPearson, A.M., and Young, R.B., 1989. Muscle and Meat Biochemistry.\n\nPenfield, M.P., and Campbell, A.M., 1990. Experimental Food Science, Third Ed.\n\nBlankenship, L.C., 1991. Colonization Control of Human Bacterial Enteropathogens in Poultry.\n\nPomeranz, Y., 1991. Functional Properties of Food Components, Second Ed.\n\nWalter, R.H., 1991. The Chemistry and Technology of Pectin.\n\nStone, H., and Sidel, J.L., 1993. Sensory Evaluation Practices, Second Ed.\n\nShewfelt, R.L., and Prussia, S.E., 1993. Postharvest Handling: A Systems Approach.\n\nNagodawithana, T., and Reed, G., 1993. Enzymes in Food Processing, Third Ed.\n\nHoover, D.G., and Steenson, L.R., 1993. Bacteriocins.\n\nShibamoto, T., and Bjeldanes, L., 1993. Introduction to Food Toxicology.\n\nTroller, J.A., 1993. Sanitation in Food Processing, Second Ed.\n\nHafs, D., and Zimbelman, R.G., 1994. Low-fat Meats.\n\nPhillips, L.G., Whitehead, D.M., and Kinsella, J., 1994. Structure-Function Properties of Food Proteins.\n\nJensen, R.G., 1995. Handbook of Milk Composition.\n\nRoos, Y.H., 1995. Phase Transitions in Foods.\n\nWalter, R.H., 1997. Polysaccharide Dispersions.\n\nBarbosa-Canovas, G.V., Marcela Go'ngora-Nieto, M., Pothakamury, U.R., and Swanson, B.G., 1999. Preservation of Foods with Pulsed Electric Fields.\n\nJackson, R.S., 2002. Wine Tasting: A Professional Handbook.\n\nBourne, M.C., 2002. Food Texture and Viscosity: Concept and Measurement, Second Ed.\n\nCaballero, B., and Popkin, B.M. (Eds.), 2002. The Nutrition Transition: Diet and Disease in the Developing World.\n\nCliver, D.O., and Riemann, H.P. (Eds.), 2002. Foodborne Diseases, Second Ed.\n\nKohlmeier, M., 2003. Nutrient Metabolism.\n\nStone, H., and Sidel, J.L., 2004. Sensory Evaluation Practices, Third Ed.\n\nHan, J.H., 2005. Innovations in Food Packaging.\n\nSun, D.-W. (Ed.), 2005. Emerging Technologies for Food Processing.\n\nRiemann, H.P., and Cliver, D.O. (Eds.), 2006. Foodborne Infections and Intoxications, Third Ed.\n\nArvanitoyannis, I.S., 2008. Waste Management for the Food Industries.\n\nJackson, R.S., 2008. Wine Science: Principles and Applications, Third Ed.\n\nSun, D.-W. (Ed.), 2008. Computer Vision Technology for Food Quality Evaluation.\n\nDavid, K., and Thompson, P., (Eds.), 2008. What Can Nanotechnology Learn from Biotechnology?\n\nArendt, E.K., and Bello, F.D. (Eds.), 2008. Gluten-free Cereal Products and Beverages.\n\nBagchi, D. (Ed.), 2008. Nutraceutical and Functional Food Regulations in the United States and Around the World.\n\nSingh, R.P., and Heldman, D.R., 2008. Introduction to Food Engineering, Fourth Ed.\n\nBerk, Z., 2009. Food Process Engineering and Technology.\n\nThompson, A., Boland, M., and Singh, H. (Eds.), 2009. Milk Proteins: From Expression to Food.\n\nFlorkowski, W.J., Prussia, S.E., Shewfelt, R.L. and Brueckner, B. (Eds.), 2009. Postharvest Handling, Second Ed.\n\nGacula Jr., M., Singh, J., Bi, J., and Altan, S., 2009. Statistical Methods in Food and Consumer Research, Second Ed.\n\nShibamoto, T., and Bjeldanes, L., 2009. Introduction to Food Toxicology, Second Ed.\n\nBeMiller, J. and Whistler, R. (Eds.), 2009. Starch: Chemistry and Technology, Third Ed.\n\nJackson, R.S., 2009. Wine Tasting: A Professional Handbook, Second Ed.\n\nHeldman, D.R., 2011. Food Preservation Process Design.\n\nTiwari, B.K., Gowen, A. and McKenna, B. (Eds.), 2011. Pulse Foods: Processing, Quality and Nutraceutical Applications.\n\nCullen, PJ., Tiwari, B.K., and Valdramidis, V.P. (Eds.), 2012. Novel Thermal and Non-thermal Technologies for Fluid Foods.\n\nStone, H., Bleibaum, R., and Thomas, H., 2012. Sensory Evaluation Practices, Fourth Ed.\n\nKosseva, M.R. and Webb, C. (Eds.), 2013. Food Industry Wastes: Assessment and Recuperation of Commodities.\n\nMorris, J.G. and Potter, M.E. (Eds.), 2013. Foodborne Infections and Intoxications, Fourth Ed.\n\nBerk, Z., 2013. Food Processing Engineering and Technology, Second Ed.\n\nSingh, R.P., and Heldman, D.R., 2014. Introduction to Food Engineering, Fifth Ed.\n\nHan, J.H. (Ed.), 2014. Innovations in Food Packaging, Second Ed.\n\nMadsen, C., Crevel, R., Mills, C., and Taylor, S. (Eds.), 2014. Risk Management for Food Allergy\n\nMatthews, K.R., Sapers, G.M., and Gerba, C.P. (Eds.), 2014. The Produce Contamination Problem, Second Ed.\n\nBagchi, D. (Ed.), 2014. Nutraceutical and Functional Food Regulations in the United States and Around the World, Second Ed.\n\nJackson, R.S., 2014. Wine Science: Principles and Applications, Fourth Ed.\n\n# Table of Contents\n\n  1. Cover\n  2. Title page\n  3. Table of Contents\n  4. Food Science and Technology International Series\n  5. Copyright\n  6. List of Contributors\n  7. Preface to the Second Edition\n  8. Preface to the First Edition\n  9. Chapter 1: The World Supply of Food and the Role of Dairy Protein\n    1. Abstract\n    2. Introduction\n    3. Hunger and the need for food\n    4. The dietary essential amino acids in proteins\n    5. Identifying the countries deficient in dietary essential amino acids\n    6. Demographic changes, aging populations, and the need for quality protein and essential amino acids\n    7. Global trade in proteins, the long-term prospects, with a focus on dairy foods\n    8. Conclusions\n  10. Chapter 2: Milk: An Overview\n    1. Abstract\n    2. Introduction\n    3. Evolution of mammals and lactation\n    4. Utilization of milk\n    5. Composition of milk\n    6. Milk constituents\n    7. Summary\n  11. Chapter 3: The Comparative Genomics of Monotremes, Marsupials, and Pinnipeds: Models to Examine the Functions of Milk Proteins\n    1. Abstract\n    2. Introduction\n    3. The echidna (Tachyglossus aculeatus)\n    4. The tammar wallaby (Macropus eugenii)\n    5. A role for milk in the control of mammary function\n    6. The fur seal\n    7. New player in milk bioactives; MicroRNA\n    8. Conclusions\n  12. Chapter 4: Significance, Origin, and Function of Bovine Milk Proteins: The Biological Implications of Manipulation or Modification\n    1. Abstract\n    2. Introduction\n    3. Origins of milk proteins\n    4. Constraints and opportunities for evolution or manipulation of bovine milk proteins\n    5. Conclusion\n  13. Chapter 5: Post-translational Modifications of Caseins\n    1. Abstract\n    2. Introduction\n    3. The caseins\n    4. Caseins from other species\n    5. Conclusions\n  14. Chapter 6: Casein Micelle Structure and Stability\n    1. Abstract\n    2. Introduction\n    3. Casein primary structure and interactions\n    4. Casein micelle properties\n    5. Models of casein micelle structure\n    6. Concluding remarks\n  15. Chapter 7: Structure and Stability of Whey Proteins\n    1. Abstract\n    2. Introduction\n    3. Bovine \u03b2-Lactoglobulin\n    4. \u03b1-Lactalbumin\n    5. Serum albumin\n    6. Immunoglobulins\n    7. Lactoferrin\n    8. Concluding remarks\n    9. Acknowledgments\n  16. Chapter 8: Effects of High-pressure Processing on Structure and Interactions of Milk Proteins\n    1. Abstract\n    2. Introduction\n    3. High-pressure-induced changes in caseins\n    4. Effects of high pressure on interactions of milk proteins involving whey proteins\n    5. Concluding remarks\n    6. Acknowledgment\n  17. Chapter 9: The Whey Proteins in Milk: Thermal Denaturation, Physical Interactions, and Effects on the Functional Properties of Milk\n    1. Abstract\n    2. Introduction\n    3. The casein micelle\n    4. The heat treatment of milk\n    5. Relationships between denaturation\/interactions of the whey proteins in heated milk and the functional properties of milk\n    6. Conclusion\n  18. Chapter 10: Effects of Drying on Milk Proteins\n    1. Abstract\n    2. Introduction\n    3. Properties of spray-dried milk products\n    4. Principles of spray drying\n    5. Process improvement\n    6. Drying of proteins\n    7. Conclusions\n  19. Chapter 11: Changes in Milk Proteins during Storage of Dry Powders\n    1. Abstract\n    2. Introduction\n    3. The formation of maillard and pre-maillard compounds\n    4. Formation of isopeptide bonds\n    5. Amino acids other than lysine\n    6. Implications for nutritional value of milk proteins\n    7. Product-specific storage trials\n    8. Conclusions\n  20. Chapter 12: Interactions and Functionality of Milk Proteins in Food Emulsions\n    1. Abstract\n    2. Introduction\n    3. Adsorption of Milk Proteins During the Formation of Emulsions\n    4. Stability of Milk Protein-Based Emulsions\n    5. Heat-Induced Changes in Milk Protein-Based Emulsions\n    6. Pressure-Induced Changes in Milk Protein-Based Emulsions\n    7. Milk Protein Hydrolysates and Oil-In-Water Emulsions\n    8. Lactoferrin-Based Oil-In-Water Emulsions\n    9. Lipid Oxidation in Milk Protein-Based Emulsions\n    10. Behavior of Milk Protein-Stabilized Emulsions Under Physiological Conditions\n    11. Conclusions\n  21. Chapter 13: Milk Protein-Polysaccharide Interactions\n    1. Abstract\n    2. Introduction\n    3. Mixing behavior of biopolymers\n    4. Phase diagram\n    5. Nature of interactions in protein-polysaccharide systems\n    6. Milk protein-polysaccharide interactions in the aqueous phase\n    7. Milk protein-polysaccharide interactions at the interface\n    8. Rheological properties and microstructures of protein-polysaccharide systems\n    9. Concluding remarks\n  22. Chapter 14: Interactions between Milk Proteins and Micronutrients\n    1. Abstract\n    2. Introduction\n    3. Interactions Between native Milk Proteins and Micronutrients\n    4. Interactions between process-modified milk proteins and micronutrients\n    5. Conclusions\n  23. Chapter 15: Model Food Systems and Protein Functionality\n    1. Abstract\n    2. Introduction\n    3. Protein functionality in foods\n    4. Role of interactions in determining food characteristics\n    5. Processing effects\n    6. Uses of model food systems\n    7. Applications of model food systems\n    8. Use of model food systems for other food components\n    9. Limitations\n    10. Conclusions\n  24. Chapter 16: Sensory Properties of Dairy Proteins\n    1. Abstract\n    2. Introduction\n    3. Sensory analysis\n    4. Whey proteins\n    5. Milk proteins\n    6. Caseins and hydrolysates\n    7. Flavor binding\n    8. Conclusions\n    9. Acknowledgment\n  25. Chapter 17: Milk Protein Gels\n    1. Abstract\n    2. Introduction\n    3. Rennet-induced gels\n    4. Acid-induced milk gels\n    5. Whey protein gels\n    6. Conclusions\n    7. Acknowledgment\n  26. Chapter 18: Milk Proteins--A Cornucopia for Developing Functional Foods\n    1. Abstract\n    2. Introduction\n    3. Functional foods\n    4. Milk proteins as a source of amino acids--specialized nutritionals\n    5. Milk proteins as a source of amino acids--specific physiological roles\n    6. Milk proteins as a source of amino acids--role in providing calories and in promoting satiety\n    7. Milk proteins as a source of bioactive peptides\n    8. Conclusions\n  27. Chapter 19: Milk Proteins and Human Health\n    1. Abstract\n    2. Introduction\n    3. Milk proteins, metabolic health, and type 2 diabetes\n    4. Milk proteins, obesity, and weight control\n    5. Milk proteins and bone health\n    6. Conclusions\n  28. Chapter 20: Milk Proteins: Digestion and Absorption in the Gastrointestinal Tract\n    1. Abstract\n    2. Introduction\n    3. Digestion of milk proteins\n    4. Milk protein hydrolysis in the intestinal lumen\n    5. Peptides released during digestion\n    6. Impact of processing on milk protein digestion and absorption\n    7. Conclusions\n  29. Chapter 21: Milk Proteins: The Future\n    1. Abstract\n    2. Introduction\n    3. Global issues for food\n    4. Consumer demands and trends for food and ingredients\n    5. New technologies and their possible effect on milk protein ingredients and products\n    6. Conclusions\n  30. Index\n  31. Food Science and Technology: International Series\n\n## List of tables\n\n  1. Tables in Chapter 1\n    1. Table 1.1\n    2. Table 1.2\n    3. Table 1.3\n  2. Tables in Chapter 2\n    1. Table 2.1\n    2. Table 2.2\n    3. Table 2.3\n  3. Tables in Chapter 4\n    1. Table 4.1\n  4. Tables in Chapter 5\n    1. Table 5.1\n    2. Table 5.2\n    3. Table 5.3\n  5. Tables in Chapter 10\n    1. Table 10.1\n    2. Table 10.2\n    3. Table 10.3\n  6. Tables in Chapter 11\n    1. Table 11.1\n    2. Table 11.2\n    3. Table 11.3\n  7. Tables in Chapter 12\n    1. Table 12.1\n  8. Tables in Chapter 13\n    1. Table 13.1\n    2. Table 13.2\n    3. Table 13.3\n  9. Tables in Chapter 14\n    1. Table 14.1\n    2. Table 14.2\n    3. Table 14.3\n  10. Tables in Chapter 15\n    1. Table 15.1\n    2. Table 15.2\n    3. Table 15.3\n    4. Table 15.4\n    5. Table 15.5\n    6. Table 15.6\n    7. Table 15.7\n  11. Tables in Chapter 16\n    1. Table 16.1\n    2. Table 16.2\n    3. Table 16.3\n    4. Table 16.4\n    5. Table 16.5\n  12. Tables in Chapter 17\n    1. Table 17.1\n    2. Table 17.2\n    3. Table 17.3\n    4. Table 17.4\n  13. Tables in Chapter 20\n    1. Table 20.1\n  14. Tables in Chapter 21\n    1. Table 20.1\n    2. Table 20.2\n\n## List of figures\n\n  1. Figures in Chapter 1\n    1. Figure 1.1\n    2. Figure 1.2\n    3. Figure 1.3\n    4. Figure 1.4\n    5. Figure 1.5\n    6. Figure 1.6\n  2. Figures in Chapter 2\n    1. Figure 2.1\n    2. Figure 2.2\n  3. Figures in Chapter 3\n    1. Figure 3.1\n    2. Figure 3.2\n    3. Figure 3.3\n    4. Figure 3.4\n    5. Figure 3.5\n    6. Figure 3.6\n    7. Figure 3.7\n    8. Figure 3.8\n    9. Figure 3.9\n    10. Figure 3.10\n    11. Figure 3.11\n    12. Figure 3.12\n    13. Figure 3.13\n    14. Figure 3.14\n    15. Figure 3.15\n  4. Figures in Chapter 4\n    1. Figure 4.1\n  5. Figures in Chapter 5\n    1. Figure 5.1\n    2. Figure 5.2\n    3. Figure 5.3\n    4. Figure 5.4\n    5. Figure 5.5\n    6. Figure 5.6\n    7. Figure 5.7\n  6. Figures in Chapter 6\n    1. Figure 6.1\n    2. Figure 6.2\n    3. Figure 6.3\n    4. Figure 6.4\n    5. Figure 6.5\n    6. Figure 6.6\n    7. Figure 6.7\n    8. Figure 6.8\n  7. Figures in Chapter 7\n    1. Figure 7.1\n    2. Figure 7.2\n    3. Figure 7.3\n    4. Figure 7.4\n    5. Figure 7.5\n    6. Figure 7.6\n  8. Figures in Chapter 8\n    1. Figure 8.1\n    2. Figure 8.2\n    3. Figure 8.3\n    4. Figure 8.4\n    5. Figure 8.5\n  9. Figures in Chapter 9\n    1. Figure 9.1\n    2. Figure 9.2\n    3. Figure 9.3\n    4. Figure 9.4\n    5. Figure 9.5\n    6. Figure 9.6\n    7. Figure 9.7\n    8. Figure 9.8\n    9. Figure 9.9\n    10. Figure 9.10\n    11. Figure 9.11\n    12. Figure 9.12\n    13. Figure 9.13\n    14. Figure 9.14\n    15. Figure 9.15\n    16. Figure 9.16\n    17. Figure 9.17\n    18. Figure 9.18\n    19. Figure 9.19\n  10. Figures in Chapter 10\n    1. Figure 10.1\n    2. Figure 10.2\n    3. Figure 10.3\n    4. Figure 10.4\n  11. Figures in Chapter 11\n    1. Figure 11.1\n    2. Figure 11.2\n    3. Figure 11.3\n    4. Figure 11.4\n    5. Figure 11.5\n    6. Figure 11.6\n    7. Figure 11.7\n    8. Figure 11.8\n    9. Figure 11.9\n    10. Figure 11.10\n  12. Figures in Chapter 12\n    1. Figure 12.1\n    2. Figure 12.2\n    3. Figure 12.3\n    4. Figure 12.4\n    5. Figure 12.5\n  13. Figures in Chapter 13\n    1. Figure 13.1\n    2. Figure 13.2\n    3. Figure 13.3a\n    4. Figure 13.3b\n  14. Figures in Chapter 14\n    1. Figure 14.1\n    2. Figure 14.2\n    3. Figure 14.3\n    4. Figure 14.4\n  15. Figures in Chapter 15\n    1. Figure 15.1\n    2. Figure 15.2\n    3. Figure 15.3\n    4. Figure 15.4\n    5. Figure 15.5\n    6. Figure 15.6\n    7. Figure 15.7\n    8. Figure 15.8\n  16. Figures in Chapter 16\n    1. Figure 16.1\n    2. Figure 16.2\n    3. Figure 16.3\n    4. Figure 16.4\n    5. Figure 16.5\n    6. Figure 16.6\n    7. Figure 16.7\n    8. Figure 16.8\n    9. Figure 16.9\n    10. Figure 16.10\n    11. Figure 16.11\n  17. Figures in Chapter 17\n    1. Figure 17.1\n    2. Figure 17.2\n    3. Figure 17.3\n    4. Figure 17.4\n    5. Figure 17.5\n  18. Figures in Chapter 18\n    1. Figure 18.1\n  19. Figures in Chapter 20\n    1. Figure 20.1\n    2. Figure 20.2\n    3. Figure 20.3\n    4. Figure 20.4\n\n## Landmarks\n\n  1. Cover\n  2. Title page\n  3. Table of Contents\n\n  1. i\n  2. ii\n  3. iii\n  4. iv\n  5. ix\n  6. x\n  7. xi\n  8. xiii\n  9. xv\n  10. xvi\n  11. \n  12. \n  13. \n  14. \n  15. \n  16. \n  17. \n  18. \n  19. \n  20. \n  21. \n  22. \n  23. \n  24. \n  25. \n  26. \n  27. \n  28. \n  29. \n  30. \n  31. \n  32. \n  33. \n  34. \n  35. \n  36. \n  37. \n  38. \n  39. \n  40. \n  41. \n  42. \n  43. \n  44. \n  45. \n  46. \n  47. \n  48. \n  49. \n  50. \n  51. \n  52. \n  53. \n  54. \n  55. \n  56. \n  57. \n  58. \n  59. \n  60. \n  61. \n  62. \n  63. \n  64. \n  65. \n  66. \n  67. \n  68. \n  69. \n  70. \n  71. \n  72. \n  73. \n  74. \n  75. \n  76. \n  77. \n  78. \n  79. \n  80. \n  81. \n  82. \n  83. \n  84. \n  85. \n  86. \n  87. \n  88. \n  89. \n  90. \n  91. \n  92. \n  93. \n  94. \n  95. \n  96. \n  97. \n  98. \n  99. \n  100. \n  101. \n  102. \n  103. \n  104. \n  105. \n  106. \n  107. \n  108. \n  109. \n  110. \n  111. \n  112. \n  113. \n  114. \n  115. \n  116. \n  117. \n  118. \n  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563. \n  564. \n  565. \n  566. \n  567. \n  568. \n  569. \n  570. \n  571. \n  572. \n  573. \n  574. \n  575. \n  576. \n  577. \n  578. \n  579. \n  580. \n  581. \n  582. \n  583. \n  584. \n  585. \n  586. \n  587. \n  588. \n  589. \n  590. \n  591. \n  592. \n  593. \n  594. \n  595. \n  596. \n  597. \n  598. \n  599. \n  600. \n  601. \n  602. \n  603. \n  604. \n  605.\n\n","meta":{"redpajama_set_name":"RedPajamaBook"}}
+{"text":" \n# A MARRIED WOMAN\n\nManju Kapur\n\nFor  \nmy daughter  \nAmba\n\n&\n\nIra\n\n# Contents\n\nTitle Page  \nDedication  \nChapter I  \nChapter II  \nChapter III  \nChapter IV  \nChapter V  \nChapter VI  \nChapter VII  \nChapter VIII  \nChapter IX  \nChapter X  \nAcknowledgements  \nAbout the Author  \nCopyright\n\nChapter I\n\nAstha was brought up properly, as befits woman, with large supplements of fear. One slip might find her alone, vulnerable and unprotected. The infinite ways in which she could be harmed were not specified, but Astha absorbed them through her skin, and ever after was drawn to the safe and secure.\n\nShe was her parents' only child. Her education, her character, her health, her marriage, these were their burdens. She was their future, their hope, and though she didn't want them to guard their precious treasure so carefully, they did, oh they did.\n\nHer mother often declared, 'When you are married, our responsibilities will be over. Do you know the shastras say if parents die without getting their daughter married, they will be condemned to perpetual rebirth?'\n\n'I don't believe in all that stuff,' said Astha, 'and I think, as an educated person, neither should you.'\n\nHer mother sighed her heavy soul-killing sigh. 'Who can escape their duty?' she asked, as she put in a steel almirah another spoon, sheet, sari, piece of jewellery towards the girl's future.\n\n*\n\nEvery day in her temple corner in the kitchen, she prayed for a good husband for her daughter.\n\n'You pray too,' she insisted as they stood before the shrine on the shelf, ordinarily hidden by curtains made from an old silk sari border, woven through with gold so pure that if the cloth was burnt, the metal would emerge in a little drop.\n\nAstha obediently closed her eyes to delicious images of a romantic, somewhat shadowy young man holding her in his strong manly embrace.\n\n'Are you praying?' asked the mother suspiciously.\n\n'Of course I'm praying,' replied the daughter indignantly, 'you never trust me.'\n\nTo prove her sincerity she fixed her gaze firmly on Krishna, Krishna the one so many had adored. He would send her marriage, love and happiness. She fingered the rope of tiny pearls around his image. On either side were miniature vases with fresh jasmine buds. There was also a picture of Astha's dead grandparents, a little silver bell and thali, two small silver lamps which were lit every evening, while a minuscule silver incense holder wobbled next to it. Whatever meal Astha's mother cooked was first offered to the gods before the family ate. She believed in the old ways.\n\n*\n\nWhile her father believed in the new. His daughter's future lay in her own hands, and these hands were to be strengthened by the number of books that passed through them. At least once a day he said to her, 'Why aren't you studying?'\n\nHow much studying could Astha do to satisfy the man? Through her school years she never found out.\n\n'Where is the maths work I asked you to do?' he would continue.\n\n'I haven't finished it yet.'\n\n'Show me whatever you have completed.'\n\nSums indifferently done were produced. The father tightened his lips. The girl felt afraid, but refused to show it. She looked down.\n\n'You worthless, ungrateful child. Do you know how much money I spend on your education?'\n\n'Don't then, don't spend anything,' she muttered, her own lips as tight as his.\n\nDriven by her insolence, carelessness and stupidity, he slapped her. Tears surfaced, but she wouldn't act sorry, would rather die than show how unloved and misunderstood she felt.\n\nHer mother looked on and said nothing. Later, 'Why don't you do the work he tells you to? You can't be drawing and painting all the time.'\n\n'So he hits me?' She didn't want her mother's interventions, she hated her as well as him.\n\n'It's his way of showing concern.'\n\nAstha looked away.\n\nThe mother sighed. The girl was good, only she got into these moods sometimes. And how much she fiddled with brush and pencil, no wonder her father got anxious, there was no future in art. If she did well in her exams, she could perhaps sit for the IAS, and find a good husband there. You met all kinds of people in the administrative services, and the girl was not bad looking. She must tell her to frown less. Frowns mislead people about one's inner nature.\n\n*\n\nThe girl's body was nurtured by walks that started every morning at five.\n\n'Get up, get up. Enough laziness.'\n\n'You will thank us later when you realise the value of exercise and fresh air.'\n\n'How can you waste the best part of the day? This is Brahmakaal, the hour of the gods.'\n\nSo Astha dragged her feet behind her parents' straight backs as they strode towards the dew and space of the India Gate lawns. Her parents arranged their walk so that they would be facing the East as the sun rose, showing their respect for the source of all life, while Astha, lagging behind, refused to participate in their daily satisfaction over the lightening sky, or the drama of the sun suddenly rising behind India Gate.\n\nWhen they came home they all did Pranayam together. Pranayam, in the patchy grass surrounded by a short straggly hedge outside their flat. Inhale through one nostril, pinch it, exhale through the other, pinch that, right left, left right, thirty times over, till the air in the lungs was purified and the spirit uplifted.\n\n*\n\nAt other times Astha's father took her for a stroll through the colony in the evenings. Away from her studies he was more amiable. He didn't want his daughter to be like himself, dissatisfied and wasted. You have so much potential, you draw, you paint, you read, you have a way with words, you do well academically, the maths is a little weak, but never mind, you must sit for the competitive exams. With a good job comes independence. When I was young, I had no one to guide me, I did not know the value of time, did not do well in my exams, had to take this job, thinking later I can do something else, but once you are stuck you are stuck.\n\nHere he grew silent and walked on moodily, while Astha linked her arm through his, feeling slightly sorry for him.\n\nAfter her father died and experience had drilled some sense of the world into her, Astha realised how emancipated he had been. At the time she felt flattered by his attention, but bored by his words.\n\nThe family counted their pennies carefully. Their late marriage, their daughter still to be settled, their lack of any security to fall back on, meant that their pleasures were planned with thrift firmly in the forefront. Once a month on a Sunday they went as a treat to the Bengali Market chaat shop. They gazed at the owner sitting on a narrow platform, cross-legged before his cash box, a small brass grille all around him. His dhoti kurta was spotless white, his cash box rested on a cloth equally spotless.\n\n'This man came from Pakistan, a refugee'' said the father.\n\n'Look at him now'' echoed the mother.\n\nAnd the shop grew glitzier every time they came, with marble floors added, mirrors expanding across the walls, extensions built at the back and sides. The tikkis and the papri did not remain the same either, but grew more and more expensive. What was in the tikki that made him charge one rupee per plate?\n\n'The potatoes he must be buying in bulk, so that is only one anna worth of potato, the stuffing is mostly dal, hardly any peas, a miserable half cashew, fried in vanaspati, not even good oil, let alone ghee; the chutney has no raisins, besides being watery, and what with the wages of the waiter and the cook, the whole thing must be costing him not more than... than...'\n\nFather, mathematician, closes his eyes to concentrate better on the price of the potato tikki.\n\n'Not more than eight annas, maximum''\n\n'Hundred per cent profit'' said the mother gloomily. 'How much does he sell in a day? Five hundred?'\n\nThey looked around the crowded restaurant, with its tables jammed together, and the large extended section behind the sweet counters. They looked at the people being served on the road. Yes, five hundred would not be wrong.\n\nThen they calculated eight annas multiplied by 500 made 250 rupees. And this was just on potato tikkis.\n\nWhat about the papri, the kulchas, the dahi barhas, the gol gappas in spicy water, the gol gappas in dahi and chutney, the kachoris with channa, the puri aloo, the channa bhatura, the newly introduced dosas, the dry savouries, sweets, and chips that he was cunningly displaying in glass cases, what about every one of those? How much money would he be taking home at the end of just one day? To top it all, he was uneducated.\n\n'I could make better tikkis at home'' offered the mother.\n\nAstha stared miserably at her plate of two small swollen tikkis, buried under sweet and sour chutney. Then she stared at the fat man behind the payment counter. He sat there with his paan-stained mouth, taking people's money, opening the lid of the cash box, calmly lifting the change katoris to add to the growing piles of ones, twos, fives, tens, hundreds.\n\n'Do you want anything else, beti?' asked the parents after they had eaten every crumb from their little steel plates.\n\n'No.'\n\n'Are you sure?'\n\n'Yes.'\n\n'Let's go home then.'\n\n'That was a nice outing, wasn't it?' they said to each other as they started the old Fiat and headed back to their flat on Pandara Road.\n\nOver these smaller worries, loomed the larger one, their unbuilt house, the place they would go to when they retired, the shelter that lay between them and nothingness. It was towards this end that they counted every paisa, weighed the pros and cons of every purchase with heavy anxiety. From time to time they drove to the outer parts of South Delhi with dealers to look at plots. Somehow, the places they wanted to live in were always outside their budget, the places they could afford seemed wild and uninhabitable.\n\n'There was a time when Defence Colony too was on the outskirts of Delhi'' pointed out the mother. 'Let us at least buy wherever we can''\n\n'No, Sita'' said the father irritably. 'How can we live so far away from everything?'\n\nHow far do we have to go before we can afford something, thought Astha, who was forced to come on these expeditions, she couldn't be left alone with the servant. She herself never intended to land in any house on any tiny plot they were looking at. Her husband would see to it.\n\nMeanwhile Delhi grew and grew, and plots they had once rejected as being too far, now became part of posh and expensive colonies, and not as far as they had once thought.\n\n*\n\nRetirement was coming nearer, the pressure to buy was growing, when in the early sixties, ministries started forming co-operative housing societies.\n\n'Thank goodness'' grumbled Astha's mother, 'at least the government will do for us, what we have not been able to do for ourselves''\n\n'It's one thing to form a co-operative housing society, another thing to get land allotted to it, and still another to build a house'' said the father, born pessimist. 'In what god-forsaken corner will they allocate land to a ministry as unimportant as relief and welfare, that too you have to see''\n\n'Arre, wherever, whatever, we have to build. Otherwise you plan that after retirement we live in your ancestral palace?'\n\nThe husband looked pained at his wife's coarseness.\n\n*\n\nThey continued to worry. When would their housing society have land assigned to it, how many more years for the father to retire, how many more working years for the mother, how long before they had to leave this government house in the centre of Delhi, so convenient?\n\nOnce the land was allotted, how much would it cost to build, how much did they have in fixed deposits, in their provident funds, how much could they borrow, how much interest would they have to pay? After discussing all this, they allowed themselves to dream a little.\n\n'I will have a special place for my books'' said the father, 'cupboards with glass to protect them from dust and silverfish''\n\n'I will have a big kitchen'' said the mother, 'with screen windows to keep flies out, and a stainless steel sink, not like this cement one which always looks filthy. I will have a long counter, so I don't have to unpack the mixi every time I need it. I will have a proper place to do puja, rather than a shelf''\n\n'We will have a study, maybe an extra bedroom for guests'' mused the father, and then they looked frightened at the money their dreams were going to cost. Maybe not a guest room, their voices trailed off.\n\nBy the time Astha was sixteen, she was well trained on a diet of mushy novels and thoughts of marriage. She was prey to inchoate longings, desired almost every boy she saw, then stood long hours before the mirror marvelling at her ugliness. Would she ever be happy? Would true love ever find her?\n\n*\n\nThen the day dawned, the day Astha saw Bunty. Bunty the beautiful, Bunty whose face never left her, Bunty whose slightest word, look and gesture she spent hours nursing to death.\n\nBunty's family lived in one of the bigger houses of the Pandara Road colony, a duplex with a large garden, and a roomy verandah. They were on visiting terms with Astha's parents, the younger sister was in her school. The boy was away in Kharakvasala in the Defence Academy and now home for the holidays.\n\nHe came over with his father. Oh, how he stood out. He had glossy black hair which he wore in a small puff over a high wide forehead. His eyes were like soft black velvet, set in pale sockets over the faint blush of his cheek. And just beneath that the bluish black shadow of an incipient beard, framing a red mouth. As she stared, steady, unwavering, he felt her gaze, looked up and smiled. His teeth were small, white and uneven, and as she lost herself in them, he raised his left eyebrow slightly. She shuddered, and weakly smiled back.\n\nThus began her torture.\n\nIf only she didn't see him so often, but Bunty was restless during his holidays. Boarding school, boarding college, as a result he knew few people in Delhi. He took to dropping in with his sister. There was the attraction of her devotion.\n\nDay and night the thought of him kept her insides churning; she was unable to eat, sleep, or study. Away from him her eyes felt dry and empty. Her ears only registered the sound of his voice. Her mind refused to take seriously anything that was not his face, his body, his feet, his hands, his clothes. She found temporary relief in sketching him, sketches that were invariably too bad to be mulled over.\n\nHours were spent in planning accidental meetings, how to bump into him in the colony, how to cross his father on his evening walk, how to fall into enough conversation to be invited over, how to borrow a book to prolong the stay, how to fall into a faint, how to die at his doorstep.\n\nOnce in Bunty's house she saw him pet his dog, who promptly put her paws in his lap, wagged her tail and salivated. At that moment she felt a keen shamed kinship with the animal.\n\nShe was too overwhelmed by her feelings to actually want to talk to him. To approach the site of all this wonder would be apostasy. To think that he would ever have anything to say to her was past crediting. Finally it was so unbearable, she had to tell someone.\n\n*\n\nGayatri, school friend, eventual confidante, decided that this affair needed managing.\n\n'What have you actually done?' she demanded.\n\n'Done?' quavered Astha, immediately feeling worse instead of better. 'Nothing''\n\n'You are such a ninny'' scolded Gayatri, 'invite him to a movie.'\n\n'How can I?'\n\n'How can you?' Gayatri stared at her. 'There is a Charlie Chaplin film at the National Stadium next Sunday morning. Ask him if he has seen it. Go on. Give him a chance.'\n\nEach day was now an exam, in which she failed daily. Gayatri was insistent. There had to be movement to the whole thing, otherwise she might as well not be in love. Astha was forced to admit the logic of this.\n\nThe day came when she stood tongue-tied before him, stammering out her request that the god come to a film with her and her friend.\n\n'Of course hell go, won't you, Bunty beta?' boomed his father.\n\n'Th-Thank-you, Uncle'' stammered Astha, not looking at Bunty.\n\nBunty seemed stiff and bored through the film. Gayatri chattered gaily in the interval, while Astha gritted her teeth and waited for the nightmare to end. Words rushed around in her head, words that would show how clever and interesting she was, but when she actually looked at him she could not speak. She wanted to never see Bunty again. She hated him. She wished his holidays would quickly end.\n\n*\n\nThey did, and Astha grew desperate. The point of getting up every morning had been the hope that she would be able to look at him, feed on a glance, a word, a smile. Now her rich inner world would become stale with nothing new to add to the store.\n\n'Suggest writing. You know, like pen pals'' said Gayatri.\n\n'No.' Suppose he laughed? Looked contemptuous?\n\n'What do you have to lose?'\n\n'Why should he write to me?'\n\n'Why not? He does drop by, and you also visit him.'\n\nAstha hesitated. 'That means very little'' she pronounced finally, thinking of those visits, the long pauses, she pulverised with emotion and Bunty shifting about in his seat, saying from time to time, 'So what's new?'\n\nGayatri pressed home her point.\n\n'He does talk to you, and objectively speaking, you're not bad looking. You have no figure, but your features are sharp, you have clear skin, and high cheekbones. If your hair was styled instead of pulled back, it would help, but still, it is thick and curly. You are on the short side, but tall men like short girls, that is one thing I have noticed, time and again.' (Gayatri herself was tall.)\n\n'I can't just walk up to him and say give me your address, I want to write to you.'\n\n'It's not anything so great you are asking. Once you write, he will write back.'\n\n'He may not.'\n\n'Then he is no gentleman'' said Gayatri severely.\n\nEventually Astha blurted out the request, shoving her diary and pen at him.\n\n*\n\nShe wrote, and he did reply, weeks later.\n\n'Who is this from?' asked her mother, holding the letter away from her.\n\n'How do I know?' demanded Astha.\n\nShe snatched the letter and tucked it into her school bag. It was from him, she knew it was. He had written.\n\nDear Astha,\n\nI received your letter a few weeks back. We do not really get time to write, we are very hard worked here.\n\nTomorrow, I am leaving for camp. There is much work to be done; we do a lot of studies on tactics and strategy of defence and attack. We leave early in the morning, first marching 20 miles, from where we will be transported another 80 miles. At the end of it all we will land in some remote village. After lunch, which we carry, we will 'dig-in' for the night to carry out a defence exercise. Digging trenches in the Deccan plateau isn't quite as easy as you might think. Each one takes 3 to 4 hours. We shall also have to climb Simhagarh, Shivaji's famous fortress, and incidentally the highest one. At night we shall ambush and patrol, the sole difference between this and a real war being that we shall fire blank rounds at each other instead of live ones.\n\nAnd so on. It was a soldier's letter, what else had she expected? If the reality of Bunty was a little flat after her image of him, her love could take it. She re-read it all day and the days to come, till she got his next.\n\nIt turned out Bunty liked corresponding. Through the year Astha heard about his friends, the war with Pakistan, Lal Bahadur Shashtri, his academic subjects, his service subjects, his feelings about the Indian Army in general, and cadets in particular.\n\nAnd Astha, Astha was witty, clever, chatty, all the things she could not be when he was in front of her. Her writing was laced with little drawings which he found ingenious and talented. She started flirting. Letters were safe.\n\nAs the correspondence established itself, so did the mother's suspicions.\n\n'Why is he writing so much to you?' she asked every time a letter came. By this time there were two people waiting for the post, Astha and her mother.\n\nIs it a crime?' Astha replied.\n\n'You are too young to be indulging in such goings-on.'\n\nShe made it sound so sordid. What words could Astha use to a woman who saw the world in terms of goings-on?\n\n'There is nothing going on'' she said, lying with great dignity. There was no need to explain the pulpiness of her heart, the wretched and permanent knot in her stomach. No doubt her mother would consider that a going-on too. How she wished she could really be gone, gone in the arms of Bunty, who would hold her close, whisper his love, confide that her letters had made him realise she was his soulmate, they would marry after he graduated, could she wait for him.\n\n'You have got your exams coming'' went on Astha's mother, staring hard and penetratingly at her daughter.\n\n'I know'' said the daughter, staring back just as hard.\n\nAstha's mother sniffed, a tight cold sniff.\n\nAstha paid no attention. She was living in a world of her own, waiting for the holidays to come, so that she could see Bunty. It would be different now, no awkwardness or shyness. They were closer, they had shared their thoughts and feelings. Hopefully they would kiss. Where and how? She imagined the places, grew lost in her fantasies.\n\nThe holidays came. The minute the mother knew that Bunty had come, she went to his house and from then on Bunty refused to have anything to do with Astha.\n\nFor a long time she didn't know what had happened, nor could she bear to find out. She lived in pain and anything that touched it was too much for her.\n\n*\n\nThe night before, on the phone, she had fixed to see him, this time she would not need Gayatri. She had spent many hours thinking about her hair, her clothes, should she wear casual or formal, new or old? How should she do her hair? Up or down? Loose or tied?\n\nDressed in her newest churidar kameez, tight around the hips, loose around the waist, Astha went to Bunty's house, at eleven o'clock as planned. His father met her at the door.\n\n'Bunty is not at home, beta'' he said politely, without asking her in, a slap in the face for Astha, standing awkwardly in her new churidar kameez, so tight around her hips, so loose around her waist.\n\n'When will he be back, Uncle?' she managed, dread making her voice heavy. Did Bunty's father hate her? Had Bunty said something to him? On the train home from the Defence Academy had he decided to loathe her instead of like her? _Was this his way of letting her know_?\n\n'I don't know, beta. It is his holidays, he has so many friends and relatives to see. You can phone him some time. Bye now.'\n\nThe door was closed before she was even down the steps. No seeing her off, no nothing.\n\nShe walked home, feeling sick. The year of writing to each other, he had said he wanted to see her, had he been lying, seeing how far she would reveal her feelings in those stupid letters before he showed them to his father? How could she have forgotten the little interest he had shown in her when he was actually in Delhi? He was amusing himself, that was why he had written, now when it was time to meet he intended to drop her. How Gayatri would laugh. Was there any way she could stop being friends with Gayatri right that minute? Dump her for ever, and never see her again?\n\nAstha had not been in the house ten minutes when Gayatri called. 'What happened?' she asked breathlessly, as though she had been the one waiting all these months to be kissed.\n\n'Oh nothing'' said Astha airily, through gritted teeth.\n\n'Nothing? What do you mean nothing?'\n\n'It's very sad. One of his uncles died, and he has to go to Bombay immediately with the whole family.'\n\n'But why didn't he tell you?'\n\n'There wasn't time to write.'\n\n'Odd'' said Gayatri after a pause. 'He might have met you for a few seconds alone. After all those letters.'\n\n'I'm telling you there wasn't time'' said Astha her voice rising.\n\n'Oh Asu, poor you.'\n\n'Not at all. I found I didn't like him so much when I actually saw him. He looked very silly. All he could say was \"So what's new\". One tends to build people up through letters.'\n\n'I suppose'' said Gayatri, sounding dissatisfied.\n\n*\n\nThe holidays passed. Astha suffered daily. Neither drawing nor reading could engage her. Her heart felt like lead, her mind like stone. She couldn't get Bunty on the phone, he was always out. Shyness, reticence, some shreds of self-esteem forbade her from persisting beyond politeness. No matter what had happened, he should also want to see her, if only to clear any misunderstanding. And so pride carried her through each miserable day.\n\n*\n\nA year later, when the pain was less, and college had made her feel more a woman of the world, she wrote, a light casual letter, 'What happened?'\n\nHe wrote back, 'I thought you knew. Your mother visited us the very night I arrived and told my father that I was distracting you from your studies. At the same time she asked him what my intentions were. My father thought it better if we had nothing to do with each other. Why create complications? I wish you well in life. Yours sincerely, Bunty''\n\nCan one die of shame twice? Astha did. How dare her mother interfere in her friendships? But then Bunty too had given in so easily, not bothered to find out how she felt, no word, no sign.\n\nWhere was the man whose arms were waiting to hold her? Till his arrival, she would walk alone, alone in college, through corridors of happy, independent, bustling girls, through classrooms devoted to the study of English Literature, alone in the colony through the dreary lanes between the houses.\n\nShe tried to put Bunty from her mind, though once or twice when girls huddled together, heads bent in the canteen, she brought out his name experimentally, to show she too had lived and knew what love was.\n\n'Yes, these boys\u2014'\n\n'Yes, there was someone, only last year\u2014'\n\n'Yes, he was handsome\u2014'\n\n'Oh, he doesn't study here. The Defence Academy at Kharakvasala.'\n\n'Yes, we still meet during the holidays, nothing special from my side. I thought it better not to have a long-distance relationship, you know how it is... '\n\nThe girls listened sceptically, how could they believe in the reality of one who was never seen hanging out at the back gate? Still, they teased her sometimes saying, 'Astha, tell us more about Bunty'' and Astha cursed her need to feel part of a group by making light of something that still tightened her chest with grief.\n\nFive years after its inception the housing society of the Ministry of Relief and Welfare was awarded a piece of land across the Jamuna.\n\nThe habitual gloom on the father's face became even more pronounced as he conveyed this news to his family. 'Other ministries, where the bureaucrats have pull, managed to get allotments in South Delhi. But what do we get? A site across the Jamuna, where there is no water, no electricity, no markets, no bus services, no amenities, no proper roads even.'\n\n'Never mind'' consoled his wife, concealing how bitter the blow was for herself as well, so much had depended on the promised piece of land. 'Once construction starts, things will change. Everything has to have a beginning. How much are the plots going for?'\n\n'How much can they go for?' replied the father. 'In the middle of the jungle with thugs, dacoits, and wild animals. 7-8,000 rupees.'\n\n'Size?'\n\n'225, 280, or 350 square yards.'\n\nTheir future home was going to be small and relatively cheap.\n\n*\n\nThe lots were chosen by draw. On the appointed day, the mother said to her daughter, 'I hope he draws a 350 plot, in the corner. There is very little difference in price.'\n\n'From?' asked, the daughter languidly. She had been paying insufficient attention to the family fortunes knowing that wherever they built a house, she would not be in it.\n\n'Don't you ever listen? After we are gone it will be yours.'\n\n'I don't want it.'\n\nThe mother trembled with annoyance. 'Don't you see our lives?' she hissed. 'Have you still not realised the value of land, that once you have it, there is always something solid to fall back on?'\n\nAstha looked at her mother, at the sallow skin with liver markings, at the carelessly dyed hair, black and white, at the hands gnarled from a lifetime of housework, the veins standing out on the backs, only fifty, despairing, shrivelled, and old. Her dream of a house was coming true in a way that made that dream for ever unrealisable. Her thoughts were now of 225, 280 or 350 square yards, of whether it would be near a park, or near the main road, near a market or bus stop, whether her husband would be happy there or not, because she of course could be happy anywhere.\n\nSlowly she took her mother's hands in her own. 'It will be all right, Mama'' she said beginning to rattle off what she had heard so often. 'Trans-Jamuna will grow, South Delhi too was once like this, it will be different once the new bridge is built. Just imagine, we will have our own house at last. A garden too.'\n\nThe mother looked at her daughter's young hands holding her own loose-skinned bony ones.\n\n'Yes darling, at last'' she sighed heavily.\n\n*\n\nThe father came home.\n\n'Well?' asked the mother, taking his briefcase.\n\n'280.'\n\nThere was silence as the family digested this. 280. They were going to live on 280 square yards. But still, that was more than 225, of which there were so many.\n\n'There were only four 350 ones.'\n\n'Only four? Then naturally we won't get one.'\n\nThe father paused before continuing, 'One of them the President drew.'\n\n'I'm sure it was rigged'' flashed the mother.\n\n'God knows. It seemed fair enough. It was done in front of us all.'\n\n'Of course it will seem fair. These people are not children.'\n\nThey consoled themselves over tea with reflections of the general perfidy of the world, and their own inabilities to succeed in the games that were demanded of life's players.\n\n*\n\nNow that they had their plot, they drove out in the direction of the wilderness to see it. Along with them was the President of the Ministry of Relief and Welfare Group Housing Society, needed to show the way.\n\nBehenji'' he turned around to address Astha's mother, sitting in the back seat with the reluctant daughter, 'in ten years time this area will be really built up. The future of Delhi is here. How far can Delhi spread south?'\n\n'It is a long way around'' murmured Astha's mother.\n\nThey were heading north, towards the Red Fort, beyond the ghats for burning the dead, towards Shahadra, across the old British-built double-storied bridge for cars and trains, the lone bridge across the river to east Delhi.\n\n'When the new bridge is completed the journey will be quicker, Behenji'' consoled the President. 'Twenty minutes and you will be in Connaught Place, heart of Delhi.'\n\nAstha's father drove without responding to any of these remarks. The privilege of owning a plot in this godforsaken place would come as a result of belonging to a ministry in which he had felt wasted all his life. The bitterness of this kept him silent.\n\nThe roads they were now passing were potholed and badly kept, establishing kinship with the dirt roads of villages. From time to time they caught sight of a brave house standing alone.\n\n'People are already constructing'' pointed out the President.\n\nThe road got narrower and bumpier, the trails of dust bloomed larger.\n\n'Nirman Vihar, Swasth Vihar, and there, Preet Vihar'' said the President, waving his hand at the bare expanses around him. A gloomy silence filled the car, they were too old and too young to regard with excitement this particular future laid out before them.\n\n'Here, turn here'' indicated the President. They left the narrowness of the main road for an indistinguishable little lane, bumped along, turned once, and there they were. The land was dry, dusty, bare, treeless and nondescript. Asman Vihar. Sky Colony.\n\nWhat had they imagined? Neat plots, lined with trees, and little lanes, waiting for owners to come and build houses? For 8,000 rupees? Were they crazy?\n\n'What's that?' asked Astha's father pointing to a village they could see in the distance.\n\n'Oh, that will go, we are dealing with them'' said the President. 'They think they have a right just because nobody has dislodged them so far. The land is vacant, so these villagers use it to farm. And the odd group of Gujjars roams around.'\n\n'Is it safe?' trembled Astha's mother.\n\n'The more people come, the safer it will be.'\n\n'And water, electricity connections?'\n\n'For water we have to dig our own tube wells. And they have promised a temporary line for electricity. It is only a matter of time when this will be like your Golf Links, Jor Bagh, or Defence Colony.'\n\nAfter this trip they did not talk about their dream home anymore. They heard stories of how, in one of the lonely houses there, dacoits had broken in at night, stolen everything, and injured the owner so much that he was in a state of semi-paralysis.\n\nWhen the future was taken out and aired they concentrated on the difference the new bridge would make, the changes in infrastructure that would come about once the area became more populated. When the prices went up, they could sell their plot and buy a little flat in south Delhi. They had to trust in God and wait, though with the father's retirement only six years away, the period they could wait was limited.\n\nNow that Astha was in college her mother focused anxiously on their primary parental obligation. Every Sunday she scanned the matrimonial pages meticulously, pencil in hand, circling ads. Later on she would show them to the father.\n\n'You have to take her to the studio to get nice photos taken. One full standing, one close-up of the face.'\n\n'She is only in second year, Sita, for heaven's sake. Let her finish her education at least.'\n\n'In the time it takes to finalise a match she will have graduated. Good boys are not to be found so easily.'\n\n'She has just turned eighteen. Let her be.'\n\n'You want her to turn out like us? Marrying in her thirties? And everybody wondering what is wrong?'\n\n'Let her settle down to a career, then we will see. I can't go around begging people to marry my daughter.'\n\n'There is a time for everything'' went on the mother. 'The girl is blossoming now. When the fruit is ripe it has to be picked. Later she might get into the wrong company and we will be left wringing our hands. If she marries at this age, she will have no problem adjusting. We too are not so young that we can afford to wait.'\n\nAstha, eavesdropping, wondered where this stream of logic would lead. She herself had tasted love and wanted nothing of an arranged marriage, but what did her father think?\n\nHer father refused to answer and refused to take Astha to the studio.\n\n*\n\nThe day the mother found a suitor, was a day when Astha came home from college, tired, stinking of sweat, her bag heavy on her shoulders, her red pointed ballerina shoes pinching her feet. All she wanted was to quickly bathe, get lunch out of the way and then lose herself in the Georgette Heyer she had borrowed from a friend.\n\nHer mother was sitting in the drawing room with a young man dressed in khaki.\n\n'Beti'' she called. 'Come here.'\n\n'Coming'' Astha shouted back, but she didn't like the tone of her mother's voice. She hid behind the curtain dividing the room and listened.\n\nMother: 'That was my daughter.'\n\nYoung man: 'Does she like sports?'\n\nMother: 'Very much.'\n\nDread filled Astha. Her mother was lying. She had somehow found a groom without a studio photo. Did her father know? She locked herself in the bathroom.\n\n'Astha.'\n\nNo answer.\n\nThe door rattled. 'Come out beta. Hurry up.'\n\n'Why?'\n\n'There is someone here to meet you.'\n\n'Who?'\n\n'Someone.'\n\n'First you tell me.'\n\n'Oho. A boy.'\n\n'Why are you so interested in a boy meeting me _now_?' asked Astha bitterly.\n\nBang, bang, bang \u2013 the wooden bathroom door shook against the onslaught of the mother's rage. Astha watched the towels hanging from the hooks shudder, she heard the tap next to the toilet dripping into the tin can below it.\n\n'I'm not coming'' she shouted.\n\n'You don't object to seeing boys otherwise. Suddenly you become so high and mighty, and refuse to even be polite to someone who has come all this way.' The mother dropped her voice to wheedle, 'Now come, what is the harm? It is just a meeting, nothing else.'\n\nAstha stared at a tiny crack in the old wood of the bathroom door. She was about to humiliate her mother in front of a stranger. She took a deep breath. 'I can't'' she whispered hopelessly, 'I can't meet anyone like this.'\n\nThe mother finally gave up, leaving Astha collapsed against the bathroom door, tears falling, crying, crying for Bunty, crying for the lack of love in her barren life, crying because she didn't want to see a dull stolid man in the drawing room who advertised for a wife and asked about sports.\n\nShe remained in the bathroom long after the suitor left. The bathroom represented her future; she had better start getting acquainted with it now.\n\nHours, years, later her mother banged irritably on the door, 'He has gone, fool that I was to try and arrange anything for you, you are just like your father, thinking everything is going to happen on its own. The food has gone stone cold, you can reheat it and clear up everything after you have finished.'\n\nOne month after this the boy appeared. In his final year of college, he was a bit older. They had been introduced by friends of friends at the University Coffee House. Like everybody they knew, they were missing classes in order to haunt these hunting grounds, their gaze sorting through the tables speculatively.\n\nAstha began to be included in groups that included him, at cinemas, restaurants, appointed meetings at the Coffee House, instead of casual ones. A boy was interested in her. With every visit he made to the back gate, her stock grew in college.\n\nShe began to lie at home about where she was going, and what she was doing. Most of the girls she knew who were seeing boys lied. It was the routine, self-protective thing. And how necessary, Astha had already seen.\n\nNow every evening Astha took a walk in the colony, announcing her intention of dropping in on old school friends on the way back. My head gets so tired with studying, she complained, I need a change.\n\nGo, beti, go. Take some exercise, and remember, walk briskly, swing your arms, and breathe deeply from the stomach, said the mother, glad that her daughter was at last beginning to understand the value of fresh air.\n\nAnd Astha would dash out of the colony, down the main road to the corner where Rohan was waiting in his old\n\nVauxhall. A quick check to make sure no one was looking, then she would jump in and they would roar off.\n\nIt was the first time in the old Vauxhall, side by side, in the front seat. The car was parked in a narrow empty lane, next to a Minister's house in the Lutyens part of New Delhi. Rohan had shut the windows, and locked the doors. It was late November and dark. The car smelled of Old Spice, Rohan's aftershave lotion, and the musty scent of ancient leather.\n\nAstha glanced at Rohan sideways. He was twisting the car keys in his long slender hands with the hairy fingers, tapping them restlessly on the steering wheel. Finally he turned and reached purposefully towards her.\n\n'Do you want to marry me?' she asked breathlessly, anxious to get this thing out of the way.\n\n'What?' he asked distractedly.\n\n'Marry me. Do you want to?'\n\nHe took his hands away and stared at her. 'Isn't it a bit early to decide that?' Astha felt she had offended him.\n\n'Well, you know, otherwise\u2014' she trailed off, trying to look as cute and disarming as possible.\n\n'Good God, is that what you are worried about? I'm not the type to let a girl get pregnant, what do you think I am?'\n\nAstha realised she was sounding very un-hep, but she couldn't help it. She had to know how safe she was. 'Marriage'' she persisted, 'you know\u2014' She inched a little further away from him.\n\n'Oh God, all right. We might get married. One day.'\n\nHis mouth closed on hers, his tongue was exploring, while Astha choked and wriggled frantically.\n\n'What's the matter?' he asked letting her go. 'Don't you like it?'\n\n'I don't know'' she mumbled non-committedly.\n\n'Well, let's find out.' Rohan was beginning to sound impatient.\n\nAstha had volunteered to go with him in the car. Her body had registered excitement when he had parked in the dark lane. When he bent close to her, she had been overcome with dread and longing. There was no going back. She offered her lips, trying not to shrink into the seat.\n\n'God you're so stiff'' said Rohan shaking her a little bit.\n\n'Sorry'' she mumbled.\n\n'This is not some kind of torture you know.'\n\nAstha didn't know what to say. Rohan let her go, also silent. Finally he said, 'Give me your hands.'\n\nShe held her fists tensely out. Slowly he moved his thumb around her wrist, stroking the closed hand open. He kissed the fingers, nails, palms, he felt the small hair on the back with his closed lips. Astha felt something flow inside her as she stared at his bent head. She had never been so aware of her body's separate life before. After a little more of this he dropped her at their secret corner.\n\nShe got out of the car reluctantly. Something hadn't happened. But then again, something had. On the whole the encounter left her anxious for more.\n\nWhen Rohan at last slid his tongue into Astha's mouth she was putty in his hands. Her neck, her ears, her throat, eyes, chin, lips, all had been explored, and this time her strongest feeling was gratitude.\n\n*\n\nThis was the start of numberless kisses in the car. The only problem was in finding a place sufficiently isolated.\n\nTo Astha all places looked the same, but no, muttered Rohan, his eyes scanning various deserted spots, his fingers kneading the palm of her hand, not that one, not there, that's not safe, while Astha burned with impatience. Finally before rolling up the windows he always put out his hand and locked the door from outside.\n\n'Why are you doing that?' Astha asked.\n\n'Precautions.'\n\n'Against what?'\n\n'Just.'\n\nAstha was not really interested. All she wanted was for him to start, so that the world could fall away, and she be lost. This is love, she told herself, no wonder they talk so much about it.\n\nOne evening, they had parked in their usual dark corner in a by-lane off Akbar Road. Rohan liked Akbar Road, he considered it among the safest places he could find.\n\nAstha had slid as far down the seat as she could go without dislocating her hip. Rohan was lying as much as he could on top of her without dislocating his own. Their eyes were closed, their breathing audible. Absorbed in what they were doing, they did not hear footsteps approaching, did not see faces pressed against the windows on either side, eyes peering down at them.\n\nThe car shook, hands banged violently on the glass, rattled the handles. Astha and Rohan jerked upright, the whole car was swarming over with threatening bodies trying to get in.\n\n'Oye, oye'' they shouted, leering and grimacing, furiously working the handles. Rohan frantically turned the key in the ignition, pressed the accelerator, the old Vauxhall shook and roared. The men fell off as he sped in reverse down the dark lane, lights off. They ran after the car for a while, then couldn't be seen anymore. Some distance down the main road, Rohan stopped.\n\n'Who were they? And why\u2014?' stammered Astha, shaking with fright.\n\nRohan took her hand. 'Some fools. I'm sorry'' he said.\n\nShe started to cry.\n\n'Calm down.'\n\n'Take me home.'\n\n'Calm down first. Look, nothing actually happened.'\n\nAstha felt worse and worse. Those men had wanted to attack. Suppose they had managed to break the car window, gang rape her because of her shameless behaviour in a public place, and beat up Rohan when he tried to intervene? And all the while her parents would be thinking she was breathing in fresh air. If her mother knew she would first kill her and then herself. Astha's tears grew copious and she began to choke in her dupatta, while Rohan took sly glances at his watch. 'Come on'' he said at last, 'it was bad, but now it is over. Don't cry, for heaven's sake. We won't go there again.'\n\n'Hoon'' sniffled Astha.\n\n'You are all right, so what exactly is the problem?'\n\nAstha only knew she felt terrible. Finally when Rohan dropped her off, she sensed eyes hidden in every bush, eyes that saw and condemned. She pulled her dupatta around her head, and hurried home trying to concentrate on the various lies she would have to tell as to why she was so late.\n\nThe day Astha's mother read her diary dawned cool and clear, beautiful like all winter days, with the flowers blooming in the garden, and the promise of basking for hours in the sun.\n\nShe was deep in a book when her mother called, 'Come here, I have something to show you.'\n\nReluctantly Astha marked her place, and went inside. When she saw her journal in her mother's hands, she wanted to instantly erase herself. There she was, with her skin ripped off, exposed in all her abandoned thoughts and deeds.\n\n'Is this you?' the mother's voice quavered, her grip like iron on Astha's arm.\n\nAstha shook her head nervously.\n\n'Then, what is it?'\n\nDesperate silence while she tried to think of something plausible.\n\n'Answer me'' screamed the mother in a whisper.\n\n'I\u2013I don't know'' stammered the daughter, 'I mean, I don't remember.'\n\nBut she did, of course. All her secret fantasies, the things she did with Rohan, painstaking details of the furtive, exciting moments in his car.\n\n'Well, look at it'' the mother waved the offending notebook in front of Astha's nose, an innocuous old brown paper covered thing with _St_ _Theresa's_ _Convent_ _School_ in a half moon on top. It had been hidden behind her college books, how had her mother discovered it? It looked awful in her hands, soaked in sin.\n\n'You have no right to read my diary'' she weakly muttered in self defence, averting her eyes.\n\nThe mother ignored this remark and continued leafing through it gingerly.\n\n'Here, what does this mean?'\n\nThe usual scene of passion. Astha went through puzzled motions of glancing, page turning, furrowed brow.\n\n'These are notes for a story I am writing'' she said, inspired at last.\n\nThe mother's body sagged as some of the tension went out. 'This is your imagination?'\n\n'Yes. Yes, it is my imagination.'\n\nThe mother was silent for a moment, then sighed heavily and held the tender young body of her innocent daughter close to her. 'My child is too sensible to do anything like this'' she whispered. The girl remained rigid, arms by her side.\n\nThey avoided each other for the rest of the day.\n\n*\n\nThere were three consequences to this.\n\nOne was that Astha stopped being able to write in her journal. She tried a few entries in an elaborate code, but an audience was now branded into every page, and she could inscribe nothing beyond a casual account of her day in college.\n\nThe second was that Astha's parents took an annoying interest in her reading matter. Her father began diligently to bring her books of moral and intellectual substance. 'You need a sense of your cultural background'' said the bureaucrat. 'Of what made this country great. Know your artistic heritage, since your interest lies there.'\n\nHer mother decided that the virtues of tradition needed to be made more explicit. 'Our shashtras teach us how to live. You will learn from the _Gita,_ __ the Vedas, the Upanishads.'\n\n'I can't read them'' protested Astha violently. 'My Hindi is not good enough, you know that. It is your fault for sending me to a convent school.'\n\n'Your father happened to think that a convent gave the best education. That doesn't mean we can't broaden your base now'' replied the mother. And she began getting Hindi books and magazines from her school library, so that Astha's Hindi could improve and the sacred texts of the Hindus be made available to her.\n\nThe third consequence was that the parents tightened their surveillance. Getting to meet Rohan proved more and more difficult.\n\n*\n\nShe didn't want to tell him of what she was going through. He was preoccupied with his final year exams and seemed to have less time for her.\n\n'Why do you keep phoning all the time?' he complained one evening when they met. 'I have to study.'\n\n'I don't phone you all the time. Once or twice a day to ask how's it going.'\n\n'It distracts me.'\n\n'If you don't want to talk to me, just say so. Don't look for excuses.' Astha's voice shook. Rohan sighed and put his arms around her. Astha snuggled into him and slid her hands under his shirt.\n\n'Baby, don't be unreasonable. A man has to do well. Get a scholarship. Go abroad.'\n\nThis was the first time he had talked of going abroad so definitely. Astha shifted herself out of his arms.\n\n'Hey, little one'' cooed Rohan, reaching out for her. 'What's the matter?'\n\n'Nothing'' she said forlornly.\n\n'Do you think I'm going to forget you?'\n\nAstha did think exactly that, but how could she admit it?\n\n'Let me just finish my exams, little one, and then\u2014'\n\nRohan's words helped bolster Astha's illusions, it was all right, she was still safe, their affair was going to end in marriage. But the cold feeling did not go away, though each time Rohan spoke, Astha clung to his assurances.\n\n*\n\nRohan did very well in his exams, and on that stepping stone began the process of his going away. Away to the West, where ordinary mortals cannot go, where even the words PPE and Oxford showed Astha how great the distance was between them.\n\n'Oxford'' she breathed in awe. Suddenly her life seemed very small.\n\nRohan looked nonchalant. 'Might as well follow in the family tradition, keep the old folks happy. My father is an Oxford man, you know.'\n\nNo, Astha had not known. 'How lucky you are, Rohan'' she said.\n\n'Well, my father is keen'' continued Rohan, his gaze centred on the middle distance.\n\n'When are you going?' she asked, and then hated herself for being in a situation where she was forced to prise answers from the man she had been so intimate with.\n\n'Soon.'\n\n'Doesn't it cost a lot of money?' asked Astha tentatively.\n\n'Lots'' said Rohan, lighting a cigarette and breathing the _smoke sexily out. 'But we are trying to manage something_.'\n\nAstha thought it might seem rude to ask for more information, and waited for Rohan to tell her. Rohan did not. He looked at her briefly and smiled. 'Come, let me drop you home'' he said, 'There are a lot of things I have to do.' He hadn't touched her once. As he turned the key in the ignition, Astha thought, he was going to Oxford, he had the resources, his father was an Oxford man. What was her father? A minor bureaucrat, who had never studied abroad, whose sole possession was 280 square yards in the wilderness beyond the Jamuna.\n\n'Wait, Rohan'' she said, 'I hardly get to see you nowadays, you are so busy, and it is still early.'\n\nRohan continued drumming his keys against the steering wheel.\n\n'How come you never mentioned your family traditions before?' Astha went on carefully, scratching and poking at the leather so hard, she could smell the car on her hands long afterwards.\n\n'Well, there was no point talking about things, until things got certain.'\n\n'Yes, but you might have told me that there was a possibility of your going away.'\n\n'You knew that'' said Rohan coldly, not looking at her. 'I never tried to hide anything. There is no need for me to hide.'\n\n'No, no, of course not. That is not what I meant'' floundered Astha. 'But you just mentioned once or twice, like people do, you know, about going abroad, and I didn't know... Why, your results are just out, and you must have been trying since last year if you are going so soon.'\n\n'Sending applications is not something to make a song and dance about. I would look very stupid afterwards if I got neither admission nor funding.'\n\nAstha felt hopeless. She sat in silence, next to this boy whom she had thought she knew. The hands that he had used on her body were now clenched around her heart, slowly squeezing, slowly hurting.\n\n'What about us?' she asked abruptly, drawing a deep breath.\n\n'We will see'' said Rohan briefly. He was waiting to take her home, waiting to get rid of her. He started the car, while Astha stared out of the window all the way to the edge of her colony.\n\n'Bye'' she said, closing the door carefully, feeling it would be her last time in the Vauxhall, which was just as well. Old cars were so ugly, so useless, so slow, why did anyone bother with them?\n\n'Bye'' said Rohan. 'Be seeing you'' he added, a remark which her dignity threw back silently, with all the coldness and contempt her falling to pieces self was capable of.\n\n*\n\n'Where have you been? Dinner has been getting cold'' came her mother's voice, as Astha tried her usual unobtrusive entrance.\n\n'Nowhere.'\n\n'Nowhere has a name or no?'\n\n'No'' said Astha, going to lock herself in the bathroom, free from voices, free from everything except the terrible things she was feeling, because Rohan didn't love her, Rohan had lied to her. Rohan was what her mother had been warning her about since she was old enough to be warned, and how pleased she would be to know she had been right all along.\n\n*\n\nIn the days to come Rohan neither called, nor sent the usual secret messages through her girlfriends.\n\nIt was over. Over. Over.\n\nAstha entered her third year with a desire to get her education over as quickly as possible. Every day was painful to her. She was constantly reminded of Rohan, in the Coffee House, at the back gate, at their secret corner of the road, every evening at home. As for old black cars, they made her physically sick.\n\nRohan went abroad and Astha enrolled in MA, bored and unenthusiastic. Three years of an Honours course in English Literature had given her all she wanted to know of the subject. Not for her did the excitement of intellectual discovery lie ahead, only more of the same. After two years were over she supposed she would drift into teaching, that is what women like her did. School or college, remained to be decided.\n\nAll through third year her classmates had been busy preparing for competitive exams, or like Rohan, applying for higher studies abroad. Those not in this category had married and disappeared, to be heard of occasionally, moving around with husband, and later baby, stamped with the marks of confirmed adulthood.\n\nNow in MA, her friends were few.\nChapter II\n\nAstha was in her final year when the proposal came. The MBA, foreign returned son of one of the bureaucrats who lived in the larger houses bordering Lodhi colony, had seen her and wanted to meet her. His father dropped in on them, and acquainted the parents with their good fortune.\n\n'I don't know him'' objected Astha when they told her the news.\n\n'He also doesn't know you, Madam'' said the mother crossly. 'That is why he wants to meet you.'\n\n'Give her the details, Sita'' reproved the father. 'This is a question of our girl's happiness. There is no hurry.'\n\n'With you retiring in one year, there is every hurry.'\n\n'That is no reason to marry.'\n\nThe mother fell into despairing silence. Retirement, father's uncertain health, finances in a meagre state, the bridge to the plot unbuilt and their dearest daughter still to be settled.\n\n'How many times can I meet him?' demanded Astha, a little excitement rising in her. Somebody found her desirable and had gone to lengths to find out who she was.\n\n'One, two times, what is the need for more?' said the mother. 'You cannot tell about a person before marriage, no matter how many times you meet him.'\n\nAstha sat silent, twiddling her thumbs, staring down at her flat feet in their bathroom slippers. Had she known Rohan? Not really, and the soiled feeling she now associated with that interlude came over her again.\n\n'Papa?' she quickly asked. 'You think this is a good thing?'\n\n'I'm not sure'' said the father uncertainly. 'The plus side is that he is the only son and both his sisters are married. The younger one, settled in the US, wanted to sponsor him, but he decided to return to his parents. He is twenty-six, five eleven, he works as an assistant manager in a bank.'\n\n'Clearly a good, family-minded boy'' said the mother complacently.\n\n'And Vadera's ministry was allotted land in South Delhi. They will be able to build on it, they won't have to wait for bridges and water and electricity connections, they won't have to worry about thugs or gypsies'' continued the father bitterly.\n\n'Isn't that a good thing for our daughter? She at least will have a decent home. God has heard my prayers'' added the mother piously.\n\n'Sita, are your prayers that the girl be married to a plot in Vasant Vihar? Why don't you go and do the pheras there?'\n\n'What's wrong, Papa? You don't like the family?'\n\n'I have heard things about Vadera.'\n\n'What things?'\n\n'He is in the commerce ministry. Nice place to be if you want to keep a certain standard of living, and licences are needed by every manufacturing unit, big or small, for anything they do.'\n\n'So?'\n\n'He travels abroad, gives his daughters big weddings, buys a car, a _new_ car every three or four years.'\n\nThis did indeed seem very bad; such high living had to have some dark reason behind it. 'How does he do it, I ask you?' went on the father. 'Must be taking bribes. Will you be happy in a house that doesn't share our values?'\n\n'Papa, you don't think it is a good idea, I won't meet the boy.'\n\nThe mother collapsed into rage. 'Everybody is corrupt, are they? Throw out nine tenths of the government, run the country yourself with your high blood pressure. Expect the whole nation to be like Gandhi. Send your daughter to an ashram, because we have neither the means nor the money to get her properly married.'\n\n'I will look after myself'' said Astha bravely.\n\nNo one paid attention.\n\n'Their sole interest is the girl, her looks, her education, her qualities. That is something'' said the harassed father.\n\n'It's more than something'' insisted the mother. 'How many people do you know like that?'\n\n'Big dowries are being offered for Hemant. He is known to be quite smart.'\n\n'Is that his name?' asked Astha.\n\n'Yes.'\n\nA nice name. Hemant and Astha. It had a certain ring to it.\n\n'Why aren't they going in for big dowries?' inquired the prospective bride.\n\n'The boy does not believe in dowry. Must be the foreign influence, couldn't be his father.'\n\nAstha felt an even greater interest in the boy. 'Let me meet him, Papa'' she declared. 'After all, the father came with the proposal, they must be thinking alike to a certain extent.'\n\n*\n\nHemant came to the house. The parents left them alone for half an hour. Astha was so nervous her palms were sweating. He had only gone by her face, she knew that was passable, but what about the rest of her? Should she tell him about Rohan, but what to tell? That though she had kissed a boy, her hymen was intact? That he had broken her heart but she hoped to find happiness in marriage? How could she say this to someone she didn't know? She looked up at Hemant and smiled tentatively, he smiled back, asked about her hobbies before continuing to talk about his experiences in the USA.\n\nA few weeks later the engagement between Astha and Hemant Vadera was decided upon. The wedding was to be held in June. By then Astha's MA exams would be over, Hemant's elder sister's children would have their holidays, and his younger sister would be able to come for a month from the States. With all this settled, Astha and Hemant began to date.\n\nThe day of Astha's wedding was like any other day in June. The heat rose and rose, dust gathered, and all activity struggled against a dull and heavy lassitude. In the morning Astha's mother brought her tea and gazed at her approvingly.\n\n'Today you are getting married and leaving for your new home'' she murmured, tears in her eyes, while relatives clustered and consoled, speaking of the necessity of this moment, the pain of a mother at parting, the joy of a mother at her duty successfully completed. These murmurs fluttered around Astha, who, restless and ill at ease, waited for the action to begin.\n\nOutside, the tent wallahs had started converting the central common ground into a wedding hall, enclosing it with shamianas, covering the stubbly parched grass with durries. In the afternoon the caterers came, putting up tandoors near the garage. Five hundred invitations had been sent out. Both the families lived in the same colony, worked for the same government, had lived for years in the same city. A huge guest list was unavoidable.\n\nIn the evening there was a havan, during which Astha's red and white wedding bangles, with the dangling chura, were put on. She sat before the fire, tossing the samagri in, feeling dazed and unreal.\n\nAfter this it was time to get ready. Her wedding sari, fresh from the ironers, was laid out. She contemplated the thick red and gold silk in which sweat and discomfort were guaranteed. Normally summer brides wore thin tissue woven with gold, but with the wedding costing so much, she had to wear the heavy bridal temple sari bought years ago at a special price by her father on a government tour to South India. Now Astha hoped Hemant would not find her dowdy or unfashionable. She hoped he would not mind that she had little jewellery, she hoped he would like her without beauty parlour bridal make-up, and that he would hate, like she did, those assembly line creations, pink and white, with black-lined eyes.\n\n*\n\nNight came, the barat arrived. Astha was called to garland her groom to the taped music of shehnais. At the auspicious hour they sat down next to each other under a small rickety pandal, with a fan trained on them. The hot air from the fan, the smoke from the fire, the sight of her father waiting to do the kanyadaan, the feel of her hand in the hand of her bridegroom, in a trance she realised this was the beginning of the life ordained for her.\n\nThe pundit was chanting. They were taking the seven steps around the fire, the steps that meant they were legally married. It was stifling, perhaps she was going to faint. Her cousins clustered around her, fussing with her jewellery, adjusting her palla and teasing the bridegroom.\n\nThis time tomorrow she would be in Kashmir, surrounded by mountains, trees, and lake, where waters rippled gently around gliding shikaras, where a book would not be her companion, but her husband bending tenderly over her, her companion for life.\n\nOn the plane to Srinagar Hemant held Astha's hand, while she looked shyly out of the window at the mountains they were flying over. A deep seed of happiness settled in the pit of her stomach, she was married, she didn't have to be the focus of her parents' anxieties any longer. She was now a homemaker in her own right, a grown woman, experiencing her first plane ride.\n\nThroughout the journey, Hemant kept touching Astha, a finger slid inside the sleeve of her blouse, a hand pressed on her knee.\n\nThe consummation took place in a houseboat on Dal Lake. Hemant undressed her slowly, gazing at her steadily, while Astha nervously looked at his stomach.\n\n'Now you undress me'' he said.\n\nShe shook her head modestly, wrapping the sheet around herself, tucking one edge in.\n\n'Come on'' he urged.\n\nShe bent her head still further and stared at his shoes planted next to her smooth, freshly waxed bare legs and pedicured feet, their mehndi patterns still a deep orange.\n\nHe took her hands and put them on his chest. She undid the buttons, and slid his shirt off. As he lifted his arms for her to remove his vest, the hair of his underarms sprang out at her, along with the smell of him. She pulled off the vest quickly and stopped. He drew her hands again to him, unzipping his pants himself in his impatience to guide her to the right spot.\n\n'There'' he whispered, jamming her hand into his underwear, swelling bulkily, 'it's yours. Do you like it?'\n\nAstha hardly knew. She snatched her hand away, and rolled face down on the pillow, while Hemant excitedly finished his own undressing.\n\nAfterwards they found a spot of marriage blood on the sheet. They both peered at it.\n\n'Did it hurt?' asked Hemant fondly.\n\n'A little'' confessed his wife.\n\nLater, in the privacy of the bathroom, Astha allowed herself to wonder whether she had been misled about the magnitude of the act.\n\n*\n\nSex. There was so much of it. The pain Astha felt between her legs was never quite absent. She could only thank God they never spent that much time actually doing it. Hemant attacked the whole thing with great urgency, gazing at her a little anxiously after each time, while she uncertainly smiled back to a look of satisfaction that came over his face.\n\nUnbidden thoughts of Rohan came. How slow his kisses had been, how infinitely long, how thorough. Then she scolded herself. Rohan had abandoned her, Hemant had married her, he valued her, he thought her pretty. The question of whose lovemaking she preferred did not arise.\n\nDuring the day they wandered around the tourist spots of Srinagar, hand in hand. People looked at the bangles on her wrists and smiled. She was a bride, and her grip on Hemant's hand grew more certain, and the blush on her face more conscious. Hemant's attention was constant. He took endless photographs and never let her read.\n\n*\n\nAstha wanted to record what she felt. This was her honeymoon, a one-time thing. She tried writing in her journal, but as usual the words didn't come. She tried sketching her surroundings, but the beauty was too overwhelming. She drew Hemant instead, his face, body, torso, arms, legs, it was the first time she had had so much nudity to work with. And when she was tired of art, she attempted writing.\n\nOne evening, sitting on the roof of the houseboat, drinking her evening tea, looking out on the lake, she wrote a poem about the sky, the shikaras, the sound of the birds, the sun behind the mountains reflected in the water. She wrote that she, the watcher, was part of that harmony, and it was fitting that her new life begin in beauty. As she put down her pen, tears filled her eyes.\n\nHer husband saw. 'What's the matter, darling?' he enquired, leading her downstairs to the bed, where they had made love last night, this morning and afternoon.\n\n'Nothing'' gulped Astha, her head laid out on the pillow. Hemant scooped her legs up, and lay down next to her.\n\n'I don't like to see my baby crying'' he said softly.\n\nAstha pressed her face into his shoulder, and laced her arms around his back.\n\n'Are you missing your mother?'\n\nShe started to laugh, the idea was so absurd. 'No, silly'' she said.\n\n'Then what? Tell me, I'm your husband'' urged Hemant. 'Tell me, wife.'\n\nAstha didn't know why she had been crying. Nothing in her present life seemed to justify tears. Finally she mumbled, a little sheepish, 'It hurts.'\n\n'Where?'\n\nAstha's hand vaguely danced over her middle. Hemant put his hand firmly between her legs. 'There?' he asked. She nodded.\n\n'Why didn't you tell me? You must tell me these things, I will never know otherwise. We are one, you know. Now promise.' He bent to kiss her.\n\nAstha responded more warmly than she had in her entire marriage. 'I didn't know what to say'' she went on whispering in the ears of her lawful wedded husband, her husband who would take care of all her hurts like he was taking care of this one.\n\n'Poor baby'' murmured Hemant, 'we won't make love till it stops hurting, all right?'\n\n*\n\n'Hemant?' asked Astha, a week after they were married.\n\n'Hum?' replied Hemant sleepily. Astha's head was on his shoulder, his arm was around her, and he had spread her hair across his chest.\n\n'Why didn't you marry an American?'\n\n'Why do you ask?'\n\n'Well, you were there a long time, you must have gone out with girls. Fallen in love, thought of marriage.'\n\n'Never.'\n\n'Never had women?'\n\nHemant side-stepped this. 'I was always sure I wanted to marry a girl from here.'\n\n'Me, you mean.'\n\nHemant's grip tightened around his wife, while Astha felt thrilled and wanted. 'Besides'' he said, playing dreamily with her hair, 'I had responsibilities to my parents. I am the only son, and I wanted someone who would fit in with our family life. American women are too demanding. Their men have to cater to all their whims and fancies.'\n\n'Is that true?' demanded Astha, visions of American women waited on hand and foot, basking in love, flashing through her mind.\n\n'You bet'' said Hemant with great certainty. 'Besides you can't be sure they won't be up to something.'\n\n'Like?'\n\n'Other men. It's not so unthinkable for them as it is for an Indian girl.'\n\nAstha fell silent. She was wondering if she liked this conversation. She turned to kiss him, but Hemant was not to be distracted. This was a topic he had considered deeply. 'I wanted an innocent, unspoilt, simple girl'' he went on.\n\nThere was a pause.\n\n'A virgin'' he elaborated.\n\n'Suppose I had not been one?' asked the wife carefully.\n\n'And the blood on the sheet, what was that? A mirage?'\n\n'Some women don't bleed, even though they have had no sex, you know'' said Astha. She had read this in a magazine.\n\n'Since that didn't happen in our case, why talk about it'' said Hemant.\n\nRohan's face bending over hers arose before Astha's eyes. Had she been a virgin? Unlike Hemant, she was not sure. She decided to forget the whole business, after all now she was definitely not one, and what was the point of thinking about the past?\n\n*\n\n'I see you are a writer', said Hemant, looking through her notebook, 'as well as an artist.'\n\n'Not really'' said Astha modestly, and waited for him to draw her out.\n\n'What are you doing now?' he asked.\n\nAstha showed him the paper on which she was writing a poem.\n\n'They say a picture's worth a thousand words'' he read, then looked up and frowned. 'But this is not a picture'' he objected.\n\n'I know'' said Astha, quickly. 'I was just looking for a way to start. Whenever I sketch the scene, it ends up looking like a post card, so I thought I'd try words instead.' She reached out to take the page, 'I'll work on it more and show you.'\n\n'No, no, let me read. Maybe I can help you. I used to read a lot when I was in college.'\n\n'Really?' asked Astha interestedly. 'What?'\n\n'Harold Robbins. Erie Stanley Gardner, Somerset Maugham, Agatha Christie, P. G. Wodehouse, all kinds of authors. I was quite a reader, you know.'\n\nAstha was silent, while Hemant's eyes quickly scanned the page. 'You certainly have a nice imagination'' he said, 'You put things well.'\n\nAstha looked pleased.\n\n'And for being so clever...'\n\nHe leaned towards her, and reached under her blouse. Astha pressed him close, and breathed my husband into his waiting ear.\n\n'My baby'' responded Hemant.\n\nAstha heard him with satisfaction. Her husband was going to encourage her writing. Maybe she could become a poetess as well as a painter. Her life was opening up before her in golden vistas.\n\n'Do you think there will be golden vistas in our life, darling?' she asked, taken with the sound of the words.\n\n'Of course, baby'' he replied. 'Golden like your body in the sunlight when it comes through the window touched by the water of this lake.'\n\n'Oh, Hemant'' laughed Astha, 'I didn't know you were a poet!'\n\nHemant looked modest. After they had kissed, fondled and not made love, Hemant told the bearer to take the drink tray upstairs to the roof.\n\nThey reclined on deck chairs facing the lake. The ice tinkled in Hemant's glass, bird sounds tinkled in their ears, water lapped around the boat. They were too high to see the sludge that had gathered around the houseboat, too high to notice the slight smell that came from the stagnant edge. Upon the roof, hand in hand, Astha's heart was as full of love as the lake was full of water.\n\nBack in Delhi, Astha submerged herself in the role of daughter-in-law and wife. The time spent in the kitchen experimenting with new dishes was time spent in the service of love and marriage. Hemant's clothes she treated with reverence, sliding each shirt in his drawers a quarter centimetre out from the one above so they were easily visible, darning all the tiny holes in his socks, arranging his pants on cloth-wrapped hangers so there would be no crease. With her mother-in-law she visited and shopped in the mornings, the memory of the night past, and the expectation of the night to come insulating her from any tedium she might otherwise have felt.\n\nEvery evening her father-in-law remarked, 'How nice it is to have a daughter in the house.'\n\nHemant looked as though it were all his doing, while Astha's mother-in-law sighed and talked of her absent daughters; Seema, so far away in America, and Sangeeta, well, now that Hemant was married, he and his wife were responsible for Sangeeta, whose troubles with her husband and in-laws were always hinted at rather than spelt out.\n\nAstha, proud that she was considered responsible enough to share the family problems invariably replied, 'Don't worry Mummy, she has us'' though she was seven years younger than Sangeeta, and had only seen her at the wedding.\n\nAfter they had had tea Hemant and Astha dropped in on her parents. 'I do not want them to feel they have lost a daughter'' Hemant insisted, as they walked through the colony to Astha's old house, while Astha thought how nice he was, and how lucky she.\n\n'Why do we have to drink tea twice every day?' she complained occasionally, for the pleasure of hearing Hemant say, 'And disappoint Mama and Papa, who are waiting? And when Mama makes snacks especially for us, no fears.'\n\n'Especially for you, you mean'' said Astha.\n\n'It is the same thing'' said Hemant drawing Astha's hand through his arm even more tightly.\n\nIn the kitchen, Astha's mother would hiss 'Happy?' and Astha would give the slightest non-committal nod, wanting to keep her happiness to herself. To share it or voice it might encourage its departure.\n\n*\n\nMeanwhile Hemant immersed himself in sex manuals. He hid them in his cupboard under rows of shirts.\n\n'Mummy might see'' Astha objected nervously. Her mother-in-law frequently visited their room, examined all the items, and straightened the covers on the bed.\n\n'So what?' laughed Hemant. 'We are married, what can anybody say?'\n\nThe number of sex manuals increased. All the books had graphic illustrations.\n\n'Why do you have to read these things?' Astha demanded for form's sake.\n\n'They are interesting. Look.' Hemant tried to show her, but Astha turned away her head, and Hemant did not persist. 'I will show you in other ways'' he murmured in her hair.\n\nAstha blushed and said nothing, too diffident to tell him that she had already noticed a change in his lovemaking, he was less in a hurry, and his focus had widened from the single point of her vagina.\n\nNew positions, timing the length of intercourse, variations on a theme. There seemed no end to what one could do with two bodies. At the suggestion of sexy clothes she balked.\n\n'What do you think I am? A whore?'\n\n'There is nothing to be ashamed of darling'' said Hemant caressing her. 'It is to increase married pleasure.'\n\nAstha looked at the lacy black thing he was offering her. 'What is it?' she asked suspiciously.\n\n'A teddy.'\n\n'So I am to be your teddy bear?'\n\nHemant was not interested in double meanings. 'I went to a lot of trouble to get it for you'' he said.\n\n'For me?'\n\n'Who else is the woman in my life?' asked Hemant, pushing her towards the bathroom. Thank God their room was slightly separated from her parents-in-law's bedroom, thought Astha, and they had a bathroom to themselves. Otherwise there was no way she could do these things. She locked the door and looked at herself in the mirror, clad from throat to ankle, neck to wrist. Diaphanous, lacy, and a soft pink she had all along thought this nightie made her look quite attractive. Slowly she took it off and looked at her body. She was in her hairless condition, the way Hemant liked her, with legs, arms, and underarms freshly waxed, shining smooth, with not an unsightly black stump in sight, only a series of pink bumps where the wax had pulled too hard and left its protest. She raised her arms and anxiously sniffed the wet place underneath. Hemant didn't like the smell of sweat, or vaginal fluids, he was a little squeamish in that respect, and she now washed and dusted herself with powder before turning her attention back to the thing. Single piece, lace and satin, slinky, with holes and slits, she could crumple it in one fist, its only stiffness the wires in the cups.\n\nShe put it on, and there from below her chin, a deep cleavage appeared with black laced mounds on either side, the dark nipples straining through black net hearts. She almost didn't recognise herself, with the sexual parts so emphasised. She raised her arms to take out the pins from her hair, watching as her breasts rose and thrust forwards, feeling an excitement that embarrassed her.\n\nAstha wrapped a dressing gown around herself, and slowly went into the bedroom locking the door quietly. Hemant was lying on the bed with the small bedside lamp on, his arms and chest shone brown and shapely. He kept his eyes on her, as she took off the dressing gown and walked self-consciously across to him, desire rising still higher, trying not to think of what she was wearing, what it was doing to him, to her. She sat next to him, and he grabbed her tightly encased body.\n\n'Sit on me'' he said hoarsely, pulling her on to him, twisting the little bit of lace aside.\n\nAstha sat on him, her breasts tight and forward, falling over him, over my husband, she thought, as they rocked together, while sensation took over, drowning thoughts even of husbands.\n\n*\n\nThe days passed. Astha had not imagined that sex could be such a master. Slightly ashamed, she kept hidden that she longed to dissolve herself in him, longed to be the sips of water he drank, longed to be the morsels of food he swallowed. The times he was away she was focused on one thing, the moment of their union. When he came through the door, she wanted to jump on him, tear his clothes off, thrust her nipples into his mouth, and have him charge his way through her. One with him, one with all that mattered.\n\nI haven't really lived, thought Astha, till now I did not know what life was all about.\n\nShe felt a woman of the world, the world that was covered with the film of her desire, and the fluids of their sex.\n\nA few months and dullness began to taint Astha's new life. What was she to do while waiting for Hemant to come home? Her in-laws were not demanding, for the housework they had help, and supervision, no matter how painstaking, still left her with enough free time to be restless in.\n\n'You need to work'' said her mother.\n\nThe teaching job she had never considered with interest loomed large. Now that she was married, Astha could see that its hours qualified it as the ideal job, a fact her mother was even now pointing out.\n\n'As a teacher you will earn some money, but you will only be out half the day so the home will not suffer.'\n\nAstha looked resentful. Her future suddenly seemed very pedestrian.\n\n*\n\nIt was some evenings later that Astha's mother brought up the subject with Hemant. 'She needs to be occupied, beta.'\n\n'Yes, Ma, I know'' said Hemant. 'I myself was thinking.'\n\n'What about your painting and writing?' asked her father. 'You can make use of these talents in journalism.'\n\nMother and husband expressed scepticism.\n\nAs they walked back through the colony to their own house, Hemant repeated, 'Journalists have to stay out late, they have very odd hours. We must see about a teaching job. You read quite a lot.'\n\n'I don't think that alone will equip me'' said Astha, briefly wondering whether all women were destined to be teachers or nothing.\n\nHemant laughed. 'You will probably know more than anyone'' he said.\n\n*\n\nWith the newly introduced 10+2 system, it was not difficult to get a job teaching elective subjects to classes eleven and twelve. In answer to the combined wishes of Astha's relatives one of her college teachers phoned with news of a vacancy at St Anthony's School, and if she was interested she should go and see the Principal, Mrs Dubey.\n\nAstha's in-laws approved. 'It is a good time pass.'\n\n'It's near enough. You won't have to spend much time on the road'' commented the mother.\n\nHer father merely said, 'It will do until you decide to develop yourself in other ways.'\n\nHer husband said, 'With a job you won't be so fidgety if I am a minute late.'\n\n'Oh, I am to work so you can do what you like?'\n\n'Who says I want to do what I like? It will benefit you to leave the house in the mornings. When the children come we will see whether to continue this.'\n\nAt the interview Mrs Dubey made it clear that a teacher at her school needed to show commitment to the institution, foster students' interests in extra-curricular activities, and make sure they did well in the tenth and twelfth board exams, the reputation of a school unfortunately depending on results. Astha agreed to everything and was hired. Later she thought that since the job fell into her lap, her destiny must be teaching.\n\nBeing a teacher meant the languor of her days was over. No longer did she have the luxury of leisurely brooding over her love, she had to get up early and go to work. She had exercises to correct, and lessons to prepare. She started a reading club, a writing club, a painting club, directed by the principal's suggestions and followed through with her encouragement. The peripheries of her world now stretched to include many schoolgirls. Life was shaping up nicely, with her mind and heart gainfully employed.\n\nHemant dropped her occasionally when she was getting late for morning assembly. Both families exclaimed at his devotion as a husband.\n\n*\n\nA day, as usual, with Hemant coming in late. Astha had been waiting the whole evening, and now took this opportunity to gaze at him, her soul in her eyes, the soul that she was waiting to hand over on a platter.\n\n'How are you, darling?' he asked, looking at her affectionately. 'How was your day in school?'\n\n'They have asked me to edit the school magazine'' she managed, but even those few words were difficult, so heavy was the passion weighing her down. Her tongue felt useless in her mouth, unless it was activated by his.\n\nHe sat down on the sofa, and Astha knelt to take off his shoes. She unlaced them, and pulled off his socks, gathering the day's dust in her lap. At that moment she loved Hemant so intensely, that every fetid, stale, sweaty smell that came from his foot was a further nail in the armour of her love.\n\n'How was your day?' she asked. 'Why are you so late? I have been waiting hours.'\n\n'The director called a meeting'' replied Hemant looking disgruntled.\n\n'At this time?'\n\n'What does he care? Slow, pompous, ass-licking fucker.'\n\n'What has happened now?'\n\n'The latest directives for distributing loans. Our target has been increased, and he is worried we might not make it. Then his head will be on the chopping block.'\n\nOh dear, this was not going to be a happy subject.\n\n'This percentage for cottage units, that for farmers, this for small scale units, that for backward classes, and without any security! No collateral, no third-party guarantor, because the government has to look good in the next election while we bear the losses. How can any bank function in this manner? This is what happens when you nationalise banks, constant meddling and interference.'\n\nHow long would it take for him to notice her? 'I kept thinking of you in school'' she started, but Hemant hadn't finished.\n\n'How are we encouraging any initiative, if these buggers get money for free? And how do you make sure someone is scheduled caste, for fuck's sake? Just a few months ago I had a branch officer complaining that the local bigwig was demanding a larger than usual cut for supplying the bank with certified scheduled caste people. He was falling short of his target and he had to give in. Bloody country, this is why we never progress. In America such interference would be unheard of.'\n\n'Well, this is India, dearest'' said Astha, not wanting Hemant to start on the subject of America versus India. 'This is the way things function. If you get angry, you will only harm your health. My father got blood pressure because he hated his job. Fire burns itself'' she added, a saying she had grown up with.\n\nHemant deflated. 'When I think of how my classmates are doing, how much money they are making \u2013 with an American MBA you can do anything, but there are no opportunities in this bloody country, none. Sometimes I wish I had never come back.'\n\n'Money isn't everything darling. Look, you have your family, me, our parents.'\n\n'Maybe we all emigrate, huh? Seema's husband keeps calling, he's willing to sponsor me.'\n\nLive abroad? 'Yes, let's go'' she said excitedly.\n\nHemant sighed, 'No, Az, I came for Papaji and Mummy, I have to stay. Papaji knows I am being wasted here, and he tries his best to make me happy, but still, what can he do about the job? This is not satisfying work, it is a clearing division, clear this loan, that loan, deal with union demands and government meddling, nothing is allowed to become efficient.'\n\nAstha's desire receded. She felt cold, dreary, and distanced from him. She had been waiting for him all day, thinking of their being together, but nothing of this was reciprocated. He was a criminal, destroying her anticipation, ruining her happiness.\n\nHer subservient position struck her. She had no business kneeling, taking off his shoes, pulling off his socks, feeling ecstatic about the smell of his feet.\n\n'What's the matter, darling?' said Hemant as her hands stopped moving. He reached out and ran his fingers through her hair. 'Leave my shoes, I'll do it.'\n\nHe got up, put them away, and catching her by the elbow sat her down next to him. Poor man, thought Astha softening, he must have had a hard day in the office, was that anything to mind? She must make his home a haven for him, not a place of recrimination.\n\n'So what were you saying about school?' he asked, passing his hand down her back, gently pressing the dividing line between her haunches.\n\nAstha sidled closer to him, and the pressure became a little firmer. 'I kept thinking of you'' she whispered. 'I missed you every minute.'\n\n'Baby'' he murmured, accepting this as his due. 'And school, how was that?'\n\n'Well, they have asked me to help with the school magazine, as I am the teacher for the senior elective English classes. And I thought, why not?'\n\n'Do they know you write?'\n\n'Of course not. Anybody with reasonable English is enough for this job. My class XI girls got really excited, they want to organise a creative writing competition. We can publish the best poems and stories, maybe even send them to the children's page in the newspaper.'\n\nHemant wasn't really listening. Astha stopped talking about creative writing as he got up to lock the door.\n\n'They are waiting'' objected Astha.\n\n'Just a quick one'' said Hemant.\n\n'They will know what we are doing'' said Astha, already imagining what was to come, even if it was a quick one.\n\n'Let them know. We are married.'\n\nAstha lay back, aware of every inch of her skin, aware of every thread she wore, now about to be dislodged. The day, with its petty vexations flowed away from her. This, what was going to happen, was the central thing in her life.\n\nThe last year of Astha's father's service drew to a close. They would have to leave their house soon. Hemant threw himself into their plans, politely suppressing his surprise at their unworldliness.\n\n'Az'' he said frequently to his wife, after visiting his in-laws, 'how come Papa didn't plan more for his retirement?'\n\n'He was planning'' said Astha hopelessly, 'in fact they were always planning.'\n\n'Then, what happened?'\n\n'They kept trying to buy, but it was always too expensive. Then this housing society thing came up and they were allotted land trans Jamuna. They thought once the bridge was built and prices went up, they could sell the plot and buy a small flat this side.'\n\nTo Astha now, this seemed like not very much planning.\n\n'As an investment, Az, this is not good strategy'' said Hemant, banker. 'The bridge is nowhere in sight, you can't depend on government promises.'\n\n'Well, I don't know, that is what they did'' said Astha pettishly.\n\n'They can still live on it, though. People are building, after all. Then when prices rise, their property will be worth even more.'\n\n'How can they? It's still so undeveloped it's not safe. No infrastructure, no nothing. You should see it, it's just a patch of mud. In one of the nearby colonies, the owner was alone in the house when dacoits broke in, stole everything, and beat him till he almost died. You want this to happen to them?' Astha's voice rose slightly.\n\n'Now, now, baby, don't get upset, of course they shouldn't go if it is not safe. We'll help them all we can.'\n\n'OK'' said the wife, feeling momentarily soothed, pushing away the knowledge that it is one thing to offer help, another to give it, still another to take it, and that her father was a very proud man.\n\n*\n\nAstha's father retired, and in six months they had to vacate the house in Lodhi colony. It had been central and inexpensive, the rent 10 per cent of the father's salary. Now they were thrown to the outside world. While the mother was at school, the father trudged around various colonies with property dealers. The private colonies near Lodhi Colony were all too posh, there was no question of trying Sundar Nagar, Golf Links, or even Defence Colony, where army officials had bought plots for a song not so many years before. Finally he found a small two-room apartment in Jangpura. Its advantage was a large terrace, its disadvantage that it was on the first floor.\n\n'You will have to go up and down everyday'' said Astha, 'are you sure your health can take it?'\n\n'Climbing stairs is good exercise'' said the father.\n\n'When you have high blood pressure?'\n\n'I will be all right.'\n\nWhat choice did they have? The flat was comparatively cheap, the location comparatively central. The landlord was kind, only demanding three months' rent in advance, not insisting on a company lease.\n\n*\n\nDismantling the house in which they had lived for fifteen years was not easy. They took the furniture the new flat could accommodate, the rest they sold. The father's books were put in boxes which were then placed so as to make two beds and a living-room divan. They would still be with him, that knowledge would have to replace the pleasure of seeing them every day. The bed linen, the small pieces of bric-a-brac that Astha's mother had stored through the years were given to the daughter. 'I don't need them anymore'' she said.\n\n'But these are brand new'' said Astha looking at the carefully preserved things, wrapped in soft, old saris. 'Why don't you use them?'\n\n'No, no, I do not need'' insisted the mother.\n\nHemant helped them to move. 'I don't like asking him to do so much for us, beti'' said the father.\n\n'He is your son-in-law, Papa. It is all right'' said Astha.\n\nAgain they had no choice.\n\n*\n\nIn the small flat, near the highway, noisy, confined, far from tree and grass, alone for half the day while his wife was at work, eating things he was not supposed to, the father wandered through his life, looking at what was left behind and what lay ahead, and decided there was no use living. Other people decide that with less success.\n\nThey had been in the new flat a little over a year when one evening after dinner he complained of a slight chest pain. That night he died in his sleep. Through the period of shock and mourning, Astha and her mother clung to each other.\n\n'It was the move'' the mother kept sobbing. 'He was never the same after he retired.'\n\n'He was a saint'' said the relatives. 'Never liking to trouble others.'\n\n'I kept telling him, do not strain, do not exert yourself, but no. He was never careful. And now he has left me and gone.'\n\n'You have me, Ma'' said Hemant.\n\n'Yes, Ma. We are all with you.'\n\nAs consolation to the widow, now all alone, the relatives said, thank God he saw his child settled, he will rest in peace.\n\n*\n\nIn the months that followed the father's death, the mother became listless and withdrawn. The evenings Astha spent with her she would desperately try and cheer her up.\n\n'You are still young, Ma, still working. Think of all the things you can do.'\n\n'You don't worry about me, beti'' said the mother dully.\n\n'You can travel, you can do social work, you can do something for the children of the poor, you always said you wanted to help other people. Now you can.'\n\n'Han, beti. You don't worry about me.'\n\n'But I do worry. Why don't you come and live with me?'\n\n'You live with your in-laws, and besides where is the room in these government flats.'\n\nThat was true enough.\n\nAstha tried to interest Hemant in the problem of her mother. He was a good son-in-law, everybody said so, his own parents in particular, closely echoed by the mother-in-law herself. If there was an illness he would call the doctor, if she needed money he would offer it, if she needed help in shifting he would provide it. But appeals beyond this irritated and annoyed him.\n\n*\n\nThen the mother met a swami. She informed her daughter of this casually.\n\n'A swami?' repeated Astha, puzzled. 'How did you meet him?'\n\n'One of the teachers in school took me. Often she has mentioned him, but when your father was alive I never felt the need for anything more in my life. Also he was suspicious of this kind of thing, your father always thought he knew best.'\n\n'With reason, Ma. Swamis are known to take advantage of women, especially widows'' said Astha.\n\nHer mother ignored this. 'Why don't you come?' she went on. 'He lectures on the _Gita_ at Gandhi Bhavan. He teaches you how to accept things, how to look inside yourself, how to deal with your wants and desires. There are lots of young girls there.'\n\n'I don't want to look inside myself'' said Astha.\n\n'Well, I am learning a lot from him. Through him I understand the _Gita,_ it is something I have wanted to do all my life.'\n\n'Really? How come I didn't know?'\n\n'Where was the time or place to say I want this or that?'\n\n'And now you have a swami? Is that what you wanted time for?'\n\nAstha's mother looked offended. 'Why don't you come and see before you start your criticising?'\n\n*\n\nThat evening Astha said to Hemant, 'Ma has found some swami. She wants me to go to him and look inside myself.'\n\n'Rubbish. These people just try and sound clever.'\n\n'That is what I said.'\n\n'Who is this man?'\n\n'I don't know.'\n\nHemant looked alert. 'One has to be careful around swamis'' he said. 'Thank God I am handling her money.'\n\n'I know'' said Astha, her wretchedness increasing. 'But what can I do?'\n\n'Somebody is putting ideas into her head. People think old women are easy targets.'\n\n'She doesn't listen.'\n\n'Don't worry sweetheart, once we have a child, she will forget all this nonsense. There will be a new interest in her life.'\n\nAstha smiled her agreement.\n\nLoving Hemant as she did, Astha longed to get pregnant. During sex she imagined his seed spurting into her womb; later she would gather his wet shrivelled penis, adoring it strong, thick and hot, or wet, limp and woebegone. 'I want to have your baby'' she would murmur.\n\n'You can't be so old fashioned'' remonstrated the progressive husband. 'This is like villagers, marry, impregnate wife, pack of children. No, no sweetheart, we need to be by ourselves. Time enough for these responsibilities later. With a young wife one can afford to wait.'\n\nAstha looked at him in admiration. Everything about him was so masculine, his decisiveness, his hairy blunt fingers, his tall heavy set figure, his muscled limbs, his moustache that tickled, the bitter tobacco taste from his tongue.\n\n'These ideas are all from America'' said his parents, refusing to see the value of bonding time for the young couple. They had married, now they should get on with it.\n\n*\n\nIt was two years before Hemant relented, two years before Astha could stop using birth control, two years before his seed found its home.\n\nAstha and Hemant drove to Jangpura on their weekly visit, full of the good news.\n\n'Ma'' said Hemant, 'You are going to be a nani.'\n\nTears filled the mother's eyes. 'If only he had been here'' she said.\n\nAstha thought of her father and felt sad. He had sent her forth, and then left, duty done.\n\n'Ma, this is a time to celebrate'' reproached Hemant.\n\n'Beta, you are right. May it be a boy, and carry your name for ever. A great son of a great father.'\n\nAstha thought her mother was overdoing it.\n\n'But Ma, I want a daughter'' said Hemant.\n\n'That's true, Ma'' repeated Astha, 'He wants a girl.'\n\n'In America there is no difference between boys and girls. How can this country get anywhere if we go on treating our women this way?'\n\nThere was no mistaking the admiration in both women's eyes.\n\n*\n\nAstha enjoyed every aspect of her pregnancy. As it advanced, she became more and more bucolic. Teaching was an effort, and she had no energy for any extra activity. At home she slept most of the time.\n\nHemant adored what was happening to her. 'My wife is becoming a woman before my very eyes'' he said passing his hands over her belly, large and full, over her breasts, certainly larger and fuller than they had ever been. 'I hope they remain like this'' he said holding them possessively.\n\n'What'll happen if they don't?'\n\n'Another baby, what else?'\n\n'You'll get tired earning for all these children you plan to produce.'\n\n'With you looking like this, never'' declared Hemant passionately. 'A real woman rather than a girl.'\n\nAstha had heard men were revolted by the way women looked when they were pregnant, but not Hemant. He loved touching her belly and breasts, her breasts especially, sucking on them experimentally, drawing a little milk when he sucked long enough.\n\n'It's very sweet'' he said with surprise.\n\n'It's called colustrum'' she informed him knowledgeably. 'It comes for the first three days, and is full of nutrients to prevent the baby from getting sick.'\n\nHemant smiled, 'How full of information my wife is'' he said. 'Where did you find that out from?'\n\n'Books.'\n\n'Our baby will be the best looked after baby there is'' said Hemant, caressing the taut stomach, gently stroking the raised belly-button, following the linea niger down to her pubic hair with his fingers, before inserting them into her vagina.\n\n*\n\nAnuradha. Born in March, after fifteen hours of labour at a private nursing home. Six pounds, eight ounces, nineteen inches. Long delicate nails, a head of thick black hair, pink, wrinkled, foetus like.\n\n'Oh'' chorused the new grandparents. 'Just like Hemant. Same nose and forehead.'\n\n'Such a straight little nose'' detailed Astha's mother, 'such big eyes. Handsome like her father. Girls who look like their fathers are lucky.'\n\nHemant leaned over the tiny baby and kissed her cone-like dome enthusiastically. Astha thought with amazement, he doesn't see through my mother's flattery, before tightening her own hold on the child.\n\n*\n\nThe first time Anuradha put her mouth to her mother's breast and started pulling, Astha was astonished. Hemant's own pullings were nothing in comparison, mild as the winter sun. Anuradha meant business. She tugged ferociously, and Astha's womb in response obligingly contracted, spurting out blood into the pad she wore.\n\nA month of wet before the blood ceased to come, before the womb had contracted all it was going to. A time of swollen aching breasts charged with milk that dribbled constantly, soaking the towel inside her nursing bra, staining her clothes, a time when she had to beg Hemant to drink from them to relieve the pressure.\n\nHemant always willingly obliged, putting a gentle mouth to the tight breast with its blue veins now clearly marked. 'It's very watery'' he said the first time, surprised once more.\n\n'Is it?' asked Astha, 'Let me see.' She cupped a hand under her nipple to catch a drop of the still-oozing milk, and tasted it. It was sweet and watery, bluish-white in colour. 'I guess we are used to cow's milk, which has more fat. That is meant for calves, this is meant for humans'' she explained pedantically, her new-found knowledge still burgeoning in her mind.\n\nAnuradha yawned in her sleep, and made wuffling baby sounds while both parents gazed at the little variations in her movements, with a joy that spilled into each other.\n\n*\n\n'Darling'' said Hemant one night.\n\n'What?' said Astha preoccupiedly. Anuradha was six months old now, and had just begun to sleep through the night. Astha was looking forward to sleeping through the night too, something she felt she had never appreciated before.\n\n'Where's the teddy?'\n\n'What on earth for?'\n\n'I wonder how it looks. It's been a long time since you tried it on.'\n\n'It's been a long time since I had a figure'' retorted Astha.\n\n'You have a figure'' said Hemant, gazing upon his wife's fullness appreciatively. 'Go on, try it'' he urged, pushing her stubborn form towards the bathroom.\n\n'No, no, I don't want to'' expostulated Astha.\n\n'Why? You think because we've had a baby, our life is over. I haven't touched you in months.'\n\n'I know, I'm sorry. Soon it'll stop hurting. And our life isn't over, if by that you mean sex, but it's not necessary to have sex with that thing on, is it? What'll happen to Anu's subconscious? She might grow up with a problem.'\n\n'Look at her. She's totally unconscious. How do you think half the country fucks? You think they have separate rooms?'\n\nAstha knew they didn't. She didn't like the leer on Hemant's face, but she could think of no more reasons for objecting. What could she say? That she was too old? She was twenty-five. That the early days of their marriage were over? They had been married three years. That Hemant should want her without her prancing around in a tight black cut-away garment? But she had worn it before, she had been turned on herself, wasn't she being rather prudish now? She threw a glance at the baby, maybe she was waking? But no, Anuradha slept peacefully, while her mother made her way slowly to the drawer where the teddy was hidden en route to the bathroom.\n\nShe pulled it on. Her breasts spilled over the top, and looked more voluptuous than they were. That was all very well, thought Astha, but the sight of her stomach bulging through the shiny stretchy lace see-through stuff, that sight was not pretty. Also she hadn't been so regular about her waxing, there was hair growing all over her limbs.\n\nThis'll put him off teddies for ever, thought Astha, surveying herself in the mirror, a little regretful that her body should have this deterrent effect. Finally she wrapped his dressing gown around her waist and emerged complaining, it's so tight, look darling it doesn't fit, I'll never be my old self again.\n\nHemant saw her point. The teddy was put away and never mentioned again.\n\n'Once we build our new house, we can start planning for our next child.'\n\n'Um'' said Astha absently, handing her husband the baby oil. Hemant poured a little into his palm and began carefully rubbing it on his daughter, her bath part of his Sunday morning ritual. He insisted on doing this, ideas about fatherhood are so antiquated in India.\n\n'I want to have my son soon'' declared Hemant, looking emotional and manly at the same time. 'I want to be as much a part of his life as Papaji is of mine.'\n\n'How do you know we will have a son?' asked Astha, feeling a little scared.\n\n'Of course we will have a son, and if we don't we needn't stop at two.'\n\nAstha silently took the oil bottle from him and closed it.\n\n'Is the water ready?'\n\nHis wife hastily tested the water in the bath-set crammed into a corner of the bedroom. 'Yes'' she said.\n\nThe father gently lowered his daughter into the water, while the mother stood ready with the shampoo, rubber toy, and soft towel.\n\nAfter the bath Astha called the servant to mop the floor and throw out the water while she hung the towel, disposed of the oil, comb, powder, toy, dirty diaper and night clothes. She then settled down to nurse the baby while Hemant went on discussing their house and their future.\n\n'Hemant?' said Astha after a while.\n\n'Yes?' replied Hemant engrossed in the soft feet and tiny legs of his child.\n\n'I thought these things didn't matter to you. What if we don't have a boy?'\n\n'Of course they don't matter to me. I was so pleased Anu was a girl. But that doesn't mean we should not try for a boy. I am the only son.'\n\n'It is not in our hands, at least not in mine. It is the man's chromosome that decides the sex, and with two sisters in your family, it may be a girl. I have read about these things.'\n\n'You are always reading'' said Hemant coldly.\n\n'I am sorry. Does it bother you?'\n\n'It fills your head with unnecessary ideas. Let us first not have a son and then we will see. Keep it simple. All right?'\n\nAstha looked dissatisfied but could think of nothing to say.\n\nIn the family she had married into Astha had ample opportunity to witness how the business of building a house and planning for retirement should be gone about. Papaji's ministry's housing society, Papaji's rank, Papaji's draw had achieved for them 633 square yards in Vasant Vihar. And for the next ten years the family watched in amazement, satisfaction, and smugness the rate at which their initial investment of twenty thousand rupees multiplied five hundred times over.\n\nAstha's marriage entitled her to the same emotions. This is what my parents hoped would happen to them, she thought wistfully every time the latest price of their plot was discussed, and it was discussed many times.\n\nVasant Vihar too was once wilderness, home to rabbits, peacocks and deer, but by the late seventies almost a third of it was under construction, a boom which the Vadera family now joined.\n\n*\n\nFor the plans Papaji contacted the chief architect of the New Delhi Municipal Corporation, who enjoyed the same secretary level status he did. A senior teacher of the Delhi School of Planning and Architecture was recommended, drawings were made, their relative living convenience minutely examined.\n\nThe house was going to be double storied, the ground floor for Hemant and Astha, the one above for Papaji and Mummy. Each floor would have a drawing-dining, kitchen, two bedrooms with attached baths, and a small study to double as a guest room. In the centre, overlooking a patch of lawn running on the side of the house, would be an open informal area where the family could congregate. There would be one large verandah beyond the drawing-dining, and small balconies outside the bedrooms. On the roof would be the servant's quarters.\n\n*\n\nA puja was done at the site, and the building started. Steel and cement could only be obtained on quotas, and construction on the house lasted nearly two years, despite Papaji's contacts, as thirty tons of rolled steel bars and thousands of bags of cement were released in dribs and drabs by the concerned ministry.\n\nPeriodically Hemant and Papaji would go shopping along with the contractor. To GB Road for cast iron and galvanised iron pipes, toilets, taps, stainless steel and ceramic sinks, wall tiles and marble chips; to Bhagirath Palace for mild steel conduit pipes, electrical cables for light and power, switch boxes, switches, fans, hinges, door locks and door handles; to Paharganj for wood and plywood; and last of all to Kotla for glass and paint, Snowcem for the outside, oil bound distemper for the inside, lime wash for the ceilings.\n\nTwo to three times a week Hemant visited the site, he was a junior officer, and he didn't have the pressures Papaji did. Sometimes Astha accompanied him, audience to Hemant's sense of himself as the child of fortune. 'This \u2013 this'' he said waving his hand at their plot, 'this is worth over a crore today.'\n\n'A crore?' breathed Astha. 'So much.' And she warmed with the pleasure of being part of a family that was in tune with the ways of the world. Now and for ever she would be looked after.\n\n*\n\nTo avoid death duties, the five Vaderas were registered as co-owners, with a letter of intention signed amongst them as to future rights. Hemant was to get the ground floor; Seema, who had contributed dollars towards the construction, was to get the first floor; and Sangeeta, who had contributed nothing, was to get the terrace, which allowed a built up area of 25 per cent. Should either of the sisters wish to sell they had to give their brother first offer.\n\nAfter the house was built, it was given on rent to an embassy, at over a hundred times the rate they paid for their Lodhi Colony government accommodation. Astha's mother listened to the details of the increase in the family finances with glistening eyes, sighing heavily, blessing her daughter, remembering her departed husband, a very simple man, with no sense of this world.\n\nThe two-year excitement and absorption of building a house over, Hemant began to get bored. On his way home from work he took to frequenting the club where, swimming, playing tennis or drinking, he met men like himself.\n\nThey were a new breed, these men. Their fathers had opted for the security and prestige of the civil services, but the sons wanted challenge and money. Educated abroad, their idealism had been exercised in their choice to return to India, now they wanted tangible returns for that sacrifice. Certainly Hemant did. He decided to try his hand at business.\n\n'Isn't it terribly risky?' asked Astha nervously. 'Business is full of bribes and corruption, headache and uncertainty.'\n\n' Az, this is the thinking of the past. Maybe a government job was all right for our parents, they wanted to serve their country after Independence. And perhaps it once was the place where you could make a difference, but no longer. The inertia, red tape and small-mindedness kills you. Now people sit on their asses and push files around all day. As an entrepreneur you can see the result of what you are doing. And it generates work.'\n\n'But we are comfortably off, you have a secure position, your work is not demanding. Even now, we spend so little time together, what will it be like with longer hours?'\n\n'I miss you too'' said Hemant absently. 'But I am not starting the business immediately. I can get a loan more easily if I am at the bank, and the company, my dear, will be registered in your name.'\n\n'And what will I be doing?' inquired Astha.\n\n'Making TVs.'\n\n'TVs? What market is there for TVs? All you get are rubbishy programmes, like _Krishi_ _Darshan,_ _Chitrahaar,_ and half an old black and white Hindi movie on Saturday with the other half on Sunday.'\n\n'You wait, Az, TVs are the thing of the future. In developed countries, TV has taken over the culture, and here too, when colour comes to India... ' He paused and, stirred by his vision of the future, put his arm around his wife.\n\nHis wife had less imagination. 'What will happen?' she asked.\n\n'Do you know how much profit margin there is on a colour TV?'\n\n'No, I don't know, and what's the point, there is no colour, even if we do make the sets.'\n\n'You wait and see.'\n\nWell, thought Astha, at least we have the security of the house if anything goes wrong.\n\n*\n\nHemant applied for a plot of land with the Uttar Pradesh Industrial Development Corporation, and he was allotted one in the ambitiously called 'Electronic City' of Noida, Sector 16. For this as yet undeveloped piece of property he had to pay nine lakhs in installments, with ten per cent down payment. His connections in his bank made applying for a loan easy, a few trips to Lucknow, and the loan was routed through the Noida branch. He registered his wife's unit as a small-scale industry, something that Papaji's position in the commerce ministry facilitated.\n\nAlong with other erstwhile factory owners, Hemant waited for Noida to develop, in the meantime hiring a factory. He made his parents board members, and started his unit with a thousand black and white TVs a month. They had the standard 20-inch screen, sold at 1,850 rupees a set, with a profit margin of 20 per cent.\n\nAll this took a year to accomplish. Hemant now left the house every morning at seven to first visit the factory, and then make the long drive to Parliament Street. 'My family comes first'' he would say, as he juggled factory, bank and home.\n\nAstha watched Hemant in his new avatar and felt moved by his grasp of the rules of getting on, by his ability to exploit situations rather than be defeated by them. Because he was her husband this meant that she too would not fall between the wide cracks of the world like her parents had done.\n\nSomewhere along the way Hemant's attitude to Astha changed. She told herself it was only slightly, but it oppressed her. Occasionally she tried addressing this directly.\n\n'Hemant, why is it that we never talk anymore?'\n\n'We talk all the time.'\n\n'About the business, the house, or Anuradha. Not about ourselves. Like we did before.'\n\n'Grow up, Az, one can't be courting for ever.'\n\n'Is it courting to be interested in the other person? Their feelings?'\n\n'Why are you so childish? I work hard all day, and when I come home I want to relax. If you are feeling something, tell me. I have no time for all these games.'\n\n'I want to be close to you, have a better relationship\u2014' faltered Astha, knowing she had lost the argument before she had been able to define its parameters.\n\n'There is nothing wrong with our relationship.'\n\n'Are you saying there is something wrong with me?'\n\n'You said it, not I.'\n\n'But I'm not happy, so how can you...' She bit back words that might seem to indicate some insensitivity on his part.\n\n'You think too much, that is the trouble.'\n\nAstha stared at him nonplussed. 'I love you'' she said lamely, but she meant something else.\n\n'I know, baby, I know'' said Hemant, drawing her to himself, caressing her. 'Maybe we should go out together more? Would you like that?'\n\n'What about Anu? I don't like leaving her with Mummy so much. She looks after her when I am at school as it is.'\n\n'We'll take her, you are the one with all the scruples. Come on, darling.' He slipped his hand under her sari, undid the first two hooks of her blouse and slid his hand over her breasts.\n\n'Poor little things'' he cooed, 'Have I been neglecting them?'\n\n'It's not that'' murmured Astha.\n\n'Cheer up, baby. Make it nice for me to be with you.'\n\nBaby. That is how he liked her. The look on his face became focused as he pulled her sari palla away and yanked at the rest of the hooks on her blouse, drawing it down from her shoulders and arms. Now he would bury his face in her breasts, pressing them against himself from either side, suck on her nipples, and they could both be babies together.\n\nShe found this soothing, and later scolded herself for being so demanding. Hemant was busy, Hemant was building their future, she had to be adjusting, that was what marriage was all about.\n\nWhen Anuradha was four, Papaji retired. The tenants left, the family moved into their Vasant Vihar house, and Astha conceived again.\n\n'God willing it will be a boy'' said her mother. 'I have asked swamiji's advice as to what offerings to make.'\n\n'Nonsense, Ma'' retorted Astha uneasily. 'These people are not like that.'\n\n'You are still such an innocent. What people say and what they do are two different things. Besides why is Hemant working so hard? For whom, if not his son?'\n\n'It doesn't matter to Hemant'' said Astha valiantly.\n\n'I hope for your sake you are right.'\n\n*\n\nA few nights later. Hemant laughing, 'Mummy is so sweet.'\n\nHemant often found the things his mother said or did sweet, so Astha paid not much attention. 'She is hiring a pundit to come every day and do some special pujas.'\n\n'Why?'\n\n'To ensure a grandson.'\n\n'But puja may not make a difference, it may still be another granddaughter'' objected Astha in alarm.\n\n'Don't worry, sweetheart, then we will try again, it's perfectly all right. Why do you get so tense for nothing?'\n\n'But Hem, I do not wish to go on trying and trying until we get a son. It's very difficult with the teaching as it is.'\n\n'Oh-ho, what is there in teaching? Hardly a serious job, you just go, talk to some children about poems and stories, organise a few clubs, and come back. If you do feel it is important, all the more reason not to mind if Mummy does some puja. Who knows it may yield good results?'\n\nBut Astha did get worked up, she couldn't help it. She tried to stay calm for the baby's sake, she took to meditation, she concentrated on peaceful thoughts. But she was not allowed to forget that everybody, her colleagues, her in-laws, her husband's friends' wives, her mother, the cook, the gardener and the part-time help all had an opinion about her baby's gender, and that almost universal opinion was that it would be a son and heir.\n\n'Baby, it's you they want to be a boy'' Astha would whisper sometimes, 'are you a boy or a girl? I'll love you no matter what'' and she soothed the foetus she imagined so troubled with her troubled hands.\n\n*\n\nWhen Astha's son was finally born she felt a gratitude as profound as it was shamed.\n\n'The family is complete at last'' said Astha's mother piously, feeling her own contribution.\n\nHemant's mother agreed, too happy in the birth of her grandson, carrier of the line, the seed, the name, to respond with her usual reserve to someone she increasingly felt was her social inferior.\n\nThe naming ceremony of the boy was carried out on a much grander scale than that of Anuradha's. Caterers were called, and they came early in the morning, setting up their fires in the narrow driveway. The priests arrived for an elaborate puja and havan. The letter taken out for the baby's name was 'h'. An auspicious sign, same letter as his father said everybody, and he was christened Himanshu.\n\nAstha was given gold jewellery and a new sari. Anuradha and the child's aunts were given gold necklaces. The newborn was given gold guineas.\n\nAstha was officially declared the mother of a son. Her status rose, and she pushed from her mind thoughts of what might have happened had she been unable to do her duty.\n\n*\n\nHimanshu was two months old when he raised his wobbly head from his mother's chest to smile at her, wet pink lips stretched over little toothless gums. Astha thought, he recognises me, and she smiled back, silently, across her chest, this human being and her connected. The baby, trying out the strength of his neck, began to laugh, which made Astha laugh too. Happiness flowed through her like a river, lapping at her mind. She never forgot this first exchange, it lived on in her memory, a link between a male and her that was joyous, simple, and unproblematic. So what if it was with her two-month son.\n\nAstha often looked at her family, husband, daughter, son. She had them all. She was fulfilled. Her in-laws frequently commented, 'Woman is earth'' and it is true she felt bounteous, her life one of giving and receiving, surrounded by plenty. Visitors to the house would say, 'A mother's love' and then trail off, words collapsing into significant silence, which in turn washed over Astha and made her feel that she had partaken of the archetypal experiences marked out for the female race. \nChapter III\n\nBetween Anuradha's birth and Himanshu's, Hemant changed from being an all-American father to being an all-Indian one.\n\nAfter he came home the last thing he wished to bother about was taking care of a child.\n\n'It's your job'' he said.\n\n'That's not what you thought when we had Anu'' replied his wife. 'I can't do everything myself. It's tiring.'\n\nIt was also boring, though this was not acknowledged.\n\n'It's woman's work'' said Hemant firmly. 'Hire somebody to help you, or quit your job.'\n\n'This is our son, the one you wanted so much. It's nice if we look after him together.'\n\n'Send him up to Mummy if you can't manage.'\n\nAstha was struck dumb. Were Mummy and he interchangeable?\n\n'And'' continued Hemant, 'my son is going to be very lucky for us.'\n\n'Oh Hemant, how?' asked Astha with an effort that wasn't noticed.\n\n'Wait and see.'\n\nHemant had invested in the future with his TV project, and was now about to witness the fruits of his foresight. Three months before the Asiad of 1982, the Minister for Information and Broadcasting declared that India would go colour: we have a certain dignity to uphold, an image to project. The games will be beamed internationally, conveying the pomp and splendour, hopes and aspirations of a developing nation. How can all this be done in black and white, when colour technology is prevalent worldwide? It is a question of marching with the times.\n\nThe Left protested: such a priority is elitist, false and a waste of precious foreign exchange. When the nation is still poor and backward, when electricity, water, roads, education and basic health care have yet to reach hundreds of villages, why should we develop a totally useless technology that will neither feed nor clothe?\n\nBut whether the technology was useless or not, whether it would help the nation or not, it was there to stay. Hemant now needed to travel to South East Asia; the indigenous black and white TV was possible to piece together locally but colour expertise was still not available in India. He resigned from the bank and security, to devote himself full-time to risk and money.\n\nHemant placed his first order in South Korea, for twenty thousand colour TV kits. Along with the order came a manager to train the workers. Local contribution involved the assembly of the TVs, the wooden cabinet, testing, selling and service. The final product was advertised as manufactured under foreign supervision, long after the initial foreigner had left.\n\nFour times a year Hemant travelled. The glamour of international references entered the house, as he flew to South Korea and Japan looking for the best deals. He always went alone, always made sure his trips included at least two weekends, which he claimed he needed in order to establish personal contacts. He invariably came back in great good humour, with generous presents for everyone: perfume, chocolate, sweaters, jeans, toys, Japanese dolls, games for the children, underwear for Astha, toiletries, soaps, creams, shampoo, kitchen and electronic equipment. Gradually their house acquired the gloss of a house with money.\n\n*\n\nAstha was now virtually a single mother. Beleaguered by job, small children and house, she sometimes toyed with the idea of resigning from school, but between her marriage and the birth of her children, she too had changed from being a woman who only wanted love, to a woman who valued independence. Besides there was the pleasure of interacting with minds instead of needs.\n\nAt school she had grown to be her principal's right-hand woman, appreciated and valued for one tenth the work she did at home, and paid for it too. Her salary meant she didn't have to ask Hemant for every little rupee she spent. With two children, family obligations, entertainment and holiday costs, the travelling involved in a new business, the uncertainty of business itself, rising prices, she knew Hemant would prefer her to bear her small expenses herself. As it was he spent enough on her clothes and jewellery that she always looked well turned out.\n\nAnd so the once looked-down-upon job had become dear. She couldn't leave it. Nor could she go on relying too much on her mother-in-law for help with the children, it led to remarks from mother-in-law to Hemant to Astha which left her seething with anger and resentment.\n\n*\n\nThus began the search for a maid. A succession of women filed through their flat, but they either came with large families, or they had insufficient references, or they stole, or they were lazy.\n\nHemant felt Astha was guilty of mismanagement, it could surely not be that no ayah was right? After all he managed a factory with four hundred workers.\n\n'Why can't you train these servants properly?' he demanded.\n\n'I do try'' she said, not liking to acknowledge how inadequate she felt with all of them. 'I was all right with Bahadur, (their cook) and the two part-timers.' (To wash clothes, clean the dishes, swab the floor, and dust the rooms.)\n\n'Then don't hire one.'\n\n'I need someone to help me'' said Astha bitterly, wondering how much her husband really knew of her life.\n\n'Have all the help you want'' went on Hemant carelessly, 'only learn how to manage it.'\n\nThe search continued till Bahadur, their cook, went home to Nepal on annual leave, and brought back a widow.\n\n'My sister'' he said, introducing her laconically. 'See if you like her.'\n\nAstha looked at the woman. She had a broad flat face, slitted eyes and a wooden expression.\n\n'Have you done domestic work before?' asked Astha, beginning with the standard questions, while wondering whether this woman was Bahadur's blood sister, cousin sister or village sister, and whether they were sleeping together.\n\n'Mala knows everything'' said Bahadur interrupting. 'Try her.' There was something about the woman's straight gaze that appealed, and she was employed. Mala's appeal grew when Astha discovered how quick and capable she was. She was fast, she was clean, she needed to be told nothing twice. When Astha and Hemant went out she made sure the children had their meals on time, and that they were in bed by nine. She even made sure Anuradha finished her homework, and this while being illiterate.\n\nMala had some bad qualities. She stole food and clothes, she answered back, she took her time coming from her quarter upstairs, she became deaf when it suited her, and on Bahadur's days off she tended to develop illnesses from which she did not fully recover till he came back.\n\nUnfortunately for Astha this usually happened on weekends, when Hemant was around.\n\n'I am going to fire that bloody woman'' ranted Hemant the last time Mala had fever.\n\n'She can't help it'' defended Astha.\n\n'She is shamming.'\n\n'How can we prove that?'\n\n'She is like this because you encourage her.'\n\n'How do I encourage her?'\n\n'I saw her going out with Bahadur.'\n\n'He said he was taking her to the doctor. Do you want me to take her to the doctor instead?'\n\n'She thinks she can get away with anything.'\n\n'I'm sure shell be all right soon.'\n\n'Where's Mala?' whined Himanshu, who was listening.\n\n'See? You make the children too dependent on her.'\n\n'She helps look after them, it's natural they should like her.'\n\n'You treat her as though she was one of the family. You have to know how to handle servants.'\n\n'I can't behave in any other way.'\n\n'She's shamming'' Himanshu piped up insistently, wanting to be heard.\n\n'She's sick darling, don't you get sick sometimes?' said Astha.\n\nIt was in this two children, husband, servants, job scenario that Astha started to have headaches. Years after she would remember the first time it happened, thinking that as a herald of what was to come, it might have announced its arrival in her life a little more gently, allowing her time to get used to this pain in her forehead, this throbbing at her temples, this stretching of the skin around her eyes.\n\nShe had laid the table for dinner, and they had all sat down to eat when she discovered she had forgotten the water. She rose from her chair, and in that moment, between getting up and standing, in the moment that hung between a bent body and a straightened back, it appeared. Just above her nose, at the inner corner of her eyebrow. She pressed the spot, and the pain promptly shot off in neat lines across her eye socket. It will disappear as suddenly as it came, she thought, carefully pouring the water into everybody's glasses.\n\nThe heaviness in her head increased as she ate. If she didn't lie down soon, she might fall headlong into her plate, banging herself against the table, startling the family.\n\n'I'm going to lie down'' she managed.\n\n'Are you ill?' asked the husband, looking at his wife's wrinkled eyebrows and drawn face.\n\n'I'll be all right. Just a little headache.'\n\nHow the children were put to bed, when Hemant came to the room, Astha did not know. Through the night the pain grew worse. Nausea came upon her, she could no longer stay lying. She got up and sat outside, maybe the cooler air would help. It didn't.\n\nAs she bent to retch in the toilet, she hoped that now she would feel better. But though the queasy feeling gradually subsided, the throbbing was still there. Her limbs were shaking, she had to lie down again. Sometimes it seemed, if she lay on the hurting side, that felt better, sometimes she felt that no, the other side was better, and she kept gingerly turning her head trying to pin the point of meagre comfort.\n\nGradually towards morning, when the sky lightened, and the pain began to recede, she fell asleep.\n\nThe next day, the whole world seemed new. She was still in one piece, that terrible thing had gone. Her head felt delicate, it had gone through some bad times and needed to be treated gently.\n\n'Are you all right now?' asked Hemant, looking concerned.\n\n'Yes, I'm better'' she replied.\n\n'What happened to you?'\n\n'I don't know.'\n\nShe took leave from school and sat around the whole day, not using her eyes to read, not using her mind to think. She dusted and tidied, mindless labour that soothed and kept her busy. She hoped that what had happened to her the night before was a one time thing.\n\n*\n\nSoon it became clear that her headaches had arrived to stay. Stress made them worse, going out in the sun made them worse, sleeping too little, too late made them worse, eating the wrong kinds of food made them worse. Slowly her life changed to accommodate her headaches. She learned to dread each small twinge, was it going to be bad or medium? Maybe she was tired, should she lie down and rest? Or maybe it was anxiety, should she meditate, shut her eyes, ignore the throbbing, clear her mind of images, and focus on a spot of light between her eyebrows? The last was the most difficult, but her GP had said there was nothing physiologically wrong with her, it was all in her mind. He prescribed some painkillers, but they only gave momentary relief, making her dull and drowsy, with greater chances of having a headache the next day.\n\nHer mother took her to a homeopath in her neighbourhood in Jangpura. 'My daughter is not well, doctor, she suffers from tension. Little things upset her, and she gets a headache.'\n\nThe homeopath, a well-known one in that area, looked concerned. 'Tension'' he stated, 'the disease of modern life. The secret of health is a balanced mind.'\n\n'I try and be calm'' said Astha earnestly, 'but still I have headaches, and the pain lasts quite long.'\n\n'Right side or left?'\n\n'Usually right.'\n\n'Front or back of the head?'\n\n'Eyebrows. One or the other, never both.'\n\n'Morning or evening?'\n\n'Any time. Occasionally I wake up with a headache, at other times it comes in the afternoon or evening.'\n\n'Which season?'\n\n'All.'\n\n'Hot or cold suits you?'\n\n'Cold.'\n\n'Sun or shade?'\n\n'Shade.'\n\nEtc. etc. etc.\n\nAstha left the homeopath clutching Sanguinaria and Belladonna, 30X. Four times a day, alternately. Come after two weeks.\n\nShe dutifully took the Sanguinaria and Belladonna four times a day alternately. She kept a diary of her headaches. Once to twice a week. Hemant felt homeopathy was mumbo-jumbo, and took her to an ENT specialist. The specialist looked up Astha's nose and informed her husband that with such a deviated septum, it was a wonder that Astha could breathe properly, in fact if you notice, her mouth is open.\n\nAstha shut her mouth quickly.\n\nAnd of course she is going to have headaches. Time will not improve her condition.\n\nAt the thought of everything going from bad to worse, all power of decision left Astha.\n\nThe family took a second opinion, and surgery was decided upon.\n\n*\n\nAstha was in hospital four days. Her nose was heavily bandaged and hugely swollen. She could hardly breathe. It was not a good beginning to a life of easy breathing, and a head that didn't pain.\n\nHemant spent a part of every evening with her, while Papaji supervised in the factory.\n\n'Poor little baby'' he murmured as he stroked her hand, 'does it hurt?'\n\nAstha nodded, and tears rolled down either side of her bandaged nose. She tried to talk, but then her nose moved, it hurt more, and the tears came faster.\n\n'Baby, don't talk'' said Hemant tenderly. Astha wished to capture his expression in her heart for ever. She looked more beseeching, more piteous, and Hemant pressed a soft kiss under the swollen lump, lingering long on the salty lips.\n\n'How are the children doing?' croaked Astha.\n\n'Do not worry'' said Hemant, head of the household, the type of person his wife could depend on, poor little thing. 'Mala is very reliable when you are not there. She knows she can't try her funny business with me. Besides they love being with Mummy, she thinks they are not dressed well enough, and has bought both of them new sets of clothes.'\n\nAfter he left, 'How good Sa'ab is'' said the day nurse with a sigh. 'Coming to see you every day. Not every husband is so nice.'\n\n'Yes, he is'' said Astha.\n\n'Love marriage?' asked the night nurse.\n\n'No.'\n\n'Arranged is best'' said the night nurse with an even larger sigh, and then proceeded to tell the story of how her husband had first seduced and then married her sister. She could hardly bear to speak to him when he came home at night, that is why she had taken up this job, otherwise she came from a respectable family where the women didn't work, but now what else could she do, it was very bad madam, her sister looked after all the children and ran the house.\n\n*\n\nAfter her operation, Astha came home, waited for her headaches to go and life to become pain free. But the headaches continued, and Hemant was naturally not as attentive as he had been in the hospital.\n\nIf that nurse could see her now, her envy would be greatly diluted, thought Astha as she fretted over absent husband, and often absent children as well.\n\nWhere were they? Upstairs. Five days had been enough to establish this pattern. When she called them down, this was seen as objecting to their being with the grandparents. She tried talking to Hemant about this.\n\n'It upsets the children's routine if they are up for so long'' she protested. 'And if they eat so much junk, their appetite is ruined for dinner.'\n\n'You fuss too much. Besides their Dada Dadi are lonely. They complain they do not see enough of the children.'\n\n'I send them up whenever I can, Hem, you know that.'\n\n'Yes, but you know how it is with old people, they think they have little time left, all rubbish of course, but if it cheers them to have the children, why not?'\n\n'What about me? As it is when I am in school Himanshu is upstairs. When I come home I want the children. I hardly have you, I should have them.'\n\nTears came to her eyes. More tears for Astha, poor thing. She was climbing a mountain, and when she reached the top her face sweating, her heart going at its fastest, all she could see was another mountain. As she gazed at the jagged edges, her head began to ache, and the blood that was pounding in her heart obliged by moving to her head and pounding there.\n\nHemant rolled his eyes, and drew out a handkerchief to dry her face. 'What rubbish'' he repeated. 'It is all your imagination. When don't you have me? You are the one who keeps wanting to stay at home with the children, or your school work, or your books when we are invited to parties, or when I want to go to the club.'\n\n'How can you say that? I always come with you.'\n\n'And hate it, don't deny it. Half these invitations I refuse because of you. I am the one who is lonely, and without company.'\n\nBy what sleight of hand had their problems become identical?\n\nShe continued with her sketching, but found herself scribbling poetry, her father's encouragement more firmly in her mind now than when it was first given. She wrote about gardens and flowers, the silent dark faces of gardeners tending plants and never getting credit. She wrote about love, rejection, desire and longing. The language was oblique, but it was her own experience endlessly replayed.\n\nWriting alleviated the heaviness within her, a heaviness she found hard to deal with. Discussing her feelings with Hemant usually led to argument, distance, and greater misery. In the struggle to express herself she found temporary relief.\n\nAfter Astha had written about two hundred poems, she felt she needed to go somewhere with them. Publication would make her work seem less futile, but how to get there? She started revising them, typing them out on the small portable typewriter Hemant had brought back from the States.\n\nAfter she did twenty she showed them to Hemant. As a man of the world, she trusted his sense of how to do things.\n\n'Poems?' he remarked, looking pleased. 'I didn't know you were still writing.'\n\nAstha smiled and said, yes, she was still writing.\n\nThe last he had seen her poems had been on their honeymoon, he reminisced, while Astha smiled some more. 'That was about a lake'' he went on.\n\n'I don't write about things like that now.'\n\n'You don't?'\n\n'I've lost interest in Nature. I'm older, I think differently'' said Astha.\n\n'But you look as young'' responded Hemant automatically. He put his arm around her for a moment before turning his attention to her writing.\n\nAstha waited nervously. It was the first time anyone was seeing her poems. Hemant frowned, shuffling through the twenty typewritten sheets. To his wife's horror he started reading one out in a puzzled voice:\n\nChanges\n\nThe eventual release from pain\n\nIn the tearing relentless separation\n\nFrom those in habit loved\n\nCan come so slowly\n\nIt seems there will never be a day\n\nOf final peace and tranquillity\n\nWho promised me, that if I\n\nDid gaze upon reality\n\nAccept it, embrace it, befriend it\n\nI would never suffer again\n\nBut no matter how many times\n\nI heave the doorways of my soul\n\nTo let the chill light in\n\nThe darkness grows silently\n\nTo hide me in the break of day.\n\nHemant stared at her. Astha cringed. 'Actually, forget it'' she said. 'They probably need more working over.'\n\n'But I am here to help you'' said Hemant genially. 'I personally thought the one you wrote in Srinagar was very good. I said so at the time, didn't I?'\n\nYes, you did, you did, you did. But now it's all changed, and I want to bang my head against the wall because you never understand anything. 'I thought you might help me in deciding what to do with them'' she said tense and calm.\n\nHemant continued riffling through the papers, sparing her the embarrassment of more loud reading.\n\n'You don't like them?'\n\n'I don't know what to make of them. Look, I am no reader, but they sound rather bleak, don't you think?'\n\n'Do they?'\n\n'Good heavens, Az, they are all about cages and birds, and mice, and suffering in situations that are not even clear. There is not one happy poem here.'\n\n'Poems are about emotions'' defended Astha. Maybe now he would ask her why she felt sad and they could really talk.\n\n'What kind of emotions? This person sounds positively neurotic.'\n\n'I don't think so.'\n\n'If others read these poems, they might actually think you weren't happy.'\n\n'No, no, they are not about me'' said Astha quickly.\n\n'I know that. But people are so quick to put two and two together and come up with five, quick to gossip, you know Az.'\n\n'Perhaps I should test that by sending them somewhere'' said Astha looking down, not wanting to see his face.\n\nHemant looked doubtful. 'Well, I don't know, it's up to you.' He held out the poems and she took them forlornly.\n\nThat night she thought long and hard of 'Changes'. How self-indulgent it had sounded when Hemant had read it out. And this was one she had considered her best, evocative and moving. Maybe he was right, they were all too alike, she would be exposing herself to the world.\n\nShe gave up writing and continued rather sadly to draw, sketching with the soft pencils and coloured charcoal that Hemant got her from Japan. Nobody could put two and two together about painting, say it was negative rather than positive, say she should paint lakes in Kashmir instead of mice, birds and cages. Maybe one day she could do something with her art, but for now her school and herself were audience enough.\n\nThat summer Astha's mother announced, 'I am going to Rishikesh for a month.'\n\n'Why?' asked Astha.\n\n'Swamiji is giving a course.'\n\n'So? You listen to him here, don't you?'\n\n'His ashram is by the banks of a river. It will be a different experience.'\n\n'I think you should stay here,' said Astha uneasily.\n\n'In my stage of life one is free from places. Soon I will be retiring. I have to think of what to do \u2013 where to go.'\n\n'You can stay with us'' said Astha, who had not learned the futility of making this statement.\n\n'Why don't you come too?' asked her mother with equal futility. 'It will help your headaches.'\n\n'I'm all right'' said Astha. She looked at her mother, who was smiling benignly. Astha became suspicious, it was not like her mother to smile, and that too at nothing in particular.\n\n*\n\n'Ma is going to Rishikesh'' said Astha to Hemant that evening.\n\n'Why?'\n\n'She says she is free of places.'\n\n'Very foolish of your mother.'\n\n'Talk to her.'\n\n'I will, as soon as I find the time'' said Hemant.\n\nWhich turned out not to be before she left.\n\n*\n\nFrom the banks of the Ganga in Rishikesh Astha's mother sent her a parcel containing a letter, a commentary on the _Gita,_ and a small booklet entitled _The_ _Purpose_ _of_ _Life._\n\nDearest daughter,\n\nHow are you?\n\nThe air here is pure, and the scenery is beautiful. Hemant, you and the children should come. I will book a room. Everything is on me. It will do you good to meet Swamiji. He is so wise, just seeing him is satisfaction. He is also asking you to come. Everything is on me.\n\nI am sending you two books that Swamiji has written. Read them every day. In ourselves alone is peace. Even when we know how difficult it is to change ourselves, still we expect others to change, and are unhappy when our expectations are not met. Remember that. It will help with your headaches also.\n\nIf you were to hear Swamiji you would realise that to keep a relationship going I should ignore the dark side, i.e. weaknesses of a person. Accept without condition if you want to live in peace. Any relationship can be beautiful if you nurture it. In time of difficulty don't lose heart. Freedom from all complexes is essential. Don't assert your ego \u2013 don't argue. Employ wisdom to solve the problem. You are committed to ME says Lord Krishna.\n\nAccommodation and acceptance keep families together. What you cannot change accept gracefully, cheerfully as prasad for the Lord. Create a home where you are. Such a person is free from sorrow. Every understanding requires composed mind. Worst thing in life is anger. Read the _Gita,_ especially chapter xiv.\n\nWith a thousand blessings for a long and happy life,\n\nMa\n\nAstha stared at this communication. Where did these thoughts come from, what was happening to her mother, a helpless widow, with her child too caught up in the web of daily life to go and free her parent from another web. If only Hemant had talked to her mother, but then why should she rely on Hemant every time.\n\n*\n\nWhen the mother came back after her month in Rishikesh, she made it a point to have her stay over often. The mother prowled around, pointing out the wasteful habits of the servants, the dirt in various corners of the house, the children's thinness and bad eating habits, and Astha's neglect of her in-laws.\n\nReduced to a nervous wreck, Astha took her anger out on the children. 'Don't scold them'' her mother's soft voice filtered unctuously through her shouts, making the children behave worse than ever. 'They are only children.'\n\nHow come, thought Astha resentfully, this thought never occurred when she was young?\n\n'Swamiji has taught me a great deal'' continued Astha's mother, reading into her daughter's silence. 'In the old days I was ignorant. Now I know better. If I made mistakes with you, I do not want you to make them with your children. All too soon this time will go. Let them enjoy their childhood.'\n\nAstha felt hunted. Nothing she could do was right.\n\nHer mother introduced her to Mrs Reddy, short, plump, grey hair pulled back, widowed colleague and original introducer to Swamiji. 'Tell her'' she ordered, 'how much going to the classes will help her.'\n\n'Behen, it is all right. When the time is right for her, she will come herself'' said Mrs Reddy.\n\n'Tell her'' insisted the mother, concerned about her daughter's happiness.\n\n'The Hindu religion'' opined Mrs Reddy, 'is wide, is deep, capable of endless interpretation. Anybody can get anything they want from it, ritual, stories, thoughts that sustain. But first you have to realise your need.'\n\n'She is always so tense and angry'' complained the mother.\n\n'I don't need religion, whatever I am'' said Astha firmly, while the two older women looked sorrowful.\n\n*\n\nThe time came for Astha's mother to retire.\n\n'I must leave this flat, beti'' said the mother. 'It is too expensive for me.'\n\n'Of course you will come and live with us, Ma'' said Astha.\n\nTradition reared its obdurate head. 'What'll his mother think?'\n\n'What'll she think? Nothing. She lives upstairs. It is not as though you are taking away her space. Besides Hemant is doing well enough for one mother-in-law not to be considered a drain on his resources.'\n\n'It doesn't look nice.'\n\n'To whom? To whom doesn't it look nice?'\n\n'To me.'\n\n'I wish you wouldn't be so stick in the mud, Ma. Why didn't you have a son to look after you when you were old, if you cannot take anything from a daughter? Why did you stop with me?'\n\n'I have talked to Swamiji'' responded the mother. 'He also thinks one must be independent.'\n\n'What does Swamiji know? Parents belong to their children.' By now Astha was grinding her teeth with impatience. When had this swami become so important that all Astha was saying meant nothing.\n\n'I am thinking of moving to Rishikesh.'\n\n'Rishikesh? You are going to live there all your life?' Astha was appalled.\n\n'Arre, who knows how long one is going to live? The atmosphere should be pure, one should lead a life of virtue and truth, where and how does not matter.'\n\n'What if you fall ill? Who will look after you? Swamiji?'\n\n'One cannot live in fear'' said the mother severely.\n\n'Nor in isolation. You will be lonely.'\n\nThe mother was silent. So was Astha, what could one say about loneliness?\n\n'Swamiji is insisting that I take my time and think about it'' said the mother finally, 'he is not agreeing for right now.'\n\n'And a good thing too'' said Astha baffled. She felt that in some way she had been tested and found wanting. She envied Hemant his relatively straightforward relationship with his parents. They demanded from him material care \u2013 which he gave, grandchildren \u2013 which he gave, emotional concern and physical presence \u2013 which he gave. Duties, responsibilities, obligations, all seemed clear.\n\n*\n\nA few weeks later Astha's mother gave up the lease of her flat, and got rid of most of her belongings. 'Material possessions are a burden'' she informed her daughter.\n\nHer daughter did not feel the same way. She loved the pretty things that decorated her home, her books, her lamps, her carpets, her cutlery, tableware, linen, furniture, everything that Hemant and she had bought together. Now she wanted to add the twelve boxes of books that had formed the beds and the divan at her parents' place.\n\n'Are you mad? We don't have the room'' declared Hemant.\n\n'We do, we can build shelves.'\n\n'Come on, Az, donate them to a library. We can't clutter up our house with a lot of old books. And you know you don't read them.'\n\n'That's not the point.'\n\n'What is the point? Books are meant to be read, and in a library they will be of use. Better looked after too.'\n\n'Please, Hemu, my father's books.'\n\n'Don't be so sentimental, Az. I will talk to Ma, you will see she will agree.'\n\nAstha's mother agreed to such an extent that the books were donated to a library before Astha even knew about it.\n\n*\n\nAstha was devastated. 'Why did you do that?' she screamed at her mother. 'They were mine as well. I loved them.'\n\n'But you never showed any great interest in them when you were growing up'' protested the mother.\n\n'That was then. This is now. Don't you care about Papa's memory? How could you do this to him, to me?'\n\n'People do not live in their things, beti. Besides'' added the mother, 'it is Hemant's house, and he said there was no room.'\n\n'Then who am I? The tenant? We could have found room, we could have built bookshelves, done something, we could at least have discussed it.'\n\n'You know how much work they were. Every year take them out, dust them, and then they get infested by silverfish, accumulate dust and space. In a library at least they will be read.'\n\n'You sound like him. At the very least I would have kept a few, or do you think I too should not be weighed down by material possessions?'\n\nThe mother sniffed, looked martyred and misunderstood. What was the use of saying anything, thought the daughter, the books had gone, and all the screaming in the world was not going to bring them back. But together her husband and her mother had deprived her of the dearest part of her father, and continued before her eyes to be oblivious of their crimes.\n\n*\n\nAstha's mother was now free to leave for Rishikesh.\n\n'When will you be back?' asked Astha anxiously, as she dropped her mother at the station.\n\n'I don't know beti, let me see how it goes.'\n\n'I wish you hadn't turned to religion, Ma'' said Astha feeling as though her mother had cheated on her, manifesting a strange turn of mind that her daughter could neither follow nor understand.\n\n'We are all looking for peace of mind'' said her mother. 'Swamiji will guide me.'\n\nThe train came and she left. Astha stood on the platform and watched her mother leave the city where she had spent all her working and married life. Now with just a bedroll and a trunk she was embarking on a pilgrimage, searching for a community she could call her own, with no possessions to weigh her down.\n\n*\n\nThe months passed. Astha's mother showed no signs of returning. Her letters, about love, peace, renunciation and knowledge, revealed nothing.\n\nDear Beta,\n\nPerform action with the full understanding that you have no control over the result. Success and failure have to be faced by everyone. By being thoughtful, reflective and prayerful we can overcome the spirit of 'I'ness that dominates all our actions. This approach keeps families intact and we don't become insecure. We have a set up to relax in, this paves the way to security, and to self understanding.\n\nThe meaning of life is struggle. There are challenges in all walks of life, how to tackle them is the question, not to run away from home, work, society and obligations. Perform your duties with detachment. Learn to give and not take. When you develop the spirit of giving intelligently, there is peace in the mind. Most of our problems are due to discontent with what we have.\n\nGive my love and blessings to dear Hemant, Anu, little Himanshu, and to your mother and father-in-law. With many more blessings to my dearest daughter,\n\nMa\n\nOnce or twice Astha asked Hemant, 'Won't you go and see her, convince her that her place is with us?' but Hemant was clearly not concerned enough for action. Astha's suspicions hardened, maybe her mother was right, it would not be so good for her to live with her daughter. She wished she had a house that was more clearly hers.\n\n'I need to go and see my mother'' she finally said to her husband. 'She might end up staying in Rishikesh. She probably feels neglected.'\n\n'That's absurd'' said Hemant, 'why should she feel neglected? Old people turn to religion. It is natural.'\n\n'It is not'' said Astha indignantly, 'only when they have no other choices.'\n\nHemant looked at her. 'Religion is a choice as much as any other thing'' he said. 'If she decides to stay in Rishikesh, it must be because she is happy there. Besides I have told you I will talk to her.'\n\n'Like when?'\n\n'When she comes.'\n\nAstha paused. She felt her mother's condition was partly Hemant's fault. Had he shown more concern... Tersely she pointed out, 'I know you had no time, but this cannot be left any longer, I need to see if she is all right.'\n\nHemant took umbrage. 'If that was the way you felt, you should have gone before'' he said. 'I have enough things on my plate.'\n\n'And so I will. As soon as the children's exams are over.'\n\n*\n\nIt was five o'clock on a Saturday morning of the following week, when Hemant took his wife and two children to the New Delhi Railway Station. He bought his wife a _Femina_ and _Stardust,_ for his children chips and chocolate, and sat with them in the compartment till the train left. 'Bye-bye Papa, bye Papa'' said his children. 'Why aren't you coming with us, Papa? See you soon, Papa.' Papa wrapped his arms around them, gave Astha a brief pat and jumped onto the platform.\n\nThe children passed the five hours to Haridwar having their breakfast, playing games, fighting, eating rubbish, dozing, and going to the bathroom, while Astha was divided between looking after them, and looking out of the window. The fields on either side had wheat growing in them. Her mother must have looked at this scene and felt alone. If she were not weighed down by children, husband, job, she too might become nothing, no different from the dots of people they were passing, lost on the flat plains of northern India.\n\n*\n\nAt Haridwar they got down, and walked across the road to the depot, from where they were to catch a bus to Rishikesh.\n\n'Bus to Rishikesh?' said Information. 'Half an hour. I will announce.'\n\nAstha and her children settled on one of the benches watching the others sitting, squatting, or sleeping on the floor. The hall was large and spacious. Already the air felt cooler than in Delhi, the breeze less polluted.\n\nThey sat and sat and watched bus after bus leave. Finally after twenty-five minutes Astha asked Information, 'When will you announce the bus that you said was leaving for Rishikesh in half an hour?'\n\n'It is already leaving'' he said languidly pointing to a bus lumbering away.\n\nThere was no time to get angry. 'Quick'' shouted Astha, grabbing the one suitcase, and shoving the smaller bags at her children.\n\nThey ran towards the slowly moving bus, their feet slamming the dust while the passengers stared at them curiously. Astha banged on its side, 'Stop, stop,' and the passengers hands echoed theirs in the banging, and the bus did in fact stop as it turned towards the exit.\n\nFeeling stupid and incompetent \u2013 you can't even catch a bus \u2013 Astha pushed her children up the steps and clambered on. Inside she distributed Anuradha and Himanshu where she found space on the hard shiny rexine seats, each packed with three to four people. The suitcase she manoeuvred in the crowded aisle, the packages she held in her lap, and with her attention wandering between her children, the green trees, the butterflies, the narrow road slowly rising, the mountains beyond, the tightly oiled plaits and shiny magenta nylon ribbons of the little girl in front, she passed the hour to Rishikesh.\n\n*\n\nThey finally stopped in a small and dusty square, which appeared to be the depot. Lugging their baggage to a group of waiting scooters, Astha gave the address of the ashram, and they bumped their way through narrow roads, lined with refuse and running sewers, the scooter wallah blaring away at every pig, cow, mongrel, rickshaw, two-wheeler and pedestrian in his way.\n\n'What's that?' asked Himanshu pointing to some enormous black creatures, rooting in the profuse garbage, ugly as sin.\n\n'Pigs, darling.'\n\n'But they are not pink'' he objected.\n\n'That is just in books, stupid'' said Anuradha. She herself was seeing a black pig for the first time, but her grasp of the difference between reality and theoretical knowledge was infinitely quicker than her brother's.\n\nIt was late afternoon by the time they reached the ashram doors, set in the middle of high walls. As they stepped into the compound, it seemed another world, clean, green, spacious, its long low buildings hidden by trees and shrubs on either side of a central open space. At the far end they could see benches, more trees and a paved terrace overlooking mountains across the river.\n\nAstha's mother was waiting with her arms open to receive her children, to show them her place, peaceful, serene, and at its centre a swami who contained the clues to life.\n\n*\n\nIn the ashram Astha could see how her mother had changed. Her movements were confident, her smile less tentative. She had made friends, she spent a lot of time walking around the terrace, and many hours reading the notes she had taken during Swamiji's lectures. 'Look'' she said, showing her notebook to Astha:\n\n*\n\nSleep, the state of being most pure. In sleep there is no thought, no emotion, no subject, no object. Sleep is the state where there is no 'I'. The state in itself no different from death, or previous lives in which we are in identical states \u2013 we need sleep not only to survive (you can't be awake if there is no sleep) but in order to understand reality.\n\n*\n\n'What on earth does all this mean?'\n\nAstha's mother looked conspiratorial. 'Ask Swamiji, he will tell you. He's a very learned man, he studied fifteen years before his own swami sent him into the world.'\n\n'But when he lectures he does so with a mike'' criticised Astha. 'That is not very unworldy.'\n\n'If you live in this world, you make it serve your aims. It is hard for him to speak continuously and loudly to such large audiences'' pointed out the mother protectively. 'So we insisted he have the mike.'\n\n'A present from one of his disciples?' inquired Astha, thinking in an idle way, that as a teacher she too could do with a mike, and she never talked as much as this man.\n\n'A present from me'' said her mother smiling that little smile again.\n\n'He asked?'\n\n'He never asks.'\n\n*\n\nThat evening Astha spent a lot of time staring at the swami upon the dais, who, after his lecture was immediately surrounded by his devotees, many women, some men, some resident and some from town.\n\nThey were sitting in the pleasant lecture hall, next to the river. All sixteen fans were whirring. Groups of people, while waiting their turn with the swami, were comparing notes on what he had said: today he explained very well, today he used a lot of Sanskrit words, difficult to understand, but then really you need ten years to understand. What was it Swamiji said, when that man asked a question about the mind \u2013 to answer will take me six years, to comprehend will take you twelve. Swamiji was in form all right, how he makes you laugh sometimes, and how my life has changed since I started coming to the lectures, yes, you get mental peace, no doubt about that.\n\nOne man in so many lives. Certainly in her mother's. She turned to look at her. 'Don't you want to ask him anything?' she asked.\n\nHer mother shook her head shyly. 'These people know so much more than me. Let them ask.'\n\n*\n\nAs they came down the steps a breeze was blowing, and a pink tinge on the water reflected the sunset.\n\n'Let us go to the temple'' said the mother.\n\nAstha stared at her. 'Since when have you started going to the temple?' she asked. Her father had not believed in going to temples, and as a consequence nobody ever went.\n\n'There is arti in the evenings, and one of the women here is a very good singer'' said the mother as Astha's question slid by her. 'Come'' she said, calling to the children, 'Anu, Himu, come, we are going to the temple.'\n\nThe temple was in another compound, small, white, with pink decorated columns and roof, facing the river front, lit with tube lights, and floored with marble. It was exquisitely clean with devotees waiting quietly for the evening prayers to begin. The mother sat at the back, Astha sat next to her, the children fidgeted and looked around.\n\nThe service lasted for forty-five minutes. Bhajan singing, praying, arti, offering bhog, receiving prasad, drinking holy water, and smoothing wet __ hand over head and eyes.\n\nIn the queue to receive prasad Anuradha asked, 'How come we don't do this at home?'\n\nAstha didn't know what to say. We don't believe was not strictly true, I don't have the time trivialised religion in a way that might be bad for her children, saying only old people prayed like this suggested that religion was only useful when you were feeble and decrepit. Instead she said, 'God is in our hearts, beti, and some of us do not believe in ritual. Maybe when Nani comes to Delhi, you can pray with her.'\n\n'We will all do it together'' said Nani firmly, her eyes gleaming with the prospect of inducting her grandchildren into puja, ritual, Vedanta, and the sound beginnings of a Hindu life.\n\n*\n\nIt was towards the end of Astha's visit that her mother said, 'I'm thinking of selling my land, and building a set of rooms in the ashram. Swamiji has agreed.'\n\n'Live with us, Ma'' Astha said hopelessly, 'it is the best solution.'\n\n'It doesn't look nice. Mother-in-law comes and never leaves.' Here the mother sighed, and looked at the waters of the river with a melancholy eye. 'It is so beautiful here, so peaceful'' she went on.\n\n'You must be lonely, Ma'' said Astha. They were sitting on one of the benches overlooking the river. The children were running up and down the steps. The heat of the day had gone, the light was gentle, the water below them was turning dark.\n\n'It is a lonely life'' said the mother, filling Astha with a dreadful sense of guilt.\n\n'It is my house too. If people mind it is just too bad. I don't believe in all this shit about parents being the responsibility only of the sons.'\n\n'After all Hemant's parents are staying with him, aren't they, not with their daughters.'\n\n'His parents can't stay with the daughters, one is in America, and one\u2014' Astha was going to say and one is staying with her in-laws, but changed it to, 'and one has a bad marriage, with a small house.'\n\n*\n\nNext morning at the lecture Astha again looked at her mother's teacher carefully. His beard was grey, there were little white spikes sticking up from his shaven head. He wore glasses, and the eyes behind them were gleaming, sharp, intelligent, she supposed compassionate \u2013 he was a swami after all \u2013 and how does one describe a swami's eyes? His legs were crossed, his foot waggled, his clothes all saffron. His voice was deliberate and quiet:\n\n'There is pain and suffering in every life. When the burden becomes intolerable, we seek distractions, which in turn trap us. We develop a craving for pleasure and sensation, till finally we are at the complete mercy of our desires, which out of ignorance we have encouraged to grow into monsters.\n\n'With desire comes dissatisfaction, and a dissatisfied man is full of misery, even if he has at hand the pleasures that the world can give him.\n\n'We mistake gratifying our senses for living in the world. We act in order to be happy, and then we are surprised that the happiness does not last, and we look for other things, and the same pattern is repeated. Discontent is the cause of restlessness.\n\n'All our pleasures are connected with our deeds. They have a beginning and an end. The fruits of our actions similarly have a beginning and an end. They are transient and can therefore never quench our longing.\n\n'We breathe to live, but every breath draws us one step nearer to our end. In our body is our decay. We cannot alter this decay, the richest man in the world shares the fate of the poorest.\n\n'Against the world we are weak. Hunger, thirst, cold, heat, flood, famine, storms, all these things create fear. We run seeking protection here and there, but the strongest protection against the world comes from knowledge that comes from within.\n\n'It is only in a state of self-realisation that we can draw from the reservoir within to gain happiness. If we find contentment within ourselves, we will find good in all things. As the sun shines so shall the contentment within us light our lives and the lives around us.\n\n'We protect our feet with shoes, we protect our body with clothes. We cannot be harmed by the stones in our paths, nor by the sun or the rain that falls on us. Similarly, those who have achieved self-realisation are contented in all circumstances. The troubles they encounter on their journey through life cannot hurt them.'\n\n*\n\nAstha listened, caught up in his words, like everybody else in the room. The swami looked beyond time, because he was bowed down by nothing. If examples were what one had to go by, he was a good example of what he preached. His face shone with non-attachment, though his disciples hung on to every word he uttered with fierce attachment.\n\nAll these people here were looking, looking for shoes to protect them from the rough paths they had to tread. She wanted shoes as well. She sat in front of the swami trying them on. For a wild moment she wanted to go up to him and beg, tell me what to do.\n\nAnd he would tell her, what? She already knew. Misery springs from desire, desire springs from attachment, and that if she gave up all these things, she would be happy.\n\nThe weight in her chest increased. She had come to rescue her mother, and yet seeing her mother in that place, the person who seemed to need rescuing was herself.\n\nShe tried no more to prevent her mother from living in Rishikesh, or from selling her plot of land. Clearly her mother needed quite a bit of money if she were to live respectably in the ashram. It seemed crazy to sell a piece of property, whose value, now that the bridge was built, doubled practically every year, but when one gave up material possessions, one also gave up speculation in the future.\n\nAnother three days and Astha left.\n\nIn October that year, the sale of the plot went through.\n\n'Dear child'' said Astha's mother, who was in Delhi for the signing of the papers, 'I have given Hemant part of the proceeds of the house.'\n\n'Why? The money is for you, Ma.'\n\n'I don't need so much. You can consider this your father's legacy.'\n\n'They why give it to Hemant?' asked Astha bridling.\n\n'Why not? He is a man, he knows about money. He will invest it for you and the children. I have discussed the whole thing with him.'\n\nHow had this happened? Hemant had found a buyer and checked the legalities of the sale, but even if he was the man of business, she wanted to participate in any decision concerning the money her mother chose to give her.\n\n'Really, Ma, don't you think women can be responsible for their own investments?'\n\n'Of course, but this was a lot. Are you suggesting I hand the whole thing over to you?'\n\nNo, Astha wasn't. The sad thing was that she herself would have felt nervous handling a large sum. Suppose she did something foolish, and it did not multiply fast enough, it would be through her arrogance that the money had not functioned in the optimum manner.\n\n'Hemant is very clever, look at the way he does business, with no background'' went on the mother. 'You yourself have said he manages everything financial. It was the same with your father, I only did the household accounts.'\n\n'You were earning too, Ma.'\n\n'Yes, yes. But he looked after my tax saving, my provident fund, decided how much we should spend, how much to save, all that. After him, Hemant took over.'\n\n'Yes, Ma.'\n\n'He has promised to double the amount in a few years.'\n\nCould Astha ever have made such a promise? Never, not even if the gates of hell opened and the stock market collapsed in her lap. She had better stick to her job, and what it earned her. Nobody thought it was anything. Nobody discussed it, speculated with it, promised to increase it at fantastic rates. She could do with it what she liked, take it to bed, chew it, shit rupees in the morning and nobody would bat an eyelid.\n\nHer mother had delivered her into Hemant's hands. If her mother was at fault, so was her father, for managing the money, and teaching his wife that this was normal behaviour, so was her mother-in-law for bringing up Hemant to never regard women as beings to be consulted in their own lives, so was the Swamiji for teaching that only in detachment lies happiness, which lesson can be read in as many different ways as there are people and attachments.\n\n*\n\nAfter Astha's mother left, the money was discussed briefly and bitterly.\n\n'Darling?'\n\n'Dearest.'\n\n'You know Ma's money?'\n\n'I have several plans for it. It will be well invested, don't you worry. Long term for the children, shorter term for you.'\n\n'Thank you darling. But I was wondering, you know, whether I could also have a say in what you do with it.'\n\nHemant began to frown. 'Don't you trust me?'\n\n'Of course, of course, I trust you. It's not a question of trust, surely. You are my husband.'\n\n'Exactly. So what's going on?'\n\n'I wish to feel\u2014' Here Astha paused and treaded carefully among the thickly laid minefields of income, expenditure, rights, responsibilities, knowledge, power, and dependency. 'I mean if I wasn't so ignorant about things concerning money, I wouldn't feel so stupid.'\n\nHemant relaxed. 'When I have finished I will explain everything to you. In fact I am glad you have brought this up. I have been thinking you should know what is going on. That way if anything happens to me, you will not be left in the dark.'\n\n'But Hemu'' said Astha, 'I don't wish to be enlightened only because you might die, which I hope will not be for a long long time, and certainly not before me.'\n\nHemant smiled, 'We will die together in old age, huh?'\n\n'Yes'' she replied, 'yes'' she repeated, 'yes'' she faltered. 'We will die together, I hope, but meanwhile, I feel so clueless about our financial situation. I know that in business things can be uncertain, so I thought that now that I have some money, it would be useful if I looked after it. That way I will gain experience.'\n\n'Your mother gave me this money to manage, I didn't ask for it'' said Hemant coldly. 'She trusts me even if you don't.'\n\n'That's not what I mean. I know she trusts you, certainly much more than she trusts me, but is it such a bad thing if I know how much is in my name and how I can have access to it?' Astha was pleading now, begging Hemant to understand. She meant nothing personal. She didn't want to feel dependent, that was all. Surely equals could relate better than master and slave?\n\n'What has gotten into you I don't understand. I will tell your mother to give the whole thing to you, you will handle it yourself. She should have consulted you first, before she handed anything to me. In fact why didn't she ask you to look for a buyer and get a lawyer to check the sale deeds? You have been missing out on so many things that life is not worth living, isn't that so?'\n\nAstha sat stunned. What kind of fool had she been to expect Hemant to understand? She had a good life, but it was good because nothing was questioned. This boat could not be rocked. She should paint that on a canvas and put it up on the wall, and stare at it day and night, so that its message burnt its way through her brain into her heart. This boat cannot be rocked.\n\nBesides if the boat could not be rocked, what need did she have of money, or knowledge of investments? Hands that had grasped money, and felt it pass through their fingers were the ones capable of rocking boats. Hers were not.\n\n*\n\nThe next morning, quickly she got her children ready and sent them off to school, quickly she had her tea, packed her breakfast to eat later, jamming an omelette between two slices of fridge cold bread and dripping violent red chilli sauce over the insides. Quickly, quickly she did all this, smiling, smiling all the while, so that no distress was palpable.\n\nIt was only in the staff room in school that Astha could be alone with her thoughts.\n\n'Why so silent?' they asked? 'Are you ill?'\n\nAstha shook her head. She looked at her colleagues, women she met every day, women whom she liked, whose lives ran smoothly, women who had no shadows between their husbands and themselves, whose husbands were 'him' and 'he'' and whose in-laws were 'they'. Whom among them could she tell that she had not been able to sleep? What reason could she give that they would not think self-indulgent?\n\n'I'm fine'' she repeated, and opened the usual stack of brown-paper covered notebooks that laced each day's work. \nChapter IV\n\nIt was early in the year 1987, that the principal of Astha's school invited The Street Theatre Group to hold a workshop on their premises. The workshop would be held in the break between the final exams in March and the opening of the new school year in April.\n\nThe staff were not pleased.\n\nAs usual the Principal wants to attract attention to herself.\n\nJust because she is interested in theatre, we are forced to be interested too.\n\nThey'll want staff volunteers, wait and see.\n\nWe have to correct exam scripts, prepare report cards, see to the merit lists, file an account of each child's progress in the school records \u2013 where is the time to do all this extra-curricular activity?\n\nA gloomy silence descended. The Principal was not known to respect the convenience of her teachers.\n\n*\n\nAstha wondered whether she would be asked, she did not relish working in the holidays while her children were at home. She was very fond of Mrs Dubey but sometimes she felt that their special relationship caused her to be exploited. She had done enough for the school, the Principal should look elsewhere, she decided, readying herself for a tussle.\n\nIn which she lost.\n\n'You need someone with more experience if an outsider is coming'' she tried objecting.\n\n'With Aijaz you don't need experience'' said the Principal. 'For him any place is a stage, any person an actor. He has performed at factory gates, outside offices, at bus stops, in front of shops. He has dramatised issues like unemployment, atrocities against women and urban poverty. Indeed he is the voice of the underprivileged. That is his genius.'\n\nHe can take his genius elsewhere, thought Astha, why is he bringing it here.\n\n'He is my brother's friend and is coming here on my personal request'' went on the Principal, somewhat coyly. 'This is a great opportunity.'\n\n'It's my children's holidays.' The woman-to-woman approach.\n\n'Bring them, they will benefit. Aijaz is a wizard. He is actually a history lecturer, but his knowledge of drama is immense. Besides writing his own plays and songs, he has adapted Brecht, Shakespeare, and Greek tragedy into Hindi. People grumble about the lack of activity in the school, but when it comes to giving our students exposure they come up with all kinds of objections. Where is the school spirit?'\n\nAstha had no option but to agree.\n\n*\n\nHemant was not pleased. He timed his trips to be free for his children's holidays.\n\n'Why can't you stay at home? And why drag the children into this?'\n\n'I had no choice'' said Astha. 'Anyway it will be good for the children to see schools not as elite as theirs. Anu was actually asking when were we taking her to Disneyland. All her school friends have been, she says. I don't believe her. Disneyland! Imagine!'\n\n'Nothing wrong with wanting to go to Disneyland'' said Hemant.\n\n'At this age! Why, I haven't been abroad yet.'\n\n'We are not talking about you. If parents can afford to show their children the world, why not?' said Hemant. 'This is the eighties. We are not deprived Indians any longer.'\n\nAstha felt there was something morally wrong with getting things without struggling for them, but she knew this view irritated Hemant. He was making more money at his age than their combined fathers at their retirement, and he didn't seem to have any intention of letting his children struggle. She turned the conversation to the topic at hand.\n\n'Apparently Aijaz Akhtar Khan, the founder of The Street Theatre Group is very well known. He teaches history, and during the holidays he performs in slums, factories, streets, villages and small towns.'\n\n'What's the point of that?'\n\n'Create empathy, generate social awareness by having workshops that involve workers and students, bridge the class divide'' said Astha glibly, replicating that morning's exchange with her Principal.\n\n'Culture-vultures'' snorted Hemant, 'why don't they do something real about the class divide, like creating jobs?'\n\n'Not everybody can be a factory owner.'\n\n*\n\nHimanshu was delighted. His face broke into a slow and gleaming smile that went straight to his mother's heart. He was always wanting to come to his mother's school instead of his own.\n\nAnuradha registered her brother's pleasure and loudly protested against the injustice being done to her.\n\n'Why should I spend my holidays going to your school?' she demanded. 'Don't I go enough to my own?'\n\n'I can't leave you here alone the whole morning. It's not classes, it's a drama workshop. You'll be doing fun things.'\n\n'I don't want to do fun things. Besides Papa said he was going to spend fewer hours at the factory and take us out.'\n\n'Well let him actually make the programme and then we will see'' said Astha with some irritation.\n\n'I won't'' said Anuradha her eyes flashing, getting ready for a confrontation that would continue till collapse or victory. 'You can't make me. I'll spend my holidays with Dadi upstairs.'\n\nHimanshu looked on piously, while Anuradha waited for the next round. 'Please beta'' said Astha, 'your Dadi then complains to me that she gets tired. You have so much energy she doesn't really know how to keep up with you. Come for a few days, if you don't like it you needn't continue. Promise.'\n\nOnce it was established that Anuradha was doing her mother a favour, it was easier to take her.\n\nAt first Astha did not pay much attention to Aijaz. He seemed quite capable of managing thirty-two children without her. He sat them in a circle on the stage. Do you know why people sit in a circle \u2013 so that there is no hierarchy \u2013 all of us have something to offer from backstage to front \u2013 what is the theatre about \u2013 communication \u2013 what kind? \u2013 drama \u2013 older than the written word \u2013 what did they think was the subject of drama \u2013 where did they find it...\n\nHow pedantic, thought Astha, is he giving the history of drama, are they going to do an exam, or is he going to get on with the workshop, which is why we are all here in the first place, I'm sure all the children are bored. And her mind wandered, till it came back ten minutes later to Aijaz explaining that the way man lived in society was politics and this affected everybody, literate, illiterate, powerful, powerless, poor, rich. He read out sections of the newspaper and asked how they would translate what was happening into drama for people who couldn't read? For example what would they do with the Babri Masjid-Ram Janambhoomi controversy?\n\nThe spot where Ram was born thousands of years ago some say is the exact spot where a masjid stands today. Is this fact or faith? If it is faith, is it sacrosanct? Are there ways in which faith can be motivated and played upon by political forces...\n\nHis voice faded, and Astha's mind turned to the religion consumed at home on one of her husband's TVs. Ever since the Ramayan was serialised, viewing it had become a ritual, insisted upon by the grandparents, strongly supported by Hemant.\n\nAnd so, every Sunday morning, the family gathered upstairs before a ClearVision TV, twenty-inch screen, manufactured by the son of the house, and watched the story of the Ramayan. Week after week they agreed, this was the golden age of India, this is our noble heritage, now thoroughly debased, when justice flourished, when Hindus had pride, when a king showed responsibility towards his people, when duty, honour, devotion, truth and loyalty had a place in Ram Rajya. And today the birthplace of this king, our Lord, is occupied by a mosque, the shame of it, dismissing as nonsense the protest that it was not possible to really place the exact spot of a man's birthplace so many thousands of years ago.\n\nSuddenly Astha saw the long arm of history twisted and refracted, till it popped out of a TV box, took them to Ayodhya and planted them on Ram Kot in front of the Babri Masjid.\n\nShe was sitting at the back of the stage, her arms around her knees, thinking all this, when she looked up and saw Aijaz looking at her. Uncertainly she smiled. 'What do you think, Astha?' he asked.\n\nHow had he found out her name? And from being indifferent to Aijaz, the single use of her name, created a pleasure in what she, unused to the ways of men outside marriage, saw as interest rather than a communication strategy.\n\n'Do you think you can write the script?' he went on.\n\n'Um'' Astha hesitated, 'I don't know anything about the Babri Masjid.'\n\nAijaz leaned towards her and said, 'Just a working script. Your daughter has volunteered your name. She says you write.'\n\n'I am not really a writer, just a few poems'' said Astha surprised, her eyes on her daughter's back, with the hair curling down the white shirt.\n\nAijaz was used to persuading people. 'Just a simple working script which we can improvise on, Astha.'\n\nHe was focusing on her. She blushed.\n\nHimanshu frowned. Was his mother being forced to do something unpleasant, but no, she was agreeing, she was participating in extra-curricular activities, doing the bit that wasn't necessary, volunteering despite her uncertainty about her capacities, because everything was worth trying.\n\nAijaz smiled, showing his even pearly teeth. Why does he smile like that, he knows he is charming, thought the newly appointed writer of scripts.\n\n*\n\nGoing back in the scooter, Astha thought of the India International Centre, where her parents-in-law were members, and the library that only she was interested enough to use. There was bound to be something on the Babri Masjid there. As if reading her thoughts, Himanshu piped up, 'I'll help you, Mama.'\n\nAnuradha snorted. 'You? You are so stupid. What can you do? Do you even know what the Babri Masjid is? Do you know where it is?'\n\nHimanshu turned around and hit Anuradha in the stomach. Anuradha hit him back twice as hard, then once on the back for good measure. Astha slapped Anuradha's hand. Anuradha glared at her mother. Himanshu began to cry. Just then the scooter took a wrong turn inside the colony, and in the middle of shouting at her children, Astha had to break off and redirect the scooter wallah through the maze of Vasant Vihar. He insisted on charging ten rupees more, and that was the end of their first morning at the theatre workshop.\n\n*\n\nLater Astha had a talk with Anuradha. 'We are going to be together for fifteen days,' she said. 'And in that time I forbid you to call your brother stupid.'\n\nAnuradha looked cunning. 'And after that?'\n\n'Even after that. You can't go on calling someone stupid. It hurts their feelings.'\n\n'But he is.'\n\n'Even if he is.'\n\nAnuradha looked victorious. 'See, you also think so.'\n\nAstha stared at her daughter, 'Anu, what's the matter with you? Four years younger, what comparison can there be?'\n\n'You are always taking his side.'\n\nWhy was it, thought Astha wearily, that love always had to be balanced by its opposite? She had a secret tenderness for Himanshu that her daughter targeted unerringly, battering her mother, shouting out her dislike, making even the love and hate in the world. She looked at Anuradha's contorted face, and angry eyes, and cajoled, 'I need help in writing a script. Himanshu can't help me.'\n\nAnuradha looked wary. 'Don't try and flatter me'' she said.\n\n'You mean what I'm saying is not true?'\n\nFor a moment Anuradha was out-manoeuvred.\n\n'So, it'll have to be you'' continued Astha.\n\n'When do we start?'\n\n'This evening. We'll go to the library and get some facts first.'\n\n'And leave Himanshu behind.'\n\n'Absolutely. I'll send him upstairs.'\n\n*\n\nThat evening Astha and Anuradha made for the library. As Anuradha looked at magazines, Astha quickly browsed through the books in the history section. There seemed to be no end of fuss around this mosque. Had there been a temple on this site, claimed to be the birthplace of Lord Ram? Had Babur ordered this temple destroyed? Had he compounded the arrogance of conquest by building a mosque bearing his name using materials from the temple? Zealous historians, pursuing evidence and rationality had gone into its structure, pillars, stones, inscriptions, had investigated Babur's diary, his religious and building habits, had cited examples of British divisive policies, but nothing had been able to quiet the controversy.\n\nAstha stared at the picture of the Babri Masjid. What was it about this monument that had created so much bloodshed and fighting over two centuries? It was not even remarkable, squat and three domed, surrounded by trees. How could she effectively present its history, long and tortured, in a manner that was simple without distorting?\n\nOver the weekend as she read through books and photo-copies she had made in the library, she thought that controversies need places, disputes need sites, not the other way around, and the Babri Masjid was one of them. Babri Masjid-Ram Janambhoomi. The amount of blood, hate, and passion for ownership these words evoked bathed each stone with a corrosive mixture, slashing through the surface so that it was no longer an old mosque. It was a temple, a birthplace, a monument to past glory, anything but a disused nesting place for bats. Despite all this it had endured for over four hundred years.\n\nIt was too much to handle as a play. She felt like giving up, but the thought of not having anything to show Aijaz drove her on. She gripped her pen, took a deep breath, and plunged.\n\nShe was still plunging when Hemant returned from the Sunday tea spent upstairs with his parents, bonding over business and politics.\n\n'Back already?' she asked.\n\n'It's been two hours'' he replied.\n\n'Oh, I hadn't realised. This whole thing is very complicated'' said Astha.\n\n'People make it so'' replied the husband. 'Otherwise what is there in an abandoned mosque? The government is too bloody soft on these Muslims, that is the problem.'\n\n'Surely that is not the issue. Power seekers \u2013 on both sides \u2013 use religion quite blatantly. How can beliefs about god be compatible with violence?'\n\n'You don't know _their_ religion.'\n\n'As though ours is so much better. Ram would have hated what was going on in his name \u2013 a man who sacrificed everything to keep his father's honour, who left his home, his palace, his kingdom in order to make sure his brother inherited, he would be the last to appreciate the fuss over his birthplace.'\n\n'Times have changed. We are preserving his honour as it needs to be done now.'\n\nAstha stared at her husband. Was he agreeing that people should be killed in the name of God? She didn't want to know what he thought.\n\n'Wasn't Aijaz going to write this play'' continued Hemant. 'Didn't you tell me he was a history teacher? Surely this is his area of expertise, not yours. How have you got so involved?'\n\n'He wants everybody to participate'' said Astha thinking quickly. 'Besides you forget I am the teacher volunteer.'\n\n'Volunteer, not donkey.'\n\n'Translating history into theatre is hardly work a donkey can do.'\n\n'Nor can you. What is your experience?'\n\n'I don't need experience.' She felt she was being denied something, not understood, throttled, and choked. And yet it was just a play. He was right, she had no experience. Though Aijaz was in a better position to write about masjids and controversies, still she would hold her own, paltry though that own might be. 'Aijaz doesn't think experience is necessary'' she went on in defence.\n\n'Oh pardon me'' he said, and his wife hated the mockery in his tone, 'he clearly knows how to get work out of you.'\n\n*\n\n'Can I speak to you a moment?' Astha asked Aijaz on Monday during the fifteen-minute break he allowed the kids.\n\n'Trouble with the masjid?' he smiled.\n\nAstha nodded briefly.\n\n'Shall we go to the canteen?'\n\n*\n\nIn the canteen she opened her bag and took out flurries of photostats. 'I don't know where to begin'' she started. 'It's such a tangled history, and leaving one piece out makes it lopsided. Besides it is used for many different political purposes in the present as well.' This Astha had only realised yesterday. So far the Babri Masjid had been something mentioned in the news with the irritating air of a problem that wouldn't go away. 'I do wish you would write it, or conceive it. I am sure you are far more knowledgeable.'\n\nAijaz looked at her clutching her photostats. 'Do you think it is only the so-called experts that should be allowed to deliver opinions? You are looking at it from the outside. Your perspective is fresh, it is invaluable.'\n\n'But I am very ignorant and I cannot possibly do it justice'' she said, quick as a flash putting herself down.\n\n'It doesn't matter, Astha'' he said. His voice was coming at her, his eyes were looking at her, any second and his teeth would glow at her. She was married, she should not be registering these things. She shifted uneasily on the hard canteen bench, clutching her bag in her lap. 'The thing is'' he went on, 'we have to create awareness. There may be differences of interpretation, it doesn't matter. If our players and our audience think for one moment about this issue, we have done our job.'\n\n'You have already created awareness in one'' she mumbled daringly.\n\n'And you will create it in many.'\n\n'I don't know'' she replied, 'I have no experience.'\n\nThe smile, the teeth, the hand that lightly touched the phototstats. 'What is all this?'\n\n'Material I gathered. I sketched out a few ideas, though I am not sure\u2014'\n\n'Let's see'' he interrupted, leaning forward. She could smell him, a faint sharp smell. She shifted uneasily again, clutching her bag still more firmly to her stomach, riffling through her papers.\n\n'I thought of starting in 1528, you know when Mir Baqi decrees that a mosque be built at the highest point in Ayodhya in the name of his most noble ruler Emperor Babur, a brief two-line scene. We could have a boy with a placard announcing dates and locations. Perhaps the same boy could double as the mosque, a mosque that just wants to be left alone thinking each fight will be the last.'\n\n'Himanshu would be good for the part. He is the youngest.'\n\nHer own thoughts exactly. She looked up and smiled, he smiled back, she quickly looked down, he must think she found her paunch fascinating, she looked at it so much. 'Do go on'' he said after a moment's silence.\n\n'Then a short scene set in 1855. The Muslims think the Ayodhya ruler is showing favours to the Hindus. They claim that the temple at Hanuman Garhi is built on a mosque, they march towards it, the Hindus retaliate by saying the Babri Masjid is built on a temple and they march upon it\u2014' she paused. 'Actually there was more but I have pared it down to the essentials, everybody thinking they have been done in, and asserting their power through temples and mosques.' She looked at Aijaz anxiously. 'I hope I have got it right?'\n\n'Absolutely. Then?'\n\n'A lot of people were killed during this time, Hindus as well as Muslims, and the whole thing became openly political. There was an enquiry committee consisting of Hindus and Muslims, presided over by the British Resident. But after 1857 power equations changed, and two years later, the British declared that access to the Babri Masjid would be bifurcated. The Hindus were to enter from the east, and the Muslims from the north.'\n\n'Then?'\n\n'This state continues till the British leave. Then in 1949, some idols appear. The Hindus claim this is a miracle, while the constable on duty states that about fifty to sixty people broke into the masjid on the night of December 22. The next day the District Magistrate declared the area disturbed and locks are put on the masjid. At this point I stopped.'\n\n'You haven't written more?' Aijaz sounded disappointed.\n\n'Well last February the district court ordered the locks open. Rajiv Gandhi is probably involved, but I don't know how far to go in showing the masjid as a tool in modern political equations'' said Astha, pleased at his tone.\n\n'We'll work something out.'\n\nAijaz took out his wallet, while Astha groped around for change. 'If you don't let me pay for one sweet and overcooked cup of tea I'll be very upset'' said Aijaz as they rose to go.\n\n*\n\nThe appreciation that Aijaz had shown moved Astha so much she couldn't help talking about it at dinner.\n\n'Aijaz liked the script'' she started.\n\n'He told us it was a wonderful script'' put in Himanshu 'but we could change it any way we wanted because we are to bring our own \u2013 own \u2013 what, Mama?'\n\n'Interpretations.'\n\n'Yes that \u2013 to our parts. And I'm to be the mosque and carry placards. I have to keep crying and getting hit. Everybody wants me.'\n\nHis parents looked at him indulgently. 'Really beta?' said Hemant. 'I must come and see you.'\n\n'Yes, Papa. We are going to do it the last day of the holidays. All our families and friends should come, Aijaz said.'\n\n'Aijaz Uncle'' corrected the father. 'He is older than you.'\n\n'No Papa, Aijaz does not believe in hi \u2013 hi\u2014' He looked at his mother again.\n\n'Hierarchy.'\n\n'The girls in Mama's school don't call him anything'' said Anuradha. 'They are so shy, can you imagine? It is a very good thing that Himanshu and I go, otherwise poor Aijaz would have a hard time with them.'\n\n'Anu'' reproved Astha, 'they are not that bad.'\n\n'Oh, Mama, you don't know.'\n\nOnly later did Astha realise that Hemant had not actually said anything about her script. Well, it didn't matter, he would see the play performed, and recognise his wife's hidden talents. At night, lying in bed, she drifted off to sleep with thoughts of Aijaz and the days ahead.\n\n*\n\nAstha loved looking at Aijaz on stage, allowing herself frequent covert glances. He was of mediun height, his body compact. His face was the clear delicate luminous brown of freshly rained-on earth. His lips were a darker brown than his skin, and his eyes were black and narrow. While working he rolled up the sleeves of his shirt, allowing Astha to view at her leisure his round strong arms, hairless, smooth and muscular. He had prematurely grey hair, which, thick and springy, fell about his face and neck in ways that suggested a good barber. He must be vain of his hair, thought Astha, knowing how attractive the grey made a young face look.\n\n*\n\nThrough those fifteen days, Astha saw the little thing she had penned transformed, and her admiration for him grew. Song, dance, mime, action, improvisation, actor involvement, he fused all these elements effortlessly into a fast-moving, absorbing piece.\n\nShe and her children talked of nothing but the play, the rehearsals, the way everybody was acting, who was good, who was bad, who came late, who not, who had team spirit, who not, and what Aijaz had said. Every day Astha was called upon to add a bit of information, to corroborate some piece of evidence, suddenly she was the Babri Masjid expert, and this she felt was Aijaz's doing \u2013 he who was the history teacher, allowing her to parade her knowledge when surely his own was greater.\n\nHe looked at her, he wanted her opinion even when it wasn't necessary, he smiled when there was no occasion. Perhaps she shouldn't think of him so much, but soon it would be over, where was the harm, it made her happy, and that in itself was worth something?\n\n*\n\nSometimes as Astha sat on the stage she absently sketched the scenes before her, wanting to capture her son as the mosque, her daughter as a rabble rouser, and Aijaz as their teacher. By now she knew by heart his perfect teeth, his full lips, the smoothness of his cheeks, the deep dimple near his mouth, the curl in his hair, the glint in his eyes. She tried to translate these things on paper, but only registered pale copies. Her activities attracted his attention.\n\n'What are you doing?' he asked during one break.\n\n'Nothing much.'\n\n'No let's see'' and he gently tugged at the papers she had turned face down on her lap. For a moment his curled fist rested on her knee.\n\nHastily she shoved the drawings at him, repeating the mandatory, 'It's nothing much.'\n\nAijaz turned the papers over. Astha drew fast and there were ten sketches in all. 'For how long have you been drawing?' he asked.\n\n'On and off since I was young. Mostly off.'\n\n'You should continue. You capture whatever is going on well.'\n\nShe found his immediate presence too disturbing for conversation.\n\n'Why don't you come to my place sometime, you can have a look at what I do.'\n\nParalysed silence on her part. After a second he dropped the papers back into her lap and shouted 'Time's up', clapping his hands to get the children's attention.\n\nWhat did it mean, did he like her, did he want to have an affair with her, why had she been so startled by his hand on her knee, why hadn't she responded, but she was a married woman, with two children and those right before her eyes.\n\n*\n\n'What was Aijaz saying to you, Mama?' asked Anuradha, the sharp eyed one, in the scooter back home.\n\n'Nothing much, beta. He was looking at my drawings, that's all.'\n\n'Did he like them?'\n\n'There is nothing much to like'' said Astha, teaching her daughter how to devalue her work, and passing on the tradition from woman to woman.\n\nAnuradha lost interest. Himanshu having just grasped their topic of conversation demanded, 'What? What? What did Aijaz say to you, Mama?'\n\n'Nothing much beta.'\n\n'Then what was Didi saying?'\n\nAnuradha cast him her usual you're so stupid look.\n\n'She wanted to know what he said about my drawings, that's all.'\n\n'What did he say?'\n\n'Nothing.'\n\n'What did he _say_?'\n\n'He said they weren't bad, that I should continue, continue to draw'' repeated Astha quickly before Himanshu could say what again.\n\n*\n\nThat night, lying awake in bed, Astha went over everything Aijaz had said, she relived that touch on her knee, his head bent over her drawings.\n\nIn a few more days the workshop would end. Would he repeat his invitation? Had it been a spur of the moment thing, or was he attracted to her? Why was she so shy? Maybe she should phone him, call him over, but how, with everybody watching, it was so difficult, after this would she ever see him? How could they meet again?\n\nShe tossed and turned, trying not to disturb Hemant. If an accidental brush against her knee was so dislocating what would anything else be? And then she felt stupid, had Aijaz asked her to elope with him? No, he had merely asked her over to look at his drawings. What connection did that have with her marriage? She was a fool, a fool, a fool.\n\nOne thing was clear though, he liked her drawings, he thought she had something. He was also an artist, he must know what he was talking about. Suddenly she glimpsed possibilities, suddenly her life seemed less constricted.\n\nShe sighed, and closed her eyes, willing sleep to come, pressing herself firmly against her husband, hoping for the comfort of habit.\n\n*\n\nThe auditorium was dark. The parents in the hall fidgeted, making allowances for the twenty-minute delay in the rise of the curtain on _Babri_ _Masjid:_ _Fact,_ _Fiction_ _and_ _You._\n\n'It sounds like a bloody political tract'' said Hemant.\n\n'Don't you like it?' asked Astha, sitting next to him in the front row. 'The title was mine. Aijaz thought it was a good one.'\n\n'Darling, you would hardly go and see a play called _Babri_ _Masjid:_ _Fact,_ _Fiction_ _and_ _You_ if your children were not in it.'\n\n'No, I wouldn't, but maybe I should. There are too many people like me in this country who are not paying attention to what is happening.'\n\nHemant raised his eyebrows. 'What is happening?'\n\n'The locks on the masjid were opened to appease Hindu sentiments. Then the Muslim Women's Bill was introduced twenty-five days later in Parliament to appease Muslim sentiments. Basically both communities were pandered to as an election ploy.'\n\nHer husband stared at her. 'Are you all right?'\n\nAstha looked self-conscious. 'Of course I'm all right, why shouldn't I be?'\n\n'You sound like a parrot.'\n\n'To have an opinion is to sound like a parrot?'\n\n'Please. Keep to what you know best, the home, children, teaching. All this doesn't suit you.'\n\n*\n\nThe play was over. Himanshu came rushing over to them. 'Did you see me?' he cried. 'I was under the sheet.'\n\n'Beta, you were the best mosque anyone has ever seen'' said Hemant swinging him up in his arms. 'No wonder everybody was fighting over you.'\n\n'And me?' cried Anuradha tugging at his sleeve, 'Did you see me?'\n\n'Of course I did. You were soooo good, sweetheart.'\n\n'Come and meet Aijaz, Papa'' went on Anuradha dragging him to where the director stood, surrounded by parents congratulating him, telling him how their children had enjoyed the workshop, how they could talk of nothing but their play, and when was he going to do another?\n\nAstha watched as Hemant met Aijaz, watched as they shook\n\nhands, exchanged a few words, as Aijaz ruffled Himanshu's hair in a parting gesture, watched as he turned towards other parents. A few months later she heard he was going around with a woman working in an NGO.\n\nHer name was Pipeelika Trivedi. She lived alone in Delhi, sufficiently isolated from conventional society to believe her choice of partner concerned only herself. Her mother was horrified when she learnt of her engagement.\n\n'You can't do this'' she told her daughter.\n\n'Why not? You're the one who is always going on about me getting married.'\n\n'But not to a Muslim.'\n\n'He's sweet. So what if he's a Muslim?'\n\nHer mother clicked her tongue. 'They marry four times.'\n\n'How do you know?'\n\n'It's part of their religion.'\n\n'Do you, you personally, know any Muslim who has married four times?'\n\n'How is that relevant?'\n\n'It shows you are speaking out of prejudice. Meet him and then decide.'\n\n'It has nothing to do with meeting him. You like him, he must be nice. But everybody knows that all they have to do is say Talak, talak, talak, and the girl is out on the streets.'\n\n'She is not.'\n\n'How do you know?'\n\n'The Qu'ran says.'\n\n'How do you know?'\n\n'Aijaz says.'\n\n'It's not true. He is lying.'\n\n'Does he know more about the Qu'ran or do you?'\n\n'I know more about the world'' said the mother, tight and tense.\n\n'Well I know more about Aijaz.'\n\nThe mother looked at her stubborn daughter. 'You kept saying no to any boy I suggested for this? For this I struggled after your father died, so that you throw yourself away?'\n\n'He is not a heap of dung, you know. Besides I am almost twenty-nine, you've always said you want to see me married, now is your chance. I'm not going to find anyone else. He's intelligent, sensitive, socially committed, a history lecturer, a theatre activist, but all you can see is a Muslim who is going to both divorce me and marry four times.'\n\nThe mother stayed silent, hating to be so opposed to her daughter. The daughter wondered at the unreasonableness of her mother. They had always been so close.\n\n*\n\nMrs Trivedi, the mother, had been a widow for much of her adult life. Her parents' apprehensions about their daughter's marriage to her Delhi University teacher, twenty years her senior, were fully justified when he dropped dead one morning in front of the blackboard in his classroom. The widow was left with two small children.\n\nShe tried for a while to manage in Delhi, but it was difficult. She was too gentle, too pretty, too meek, too young and her circumstances too straightened. Her parents called her to live with them in Bangalore.\n\nMrs Trivedi went. Gentle and meek though she was, she had also been a wife, and she found it galling to fit into the daughter mode again. Added to this was her parents' obvious sense of her doom. The years with Jyotin had been the best in her life, it was an insult to his memory to be treated like a cornucopia of tragedy. She made enquiries about a teaching job in a boarding school where she could live with her children.\n\n'I am moving to Shiksha Kendra'' she announced. 'Board, lodging, and the children's education all will be taken care of.'\n\nThe parents were opposed to further movement. 'You have not suffered enough?' they asked. 'These poor children have not been knocked around enough?'\n\n'We will manage. Their home is with me, their mother, not in a place'' said the widowed daughter, showing all the signs of marriage to an intellectual husband.\n\n'On what money?'\n\n'At Shiksha Kendra I won't need to spend on the essentials. My salary will pay for the extras.'\n\nShiksha Kendra, set in a forest, miles away from nowhere, the brainchild of the philosopher S Swaminathan, a school which emphasised harmony with nature, respect for every form of life, and the all-round development of body and mind. All this the brochure said, and all this Mrs Trivedi felt when she visited the school. A home for herself and her children was what she was looking for, and at Shiksha Kendra she found it.\n\n'This school will not equip Ajay and Pipeelika for the competitive world'' warned the grandparents. 'They need to get ahead. They have no father, they are starting out with a disadvantage.'\n\n'Swaminathan got ahead'' said their daughter, somewhat elliptically, 'if fame and reputation are anything to go by.'\n\n*\n\nAjay, the son, showed his determination to succeed from a very early age. No need to tell him the disadvantages of his situation, he felt them all himself. A boy with competition in his blood, he stood first all his life, in school, in IIT, making straight to the US as soon as he possibly could with a wonderful scholarship to MIT. His success evoked tears of joy in all concerned. The widowed mother's sacrifices had borne fruit. He departed amid great jubilation, and never came back.\n\n*\n\nPipeelika, the daughter, was left to fulfil the hopes of her mother on native soil. After school, she moved up North, to Miranda House, college and hostel, to do an Honours degree in Sociology. After that an MA from the Delhi School of Economics.\n\nHer brother thought she should come to the States and do a PhD, increase her market value, he would sponsor her, but I do not wish to join the diaspora, and what about Ma, said Pipee, morally the superior. Instead, after a brief teaching stint, she joined an NGO run by three women, dealing with alternative education for slum children.\n\n*\n\nPipee had been working in Ujjala for four years when she met Aijaz Akhtar Khan, at a conference. She was reading a paper on the effects of communalism on the education of Muslim children in the basti. They were discriminated against, made to feel stupid and backward, were told their loyalties were to Pakistan, and looked upon with suspicion. Aijaz was reading on the use of street theatre in the dissemination of social and political awareness in educational institutions. Clearly their interests were similar.\n\nAfter the sessions were over, he sought an introduction, inviting her to a nearby dhaba for a glass of tea if she were free. She looked at him, the clear warm reddish light brown colour, the long thin nose, the gleaming even teeth, the thick grey hair, and then she smiled, her mouth turning in, making dents at either edge. Yes, she was free.\n\nThey talked for hours, it became dark and Aijaz insisted on escorting her back to her flat. She lay awake at night thinking of him. He seemed so gay and lighthearted, with many interests besides teaching. Not only did he manage and encourage drama activities in his college, but he was the prime mover and shaker of The Street Theatre Group. Drama was an effective way of addressing communal issues and dealing with social evils, if she liked he could bring the group to her basti. Pipee liked, and she was sure Neeraj, her friend and colleague would also approve. Ujjala was already involved in introducing drama to their children through the helpers, it would be a wonderful opportunity.\n\n*\n\nThe courtship took six months. Now Pipee wanted to marry him. Mrs Trivedi tried an old tack.\n\n'Your father would not have liked it'' she said.\n\n'He would have. My father was a secularist'' said the daughter firmly. 'Any father who names his daughter after an ant proves that.'\n\n(Pipeelika? That's not a proper name, that's a word.\n\nWhat does it mean? How do you spell it, pronounce it?\n\nIs this a real name? Never heard of it.\n\nIsn't that the Sanskrit for ant? How can you be named after an ant?\n\nAnd so on through the years.)\n\nPipee saw her mother momentarily at a loss and pursued her advantage. 'You can't deny it'' she said, 'my father didn't want his daughter's life cluttered up with references to goddesses, and now I am going to live up to his legacy. He married whom he liked, so did you, now it's my turn.'\n\n'You don't know what you are letting yourself in for. It is only later that you will realise.'\n\n'We'll wait for that day. Right now we are getting married.'\n\n'What does his family say?'\n\nPipee hesitated.\n\n'See. They are not happy either'' her mother quickly pointed out.\n\n'You are all the same. We don't care.'\n\n'Oh, Pip, everybody else will care'' sighed her mother.\n\n'Neeraj doesn't. She likes him, he is so charismatic it is hard not to like him, you would too if you gave yourself the chance.'\n\n'I keep telling you it is not him.'\n\n'So much the worse for you, Ma. Besides _Neeraj_ thinks, even if _you_ don't, that I will be happy with him, she encouraged me.'\n\nMrs Trivedi was silent for a moment. Neeraj was somebody she respected, whose interest in her daughter she had been hitherto grateful for. Now she said bitterly, 'You all work in your own NGO, and think you don't have to answer to anybody. My child you are swimming in a very small pond.'\n\n'Small pond! When I've been working with women for five years, going to all kinds of slums, seeing all manner of injustices done to people I have actually met. If we help them too overtly we alienate the community, and lose whatever influence we have. It's so frustrating. Ma, you haven't even seen a slum.'\n\n'I hope my daughter will not judge her partner by the men in slums'' said Mrs Trivedi crossly. 'And don't tell me what Neeraj thinks. I had no idea she would encourage you to go against your family and religion.'\n\nThis was not the time for Pipee to point out that she didn't give a shit about religion. 'Come on, Ma'' she said instead, wrapping her arms around her mother's neck, 'the world has changed, you don't realise it living in this tiny place. When you meet Aijaz you will love him, you'll see.'\n\n'At least make sure my grandchildren are Hindus. Once you marry God knows what he'll make you do.'\n\n'Ma! They will be his children too. He's not that sort of person, and do you think I would love him if he were? He never mentions religion, except politically, never suggested conversion, nothing. In fact you are the one obsessed with the whole thing.'\n\n*\n\nAijaz to Pipee in Delhi, 'Was it bad?'\n\n'It could have been worse.'\n\n'Poor thing, it must be very hard for her'' said Aijaz, shifting Pipee's head more comfortably on his shoulder. They had finished making love, and were talking about their marriage.\n\n'When she sees you, she will come around.'\n\n'The whole world may not have your faith in me.'\n\n'But my world does, and she is a big part of it.'\n\n'You are very close to her, aren't you?' asked Aijaz wistfully.\n\n'Of course. She is all I have. My father's family don't like my mother, we are not in touch with them, my grandparents disapprove of my lifestyle, and Ajay shows no signs of coming back. What's the use of having a son and brother if all he does is write patronising letters from the States?'\n\n'Well, you have me now, and so does your mother.' Aijaz pulled Pipee on top of him, and pushed his hands through her hair, pulling her head back so that he could look at her milky skin, and pink mouth with its indented corners that smiled in a peculiar way. She smiled now, loving the feel of his hands in her hair, the way he massaged her scalp.\n\nPipee had a lot of hair, it sprung up all around her head in waves and curls and frizzes. Aijaz loved it, loved it almost as much as he loved her breasts, large and full of give. He shifted his hands to them, and Pipee squirmed a little. She was still not used to how much sex Jazu seemed to need, but men were like that, and all the time before and after was the stuff of happiness, when they were talking, wrapped in each other's arms.\n\n*\n\nAfterwards Aijaz cast a nostalgic look around, 'I will miss this room'' he said.\n\n'I won't'' said Pipee. 'The landlord is an extortionist. This must be the smallest room in all of Delhi.' In fact it was one of six tiny ones, built around a spiral staircase in the back spaces of one of the larger houses of Greater Kailash II.\n\n'Our love grew here'' pronounced Aijaz.\n\nPipee laughed, 'Well it can flourish somewhere larger.'\n\n'Yes'' said Aijaz thoughtfully. 'Sometimes I wish I had my own flat \u2013 but out of the house all day, teaching, travelling, theatre \u2013 being a paying guest was the most convenient thing.'\n\n'Soon both of us will have a proper home. I am sick of living in hostels and rented rooms. It's been almost ten years, but now all of that is over.'\n\nPipee flung an arm out, the future glinting in her eyes. Aijaz smiled and kissed the waving arm.\n\n'You have to promise to spend more time with me'' went on Pipee. 'I refuse to be a nagging wife. You have to promise and keep your promise, and never break it, without my saying a single word.'\n\n'Of course. Why do you think I am getting married?'\n\n'Sex every minute, seems like.'\n\n'You think one needs to get married for that?' laughed Aijaz.\n\nPipee remained silent.\n\n'What's the matter?'\n\n'Nothing.' But the number of women Aijaz had had bothered her sometimes.\n\n'I want to settle down, I want a home, I want you'' said Aijaz turning to Pipee impatiently again.\n\nPipee pushed him away, 'Really Jazu, sometimes I think you just have one thing on your mind.'\n\nAijaz looked proud and manly. 'Wait till we are living together \u2013 then you will really see.'\n\n'Yes, let's see if marriage will cool your ardour.'\n\n'My ardour, as you put it, will never be cooled. And we must really start looking, it's very difficult without a company lease, or months of rent in advance.'\n\n'I've a surprise for you.'\n\n'You've found a place!'\n\n'In a way.'\n\nAijaz looked wary. 'What way?'\n\n'Now listen \u2013 listen properly\u2014'\n\n'I'm listening, I'm listening.'\n\n'You know what Premlata said to her father, when he was going to marry her off? She said thirteen was under-age and against the law, and if necessary, she would call the police! Wasn't that brave of her?'\n\n'Very. But what's the connection?'\n\n'Our efforts are bearing fruit, that's what. After three years of going to our centre at Salempuri, more children have reached literacy level, more girls are going to school, and you wouldn't believe how some of them have changed! They always worked hard, these girls, they cook, wash clothes, look after the cows, buffaloes, younger brothers and sisters, send them to school, help in the family business, they embroider, make envelopes, necklaces, sew sequins on, but are often made to feel worthless. But at the centre they develop self-confidence, look at Premlata! We want to open more centres.'\n\n'I still don't see what that has to do with us'' repeated Aijaz patiently. He had heard Pipee about her work many times.\n\n'Since we are expanding, we are going to apply for permission from the home ministry for foreign funding. Then Ujjala will hire me a flat in lieu of a raise in pay. They know I'm getting married\u2014'\n\n'I take it they don't disapprove of me, no don't tell me, our marriage is a strike for communal harmony.'\n\n'What's wrong with their approval?'\n\n'Your mother hates me because I am Muslim. Your friends love me because I am Muslim, I don't know which is worse.'\n\n'How does it matter? Look what they are doing for us, isn't that nice of them?'\n\n'Very'' said Aijaz with reserve. 'And I am sure they will extract their pound of flesh. Make you work ten times harder, demand your presence so much you will hardly be in your precious flat.' There were times when he resented the women in Pipee's life, especially Neeraj.\n\n'You don't know how women operate. Just think, we will have enough space to have my mother visit us in Delhi during her holidays, and of course your family too.'\n\nAijaz yawned and turned away. Pipee tried to suppress her annoyance. Why was the man so unwilling to discuss his family?\n\n'Have you told them yet?' she demanded.\n\n'I'll tell them, I'll tell them, what's the hurry?'\n\n'They're your family, I want to meet them, know them.'\n\n'You are so idealistic'' remarked Aijaz.\n\n'It must be nice to have so many people belonging to you.'\n\n'It's a total pain in the ass. You can deal with one person's expectations, but here there is the whole community.'\n\n'So?'\n\n'So they all take my father's side. He has never accepted my theatre activities. If his eldest son wanted to be a lecturer, the least he could do was help with the mango orchards in Shahjehanpur during the summer, instead of getting involved in some silly drama-shama. It doesn't even pay, which makes it that much harder to understand.'\n\n'We can both help with the orchards from time to time'' said Pipee enthusiastically and ignorantly.\n\n'That's not all. They wanted me to marry my cousin Azra. My mother was especially keen since she had brought Azra up after my aunt died. I suppose they were trying to make sure I eventually returned. They don't understand my life, they don't realise I have no time for all this fuss.'\n\n'I am sure they will hate me'' said Pipee in a small voice.\n\n'We'll take it as it comes. Why worry now?'\n\n*\n\nIt took six months for the grant to come through. The accommodation they ended up with belonged to Neeraj's sister's husband settled in the States. He had bought a flat in Vasant Kunj as an investment, and was now looking for a reliable tenant (i.e. one who would leave when asked). Neeraj convinced him that Ujjala and Pipee were what he was looking for.\n\nIt had two bedrooms, two bathrooms, a kitchen with built-in closed shelves, a dining area at right angles from the sitting area. Outside the sitting-dining there was one big balcony, outside the bedrooms there were two smaller ones.\n\n'I'm going to have a potted garden here'' said Pipee, stretching out her arms to the hot white sky above the verandah. 'I'm going to have everything. I can't wait to show it to my mother.'\n\n'Look at all the space! How clever you are, darling'' exclaimed Aijaz, putting his hands under her shirt, and unhooking her bra as she walked about.\n\n'Well it was really Neeraj. She always manages to find solutions to problems.'\n\n'I prefer to think it was you.'\n\n'Are you jealous?' laughed Pipee. 'You shouldn't be, she loves you.'\n\n'She hardly knows me.'\n\n'I talk to her sometimes.'\n\n'About us?' Aijaz looked appalled.\n\n'One can't be talking of work all the time'' temporised Pipee, and then to change the subject, 'Oh, I never told you, we will also have a phone, think how nice that will be.'\n\n'We could have got that on our own.'\n\n'We would have had to wait years.'\n\n'I have connections too, you know, I could have got us a phone. Now stop moving.'\n\n'Jazu, do you ever think of anything else?' murmured Pipee, as she so frequently had to.\n\n'No'' said Aijaz pinning her against the wall, seriously this time.\n\nIt was in September 1988 that the marriage between Aijaz Akhtar Khan and Pipeelika Trivedi was solemnised in Tees Hazari. The bride and groom paid for their own wedding, the whole thing came to five hundred rupees. No relatives were present from either side, a colleague of Aijaz's and Neeraj acted as witnesses, while the theatre crowd, a few of Aijaz's colleagues, and the staff of Ujjala, later gathered at Karim's to complete the celebratory aspects.\n\nPipee had arranged her work so that she would be free the two weeks of Aijaz's autumn break. They were going to Shiksha Kendra, and as Mrs Trivedi's winter holidays started in mid October, they would all come back together.\n\n'I think we will avoid my grandparents, they needn't really know we are coming. Besides they are very orthodox, and will fuss like mad over the Mozzie issue.'\n\n'I can pretend to be a Hindu if you wish.'\n\n'I wouldn't dream of it, why should you? You are not a pariah, after all.'\n\n'It's not a question of pariah, what difference does it make? Old people need to be treated carefully.'\n\nPipee needed only a second to realise the possible personal application of this remark. 'Will your family look upon me as a pariah? Shouldn't we visit them so that they get to know me?'\n\n'No, let's give them time to get used to it first'' said Aijaz. 'Besides your mother is coming back with us, and we can't complicate matters.'\n\n'If your mother came too, they could be company for each other'' said Pipee, showing how little she knew of the science of in-laws.\n\n'Another time.'\n\n*\n\n'You have to travel quite a bit to this school of yours, Pip'' said Aijaz on the second day of their train ride to Bangalore.\n\n'It's the best school in India'' said Pipee proudly.\n\n'And like all shrines, difficult to reach'' replied Aijaz looking deadpan.\n\nPipee smiled in the way Aijaz loved to see, the corners of her mouth turned in, the deepening dimples. She pinched his side several times in the crowded second class compartment. 'You'll see'' she said loftily, 'I will say no more.'\n\n'Promise?'\n\nThis time Pipee pinched him so hard, he cried out, and everybody looked at them with curiosity and disapproval. Young, alone and enjoying themselves.\n\nIn Bangalore they took a bus to Madanapalle. 'From there we will take a taxi'' said Pipee. 'It is sixteen kilometres.'\n\n*\n\nAs they drove away in the taxi towards Shiksha Kendra, Pipee grew thoughtful, the dimples and the smile went.\n\n'Why so quiet, dearest?' asked Aijaz, 'I'm not used to it.'\n\n'Nothing much.'\n\n'Come on, tell me.'\n\n'It was in school that I first fell in love, and now I am coming here on my honeymoon. I feel strange when I think about it, that's all.'\n\n'Your first love! You never told me.'\n\n'There was nothing to tell.'\n\nAijaz ignored this. 'Who was he?' he went on.\n\n'She.'\n\n'She?'\n\n'Her name was Samira.'\n\n'You were in love with a woman?'\n\n'Woman? Hardly that. Schoolgirl really. She was only seventeen.'\n\n'That's not so young. In my village girls marry at sixteen, how old were you?'\n\n'Well Shiksha Kendra is not Shahjehanpur'' said Pipee a little coldly. 'And what does it matter how old I was? It was so long ago I do not remember.'\n\n'Did your mother know?'\n\n'What was there to know? We were schoolgirls'' said Pipee withdrawing from the conversation.\n\n'Where is she now?'\n\n'She married'' said Pipee shortly. 'We lost touch after college.'\n\nAijaz fell silent. Pipee was so unlike her usual self that he didn't know what to think. It must have been like those crushes that girls had on filmstars or their teachers. She was young and inexperienced and imagined her feelings to be love.\n\nHe looked sideways at her, she was still looking remote. Did she think he was narrow minded enough to disapprove of a schoolgirl crush, he who knew of the strong ties that existed between women in the zenana? He reached for her hand. 'Don't feel sad, Pip. I am here. After all this is our honeymoon.'\n\nPipee smiled at him and thought there were some things that could not be shared, no matter how understanding the other person. All said and done she was lucky to have found him. So many of her acquaintances were still struggling, looking for love and companionship, rejecting arranged marriages, only to experience a series of heartbreaks on their own.\n\n*\n\nAt the gates of Shiksha Kendra, Pipee stopped and paid the taxi. 'I want to walk'' she said to Aijaz. 'I want you to see it slowly, take it all in.'\n\nThe path leading inside was wide with thin trees lining the way, shady, green. 'The school planted these'' said Pipee gesturing around her. 'This is a drought area, even now the leaves are drooping.'\n\nAijaz looked carefully and could see that indeed the leaves were drooping. 'They have their own dairy, bakery, their own gardens, fruit trees, imli trees, mango trees, which they lease out'' continued Pipee.\n\n'U-huh'' said Aijaz.\n\n'We were not allowed to touch any of the fruit because it wasn't the school's.'\n\n'But of course you did.'\n\n'Of course.'\n\n'Where are we going?'\n\n'They will be eating now'' said Pipee looking at her watch, 'It's one.' And she started to walk faster, though laden with bags, afraid to miss her mother in the dining hall, anticipating the surprise and pleasure on her face.\n\nThe din in the dining hall was deafening, though Pipee didn't seem to notice. Aijaz hung back as she scanned the rows of tables and benches.\n\n'There she is'' she said, making unerringly towards a particular back.\n\nHer husband remained in the doorway watching the reunion.\n\n*\n\nLater in Mrs Trivedi's two rooms in Peacock House. 'Mama, don't you like Aijaz? Isn't he all I promised?'\n\n'Very much beta'' said the mother. 'He is your husband after all.'\n\n'Let's show him where I grew up'' Pipee went on.\n\n'What is there to show? These two rooms.'\n\n'And where Ajay didn't grow up.'\n\nMrs Trivedi shot a glance at her son-in-law, who was careful to look as bland and harmless as possible. 'My son stayed at the hostel'' she said. 'It was better, he could participate more in the activities, and of course he came every day.'\n\n'For fifteen minutes'' said Pipee.\n\n'And every weekend.'\n\n'Just to eat.'\n\n'Pipee thinks he should have stayed with me like she did'' went on Mrs Trivedi. 'She doesn't realise boys need to be with their own kind. He had a housemaster who was like a father to him. And I had Pipee.'\n\nPipee pressed her cheek against her mother's, 'And now you have Jazu.'\n\nJazu looked charming.\n\n'Indeed'' said Mrs Trivedi.\n\n*\n\nThey spent ten days at Shiksha Kendra. Pipee took Aijaz over the entire campus, the banyan tree, the rocks they had to climb, the place where they watched the sun rise every morning, the art, music and dance rooms, the playgrounds, the senior school, the library, the lab where she had sat for her exams.\n\n'It's a whole world in itself, isn't it?' wondered Aijaz.\n\n'Some parents are not happy about how cut off it is, they think their children will not be able to survive outside. But look at me. I've survived perfectly well.'\n\n'Indeed you have'' said Aijaz kissing her. 'A perfectly untouched specimen.'\n\n*\n\nPipee was right, once Mrs Trivedi came to know Aijaz, she loved him. They had been in Delhi two weeks when Pipee said triumphantly, 'Well Ma, what do you have to say about Muslims now?'\n\n'He is a very good boy, beta'' responded Mrs Trivedi.\n\n'Then?' said Pipee sharply for she knew what was coming.\n\n'I am sure his family like you equally'' said Mrs Trivedi smoothly.\n\n'They will when we go to meet them in the holidays. They are a very large family, and his mother is old and cannot travel easily'' said Pipee, with a guile to match her mother's.\n\nLater to Aijaz, 'I've told her that we are going to meet your family in the holidays.'\n\n'What was the need to do that?'\n\n'She happens to think the lack of your family presence in our marriage very strange. Don't mind, she is just an old worry pot.'\n\nAijaz said nothing. Pipee felt a pang of guilt. What did it matter about his family anyway? Let them think whatever they wanted, she should not make it more of an issue than he did. Besides Aijaz had been so sweet to her mother, coaxing her from her prejudice, never seeming to mind her oblique references to Muslims, four wives, large families, instant divorce, inter-community marriages, the religion of babies from such unions. The more she relaxed with him the more she wanted to know.\n\n'There is a limit to your questions'' Pipee shouted one day. 'Is he your son-in-law or the whole Muslim community dating from Babur's time to now?'\n\n'It's all right'' said Aijaz soothingly. 'Let her ask. After all I am the first Muslim she has had anything much to do with.'\n\n'Still.'\n\n'I'm used to this. She is not alone.'\n\nSuch gentleness deserved to be rewarded by total belief. Pipee vowed that she would never mention Aijaz's family unless he himself brought them up.\n\nAfter Mrs Trivedi left, the young couple settled into the joys of living on their own.\n\nPipee's hours were flexible, and she tried to be home by the time Aijaz arrived. This was usually not difficult. After a morning of teaching Aijaz was often at college rehearsals, or working out programmes with The Street Theatre Group. And, thought Pipee indignantly, everybody imagines academics to have nothing but free time.\n\n*\n\nUjjala was by now in the process of establishing a community centre at another basti. This second place had a greater number of facilities. It had sewing machines for women to acquire a skill to increase their earning potential, it had a library, toys, and art and craft supplies for the fifteen to twenty children who came every afternoon from three to five.\n\nSoon they extended their activities by organising trips outside Delhi. The results were encouraging. Girls, helpers and administrators bonded, and the girls' sense of themselves strengthened. Each of them wrote a piece on how she had experienced the trip, what she had felt being away from home with others from the basti, for the first time not part of a family structure. Pipee put these together in a series of booklets called _Yatra_ _aur_ _Vichar_ that she spent many hours over. She was filled with a sense of achievement, all day with Ujjala, every other moment with Aijaz, she thought life could have no more to offer.\n\n*\n\nIt was almost a year after their marriage that Aijaz made a casual announcement. 'We have to go to Shahjehanpur. They want to see you.'\n\nPipee, lounging on the cushions of the cane double-seater they had recently bought, looked up, astonished. In all this time Aijaz's family had shown no signs of her existence.\n\n'That's nice'' she said carefully.\n\nAijaz stared moodily at the balcony. Pipee gazed at him, and for the thousandth time thought how she loved the way he looked. His wavy grey hair, his clean brown colour, his sharp nose, his warm eyes. 'You know my mother also had her reservations'' went on Pipee encouragingly.\n\n'Mine would have too, had she known'' muttered Aijaz.\n\n'What? What did you say?'\n\n'You heard me.'\n\n'Do you mean your mother \u2013 family \u2013 didn't know we were getting married.'\n\n'Something like that.'\n\n'You didn't tell them?'\n\n'How could I tell them?' demanded Aijaz. 'You knew the problems.'\n\n' Still, you could have _told_ them. They must be feeling awful now, much worse than if you had _told_ them.'\n\n'For God sakes, Pip, stop going on.'\n\n'You hide things from them, from me, and you accuse me of going on'' shouted Pipee. 'How do you think I feel?'\n\n'You have to take me as I am'' shouted Aijaz back. 'Me, alone. If I didn't tell them it was to spare them pain, and you trouble.'\n\nPipee tried to tell herself that Aijaz was an exemplary human being, socially committed, personally tender, but this palliative irritated her further. He had no moral right to behave in a way that didn't add up.\n\nAll the things her mother used to say about Hindu-Muslim marriages came unpleasantly to her mind. For a moment she stared at him with revulsion. What was the use of him looking like a dream if he could behave like a nightmare?\n\n'What the hell, Aijaz'' she said, 'you have a poor idea of trouble. You have not been fair to your family or to me.'\n\n'I'm sorry, Pip, I really am, don't be angry. My family is not like yours. There are so many, and they all want to be part of things, they would never have tolerated a Tees Hazari wedding, we would have had to go there and get married amid five thousand people at least, God it's enough to put anyone off. And then there might have been fuss about the conversion thing \u2013 I didn't wish to put you through all that.'\n\n'Or yourself'' said Pipee dryly.\n\n'Whatever'' said Aijaz, looking charming.\n\n'Well, why now?'\n\n'They heard rumours. Made enquiries.'\n\n'So I am going to meet them with the guarantee that they will hate me.'\n\n'They'll adore you.'\n\n'With this background?'\n\n'You don't know my family. Once they know they can't change things, they just accept them.'\n\nThis time Pipee kept her reservations to herself. 'When are we going?' she finally asked.\n\n'We are on the waiting list. As soon as I can get confirmed bookings.'\n\n'What about my work? We have a big meeting with the community helpers from both centres next week. Neeraj, the others, won't like it.'\n\n'Tell them you are going to visit your Muslim in-laws. They will love it.'\n\n*\n\nTheir reactions were reserved, which was just as I expected, thought Pipee, and shows how little Aijaz knows of families in general.\n\n'I hope they like me soon'' she said to him on their first night in Shahjehanpur.\n\nIt was summer, and their beds were spread on the terrace, in deference to their married status, a little separately to one side of a storeroom. As soon as it was late enough they squeezed together in one under a mosquito net.\n\nAijaz yawned. Pipee poked him. 'Do you think they will?' she asked.\n\n'Give it time, Pip, now let me sleep.'\n\n'I told you they wouldn't like me.'\n\n'They are so glad I'm married, they would have liked anyone.'\n\n'But they would have preferred a Muslim?'\n\n'Come on, Pip, be reasonable. After all your mother would have preferred a Hindu. Anyway who has the time to worry about such things?'\n\n'I suppose'' said Pipee forlornly, thinking of his mother and the jewellery box she had pushed in front of her.\n\n'For my eldest son's wife'' she had said.\n\n'No'' said Pipee, embarrassed, yet dying to look at what was inside.\n\n'Take'' said Ammi, with a trace of reproach, her hands busy with the lid. Pipee gazed at the plump, rounded fingers, studded with gold rings, the short nails which gleamed with clear nail polish, the kurta sleeves long and fitting, and the many bangles that tinkled at her wrists. She looked very sure of herself, unlike her own poor mother, who lived in two rooms at Shiksha Kendra, with no one to boss over except some very small children.\n\nEventually, since she would not take, she was given a heavy gold necklace, thick gold bangles embossed with flowers, and a set of jhumkas set with pearls and rubies.\n\nShe held them, admiring their beauty, marvelling at their heaviness before returning them, I have no locker, I will have to worry about their safety, keep them for me please.\n\n*\n\nIn the days that followed, Pipee realised for the first time she had married a Muslim. Everything was strange, the large haveli, the dishes they ate from, the food they ate, their paan making, the way they dressed, the way they greeted each other, As salamalaikum \u2013 Wa Alaikum Assalam, their manner of speaking, the kh's that made her Hindi tongue seem crude and unsophisticated.\n\nAnd then there were so many relatives. How many people lived in that house, till the end of her visit she did not know. They were a world complete unto themselves, so different from anything she had known while growing up. Occasionally when eating in the long dining hall, she would gather as many as she could within a single glance and feel a great longing for the day when she would be completely accepted as one of their own.\n\nIt was the year 1989, and bricks were being collected for the Ram Mandir \u2013 collected, worshipped, and escorted out of towns, wrapped in silk and saffron, on their way to Ayodhya. If communal disturbances occurred in the wake of these processions, that was not the fault of the bricks, but the fault of the narrow-mindedness of minority communities, who couldn't bear to feel that their domination in this country was over.\n\nIt was in this atmosphere that Aijaz and The Street Theatre Group travelled to Rajpur fifty kilometres outside Delhi to put up a play.\n\n'I wish you wouldn't go'' said Pipee, 'Rajpur is a sensitive area. It is not safe.'\n\n'If I only went to places where it was safe, I would never go anywhere'' said Aijaz. 'Theatre is a limited medium, but what else do people like us have?'\n\n'Then don't go'' said Pipee, 'don't go if it is no use.'\n\nAijaz looked depressed. 'One has to do what one has to do'' he said. 'Of course it is so much easier working with people from schools and colleges, they even write the scripts, and do the research.'\n\n'Well stay here, and go to schools and colleges, instead of dashing out on weekends to some town or mohalla, or factory, god knows where all. Now you are married you have a responsibility to me, to us'' said Pipee, and then felt guilty. Here she was sounding like a nagging wife. Would she like it if Aijaz stopped her from going to the bastis? Or decided that her work with Ujjala led her into dangerous situations?\n\n'What is the use of confining oneself to the middle classes where it is safe \u2013 safe and cowardly'' went on Aijaz reflectively.\n\n'At least wait till the whole fuss about the bricks is over'' amended Pipee. 'I will come with you next time. I've never travelled with you. Besides you will be leaving me alone on New Year's Eve.'\n\nAijaz looked at her in astonishment, 'I never knew this day meant anything to you, Pip. It's just a capitalist device for making money.'\n\n'If it can keep you home, then I am a committed capitalist.'\n\n'You are being totally neurotic. When I go somewhere nice, then you come. The mohallas of this township are dirty and crowded, there is nothing much to see or do. I'll be worrying about you, instead of concentrating on the play and the group.'\n\n'I can take care of myself'' said Pipee with great dignity.\n\n'So can I'' said Aijaz ruffling her hair.\n\n'Didn't you know this man?' asked Hemant looking through the papers three days later.\n\n'Which man?' asked Astha indifferently, her life an arid desert so far as men were concerned.\n\nHemant flapped the papers in front of her. There, in the middle of page three were the headlines, THEATRE GROUP BURNED ALIVE IN VAN, and below the story:\n\nA horrendous incident took place here last night, in the township of Rajpur. Aijaz Akhtar Khan, noted theatre activist, and his troupe were dragged from the site of their performance, and taken away in a Matador. Later the charred remains of the Matador along with the bodies were found near the river. The culprits are still absconding.\n\nIt is surmised that rising tensions between two communities led to this action. Aijaz Akhtar Khan, leader of the well-known Street Theatre Group was in town to perform in the mohallas. The issues dealt with were of a sensitive nature, and it is surprising that in this time of communal unrest he got permission to stage a piece involving the Babri Masjid-Ram Janambhoomi controversy. The District Magistrate says he was deliberately misled about the contents.\n\nAccording to our sources, a procession containing bricks for the proposed Ram temple in Ayodhya was routed through a gully adjacent to a minority community mohalla earlier in the afternoon. Despite the presence of the police, slogans were shouted. Untoward incidents were then avoided, but that evening violence, possibly premeditated, broke out during a performance by The Street Theatre Group. Unruly elements in the crowd started heckling the actors. Other elements responded. In the confusion the members of the group were driven away in a van, ostensibly for safety. This seems to have been a ploy.\n\nAijaz Akhtar Khan has left behind a wife.\n\nThere followed a list of the other members of the theatre group, along with their survivors, but Astha could not read further for the tears in her eyes. What a way to die, what a horrible, horrible way to die \u2013 and for what? Because the man was trying to reach people and do some good. She hadn't even known he was married. She turned away her head to cry some more.\n\nHemant, watching her, immediately lost his temper. 'Why are you crying?' he demanded. 'What was he to you?'\n\n'Some murderers trap and burn a whole theatre group in a van and you ask me why I am crying?'\n\n'This kind of thing happens all the time, I don't see you wasting your tears.'\n\n'I can't weep for the whole world, only when it means something to me. Maybe I am deficient, but I knew him, he was always working for everybody's good, even the children loved him. And he has been burnt to death. Isn't that reason enough?' she sobbed rocking to and fro with rage and grief.\n\n'Don't get me wrong, this should not have happened. But if you meddle in things that do not concern you, you have to take the consequences. He was a Muslim, he should have kept to the issues within his own religion.'\n\nAstha stared at her husband in revulsion. Ten men had died in the most ghastly way possible, and this was all he could say. Did he have no feelings?\n\n*\n\nAfter Hemant left for work she started phoning. Identification of the bodies was being done at Willingdon Hospital, they would probably be released the next day. A condolence meeting was being held that afternoon at the Constitution Club. The next day there would be a funeral procession that would start at the Club and go all the way to the electric crematorium.\n\nNumbly Astha put on a white sari, she would go straight from school to the meeting, at least she would be with people who felt as she did. She would meet his wife, what would it be like to be her at this moment, and to have your husband dead like this. Could you ever get over it, should she arrange for the driver to bring her children there after school, they had known Aijaz, they would grieve with her, they should be exposed to the political realities of this country, but then to be exposed to such violence, such mindless hate, how could she explain it, she could barely deal with it herself. Political realities could wait, Mala would look after them, if she was late they could go upstairs.\n\n*\n\nAt the Constitution Club mourners were gathered on dusty lawns, standing on sidewalks, dressed in white, with black armbands, sombre faced. There were many speeches:\n\nWe are witnessing crimes deliberately stoked by the forces of communalism. Neutral voices are seen as threatening, the voice of secularism is not tolerated. Can ten men be burned alive, taken from the mohalla in full view of everybody without connivance from the authorities? What has the State done so far, what have the police done so far to apprehend the criminals? Is this the message for the citizens of this country, live in fear, do not raise your voices for they will be stifled by fire, murder and violence.\n\n_This_ is what the state provides, _this_ lawlessness, _this_ disregard for life, _this_ brute force. _This_ is its protection for its citizens.\n\nTo speak and be heard is the freedom that is at the heart of a secular nation, this is the right for which these brave young men gave their lives. Now we must carry on as though they were in our midst, forcing us to resist repressive fascist forces. This is the struggle that lies before us.\n\n*\n\nAstha saw Mrs Dubey, her eyes damp and swollen, she went to her and touched her on the shoulder, they stared wordlessly at each other, and then Astha's own tears, soaking her hanky, her nose running.\n\nIt grew dark. Candles were lit and passed around. They started singing. Songs of protest, songs that Aijaz had penned, songs that many had sung in different circumstances. They ended with _We_ _Shall_ _Overcome_ in Hindi. Word went around about the funeral arrangements. Tomorrow they would start at noon from the Club and walk all the way to the Crematorium with the ten bodies. Let the city see the atrocity that had been committed, let the traffic come to a standstill, let the line of death be visible in slow motion.\n\n*\n\nNext day there was a crowd of thousands waiting for the bodies to be released from the hospital. Many had not known the ten men, but it was not necessary to have known them. They came to protest an outrage, to arouse similar protests from an anaesthetised public. Artists and innocent men have been murdered without any provocation during a performance in broad daylight. Today them, tomorrow us. How can this happen? What can we do?\n\nFinally the procession started. On and on they walked, blocking traffic, creating havoc, silent, disciplined and determined. The police tried to stop them, they did not have permission, they would have to turn back. The news spread \u2013 they are trying to stop us, we are going to defy them, nothing can turn us back, we will fight if necessary and then the police had to give in, escorting them instead, as they walked down the streets of Daryaganj, past the Jama Masjid, turning right towards Ring Road, then on to the electric crematorium, where thousands more were waiting to receive them.\n\nIt took six hours to reach their destination. The vast room quickly filled while the rest of the crowd waited outside. The families of the men laid the bodies out, and two by two their charred remains, indistinguishable from one another, were slid into the massive fires and the doors clanged shut. They had been together in life, and they were together now. Silence occupied the hall. Astha watching from a squeezed-in place near the door remembered the Aijaz she had known, and that once she had thought he smiled too much.\n\n*\n\nFour days later a massive protest rally was organised from the Red Fort to the Prime Minister's house.\n\n'I shall be late coming home from school today'' said Astha to her husband that morning. Her tone was cold; she had still not forgiven him.\n\n'Why?' he asked busy with his own preparations for the factory. 'Where are you going?'\n\n'To a rally to protest the circumstances of ten men's deaths.'\n\nHemant looked at Astha. Astha returned the look.\n\n'Whenever did rallies do any good? Goondas hire people from neighbouring villages at ten rupees a day to come and make trouble, block traffic and show their muscle.'\n\n'It's not the political, made-up kind of rally. We want to draw attention to what has happened. How does one speak so that one is heard? You tell me a better way.'\n\n'Rallies!' snorted Hemant ignoring the question. 'No matter how big \u2013 who cares \u2013 who remembers what they are about?'\n\n'Besides, we don't want their memories to die.'\n\n'I'm sure you don't.'\n\nAstha left the house without a further word.\n\n*\n\nBy the time school had finished and Astha reached Red Fort, the air was thick with banners. Some of the marchers were carrying posters with Aijaz's photograph hugely blown up. Some were carrying banners with Leftist slogans. Black armbands were being passed around.\n\nThe rally set off. Down the road, shouting slogans, they marched, blocking traffic in a way that Astha found most satisfying. Cars were standing still, motorists were fuming, and people were getting late because of her. She shouted with the others:\n\n_Sampradayakta_\n\n_Down_ _Down_\n\n_Down_ _Down_\n\n_Communalism_\n\n_Will_ _not_ _succeed_\n\n_Will_ _not_ _succeed_\n\n_The_ _Street_ _Theatre_ _Group_\n\n_Martyrs_ _All_\n\n_Aijaz_ _Akhtar_ _Khan_\n\n_Remembered_ _Forever_\n\nWhy did they have to die like this, thought Astha, trapped in a van, what were his last thoughts, he who had lived for others. How was there any fairness in the world when such a man could be murdered so brutally? Tears came to her eyes, but tears were not an adequate tribute to Aijaz, they were too ephemeral.\n\nHe had seen talent in her, what was it like to live with a man who saw you as having something to offer? If only there was some cause to which she could devote herself, maybe she would not feel so lost and dissatisfied, but what, and how? Knowing what to do was so difficult, and brooding over her life she continued to shout and raise her fist with the others. Down Red Fort Road, past the Asian Circus, past the Centre for Tibetan Refugees, past the Kashmiri outlet for woollen shawls, past the police chowki, past water sellers, lemonade sellers, past bhelpuri wallahs down Connaught Place and Janpath marched the procession. Compressed into half the road, cars were inching along, staring at them, curious, sympathetic, frustrated, annoyed.\n\nThey reached the boat club. Astha sank under one of the trees, extremely hot and tired. She had not realised her clothes were unsuitable for marching in the sun, she was wearing a thick black polo neck sweater, with Hemant's vest on underneath. This meant that though damp and hot, she couldn't possibly take it off and be exposed in her underwear.\n\nThe speakers on the stage were beginning to talk about state atrocities, an endless list. After that were impassioned recitals of Brecht's poetry in Hindi. Fists were clenched, defiance was hurled towards parliament looming above the tree tops behind the boat club.\n\nAn hour later the procession set off towards the prime minister's residence. Three roads away they met a police block. 'No further'' said the policemen. 'Question of security.'\n\nThey handed over their memorandum, and were forced to disperse.\n\n*\n\nAs Astha was leaving, her principal stopped her. 'Astha meet Reshana, she used to be a singer for The Street Theatre Group. She was especially close to Aijaz.'\n\nAstha stared at the direct eyes, the face still with sorrow. Especially close \u2013 how close was that? What about his marriage \u2013 was she close before or after?\n\n'I am trying to meet all those who worked with him'' Reshana was saying through swollen lips. 'We have to make sure his memory does not die, are you interested?'\n\n'There is nothing I wouldn't do for him'' breathed Astha.\n\n'Good'' said Reshana. 'I will inform you of our first meeting.'\n\nAs Reshana left, Astha turned to Mrs Dubey, 'Who is she?'\n\n'Reshana Singh. She is a classical singer from an old and established family. She has many connections, it is good she is taking such an interest in this cause.'\n\n'Is Aijaz's wife not here?'\n\n'Poor thing, I only saw her at the funeral. I don't think she is able to cope with the shock of it all.'\n\n'I would have liked to meet her.'\n\n'When she recovers, we can arrange something.'\n\n*\n\nIn the evening Hemant asked somewhat testily how it had gone. Astha was too full of the day to continue angry with him. If he was limited, that was his misfortune, she could be generous. Where should she begin, the crowd shouting slogans, the palpable determination to do something, singing _We_ _Shall_ _Overcome,_ the sense of togetherness, her excitement at Reshana asking her to be part of the new society.\n\n'The traffic arrangements were terrible as usual'' said Hemant, not realising she had an answer. 'I had a meeting with the distributor in Connaught Place, and getting there was totally impossible. Why do they allow rallies in the middle of the day, in central Delhi, I'll never know. Arre, you want to protest, protest, who is stopping you? Let the ordinary tax-payer lead his life, that's all I ask, but no.'\n\nAstha's generosity was not required, her sharing could keep. She could not enter into his frustrations, he could not share her enthusiasm.\n\nFor the rest of the evening, they talked of the children, Hemant's concern about his mother's arthritis, his father's blood pressure, his forthcoming trip to South Korea, and maybe they could all go abroad next year for a holiday, and finally something that was beginning to bother him more and more, the increasing competition in colour TVs.\n\nIn Noida alone where Hemant had his factory, eight others had come up. He was making 1,500 black and white, and 1,200 colour TVs a month, but the market had become so cut-throat that he was forced to reduce his profit margin to maintain his position.\n\nNever mind, Astha tried to console, dragging her mind to business concerns, now that the government has allowed religion on TV, there will be no end to the shows that will have the same kind of popularity as the _Ramayana_ and the _Mahabharat._ There was a captive audience of millions, with a big enough market for all.\n\nHemant grunted. That very day he had heard that a rival factory was trying to instigate a strike amongst his workers. He had managed to bribe the men in question, but the general atmosphere of suspicion made it difficult to go on bribing. He would discuss the problem with his father in the morning. \nChapter V\n\nA few days later at the meeting of the Sampradayakta Mukti Manch, a forum set up in memory of The Street Theatre Group, it was decided that painters should donate a painting for an exhibition devoted to worker unity and secularism. Astha was busy staring out of the dirty windows, where was his wife, was she still getting over her grief, how come she wasn't there, would she never get to see her, when she heard Reshana address her.\n\n'Astha, what about you?'\n\nAstha panicked. Why was Reshana asking, had Mrs Dubey made claims about her talent, but she was no good, she was a beginner, a drawer, a sketcher, nothing serious, her support was absolute, but she could do nothing on her own.\n\n'I'm not sure'' she managed.\n\nReshana smiled warmly, 'Just try'' she suggested.\n\nAstha felt the disapproval of the gathering at her delayed consent. There were too many people looking. She nodded, and sank back into her chair.\n\n*\n\nHer anxiety over her task was so great, she had to start immediately. After school the next day she sketched crowd scenes, patterning them on Rajasthani miniatures, trying to choose between a funeral and a procession. Finally she decided there would be more colour and interest in a procession.\n\nShe painted a broad road, on one side lines of figures, dots of black hair, holding banners, on the other side, rows of cars, scooters, taxis, cycles, and bordering this the white shops of Connaught Place, the trees in the central section, the massed pedestrians, the large blue sky.\n\nShe could think of nothing but her painting. When she was teaching, her mind was on her figures, the spaces, the colours of her canvas. At home, after lunch, she painted, and as a result there was no time to take the naps she had been used to. Her headaches became worse and often in the evening, after the children's homework, she lay on the sofa, balm smothered, dopey with pain killers. When the pain was very bad she threw up. She tried to keep this from her husband, to participate enthusiastically in his social life. But he did notice, and he did mind.\n\n'Why are you doing this to yourself?' asked Hemant one evening, when it was obvious she was in pain, the smell of balm all pervading, eyes drooping, brow furrowed, face contorted. 'You can't paint and teach, every time I come home you are lying on the sofa. You are suffering, we are all suffering.'\n\nCertainly suffering was involved, it was true. Astha remained silent, her shoulders hunched. Assent and helplessness.\n\n'Your body cannot stand the strain. Mummy said you are neglecting the children, you do not sleep in the afternoons, you are exhausted in the evenings, you are spreading mess in the house, everything smells of turpentine. And all for what? Some dead man.' He never mentioned the nine others.\n\n'It's not for some dead _men,'_ flashed Astha, 'it's for a cause. And I'm sorry your mother found it more convenient to complain to you instead of me.'\n\n'What is it to her? She has your interests at heart.'\n\n'I have a better idea of my interests.'\n\n'It seems not. You can't do everything. Leave your job if you insist on painting. It never brought in enough money to justify your going out of the house.'\n\n'You were the one who thought I should work.'\n\n'But now you need not, dearest, I am making enough money.'\n\n'I want something of my own'' murmured Astha.'\n\n'What?'\n\n'My own money'' though she knew it was contrary to the spirit of good marriages for a wife to hang on to things and say they were her own.\n\n'You make me sound like a stingy husband, Az'' said Hemant in some hurt.\n\n'No, no, that's not what I meant'' she said weakly.\n\n'Then? Quit for heaven's sake.'\n\nBut she was not yet enough of a painter to risk giving up a job she had had for ten years. It represented security, not perhaps of money, but of her own life, of a place where she could be herself.\n\n*\n\nReshana phoned frequently, inquiring about the progress of the painting, once dropping by, flattering Astha by her interest and her praise.\n\nAt this visit Astha asked, 'Reshana, how come I never see nor hear anything of Aijaz's wife? She must still be really devastated, poor thing'' she added in case her remark was construed as criticism.\n\nReshana made a face, 'Just between you and me Astha, some people have a problem working with others. I do not wish to say more.'\n\n'Oh.'\n\n*\n\nSix months after the massacre, the exhibition was ready to be held.\n\n'Ten thousand rupees'' said Reshana.\n\n'Ten thousand!'\n\n'We need the money.'\n\n'But will people buy? I'm not known.'\n\n'It's a large canvas. It is good. If you were known I would have priced it at fifty thousand.'\n\nTen thousand rupees \u2013 the outrage among spectators, each one saying, I wouldn't take this canvas if you _paid_ me ten thousand. She would be tested, tried and rejected. But the money was for the Manch, she couldn't protest too much.\n\n*\n\nReshana was right. The painting sold on the second day. The crowd was large, many people wanted to help the anti-communal cause, especially if they could get something in exchange.\n\n'I have told all my friends that my mother has sold a painting'' said Anuradha, looking important.\n\n'Only one, darling'' said Astha.\n\n'So what? They were very impressed. None of their mothers is painting.'\n\n'Well don't say too much about it, it was not because of me. People bought it to help those harmed by state violence.'\n\nAnuradha's face darkened as she stared at her mother, and Astha knew she had ruined her satisfaction. She wanted to say yes, I have done it, I have sold my first painting, I have achieved something, let us celebrate, but the number of 'I's' involved ensured that the words refused to leave her mind.\n\n*\n\nThe Manch was anxious not to lose the impetus it had gained and efforts were made to chalk out a long-term plan of action. Unfortunately the Manch also had its fair share of members who could not agree, and valuable time was spent in arguing. Some talked excitedly of the international recognition their cause could get with a film that would document communal atrocities in the villages of North India. It could end with the murder of The Street Theatre Group so that the middle class could also relate to the theme.\n\nSome wanted to start at a more grass roots level, doing the kinds of things Aijaz had done, street plays, slogans, posters, meetings, pamphlets, consciousness raising.\n\nSome wanted to bring anti-communal activists and academics together to exploit the forum of the written word, maybe start a journal. Others thought this was too elitist, too far from the spirit of the theatre group.\n\nSome wanted to concentrate on bringing out a collection of the writings of various members of the group, while objectors felt that since Aijaz was the main person who wrote, it would be like bringing out his writings, and such individualism was inimical to the spirit of the Manch.\n\nSome felt that all their energies should go towards bringing the killers to book. Not a single arrest had been made so far, and this just mirrored the complicity of the police in communal riots and murders.\n\nMost felt this would only end in frustration, and with the rampant corruption of the government they might as well bang their heads against a brick wall for the rest of their lives. The need of the hour was for positive action.\n\nAt last a sub-committee was formed. They would present a report, everybody would meet again.\n\n*\n\nAstha sat silently at the back, her head bent steadily on the moving hands of her watch, and as the hour advanced so did her alarm. It was getting late, the children were upstairs, their homework had to be attended to, Hemant would be coming home.\n\nAs she got up to go, Reshana, near the door, put up her fingers. 'Five minutes'' she mouthed. Astha sank back in her seat. She felt the familiar pain marching across her temples to the tune of what were five minutes.\n\nIt took twenty-five. Astha was in agony. Reshana turned to her once in the middle, winked and smiled, enclosing her in a conspiratorial glance from which Astha was powerless to escape. She thought of the dinner, they could order some chicken from the neighbourhood restaurant. Rice, a salad, potatoes fried in cumin and coriander, it would only take a minute, there was the dal from the afternoon.\n\nThe meeting over, Astha made her way to Reshana. 'I have to go'' she whispered, 'it's getting very late.'\n\n'Stay a moment, I want to introduce you to someone who really liked what you did.'\n\n'His wife?'\n\n'No. This man is a film maker.'\n\nPyjama-kurta, grey beard, grey hair, 'I loved the emotion portrayed in your painting. I wish I could have afforded it, but Reshana here had priced it too high.'\n\n'Very funny, Arjun'' said Reshana distractedly, 'If we start buying our own work we might as well kiss the Manch goodbye. And here, Astha, meet Kabir, he was a good friend of Aijaz's, they used to perform together. He is on the sub-committee.'\n\nKabir blew smoke through his cigarette, and smiled at Astha, 'Tell me about your other work.'\n\n'I have not exhibited anything else.'\n\n'You must do more.'\n\n'Thank you'' said Astha in some confusion. She could barely keep her voice from trembling.\n\nReshana looked at her. 'Are you all right?' she asked.\n\n'I have a headache'' said Astha, clenching her teeth, and carefully enunciating her words.\n\n'Oh you poor thing. Why didn't you say? Come let me walk you to a scooter stand.' On the road Reshana gave Astha a brief hug. 'Take care.'\n\nAstha replied, feeling foolish, 'You too, see you, bye'' and turned to a scooter man who was cleaning his teeth with a neem twig.\n\n'How much to Vasant Vihar?'\n\n'Thirty.'\n\n'Too much.'\n\n'That's what it costs.'\n\n'I'll pay by the metre.'\n\n'Metre not working.' The scooter wallah spat on the road to emphasise his point.\n\n'Twenty-five'' argued Astha, 'I pay twenty-five every time.'\n\n'Thirty. At this time I won't get a fare back'' he added to make the defeat easier for her.\n\nAstha sat inside. The scooter wallah, galvanised into action, threw away his neem twig, and jumped vigorously up and down on the pedal. It didn't start. He flung open the seat, took out his tools along with a rag, fiddled with something underneath, carefully wiping his hands every five seconds.\n\n'I can take another auto'' Astha pleaded. She didn't dare look at her watch.\n\nThe scooter wallah glared at her. 'I'm fixing'' he stated. 'Nothing wrong. Just fixing.'\n\nFinally the vehicle coughed and shuddered. Astha's head throbbed along with everything else.\n\nAt the next red light more stalling. With cars furiously honking behind it, the scooter was reluctantly pushed to the side of the road, and tinker, tinker, on and on before it sputtered into life, only to collapse on the bridge over the tracks of the rail museum. Astha could stand it no longer. She jumped out, opened her purse defiantly, and thrust fifteen rupees towards the man's hand.\n\n'What's this?'\n\n'Your fare. Or don't you want it? This is the worst scooter I have seen in my life. You have made me late, very late.'\n\n'What can I do? The scooter is about to start. Just fixing.'\n\n'No. Here.'\n\n'Twenty.'\n\n'You make me late, and now you are arguing about the fare.' Astha was almost beside herself.\n\nThe scooter wallah was not impressed. 'I'm a poor man'' he insisted, scratching his balls. 'What can I do? The fare is twenty till here.'\n\nAstha lacked the courage to throw fifteen rupees at the poor man and walk away. She thrust another five towards him, and walked down the bridge towards the next stop light, where there was a cab stand. She was coated with dust. The sound of traffic roared in her ears, there would be the problem of dinner waiting for her and the children's homework which would not have been touched.\n\n*\n\nIt was clear from the moment she stepped inside that she was in trouble. Hemant received her frostily, no question as to how was the meeting, you are looking tired, are you all right, I will look after things, you go and lie down.\n\nInstead there was silence through the hastily put together meal, silence as she went through the children's notebooks after dinner.\n\nHimanshu wrinkled his nose at the balm on her forehead. 'You smell, Mama'' he complained.\n\n'Sorry darling, my head is hurting'' murmured Astha.\n\n'Shall I press it?' he asked. Himanshu liked pressing his mother's head, and she liked having him do it, the touch of his small inept hands soothing to her.\n\n'All right'' she allowed. He scrambled into her lap, and put his face next to hers, managing to jab her eye with his finger. The discomfort was slight, but the tears still came. The day had been too much.\n\n*\n\nThe homework was finished at last, the school bags packed and the children asleep. Before sinking her head onto her own pillow and blanking out the whole horrible day, Astha had to try and make amends with Hemant. He had come home, she had not been there, he must have been surprised, wondering, maybe even worried.\n\nFrom the passageway she could see him in his reclining chair, with his newspaper, feeling lonely. She was his wife. Still she looked, feeling exposed in her thin nightie, breasts hanging loose and obvious, eyes watering with fresh balm. She lifted her feet to go towards him, but found herself walking to her bed. She was tired, her feet were telling her, and tired women cannot make good wives.\n\nThat night as the pain receded and she fell asleep, she dreamt. She and another person were riding close together in a scooter \u2013 rickshaw. The person turned, it was Aijaz with long silky hair, which brushed across her face. Astha leaned closer, the corners of their mouths met and pressed, alone against the commotion of the street. Slowly Astha opened her mouth, and bit on the hair. She didn't let go, even when the scooter stopped, and they got out, her mouth firmly clamped on the rich, long, black, thick, sweetly smelling, dusty hair. This made her dumb, she could not argue with the scooter wallah, who was charging too much, but Aijaz took care of him. Aijaz took care of everything. Together, they walked into a room full of doors and windows, with a huge double bed in the centre. Blue and white curtains waved in the breeze, sunlight came flowing through, the bed was covered with soft, printed Rajasthani quilts. Doors opened, people walked in and out, but they were invisible.\n\nSlowly they fell on the bed, kissing all the while, when Aijaz, entwined around her, turned into Reshana.\n\nReshana?\n\nShe woke. It was early morning, the sky was lightening, she could hear the birds beginning outside. Deeply unsettled, she turned to Hemant, opened his pyjamas, gave him an erection and climbed on top.\n\nHe forgave her sins of the evening before by responding.\n\n*\n\nThe disturbance lingered with Astha all next day, the vividness and strong emotions of her dream demanding some kind of recognition. Hesitantly she started making sketches. Two women faced each other in a scooter, their noses covered because of the pollution, only the eyes visible. The scooter wallah was a dark Sardarji with a striking red turban. Perched next to him was a young man, taking a ride. Around the edges of the canvas, traffic, buildings, road, but in the centre the scooter with its passengers bent towards each other, the devouring eyes, the Sardarji and the young man.\n\n'How's it going?' Reshana phoned to ask.\n\n'Fine'' said Astha briefly, not wanting to engage with Reshana when her head was full of other things. She would think about the Manch canvas when she had finished this one.\n\n*\n\nNow that Astha was devoting practically all her afternoons to painting she found it difficult to work inside the house. There were too many interruptions, the servant, the children, the phone, the kitchen, her own restless mind. Besides which she was continually observed by whoever happened to be around, watched intently as she made preliminary sketches, prepared canvas, squeezed paint, mixed and applied colour, cleaned brushes. She could not say go away, that was rude as well as selfishly withholding of herself.\n\nThe canvases also meant that when they entertained guests, certain conversational sequences were invariably set in motion \u2013 who paints, my wife, oh really, very good hobby for a woman, my daughter also paints very nicely, or my sister, or wife's sister \u2013 you name it, there was always somebody who knew somebody who painted. Each time this happened Astha was forced to make her work the subject of idle gossip, a thing she hated doing.\n\n*\n\nShe mentioned this to Hemant one weekend. They were in the bedroom lying off a heavy lunch eaten upstairs.\n\n'I need more space.'\n\nHemant drew her close. 'The whole house is yours, Az.'\n\n'I was thinking of something more specific. You know, a place to work in peace, spread my stuff about.'\n\nShe knew it sounded presumptuous and unfamily-like to want space that was hers and hers alone. Hemant clearly thought so too, as he said, 'You don't need more, you have all you can use here.'\n\n'Not quite. I get in everybody's way.'\n\n'Many women would die to have the space you do. We could never afford anything like this now. If only your father had done the same\u2014'\n\n'Maybe I could have the other room on the barsati?' Astha interrupted in a rush, a room so uncomfortable, distant, remote, and undesirable that she could ask with equanimity, and hopefully be given without hesitation.\n\n'What?'\n\n'Nobody is using it.'\n\n'But it belongs to Sangeeta, she may feel insecure. You know how touchy she already is.'\n\nA wave of anger hit Astha, Sangeeta sitting in Meerut was to be given greater consideration than herself.\n\n'I will vacate it whenever necessary, besides the servants are already there, and presumably she tolerates that.'\n\n'But darling, it has no electrical connections, how can you use it?'\n\n'I'll get it wired, all we have to do is extend the connection from the servants' room.'\n\n'It'll kill you, with your headaches, that's for sure.'\n\n'Please, please, please.'\n\nHemant looked distinctly annoyed. His wife on the roof, next to the servants' quarters, painting.\n\n'What is wrong with working down here? I let you work \u2013 I don't stop you \u2013 I say nothing about the smell, about the canvases all over.'\n\n'The smell, the canvases, the inconvenience are exactly why. Please, darling.'\n\nHemant talked to his parents. They did not agree. Sangeeta would be very sensitive to a family member encroaching on her territory, servants were different.\n\nAstha vowed bitterly to earn enough money to rent her own studio one day. In the meantime if there was no area available to her, she would try and make do with the wide ranges inside her head. Constantly reminded of the space nobody thought enough of her to give, she became very bad tempered during interruptions. Finally she steeled herself, she shut the door, and if disturbed too often locked it. In this way a certain uneasy privacy was granted her.\n\n*\n\nAfter _Women_ _Travelling,_ Astha's imagination increasingly worked in pictures. For the Manch painting she decided to experiment with an issue she felt strongly about. She would deal with the Rath Yatra, with the journey a Leader was making across the Hindu heartland in the name of unifying the nation. Like the religious leaders of old, he drove a chariot, identical to Arjun's in the serialised _Mahabharat,_ familiar to millions of viewers. That the chariot was really a DCM Toyota was a necessary concession to the 10,000 kilometres to be done in thirty-six days. His journey was to start from Somnath, one of the first places to be destroyed by Muslim mauraders (Mahmud of Ghazni) in 1025, and end in Ayodhya, where Lord Ram was born, the hallowed spot that needed to be reappropriated to assuage the feelings of 700 million Hindus. It was also a journey to political prominence.\n\nTo portray this Astha chose a large canvas, four by six, and again drew inspiration from Rajasthani miniatures. On one end was a temple, on the other was the Babri Masjid, on its little hill. Between the two the leader travelled, in a rath, flanked by holy men, wearing saffron, carrying trishuls, some old, some young, their beards flowing over their chests. Besides the rath on motorbikes were younger men, with goggles and helmets, whose clothes she painted saffron as well, to suggest militant religion. She sketched scenes of violence, arson and stabbing that occurred in towns on the way, people fighting, people dying; she showed young men slashing their bodies, and offering a tilak of blood to the Leader; she showed young men offering even more blood in a vessel; she showed the arrest of the Leader as he approached Ayodhya.\n\n*\n\nThe day Astha finished her Manch canvas, called simply _Yatra,_ she took a deep breath and stared at it for a long time. This was good, she felt it was. The Manch had promised her half the money for the painting, she wondered how much that would be.\n\nThis time Reshana priced Astha's painting at 20,000 rupees. 'It's very strong. A bit bloody, but the scale is so small it is not offensive. And it certainly adds to the colour.'\n\n'Thanks'' said Astha, feeling warm and glowy.\n\n'I had no idea you were doing the yatra. A controversial issue will be noticed in the reviews.'\n\nAstha saw respect on her face, which pleased her, but unfortunately it also made her remember her dream. Desire for Hemant darted through her, the safe, solid, stable, secure thing in her life.\n\n'Come back tomorrow and see where we have put it'' continued Reshana, and Astha returned from the exhibition hall with an empty feeling in her chest. The canvas she had worked on and thought about all these months was gone.\n\n*\n\nAgain Reshana proved right. Astha's painting was mentioned in the reviews, one paper even printed a photograph of it, and it was sold before the end of the exhibition.\n\nHemant said, 'Congratulations, you must be really pleased, I am happy for you'' as though they had met at a party, instead of sharing the same bed for years.\n\nAstha said, equally politely, 'Thank you, Hemant.' She put out of her mind an idle romance, that he would be the one to buy it, give it pride of place in house or office, and tell everyone that this was an example of his wife's work. She knew this was impossible, and that people who expect the impossible are setting themselves up for misery, and Astha would rather die than be such a pathetic woman.\n\nInstead she hugged the vision of herself as a woman who had sold two paintings in one year, sum total thirty thousand rupees, of which ten thousand was hers. She felt rich and powerful, so what if this feeling only lasted a moment.\n\nOne day she would get so famous that Hemant would feel obliged to display something she had done, and somebody, friend? banker? associate? would see it and, impressed, would ask to meet her. Unlike Hemant, he would find her fascinating. Would he want to have an affair with her? What would he be like in bed? Here Astha firmly drew a line across the remaining part of her fantasy, it exceeded anything remotely credible.\n\nSummer holidays. Everything that was touched or breathed was dust laden. The heat was its usual, intense and unbearable.\n\nThere was no question of Astha painting, her children were all over the place, she was busy with things to occupy them, summer workshops, the transportation involved, and the impending visit of Sangeeta with her children.\n\n'Will you show Sangeeta Bua your paintings, Ma?' asked Anuradha.\n\n'Right now I have nothing to show.'\n\n'You have the picture of it from the newspaper, and the mention in the review.'\n\n'Let it be, babu, she might think I am showing off.'\n\n' So? Shefali is always boasting about all the things she has.'\n\n'Poor thing. Sweetie, there is a lot of trouble in Shefali's house, her parents fight, and maybe she talks like that because she is insecure. Let's not say anything about my paintings it might make Sangeeta Bua feel bad.'\n\n'You mean jealous.'\n\nThat was what Astha meant, but this was the child's aunt they were talking about. 'Painting is not everybody's cup of tea'' she temporised.\n\n*\n\nThrough the summer, and the trials with Sangeeta, Sangeeta's children, Shefali and Samir, and her own children, her painting remained with her, at the back of her mind. She yearned for the moments when her hand, her eye, her brain fused into one, and her daily life was blocked out. She had experienced this increasingly with the second and third canvas, and she was impatient to experience it again.\n\nMeanwhile the six of them shopped, went to the zoo, went to films, went to restaurants, went to Appu Ghar, went to the science museum, went to the crafts museum, went swimming. For a week the nine of them went to Nainital, where Hemant rented a cottage. Here they boated, roamed around the lake, took long walks, had pony rides, and Astha was wife, mother, sister-in-law, daughter-in-law.\n\nHemant was happy with her. He found this easier when his relatives were there, and Astha spending so much time with them. When their anniversary came, he bought her a ring, an emerald surrounded by tiny diamonds. The quality was excellent, and the ring looked well on her hand.\n\n'Brings out your colour'' said Hemant turning the hand around in his own, smiling at her.\n\n'Such a husband'' murmured her mother-in-law in the background. Sangeeta looked on registering each gesture.\n\nThere was no need for Astha to say anything.\n\n*\n\nThe summer over, Sangeeta and her children departed, school about to start. Astha stood on the verandah overlooking her tiny garden, thinking her forced exile from paint, turpentine and linseed oil was at last over. She looked at the scene in front of her, wondering how she could catch even a fraction of it on canvas. The sky was heavy with dark clouds, the air had a grey yellow quality to it that made the grass and trees more luminous, the red flowers of the gulmohar tree more vivid, the waxy white flowers of the champa tree more arresting against their large dark green leaves. There was so much moisture in the air, that as the breeze blew, it brushed her face with dampness.\n\nMughal miniatures were full of monsoon scenes, lovers on the roof, the man's hand fondling the woman's breast, while the woman leans heavily against him, a grey sky above with white birds flying in a V-formation against the clouds. How about a monsoon urban scene, children splashing in puddles, kites flying, jamun, bhutta and phalsa sellers squatting in front of their baskets on pavements, and on the roof, a solitary woman looking towards the heavy darkness above. Melancholy filled her. After the deadness of summer, the monsoon was a time of awakening and desire, but what was one to do with one's longing?\n\nShe wished she could share her feelings with someone, but with only Hemant to fall back upon it was certain that her loneliness was secured. Still he was all she had, and she made an attempt when he came home and settled down to his drink.\n\n'It was really pretty today.'\n\n'I suppose. I didn't have time to notice.'\n\n'That's why I am telling you. I want to share it.' But already the tone was edgy, and Hemant responded promptly.\n\n'Yes, it's nice when you have time to admire nature.'\n\nThe offensive implications were clear. Astha forced a sketchy smile to her lips, then turned to study the label on the whisky bottle. More than this she could not lie.\n\n'I have a surprise for you'' he said.\n\nShe was grateful, 'Oh, really? What?'\n\n'We are going to Goa.'\n\n'Goa! Why Goa? The monsoon has begun there.'\n\n'Arre! You were the one who wanted to go.'\n\n'That was in winter. In season.'\n\n'Exactly. And do you know how expensive it is in season?'\n\n'Not if we had stayed in a cheap place. There are plenty of those.'\n\n'Why go if we have to slum it. Now I've got an excellent package deal.' His eyes softened and he squeezed Astha's arm. 'It's been fifteen years since we married. It's an anniversary present for you.'\n\n'Our anniversary is over.'\n\n'O-ho, May-July same thing. Either it's hot or it's raining. And the rates are off-season. I've got reservations for the Taj. When one goes to a five-star, the hotel becomes the destination then you really get your money's worth.' Hemant looked pleased with himself. 'Off season rates'' he repeated as they settled down to dinner.\n\n'But Hem'' said Astha, managing to get excited at the idea of staying in a five-star hotel, if it was raining outside, so what, five-star was five-star. 'It will take two days to go there, two days to get back, almost as long as the stay itself, is it worth it?'\n\n'We are flying'' and pride swelled his chest, and filled the room.\n\n'What? Have you won a lottery?'\n\n'I have to go to Bombay to see a dealer, the children's tickets will cost half, yours is the only ticket we have to pay for. We will spend the money you earned for your painting.'\n\n'But darling, you could have asked me if I wanted to spend the money on a plane ticket, and that too when it is off season.'\n\n'You have a bee in your bonnet about seasons. I am telling you it will be very nice, you don't trust me.'\n\n'I do, really I do.'\n\n'Then show it.'\n\n*\n\nIt was fair, she told herself later, that her money should go towards paying for a family holiday, after all why should Hemant have to pay for everything. There was no question of any choice in the matter. Everything was already decided. They reached Goa in the rain, they drove to the hotel in the rain, the children ran towards the beach in the rain.\n\n'Why don't we go too?' asked Astha. 'It might never stop raining.'\n\n'No, you go.'\n\n'I don't want to go without you,' said Astha. There was a possibility he would remind her they were on holiday, and why did they holiday if not to be together? She glanced wistfully outside. In the distance was the sound of the sea, and she could make out a thick grey and white undulating line.\n\n'You are such a child'' said Hemant indulgently moving to her. 'Remember this trip was to celebrate our anniversary?' He started tugging at her sari.\n\n'What are you doing? The children may come in any minute.'\n\n'Just a quickie. They won't come for another fifteen minutes at least.'\n\nQuickies. It seemed that was all they ever were. They completed the act within the specified time, the sound of the rain and the more distant noise of the sea mingling with Hemant's breathing in Astha's ears.\n\n*\n\nThe next day it was clear in the morning.\n\n'I have been talking to reception and they say that we should sight-see now as it will probably rain in the afternoon. I have hired a taxi.'\n\n'Where are we going?' demanded Anuradha.\n\n'Mapusa, and then some beaches.'\n\nThey set off. Husband, wife, two handsome children, riding in a taxi, sightseeing in Goa.\n\nThe town of Mapusa was small and barring a few traces of Portuguese influence, not very interesting. The Mediterranean colonial style of architecture could be seen here and there in old houses surrounded by lush green gardens, colourful bougainvillaea and hibiscus spilling over boundary walls, or flinging themselves with abandon on the houses.\n\nAfter driving them around a bit, the taxi stopped in front of a hideous shopping arcade with concrete circles plastered all over for decoration. The traffic was chaotic and noisy, taxis, cars, cycles and motorbikes driven by scantily clad foreigners whizzing around.\n\n'Cashew nuts, Goan wines'' said the driver firmly as the family hesitated inside the car. 'Antiques, silver, jewellery'' he continued gesturing at the dark spaces behind open doors.\n\n'Might as well see what this town has to offer'' said Hemant.\n\n*\n\nPerhaps that was a mistake. Because one of the things the town offered was an antique silver box, priced at five thousand rupees. It was so beautiful Astha fell in love with it immediately \u2013 old, blackened, intricately carved, and totally useless.\n\n'Please, can I have that box?' she asked Hemant.\n\n'You must be out of your mind'' said Hemant.\n\nThe tone, the refusal both hurt her. She was an earning woman, why couldn't she have a say in how some of their money was spent? She never said anything when he chose to squander money on airline tickets, why couldn't she buy a box she liked? Maybe it was too expensive, but she was sure if they bargained, it would become cheaper. Besides silver was silver.\n\n'It's so pretty. It would also be a memento of Goa.'\n\n'It's too expensive, these people are all cheats.'\n\nThe shopkeeper sensing indecision, urged the box upon them, very nice, old box, old price, now it will be twice as expensive if you go to buy.\n\n'See?' said Astha. 'Old prices.'\n\n'How can you believe him? They all lie.'\n\n'I also earn. Can't I buy a box if I want, even if it is a little overpriced?'\n\n'You earn!' snorted Hemant. 'What you earn, now that is really something, yes, that will pay for this holiday.'\n\nI have earned for my ticket she thought, but this was not the place to bring it up. The children pottering about in the shop had fallen silent. Anuradha went and stood at the doorway staring at the traffic. Himanshu was fiddling with the cashew nuts they had bought to take back to Delhi.\n\nAstha let out her breath in jerks so that nothing was audible. 'Let's go'' she said almost to herself.\n\n*\n\nThey went to see the other beaches, and on the way back from Vagator, Hemant put his arm around her for a conciliatory moment in the taxi. She could feel the solidness of his body next to hers. She felt limp, attacked and baffled. She didn't want his touch, his nearness to compete with the pureness of her despair.\n\nShe got through the rest of the day somehow, sick and wretched. The beaches were lovely, and she felt resentful of their beauty, resentful at being forced to register anything besides the pain within.\n\nBack in the hotel, the children beat against her mind, forcing attention from her through their shells. 'Look, look at this one \u2013 you're not looking \u2013 see, mine, put it to your ear, can you hear the sound? \u2013 not like that, you have to put it like this, can you hear the sea now? \u2013 I want to take all these shells to Delhi, they will look so pretty \u2013 I'm not putting sand everywhere, they are perfectly clean \u2013 that's my shell \u2013 she took my shell \u2013 it's mine \u2013 I saw it first \u2013 no he did not \u2013 she's always taking my things \u2013 you are always taking his side...'\n\nAnother hour and Astha's head was splitting. By the time the children had eaten their dinner and changed she was ready for the waves of pain that submerged her consciousness.\n\nThe night passed. Twice, thrice she staggered to the bathroom, clutching the walls for support to retch into the pot. Each time she hoped the pain would lessen, but it didn't, and her nausea continued until the birds started chirping, and the dark sky turned silver with the day. Finally with nothing left in her stomach, nothing left of her, she managed to close her eyes and sink into a calm exhaustion.\n\nOnce or twice she was aware of Hemant asking from his side of the bed, expressing concern in a strained voice, 'Are you all right?'\n\nShe acknowledged its tokenness by replying in a voice hoarse from vomiting, 'I'm fine.'\n\n'Are you sure?'\n\n'Yes. The pain will soon go.'\n\n*\n\nIt was late next morning. Hemant had given the children breakfast, and he was now sitting with Astha on the verandah, in front of a tray of tea and papaya. Astha looked over the undulating grassy patches to the sea line. She could hear the thundering of the waves. Above, the sky was rolling with heavy grey clouds. She couldn't remember seeing a miniature of the sea. Maybe miniature painters traditionally lived inland.\n\n'Feeling better?' Hemant asked. She nodded. He held out his hand, and she put her own in it. The feel of it was dry and warm. There was a certain comfort she associated with this hand.\n\n'I had hoped that with the sea air your headaches might become better, not worse'' he continued, a careful, mock blame in his voice.\n\n'That would have been nice'' she managed just as carefully.\n\nInside, the children's voices could be heard trading shells, with a few brief snatches of argument.\n\n'I think the children really like it here'' remarked Hemant, letting go of her hand to pour her a cup of tea.\n\nIt was tepid, but Astha sipped it gratefully, aware of the residual heaviness in her head with every motion.\n\n*\n\nThe bill for five days and four nights was nine thousand five hundred rupees. Hemant was triumphant.\n\n'That was money well spent'' he said as he came back to the room after settling all the accounts at the front desk.\n\nNine thousand five hundred rupees spent on one of the worst weeks of my life, thought Astha, as she stepped into the hotel bus for the airport. She thought hopelessly of all the things she could have done with that money, of the beautiful silver box she could have possessed and admired for ever. But their money spending was decided by him, not by her.\n\n'Oh, God, you look terrible. Have you been ill?'\n\nThus was Astha greeted by her colleagues on the first day of school.\n\n'The holidays were tiring'' she replied. 'My maid was away.'\n\nEverybody understood what this meant.\n\n'I tell you, after one's servant takes a holiday, it should be understood that we get a holiday too. Look at Astha, poor thing, it is obvious she needs a break.'\n\nTake a break, how they all said it like a mantra, as if taking a break would make any difference when you always came back to the same thing.\n\nColleague two was talking of her sister-in-law, settled in America, who had discovered that her husband was cheating on her, and who now wanted a divorce. This brought about the usual virtuous reactions centring around Us and Them, East versus West.\n\n'There they go on divorcing \u2013 marrying till the age of 60\u201370.'\n\n'They do not understand the concept of family. They only think of themselves.'\n\n'The divorce rate is three out of four.'\n\n'They don't know what it is to be a woman, what it is to sacrifice.'\n\nWell, Astha was a woman, and she was sick of sacrifice. She didn't want to be pushed around in the name of family. She was fed up with the ideal of Indian womanhood, used to trap and jail. Excuse me, stop the juggernaut and let me off. I have had enough.\n\n'It may not be a bad thing'' she said tentatively. 'If a marriage is terrible, it is good to be able to leave.'\n\nEverybody stared at her. Astha fiddled with her notebooks. They would be wondering whether her marriage was all right. 'Take my sister-in-law, for example'' she added quickly. 'Her only time off is with us in the summer. She is not allowed to work, rather her in-laws make her slave inside the house, she is nothing but an unpaid servant. If she complains, her husband sides with his parents. If she were in the West she could contemplate divorce without the social and economic death that would follow here.'\n\nThe bell rang. Astha got up carrying the forty notebooks of her students and headed for class. She had a job, there was no doubt as to that, but she doubted whether that made her any less trapped than poor Sangeeta. She should have kept her mouth shut about divorce. Its sole result would be speculation about her.\n\n*\n\nMeanwhile Anuradha turned thirteen and started menstruating. She did not take kindly to this, and Astha grew to dread her periods, interspersed as they were with bouts of rage, pain and depression. She could not remember ever attracting so much attention to herself during these times, even when it had hurt unbearably.\n\n'It is a woman's lot'' she explained.\n\n'Why, why is it a woman's lot, it's not fair'' moaned Anuradha, as she clutched her hot water bottle, tears flowing from her eyes, wetting the corners of her face, disappearing into her hair.\n\nWhere does fairness come into it, thought Astha. It hurts, you bear it. That was the end of the matter. 'It happens so you can have children'' she tried again.\n\n'I'm never going to have children, I'm going to adopt.'\n\n'We'll see when the time comes. You might want your own children.'\n\nAnuradha glared at her mother and did not deign to reply.\n\n'What's the matter with Didi?' piped up Himanshu, who was watching his sister wail and scream with great interest.\n\n'She's got a stomach ache. Go and see what is on TV, beta.'\n\n'Nothing is on TV. Why can't we get a dish and watch the Gulf War?'\n\nAstha turned to stare at her son. Anuradha forgot her pain long enough to point out how spoilt he was.\n\n'What's wrong with _Chitrahaar?_ You have always liked it.'\n\n'I want to watch the Gulf War. In school everybody watches the CNN and the BBC.'\n\n'I doubt everybody in school has a dish. You can talk to your father when he comes.'\n\n*\n\n'How was the day?' asked Hemant when he came home that night.\n\n'Terrible. Anu has her period.'\n\n'Oh? Poor little thing. Was it very bad?'\n\n'Yes. I had to give her two Brufen. I hope she doesn't become dependent on them. How will she bear pain in later life?'\n\n'She's still a little thing. Why should she have to suffer so much?'\n\n'She's not so little, and it's part of nature.'\n\n'Where is she now?'\n\n'In bed with a hot water bottle, reading Nancy Drew. And she has a test tomorrow.'\n\n'Poor baby, let her be'' repeated Hemant quickly, pouring himself a drink and making for Anu's bedside.\n\n'Oh Papa, I want some chocolate'' murmured Anuradha in a babyish voice, snuggling next to her father.\n\n'Tomorrow, all right?'\n\n'All right.'\n\nHe never sounds or looks like that when I have a headache, thought Astha, and then struck that thought from her consciousness. The father-daughter bond could not be compared to the rocky terrain of a marital relationship.\n\n*\n\nA few weeks later a dish appeared on the terrace. Astha was informed of this casually the night before.\n\n'A dish? But it is so expensive.'\n\n'It is good for the children. They will see the BBC, the CNN, they will know what is happening in the world. Who can watch Doordarshan? Two channels, I ask you. Now at least they will have competition.'\n\n'But such a lot of money, to have a dish in our own house. We are not a hotel, or something.'\n\n'Arre, I am in the TV business, I have to keep up with these things.'\n\nAstha's mind travelled to the little silver box in Mapusa, only five thousand, while the dish was at least eight times that. But it was useless to say or feel anything, the children and the business ensured the non-comparable nature of any\n\nargument. If she knew how much money they had, she might be on surer ground, but she never did.\n\n31 December. Constitution Club. 6.30 p.m. A slight mist was beginning to add to the general chill. Astha had not realised it would be this cold, and she stood shivering in her sweater and shawl. Nearby was a peanut seller, roasting his peanuts over a small fire but she didn't dare advance towards him, in case it looked as though she thought more of her appetite than the cause.\n\nIt was the anniversary of the massacre of The Street Theatre Group. It was also a protest against the Hindu Samaj Andolan decision to construct a temple at the site of the Babri Masjid.\n\n'Come and help me, Astha'' said Reshana, approaching with two big plastic bags. Squatting on the pavement, they poked candles through tiny foil-coated plates to prevent wax from dripping onto the hands that carried them. Absorbed, Astha could forget the scene she had had with Hemant before she left.\n\n*\n\nTen days ago, Hemant had asked, 'What shall we do this New Year's Eve?'\n\nAstha looked wary. Last year they had spent over two thousand to go to a five-star hotel with friends, and Astha had disgraced herself by getting a headache and throwing up at one o'clock in the morning with the discomfort of everyone's concern directed towards her. 'Leave me at home'' she had pleaded when Hemant had taken it up with her. 'I can't help myself.' But that was not socially acceptable either.\n\n'I don't know'' she now said. 'What did you have in mind?'\n\n'I'm not sure'' said Hemant leafing through brightly coloured invitations sent by various hotels and clubs about Xmas Nite, New Year's Eve Nite, dinner and dance. 'The Delhi distributor has invited us, they have booked a hall at the Sayonara club.\n\nBut it's not very personal, they call all their clients, and it is one big tamasha'' said Hemant looking disgusted.\n\n'Can't we stay at home'' asked Astha tentatively. 'That's really personal.'\n\n'Stay at home on New Year's Eve? No thank you.'\n\n'Tell me then, where are we going?'\n\n'We've got several invitations, let's see how many we can take in'' said Hemant, his pride at being socially sought after showing in his voice.\n\n'All right'' said Astha, not bothering to ask who the invitations were from. Some friends, some place. Eating, drinking, laughing, talking. It made no difference to her. Her mind was always not quite there.\n\nShe didn't tell him about the demonstration, also planned for New Year's Eve. She felt this information would not be well received.\n\n*\n\nNow she was about to be proved right. Hemant saw her getting ready to leave and demanded, 'Where are you going? I am free, you know that.'\n\nAstha thought of all the evenings she had been free and waiting, and wondered if there would ever be a day when she could feel the same right to complain that Hemant did. Now she tried to be conciliatory, she didn't want tension on a night of heavy duty partying. 'I am not going to be away long, just an hour.'\n\n'Where are you going?' Hemant repeated.\n\n'To a demonstration outside Rashtrapati Bhavan. It is the anniversary of the massacre.'\n\n'You seem to forget that your place as a decent family woman is in the home, and not on the streets. You also forget that this is New Year's Eve, and we are going out.'\n\n'No, I do not forget. I will come back in time, what does it matter what I do one or two hours before?'\n\nHemant's face assumed its shut-in aspect. Astha knew she was equivocating. It mattered because going out with her husband must be the highlight of the day, not something she was squeezing into the rest of her activities, unregarded, unimportant, done for the sake of doing. She left the house, hoping the anticipation of parties would do its bit in removing Hemant's ill humour.\n\n*\n\nBack at the Constitution Club. By 7 p.m. about three hundred people had gathered. 'Good turnout'' said Reshana to Astha as they finished with the last of the candles, and gathered themselves up from the pavement. 'And that too on New Year's Eve. We did contact everybody but you can never be sure.'\n\n'Many might think this is the best way to spend it'' said Astha with feeling. 'To do something you believe in makes other things a little easier.'\n\nReshana drew back. Astha flushed. There she was trying to give Reshana her heart and soul, behaving inappropriately. She must remember that everybody was here for the cause, and if the cause also had a personal impetus, discretion demanded this be shrouded in silence.\n\n*\n\nDown Rajpath they marched, candles glowing. They carried placards that declared they were for a united India, that secularism was part of our Constitution and traditions, that communalism was the scourge of the nation.\n\nThey chanted as they went:\n\n_Raise_ _your_ _voices_ _\u2013_ __ _We_ _are_ _one_\n\n_We_ _will_ _fight_ _injustice_ \u2013 _We_ _will_ _fight_ _together_\n\n_Communalism_ _will_ _\u2013_ __ _Never_ _succeed,_ _never_ _succeed_\n\n_These_ _are_ _false_ _weapons_ \u2013 _Of_ _the_ _true_ _god_ __ _Ram_\n\n_False_ _Ram-lovers_ __ \u2013 _False_ _weapons_\n\n_Temple,_ _church_ _\u2013_ __ _Mosque,_ _gurudwara,_\n\n_All_ _the_ _same_ \u2013 __ _The_ _same_ _for_ _all_\n\nUp towards Raisina Hill, candles dripping wax on the paper plates, holding a memorandum, on which they had been gathering signatures for the past month, protesting the attacks on the Muslims, protesting the bid to demolish the Babri Masjid.\n\nAround them swirled cars and pedestrians, irritated at having to stop in front of aggressive placards and glowing candles, while the procession marched across as many roads as it could, hindering traffic, drawing attention to its message.\n\nTowards Rashtrapati Bhavan, home of the President, home of previous Viceroys. Huge, mammoth, it towered in the distance, far from the high and massive wrought-iron gates that barred unauthorised entry. There the processionists halted, lit by TV cameras that dimmed the candles they were focused on. From Raisina Hill Astha could see the lights of cars swishing up and down Rajpath. How few we are, how many indifferent on this one road. She looked towards the former Viceroy's Lodge. Designed as a regal sandstone testimony to British glory, it had served its purpose for only seventeen years, before becoming a testimony to illusion.\n\nThe protest songs and slogans continued. Finally an official arrived, and a side gate opened a suspicious crack. The memorandum along with two spokespersons squeezed in; two people and a thousand signatures of mainly school and college teachers, artists, painters, and film people. A lot of the marchers had brought their children, who looked as convinced as their parents as to the justice of their cause and the usefulness of their protest, never mind how few they were.\n\nWhile they were waiting a letter was read out. Worded in English \u2013 objection \u2013 it should be in Hindi. The writer, an English academic, quickly explained in Hindi that the letter would be in both languages, and sent to all the leading newspapers. For now he begged their indulgence, he would read the letter in English, pending its immediate translation.\n\nThe letter proclaimed that the Sampradayakta Mukti Manch, and the teacher and artist community were united in condemning both the BJP and the Congress in encouraging Fascist forces in the country, and in failing to take quick action against the threats to the Babri Masjid. Were these threats actualised, secularism would be at grave risk, and communal hatred unleashed on a scale that would be difficult to control. To take no action was tantamount to encouraging social divisions along religious lines. Weaker sections would suffer. This was not to be tolerated. We appeal to the government to do something before it is too late.\n\nSigned\u2014\n\n*\n\nA resolution was then formed to establish a core group that would see to further action, the first being to circulate more petitions.\n\nThis done, the songs resumed:\n\n_For_ _how_ _long_ _will_ _they_ _loot_ _my_ _village?_\n\n_Taking_ _a_ _torch_ _I_ _will_ _go_\n\n_Through_ _the_ _world_ _I_ _will_ _wander_\n\n_To_ _make_ _my_ _village_ _safe_ _for_ _me._\n\nHalf an hour passed without any sign of the spokespersons. The last night of the year was wearing on. Astha kept surreptitiously looking at her watch.\n\n'Arre, will we ring in the New Year here?'\n\n'Let's go, they can come later.'\n\n'No point hanging around.'\n\nThe TV crew began to pack up. The candles had burnt down. As the procession started back towards the Manch office, Astha lagged behind, keeping a sharp look out for an empty scooter. She had to be home by eight-thirty, or things would be worse with Hemant. At one point where the procession had stopped the traffic she found one, and by quickly agreeing to pay his price, bumped her way home.\n\nIt turned out they were going to at least three of the parties they had been invited to. Senior bank manager, dear friend, and American NRI come to visit his parents.\n\nAstha hurried towards a thick dressy silk sari, peeling off her woollens. The sari was green with a broad red and gold border with woven flowers, hearts and peacocks. A matching deep green blouse was dotted with tiny gold paisleys. Her ordinary jewellery would have to do, she hadn't had time to go to the bank locker to take out some of her heavier stuff. Hopefully Hemant wouldn't notice. She threw her mother-in-law's maroon pashmina shawl casually over her shoulder, and thus guaranteed to freeze in the manner of women partying in Delhi winters, she was ready.\n\nReady to feel cold, ready to drink, dance, smile, laugh, talk, ready for anything the last day of the year might bring. She had made a gesture of some significance before Rashtrapati Bhavan, it made her more amenable to the evening now. Having something of your own makes you strong, she thought.\n\n'All set?' asked Hemant picking up his wallet and car keys.\n\n'Yes'' replied Astha. She was pleased that he had put the unpleasantness over her involvement in the demonstration behind him. They were going to have a nice time.\n\n'You look very nice'' he commented admiringly.\n\n'Thank you'' said Astha, feeling a small flush of pleasure.\n\n'I thought we would go to the manager's house first. It is bound to be the dullest, we can leave quickly.'\n\n*\n\nThe senior bank manager lived in a large house in the older part of South Delhi. Clearly he believed in doing things big. The little front lawn, shamiana draped, the verandah, the drawing, dining room, all were devoted to the party.\n\n'Gosh'' said Astha, as they were bumped constantly by people, avoiding glasses, and lighted cigarettes, 'there must be five thousand people here.'\n\n'More like two hundred'' said Hemant smiling indulgently.\n\nAstha could see his mood had further lightened. It had taken two hundred people but still she was glad.\n\nShe put out her hands to warm over an angeethi and tried not to breathe the thin spirals of acrid smoke coming from it, the coals were obviously wet. Other women holding soft drinks and glasses of juice were also standing around the angeethi. Astha smiled at them uncertainly, noticing the jamawars and pashminas flung over their shoulders, their smooth white waxed arms, glittering jewellery, and beauty parlour done hair. They looked perfect, perfect in a way she could never hope to look, lacquered and finished. She wished she could say she despised that look, and she did despise it, in theory, while crumbling wordlessly before it in practice, never able to hold her own.\n\nShe took a deep breath, turned to the woman next to her, and remarked, 'Cold isn't it?'\n\nThe woman smiled her agreement, 'What does your husband do?' she asked in her turn.\n\n'He manufactures television sets. And yours?'\n\nAnd so the phrases flowed on, till Hemant, one double whisky down, gestured to her that it was time to leave.\n\n*\n\nThe next party was at the house of the parents of the NRI. The place was full of men slapping each other on the back, counting the years they had been acquainted, walking down memory lanes, those lanes always so evident at this time of year with the foreign returned, the come back for two-three weeks when the weather is good and the kids can stand it, returned.\n\nThe food was mostly chaat and snacks. There was all kinds: papri, gol guppa, and for those who couldn't eat cold things in cold weather, hot tikkis, with green sour chutney and red sweet chutney, fat and swollen bhatura served with spicy channa, laced with halved green chillies and onion rings, dosas, idlis and vadas, and finally jalebis floating in hot oil, crisp, sweet, inviting to be crunched up ring by ring. There was even tea in earthen mugs which all those who weren't drinking sipped gratefully.\n\n'Oh, can't we go home, now?' moaned Astha, who thought she would burst if she had to eat another thing.\n\n'One more party, darling'' said Hemant over his whisky glass. 'Chin up.'\n\nI hope he is not too drunk to drive, thought Astha, the glow the food had given her fading, as she thought of the drive to Greater Kailash II where Hemant's closest school friend resided.\n\n*\n\nAnkur's party was on the terrace of his two-roomed barsati. Ankur had divorced after ten years of marriage, and was now discovering the joys of an affluent single life in an emphatically male environment. He fancied himself a cook, and with flushed face offered earthen mugs of mulled wine, mulled wine going ethnic, he said genially. On one side of the terrace a barbecue was set up. Seekh kababs, paneer, mutton and fish tikkas were being served with thin romali rotis, folded into triangles, on flimsy silver paper plates. There were four dead-looking salads, all smothered in shiny glutinous mayonnaise: pink (thousand island?) green (herb?) two whites (garlic? yoghurt?). As Astha jabbed at bits of paneer, it was easier to seem to eat than to argue with her host, she wondered it was only five hours ago that she had stared at wax dripping onto an identical foil plate.\n\nInside the music was loudly drowning everything out.\n\n'Come, darling, let's dance.' Hemant's alcohol-aided spirits were high.\n\nAstha obediently swayed, her sari palla slipping, looking covertly at the others doing their stuff to popular numbers pounding through the dimly lit smoky room.\n\n'Are you OK?' shouted Hemant above the din.\n\n'Yes'' she shouted back, automatic response-cum-smile.\n\n'Good'' he said, his voice slightly slurred, and in the dark he came towards her and pecked her cheek. His breath smelt of whisky, and she let her head tilt towards him, imitating reciprocity, before a couple bumped into them and forced them apart.\n\n'Now, now'' they shrieked, 'no kissing between husbands and wives.'\n\nHow stupid they all are, thought Astha. No kissing between husbands and wives. As though we were something besides conservative, strait-laced, middle-aged Indians. Should an unmarried couple kiss, I would like to see the reaction, I would just like to.\n\n*\n\nEarly next morning Astha rose, made herself a cup of tea and went out. It was another year, and she wanted to mark it in some way special to her. New Years should be private affairs, she thought, thinking of all the partying she had done last night. They had screeched Happy New Year, hugged, kissed, danced, eaten relentlessly, drunk this and that, and finally at 2 a.m. made their way home, Hemant slow and careful because he was trying to appear in control, and Astha silent, because she knew Hemant had drunk more than he should have, and there was no tone sufficiently neutral in which she could convey this.\n\nIt was cold in the tiny garden. Astha grabbed the mali's jhaaroo, and began to sweep the dead leaves into a pile. She wanted to make a fire. A fire was a good New Year's thing. Burning all the old year debris away.\n\nAs the flames smoked through the wet leaves, Astha cupped her hands around the mug of tea. It was Flowery Orange Pekoe, a delicate and flavourful smell. She smiled, thinking of the year ahead. She had found what she really wanted to do, something she was good at, she was lucky. She now felt established enough as a painter to give her art the time and energy that was its due. She was ready to leave her job. She had been teaching almost fifteen years, staying because it had been a good occupation for a woman.\n\nShe finished her tea, and went into the spare room. It was early, but she wanted to begin the first day of the New Year with work important to her. She took out her file and started visualising scenes for a March for Justice. The idea had grown last night among the candles. The canvas would be dark, with a group of people huddled before the gates of Rashtrapati Bhavan, which loomed remote and massive in the background. The bright spots were going to be the candles the marchers held, the yellow of the halogen street lamps and the red and white lights of the cars on Rajpath. The rest would be in shadow. Astha hummed as she worked. There was no one to tell her how tuneless her singing was. \nChapter VI\n\nPipee stumbled into the New Year alone in her flat, staring at the two-rod heater, nursing a small rum and coke. It had been a year since Aijaz's death, and as every day in the past year, she had been fierce in her desire to be alone, turning down well-meaning invitations that friends, colleagues, relatives and acquaintances showered her with.\n\nHer mother-in-law had phoned from Shahjehanpur asking her to visit. But she couldn't. Not yet. The one time Pipee went, she had hardly been able to stand the memories that swept her every inch of the way. In every face she saw traces of Aijaz, and their sweetness to her had made it even harder.\n\nShe and her mother-in-law had cried and cried together, but conversation had been difficult, everything they had in common was in the past. She only stayed a few days, and as she was leaving, the mother-in-law gave her a cheque for one lakh. 'I didn't spend on your marriage, now you take this.'\n\n'I don't want it'' Pipee's voice trembled, there seemed no limit to the number of times they could cry together.\n\n'Please, for him'' replied the mother. 'He would not like to think we did not look after his wife. I want you to know you will always have a home with us.'\n\nDully, and with Neeraj's help, Pipee bought a flat in Vasant Kunj. She had the money from Shahjehanpur, life insurance, and dollars sent to her by her brother.\n\nIt usually takes a lifetime to possess a place of one's own.\n\n*\n\n'You can't go on like this'' Neeraj had remonstrated on New Year's Eve at the office. 'It is not healthy. You are still young.'\n\n'I don't feel young. I don't feel anything.'\n\n'Make an effort, you are not even trying.'\n\nPipee turned away. What did Neeraj think, that she liked feeling the way she did? In fact she would give anything to be free from the thoughts that haunted her. Only since Aijaz's death did she realise that how you die is as important as the loss itself, and can make all the difference to the ones left behind.\n\nThere was no relief from the pain of his final moments. She couldn't get rid of the thought of him trapped in the Matador, suffocating with the heat, burning bit by bit, screaming for help perhaps, trying to break the windows, wrench open the doors, and then the terrible moment when he realised he was going to die, him along with nine others, those nine there because of him. What had it felt like? Had he been able to think of her, their love, their lost future?\n\nTill now not a single culprit had been brought to book. Perhaps if the assassins had been identified and punished, a bit of the horror might be stilled; she didn't know, she only knew it wasn't likely to happen. As for that Sampradayakta Mukti Manch, she hated it more than anything. What had they done to ensure justice? Had they worked on bringing pressure on any government organisation? No, they had a platform in his name which they called freedom from communalism, and all they did was hold exhibitions, raise money, and indulge in cultural nonsense. She hated them, each and every one of them individually, but above all she hated Reshana Singh, who had surfaced out of the woodwork, from god knows where, after Aijaz's death and taken over his memory. She managed to imply that theirs had been a deep connection, she was practically masquerading as his wife. How well had she known Aijaz, she was so much older than him, that any attraction on her husband's part must have been a purely passing phase.\n\nWhat should she do, should she leave Delhi? Her mother had tried to get her to relocate in the south, you can find an NGO in Bangalore or Madras, there are slums here as well. You need to put the past behind you, start a new life, you will be near me, come, come, she persuaded in letter after letter. Pipee now looked at the last one:\n\nMy darling daughter,\n\nEvery day I miss you, think of you, pray to God for your well being, and the courage that will see you through this crisis. Aijaz was a wonderful man, a loving husband, and you were lucky in your marriage. I say this despite the terrible tragedy, because what you two had can never leave you. You have been a wife, you have been loved, and this will stay with you for the rest of your life.\n\nI know what you are going through and darling I would have given my right hand for the same thing not to have happened to you that happened to me. But it seems we cannot escape our destiny, whether our husbands are young or old.\n\nMaybe it is a blessing in disguise that you have no children. When your father left me, I had my Pipeelika, and my Ajay, I needed no one else, but you with your youth, your intelligence, your personality, you need other outlets. Aijaz would not want you to be unhappy or alone. I know that. Life has its own laws that will be heard and felt.\n\nYou are always, always in my heart,\n\nYour loving Ma.\n\nMaybe, she thought, staring at her mother's letter, she really should make more of an effort to go out. Although it had been a year she didn't feel any better, perhaps she never would. But to go on refusing to meet people, always to be alone, that was not the answer either. Her life stretched before her, long and dreary. What her mother was advising was to form a new relationship. But how? Aijaz had been hard enough to find. And there had been Samira when she was young. She had never loved anybody else.\n\nPerhaps she should go to the States, leave Aijaz to the Reshanas of this world. The whole of last year Ajay had been calling her insistently for a Ph.D. programme, you will be surprised what a difference a complete change of place will make.\n\nYes, she would be surprised. Ajay had no imagination, but still she, who had lost everything and had nothing more to lose, could give it a try. In the meantime she might travel with Ujjala.\n\nWith these thoughts, in front of the heater, eating her dinner of scrambled egg on toast, Pipee passed into the new year.\n\nWaving saffron flags, Hinduism marched across the country in the following months, marched in time to film songs converted into bhajans, to Leaders trying to convince the masses that the glory of an ancient land could be resurrected by their united hands. Young men, show your manhood, rescue mother India from the influence of the Muslim invaders, whose long shadow falls over us even now. The wrongs of the past have to be righted.\n\nThese hoards, gathered mostly from the Hindi heartland, become the face of militant Hinduism, armed with tridents, swords, and a determination to die if necessary for the cause entrusted to them. This behemoth turns it head towards Ram's Janambhoomi. A temple needs to be constructed on the sacred soil of Ram's birthplace, burdened for so many years by a mosque. A date is fixed for the event.\n\nAs they converge upon Ayodhya, a cordon is drawn around the city, roads are blocked, trains and buses denied entry, any leader suspected of creating trouble is carefully watched.\n\nBut there are always the fields and villages, always people to give food, water, rest, and show the way.\n\nAnd likewise there are leaders to hide in the lanes of Ayodhya to mastermind the breaking of the cordon around the city, there are officials in the state police who feel it their duty to personally assist all those similarly inclined.\n\nThe kar sevaks declare that neither guns nor bullets can stop them. They prove this when in defiance of all barriers they climb the mosque, plant a saffron flag on the highest dome and claim it for their own. They are fired upon by the police, hundreds of them are injured, many are killed. Videos are made of this, and are later shown around the country as an example of the threat to Hinduism.\n\nThe government falls, and for the time being further crisis is averted, but only for the time being, promise the forces for Hindu Restoration in India.\n\nGive us three places in India, that is all we want, Ayodhya, Varanasi, and Mathura where the Muslim invader built mosques on our sacred sites. If necessary we will bathe these mosques in blood. Why should Hindus give up their position of dominance in the only Hindu country in the world? If it is mosques the Muslims want, let them go to the many countries where Islam is the official religion, we are not stopping them.\n\n*\n\nThe Sampradayakta Mukti Manch were doing what they could in the face of resurgent communalism. They prepared pamphlets, organised marches with other Left groups, and decided to go to the banks of the Saryu to talk directly to the people of Ayodhya to counter the growing rhetoric of religious fanaticism. As they planned their trip, Reshana suggested that Astha also come. 'Between you and me I wonder if academics sometimes have the impact we desire. How I wish Aijaz was with us today. He could capture a crowd like no one else.' She sighed and continued, 'If you could give a small five-ten minute speech? I think it might make a difference to the women. If they realise they have some kind of voice, it will be a useful counter thrust to violence and aggression. After all they stand to lose the most. It's worth a try.'\n\nAstha agreed. Now that she was no longer teaching she welcomed brief respites from the house. And yes, any contribution to the cause was worth a try. In her association with the Manch she had been exposed to detail after detail of atrocities perpetuated in the name of religion. She had made paintings for this cause, she had been part of debates that worried about the far-reaching implications of fundamentalism, she had seen the spread of the worst kind of jingoistic rhetoric and it gave her both a platform and a focus around which she built her work. When she looked back it seemed amazing that she had come such a long way in two years. The detour she had taken between home and school had now become the road she travelled.\n\n'I hope it won't be a problem, leaving your children'' Reshana ended.\n\nIt was a rhetorical statement, but Astha responded with a dry laugh, 'Since when has the personal been allowed to interfere with the need of the hour?'\n\n*\n\nSo far her mother-in-law had not commented about her activities. But Astha's going to Ayodhya was a different matter.\n\n'You know I never interfere in whatever you decide to do. Today young people feel they must live their own lives. But there are times when it is necessary to listen to the advice of elders. What is the need to leave your family, and roam about like a homeless woman on the streets of some strange city?'\n\n'To protest.'\n\nMummy looked nonplussed. 'But why go to Ayodhya?' she returned after a pause. 'You want to say something you write a letter to the newspapers. That is much better. People get to hear. You used to write.'\n\n'Long ago.'\n\n'This is all politics, you should not get involved. Besides have you thought about what you are going to protest? Lord Ram's Janamsthan is in Ayodhya, is there any country in the world where the birthplace of their god is not honoured? Hindu tolerance does not mean you accept everything and anything. Is this the pride we have in ourselves?'\n\n'But Mummy, if the temple is constructed, thousands of people will die agitating over it. Why they could feed hundreds  of poor children on the money they are collecting for the bricks.'\n\nHer mother-in-law looked at her. 'It is not a woman's place to think of these things'' she said firmly.\n\nAstha remained silent, her mind full of her husband. She had mentioned her trip as a possibility in a casual conversation with Hemant. Was this his way of letting her know he did not want her to go? He did not even have time for a discussion with her.\n\nMeanwhile Mummy was repeating, 'You know I never try and stop you from doing anything. Even when you neglect the children, and are busy in your paintings and meetings, I do not say anything. I am not the type to interfere. I am glad my daughter-in-law does not feel she has to sit at home. Till I have the use of my hands and feet I will help you, but it is my duty to point out that you are going too far.'\n\n'You won't have to help with the children this time, I will take them'' said Astha wildly thinking of Anuradha's sulky face, and Himanshu's bewildered expression. 'It is good if they are exposed to such things early.'\n\n'Exposing them to what? Filth and crowds? Don't you care about your children or husband? But he is too good, he will say nothing. If you were living in the conditions Sangeeta is, you would better value what you have. I hope you never regret this.'\n\nAstha was struck dumb. Her mother-in-law had never spoken so openly before. And where did Hemant have the time to notice what she was doing, let alone mind? But he had noticed, he had minded, and so had others. Mumbling something non-committal she retreated downstairs shaking with rage and hopelessness. With a mother like that, what chance that Hemant would ever support her? She dreaded trying to convince him and the possible scene. And because she dreaded these things, she became all the more determined to go.\n\n*\n\nThe argument started that night when they were getting ready to go to bed.\n\n'I have decided to go to Ayodhya'' she said.\n\n'As my wife, you think it proper to run around, abandoning home, leaving the children to the servants?'\n\nAstha went into familiar distress. As his wife? Was that all she was?\n\nShe tried to interest him in the issue, pulling out a pamphlet from her bedside drawer, 'Look at the stuff they are publishing. It's so inflammatory but people fall for it.'\n\n'You should see the stuff they publish against India and Hindus in Pakistan. Why don't you protest against that?'\n\n'I do protest. I happen to think that any religion that incites violence is bad, ours, theirs, everybody's. Listen to this:\n\nThis is not a 'new' political struggle. It is the 77th attempt in the history to restore the Ramjanambhoomi, our heritage. Thus far over 300,000 kar sewaks have laid down their lives in the 400 years.\n\nPseudo-secularists want the mosque declared a national monument forgetting that Ram was an Indian and Babur an invader. It is a national dishonour if a symbol of invasion is so declared:\n\n'Now Ask Yourself!'\n\nCan even the most tolerant, most reasonable and peace-loving Indian run away from his pride \u2013 the reason for his being? The time has come to fight for our threatened faith.\n\n'Hindus unite! Act as one.\n\nNot against anyone!\n\nBut in defence of our motherhood.'\n\nShe watched as Hemant reached out and turned the Ramjanambhoomi Nyas pamphlet over in his hands. She liked his hands. They were so square, so competent, they smelled nice, they felt nice on her. His palms were soft and pink, his nails always short and clean. Why was it like this between them? She sidled next to him, and put her hands under his kurta, rubbing his soft stomach. 'I do so wish they hadn't planned it around New Year's. I hate to leave you alone, but darling what to do?' Plaintive, appealing, emphatic.\n\nHemant grunted. 'Say no, what else is there to do?'\n\n'But I have committed, it'll look bad.'\n\n'They don't own you.'\n\n'Just for two days. I'll be back so soon, you won't realise I have gone'' said Astha trying to be playful.\n\n'I won't be here.'\n\n'Why?'\n\n'I have to go away.'\n\n'But you never said.'\n\n'I only knew of this today.'\n\nShe did not believe him. How would she leave the children? She would have to move them upstairs, and that too after her dignified statement of taking them with her. He was doing this to punish her.\n\n'Where are you going?'\n\n'Bombay. To see a dealer. It's important.'\n\nAstha did not ask how and why, and nor did Hemant elaborate. 'What about the children?' she asked a little forlornly. They had never been without both parents before, without her really, Hemant was frequently away.\n\n'That's your responsibility'' he replied. 'I have work to do, a factory to run, I can't be both mother and father.'\n\nShe would have to be conciliatory with Mummy, she would have to sit down and explain why she was going instead of getting angry, she would have to tell the children she was leaving them with their grandmother, and hope the grandmother would not bad mouth her while she was away. She might as well have spared herself the worry of what Hemant was going to do, he was going to manage just fine.\n\n*\n\nThat night she couldn't sleep. Her mind refused to rest, roaming restlessly among the things that made up her life, her home, children, husband, painting, the Sampradayakta Mukti Manch. Was it too much for a woman to handle; was her mother-in-law right? But why? Her children were well taken care of, she had trustworthy servants, she had someone who cooked better than she, she had left her teaching. And yet she was chained.\n\nHer thoughts grew darker and darker. Restlessly she tossed to and fro, looking for a position that would force her mind to imitate her closed eyes, and free her into sleep. Hemant snored next to her, and his impenetrability irritated her further.\n\nNext morning, tired and bewildered, she got up, looked at her husband, who appeared fresh and lovely. He glanced at her, and she smiled, her lips stretched across her face, cracking her skull, but still her lips would stretch, and her eyes would look up at him.\n\n*\n\nHemant left for Bombay, departing one day before she did, destroying the fantasy she had had that he might drop her at the station, and they could part tenderly with many expressions of I will miss you, hurry back, phone me when you reach.\n\n'You are also leaving?' Himanshu asked, round eyes.\n\n'Yes darling, only for two days.'\n\n'But why?'\n\n'I have some work.'\n\nBut this explanation did not resonate the way the father's did, and both mother and son felt a little unconvinced.\n\n*\n\nNext night, the train to Ayodhya from Old Delhi at 9.30 p.m. Both children insisted on accompanying her to the station. Mala was taken to escort them back. They left the house at 8.00 and at 8.30 were caught in a religious procession starting from a gurudwara.\n\nMother, son and daughter watched the green dial of the dashboard clock tick the minutes away as they waited and waited. For the first time Astha felt the impatience Hemant did in traffic, but there was nothing she could do, blaming the government did not come so easily to her, nobody to blame in fact, but God above who had made them Indians in an overcrowded land.\n\nPeople darted in and out of the traffic, bumping against rickshaws, cars, buses, weaving in and out all over the road. From time to time cars, scooters and scooter-rickshaws inched forwards squeezing themselves wherever they could, but they could not squeeze themselves as small as people did.\n\n'Will Mama miss the train?' asked Himanshu interestedly.\n\n'Don't be stupid'' said Anuradha.\n\nAstha clenched her fists. 'I think I can see the traffic lights now'' she said after the car had crawled along for twenty minutes.\n\nIt was five to nine. There were the traffic lights visible at last, the end of the intersection was almost in sight, it was the last major light before the station.\n\nFinally they reached. Station. Parking lot. Platform. They might as well have saved themselves all that anxious clockwatching. The train was one hour late. They hung around the platform, surrounded by standing, sitting, squatting, lying, waiting people. There was hardly any room to move. By the year 2010 standing room only in India. Make way, make way, squeeze in more, that year is lurking around the corner.\n\n*\n\n'How long do we have to wait for that stupid train?' complained Anuradha, while Himanshu clung to her. Astha felt his body through her sari, felt his arms around her waist, his hand resting on the bit of bare back between her sari and her blouse.\n\n'Do you want an orange?' she asked.\n\nHe nodded. Astha reached into her sling bag and started peeling one.\n\n'I also want'' said Anuradha indignantly. Astha handed her half.\n\nAnnouncement. The train was delayed another hour. The people on the platform stirred, rippled, and then settled down to waiting again.\n\n'Go home'' said Astha, 'Mala take them home. It is getting very late.'\n\n'No, I'm not going, I'm waiting with you'' wailed Himanshu.\n\n'We will wait, it's all right'' said Anuradha gruffly. She demanded some money to buy _Stardust,_ and settling herself on her mother's suitcase, began to read. Himanshu picked his nose, and looked vacantly at the train tracks beyond his feet.\n\nAt last the whistle, the clang, the arrival. The platform woke, and a huge beast sprang into motion. It pushed, it shoved, it jostled. Sharp cornered boxes and heavy suitcases were lugged onto the heads of coolies while the parcels and bags slung from its arms jut, poke, obstruct, protrude, and threaten with injury. Astha clutched Himanshu with one hand and dragged Anuradha along with the other, trying to keep up with the coolie looking for her compartment.\n\nThere was her name and berth number, pasted outside a second class AC coach. More squeeze and push till they reached the berth.\n\nFinally. The coolie was paid, and Mala and the children sat around in a listless sort of way, listening to more announcements of delayed trains, before they all agreed that the family had seen Astha off and now they could go home.\n\n'Bye darlings, bye dearest ones'' she said, 'I'll be back before you know it, and I will phone, all right. Be good, don't give Dadi any trouble.'\n\nThe children jumped off and led by Mala fought their way out of the crowd.\n\nEventually, three hours after it was supposed to, the departure whistle blew, and the train gave a little jerk.\n\nAstha sank back into solitude. She laid out the pillow and sheets that an attendant had thrown at her, and settled down for the night, rocking with the quickening rhythm of the train, not yet wanting to close her eyes and go to sleep.\n\nNext morning, and the U.P. landscape through the purple film plastered over the train windows. The land on either side was flat and dry, with patches of green fields. Uttar Pradesh, home to eighty million people, many of them leading poor, illiterate, and harsh lives, but ready to leave their fields, villages, and towns to converge upon the Babri Masjid, to protect their faith and motherland, something that would not have occurred to them before.\n\n*\n\nFaizabad, Ayodhya's twin city, 11 a.m. The Sampradayakta Mukti Manch had made arrangements for the women to stay at a guest house they frequently used. Astha got into a rickshaw and gave the address.\n\n'Have you come to do Ram darshan at the masjid?' asked the rickshaw wallah, as Astha put her feet on her bag to prevent it from falling on the road.\n\n'Yes'' she answered cautiously.\n\nThe rickshaw wallah nodded, it was the expected answer, Astha could see.\n\n*\n\nThe guest house was a large white washed bungalow set away from the road, in what used to be the Faizabad Civil Lines. A middle-aged lady came out to greet her.\n\n'Astha Vadera? The others from your organisation are out. They will be back soon.'\n\nShe was taken to a high-ceilinged, dark drawing room and served tea. The lady launched smoothly into a brief history of her life, she owned the house, she didn't really need the money, running a guest house was a time pass, one must be active, her son and daughter were in America, she didn't want to burden their lives. See, here they are, gesturing at pictures in ornate silver and wooden frames on the massive Burma teak sideboard.\n\nA house, thought Astha, if my mother had a house, she too could have done something like this, instead of going to Rishikesh and losing herself in an ashram.\n\nThe widow got up, adjusting her sari palla around her head. 'Your room is upstairs. Come.'\n\nThe uncovered staircase was next to the outer wall, and led up to five small rooms in a row. There was a verandah running the length, a nice view of the garden, in one corner were the bathrooms, in another, a bit of terrace to sit on.\n\n'Food to be ordered two hours in advance'' said the widow, unlocking the door of a small room, one little window, red floor, one bed, chair, table and cupboard.\n\nAs Astha sat there, eleven forty-five in the morning, the sense of adventure she had experienced in the train fell away. The room was neat, clean, without character and totally remote from everything that made up her days. She felt strange and dislocated. What would her children be doing? She missed them, she hoped Anuradha wasn't fighting too much with Himanshu, she hoped that their grandmother wasn't feeding them too much rubbish, but it didn't matter, it was just two days, she hoped they weren't watching too much TV, but then that didn't matter either, it was just two days.\n\nTonight will be better, she thought, trying to argue away her depression, tonight at the function she would be where the action was, she would make her speech, feel the purpose of her visit more.\n\n*\n\nA little later, when Astha washed and went down she discovered that in the widow's estimation there were seven thousand temples in Ayodhya.\n\n'Seven thousand? Are you absolutely sure?' she demanded incredulously.\n\nThe widow looked at her sternly. 'It's Ram's birthplace. There is a need for so many. When there is a festival like Ram Navmi, lakhs of pilgrims visit. Many temples double as dharmsalas. They charge one rupee to ten, and the pilgrims sleep wherever they can.'\n\n'So many temples. And they want one more.' The figures startled her into being na\u00efve, she knew the agitation had nothing to do with numbers.\n\n'Of course'' said the widow. 'Ram was born right on the exact spot where the Babri Masjid is. You can even see from the pillars inside that there was a temple there. Eight pillars with Hindu carvings, mango leaves, goddesses, apsaras, kalash in black stone. Where did they come from? They built the mosque around them to mock us.'\n\n'Mock us?'\n\nThe widow glanced at her pityingly, and spelt it out. To remind us that they have the power to destroy our temples.'\n\n'I don't think it's quite like that'' began Astha when the widow interrupted. 'Even now, Muslims living here really have their allegiance somewhere else. You will see during cricket matches they want Pakistan to win, this is not their soil.'\n\nAstha knew it was useless to protest. Opinions like this, based on preconceptions, did not change. What did it take, a lifetime? A whole new history? What?\n\nThe widow seemed nice, even educated, she would not condone violence, no. Hers would be the gentle voice declaring 'they' were all the same, and these were words that would have a longer reach than any missile thrown.\n\nWas it like this in Pakistan, Astha wondered. Did Muslims look upon Hindus with suspicion? Ah, but where were the Hindus in Pakistan? All dead or gone, leaving scars that rankled even now.\n\n*\n\nReshana came, 'Have you been waiting long?' and didn't wait for an answer. 'They are escorting a fresh lot of bricks into Ayodhya, bricks wrapped in saffron, silk, cotton, with tikka on them, stamped with the name of Ram, as though they were an object of worship, bricks to build the temple, high hysteria around the whole thing. We have been trying to make sure our function tonight is well attended, but\u2014' her voice trailed off, a little hopeless.\n\nAstha understood. As an artist the visual and symbolic appeal of saffron clad bricks would be far stronger than any appeals to reason and history. Still one had to do what one had to do.\n\nAfternoon shaded into evening, as not far from the banks of the Saryu, on a platform in front of a mike a thin academic gave the history of the Babri Masjid.\n\nThe audience spread before him had been gathered through posters and advertisements, with the promise of entertainment and songs. Despite Reshana's fears the turnout was large, though it was debatable whether they had come for the spectacle or from a willingness to be converted to a historical point of view.\n\nThere is no evidence, thundered the academic, punctuating the air with an excited fist, no evidence that Babur, busy fighting the Afghans, ever came to Ayodhya, let alone destroyed a temple.\n\nDo you think Babur, founder of an empire in India, would have come here to build this little mosque? Yes, there is an inscription inside saying he ordered it, but the close set writing is of a much later style, carved to strengthen rumours of imperial destruction. The wooden beam below the arch is not a remnant of a temple, but put there by local masons, using local materials, unskilled in building arches. There are others like it in Jaunpur.\n\nBrothers and sisters, I have not come from Delhi to bore you with historical details, only to show you that for every bit of evidence used to prove there was a temple to Lord Ram here, there is a counter-argument to prove there wasn't.\n\nHistory can be used to build or to destroy. We choose the lessons we wish to learn from it. For years Muslims and Hindus have lived peacefully together. It is the British who suggested that an ancient temple was destroyed so that Hindu would turn against Muslim. Brothers and sisters, we have seen what the British succeeded in doing. They believed in Divide and Rule. They ploughed rivers of blood through our country. The same dark forces threaten us now. It is politicians who are creating religious insecurities to get votes. Do not let them succeed.\n\n*\n\nAstha was sitting in front, nervously waiting her turn, clutching in her cold palm the piece of paper on which her rehearsed points were written. She looked around to see the reaction of the audience. He may have been passionate, but he was still an academic. 'Do you think they understand what he is saying?' she whispered to Reshana.\n\n'It's all we can do, though I doubt we are any match for organisations that have been working the fundamentalist rhetoric at the grass roots level for years.'\n\n'I think this speaker should appeal to their emotions, instead of talking about beams, arches and inscriptions'' said Astha.\n\n'He's a very respected historian'' replied Reshana stiffly. 'And he is showing the relevance of beams and inscriptions.'\n\nHer tone annoyed Astha, Reshana was so easily offended. How come love for the people did not translate itself into tolerance for individuals? She looked around for a more congenial sight.\n\nThey were in front of a canal, next to a bridge. Across the modern park on the other side of the water lay the old town, its interspersed domes and spires clearly conveying its mixed heritage. It looked old and fragile in the yellowish rose of the falling light.\n\nFinally the academic finished. 'It's your turn now'' whispered Reshana.\n\nAstha got up. Her irritation had given her energy. When she spoke her voice was firm and clear.\n\n'Brothers and sisters'' she started, 'In essence women all over the world are the same, we belong to families, we are affected by what affects our husbands, fathers, brothers and children. In history many things are not clear, the same thing that is right for one person is wrong for another, and it is difficult to decide our path of action. We judge not by what people tell us, but by what we experience in our homes. And that experience tells us that where there is violence, there is suffering, unnecessary and continuous suffering. When we look to righting wrongs committed hundreds of years ago, we look to the past. But that past cannot feed us, clothe us, or give us security. History cannot be righted easily, but lives are lost easily, pain and trauma to women and children come easily. Tomorrow your sacrifice will have been forgotten because the duty of life is towards the living.'\n\nShe saw some people nodding, and she ended by repeating that nothing except misery and suffering were to be gained by violence.\n\nA song, followed by a street play, and the evening concluded with an invocation to Gandhiji:\n\nGandhiji was a devout Hindu \u2013 none more devout than he. But he knew the true meaning of religion. All men are brothers. Hatred between communities led to his death, and in listening to the voice of hate we kill him all over again.\n\nThe speaker was speaking in terms everybody could understand \u2013 Gandhiji, father of the Nation \u2013 love \u2013 hate \u2013 oneness. But were these strong enough to drown out \u2013 exploited for centuries \u2013 awake \u2013 defend \u2013 protect \u2013 Motherland \u2013 Ram \u2013 God, faith, and Love of Him?\n\nIn the middle of all this Astha looked up and saw someone staring at her. The woman caught in the act, went on staring instead of looking away. Then she smiled slowly, squirrel front teeth advancing slightly from the rest.\n\nI wonder who she is, thought the one being stared at.\n\n*\n\nLater. 'I really liked your speech.'\n\n'Oh, thank you. It was nothing much.'\n\n'It made sense. The basti women who are with me related to it.'\n\n'It was my first. I am not used to making speeches.'\n\nA pause.\n\n'What is your name?'\n\n'Pipeelika.'\n\n'Pipeelika? Ant?'\n\n'Yes. My father's legacy. He liked the sound and he had a sense of humour. It does mean I have spent my life explaining it. Maybe it has affected me, I don't know. What's your name?'\n\n'Astha.'\n\n'Faith?'\n\n'Yes. I don't know if I have been touched by it. The faith in my family centres around my mother and her swami.'\n\n'Don't you have faith in anything?'\n\n'I don't know. Perhaps my brush.'\n\n'You paint?'\n\n'Yes.'\n\n'Can I see?'\n\n'Any time.'\n\nThey arranged to meet the next morning. 'Do you know the place?' hinted Astha at parting. 'I thought I'd see something of Ayodhya before my train leaves tomorrow night.'\n\n'Of course I know the place. I'll show you around.'\n\n*\n\nShe thought about her later. Her hair was like a halo round her face, springing away from it, black, brown, red, orange, and copper, her skin was a pale milky coffee colour. She liked the way she smiled, but she looked sad at the same time, why was that? Had she herself sounded interesting, why hadn't she brought something nicer to wear, suppose she didn't come to the park at ten like planned, why hadn't she asked her where she was staying?\n\nShe tried shaking herself, if she didn't come, she would see Ayodhya on her own.\n\n*\n\nThe next day as she hurried in a rickshaw to the meeting place, she saw her waiting under a tree. Immediately she felt stupid. A stranger she had hardly spoken to, to bother about her clothes, what was wrong with her? They would meet, they would part, she would catch the evening train home.\n\n'Hi.'\n\n'Hi.'\n\nThey said nothing much as they walked through the small town. In every lane were shops crammed with representations of gods, pictures and figures, small, medium, large kalashes, bells for doing arti, prayer beads, green, yellow, black, blue with pearls, and the mandatory rudraksha in every possible size and colour from pale blonde to dark brown. This was a town of serious religious buyers judging from the number of shops.\n\n'We are near Hanuman Garhi, it's on the way to the masjid, do you want to see it?'\n\n'If you think it's worth seeing,' said Astha, humble in her being guided mode.\n\n'It's one of the biggest and richest temples here. Hindus and Muslims fought over it too, though that is not so well publicised.'\n\nThey climbed steps lined with beggars, mostly old people dressed in white or saffron, begging bowls in front, in which people were dropping money, coconuts, sweets, prashad. Overtaking them were eager pilgrims bounding up, shouting, 'Jai Shri Ram, Jai Siya Ram'.\n\n'This is supposed to be the temple with the most steps,' said Pipee, as they passed an old lady bent over her cane, her eyes on her bony, bare brown feet, with their spread-out toes. They could hear her murmuring Ram, Ram, with every step she took.\n\nInside Pipee hung back as Astha advanced towards the shrine flanked by huge donation boxes. A long line of devotees queued before the priest, clutching their offerings, boxes of sweets, coconuts, flower garlands, small thalis with tikka and incense. The priest, swift and practised, set aside their garlands and coconuts, deftly opened each box, dumped half the sweets in the bucket next to him, and returned the rest. The devotee then took a parikrama of the shrine, lingered in the courtyard and rang the bell while leaving.\n\nIf only I could feel like that, thought Astha, looking at the expression on some of their faces, coming to this temple would mean so much. Her eyes fell on the daan box. She opened her purse and took out five rupees, I wish I had something more in my life, I wish an end to this hollow feeling. She shoved the money in the box and rejoined Pipee.\n\n'Do you not believe?' asked Astha as they passed through the inner courtyard, and down the steps.\n\n'No,' she spat out. 'I believe in nothing. I hate religion. You wanted to see, and I am showing you,'\n\n'I'm sorry,' said Astha, a little alarmed, 'that you are doing something you don't want to.'\n\nThe woman drew a breath, and touched her arm briefly, 'No, I'm sorry I was like that, it's nothing to do with you. Come, let's go to Kanak Bhavan.'\n\nOn the way, Astha hesitantly, 'If you hate religion, doesn't it upset you to come to places like these, where there is nothing but?'\n\n'Oh, who cares how upset I get?' she said flippantly. 'I have to come. We are based in a slum, and this kind of field trip works very well to sensitise women to communal issues, which in moments of crisis get totally out of hand. Besides I don't like staying in Delhi much.'\n\n'Why is that?'\n\n'No particular reason. I live alone, I like to travel.'\n\nAstha looked sideways at Pipee and encountered nothing but her hair.\n\n*\n\nIn Kanak Bhavan a small guide greeted them.\n\n'Five rupees, I show you.'\n\nAnd for five rupees they saw the room where Ram slept, where Sita played her sitar, where they played chess, where they bathed, where they dressed, the cupboard where those clothes were kept, where Sita got ready to receive Ram in the evenings, where Kekayi did Sita's muh dekhayii when she came a bride into this house.\n\n'Wasn't all this some thousands of years, BC?' whispered Astha to Pipee, amazed that such anachronisms could be taken seriously.\n\n'Nothing here is archaeologically or historically accurate,' whispered Pipee back.\n\nThe boy gauged what they were saying, though in English.\n\n'Who knows what is real or not?' he said, smooth beyond his years. 'What matters is the feeling of devotion,'\n\nAstha felt ashamed of herself, and tipped him ten rupees as they climbed down the narrow stairs, into the main courtyard below, repeating her earlier wish for something, she knew not what.\n\n'I take it you are religious?' asked Pipee, observing the size of the tip.\n\n'I gave because I want something.'\n\n'He's not a wishing well.'\n\n'He will do for one.'\n\n'What did you wish for?'\n\n'There are many hollows in my life, and I wanted them filled.'\n\nPipee fell silent, and Astha wondered about her empty spaces, with eyes like that, there could be many. 'Are we going to the masjid now?' she ventured as they left Kanak Bhavan.\n\nPipee sighed, 'We should have gone there first, but I always find it so depressing.'\n\n'Why?'\n\n'You'll see.'\n\n*\n\nThey walked up Ramkot, the slight incline that led to the mosque. The way was lined with temples. Temples, houses, houses that doubled as temples, temples that doubled as dharamsalas, all needed for the lakhs of pilgrims that descended on holy occasions. Ramnavmi, Diwali, Navratra.\n\n'It makes me sick the way Ram is being associated with Hindu-Indian-nationalism. It was terrible when the locks of the masjid were opened some years ago. The Muslims were not even given a hearing, considering this is waqf land. Millions of pilgrims poured in to see statues they believed were placed there divinely because God wanted his birthplace back. People will believe anything.'\n\n'We did a play about it,' put in Astha.\n\n'Really?'\n\n'With Aijaz.'\n\n'You knew him?'\n\n'He came to our school. He put together a brilliant piece about the Babri Masjid. Then I never saw him again.'\n\n'His life was short.'\n\n'Yes. That's why I am part of this group.'\n\n'The S double M?'\n\n'Is that what you call it?'\n\n'When I call it anything. I prefer never to think about it.'\n\n'Why? Don't you think they do good work?'\n\n'It's so elitist, and Aijaz was nothing if not one of the people. Now they sell art in his name.'\n\n'I did something they sold.'\n\n'You are part of their core group?'\n\nAstha laughed dryly. 'Hardly that. I can barely make it to a few meetings. But the Manch was happy to have my canvas, I was happy to sell it, and the cause benefited, surely.'\n\n'I wonder. Preaching to the converted. Working through songs, art, literature.'\n\n'But that was what he himself did, I saw him, and he was very effective.'\n\n'There is now such strong feeling about Hindu manhood, pride, valour, protection of the motherland, redressing the wrongs of history, that I wonder whether any street play, song or poster can make a point beyond general entertainment.'\n\n'There were lots of people last night.'\n\n'Of course they were there. They know how to promote themselves all right.'\n\n'If you think like this, why are you here? Why did you bring your women?' challenged Astha.\n\n'To attend the picnic,' said Pipee facetiously before lapsing into silence, leaving Astha to wonder how much to tread in these murky waters.\n\nShe turned her gaze to the bare feet of the women in front. It was not necessary to walk barefoot so far from the shrine, but for these women the very hill was sacrosanct, and their bare feet honoured a faith Astha could never have.\n\nIf she did, she would not be throwing money around, wishing for elusive fulfilment. Faith would do it. She too would walk barefoot up Ramkot not minding the stones, the heat, the germs, the piss of dogs, the shit of monkeys, the spit of people. Wearing no skin of dead animals to pollute the purity of the place, no leather, no shoes, no belt, no bag, no wallet.\n\nBy this time they had reached the top. The nicest thing about the mosque was its location. On the highest spot in Ayodhya, it overlooked the town, with its collection of spires, domes and houses crowded together. Beneath them swayed the trees, a mild calm breeze blew about, a breeze that seemed to suggest that there were many ways to worship.\n\nIn a mosque built in 1528 there was now a Hindu image. Was this not enough to make it a temple? Courts had declared that Hindus had the right to worship here. But now the worship had extended beyond the deity, so that the shape of the enclosing structure had become an obstacle to faith, and every barefoot pilgrim a warrior.\n\nAt the entrance they stopped to take off their shoes. Outside bhajans were blaring on loudspeakers, declaring that the name of god is more effective if all can hear. Inside, under the central dome, hardly visible under a mound of flowers, were the images flanked on either side by men in khaki armed with guns. In front of them a line of devotees streamed past, stuffing money into large donation boxes. Pipee refused to join the line while Astha, made uneasy by the guns, hurried past the little figure that had suddenly appeared on the night of December 22nd, 1949.\n\nThe two women walked down from Ramkot to a song shrieking from a cassette on full volume, _'We_ _will_ _go_ _to_ _Ayodhya_ _\/_ __ _We_ _will_ _build_ _Ram's_ _temple'._ They neared the main road and a waiting rickshaw wallah started cycling towards them. As he did so a policeman detached himself from a patrol group. His khaki clad belly hung over his belt, his truncheon swung from a thick hand. Leisurely he walked over to the approaching rickshaw wallah, grabbed him by his kurta, pulled so hard that the women could hear a tearing sound, forced him from his seat and kicked him. Once, twice. The grizzled rickshaw wallah looked around, smiled and slowly, quietly, began to pull his rickshaw away. Not a word was exchanged. The policeman walked back to his group.\n\n'God! Did you see? How could he behave like that?' demanded Astha, her tone shrill and naive.\n\n'Maybe he thought he was a Muslim,' shrugged Pipee.\n\n'So?'\n\n'So? I don't know. Perhaps the cops think Muslims shouldn't tread on this sacred soil. At any rate they generally don't come here. That man must have been desperate for customers.'\n\n'And why shouldn't Muslims... it's a mosque as well. He should have hit him back.'\n\n'And be beaten into pulp?' inquired Pipee. 'No, I don't think so.'\n\nAstha stared at the ground moodily, and pulled her sari palla further over her head to protect herself against the sun.\n\n'But why are you so upset?' demanded Pipee in her turn. 'These things happen all the time. Surely you know that.'\n\n'He looked like Himanshu,' said Astha suffering into her sari.\n\n'Himanshu?'\n\n'My son.'\n\n'Your son is so old?'\n\n'Of course not. But that look \u2013 when he gets out in cricket for example \u2013 he is not very good, and when he gets out \u2013 that is when he smiles. Just like that.'\n\n'Mother-son,' said Pipee somewhat gloomily. 'An obsessive over-protective phenomenon.'\n\nAstha felt defensive. 'Hardly. I am careful not to smother him. He is the one who clings to me.'\n\n'Despite yourself, you must be liking that.'\n\nSomehow Astha didn't mind this comment from Pipee, it was so non-judgemental. 'I do rather,' she confessed, and then, 'he is the only one in the whole world who smiles whenever he sees me, no matter what.'\n\n'Women. So pathetic in their hunger for love,' remarked Pipee sapiently, guiding Astha into a tea stall.\n\n'Isn't there someone you love?' asked Astha seizing this opportunity, hoping Pipee wouldn't draw back, that it was not too early to be exchanging of themselves.\n\n'I married for it,' said Pipee, and again that reserve. Yesterday it was 'I live alone'. Was she divorced, had her husband been unfaithful, or cruel, was it a problem of in-laws? How soon before she could ask?\n\n*\n\nThey left the tea stall, and had stopped for a moment under the shade of a tree to exchange phone numbers, when a monkey jumped on Astha's back. The sudden weight, the shock of her sari being pulled from her shoulder, her own scream, left her collapsed with fright.\n\nPipee grabbed Astha and examined her arms, her back, her neck, pushing the hair to one side, looking minutely for any scratch the monkey might have left.\n\nAt last she drew her away from a crowd that was beginning to gather, curiosity gleaming in their eyes. 'It's all right,' she declared, 'no scratch, you won't need rabies injections.'\n\nRabies injections. This thought had not occurred to Astha, she had registered nothing besides panic and the fact that Pipee's arm around her had tightened for an unnecessary second.\n\n*\n\nIt was getting on for five in the evening when they took separate rickshaws to go to their separate guest houses. Looking at the face before her, Astha said, 'Please keep in touch.'\n\nThe eyes crinkled. 'Of course. I'll be visiting my in-laws for a few days, they are not far from here. So sometime next week?'\n\nI'll look forward. I mean it,' Astha went on babbling, hating herself. Why did she always have to sound so stupid? And how come she was visiting in-laws when she was no longer married? Perhaps it wasn't divorce but death, and she so young and attractive, with her smile, her hair, her skin, her eyes.\n\nThe mouth folded inwards. 'Well, see you then.'\n\nThey smiled at each other and parted.\n\n*\n\nBack in the guest house. 'I see you have met Aijaz's wife,' said Reshana.\n\n'No, I haven't,' replied Astha apprehensively.\n\nPipeelika \u2013 wife. The one you were talking to last night.'\n\nOh no. What must she have thought, why didn't she say, why didn't she guess, what kind of impression did she make \u2013 oh no, oh no, oh no. Aijaz. Aijaz's wife. What must it be like to be Aijaz's wife? Widow \u2013 widow, not wife. So that was why she looked like that, and spoke like that.\n\n'It's obvious she didn't tell you,' Reshana continued. 'Just like her. Very strange woman. After his death she became totally neurotic. Wanted to own his memory. How can anyone do that? Aijaz was one of the people, if he was anything, but she resented everything we did to keep his memory alive, accusing us of all sorts of things. It was so perverse.'\n\n'To have your husband die like that must be very difficult,' murmured Astha.\n\n'It was hard for everybody, not only her,' shot out Reshana.\n\n'But why didn't you tell me?'\n\n'Me tell you? Why should I?' demanded Reshana annoyed. 'When I saw her with you, I imagined she must have introduced herself like any normal person. Besides she avoids us''\n\n'She did, she did introduce herself,' Astha was already defending Pipee.\n\nReshana looked at her, 'Then how come you didn't know she was Aijaz's wife?'\n\n'She only said her first name, and that was so unusual I started commenting on it \u2013 oh, I don't know.'\n\n'Huh. She never forgets she was his wife when it comes to the Manch. Always bad-mouthing us. I'm surprised she didn't try it with you.'\n\n'She said nothing about the Manch.'\n\n'Surprising. She usually does. Probably trying to impress you with her tolerance. Or maybe she is a little better now, I hoped so when I saw her in the audience,' said Reshana briskly packing her last item, her bathroom slippers, and then sitting on the suitcase to shut it. She was leaving by bus for Lucknow to do some field work.\n\n*\n\nAlone Astha sat in a daze. She had met Aijaz's wife. She couldn't believe she had spent so many hours with her without knowing. She didn't think Pipee would phone her in Delhi, it now seemed too improbable. But she felt wretched, and in this mood passed the remaining few hours before leaving for the station.\n\n'What is your train, beti?' asked the widow when she went down.\n\n'The Sarva Yamuna.'\n\nThe widow sighed.\n\n'It is not a good train?' Astha asked uneasily.\n\n'It will be late,' said the widow.\n\n'How late?'\n\n'Two \u2013 three hours. Maybe four.'\n\n'How can you be so sure?'\n\n'It comes through Bihar. No law and order in Bihar. So the train is always late.'\n\n'Always?'\n\n'Without fail,' said the widow looking ghoulish and satisfied. 'These Biharis keep pulling the chain when and where they feel like it, getting off, getting on, so the train gets later and later. Simple.'\n\nThere was nothing Astha could do, but continue as she had planned. She was afraid of waiting at the station so many hours alone, she thought of Hemant who had not wanted her to come, and who might consider himself justified if she was dragged into a corner of a deserted platform and raped.\n\nShe gritted her teeth against her unreason, while the widow sent her gardener, Hanif, to call a rickshaw.\n\n'Here is your packed dinner,' she said handing her the box Astha had forgotten she had paid for.\n\n'Thank you,' she said and at nine-thirty departed into the still small-town night. The stars above were more brilliant than they ever could be in the polluted skies of Delhi.\n\n*\n\nThe usual damp, stale, wet coal, urine station smells met her as she stepped onto the platform. She sat desolately on her suitcase and waited. Soon, it would be ten. Maybe the widow was wrong.\n\nAt 11 p.m. the train was announced to be three hours late, and the widow was proved right. The numbness that had been seeping into Astha during the past hour intensified. Mosquitoes big as flies buzzed around her. She slapped them off, and started walking up and down the platform. There were mostly people sleeping, covered from head to foot in sheets, shawls, durries or sacking to keep the mosquitoes away; shapeless bundles, unidentifiable as man and woman, the length alone indicating child or adult. Water pipes strung along the side of the track dripped through the hoses that were attached to each one.\n\nShe drank a kulhar full of sweet tea and went on walking up and down the long platform in a kind of daze. There were a few other passengers waiting, but most were the bundles scattered abundantly about. At the far end in the darkest corner of the station, the only sign of life, a bundle with an elbow raised, jerking frenziedly and rhythmically beneath the cloth, in an action Astha immediately recognised. She hurried back to the tube lights of the central platform, where amid the mosquitoes and the tinkling of water dripping onto rail tracks, she had another kulhar of sweet earth-smelling tea, and then waited, waited in one spot for the train to come.\n\n*\n\nAstha passed the night in the train restlessly. Not for her the easy sleep on the way out. Was it only two days ago that she had left? She thought of the meeting, the speech she had given, all the temples she had seen, the security around the masjid, Pipee protecting her from the monkey, thinking of rabies injections. She wished she had known her connection to Aijaz, she wouldn't feel so foolish now, but Pipee clearly hadn't wanted to tell, that much was obvious. If she had had the foresight to take her number she could at least phone and apologise.\n\nHer thoughts turned to home. When would Hemant come back? When the work finishes was all he had said, maybe he would have called his mother and informed her. How would it be between them? Would he still be annoyed that she had gone away? Had he missed her?\n\n*\n\nNext morning, Delhi. Quickly she hired a coolie and jumped into a three-wheeler with her suitcase. She hadn't seen her children for two days and three nights, and now every thought was fastened onto them.\n\nVasant Vihar at last. She rang the doorbell, and there they were, her precious children.\n\nHimanshu rushed to hug her, clutching a drawing in his hand that said _Welcome_ _Home_ _Mama._ Anuradha complained about the book she had to read in the holidays.\n\nI am home, thought Astha. Emotion grabbed her firmly by the throat.\n\n'Do you like my drawing?' shouted Himanshu, tugging her hand, feeling not enough attention had been paid to it.\n\n'I love your drawing,' responded Astha automatically. She lifted it close to her face to illustrate her total concentration. 'Such peacocks, so colourful, and my, what a sun. And so many cars, you have done well with the cars. Are these blue streaks rain?'\n\nHimanshu nodded.\n\n'So interesting. My goodness, Himu, you've got everything in this drawing.'\n\n'Didi said you wouldn't like it,' said Himanshu.\n\n'Well, Anu doesn't know everything, does she?' replied Astha.\n\nHimanshu beamed, and lost no time in informing his sister, 'Mama likes it. So there. You were wrong.'\n\nAnuradha did not even have to think. 'She's saying that to be nice to you, stupid.'\n\n'That was an entirely unnecessary thing to say,' Astha informed her daughter.\n\n'You mean you are not being nice to him?'\n\n'It's not that. I like the painting irrespective of whether I am his mother or not.'\n\nAnuradha did not bother to reply, while Himanshu said nothing more about his art work.\n\n'Have you heard from Papa, Anu? When is he coming home?'\n\n'Don't you know?' asked Anuradha, puzzled.\n\n'Of course,' said Astha quickly. 'I just wondered whether he was sticking to his original plans.'\n\n'Well I don't know. Dadi might. Ask her.'\n\n'I will. Now I'll just go up and see her, all right?'\n\nAs she climbed the steps to make sure no cause for offence would be found in postponed expressions of gratitude, or delayed news giving, Astha thought yes, indeed, she was home.\n\nHemant arrived next evening, smiling, genial and pleasant. Astha was relieved. Clearly their misunderstanding was a thing of the past. 'So,' he asked, after her questions about Bombay, 'the mosque still standing?'\n\n'Yes indeed.'\n\n'And Ayodhya? How was that? Did you see anything?'\n\n'It was very nice,' said Astha, pleased at his interest. 'I met a girl. She showed me around.'\n\n'What kind of girl?' smiled Hemant. 'You are no more than a girl yourself.'\n\n'Oh Hemant, don't be silly, I am old. She is much younger than me.'\n\n'Well, what kind of girl?' he repeated indulgently.\n\nAstha changed the topic. 'The mosque is quite unremarkable, you know. It is old, but that's it. Half its beauty comes from the little hill where it is, overlooking the town. But it's full of policemen with guns, it's not possible to worship there, though people do \u2013 lots.'\n\n'I told you there was no need for you to go.'\n\n'I had to make a speech, don't you remember?'\n\n'Why do you have to travel to Ayodhya to make a speech? It's not as though you were a religious leader, or a politician or a public figure.'\n\n'But it is important for everyone to do what they can, to make things better, you have to try, whether ultimately it makes a difference is not in your hands,' said Astha earnestly.\n\n'Well, I hope you are not going to indulge in more rabblerousing.'\n\nHis fingers were twisting the ring he had given her so that her hand hurt. Hopelessness settled in its familiar place in her chest. He belittled her, yet if she pointed this out, he would deny it. It was better to stay silent.\n\nLater they made love.\n\nThe ritual enacted before partings, after homecomings, this establishment of the marital tie, this coming together of flesh that had been sundered.\n\n*\n\nOr so Astha thought, until next morning, while unpacking his suitcase, she came across a condom.\n\nShe stared at it for a long time, its implications running through her head. What should she do? Leave it in the suitcase, throw it, or confront him? Who had he slept with, he who was never in any place for very long, it could not be that he was in love \u2013 or had a relationship \u2013 or maybe he did. Some woman might travel with him, how would she ever know? Maybe the distributor had supplied him with someone, she had read somewhere that women were often a part of business deals.\n\nBut why now? Was this his message to her? Was this why he seemed in good spirits? Why he had asked her about Ayodhya, and expressed an interest in what she had seen?\n\nFinally she left the suitcase on the bed, the lid closed and buckled, the children should not see and ask what is this, the servants should not see and jump to unnecessary conclusions.\n\nShe waited till he came home. It was 9 p.m., he was late as usual. As usual he first poured himself a drink before settling down in the drawing room. The children were interacted with, while Astha moved between the drawing room, kitchen, and dining area, unable to sit anywhere, the condom firmly in her heart.\n\n'How are things at work?' she asked after a while. Not that he ever discussed business with her. For that he had his father.\n\nTo her surprise he didn't brush aside the question. 'There is trouble in some factories in Noida, all the TV ones.'\n\nAstha had to drag her mind to this. 'Are you worried about ours?'\n\nHemant got irritated. 'Obviously I am worried. Different unions compete for power over the workers, and we get caught in the middle. Everybody suffers but who sees that?'\n\n'You pay a fair wage, the workers will realise that making trouble will benefit no one.'\n\n'Even if they don't come to their senses, I can't pay more than I do. Five thousand for the men with overtime, and four thousand for the women with benefits. Paying four hundred and fifty salaries is no joke,' brooded Hemant, 'and these are the rates.'\n\n'Then what is the problem?'\n\n'The Communist Party Union tells them they can ensure they get more benefits and a higher wage. Well, let's see. So far they have not been able to make inroads.'\n\n'Maybe nothing will happen.'\n\nHemant grunted, slowly sipped his drink, threw back his head on the sofa and closed his eyes. It seemed a bit difficult to bring up the condom in these circumstances, yet it had to be done. There were problems in her life as well as his.\n\nThere's something in your suitcase,' she said. 'Perhaps you would like to take it out yourself. I left it on the bed.'\n\n'Yes?' he said indifferently. 'What is it?'\n\n'A condom.'\n\nAt this he opened his eyes. 'Ah, yes.'\n\n'I take it when you travel you have sex, and that is why there are condoms in your suitcase,' Astha could barely keep her voice from breaking.\n\n'As usual, your imagination runs away with you.'\n\n'That is not an answer.'\n\n'For your information, I don't.'\n\nShe didn't believe him, and yet the hurt eased a little. 'You carry condoms just like that?'\n\n'Of course not. The dealer wanted to give me a girl, was very insistent, forced this condom on me, but I'm not that kind of guy, I left for the bar before the girl came. As you can see, it is not used.'\n\nAs a story it was thin, but yes, the condom was not used. Hemant got up and stroked her cheek. 'Even if you behave badly I love you, he said.'\n\nAstha forced herself to be content with this. It was too dangerous to venture further. \nChapter VII\n\nPipee called a week later.\n\n'I'm sorry, I'm sorry, I didn't know. You must have thought me horrible. I'm sorry,' Astha rushed to say.\n\n'How were you to know? I didn't tell you. You could be the one angry with me.'\n\n'Of course not, how could I be angry with you? You spent so much time with me, you showed me places you hate, you protected me from the monkey.'\n\n'Hardly,' she laughed, 'I can't stop monkeys from jumping onto people, much as I would like to.'\n\n'I have been waiting and waiting to tell you I'm sorry if I upset you in any way.'\n\n'Well now you've told me. And you didn't upset me in any way \u2013 I'm over that kind of stuff. Don't worry about it.'\n\nA pause. Then, 'You were going to show me your paintings.'\n\n'Please, come over. I would love that.'\n\n'Tomorrow?'\n\n'Please.'\n\nShe had called, she had called, and for a moment despite the condom, and all the wretchedness of the past week, Astha felt a little lighter.\n\n*\n\nShe looked at the work she was doing for the Manch, trying to prepare a readable memorandum that would combine historical accuracy with emotional appeal. It was proving uphill work. How could she make the nation care about the fact that no destruction of a temple had been chronicled in Babur, or in any other contemporary source, be it Abdul Qadir Badauni of the 16th centrury, or Goswami Tulsidas, in Ramcharitmanas.\n\nAstha stared rebelliously at her writing: _The_ _un-Islamic_ _black_ _stone_ _pillars_ _within_ _the_ _mosque_ _are_ _not_ _proof_ _that_ _a_ _temple_ _was_ _destroyed_ _on_ _the_ _Babri_ _Masjid_ _site._ _As_ _they_ _are_ _not_ _load_ _bearing,_ _they_ _were_ _probably_ _taken_ _from_ _a_ _Hindu_ _or_ _Jain_ _temple,_ _ravaged_ _by_ _Shah_ _Juran_ _Ghori_ _and_ _brought_ _for_ _decoration._ _Seeing_ _their_ _location_ _as_ _a_ _sign_ _of_ _contempt_ _for_ _Hindu_ _feelings_ _is_ _a_ _political_ _interpretation._\n\nIt sounded so uninteresting. Yet she had to go on sifting, sieving, fact from fact, fiction from fiction, and in the end not be sure of anything. It was lonely working on these pamphlets, it was not like painting where she required no mind to bounce her thoughts off. If only she had some of Aijaz's magic.\n\nAs she looked at what she was writing, her old hostility to words rose in her. She couldn't do it, she was a painter, not a writer.\n\n*\n\n'But it's not bad,' said Pipee the next day, when Astha showed it to her.\n\n'Pedantic, dry and boring,' said Astha.\n\nPipee pulled in the corners of her mouth while Astha stared in fascination at the dents it made in her cheeks. 'Now don't bother so much, just finish it. No one will read it anyway. The Manch excels in preaching to the converted.'\n\n'But it's for the nation.'\n\n'Please. Give me a break.'\n\nThey went to Astha's work room. Pipee's eyes flickered over the canvases. 'I know nothing about painting,' she said. 'You must teach me.'\n\n'There is nothing to learn. I've always responded to colours. It's words I find so slippery,' said Astha, the burden of _The_ _Testimony_ _of_ _the_ _Black_ _Pillars_ lying heavy upon her.\n\n'How do you manage to fit so many people in?'\n\n'It's something I learned from the miniatures. They are both very full and very detailed, I love that.'\n\n'It must take for ever.'\n\n'It does, rather. The one the Manch sold took almost six months. Now I am getting faster, but still \u2013 I can't work on them as much as I wish.'\n\n'You've got a pretty fancy set-up, it couldn't be that difficult. Doesn't your husband help you?'\n\n'My husband spends a lot of time at the factory and he travels too, so he can't really help with the children. And the setup...' her voice trailed off miserably. It was hard to explain her life, especially when she herself barely understood it.\n\nThe usual female trap, it's all right, you are not alone, we all experience it in one way or another,' said Pipee putting her hand on Astha's and pressing it gently. 'So if you want to do anything of your own I guess you have to work your ass off. You are like an ant too. I shall call you Ant, I'm not sure I like this faith business.'\n\nAstha blushed with pleasure, 'So we can be ants together.'\n\n'Exactly.'\n\n*\n\nAnuradha and Himanshu stared at Pipee over lunch.\n\n'Is that your scooter outside?' asked Himanshu.\n\n'Yes.'\n\n'How come you ride a scooter?'\n\n'To get around. Do you think only men should drive scooters?' asked Pipee.\n\nAstha felt embarrassed at her son's ideas, maybe she hadn't been sensitizing him to gender issues. She blushed into her roti, while Anuradha asked accusingly, 'How come you are called ant?'\n\n'My father thought I should work like an ant for the good of the community.'\n\n'And do you?'\n\nPipee smiled at the assembled mother and children. 'Sometimes,' she said, 'when I feel like it. I'm not a very good ant I am afraid.'\n\n*\n\nPipee left after tea when Astha began to worry about her children's homework. Driving home on her scooter, she thought I want to know her better, at least she doesn't remind me of Aijaz. Her house is quite near mine, that is convenient, I wonder if she realises she is attractive. Her marriage sounds horrible. I'm sure her husband is a jerk.\n\nShe thought of Astha's painting. She clearly had a political sensibility, which made her acquiescence in a traditional domestic set-up even stranger. Maybe she was just unawak-ened. And she loved her hair, it was so thick and curled around her face even when tied back, and her skin was so pretty, clear pink and white.\n\nAs for Astha who had shown such eagerness to know Pipee, how was she to realise that given certain circumstances, there was no aphrodisiac more powerful than talking, no seduction more effective than curiosity.\n\nThey began to meet more often. Astha was circumspect in revealing the amount of time she spent with Pipee. She knew it would be frowned upon as excessive. When the boundaries of what might be considered normal interaction passed, she started to lie. Thus an element of secrecy entered the relationship and gave it an illicit character.\n\nThey met on weekdays; evenings and weekends were out. Still Hemant caught a whiff of this new interest in his wife's life and was free with his disapproval. Since Pipee was a woman this disapproval was tinged with contempt, and the assurance of no real threat, indeed had Pipee been a man, Astha would have found it impossible to stray so far down the road of intimacy, or be so comfortable on it.\n\n'Women,' said the husband emphatically after a somewhat long phone conversation the wife had had with her friend, 'always mind-fucking.'\n\nAstha cringed. Mind-fucking. Not the excitement of the real thing. The organ penetrated, the ears, the weapon of penetration, words. Words, that left no mark but in the mind,where they mingled with others that had been used to describe someone else's past, till those experiences became your own, and you saw with other eyes, because you were no longer one person, but two. Listening upon listening, fucking upon fucking. In full view.\n\nThen she grew angry. How dare Hemant be so derogatory. Would he prefer her to be like him, with condoms in her suitcase, which a friend had put there by accident? She refused to engage with him on any issue, he was capable of nothing but the very crudest understanding. Instead she related the whole to Pipee who said that men were so pathetic, so fucked up themselves, they only understood the physical, and in this way she felt soothed.\n\n*\n\n'Have you ever been in a relationship with a woman?' asked Pipee one day.\n\nThey were lingering at the caf\u00e9 at the Tagore Arts Centre, after a lunch of kebabs and roti. It was late February, there were people sitting on the steps of the lawn next to them, on the walls white rose creepers were blooming. It was almost four, and the sunny spot they had originally chosen had long gone cold. A little boy was swabbing at the tables with a dirty cloth, a waiter was tilting the chairs against the tables, to enable the sweeper to clean properly. Pipee's voice had dropped to a murmur, Astha leaned forward to catch her words.\n\nAstha felt uneasy and didn't answer.\n\n'Well?'\n\nShe tried to laugh. 'I'm married,' she said.\n\n'So? Are you telling me you are happy, fulfilled, and what have you?'\n\nUnexpected tears came to Astha's eyes. Pipee was instantly contrite. 'I'm sorry. I didn't mean to make you cry.'\n\n'It's all right,' said Astha wiping her nose on the edge of her sari palla.\n\n'No, it's not,' said Pipee. 'If you are unhappy, it's not all right.'\n\nAstha went on sniffing. 'I don't usually think about it,' she offered.\n\n'Who would think about anything if they could help it?' said Pipee gloomily, 'God knows I have tried...'\n\nThere was a silence while Pipee drew squiggles in the rings of water left by their glasses on the table, and Astha watched her fingers. 'Have you?' she finally asked.\n\n'Once. Met her in school, continued in college, on and off for three years. Eventually she got married. Much later I did too.'\n\n'Oh.'\n\n'What was her name?'\n\n'Samira.'\n\n'Was she nice?'\n\n'Not often. She seduced me, and then when I fell in love, triumphed in that power. It was not so different from being with a man, though I am sure it can be.'\n\n'Oh?'\n\n'It is more a question of choice than people make out. That is what I believe at any rate. Besides sex is sex, don't you think? It is other things that become important.'\n\n'Yes, yes \u2013 of course. Did your husband know?'\n\n'I told him. But you know what men are like\n\n'No, I don't think I do,' said Astha forlornly. 'I have actually only known my husband, and now I am not even sure of that.'\n\nShe thought of the condom again \u2013 would it go on coming up in her mind at every point of sadness in her life, she wondered. She could tell Pipee about it, but Pipee might think she was inadequate in her responses, or weak in her understanding, or a fool. For now she preferred to keep this wound to herself.\n\n'Does your husband have affairs?'\n\n'I don't know.' Then quickly, 'Did yours?'\n\n'Well there were several women before we got married, I knew that.' Astha thought of the little gesture Aijaz had offered her, and now realised that it was in fact an invitation. 'I think he must have had an affair with Reshana Singh, the way she goes on. I know she thinks I am jealous, and maybe,' went on Pipee reflectively, 'I am.' She shook her head. There is no escape from jealousy, is there? We are all embryonic Othellos.'\n\n'I know what you mean,' said Astha gloomily.\n\n'Yes, well. I don't know why I am delving into the past today,' said Pipee, hauling her heavy bag onto her shoulder and getting up to go as the sweeper reached their table.\n\n'Maybe so I can get to know you.'\n\n*\n\n'You're so pretty, Ant.'\n\n'Do you really think so?'\n\nThey were sitting in Pipee's flat drinking beer before an early lunch. Pipee had made arrangements to go to work late, and now she pulled Astha by the hand and led her to the bathroom mirror.\n\n'Are we going to do mirror, mirror on the wall \/ Who is the fairest one of all?' laughed Astha nervously. She often felt an underlying tension when talking to Pipee, as they swooped and dived among their lives, offering bits to the other to share.\n\n'A modern version of it,' said Pipee putting on the light and pushing Astha's head gently forwards. 'Look.'\n\nAstha tried to turn away, 'I don't like looking at my face, especially so close.'\n\nThen she felt Pipee's hands in her hair, her clip undone, her hands framing the oval of her face. Lightly from behind she traced her eyebrows with her fingers, her nose, cheeks and mouth.\n\nThe two women said nothing looking at their reflections in the small water-stained mirror. 'See?' whispered Pipee.\n\nAstha saw nothing, and abruptly left the bathroom. Later taking a scooter-rickshaw home, she felt lost and confused, the image of the two of them in the mirror often returning when she thought of Pipee.\n\n*\n\nOne day, in Astha's house, a rare occasion. Pipee preferred to meet Astha anywhere else than in her house.\n\n'So this is the marital bed,' said Pipee, surveying Astha's room, full of double bed. 'The marital bed in the marital room.'\n\n'Like in most people's houses,' replied Astha, not particularly liking Pipee's tone.\n\n'I know. It's how I used to live. Are you happy here? Do you have good sex?'\n\n'Good enough, I suppose.'\n\n'Don't you know?'\n\n'Well he was my first, and only.'\n\n'You're joking.'\n\n'Not really.'\n\n'What about the other two?'\n\n'They were crushes. One I kissed a lot, with the other there were only letters.'\n\n'Have you ever wanted more lovers?'\n\nWhat could Astha say? She was living, the way people like her lived, where was the question of more lovers, or love for that matter?\n\nPipee stretched out her palm for Astha's hand. Gently she held it, fingering her thumb nail. Round and round the stubby nail Pipee's finger went, lightly tracing the pink part, the white part, the skin part. Astha looked at their two hands together, and inched a little closer to the woman on her bed.\n\nPipee took a firmer grip of the hand in hers, and turned it over, stroking the back of it, gently sliding her rings off, and putting them on her own fingers, manoeuvring her bangles off and slipping them on to her own more narrow wrist.\n\n'I look so bare without them,' murmured Astha.\n\n'All the better,' murmured Pipee even more softly. Her breath quickened, and she pressed the tips of Astha's fingers into her mouth, sucking each one gently before letting them go. Astha hardly dared breathe.\n\n'What would your precious spouse say, if he saw us together now?' asked Pipee.\n\nAstha swallowed and did not reply.\n\n'Did you say he was a faithful husband?'\n\n'I didn't say anything.'\n\n'Is he good in bed?'\n\n'I suppose.'\n\n'If you have to suppose, he is not,' said Pipee severely.\n\nAstha decided she knew nothing about love making, that she was inexperienced and stupid. 'What about you?' she responded in a low tone, 'You yourself have only had two lovers.'\n\n'Yes, that's true,' sighed Pipee. 'But I'm looking for a third.'\n\n*\n\n'Why so silent?' asked Hemant that evening.\n\n'Silent? Am I?'\n\n'You need me to tell you that?'\n\n'Sorry. I hadn't realised.'\n\nMore silence. What should she talk about? What had she talked about before silence came upon her? Their days, his day certainly. Now she made enquiries.\n\n'I have managed to bribe our union leader this time, but bribing is difficult, the workers are watchful and suspicious, I won't be able to do it again.'\n\nAstha hated it when Hemant talked about bribing, and yet the way he described it, it seemed necessary.\n\n'Pipee came over today,' she said, changing the subject.\n\n'That woman,' said Hemant.\n\nAstha's heart sank. Things would be difficult if Hemant became violent about his dislike. She tried to change the topic again, but Hemant was having none of it. 'What did you say she did?' he continued.\n\n'She works with basti children,' said Astha proudly. 'She helps them get through school, she gives them a sense of self-confidence, and strength.'\n\n'Who finances this?'\n\n'She's part of an NGO called Ujjala.'\n\nHemant grunted, 'One of those types.'\n\n'What does that mean?'\n\n'Take money from here and there, and pretend they are working.'\n\nIt seemed there was nothing Astha could say, and yet he wanted her to talk. She started on the children. That was always safe. It was what they were united upon, and it served its purpose now.\n\n*\n\nThat night, Hemant started his sex routine.\n\n'No,' said Astha, 'I don't feel like it.'\n\nHemant paused. This was the first time his wife had not felt like it. 'What's up?' he demanded.\n\n'Nothing.'\n\n'Then?'\n\n'Then what? Do I have to give it just because you are my husband? Unless I feel close to you I can't \u2013 I'm not a sex object, you have others for that.'\n\nHemant relaxed. That old thing. He took her face in his hands. 'Sweetheart, why do you upset yourself over nothing? You are my wife, I love you, there has never been another woman for me, never. On business trips people don't understand commitments to wife and family, they assume their clients want a good time. If I had had sex, would the condom not have been used? You only tell me,' he whispered, his hands falling to her breasts and circling them in the way that was so familiar, kneading them, pressing them, as he continued, 'you only tell me,' then pulling up her nightie, and fondling her, 'does what you imagine have any logic?'\n\nWithout her willing it her body responded. Hemant became even more ardent. 'Baby, you are the only one for me, what's the matter, are you jealous?'\n\n'No,' she said, trying to push him away, but it was of no use.\n\nAfter the marital function had been performed, Astha got up to wash herself. Looking up from her wet and soapy hands, she caught sight of a sad and haggard face. How old she looked, and yet she wasn't old. She was thirty-six, but all the life seemed gone. She leaned over the sink, and examined her face more carefully, certain to increase her wretchedness. Around her eyes tiny wrinkles were beginning to form. She stretched her mouth in imitation laughter, and they became more pronounced. She stared at her nose and saw the blackheads there. Her skin looked yellow and sallow, when she put her head up to look at the folds in her neck more clearly, she could see the white line at the base of her scalp, where the new hair had come, and the dyed parts grown out.\n\nWhy should anyone love her, she thought hopelessly. She was so ugly. She thought of Pipee sucking her fingers. She looked at them, and put them experimentally in her mouth. They didn't taste very nice \u2013 of soap and sex. What had Pipee thought of them? And what would Pipee's own fingers taste like? What had Pipee seen when she had pushed her face towards the mirror? Certainly not what she saw now. Slowly she went back to bed.\n\n*\n\nHer meetings with Pipee increased. When she was alone in the home in the mornings Pipee dropped by on her way to work, she phoned her at least five times a day, short brief conversations, but which drew each of them firmly into the nitty gritty of the other's life. And the days when she didn't see or talk to her were days with something missing, and not even extra hours at the canvas could fill the vacuum Astha felt. She started to fantasise about touching her, imagined her hair between her fingers, her skin beneath her own, her hands on the back of her neck.\n\n*\n\nAstha frantically trying to reach an appointed meeting place.\n\n'I'm sorry, I'm sorry, I'm sorry.'\n\n'I was about to go.'\n\n'I'm sorry. I couldn't help it.'\n\n'What happened?'\n\n'I forgot it was a bandh. Not a single scooter wallah agreed to come. Not one. They said they would be beaten up. I even offered fifty bucks.'\n\n'Then?'\n\n'In the end he took eighty.'\n\n'Eighty! Three times! You shouldn't have paid it, Ant.'\n\n'I had no choice. I would have given him anything.'\n\n'He was taking advantage of the situation,' said Pipee sternly.\n\n'What could I do? You were waiting, I kept thinking of that, but I was on the road, and there was no way to tell you.'\n\n'Oh sweetie, it's all right. Now, forget about it. I thought you must be having a problem. I can't imagine where I'd be without my scooter.'\n\n'I don't see why you haven't bought a car,' said Pipee later, as she was stirring her cold coffee. The meal had ended, and Astha was worrying about how she was to get home. 'One needs to be mobile. I learned how to drive a scooter, it was all I could afford, but with you it's different.'\n\n'We have a car.'\n\n'Hemant's.'\n\n'Which I use.'\n\n'Only when he doesn't.'\n\n'He sends it back from the factory with the driver whenever I need it.'\n\n'I'm sure he does, but you can be more independent with your own.'\n\n'A car costs over a lakh.'\n\n'You could hold an exhibition, and earn.'\n\n'Not lakhs.'\n\n'Ant, why are you being like this? Didn't you tell me your mother left you some money?'\n\n'With Hemant.' And the old hurt comes to choke her.\n\n'Hemant is not a monster. Have you tried asking him? Since ask you must.'\n\n'He'll say whenever I want the car, I have it. Also I can ask my in-laws for theirs.'\n\n'If Hemant can keep a car for his parents, he can keep one for you.'\n\n'Well, I, the children that is, use it as well. For tuitions, classes, and stuff.'\n\n'The point is'' went on Pipee patiently, 'if you had a car you would not have to do all this asking business.'\n\n'I can't ask for a car for myself.'\n\n'Why not?'\n\n'Hemant says there is going to be trouble in the factory.'\n\n'How long has this factory been running?'\n\n'Ten years.'\n\n'I imagine he has made enough money to buy you a car.'\n\n'He is very generous to me.'\n\n'Good. Now come let me drop you home otherwise you'll be cheated all over again.'\n\nAs they roared through the streets of Delhi, Astha leaned against Pipee, with her arms around her waist. Once or twice Pipee turned to ask, are you all right, Astha merely nodded, too happy to speak, even had the sound of the vehicle allowed it.\n\n*\n\n'But why? The car is there for you whenever you want it.'\n\n'Please, Hemant. I am thirty-six. I need to be independent. I am always adjusting to everybody else's needs.'\n\n'And the money?'\n\n'We could use what my mother gave.'\n\n'You know I have invested that for the children, and in five years the amount has grown nicely.' Hemant looked satisfied. Astha had heard all this before, heard when the bonus shares came, heard when the dividends came, when the debentures were bought, heard as it doubled, trebled, quadrupled. There was no question of touching it, she knew that. Only somewhere surely there was money she could touch? She said as much.\n\nHemant looked at her. 'Who is putting these ideas into your head?'\n\n'Nobody'' said Astha offended. 'Does somebody have to tell me to want a car?'\n\n*\n\n'Mama, Papa is getting you a car?' Anuradha. Hemant had told her first.\n\n'It is also my money'' said Astha suddenly angry. The children turned towards her, slightly shocked. Only prices were discussed in their house, never money, and certainly not whose money was whose. It was all common money because they were a family.\n\n'Papa's money too'' said Anuradha quickly.\n\n'Of course Papa's money too. But if necessary I will hold an exhibition to help pay for this car.'\n\nHimanshu put his hand into hers. 'When I earn I will buy you a car'' he proclaimed. Astha tightened her hold on his thin interwoven fingers and stared at the overgrown nails, at the fine hair glinting blondly, at the sun exposed brown skin.\n\n'And I will buy a car for Papa'' said Anuradha.\n\n'But Papa and Mama are not separate'' said Astha, quickly. 'Whatever you buy will be for both of us. Don't I use the car we already have? It is not Papa's or mine. And now we will have a second car, besides the one upstairs, neither Papa's nor mine, but for everybody. We are a family with growing needs.'\n\nIt was the end of the term, before the summer holidays, and PTA day. Astha was in her children's school, trying not to stare at the fathers and mothers around her, united and content.\n\nThere was a whole list of teachers she had to see.\n\nHimanshu had done badly in his mid-terms. He hadn't finished his papers, but really he knew everything. Astha was waiting to tell his class teacher this, something she had been saying since nursery. The teacher in turn would tell her that even so, he had to increase his speed, other children managed. If he didn't, he was going to find it very difficult at the higher levels. Astha could predict the conversation verbatim, but these motions had to be gone through.\n\nAnuradha was doing badly in science and maths. She didn't understand the method of explanation, and Astha had to find out why in a way that didn't compromise her daughter's intelligence, attentiveness, or abilities. Both her children were dead against her discussing any of their problems in school. Before she had left Anu had screamed, don't say anything, don't Mama, then in class the teacher says, so you are having difficulties, why don't you ask me during the lesson instead of complaining later, and the kids stare at me, as though I am a moron. Besides, I keep telling you, there is no _point_ asking for explanations, they all repeat the same thing in the same way, only slower.\n\nHimanshu had looked equally worried, you are not going to say anything to my teacher, are you, Mama? In class, she'll say something to me, she will get angry.\n\n*\n\nWith the futility of it all firmly established, she waited in line after line to see various instructors, behind parents busy pumping them for the secret of success in that particular subject. She was going to be late for her meeting with Pipee, why had she, against all experience, allotted two hours to school? Now precious minutes were being wasted in the corridors of this huge and unfeeling institution.\n\nThe maths teacher, Mr Sharma, before whom there was a line half a mile long. Anu obviously not the only one having problems. After an hour of waiting, her turn.\n\nAnuradha? Yes, a very bright child, but she should work harder, if she has a problem in following, she should ask me, I tell them, ask me, there is no excuse for not understanding. She should do one hour timed exercise every day, maths is only practice.\n\nAstha pulled out Anu's mid-term exam from her bag, 59 out of 100. Mr Sharma's temper rose the moment he saw it, look at this, correct method, but a mistake in adding and the answer is wrong. And here, she has copied the sum incorrectly. How will she get marks? Crooked margins, untidy rough work. Careless, careless.\n\nAstha stared at the paper, she understood nothing of it. Were Anu's crimes so bad? A crooked margin, a sum added wrong, another carelessly copied, did that result in 59 and feeling a failure?\n\nSurely it doesn't matter if the rough work is not neat? she queried.\n\nIt is the attitude that matters, the attitude, thundered Mr Sharma.\n\nIn the face of this, further comment seemed redundant.\n\n*\n\nFinally, all the teachers met, the Vadera children ticked off in various registers, her participation in the learning process marked, her children's faults pointed out and noted, and she was free. Frantically she ran out of the school gates, she was over an hour late already. Pipee would have cooked for her, she would be wondering.\n\nAs she rang the bell to Pipee's apartment, she could hear footsteps coming towards the door. Her heart beat faster, explanations trembled on her lips. The door opened, and before her, the face, always in her mind, always indistinct, the long narrow eyes, the hair which sprang back wild and unruly, the voice she could drown in, the mouth that pulled inwards as she smiled, the little mole hanging under her nose like a dew drop.\n\nThere she was, and there Astha stood, and nothing else mattered. Silently Pipee motioned her in, took her bag, and closed the door.\n\n'What took you so long? I was getting worried.'\n\n'Sorry, Pip, I'm sorry, I couldn't help it, there were millions of parents, and the teachers took ages. I kept thinking of you waiting, I felt terrible the whole time.'\n\nThey were standing. Slowly Pipee put her arms around her. She could feel her hands on the narrowness of her back, on the beginning spread of her hips. Gently she undid her blouse hooks, and her bra, looking at her face as she did so and slowly she continued, feeling her back with her palm, coming round up towards her breasts, feeling their softness, especially where the nipples were, feeling them again and again, in no hurry to reach any conclusion. They were enclosed in a circle of silence, the only sound, the sound of their breaths, close together and mingled.\n\nIn the small bedroom, Astha tense with nervousness. She was afraid, yet there was no going back. Sensing how she felt, Pipee took her time, touching every crevice of her body with her mouth. The sweaty patches of her armpits with small stiff hair beginning to poke out, the soft fold of flesh where the arm joined the torso, the hard bony part behind her ears, the deep crease between her buttocks, the hairiness between her thighs.\n\nIn between they talked, the talk of discovery and attraction, of the history of a three month relationship, the teasing and pleasure of an intimacy that was complete and absolute, expressed through minds as much as bodies.\n\nAfterwards Astha felt strange, making love to a woman took getting used to.\n\nAnd it also felt strange, making love to a friend instead of an adversary.\n\n*\n\nShe returned home in a daze. As she neared her house, she succumbed to panic, she was a mother, nothing should disturb that. For a brief and guilty moment she wished she was like Pipee, alone and free, but she checked herself. A large part of her belonged to her children, that was how she lived her life. She couldn't imagine any other way.\n\nShe was a wife too, but not much of her was required there. A willing body at night, a willing pair of hands and feet in the day and an obedient mouth were the necessary prerequisites of Hemant's wife.\n\n*\n\nA few days later. 'Hemant should be pleased'' said Astha to her lover, 'he says women are always mind-fucking.'\n\nThey both laughed at the wife's revenge.\n\nAstha was in love. All day she thought of her, visualising the turn of her neck, long, sloping, unornamented, the collar bones on either side of the small hollow at the base of her throat, the screws of her hair latticed, as she had once seen them against the dark, heavy, green of the trees of the Tagore Arts Centre. And her fingers, long and so narrow the bones showed, with stubby nails, and a snake ring, three silver bands, two small turquoise eyes, two black painted dots for a nose. And her eyelids, that fragile tender area where she wanted to press her lips, compressing them to the size of peas to enclose that space. And the mouth with its inward-turning corners, she could gaze at those dents for ever.\n\nFrom time to time she brooded about her own sexual nature, but her desire for Pipee was so linked to the particular person, that she failed to draw any general conclusions. So far as her marriage was concerned, they were both women, nothing was seriously threatened. Meanwhile her best time at home was when she was fantasising about the one she loved without interruptions, lost in her thoughts, wallowing in her feelings.\n\nAll this made it difficult for her to focus on what was going on around her. She was able to forget she had another life only when she was absorbed in her painting or her children's homework, an echo of an earlier simplicity that now appeared to have some advantages.\n\n*\n\nAstha was surprised when Hemant noticed.\n\n'You seem distracted'' he pointed out. 'What is it?'\n\nShe felt a flash of fear, but then an affair with a woman was not an easy thing for a husband to suspect. Caution drew her lips into a smile, and put a hood across her eyes. 'Nothing'' she said. 'What do you imagine?'\n\nA look of dissatisfaction that it seemed must always be on one of their two faces, crossed his brow, giving her a momentary sense of control. She was not there. How right he was. But when had he acquired the sensitivity?\n\nWhat about the times he had not been there, and the reasons had always been such that her own claims seemed selfish. Now sexually involved with another, she realised how many facets in the relationship between her husband and herself reflected power rather than love. Hemant had managed to ignore her because ultimately he filled his own landscape. That her discontent had been expressed in nuances that were minor, only helped him in his disregard.\n\nIn the days that followed, Hemant began to watch Astha. Let him watch, thought Astha, he who had not looked since the early days of marriage, was now looking and found that what he saw did not add up.\n\nHer lies grew skilful. Her desperation and her need ensured that they tripped off her tongue, as though she had rehearsed them for hours.\n\n*\n\nFed by right-minded parents, Astha had believed that never, ever must one lie. There was a Pinocchio lurking in her moral self, waiting and watching. Her nose would grow, her eyes cross themselves in vain attempts to hide the gruesome deed, her skin would turn yellow and pimples sprout all over her. Her inner ugliness would be reflected for all to see.\n\nShe had lied about the boys she had known, and each time she had been punished. They had left her, she had not deserved better.\n\nWhen she married she had wanted to tell her husband about those boys, but she had been afraid he would not accept her, and that tiny seed, usually forgotten, was still inside, telling her she was unworthy. She had compromised by being excessively truthful; she knew her husband trusted her implicitly.\n\nNow, she lied all day. The strongest thing in her was the most secret. Pipee encouraged her. Not for her the moral values of George Washington, the boy on the burning deck, Eklavya, Ram, Sita, Lakshman, those for whom words translated into codes of honour never to be broken no matter what.\n\n'Of course you have to lie. They don't own you.'\n\n'I know, but... I wish I didn't feel the way I did.'\n\n'What way?' asked her lover very naturally, and when she didn't reply, very insistently, 'what way?'\n\n'Oh you know'' Astha became vague. 'So much of the real stuff is with you, and since I can't talk about it with my family, it makes me feel pretty schizo.'\n\n'Why do you have to talk about it with them? You talk about it with me. Are they your guardians or something?'\n\nIt was hard to explain. Pipee lived on a grander, more open scale then she did.\n\n*\n\nMeanwhile this grand and open creature was growing jealous of other claims. She had even wondered, to Astha's horror, when she was going to inform Hemant about them.\n\n'He is not your owner, you know, he'll have to face up to his inadequacies.'\n\n'No, no \u2013 I can't do that.'\n\n'Why not?'\n\nIt seemed so unthinkable, how could she explain. 'Maybe I'm a coward'' she remarked thoughtfully.\n\n'Oh dearest'' sighed Pipee, 'Don't be so hard on yourself. You've lived a certain way, you are used to certain ideas, you can't suddenly be different. If I am impatient with your situation, it's because I want you to be happy.'\n\nShe turned the other's face towards her, took out her pins and stroked her open hair, reaching into the scalp, in a way that reminded Astha of her mother oiling her hair every Sunday when she was young. She closed her eyes and sank against her, feeling as though she were in a warm bath. With Pipee there was no battering against something hard and ununderstanding, she was all warmth and intuition. She thanked God again for this love in her life, when she had thought all chance of love was over.\n\n*\n\nIf God had given her love, there was no time supplement with this gift, so Astha often found herself wishing despairingly she could live each day twice, once with Pipee, and once on the ordinary plane.\n\nShe dreaded the occasions when her lives clashed, and was at no time more at the mercy of her circumstances than weekends.\n\nOne Wednesday Pipee said firmly, 'There is a gay and lesbian film festival this Saturday. I know, I know, Saturdays are difficult for you, but I want to go, and I think you should too.'\n\n'But Pip...'\n\n'Don't say anything. I want to see the films with you. Don't you think they have a special relevance?'\n\nThis was undeniable.\n\n'Do you mind if I ask Hemant? Don't worry, he is bound not to come.'\n\nPipee made a face. 'Then why ask him?'\n\n'He's going to be home this weekend.' went on Astha hurriedly. 'He will find it strange if I make a programme without him.'\n\n'Let him.'\n\n'He is beginning to complain.'\n\n'Of what?'\n\n'Oh, just'' said Astha, avoiding specifics. 'You know. He feels something is not quite right.'\n\n'Well, it's not. It's time he woke up.'\n\n'I wouldn't go so far'' said Astha quickly.\n\n'You wouldn't, huh?'\n\n'No. Everything is all right the way it is.'\n\n'Don't ignore the obvious, Ant'' was all Pipee said.\n\n*\n\n'Go with you and Pipeelika Khan to a gay film show? Are you out of your mind, Az?'\n\n'Well, I am going with her. You can for once come to something I am interested in.'\n\nHemant stared. 'I'm not interested in homosexuals'' he said. 'And I thought neither were you. But I'm learning something every day.' He held out his hand, and Astha slowly put her own in his. 'Stay home. We can rent a video. We haven't done that in a long time.'\n\nIt was not fair. It needed his wife's having an affair for Hemant to promise to see a video with her, something he knew she loved. Such an evening might have made her happy a year ago, now it seemed like blackmail.\n\n'I've promised Pipee...' she said.\n\n'So? Unpromise her.'\n\n'Maybe next Saturday, but not this.'\n\nA sullen look settled on Hemant's face. Astha could see resentment, and she felt sorry. But not half as sorry as she would feel if she didn't go, didn't sit next to Pipee in a dark hall, with their arms, hands, knees touching.\n\n'Why can't we do this some other time?' she went on, 'often you have not been here for me. I think you should be understanding about one day.'\n\n'I was busy, Az, I was establishing myself.'\n\n'For ten years?'\n\n'It takes that long, you knew that, you supported me.'\n\nTheir disagreements had the history of their marriage hanging onto them, and Astha had no time for this now. 'We can continue our discussion when I come back'' she said.\n\nHemant refused to respond, and Astha could think of nothing further to say. She didn't want to leave him like this, but he was giving her no choice. Quickly she put on the first sari that came to hand, grabbed a shawl and left.\n\n*\n\nIt was only when she walked down the road to the scooter stand that she realised it was in fact a chilly day, and she was going to feel cold. Should she go back for a sweater? No, she didn't want to encounter Hemant again, better to freeze. She wrapped her shawl and palla firmly around her, and hoped it wouldn't rain.\n\nThe cold bit through her in the three-wheeler, and her nose began to run. She looked through her purse for a hanky \u2013 no, she had forgotten to bring a hanky as well. She bent down and wiped her nose on her petticoat.\n\nAt the community centre, Astha made her hesitant way into the hall. She was late, and it took her a few minutes to adjust to the darkness. She felt her sari being tugged, and there was Pipee leaning against the wall by the entrance, waiting. She sank down next to her, feeling exhausted after the battle with Hemant, looking with cursory interest at the screen, registering indifferently the men and women, speaking broad American about the discrimination they faced as gays. In between some social scientist gave his opinion, in between that there were clips of marches and demonstrations. Astha looked at the faces on the screen. All of them open, none of them living a life of lies.\n\nPipee's voice breathed through the craven recesses of her mind, 'We have to struggle for acceptance and the right to love as we feel. Don't you think so, Ant?' But try as Astha could, she could not connect to what she was seeing. Her own situation was different, though if Pipee didn't think so she would keep that information to herself.\n\nIn the intermission, Astha broke the news to Pipee as they were drinking coffee, 'I have to go.'\n\n'Why?' she could hear the concern in Pipee's voice, feel her hand as it lay in the crook of her elbow. 'Are you feeling all right? Don't you find it interesting?'\n\n'Anuradha has a test. It was difficult for me to get away today.' She didn't want to go into the whole Hemant thing.\n\nAt the sounds of domesticity, Pipee's face twisted slightly, but she merely said, 'Why can't the father sit with her for a change? Why do you have to do it all the time?'\n\n'That's the way it is.'\n\n'Go then.' She gave her a little push.\n\n'Dearest, don't be offended, I'll make it up to you, I swear.'\n\n'How will you go home?'\n\n'Scooter'' said Astha dully. How did it matter how she got home? Maybe she should crawl to that shrine of marital and maternal bliss on her belly, or drag herself on her behind with the stumps of her legs sticking out straight out in front of her, pulling herself along with her hands the way lepers did, begging for alms around the crowded intersections of Delhi.\n\n'Pipeee'' Astha could hear somebody shouting. 'Hurry up, the next film has started.'\n\n'I'll call you when I get home.' She squeezed Astha's arm again, kissed her cheek, and vanished into the hall warm with its comradeship for her, cold with its indifference towards Astha.\n\n*\n\n'How come you are back so early?' asked Hemant in a jocular tone, as he saw her descend from the scooter, her ears red and her nose now running like a river. Without looking at him, Astha held out a hand and said, 'Hanky.' Hemant took out his own and gave it to her. She blew her nose, at long last, through the tortures of the day, she was able to blow her nose.\n\nThen she looked at him. He was smiling. He thought he had won, and now was trying to be nice to her.\n\n'Just like that'' she said.\n\n'Gay and lesbian films not your cup of tea, huh?'\n\n'Not at all. They were very good.'\n\n'Then?'\n\n'Then what? The hall was crowded, and I didn't have enough to wear, and I was feeling cold, and I came home because I didn't want to sit on the floor too long.'\n\nHemant looked disbelieving.\n\n'Have the children had their lunch?' asked Astha.\n\n'Yes, for all you care.'\n\nIt didn't bother Astha, his tone, nothing bothered her. She went inside, she was hungry, she had had no breakfast, and she now ate some leftover lunch. Then she made herself a cup of tea, she felt a sore throat coming, and for now would think of nothing but her physical wellbeing.\n\n*\n\nThat evening Hemant solicitously offered her a brandy for her cold. He talked of her painting, he talked about the children, he talked about her mother, his parents, he said maybe next year they would go on a holiday to America, by then the factory problems would be sorted, he worked very hard, and he needed to take it easy. And the car, what about that?\n\nShe, which car?\n\nHe looked hurt. The car she had demanded, had said she needed in order to be independent, he had arranged to get one in the company name.\n\nIn the company name. It was that easy after all.\n\nWhat did she think of a white Maruti?\n\nAstha's feelers went up. She could not remember when her opinion had been sought about a major purchase. Why was he being so considerate? Was he trying to buy her? True, she would be able to rush to Pipee's whenever she liked, in the car her husband had bought for her, but how was that going to make her feel? She didn't want a car, she realised, it would end up making her feel more guilty, and were she to express all this to Pipee she would say, but it is your car, why do you feel you have to pay for it with mind, body and soul, she could hear her voice even now.\n\n'I don't know how to drive'' she temporised.\n\n'I'll teach you, we can learn every Sunday'' he said, caressing her. 'We will be together.'\n\n'Yes, yes, I suppose.'\n\n'Try and sound a little enthusiastic, will you?'\n\n'I am enthusiastic, why do you look for meanings in everything I say? They say husbands should not teach wives, the relationship deteriorates'' said Astha irritably.\n\n'We'll see. I don't want to waste money on lessons.'\n\nThe car came. Hemant in fact had no time. In the end it was Ram Singh, the driver, who taught Astha in the colony lanes.\n\n*\n\nThat summer Rajiv Gandhi was assassinated by a suicide bomber in Tamil Nadu during an election campaign. Political uncertainty meant that Pipee's work in the bastis grew more demanding and she could not see Astha much in the day. Astha felt her absence every minute, and when Pipee called her in the evenings, she went, but her home situation was such that the meetings had to be hurried, not more than an hour, not much for lovers, certainly not much for Pipee.\n\n'Ajay has written. He is keen to sponsor me. He has been suggesting it ever since I finished my MA.'\n\n'What?'\n\n'A Ph.D.'\n\n'You never told me.'\n\n'It was before I met you'' said Pipee.\n\n'How come you never mentioned it? And why are you telling me now?'\n\n'Because his letter has come'' said Pipee in limpid accents.\n\nAstha slid Pipee's hand from under her shoulder and looked at it. There was the ring, there were the bones, there were the long thin fingers. She placed their palms together, and thought it was an illusion, you could never be one with another, no matter how hard you tried. It was better to realise and accept that, life became easier once you did.\n\n'Yes'' she said, 'you better explore doing a Ph.D. It's a good thing for the future.'\n\n'Well let me see. It will mean leaving you.'\n\nAnd Astha's poor heart rejoiced to hear she was important.\n\n*\n\nDriving home, Astha brooded over Ajay's letter. She was not stupid, she knew why Pipee had brought up the letter. She wants a full life, after six months she wants commitment, if I can't give it to her, why shouldn't she look elsewhere, but she didn't want Pipee to look elsewhere, she wanted her to stay with her for ever, as she was, as they were.\n\nResentfully she thought of Pipee's Ph.D. Suddenly it was a burning desire. Well, she knew why. She was saying if Astha had her children, she had her Ph.D., as though you could equate the two.\n\n*\n\nAstha: I have a fantasy, listen my love, and do not laugh. It is not much, I think it is not much.\n\nI have a room, small but private, where my family pass before my eyes. It is very light, before me is a wall which divides the house, but I can see my children, that satisfies me, though to them I am invisible, that satisfies me too.\n\nThis room will be our room, you with me, living in harmony. Our lives are separate, different things call to us, different demands are made on us, but always that solid base beneath us, like two flies caught in a sticky pool they cannot leave.\n\n*\n\n'Sticky flies? You must be mad.'\n\n'All right the image is bad. Still, you know what I mean.'\n\n'You are a hopeless romantic. You want me and you want not to leave your old life. It's a nice fantasy, I wish it were possible. I also wish'' added Pipee after a little thought, 'that it had taken place in my house.'\n\n'Does that show something?' asked Astha, wishing that she with whom she shared everything, was not quite so into analysis.\n\n'Well, what do you think?'\n\n'I think nothing. It was a dream, an idle dream, for God's sake, something I know can never happen.'\n\n'But it can, don't you see, even in a dream you are in your precious Vasant Vihar. There are other places in the world, Ant, if you would only consider them. Instead you allow yourself to be shut up by that man, who neither knows nor appreciates you, and for what? I do not understand.'\n\n'There are my children.'\n\n'Your children don't have to be stuck in that house any more than you do.'\n\nAstha's mind boggled. What about their school, their routine, their friends in the colony, their grandparents, their father, who whatever his faults, did love his kids? Maybe she was deeply conventional but for her the business of raising children had a set of dynamics that were the standard ones. That those dynamics did not include companionship and understanding was regrettable, but she had grown used to it. She saw herself as a bird pecking at a few leftover crumbs from the feast of life. She said as much. Pipee stared at her.\n\n'I never thought of myself as a crumb'' she said dryly.\n\nThis drove Astha on to further explanations.\n\n'I love you, you know how much you mean to me, I try and prove it every moment we have together, but I can't abandon my family, I can't. Maybe I should not have looked for happiness, but I couldn't help myself. I suppose you think I should not be in a relationship, but I had not foreseen... Oh Pipee, I'm sorry I am not like you.'\n\n'What do you mean? Don't you want an honest aboveboard life?'\n\n'You are being unfair.'\n\n'When do you ever think of me? Always their needs, your needs, before mine.'\n\n'That's not true.'\n\n'It is. You can't see me in the evenings\u2014'\n\n'How can you say that? Just the other day I spent the whole evening with you, I went home at twelve, I told endless lies\u2014'\n\n'Who asked you to tell lies? I didn't. Don't you see, Ant, I want an end to all this deception.'\n\n'My whole life is a fabric of lies'' said Astha sadly, 'you are the one true thing I have.'\n\n'And you don't want to change it. That's the trouble with married people'' said Pipee gloomily, 'there are always others involved. Why did I think with a woman it would be different?'\n\nPanic rose in Astha. Tears came to her eyes, and she felt a headache coming on. All she wanted to do was drive back, shut herself in her room, and sleep till the end of time. She got up.\n\n'What are you doing?'\n\n'Going home, since you ask.'\n\nPipee reached out and pulled her dupatta. 'Don't you get it? That I love you, I want you, I miss you?'\n\n'What about your other friends and your work?' asked Astha in a small voice.\n\n'What about it? Work never kept one warm at night, and yes, I have friends, but they are not people I choose to be intimate with. Either I spend my time here moping, or I go out with them, talk, laugh, then come home to a flat which holds the moments I have had with you. It reminds me\u2014' Here she paused, Astha looked tortured, and Pipee continued quickly, 'whatever it is, I don't wish to experience that kind of emptiness again. Sometimes I go crazy with longing, and I can't even pick up the phone.'\n\n'You can.'\n\n'I can't. I don't want to hear your husband's voice, I don't want to put the phone down if he picks it up, I don't want to share your life of lies.'\n\nAstha thought that if husband and wife are one person, then Pipee and she were even more so. She had shared parts of herself she had never shared before. She felt complete with her. But this was not the time to say these things.\n\n'I'm sorry, I don't mean to be harsh'' said Pipee contritely. 'Leaving a marriage, even like yours, could not be easy. I do feel that away from that house and those people you will be able to lead a fuller life. You have so much in you, so much to give, but take your time. Whatever you do it'll be all right.'\n\nAstha turned towards her gratefully. That her lover should understand how she was feeling was enough. She sank down to the sofa into her arms.\n\n*\n\nIt was dark before Astha got up to leave. With the thought of Pipee in her mind, the scent of Pipee on her body she moved trance-like towards her car, driving slowly and automatically down the roads of Delhi.\n\nAs Astha parked the car outside the gates of her house Anuradha came rushing out. 'I'm failing'' she gasped. 'You have to see my teacher. Where were you? I have been waiting hours and hours.'\n\n'In what subject?'\n\n'Maths.'\n\nOf course. It had to be maths.\n\nAnuradha was biting her nails. 'I'll fail the year, I'll fail the year, I'll fail the year, I'll fail the year'' she chanted in a frenzy.\n\n'Can we wait for the results before you decide that?' demanded Astha.\n\n'You don't care! Why can't I have tuition, all my friends have tuition, but you want me to do it by myself, because you did.'\n\n'Anu, don't be unreasonable. When did I say...?'\n\n'You did, and now you have forgotten. You want me to be like you. I am not, but you don't care.'\n\nAnuradha stared at her mother, tears streaming down her young cheeks. Dear God, thought Astha, when did I say she had to be like me? When did I say she couldn't have tuition because I hadn't? When?\n\n'Sweetie, can we talk about this later? I have just come home, I am tired, I don't remember what I said and when and why. If it is necessary of course you will have a tutor, but you also have to learn to do things on your own.'\n\nAnuradha ran inside, scowling. 'I had to phone Papa, you weren't here, he said to wait till you came home, you would know what to do, you know the teachers, but what's the use? I have been saying I need tuition. No one listens to me.'\n\nAstha walked in wearily. In the face of Ami's maths her own feelings seemed an indulgence rather than a necessity. They would have to wait, wait in the wings, wait on a more permanent basis.\n\n*\n\nAfter dinner Astha watched her daughter sit in her favourite chair and read unconcernedly. The lamp shone on her hair, highlighting its copper shades. Her socks lay untidily on the carpet, she was wiggling her toes in front of the heater, the light catching the lurid colours of her nail polish. Her troubles were over, her friend's tutor had been phoned, he was going to start from next week.\n\nAstha sighed and took another sip of her tea. On the sofa next to her, Himanshu laboured over his homework, his notebook getting more and more smudged as he continually rubbed out what he had written, shredding bits of the page.\n\nHer thoughts wandered, to the series she had imagined on mosques and temples in Ayodhya, Kashi and Mathura. Pipee had thought it was a brilliant idea, but there was no space in her head to execute any idea, be it ever so brilliant. \nChapter VIII\n\nNovember 26th\n\nPip called. I want you to come with me for three weeks. Will you?\n\nWhere?\n\nThe Ekta Yatra, from December 10 to January 26. It starts from Kanyakumari and ends in Kashmir.\n\nI thought of the Rath Yatra canvas I had painted for the Manch. Here was another journey, taken by another Leader.\n\nWhat's this Yatra all about I ask her.\n\nThe usual. This Leader says he wants to unify the country.\n\nThey all say that.\n\nWell, he is going to spread his message from north to south, east to west. I know it's a political stunt, yatras like this create nothing but trouble, but it's an excuse to be together. Will you come?\n\nOf course.\n\n(My heart is beating, my hands begin to sweat, of course I will come as though it is the easiest thing in the world, of course, as though I can get up and go anywhere I like, anytime I like, of course, because I love you, and at times love makes life simple because it demands you worship at its altar, dragged though you may be kicking and screaming.)\n\nGood. Talk to you later. I have to work on this, her voice breaks in on my thoughts.\n\nI put the phone down. Three weeks with her. My mind is whirring, how will I manage it, what will I say, but I have to go, I have to. She has never asked me to do anything so directly. Is this a test, I wonder?\n\n*\n\nNext day\n\nWith P. Why a Yatra? I ask. It's not like Ayodhya. You are not taking Ujjala helpers to sensitise them to communal issues.\n\nI thought of this as a way to be together. Then she hesitated. I am learning the language of her voice, she was about to say something she thought might hurt me.\n\nAnd?\n\nShe was silent.\n\nCome on, Pip, tell me, though actually I don't want to hear. I want to stay with the pleasure of proof that she wants to be with me, but I force myself to ask because shadows between us are ten times worse.\n\nShe started talking of communal issues as an area of research, something that now interests her. (Due to Aijaz's death, I imagine.) With an interpreter she could get some field work done.\n\nSo she thought I might be upset because she is still thinking of her wretched higher studies.\n\nNice, I said enthusiastically, what a good idea.\n\nShe looked pleased, and then told me how she was arranging it. I waited for Neeraj's name to appear, which it did. I am sure Neeraj is pushing her towards this Ph.D. I hate Neeraj, though we met only once. She is fake arty, has a deep voice, smokes endless cigarettes, and of course has a marriage where her husband is deeply involved in all she does. He is a lawyer, and helps her when necessary, and in Ujjala it is always necessary. She looks upon Pipee as her prot\u00e9g\u00e9. Pip says she gets on her nerves sometimes, but she has been so kind to her, she can never say anything. I personally think she is insensitive and power hungry. I wonder if Pip has told her about us.\n\nNeeraj's cousin is a journalist, she is arranging that we go in the accompanying bus. Pip has to produce articles for her newspaper (I am sure Neeraj thinks it will help her CV). The cousin herself is going to make an initial brief appearance. After a week or so we go to Bangalore, then to Shiksha Kendra.\n\nShiksha Kendra, her school, her past, her mother. I want to absorb everything to do with her, because it is her.\n\n*\n\nNext day\n\nDreading talk with H. Yet why should I be nervous, hasn't he travelled, it is my turn, but even as I think this, I know it is the wrong argument to use. I shouldn't seem to want justice, it will create endless arguments, I must seem to want his compassion, his magnanimity. He is doing me a favour, but I must also be firm, he is not going to be compassionate and magnanimous if he has a choice.\n\nYesterday. Pip, why can't the two of us go on a holiday for the weekend, why the Ekta Yatra?\n\nI want more time with you. After this we will go on a holiday if you wish.\n\n*\n\nNight\n\nHemant could not believe his ears. What, go where? Do what?\n\nGo on the Ekta Yatra, cover it for the Manch. (Since he has never taken much interest in the Manch, he doesn't know this is not the kind of thing they would do.)\n\nThen he started. And went on and on. I was running off on a wild goose chase, neglecting my family and burdening his poor mother with my responsibilities. I had no sense of what was fitting for a woman, I hadn't bothered to ask him whether it was appropriate or convenient. Ever since Aijaz had died, and I had started being exploited by the Manch, and gone to Ayodhya, and met Pipeelika Khan, I had no sense of home, duty, wifehood or motherhood.\n\nI said nothing. What should I reply to? The text or the subtext? How calm my relationship with Pipee has made me! There was a time when had he said half so much I would have started crying. Now all I said was I am leaving on December 8th, and will call my mother to help with the children, I didn't want to bother his mother, or even him.\n\nThis made him even angrier. 'Why stop at this yatra?' he practically shouted. 'The Dalits have called a Nyaya Yatra, they want justice, some mill workers have called a Roti Yatra, they want employment, the Indian Save the Cow Federation has called a Cow Yatra, to prevent cow slaughter, every Tom, Dick and Harry is going to march up and down India demanding something. Join them all.'\n\nAgain I said nothing.\n\n'Who will protect you? Suppose you get raped?'\n\nHe doesn't care how low he hits. 'Why would I get raped?' I asked after a moment.\n\n'Anything can happen. All these yatras have goondas attached to them. You think everybody who is going is so moved by the desire to unite our country? Our country is better united by you staying at home, so that there is one less incident to cope with.'\n\nEvery day the papers are full of crimes against women. Yet I have to learn to not be so afraid. There are other women in this world. They live.\n\n*\n\nNovember 27th\n\nWhen I told P. about the rape she got quite angry. Tell that sod to stuff his fantasies of rape up his ass. What does he mean by scaring you like this? It is his way of keeping you at home.\n\nShe takes what I feel, clothes it into words, and there it is, for us to look at, and for me to feel better.\n\nHave to phone my mother tomorrow. Another session of blackmail and guilt.\n\n*\n\nNovember 29th\n\nFinally phoned. She was all against my going, of course. Little does Hemant realise how much her thinking matches his.\n\nShe started out by blessings \u2013 from both God and the swami, swiftly moving to blame. 'I have been expecting your call. It has been a long time since I heard from you, but then I know how busy you always are.'\n\n'I'm sorry, Ma.'\n\n'Give my love to Himanshu and Anuradha, give my regards to Hemant'' she went on, messages too important to be left to the end.\n\n'I'm going\u2014' I started.\n\n'With the family?' Sharp as a knife, when it comes to protecting their interests.\n\n'Is that the only reason you can think of for going somewhere?'\n\n'Then why are you going?'\n\n'For an assignment.'\n\n'With who?'\n\n'By myself. Please come and stay here.'\n\n'How long?'\n\n'Three weeks.'\n\n'Three weeks! Why are you leaving your family for three weeks?'\n\n'It's an assignment, I told you. Assignments don't adjust themselves for my convenience.'\n\n'Then don't take such assignments.'\n\n'Ma, will you come or not?'\n\n'I'll see. What does Hemant think?'\n\n'Why don't you ask him?'\n\nLet them both see together.\n\n*\n\nNovember 30th\n\nTaking large supply of headache medicines, couldn't bear to have a headache even one day. Just imagine in two weeks, I will be away from pollution, stress, tension, strain, I will be rolling along in a bus, staring out of the window, sitting next to her, our bodies touching.\n\n*\n\nDecember 1st\n\nPip talks about nothing else but the Yatra, I thought education of slum children was her speciality, but it seems she is diversifying. She is full of this as a political ploy, the Hindu vote bank under the pseudo secular banner of national unity, the Rath Yatra last year, the increase in communal tension, the rise in violent incidents, the number of towns under curfew. And incidentally, one Leader trying to replace another by doing his own journey.\n\nMaybe I can do another canvas on this Yatra \u2013 it will be fun seeing first-hand what it is all about.\n\n*\n\nP. has visited Neeraj's cousin twice. You also come.\n\nNo, you go. I shall see her on the trip.\n\nWhen I am with her and others I feel marginal and excluded. It's stupid, I know, but what we have is so intense I can't bear for it to be diluted, I can't bear for her not to give me her full attention. This is not _good,_ I know. Maybe if we were together all the time, it would be different.\n\nBut we are not because of me, not her, then I am the one who complains.\n\nHow do people have affairs? They seem very complicated businesses.\n\n*\n\nNext day\n\nTold H. my ticket has come, hoping to involve him in my going. He demanded to see it. 'Why do you want to see it?' I asked suspiciously. 'I don't have it.'\n\n'Why don't you have it?' he asked suspiciously in his turn. (The perfect marriage.)\n\n'The Manch has it.'\n\n'I want to check if it is all right'' he said.\n\n'Don't worry, it is all right. But thank you for your concern. I know you want me to develop myself and stand on my own two feet.'\n\n'Since my wife understands her duties so well, why should I worry?'\n\nHa, ha. Why don't we get divorced.\n\nI hope my children aren't tainted by his idea of my duty. I don't want them to think I am abandoning them. What if they are taught that while I am away?\n\n'In this day and age no child can think anything if their mother travels once in a blue moon'' said Pip.\n\nI wonder.\n\n*\n\nDecember 3rd\n\nAs the time comes to go I am tense and anxious. I have never left the children for so long. I told them this evening I was going for three weeks, and I'll phone you every day \u2013 I promise.\n\n'I don't care'' said Anu flicking her hair around. 'You can go. It doesn't matter.'\n\nI wanted to slap her. It is so difficult to reach her in her adult mode.\n\nHimu said, 'Go, Mama, we should learn to be without you.' (!) Sometimes he sounds so grown up.\n\nI wish things didn't seem so muddled and confused. Nothing is sure except that I might be raped. I walked to the church nearby where it is usually peaceful.\n\n'Teach me how to live, God'' I prayed, casting an uneasy eye at the Christ hanging bloodily before me. His own life had been short and violent, but presumably successful. 'I am not asking for happiness, but I would welcome some stability, so I need not run all over the place looking for love and confirmation. Give me substance, God, give me a life that has not been lived for nothing. And protect my children'' I added as I got up to leave.\n\nI thought of Pip on the way home. She has her future plans, her study, Ujjala, Neeraj. Is there really a place for me in her life? Though even as I write this, I can hear her saying you have your painting, your children, your home. If there is neediness in love, is it more or less genuine? If you need, you want, you search, you cling. You reward the person you have found with all your feelings.\n\n*\n\nDecember 5th\n\nMy mother has come radiating disapproval. She considers the whole trip unnecessary. She who has turned to God, while her daughter is running after human love, how can I reassure her?\n\n*\n\nDecember 6th\n\nYesterday Pip asked, 'Does Hemant think you are having an affair? Why else would he be so suspicious about your ticket?'\n\n'I don't know. I don't care.'\n\n'Maybe he indulges himself when he travels.'\n\nI felt a tightness in my chest, and then annoyance. Why does she keep bringing this up? 'I don't know what he does. It could be, I suppose.'\n\n'Most men do.'\n\n'Do they?'\n\n'Don't be such a child.'\n\nI thought of all the late nights at the factory, the trips out of town, the extended trips to South East Asia, the condom, the many opportunities there must have been, but I said nothing.\n\n'Sometimes one doesn't want to know. It's painful or inconvenient. But now you are not so dependent on him, now it is all right to see.'\n\n'I suppose.'\n\n'Does he suspect you are having an affair?'\n\n'It's not the same thing.'\n\n'Why not?'\n\n'You're a woman.'\n\n'And that makes you a faithful wife?'\n\n'No. But it is different, surely.'\n\n'What you mean is you don't feel guilty.'\n\nWhat could I say to this? This love of mine would have not been possible had she been a man, and yes, I don't feel guilty.\n\n'Would you mind if Hemant was having an affair?' she went on, probing.\n\n'Of course not. He can do what he likes.'\n\n'After all you do, don't you?'\n\n'Yes. Yes' I do.' __\n\n*\n\nTook the children and my mother to a restaurant in the evening. A last outing before I left. They wanted dosas so we went to Sagar. Hemant was working late as usual.\n\nIt was not a nice meal. I was not giving the children my full attention; they felt it and began to fight. My mother fingered everything unhappily. No doubt she was calculating the owner's profits, seeing how the place was jammed with customers. But because she is so spiritually oriented she was forced to remain silent. I couldn't wait to get home.\n\n*\n\nDecember 7th\n\nMa keeps saying in a puzzled way, why doesn't the Manch send a man, it's not safe for a woman, what kind of place is this, should I talk to them and explain the situation, you have a family, maybe they don't have families, why isn't Reshana going, why is she so keen to send you? Finally I lost my temper, and had to shout are men the only ones who can do things, nothing is going to happen to me, will you stop talking like this, you are making everything worse.\n\nShe continues in a different register, don't talk to strange men, don't wear any jewellery on your trip, not even your watch, be careful of what you eat and drink, keep on phoning.\n\nI have to remind myself of my three weeks with P.\n\n*\n\nLater.\n\nAm leaving shortly. More lies as to why they can't drop me at the station, Reshana, Manch, gathering early, train leaving late. I want to leave before Hemant comes home. Remember journey to Ayodhya, when the children came to leave me at the station, and waited for hours with me, the pre-Pipee time, the non-lying, looking for the key to happiness time.\n\n*\n\nStill later\n\nAt P.'s place we prepare for departure. We shut the windows, shut the fridge, leave two lights burning. I have had puris made for us and aaloo ki sabzi, along with pickles. We take all the fruit she has, plus water, glasses, steel plates, napkins, a knife.\n\nWe don't say much, but already I feel she and I are enclosed in our own special world. Is this feeling on call to those who are happily married?\n\nThis is what she is offering me if I leave Hemant, this togetherness. Dearest, is this why you were so insistent that I come? You have already proved your point, we don't have to get on that train at all, don't have to go to Kanyakumari via Madras, with my supposed Reshana at all.\n\nI think we are ready she said. The taxi will come soon.\n\nWe lock up and go down.\n\n*\n\nDecember 9th, night\n\nAt last, Kanyakumari. The train to Madras took for ever, and from there a bus. Felt complete and peaceful the whole way; I think she felt the same. No wonder marriages start with going away, cutting off from the old, entering the new with a journey, just the two of you \u2013 even in an ocean of people \u2013 just the two of you. It seemed so wonderful, we kept looking at each other and smiling.\n\nI am waiting for her to finish her bath, then we walk down to the beach. The Yatra starts tomorrow.\n\n*\n\nLater\n\nThe beach half a kilometre from hotel. We could see the gulls, smell the sea air. As we leave the hotel after tea, I babble, the tip of the continent, the tip of the continent. P. laughs at me, grabs my hand, and we run, our feet sliding in the softness of the sand. We run to the shore line, where we can see the waters of the Arabian Ocean, Bay of Bengal, and Indian Ocean merge, grey, blue and green. The sands are three distinct\n\ncolours too, red, black and pale yellow flowing into one another. There is something about the sea, its smells, its sounds, you feel small but liberated. There it is before you, vast and eternal. My troubles felt trivial.\n\nWe were together, we were happy. We walked along the water, me with my polyester sari tucked high into my petticoat, handbag with our money heavy but safe under arm, chappals in hand, P. with her salwar rolled up, taking turns with the bag.\n\nLittle boys ran up and down hawking packets of the separate coloured sands. I bought some for Anu and Himu.\n\nP. pointed out the Vivekananda Rock in the water. Apparently that is where Vivekananda stood one December a century ago and moved to great emotion by the sight of India across him, pledged to work for the upliftment of the masses and the unity of the country. The Leader of this Yatra is also big on unity and saving the country, still not unified; the masses, still not uplifted.\n\nI stare at the sunset as though I had never seen one before. I felt every second of its sinking in my bones. I am scared. No one can be so happy and have it last. When am I going to pay?\n\n*\n\nEarly morning, December 10th\n\nWe stayed awake the whole night. I kept telling Pipee she had to go to sleep, for me it was a holiday but she was here on work. She looked at me and said when will you learn anything, the whole thing was a way to be with you. She closed her hands over me, and I could scarcely breathe with the pleasure. I often find it hard to accept that she could desire someone like me, but when I am with her the doubts fade, and I feel strong and loved.\n\nMuslim and Sikh relatives of martyrs who have died for the country are gathered here to hand the Leader the flag that will be hoisted in Srinagar's Lal Chowk, 47 days, 14 states and 15,000 kilometres later.\n\n*\n\nDecember 10th, night\n\nThe mood of last night completely gone. Five hours in the hot sun. P. was a wreck. If she arranged this trip to be with me, if I need this kind of plan to leave home, then we pay for our sins in sweat and irritation.\n\nBut we are together \u2013 no denying \u2013 would I have had the imagination to think of something like this? Why am I so passive, why can't I bristle with initiative, maybe this is what she hates about me.\n\nHer Ph.D. rears its ugly head whenever I see her talk to someone or take out her notebook. She has already made contact with several journalists while I watch her.\n\nThe Leader was late, the auspicious moment came and went, and still we waited, sweat pouring down, 10,000 of us boiling away. Then finally the Leader spoke for one hour, then all the martyr's relatives spoke, then every Tom, Dick and Harry took his turn.\n\nAt 1.47 we started. The coconut was broken, lemons put under the wheels of the two vehicles made to look like a temple and a houseboat. South and north. Inside there are two rooms, storage, water tanks, etc. The Leader refused air-conditioning, he was taking this journey not for his comfort, but for the unity of India. We could have done with some air-conditioning, but then we are not leaders.\n\n*\n\nDecember 15th, night\n\nWe cross at least five villages or towns a day. Whenever I can I phone home from an STD booth. At appointed stops, the Leader emerges to the front of the houseboat he is riding in and addresses the people over loudspeakers. He indicates the flag in the Bharat Mata Temple perched on the bonnet of each vehicle. He tells them about the pride every Indian must have in his nation, the pride that has been trampled upon in the past. He announces that India is one, and that is the meaning of his journey. He declares that India will not tolerate terrorism in Punjab or Kashmir. He reiterates that no Indian can accept the separate status given to Kashmir, that Article 370 of the Constitution is now irrelevant. He describes the water he is carrying with him, the water of all of India's sacred rivers; the soil he is carrying belonging to the birthplaces of India's noble sons. He allows them to have darshan of the vessels in which the water and the soil is kept. Amazingly they want to. They rush to touch them, to put tikka on them, to garland them. They also want to touch the Leader's feet, but this the security men do not allow.\n\nAt night we eat what has been arranged for us at the circuit house or dak bungalow, and fall into bed, weary as hell. Perhaps it is just as well we are so tired for we do not have a room to ourselves. All intimacy is confined to the bathroom. In the bus our hands enjoy a limited freedom, no one can see what we do, but still, was there an easier way to be together?\n\n600 kilometres in 4 days.\n\n*\n\nDecember 18th\n\nWho would have thought one state was so large? We are still in Tamil Nadu. We are visiting, glimpsing rather, all the temple towns in a cavalcade, flanked by two security jeeps, rifle butts poking out through the windows. The Leader has to be protected. The heat of the air is sharp, this is their winter, so strange to never be cold. From the bus window, the landscape flashes by, the greens and the browns brighter than the ones I am used to, with an occasional rock or hill. I think of the flat plains of the north, and I think Ah, the diversity of India. Soon I will talk like the Leader, of Unity in Diversity, of The Oneness underlying The Difference.\n\nI fantasise about food constantly. The food provided for us is too hot, and I am forced to eat dry rice. Whenever I can I buy fruit for both of us. P. doesn't care what she eats, but if I go on with this stuff, I shall be sick.\n\nToday she gave a banana I had kept for her to a journalist. I wanted to kill that woman. In the bus P. said, I didn't want it, and her stomach is upset. What could I say? I kept my jealousy to myself.\n\n*\n\nDecember 20th\n\nWe are now in Karnataka. Phoned children from an STD booth near the tea stall where we had halted, while P. finishes her cold drink. We then walk down the road bordered by red earth. The cacti on the edge come up to my shoulders. There are fields and fields of tomatoes, light green against the leaves, supported by trellises, or simply sticks. I can see women picking them. Green tomatoes wait in piles next to the road, for buyers. They are obviously reddened somewhere else. I remember my father used to like green tomato chutney, a recipe he taught my mother. My own children will never be able to think of my cooking, only Bahadur's. I don't care, I am too happy to worry about anything.\n\nThe Deccan Plateau. Hills popping out of the landscape. The bus weaves to and fro and I feel sick. I take Avomine, and drowse against P.'s shoulder. I love her smell.\n\nDays merge one into another, the landscape changes, I too have fallen into the rhythm of the journey. My mind is stilled. At night we roll into beds that are provided for us at the circuit house or dak bungalow. How many more days before we can share a bed???\n\n*\n\nDecember 22nd\n\nThere are two buses following the Leader. One is security, aides and party workers. The other is publicity and journalists.\n\nThe woman who had a stomach upset continually hounds us. She is a correspondent for a paper based in Madras. Periodically, when the convoy stops, Pip and she disappear for their interviews. At these times I take out my pad and sketch. It will be a record of our journey when I return, and maybe a base for a canvas. I want to feel productive, that I did something besides stare besottedly at one woman all day. It's not easy being in love every single minute. Resentment creeps in, especially when the other person is talking to someone else.\n\nMeanwhile we pass through Mother India, who impassively stares at this cavalcade of temple, houseboat, and gun-toting security men. Nothing is new for India. Doesn't the Leader say that again and again, India is our mother. Her qualities are patience, tolerance, love and resignation. Her rewards are that she is forced to suffer over Kashmir the recalcitrant child, Punjab the rebellious one. The father \u2013 i.e. the Leader \u2013 will not stand for this any longer. Time to take a firm hand.\n\nHow can we listen to this rubbish day after day? I complained to P. when she was looking at my drawing pad that night.\n\nOnly three more days before we take the bus for Bangalore. Then it will just be you and me, she replied, carefully examining each sketch. I am continually flattered by her attention and comments:\n\nAre these scenes for your Ekta Yatra canvas, I like the houseboat and temple and the way you have captured these crowds, but isn't this a lot for one painting, and so on.\n\nLiving with someone interested in the details of your work is companionship at the deepest level. I long to create the canvas I have in my head so she can see it too.\n\n*\n\nDecember 24th\n\nBangalore at last! In the guest house of the Y. Our room, our bed, on which we spend hours. Maybe this is what good marriages are like. To be able to express what comes into your head, and know it will be understood as you meant it. To be more yourself because all of you is able to love in a way the other responds to.\n\nShe goes to sleep, and I pass my hand over her breasts. At first it had seemed odd, after years of being made love to by a man, to have one's breasts met by a similar pair, though larger. No wonder men like them so much. You can do much with a pair of breasts. These loose, hanging, swinging items, breasts, penis \u2013 objects of passion and anxiety. Stuff you can hold in your hands, squeeze, maul, make yours, like playing with clay \u2013 taking you back to your childhood.\n\nThe rubber trees are enormous and green outside, the bougainvillaea is blooming, it is warm, fragrant, pleasant, far from the cold of Delhi. Why can't I live here for ever with her, forget I have a life outside this room, this bed, these arms, this mind that sees me the way I am and loves me still.\n\nShe looks at my face, puts her arms around me, don't look so sad, we have each other, we are the lucky ones.\n\n*\n\nDecember 25th\n\nChristmas.\n\nShe pointed out her grandparents' house as we passed by in a scooter.\n\n'Can't we visit?'\n\n'No.'\n\n'Why? I want to see them. I want to see where you spent your childhood.'\n\n'Well, you can't. They'll pester me to stay, and ask a lot of questions.'\n\nI could make out a small house, a little garden and a huge tree, studded with white champa blossoms. Pretty, but I couldn't imagine Pipee in it, the antithesis of suburban.\n\n'What was it like, growing up here?'\n\n'All right'' she said non-committally. Getting into her past is sometimes a problem. Especially the death of her husband. She never talks about that.\n\nSpent the day roaming Bangalore. Talking, talking to fill the time our lives were separate \u2013 oh this happened when I was here, didn't I tell you, and she said and he said, tell me, tell me how it was?\n\nWe laugh because we are together, doesn't matter where, or how, cemented by our nights and words together.\n\n*\n\nJanuary 2nd, 1992\n\nBack from a week at Pip's school. Idyllic place, with all the usual about idylls. Trees, millions of butterflies, thousands of birds, lap of nature, the works. The most miraculous thing about the place, I had no headaches. Pip, no headache, I said every evening, and she smiled, the corners deepening, dimple appearing, eyes warming. My painlessness I offered as a gift, she accepted it as her due.\n\nShe showed me her butterfly tree, the walks her mother and she used to take, her classrooms, her library, she was even nostalgic about the din in the dining hall.\n\nI had no idea P. was so involved in her school. The usual pangs with every teacher she threw herself on, with every old friend she talked about. This kind of jealousy, however slight, makes no sense. I think I need my head examined.\n\nWe stayed with P.'s mother. I slept on the divan in the big room, she with her mother in the bedroom. Her mother didn't say much to me, she is a house parent, besides being a middle-school class teacher and quite busy. Does she know we are lovers??? I ask P.\n\n'I think so.'\n\n'You told her?'\n\n'She has eyes.'\n\nSo does my mother but even if I told her, I bet a thousand to one she would not believe it. I said as much.\n\n'She has always known how I am feeling, that is the important thing.'\n\nIt seems Pip has the ideal mother-daughter relationship, just as she had the ideal marriage. I wonder how these things operate?\n\nPip organised a street play around interpretations of history. Among other things she used my pamphlet, _The_ _Testimony_ _of_ _the_ _Black_ _Pillars._\n\nLast night we went to P.'s favourite restaurant in Bangalore, a great relief after school food, though to listen to P. it was manna from heaven.\n\n(n.b. If I am jealous of every thing about P. that doesn't include me, perhaps I should not mind so much her attitude to my family.)\n\n*\n\nJanuary 4th\n\nWe are leaving on the Karnataka Express. The Yatra has reached Gujarat, then it is going to Rajasthan, Madhya Pradesh, Uttar Pradesh.\n\nIn the evening P. said, I don't want you to go home.\n\nWhat is she saying, it is almost a month, is this another test? Did I not pass the first one?\n\nStay with me a few days in Delhi, please. You can always go back to them a bit later.\n\nI agreed, but for the first time, the thought crossed that perhaps P. was not always wholly reasonable. Maybe I should assert myself. (How?)\n\n*\n\nJanuary 7th\n\nWe came yesterday. Took a scooter to her flat, stopping at the market on the way to buy provisions: milk, bread, eggs, fruit and vegetables. It seems strange to come to an empty place, no one waiting, nothing done. You have to do everything yourself the minute you come, clean, organise, buy, cook. If it is so tiring in winter, what will it be like in summer? But this is Pip's life, and she doesn't complain.\n\nPip has gone to Ujjala, I make the bed, dust, clean the cobwebs, cook lunch, and then haul out this diary to write.\n\nI wonder how Anu and Himu are managing. I can't tell on the phone. Their school is opening today. Did they finish their holiday homework? Does my mother manage to get them up and off in time? Are they all right? They say yes to everything. P. says I worry to feel needed.\n\nI feel disturbed here. Why isn't Pip coming? She promised to come quickly, she might have gotten caught up with meeting her colleagues, Neeraj probably, while I am here waiting. It was much better in Bangalore.\n\n*\n\nJanuary 8th\n\nAwful, awful. Couldn't sleep. Last night we fought. She left this morning without telling me where she was going. What did I do, it was nothing.\n\n'In a few days you will be gone'' she started over the dinner we had cooked together.\n\nOh no. 'Yes.'\n\n'And then?'\n\n'Then?'\n\n'Back to the way it was?'\n\nShe was spoiling for a fight. I was determined to say nothing, but she went on, 'You don't really want to be here.'\n\n'I do'' I said quickly.\n\nShe started withdrawing. Leaving a trail that I followed. 'Would I be staying if I didn't?'\n\nShe glared at me, pointedly left the table and began clearing away the dishes. Doesn't she realise what I go through because I want to be with her? I am in the same city as my children and I cannot meet them. Still she broods. Is this how she wants to spend our time in Delhi? To fight, sulk and turn away from me?\n\nWhy is she like this? I wish Aijaz were still alive, but then she would never have been interested in me. They had the perfect marriage, she hankers after that wholeness. What can I do? I live my life in fragments, she is the one fragment that makes the rest bearable. But a fragment, however potent, is still a fragment.\n\nThis morning I got up, made her breakfast, but she would not relent, continued cold. If she wants to punish me she certainly doesn't have to try very hard. I am in such misery, I don't care what I do. To be with her, yet distant, anything is better than that. She has left me alone here, God knows for how long. I might as well go home.\n\nI wish I had the energy to hate her, but I don't. I feel sick.\n\n*\n\nJanuary 9th\n\nHome. They all exclaimed how thin I was.\n\nI left without saying goodbye, or leaving a note. What will she think when she comes back and finds me not there?\n\n*\n\nJanuary 13th\n\nJaundice. I vomit all the time.\n\nP. is all right, then how come me? We drank the same things, but some germ from some water drop has lain inside me, waiting for me to be safe at home before moving in for the kill.\n\n*\n\nFebruary 15th\n\nWhat was the point? I can still barely eat. I look yellow and horrible. I smell.\n\nI have travelled from P.'s house to my own via the tip of the continent, a long detour.\n\nThis is what happens when you leave your home. The in-laws, the mother, the husband, the servants all unite on this.\n\nI feel exhausted.\n\nMy mother is still here because I am ill.\n\nH. grates on my nerves. It's all my fault, does he never get tired of finding different ways to say this. He likes me to be ill and dependent.\n\nP. comes to visit in spite of their hostile attitude.\n\nI am sorry, she said, I'm sorry I left you like that.\n\nI am sorry, I replied, that I didn't wait for you.\n\nWe talk of other things.\n\nShe told me there was a bomb blast attacking the Yatra in the Punjab, two people were killed.\n\nSuppose it had been us?\n\nHave I been struck by this dreadful illness because I left my home to be with the one I love? I feel so weak I can't get out of bed. When Hemant comes home and puts his heavy arm around me, I want to tell him everything just to see the look on his face. But then I'll have to cope with the rest of it.\n\nMy children draw pictures with huge Get well soon Mamas on them. I keep them by my bed and look at them often. Pip calls, concern in her voice.\n\nI can't deal with my life. I want a safe place, a warm place, a loved place.\n\n(n.b. Who doesn't?)\nChapter IX\n\nGradually Astha's bilirubin count came back to normal, as did her diet. Her mother departed for Rishikesh, yet she remained tired. When Pipee dropped by on her way to work, she did her best to be amusing and interesting. Pipee should not feel she was in love with an invalid, but it was so much effort, she almost wished she wouldn't come. Yet the days she didn't, she felt unloved and anxious.\n\nEvery morning she gazed piercingly and objectively in the mirror. She looked haggard, yellow, ugly and undesirable, she would perfectly understand if Pipee never wanted to see her again. When her lover left, she again checked the mirror, despite her better judgement. Maybe in the interim she had grown more beautiful, maybe Pipee had spotted something attractive that had missed her eye in the morning.\n\n'I wish I didn't feel so exhausted'' she permitted herself to moan occasionally.\n\n'It's only natural.'\n\n'Yes, but it's so boring for you.'\n\n'Let me decide that.'\n\nA pause.\n\n'How's your work going? How is Neeraj?' asked Astha to cover up the anxiety of the silence.\n\nEverything was fine, Pipee assured her, as she got up to leave.\n\nAfter these visits, Astha felt depressed and gloomy \u2013 why can't we be like we were during the trip \u2013 what's the point \u2013 I wish I were dead \u2013 while her family put her listlessness down to her fragile state of health.\n\n*\n\nMeanwhile tension in the house gathered. The workers of the factory went on strike, despite ClearVision offering fifty thousand rupees to the strike leaders, couched as temporary relief measures. It was clear that the rival union meant business, and soon another six TV factories in the area saw labour unrest.\n\nThese factory owners were not united. Meetings ended acrimoniously. They could not decide on an incentive package, though all of them felt that the demands of the union were unreasonable.\n\nEvery day that passed meant greater losses for the company, as well as an erosion of their market share. It was more than the owner could bear.\n\n'Half pay'' Hemant fumed, 'we still have to pay them half their wages. Where do they think I am going to get this money if there is no production? The company will be ruined. Bloody fuckers.'\n\nHe spent his days running around looking for a solution, meeting lawyers, representing his case before the Labour Commissioner of Noida, trying to get the strike declared illegal. Meanwhile they were losing their share of the market at a time when there were over four hundred TV manufacturers in India.\n\n*\n\nTwo months later the Labour Commissioner declared the strike to be a lock out. No work, no pay.\n\nTriumph reigned in the Vadera household, it was seen as the silver lining in the dark cloud that had lain across their home.\n\nThe next day the manager's car was damaged, and every window of the factory broken. The number of guards were increased, but a few days later a fire broke out on the premises. It was detected before great damage could be done, but Hemant could not risk further vandalism and was forced to hire a private security agency, with instructions for twenty-four-hour surveillance. More money spent without any sales to cover the costs.\n\nDespite being declared illegal, the strike continued. Too many workers, owners, factories were affected for there to be any immediate resolution.\n\n*\n\nHemant developed chest pain. The doctors diagnosed hypertension, told him change your food habits, quit smoking, cut down drinking, exercise every day, and avoid anxiety. The early forties was a vulnerable time for men with stress.\n\nHemant was seeing the work of the past eleven years go down the drain, and he wasn't able to respond to this advice.\n\nHis parents went into damage control.\n\nIt was decided that as soon as school shut for summer, he, his wife and children would go on a holiday, and spend a relaxed time with Hemant's sister Seema in the US. When Hemant came back, they would work on the lifestyle-food habits-exercise thing. Meanwhile Papaji would manage things in the factory.\n\n*\n\nAstha told Pipee of these plans while they were having lunch at a restaurant in Connaught Place.\n\n'How long will you be away?'\n\n'I don't know yet.'\n\n'I suppose you have to go?' asked Pipee a little hesitantly.\n\nAstha remained silent. If only she didn't have to put her husband's health over the companionship of her lover. But not going was like getting divorced, a public statement of difference and separation.\n\n'Look, it's not working out'' said Pipee suddenly.\n\n'What is not working out?' asked Astha desperately.\n\n'One should never have affairs with married people, they are the worst.'\n\nAstha looked at the face she had kissed lovingly and in such detail at least a thousand times, and said resentfully, 'Why did you, then? You want to spoil what we have.'\n\n'I had thought that with a woman it would be different\u2014'\n\n'So did I. With a woman\u2014'\n\nA silence fell, in which the air-conditioners fought audibly against the April heat. The glass on the windows let in blue-tinted light. At certain places the glaze had peeled and spots of glare came through. Astha dabbed at the breadcrumbs left on the table from their soup rolls. Pipee looked moody. 'You can tell me all about your nice little domestic holiday when you come back'' she remarked coldly.\n\nAstha stared at Pipee anxiously, 'You know how it is. The workers are on strike, he has got high blood pressure'' then she stopped, hearing the words of a devoted wife in her ears.\n\nPipee concentrated on her empty glass. 'No. I don't know how it is.'\n\n'You are independent'' said Astha bitterly, 'so you can talk like this.'\n\n'And somebody is holding your hands, preventing you from being the same?'\n\n'You need money'' flashed Astha, 'or do you think I should be independent on his money? Stand in the streets with a begging bowl? Live in an ashram like my mother? What about my children?'\n\n'Your children, your children, don't hide behind them. Live with me. Bring them.'\n\nThat old thing.\n\n'But no \u2013 you don't even try \u2013 Ant why don't you even try?' Pipee swallowed once or twice. 'Have an exhibition, do something on your own, or are you waiting for Hemant to give you permission?'\n\n'You are not being fair.'\n\n'Yes. Well.'\n\n*\n\nThe anticipated vacation split Astha more decisively than anything else since she had got to know Pipee. There was her lover and her lover's feelings. But there was also the visas for the USA and the UK, the foreign exchange, the getting ready, choosing suitable clothes and shoes, the packing and shopping for presents.\n\nWith their holiday abroad Hemant and Astha joined the have-gone-abroad club, whose denizens created envy and ill-concealed curiosity about how much money they were going to spend, where had they got it from, even with the factory in trouble they can afford to go, they must have stashed it away all these years.\n\nMany people took their proposed trip badly. The most immediate was Sangeeta who was there as usual for the summer holidays. She insisted on being part of the discussion and planning that revolved around itineraries, addresses of friends of friends, cheap fares, cheap central hotels, foreign exchange. Astha had to brace herself against the flow of her resentment and curiosity.\n\n'One day I too will go abroad. Seema is always inviting me'' she said.\n\nIt has nothing to do with me, thought Astha, if she is angling for a trip let her angle directly. Sangeeta sighed, announced Poison was her favourite perfume and disappeared upstairs for the day.\n\n*\n\nAnuradha said now her friends would not be able to act so superior, she too could tell stories of abroad, and Himanshu said now he could have the latest in Nintendo and Sega, and could they please go to Hamleys.\n\n'Hamleys? What is Hamleys?' asked Astha.\n\n'A shop in London'' said Himanshu. 'Everybody goes there.'\n\n'He is so retarded'' said Anuradha.\n\nAstha hoped the trip wasn't feeding into her children's materialist desires.\n\nAstha's mother was delighted. She wrote from her ashram: God bless you my little one and your family. Poor Hemant needs a break from all his troubles. You do not give him enough attention. Remember men have to bear the burdens of the outside world, home is their refuge.\n\n*\n\nPipee retreated further into herself, getting ready for her summer, Shahjehanpur, Shiksha Kendra and Ayodhya, we'll compare notes when I get back, bye, no need to drop me to the station, have a nice time, call me on your return.\n\nAstha felt Pipee's abandonment, but maybe she thinks I have left her, she brooded in the middle of the night, when the electricity went, and the couple lay sweating.\n\n'I will be glad to leave this fucking country'' muttered Hemant.\n\n'So will I'' muttered his wife.\n\nDelhi, the trap in summer, with power cuts, water shortages, heat waves, dusty winds, and pollution emanating from all its pores. Not the garden city of their youths, but fourth, third, creeping up to second, now coughing and wheezing its way to first, yes, almost the first most polluted city in the world.\n\nA trip abroad would be nice, no matter whom one loved and whom one left behind.\n\n*\n\nFinally the family took off on their cheap flight to Miami, Florida, with a stopover at London on the way back.\n\nHour after hour into the dark night they flew. Four abreast, in the central section of the plane: father, mother, daughter, son going to holiday on Western shores.\n\n'Are you all right?' Hemant would ask from time to time. Astha nodded, her eyes closed. She wondered at the great silence concerning the discomfort of planes, the torture one had to undergo to get to the lands of milk and honey. Her knees were hurting in the small cramped space, her shoulders and back were aching, a headache was coming on, would she make it to the bathroom to throw up if she had to. Excuse me, I am sick, I have to throw up, madam use the bag in the pocket in front of your seat, ah, there it is, sorry, not at all.\n\nThe rest were enjoying themselves. Himanshu was absorbed in the child kit the airline had given him, Anuradha had her headset glued to her ears, and fiddled with the dials constantly. Hemant was nursing his drink, chewing with relish on the peanuts that came with it, tinkling the ice and the alcohol in his glass, twitching his toes in the airline socks, his shoes neatly stowed away under the seat in front of him.\n\nHe shouldn't be drinking, thought his wife, but she was in too much pain to comment or persuade.\n\n*\n\nThey stayed for three weeks in Florida. Hemant talked incessantly of his life as a student, and how he had slummed it, how he had worked to earn a little extra money, how he had slept two hours a night, how the great American tradition encouraged self-reliance from babyhood, how you had to sink or swim, how the whole society was geared towards meritocracy, not towards blackmailing people by going on strike. Loafers wanting something for nothing were not tolerated here.\n\nSeema and Suresh sympathised completely, never mind, you have family, family still means something, and they talked of here and there, there and here, till Astha felt her ears would fall off.\n\nThree weeks crammed in their guest room, three weeks of Anuradha feeling jealous of everything that Sushma (the daughter) had to show her.\n\n'School in the USA is like no school at all'' she announced to her mother. 'They get hardly any homework, they choose what they want to study. Her maths, I can do it with my eyes shut.'\n\n'I am sorry, darling'' said Astha looking at her daughter's angry face.\n\n'Why should you be sorry?' said Anuradha turning upon her mother, the easiest person in the world for her to turn upon.\n\n'The system here is not so demanding, that's all I meant.'\n\n'She thinks she is so clever, but she is not, Mama, I know much more than she does. Her handwriting and spelling are so bad, you wouldn't believe, but she doesn't care, and neither do her teachers. She says in the computer everything comes out OK, so what is the point? Imagine!'\n\n'You are better off beti, you can write, you can spell, you can do maths, when you come here for higher studies you will be at an advantage.'\n\nAnuradha looked mollified. 'I'll show her'' she muttered.\n\n'Quite'' said Astha, 'and while you are about it, do remember that we are guests in their house, and that she is your cousin.'\n\n'She has an American accent.'\n\n'That is not something she can help, she only knows this country, poor thing.'\n\nMother and daughter smiled slightly at one another. Nothing is so much a bond as criticising relatives.\n\n*\n\nThe marriage of Seema and Suresh was a source of great amazement to the brother and sister-in-law. Seema and Suresh constantly deferred to each other. Suresh cleared up after meals, ran the dishwasher, did the grocery shopping, mowed the lawn on weekends, and went to the park with his son to kick a few balls in the evening, almost as a duty.\n\n'What has happened to Suresh'' wondered Hemant. 'He was never like this at home.'\n\n'This is not home'' replied Astha.\n\n'Poor chap'' went on Hemant. 'You should have seen him when he was just married. Boozing and smoking with the rest of us. Now he doesn't even touch a cigarette.' Hemant fumbled for his own packet and lit one, to further express his disgust.\n\n'Perhaps it would be better if you took a leaf out of his book'' said Astha. 'Suresh looks just fine to me, at least he is not a source of worry to his family.'\n\n'He is ashamed to look me in the eye'' declared Hemant, surrounding those very eyes with smoke.\n\n*\n\nThe high point of their US holiday was a trip to Disney World.\n\n'It's built on 27,000 acres. Acres of fun'' said Suresh, while Seema sketched the delights of the fairy tale park, water park, animal park, future park, past park, sports park. She spoke with all the pride of ownership.\n\nThey planned to drive to Orlando and spend three days there. The hotels were expensive, but to absorb such wonders money was necessary.\n\nHemant offered to participate in the driving, but Suresh did some more back slapping, this was America, not your India, where a visitor could drive without an International Driving Licence or indeed without any kind of licence at all, just a bribe.\n\n*\n\nDisney World, Orlando, Florida, USA.\n\nIs such a thing possible in your India? There was no end to this question, as Hemant was forced time and again, to say no, such a thing was not possible in their India.\n\nSo organised, such crowds, such a money-making machine, such technological marvels, such fantasy, such going through tunnels, haunted houses and castles, such an onslaught of souvenirs, such marvelling, such eating of hamburgers, hot dogs, Kentucky Fried Chicken, tacos, and thick milkshakes. Around they wandered with those milkshakes which never seemed to end, sipping the cold sweet stuff through giant straws. Was there anything in this country that wasn't big?\n\nAnuradha and Himanshu loved it, Hemant loved it, Suresh, Seema plus two kids, their millionth visit with Indian tourist and wonder seeker in tow, they loved it all over again. Even Astha managed to be caught up in what she saw and experienced. They were all children together, all Mickey Mousers in a Disney World.\n\nBesides families everywhere there were couples embracing, couples walking with their hands in each others pockets, kissing, eating, conversing, laughing.\n\nSuresh and Seema became even more of a couple here. They walked holding hands. For our benefit, or because they are on vacation, or because they have lived in America so long, or because they love each other so much? It was the last possibility that Astha could bear the least. Anything but that Hemant's sister should live in bliss while she lived in misery.\n\n'I thought Disney World was for children'' she remarked to Seema.\n\nSeema and Suresh both grinned at her.\n\n'Arre, people come to enjoy'' said Suresh.\n\n'Relax, have fun, spend quality time together'' clarified Seema for Astha's greater understanding.\n\n*\n\n'Well, wife'' said Hemant, the second night in the hotel, at his most affectionate, swept by emotion at having seen Disney World, and recorded it on a thousand pictures taken for the benefit of back home, 'it's been quite an experience, no?'\n\n'Yes, it has.'\n\nIt was late, the children had fallen asleep, exhausted by so much pleasure and walking around. Hemant sat next to Astha, and put his arm around her.\n\n'How's your head?' he enquired tenderly.\n\n'OK.'\n\nThey sat on in silence. After a while Astha dislodged herself. 'I have to pee'' she said.\n\n'OK'' said Hemant, getting up as well.\n\n'What are you doing?'\n\n'Coming with you.'\n\n'Don't be silly.'\n\n'What's so silly about it?'\n\nIt was easier to let him come, and Astha sat on the toilet seat, feeling a bit strange. It had been a long time since they had shared any intimacy.\n\n'Go away'' she said at last, 'I can't pee.'\n\nHe ran the tap.\n\n'Now?'\n\nA small trickle. Hemant tore a piece of toilet paper and advanced his hand towards her legs. The trickle stopped. Her legs tightened. 'Please leave the bathroom'' she stammered.\n\n'Why? I'm your husband.'\n\n'So what?'\n\n'So everything.'\n\n'You think marriage is just sex.'\n\n'Of course I don't. What do you want that I don't give you?'\n\n'Interest. Togetherness. Respect.'\n\n'Baby, I respect you'' said Hemant soothingly, 'you are my wife. As for togetherness, that's just what I want.'\n\n'Why all of a sudden?'\n\n'We are on holiday. This is what people do on holiday.'\n\n'I don't want to. I am out of practice.'\n\n'Well, let's get into practice'' said Hemant stretching out his hand again towards her legs.\n\n'I am not able to switch on and off like you'' said Astha.\n\n'It is not as though you were the most willing creature. Each time I try and come near you, you say you have a headache. A man is tired, he can't be doing the chasing all the time.'\n\n'Is that what you call it, chasing? Not having sex on demand? There has to be something more between us. I have to feel it is me you want.'\n\nHemant looked baffled. 'Of course it's you I want. You are my wife'' he repeated.\n\n'That's the problem. Anybody could be your wife.'\n\n'What rubbish. I picked you, didn't I?'\n\n'Picking is not the same as knowing.'\n\n'Why do you always make things so complicated? You are my wife, that is enough for me, I would have thought it is enough for you. Or is it someone else?'\n\n'Are you referring to my life or yours?' asked Astha flushing slightly.\n\n'Come on darling'' replied Hemant, ignoring her barb, 'we are on holiday. I want this to bring us closer, as a family, as a couple.'\n\nHe had felt her distance, he wanted her back. There seemed to be no way out, unless she decided to leave the marriage there and then. Slowly she moved towards him. With sleeping children in the room they would of course have sex in the bathroom. He spread a towel on the mat and waited for her to undress.\n\nOn and on marched the holiday, relentless, inexorable, eating up money, energy, rolls of film, pushing them to cheap eating places, and suitcases that grew heavier by the day.\n\n'Shopping on the way back, shopping on the way back'' Hemant kept saying but it didn't quite work like that. There were so many souvenirs, the Disney World ones alone filled half a suitcase. Besides there were the presents Seema and Suresh were sending back for the rest of the family, and clothes for everybody, so much cheaper in the States than anywhere else.\n\n*\n\nLondon. They were met at the airport by Hemant's cousin.\n\nAstha had always liked this cousin. He had gone abroad to do well, since he couldn't do well in India, and ended up owning a shop in the suburbs of London. Just what this meant was only now becoming clear as they drove, drove and drove, and finally stopped in front of a house, which was a double storied, very narrow building, identical to the entire row on the street. Naked houses on a treeless street.\n\n'Welcome to my humble abode'' said Jagdish, edging the car near the curb, and jumping out to take their suitcases. 'I'll see where Liz is'' he panted, lugging them inside.\n\nLiz, the unenthusiastic wife. 'Hello, would you like a cup of tea?' she asked, and they could feel the indifference, and they could understand why Jagdish was being so effusive.\n\n'Their house is so small, Mama'' whispered Anuradha, awed by such discomfort in the West.\n\n'They don't have much money'' whispered Astha back.\n\nThe bags, the guests, and the host struggled up the narrow stairs, what a nice house you have Jagdish, well, it's all right, and they went down to have the tea that Liz had prepared.\n\n*\n\nOne week in London, of learning how to take the Tube, of don't worry, Jagdish, we will take care of ourselves, no, no, please do not bother, Liz, we will manage, and Jagdish's reply, well, if that's all right then.\n\nEvery morning Astha got up and made sandwiches so they could save money on eating. They bought the ingredients and the drinks at the corner store, because Liz clearly did not understand the imperatives of Indian hospitality, and they didn't want to burden Jagdish's marriage further. They gritted their teeth and managed to not all bathe every morning, the house only had one complete bathroom.\n\nThere was some disagreement as to how they would spend this precious week. Astha wanted to see all the art treasures London had to offer, she was willing to go on her own while her family did whatever they wanted. But Hemant would not hear of this \u2013 we are here to be together \u2013 and as a compromise they spent a morning at the Tate, a morning at the British Museum, and then covered the famous sights of London in a couple of day tours. Many photographs were taken as proof of the good time they were having.\n\nAll this over, they devoted themselves to shopping. There had to be much looking, exclaiming, comparing, soul searching, and converting of currencies before they could buy.\n\n'I must say London is a very expensive place'' said Hemant, as they emerged from Marks and Spencer, arms laden, a light rain falling, a cold wind blowing.\n\n'I wish we didn't feel the need to buy everything we see'' moaned Astha, exhaustion reducing her to the desire to lie in front of the department store door, and be trampled to death by all the Indians rushing in and out, buying, buying.\n\nAnuradha and Himanshu looked at her reproachfully. They could hardly contain themselves in this material paradise. Floors and floors of merchandise with Hemant the indulgent father. The trouble, thought Astha, was that she too could hardly contain herself when she saw the kitchenware, gadgets, art supplies, bed linen, children's toys, clothes, underwear, stationery. Was there anything that did not move her with the urge to possess? No, such shopping was not morally good, she felt her sense of perspective and focus vanish amidst its successful assault on her greed. It was just as well these trips were rare.\n\n*\n\nOn the evening of their fifth night. 'There seems to be trouble in India'' said Jagdish, a held back pleasure edging the notes of concern. He was entitled to a revenge so small, that he was in the safe place, the sane country, something in return for his unsatisfactory house, job, career, marriage and neighbourhood.\n\nAstha and Hemant looked at each other. At home trouble was part of the atmosphere, outside it assumed more sinister proportions.\n\n'What's happened?' asked Hemant\n\n'On the BBC. They are going to build the temple'' continued Jagdish.\n\nHemant relaxed. Oh, the temple. 'These politicians keep stirring things up'' he replied, uninterested.\n\nWhile the family ate, Astha hung around the TV waiting for the news.\n\nThere it was. A brief visual of the Babri Masjid at night, floodlights beaming, sounds of bhajans in the background, thousands of kar sevaks surrounded by security forces, clearing the ground, laying the foundation for the temple, working, working, round the clock.\n\nThings are tense in this ancient temple town, said the commentator, where a mosque stands on the site that Hindus claim to be the birthplace of the Lord Ram. While six thousand pilgrims work day and night, an estimated fifty thousand more have assembled here. The kar sevaks swear that this time they will rather die than stop. There have been protest marches by groups concerned with saving the Babri Masjid but so far the laying of the temple's foundation continues at a lower spot on the hill. The Prime Minister has called Hindu holy leaders to Delhi to discuss the issue.\n\nHow awful, thought Astha, what was going to happen? She wanted to go home. Her political self, her intelligent self functioned best there, here she felt isolated, saturated with things rather than thoughts.\n\nWhat was Pipee doing? Each day she had been aware of her absence, yet she had enjoyed being with her family, enjoyed the comparative ease between Hemant and herself.\n\nShe dreaded what Pipee would say when she sensed this. As she tried to defend herself, I am married, she felt the betrayal Pip would feel, but by now betrayal was a second skin.\n\nAstha had often imagined the breaking of her relationship with Pipee. What she hadn't realised was how slow the process would be, and in what infinitesimal stages.\n\nThere were differences, she thought miserably, but they hadn't seemed so important. This was no longer the case. After she came back they were clearly not in harmony.\n\n*\n\n'You won't like abroad'' remarked Astha to Pipee. 'It is awful.'\n\n'Who would have thought it?' said Pipee dryly.\n\n'You know what I mean'' said Astha impatiently.\n\n'No, I don't. How could I? And anything is better than the things I saw.'\n\n'What did you see?'\n\n'For ten days total frenzy, policemen jeered at, control rooms smashed, loudspeakers blaring out prayers and bhajans \u2013 in such an atmosphere \u2013 pandemonium at the building site, and kar sewaks all over.'\n\n'You mean you went to Ayodhya?'\n\n'Yes.'\n\n'But why? You didn't tell me.'\n\n'Where were you to tell?'\n\n'It might have been dangerous, Pip.'\n\n'Oh Ant, one can't always be safe. It was no more dangerous for me than for all those other poor women there. Besides I wanted to go. I am thinking of a conference on how families are affected in communal riots.'\n\nMore PhD stuff, thought Astha. 'Well, how was it?' she asked.\n\n'They are going to build the temple in the masjid area. That kind of energy, so deliberately stoked doesn't go away. It's only a matter of time.'\n\nThere was a silence. Pipee leaned back in her chair, and stared at the clouds that were running against the sky of her Vasant Kunj flat. Astha looked at her, she seemed so distant. She had felt closer thousands of miles away, thinking of her, writing to her.\n\nThen Pipee said, 'Enough about Ayodhya. How was Disney?'\n\n'Fine.'\n\n'And you and Hemant?' she asked. 'How was that?'\n\nAstha kept her face still. 'Also fine'' she said.\n\nPipee looked at her sharply, 'You have had sex with him'' she stated flatly.\n\nHemant's face rose before Astha's eyes, the moments in the bathroom, the appeal he would never verbalise, her own realisation that somewhere he still had the power to affect her. She felt her face going red.\n\n'You've never really liked it any other way, have you?' persisted Pipee, her voice dry and hard.\n\n'That's not true'' pleaded Astha.\n\n'Yes it is. What you really want is your husband's cock.'\n\nAstha winced and tried to retaliate. 'It's not that. You resent that I am not leaving him. You want a full-time partner. I understand that.'\n\n'You would. It is what you have, after all.'\n\nAstha was silent for a moment. If her husband represented more than just a cock, so much the worse, but how was she to explain to Pipee? It was better not to advance into these murky waters. She went on, 'It has nothing to do with us.'\n\n'You went away with your family, that was bad enough, and I didn't say anything, because it's no use, and then you do this, why have me?'\n\nEverything Pipee said was a distortion. Words were raising their ugly heads, and Astha could do nothing. No matter how hard she tried, she was not going to succeed.\n\nPipee kept that transgression in her heart and used it as a foundation for the separation she saw ahead. A good memory is always useful when something needs to be destroyed.\n\n*\n\nPipee and Astha continued to see each other, but there was now a carefulness between them. For Astha everything became dull, the grass looked ordinary, the sky looked bleak, the paint on her canvas colourless.\n\nA thousand times she said to herself, confront her, tell her you want it like it was, or not at all, but she was too afraid. Pipee might say not at all, then what would happen to her, worse than this, much worse.\n\nThings would become all right on their own. Love would triumph, even in circumstances like these. Love had to, that was its nature.\n\nBut Pipee behaved as though love had had its day. Even moments of affection contained references to endings. Pipee to Astha, tucking her hair behind her ears.\n\n'I'm so grateful to you, Ant, never forget that, no matter what happens. From you I got the energy to go on.'\n\n'To do what? Leave me?'\n\n'You really want to go into who left whom?'\n\nAstha couldn't say she did. 'You see?' said Pipee. 'We both gave each other something. Let us leave it at that.'\n\nAshta couldn't say anything. Words made what was between them so small.\n\n'And of course whatever happens, we will always be friends'' went on Pipee.\n\n'Yes, always'' replied Astha gratefully, too inexperienced to know that that is what breaking up people say to each other to make it more bearable.\n\nMeanwhile the strike was resolved after six months. There was a tremendous backlog to be made up, and a market share to be recaptured. Hemant made an effort to resume his previous pace of work, when the chest pains started again. The doctors were very severe, he was not paying enough attention to his health. Collapse was imminent if he continued smoking, drinking, not exercising, eating red meat and heavy food. Furthermore he had to try and control his levels of stress, a very modest working day was all his body could tolerate. Angina had to be taken as a warning, a serious warning.\n\nThe whole family was alarmed. His father insisted another manager be hired, money wasn't everything. As for Astha, a brief survey of the literature on heart disease established that permanent changes were required in their living habits. Diet \u2013 exercise, diet \u2013 exercise, there was no getting away from these pillars of health and longevity. It was up to her, Hemant was not going to change on his own.\n\nEvery morning she made sure they went for a walk. All those years ago, exercising and resentful with her parents, she was now doing the same with her husband, with feelings so much more complicated with the years that had passed. Was this where her life had led her, this the space she had travelled between those walks and these? Striding briskly to still the thoughts in her head, speaking to mask the feelings in her heart. She looked at Hemant, swinging his arms, concentrating on getting his heart rate up. Perhaps he was disappointed too, perhaps he had looked for something different in marriage. They didn't talk about such things, she would never know.\n\nShe changed her family's way of eating. She bought books on low cholesterol diets, she studied recipes demanding no fat and little salt. When the children complained they were compensated privately during lunch and tea.\n\nHemant was bad tempered about having to give up his favourite foods, but he had no choice. And if he had to eat porridge for breakfast instead of his usual green chilli-onion-tomato omelette, he could not complain, his wife was eating the hated porridge too.\n\nAstha spent a lot of time thinking about herself. Was she a traditional wife as Pipee had alleged? She flinched at the idea, but she was certainly doing what devoted wives did, putting a great deal of effort into protecting their husband's insides. When she saw him tired, afraid, depressed at having to change, unprepared mentally for the betrayal of his body, she felt sorry for him, and wanted to help him live. She told herself it was for the children, but sometimes she wondered bleakly at the nature of the bond between them.\n\nHemant was touched by her efforts. Occasionally he would enquire, 'Well wife, how are you?' in a proprietary kind of way.\n\n'Fine, fine'' Astha replied in a monotone. Hemant was not adept at noticing discrepancies between the apparent and the stated, and this quality was conspicuous now.\n\n*\n\nThe monsoon came and went. The muggy days marched into October to become more human.\n\n'I'm going to get an air-conditioner'' said Pipee. 'That is if my scores are so lousy I get no aid, and am forced to remain in this dump.'\n\nAstha turned absolutely still. 'The PhD?' she asked.\n\n'I give my GRE next month.'\n\nOh Pip, you didn't tell me and not telling used to be felt a deception between us, but I see no more, mourned Astha silently, as Pipee continued, 'I need a change.'\n\nAstha made a heroic effort. 'Yes. I'm sure you do. In an academic environment you are bound to flourish. They'll love the work you do.'\n\nPipee looked at her and smiled, 'I'll miss you, Ant.'\n\nAstha didn't believe her. Pipee went on talking, and Astha heard all the things she wasn't saying, her loneliness, her desire for steady companionship, the need for commitment.\n\nThey smiled at each other. Astha said she understood. They drank tea, they exchanged a goodbye kiss, they did all this before Astha ran to her car, buried her face in the steering wheel, and took a good, long look at the void she had desperately tried to plug through loving Pipee.\n\nWhat would it be like to be painfully separate having known togetherness?\n\nHow would she live? But she had to, she had that rock of stability women had, her husband and her children.\n\nDrearily she turned the ignition and let out the clutch. The car rattled and jerked. She had to start it three times in the two metres she backed it. The clutch seemed to be slipping, the car's servicing was long overdue. This knowledge had hovered on the edges of her mind for a long time, but the imperatives of her life had not allowed her to pay attention. No longer. Her life was made up of these things.\n\n*\n\nAt home she threw herself into a frenzy of house cleaning. Every nook and cranny, every book, every mote of dust, layer of dirt, every inch of carpet, every remote cupboard high and low she attacked.\n\n'Mama's gone mad'' Anuradha informed her father conversationally, 'all she does is clean. And she makes me polish and clean too.'\n\n'You will thank me for it later, when you have your own home'' snapped Astha. Everybody looked surprised.\n\n'Do you have a headache?' asked Hemant.\n\n'No.'\n\n'What is it then?'\n\n'Nothing. Nothing.' And without her wanting or willing, the tears started pouring down her face.\n\nAnuradha's face contorted. 'Why are you crying, Mama?'\n\n'No reason, sweetie'' gulped Astha.\n\n'Shall I get you your medicine?'\n\nAstha continued to sob, while Hemant said, 'Do, Anu, there's a good girl'' and as the daughter ran off, he turned to his wife and said, 'Don't cry. You are upsetting the children. They will think something is really wrong with you.'\n\n'Let them'' wailed Astha. 'Let them know mothers also can feel.'\n\n'Az? Are you all right? Stop cleaning, if it upsets you.'\n\n'You always say how dirty the house is.'\n\nShe sounded unreasonable to her own ears. Hemant sat quietly by. Anuradha ran back with the pills and a glass of water, dragging a worried Himanshu with her.\n\n'She says you are crying'' he accused.\n\n'It's all right, baby'' said the mother, swallowing the pills, though she had no headache. 'It's nothing really.' Himanshu stared at her, his face opaque. 'It's nothing, baby'' repeated Astha. The boy turned and walked away while Anuradha looked at him with contempt.\n\n'He's so thick'' she said. 'He never understands anything.'\n\n*\n\nInto Astha's mind came a memory, dredged from her subconscious. Her mother coming from the bedroom, the bedroom that had been locked, unusually, for a whole hour.\n\n'Why are you crying, Mama?'\n\n'It's nothing.'\n\n'How can you be crying for nothing?' persisted Astha.\n\n'I am not crying. What gave you that idea, beta?'\n\nOh well, if she wasn't crying, then those couldn't be tears, nor could those be signs of grief around her eyes and mouth. They could go on being happy, everything in its place.\n\n*\n\nAstha was amazed at how much work Pipee devoted to applying for a Ph.D. She spent hours at the US Educational Foundation studying profiles of universities, their faculties, their requirements, the ones most likely to give aid, the cost of living in small towns, cities, East Coast, West Coast, balanced against the cost of living with Ajay if she got into his university, the cost of clothing in cold places, hot places.\n\n'I can afford to apply to only five'' she said, looking at her finances, calculating how much for GRE, how much for application money, how much for postage. 'Though that is taking a risk. Well let us see. Maybe I won't get aid, or maybe I won't get a visa.'\n\nShe would talk of something else, and Astha would seize these flimsy possibilities, clutching them in her grip, where they lay mangled and inert, of no real comfort.\n\nWho has seen tomorrow, she often thought, and this with tomorrow staring her in the face.\n\nWhen she was with Hemant she felt like a woman of straw, her inner life dead, with a man who noticed nothing, with whom for that very reason it was soothing to be with. Her body was his, when they made love it was Pipee's face Astha saw, her hands she felt. She accepted the misery of this dislocation as her due for being a faithless wife.\n\n*\n\nPipee's GRE scores were announced. She had got 23.4 on 24, and could now walk into any university she chose, or so her congratulators said. Astha hugged her, and felt she hadn't known misery till then.\n\n'Oh, Ant'' was all Pipee said, but Astha could see how happy she was, how vindicated she felt with her score, how much further down the road towards the USA she was treading.\n\nThen Pipee began to talk. 'You can join me for a bit if you feel like it.'\n\n'Of course I'll feel like it.'\n\n'No, I mean it, Ant. I know you'll be sad without me. You come for a holiday.'\n\n'Sure.'\n\nBut they did not explore this topic further. Too much water had flowed under this particular bridge for it to be a comfortable one to tread on now.\n\n*\n\nIt was December. The initial response from American universities was positive. 'I'm so glad'' said Astha looking at the face before her, and the shy glow on it. She re-read the letter Pipee had shown her. The University of Illinois had received Pipeelika Khan's application, and were acknowledging this. She would hear from them in the very near future. 'They have realised how bright you are'' went on Astha, thinking true love had no element of self in it, and she had better measure up.\n\n'Not everyone thinks of me like you do, Ant.'\n\n'Your application, your recommendations, your NGO work, your academic record, that paper you wrote and published, your GRE scores, why else would they reply so fast?'\n\n'You're biased.'\n\n'Not without reason, surely?'\n\nPipee laughed, and took the letter back, carefully folding it along the original creases, before sliding it into the browny-yellow envelope. Astha watched her.\n\n'When will you finally hear?' she asked.\n\n'Hopefully by January.'\n\nMaybe by January a bomb would fall on Urbana Champaign and blow the university off the face of the earth, thought Astha, but what would be the point, there would be other places. The real act of leaving was in the decision, not in the departure.\n\n'Maybe they won't give me a visa'' Pipee broke in on the pause. 'After all, I'm single.'\n\n'Yes'' said Astha slowly, 'you're single.' How soon before she would find someone?\n\n'You've not asked me what I'm going to work on.'\n\n'Education of slum children, I imagine?'\n\n'No. The politics of communalism and how it is represented. I am more interested in that now, maybe because of what is happening around us. It might also help me come to terms with things in my life. If you realise you are not alone...' She did not complete her sentence, and Astha felt more than ever removed from her life, from a pain so horrifying she bowed before it and shut her mouth.\n\n'The only trouble is there are so many aspects, all of such relevance that it is a bit hard to choose a specific area'' went on Pipee.\n\nFor a moment Astha felt an intense stab of envy, not just for Pipee, but for anyone who had the possibility of a new life. She had to remind herself sternly that if she wanted, she too had choices. \nChapter X\n\nBy the end of the year, there was plenty of material being generated for Pipee's thesis. Kar seva had been stopped in summer on the condition that the Prime Minister solve the Babri Masjid problem in four months. Those four months were up with no solution in sight.\n\nThousands of kar sevaks were again being mobilised for what was termed symbolic kar seva, starting 6 December. The central government sent 135 companies of its security forces to Ayodhya and Faizabad despite the protests of the U.P. government, who claimed the law and order of their state was their responsibility.\n\n'This time they are not going to give up easily, they have been stopped twice before'' said Pipee worriedly.\n\n'Are you going?' asked Astha.\n\n'Too much to do. I hope nothing happens.'\n\n'The Babri Masjid has survived almost five hundred years. Why should something happen now?'\n\n*\n\nMeanwhile Ayodhya is witnessing the unprecedented influx of thousands of kar sevaks from all over the country. Religious leaders issue press statements declaring that religion is above politics, above nation, above courts and any restraining orders passed.\n\nBy 5 December the city has swelled by 200,000 kar sevaks, and there are not enough places to put them. Schools and colleges are declared shut, while the kar sevaks storm various institutions for accommodation. The area around the masjid is littered with garbage and human excreta. Food prices go up, the U.P. government declares they have in stock 8,ooo tonnes of rice, 100 tonnes of sugar, 45,000 litres of kerosene, as well as an ample supply of life-saving drugs.\n\nThe BJP declares that no harm will come to the masjid, the kar seva will only be symbolic.\n\nThe Union Minister sends extra paramilitary forces to Ayodhya.\n\n*\n\n7 December, Astha's house. Headlines: A NATION'S SHAME there on the folded newspaper lying on the verandah, waiting to be read, digested, somehow understood.\n\nAstha picked it up and stared at the front page. It was not possible, this could not have happened, but there it was:\n\n*\n\nA NATION'S SHAME: BABRI MASJID DEMOLISHED\n\nCentre sacks Kalyan Singh's Government. 500,000 kar sevaks armed with pickaxes, crowbars, pipes and uprooted barbed wire barricades, attacked the disputed site yesterday. All domes collapsed under the onslaught. Between 11.50 to 4.50 the central dome collapsed. Between 2.00 to 4.00 p.m. the two side ones were destroyed. 50 people injured. Hundreds of kar sevaks carted away bricks, pillars, and large stones. BJP leaders urged restraint through megaphones.\n\nAngry kar sevaks singled out photographers and foreign correspondents beating some brutally with sticks and leaving them bleeding on the road.\n\nCurfew in many U.P. towns. Muslim MPs seek the Prime Minister's resignation. Muslim houses set ablaze. Kar sevaks not allowing fire engines in many places.\n\nArmy alerted in six states.\n\n*\n\n'They've broken the mosque'' she found herself telling Hemant. 'They have done it at last.'\n\n'I know.'\n\n'You know?' Astha stared at him.\n\n'It was on the BBC last night.'\n\n'Why didn't you tell me?'\n\n'What was there to say? I didn't know you were interested.'\n\nHe was lying. She had gone to Ayodhya twice, painted the masjid at least five times, scripted a play about it, and he didn't know she was interested? This was his revenge for being concerned in things other than him.\n\nShe turned away, sickened by everything.\n\n'I never knew you were such a Muslim lover'' said Hemant watching her. 'Do you know what happens to our shrines in Pakistan, Bangladesh, not only to our shrines but to Hindus? Why doesn't your precious Manch ever protest about that? Or any of your activist friends?'\n\n'The fact that shrines are desecrated there, doesn't make it acceptable here. It's not a Muslim thing, it's a secular thing, a human thing.'\n\n'It's a cowardly thing, a fool thing'' he said mockingly.\n\n*\n\nThere was no point talking to him. Her one thought was to call Pipee, Pipee who felt like she did, with whom there would be no arguing at this moment. Quickly she dialled her number.\n\n'Have you heard?'\n\n'Yes. They've done it.'\n\n'You were right.'\n\n'What are we going to do?'\n\n'What can we do?'\n\nBoth women fell silent, their own lives dwarfed by what was happening around them.\n\n'Neeraj phoned. There's a demonstration on at the BJP office, we might as well go. It must have been planned, such a thing cannot happen without careful planning.'\n\n'You said it was only a matter of time.'\n\n'It was just a thing to say. I had no idea I would be proved right so quickly.'\n\n'Don't cry, sweetheart. Come to the church crossing. I'll pick you up in half an hour.'\n\n*\n\nThey talked little as they drove. As they came nearer Central Delhi they could see that the streets were lined with throngs of weeping men and women, dressed in black, faces covered. Near the BJP office, they were forced to walk, the whole area was cordoned off, lined with policemen, ready to lathi charge at any provocation.\n\nJournalists were there, TV crew, academics and activists, all shocked and numb. They shared their information in broken sentences: paramilitary forces hand in glove with the kar sevaks \u2013 the police were helping \u2013 the leaders shouting on megaphones don't destroy the mosque \u2013 but pre-arranged that such messages to be ignored \u2013 many killed in the falling rubble \u2013 absolute pandemonium with 500,000 kar sevaks \u2013 the situation going to worsen \u2013 the government in U.P. had no political will to protect the mosque \u2013 only a matter of time before something like this happened\u2014\n\nThey waited for one hour, two hours. Nobody came out of the BJP office to address them. Was there anybody there? They courted arrest, were put into waiting buses, taken to the local police station, kept for half an hour and sent away.\n\n*\n\nThree days later the United Left Front organised a march to protest the demolition of the masjid.\n\nOn the morning of 10 December Astha said to her husband, 'There is a march today.'\n\nThe husband said nothing.\n\nAstha persisted with her information. 'It's going to be a tremendously big march. Traffic will be blocked around Red Fort and Connaught Place for hours. Do try and avoid those places if you wish to save yourself trouble.'\n\nNothing.\n\n'OK?'\n\nThe husband saw a female bull charging from the distance and his body tautened. He lifted a wary face and looked at his wife. Astha carefully patted the tea tray cloth.\n\n'I always admired your sense of proportion'' he said at last.\n\nAstha raised her eyebrows and looked inquiring.\n\n'Out in the streets, jostling with goondas, neglecting your family, all for some fool masjid you didn't even know existed before your great friend Aijaz chose to educate you.'\n\n'It has nothing to do with Aijaz'' said Astha, choking on the rage she had kept inside her the last three days.\n\n'Then his widow.'\n\n'I suppose I have no mind of my own.'\n\n'I didn't say that.'\n\n'You meant it.'\n\n'I refuse to talk to hysterical women'' said Hemant, 'especially when I have got a busy day ahead. Some people, work, you know.' He got up and went into the bathroom, firmly closing the door.\n\nWhen Astha reached the Red Fort her eyes were red with the hour-long cry she had had after Hemant left. Pipee saw her and linked her arm through hers, lacing her fingers through Astha's own clammy hand. 'Dearest, don't be this upset, it's terrible, but you can't afford to take it so personally.'\n\nAstha nodded dumbly. Everything in the world was terrible.\n\n*\n\nA mild winter sun shone on the gathered marchers as they stood around waiting, while truck after truck of United Front activists and associates drove in.\n\nThe line started. It was so long that by the time it was Astha's turn, forty minutes had passed.\n\nThey marched out of the Red Fort into the middle of the road, blocking all traffic. As she walked Astha could see various people, Pipee included, handing out leaflets to onlookers, scooter wallahs, passengers in rickshaws, men scratching their balls, women holding children on their hips, women with plastic shopping baskets in their hands. Down the line the familiar slogans were shouted: _Down_ _with_ _communalism,_ _down_ _down;_ _BJP_ _down,_ _down;_ _False_ _followers_ _of_ _Ram,_ _you_ _will_ _never_ _succeed;_ _Mandir_ __ \u2013 __ _masjid,_ _all_ _one._\n\nAstha was overcome with futility. Maybe Hemant was\n\nright. What was the use of forcing motorists, passengers and pedestrians to listen to the voice of tolerance and peace? It had not prevented anything. Maybe the true victory of fundamentalism was the total despair of the secularist.\n\nThe line reached Delhi Gate, and turned right towards the Ram Lila maidan. Down another empty street with bad-tempered traffic gathered on the other side of the divide, and then the line poured into the Ram Lila grounds, to mill around a platform erected for the speakers.\n\nOne after the other they spoke, leaders from the Congress, from the Left parties, activists who had seen what had happened in Ayodhya. They expressed anguish, regret, sorrow, they issued warnings, predicted consequences:\n\n*\n\nWhat had happened was a betrayal of trust. Millions of Muslims would now feel insecure in their homeland. The assurances of the U.P. government had meant nothing, the assurances of the central government had meant nothing.\n\nThe law had been blatantly, openly flouted, what was going to prevent it from being flouted again? What was going to prevent the two disputed sites in Kashi and Mathura from going the way of the Babri Masjid? Was this a government or a passive instrument in the hands of thugs? Without delay the government should acquire all the land around the Babri Masjid.\n\nIt behoved every citizen in the land to be vigilant so that anti-communal forces did not gain ascendancy. How was it possible to demolish a masjid in broad daylight in little over four hours? And that too with home-made tools, pickaxes, crowbars, the implements of farmers and peasants. No, there was organisation and planning, there was the connivance of the authorities.\n\nVarious Leaders had been arrested, but was it all for show, like the security forces that were sent to protect the Babri Masjid, and helped in its destruction?\n\nThe nation and its people demanded answers.\n\n*\n\nIt was late afternoon by the time Astha left. When Hemant came home he did not ask about the rally. And Astha was only able to sleep towards morning.\n\nAfter the demolition:\n\nNationwide, 1,801 people were murdered in communal clashes in the next two months. 226 places in 17 states and 1190.18 lakh people were affected by curfew.\n\nIn Pakistan 240 temples were targeted by mobs.\n\nIn Bangladesh attempts were made to destroy 305 temples, 1,300 houses, and 270 shops belonging to Hindus.\n\nIn the United Kingdom 18 temples and cultural centres were damaged.\n\nIn Afghanistan 4 temples were attacked.\n\n*\n\nOver the next two months major riots broke out in Bombay. 41 areas were affected, 31 per cent of the deaths were caused by the police. Pipee decided she needed to gather first-hand material.\n\n'Why are you going?' asked Astha.\n\n'I have to go. Awful things are happening there.'\n\n'I know. I also read the papers'' said Astha irritably, 'and that is why I wish you wouldn't go. It's not safe.'\n\nPipee looked at her for a moment, then gave a strange laugh. 'One has to do what one has to do.'\n\nAstha looked bewildered.\n\nPipee ruffled her hair with a slow unsteady hand, 'Don't worry, I can take care of myself.'\n\n*\n\nShe came back unharmed but terribly shaken. 'It's worse than you realise, the police are actually firing on the innocent, making false arrests, and refusing to register complaints. How can Muslims have any security or protection when the forces of law are among those who beat and kill? Go back to Pakistan they keep taunting, when were they ever in Pakistan, that they should go _back?_ And nothing is done, nothing. What kind of country can these people feel part of? To be a Muslim here is a curse.'\n\n*\n\n'They started it'' said Hemant. 'After the Babri Masjid fell they were the ones who first took to stoning temples in Bombay. What did they expect, that this is the time of the Muslim rulers, where Hindus will sit down and not retaliate? Who set fire to the temple in Govandi? Who started throwing stones at buses, and police stations, and the BMC offices?'\n\nAstha listened. If it was quite clear that there were many ways to regard what was happening, it was equally clear that she and Hemant held opposite views. Whose voice would be stronger remained to be seen.\n\nThe most effective way she had of making a statement was with paint, and she focused on that. It took her mind off her personal predicament, with such violence around her, her problems seemed small. She turned to brush and canvas to make her contribution to her country, she hoped it would be noticed. It was only a drop in a large, large ocean, but drops added up.\n\n*\n\nPipee ended up making several trips to distressed areas. Work, she said briefly, and maybe, thought Astha, she keeps coming back because she misses me, though she knew Pipee would never say anything to this purpose. Once she had decided that they were going to break up, that was it.\n\nShe called Pipee over one day, and had the pleasure of her approval when she saw her canvases.\n\n'They are strong and make a very effective statement. I can see how you have evolved, Ant.' So what if Pipee was leaving, at that moment Astha felt they could never be parted.\n\n'They really are good'' continued the friend and activist, 'perhaps you can hold an exhibition on your own. It's time you emerged from the shadow of the Manch.'\n\nThen she returned to her travels, so much is happening.\n\nAstha noticed Pipee didn't ask her to join her even for a weekend. She would have gone anywhere if Pipee had only asked her.\n\nIt was in January that Pipee got the letter confirming her admission to the University of Illinois, Urbana Champaign.\n\n'So soon, you have got to hear so soon, how wonderful'' said Astha over the phone, glad that Pipee could not see her face. 'They must really want you.'\n\n'I don't know about that. The amount they offer will show.'\n\n'When will you know?'\n\n'Soon, I hope.'\n\nAfterwards Astha scolded herself, this is the dress rehearsal for the real thing, why should I care, what is she to me, someone I loved, but we both have our own lives. She has chosen larger horizons, it's her life, this is mine. She told herself this firmly and repeatedly and was surprised that the information did not make her feel better.\n\n*\n\nNext morning she woke up with a headache. So, what's new, she asked herself, quickly swallowing a decongestant and a painkiller.\n\nAs the pills took effect and the pain receded, she drearily made for the spare room, to try that other cure, work. She pressed her turpentine rag against her face and breathed its sharp smells. She imagined it around her eyes, running under her eyebrows, through her temples, pushing all the throbbing in front of it, sweeping it away, throwing it out, so that it lost the power to affect, now and ever after.\n\nIt was a month before the axe finally fell. 'Oh, Pip, I'm so glad. How much are they giving you?'\n\n'A full fee waiver, and twelve hundred dollars a month.'\n\n'You deserve every bit of it. I hope you will be very happy.'\n\n'Hey, I'm not going yet. And I want to see your exhibition before I go.'\n\n'I'm doing my best.'\n\n*\n\n'I will get Ravi's wife to review it'' said Hemant. 'She is an art critic for _The_ _Indian_ _Express._ She probably knows others as well.'\n\n'Thank you.'\n\n'I'm sure they will be impressed'' said Hemant smiling at Astha.\n\nAstha knew Hemant was being helpful because the Manch was not involved and he welcomed the breach between her and any activism, but she had been too long married to linger over the source of his appreciation.\n\n*\n\nEvery morning, the children in school, the servants supervised, Hemant safe with his diet lunch in the factory, and Astha would shut herself inside her painting room. She needed to feel closed in and protected, if by nothing else than walls. There she was with shrouded canvasses, bottles of turpentine and linseed oil, tubes of colour lying in baskets around the easel, and grey rags stiff with dried paint. These were the tools of her trade, these were the things that established her separate life, touching them was comfort.\n\nAs her brush moved carefully over the canvas, her hand grew sure, her back straightened, she sat firmer on her stool, her gaze became more concentrated, her mind more focused. A calmness settled over her, tenuous, fragile, but calmness nevertheless. She thought of her name. Faith. Faith in herself. It was all she had.\n\n*\n\nPipee too was working. She was looking for a tenant, getting her papers ready, packing away her books, winding up her affairs. She required no interaction with Astha in arranging these things.\n\nThere was a time when Astha's day revolved around being\n\navailable to Pipee. The children, Hemant and all her obligations were frantically juggled so that she could see and talk to Pipee whenever possible.\n\nNow they talked, but it was on the surface, both of them reluctant to work at letting go of a connection that would naturally cease when Pipee left.\n\n'I can't wait to see your work.'\n\n'Come.'\n\n'I'll come, I'll come. I'm going to Shahjehanpur. They have called me. I think I ought to go before I leave.'\n\n'How come you didn't tell me?' quavered Astha, the weaker of the two, remembering the time when she knew what Pipee was going to do the second she thought of doing it.\n\n'You are so busy these days.'\n\nAt this prevarication, for the first time Astha felt relief that in a few months she would not have to talk to Pipee anymore.\n\n*\n\nIt was May. The amaltas trees were blooming. Every morning when Astha stepped onto the road, she was forced to step on fallen, perfect, clear yellow flowers, pale green buds, and scattered curving yellow stamens. She stepped on them because there was no way to avoid them, and the flowers forgave her by looking just the same.\n\nThis morning Astha was going to Vasant Kunj to pick up Pipee, despite her protests, to take her to the station for the train to Shahjehanpur.\n\nAs she drove her hands felt heavy on the wheel. How many times had she travelled down this road in hope and longing, and then rushed back dreading the demands and questions of her children, husband, in-laws. Where have you been, we were waiting for you, this that and the other happened, and you weren't here to fulfil your place in this house. Soon nobody would have cause for complaint, if there had been neglect, she would make up for it now.\n\nIn Pipee's flat, third floor, no fans or cooler because the electricity had gone. 'How hot it is'' remarked Astha for something to say.\n\n'Thank God, I'll soon be leaving'' said Pipee carrying a small suitcase and locking, then double locking the doors.\n\n'Indeed.'\n\nPipee looked at her. 'I don't mean I want to leave you. You know that.'\n\nAt these words hope sprang up in Astha's breast. The eternal stuff, hope. She looked at it in disgust. Hope looked back coy and stubborn. It did not have to say anything. Its presence spoke for itself.\n\nThe suitcase thrown in the car, the two got in and started the long drive to the New Delhi Railway Station.\n\nThat summer was the hottest Delhi had ever known. The temperature hit the 40s and stayed there day after day. Astha only felt fresh enough to work in the early mornings. She now woke at 4.45, made herself a cup of tea, and was at her easel by 5.00. As a consequence it became necessary to sleep by 10 p.m. Every day she and Hemant fought about this.\n\n'This is crazy, you are crazy, your life revolves around those canvases.'\n\nHow could she make him understand? Work was the only place she could forget everything, where she could become her mind, her hand, and the vision inside her head. At any rate she was sleeping badly, only by working hours every morning before the demands of the house took over did she know some peace. All this was not explainable.\n\n'Only for a little while more'' she tried coaxing.\n\n'You've been saying that for ages.'\n\n'You were the one who encouraged me to hold an exhibition, you showed interest, you said you would speak to Ravi's wife.'\n\n'But what's the big hurry? You can have your exhibition later, anyway winter is a better time.'\n\n'I want it now.'\n\nAstha was feeling too sick at heart to give being sweet and coaxing more than the briefest try.\n\n*\n\nThe hall at the Tagore Arts Centre was rented for five days. There were twenty canvases in all. It was two years work, and from December on, she had worked almost every day. Six canvases were devoted to the Babri Masjid and different forms of protest, another six to various aspects of Pipee and herself, though she hoped they were so disguised no one would be able to identify the women. There were four of her children, and two of men she had modelled on Hemant, one of Mala and Bahadur. Basically my life, thought Astha as she, Hemant, and the children worked in the gallery, putting them up, placing them to the best advantage.\n\n'Why is this so small?' asked Hemant picking up one the size of a sheet of paper.\n\n'It's for Pipee to take with her'' said Astha. 'I made it small on purpose.' Then as Hemant said nothing, she continued, 'Do you think she will like it?'\n\nThe painting was an interior, two women sitting on a charpai. The patches of colour came from a red cushion, an open window, the white of a pillow on the bed, the bangles of one, the bag and chappals of the other thrown on the floor. The figures themselves were indistinct and shadowy, one had a drooping head, the other had her face turned away. The small canvas added to the sense of claustrophobia.\n\n'It's kind of sad'' said Hemant.\n\nAstha was always surprised when Hemant said anything she could remotely relate to. 'I suppose it is rather'' she said. 'Maybe she should only be surrounded by happy things in Illinois?'\n\n'I'm sure she'll like it. Are these women you two?'\n\n'Of course not'' said Astha quickly. 'They are imaginary. You can't see their faces. Could be anyone.'\n\n'Hummm.'\n\nThe last paintings to be hung were the Babri Masjid series, six in all, ending with a bare hillock, a trishul and a saffron flag planted on empty earth amid scattered stones, a peepul tree hanging forlornly on one side.\n\n'I hope it is not too obvious'' fussed Astha, knots gathering inside her from the stress of exhibiting, displaying, exposing.\n\n'Not at all'' said Hemant. 'You need obvious symbols to say obvious things.'\n\nWhy is he being so nice to me, thought Astha. He even seems to have changed his political opinions. Is it because all the work is over, no more early nights, early mornings, is it because she's going in another two weeks?\n\n'Mama, I'm going to give out the price lists'' said Anuradha firmly.\n\n'Of course darling, I shall depend on you.'\n\n*\n\nPipee had returned from Shahjehanpur, but she still had to get her tenant, and decide what to do about her possessions. She couldn't help Astha with the setting up of her exhibition, Astha couldn't help her with the disposing of her things.\n\n'It is rather badly timed'' said Astha on the phone, 'but I am holding this exhibition for you, Pip. So that, before you go...' She stopped. She didn't need to finish her sentences with Pipee.\n\n'I know, I know, Ant. I wish I could be more there for you.'\n\n'And I for you.'\n\n*\n\nPrivately Astha thought perhaps it was just as well, she couldn't bear to witness the disbanding of Pip's house, where they had been skin on skin, mind on mind with nothing in between.\n\nAnd Pipee thought, it is just as well Ant is not here when I am packing to leave. I don't think she could take it, and I couldn't take her not taking it. I wish she hadn't come with so much baggage, but she did, and well, there it is.\n\nThe opening of the exhibition, 1 August.\n\nPipee said, 'I had no idea you had been painting so much. It's wonderful, just wonderful.'\n\nAll Astha could say was, 'Did you like no. 12? It's for you.'\n\nPipee looked at her, squeezed her hand, and after half an hour of hanging about, affectionately said, 'I have to go dearest, I will see you later.'\n\n'So soon?'\n\n'I have to. A tenant is coming to see the flat, and could come at no other time. I am getting frantic, I hope this one works out.'\n\nAstha thought of the very long distance between Pipee's flat and the Tagore Arts Centre, of the rush hour traffic, of the hour it would take her to get back home, and decided she could only be grateful that Pipee had come at all.\n\n'Bye, see you.' She blew her a kiss and was gone, leaving Astha to listen to what Hemant was saying and who he was introducing her to.\n\nMore than half the paintings sold. Astha made almost two lakhs.\n\n'It is a good beginning'' said Hemant, quite the manager of his wife's career. 'Ravi said his wife is going to give it a positive review, and talk to some other art critics so they mention it too. Exposure is what counts at this stage.'\n\n*\n\n'You must mail me the reviews'' said Pipee. 'I'm sure they will be very good. I look forward to reading them.'\n\nOh, Pipee, don't talk like a stranger to me, I can't stand it. I only want to talk about how sad I am feeling.\n\nBut the wall between them was by now quite high, and from time to time they both threw another brick on it. They were doing this now.\n\n'How is the tenant?'\n\n'Just what I wanted.'\n\nAnd so on.\n\n*\n\n'I will take you to the airport'' said Astha on the phone.\n\n'Are you sure?'\n\n'Please, Pip, don't be insulting.'\n\n'I only meant what about Hemant, won't he mind?'\n\n'Hemant will understand.'\n\nWith Pipee about to go, it was guaranteed that Hemant would understand anything.\n\n*\n\nThe night of 6 August. The last time Astha would drive to Vasant Kunj. The weather was hot and still, it hadn't rained since the night of her opening. She parked, climbed the three flights to Pipee's flat, rang the bell, and contemplated the bars, bolts and locks on the wooden and screen doors. Last time, for the last time, rang irritatingly in her mind. Was there anything about this night that was not going to be drenched in significance? She wished it was over, that she did not have to go through it step by painful step, Pipee's departure from her life.\n\nShe thought of how they had both been ants together. And now Pipee was journeying eight hours to London, ten hours to Chicago, two hours by bus to Urbana, to be an ant somewhere else.\n\n'Hi!'\n\n'Hi.'\n\nShe entered. The flat was bare, with just the things Pipee had sold the tenant. The bed, the cane chairs, the small wooden dining table.\n\nAstha sat down and looked around. 'What'll you do when you come back?' she asked. 'About bedding and stuff?'\n\n'Oh'' Pipee sounded vague. 'Buy new, I guess.' Astha could tell it was far from her mind, her return, why was she hurting herself by looking for clues?\n\n'Are you ready?' she asked.\n\n'The chowkidar is coming. I have to give him the keys and he'll take down the stuff.' Pipee wasn't quite looking at her, and Astha realised she was making Pipee uneasy, the way she sounded, sad, heavy, teary. She said nothing more as she watched Pipee doing last-minute things.\n\n'It's a good thing that weight is not important when you fly to the US'' said Pipee as the chowkidar staggered out first with one heavy suitcase, then another. 'They go by the number of bags.'\n\n'Yes, I suppose.'\n\n'Come, let's go.'\n\n*\n\nThe long drive, their car one of a stream going to the International Airport late at night, all saying good-bye to people they loved. As Astha drove, she imagined the misery in the cars around her. Join the queue, Astha, join the queue.\n\nThe crowd at the Indira Gandhi International Airport was as usual overwhelming. Astha drove up the ramp to Departure, nosing through the cars, coaches, taxis, and thousands of people.\n\n'I'll get a trolley'' said Pipee, jumping out.\n\n'I'll get the luggage'' said Astha moving towards the dickey, and fumbling with the key. There was Pipee with the trolley, the luggage unloaded, there was a policeman waving her car away \u2013 no standing allowed, and Pipee saying go, sweetie \u2013 where will you park \u2013 it's so crowded, and Astha, wailing but I want to see you inside \u2013 they won't allow you \u2013 and the policeman \u2013 not moving towards any of the other parked vehicles on the ramp but threatening her with traffic violation \u2013 Pipee propelling her into her car \u2013 a last kiss, goodbye, goodbye, take care, and she was lost to the eye even before she had wheeled her trolley through the entrance door.\n\n*\n\n'So, she's gone'' said Hemant when Astha returned. Awake at that late hour and witness to his wife's face and eyes.\n\n'Yes.'\n\n'Was the plane on time?'\n\n'I don't know. I didn't stay.'\n\n'How was she?'\n\n'Who?'\n\n'Your friend, who else?'\n\nAstha could not reply. 'I'm tired'' she said, 'I want to sleep.'\n\nMechanically she changed, brushed her teeth, put cream on, got into her side of the bed, pulled the sheet up, and turning to the very edge lay absolutely still. Motion of any kind was painful to her. Her mind, heart and body felt numb.\n\nIt continued like this for days. She felt stretched thin, thin across the globe. \n\n# Acknowledgements\n\nMany people have been particularly kind in sharing information that I found invaluable. In this regard I would particularly like to mention Anshu Balbir, Mita Bose, Ranjan Dhawan, Jamal Kidwai, Vinay Minucha, and Jay a Srivastava. This book could not have been written without their help.\n\nMasooma Ali, Sunanda Ali, Bharati Bhargava, Nidhi Dalmia, Nilanjana Dalmia, Christopher Fruean of Walt Disney World, Vijay Kapur, Vimla Kapur, Fauzia Khan, Angela Koreth, M. K. Raina, Maseeh Rehman, Saswati Sen Gupta and Jaya Sharma were badgered about numerous details, and responded with patience and generosity.\n\nJaya Srivastava, Sharmila Purkayastha and V. Karthika were generous with pamphlets and books I would not have been able to get otherwise.\n\nPenelope Anderson, Janet Chawla, Katyayani Dalmia, Anuradha Marwah, Ira Singh, Ramya Sreenivasan, and Addison Ullrich contributed encouragement, interest, enthusiasm and criticism.\n\nAnuradha Marwah helped in crucial ways during the final stages.\n\nJulian Loose, my editor at Faber, bestowed a clarity and vision upon these pages that much benefited them. Heather Schroder showed faith in me by becoming my agent on trust.\n\nRoma Bhagat Baraya helped with legal aspects of the text. Sanjeev Saith, my publisher at Indialnk was a model of patience and tact. His meticulous attention to detail was greatly appreciated.\n\nIra Singh's repeated readings and comments helped shape the characters. Her other contributions defy exact description.\n\nGun Nidhi Dalmia, my husband, was astute and reassuring in his reading of my manuscript. My hours at the computer would not have been possible without his support.\n\nMy children, Maya, Amba, Katyayani and Agastya were with me throughout in body and spirit.\n\nDuring the research for my novel I consulted the following books for their spiritual commentary: _The_ _Secret_ _of_ _the_ _Kath_ _Upanishad,_ Swami Krishnananda, The Divine Life Society: Tehri-Garhwal, UP, India, 1974; Maharishi Mahesh Yogi on the _Bhagavad-Gita,_ _A_ _New_ _Translation_ _and_ _Commentary,_ Chapters 1-6, Penguin, 1967.\n\nFor the political events that form the background of the novel I consulted: _Ayodhya_ _Imbroglio:_ T. P. Jindal, Ashish Publishing House, New Delhi, 1995; _The_ _Demolition:_ _India_ _at_ _the_ _Crossroads,_ Nilanjan Mukhopadhyay, Harper Collins, 1994; _The_ _People's_ _Verdict:_ An inquiry into the December '92 & January '93 riots in Bombay by the Indian People's Human Rights Tribunal Conducted by Justice S. M. Daud & Justice H. Suresh published by The Indian People's Human Rights Commission, August 1993; Pradeep Nayak, _The_ _Politics_ _of_ _the_ _Ayodhya_ _Dispute,_ Commonwealth Publishers, New Delhi, 1993; and _Cry_ _the_ _Beloved_ _Country,_ a PUDR pamphlet.\n\nIn section VI, the advertisement by Ramjanambhoomi Nyas (a non-political body affiliated to the VHP) in The Pioneer, May 11, 1991, has been taken from Pradeep Nayak, _The_ _Politics_ _of_ _the_ _Ayodhya_ _Dispute,_ (details mentioned above) p. 167.\n\nThe Hindustan Times was consulted on microfilm, accessed from the Teen Murti Memorial Library. I am grateful to the library for allowing me this facility.\n\n*\n\nThe actual events leading to the destruction of the Babri Masjid have either been fictionalised or used in imaginative reconstructions.\n\n*\n\nThis book went through several of its many drafts during a three month stint at the Universities of Kent and Stirling in the UK. I am indebted to the Charles Wallace Trust, India for granting me a fellowship to these places. \n\n# About the Author\n\nManju Kapur is the author of four novels. Her first, _Difficult Daughters_ , received tremendous international acclaim, won the Commonwealth Prize for First Novels (Eurasia Section), and was a number one bestseller in India. Her second novel _A Married Woman_ was called 'fluent and witty' in the _Independent_ , while her third, _Home_ , was described as 'engaging, glistening with detail and emotional acuity' in the _Sunday Times_. Her most recent novel, _The Immigrant_ , was called 'intensely readable' in the _Daily Mail_ and 'admirable and enjoyable' by the _Guardian_. She lives in New Delhi. \n\n# Copyright\n\nThis ebook edition published in 2012  \nby Faber and Faber Ltd  \nBloomsbury House  \n74\u201377 Great Russell Street  \nLondon WC1B 3DA\n\nAll rights reserved  \n\u00a9 Manju Kapur, 2003\n\nThe right of Manju Kapur to be identified as author of this work has been asserted in accordance with Section 77 of the Copyright, Designs and Patents Act 1988\n\nThis ebook is copyright material and must not be copied, reproduced, transferred, distributed, leased, licensed or publicly performed or used in any way except as specifically permitted in writing by the publishers, as allowed under the terms and conditions under which it was purchased or as strictly permitted by applicable copyright law. Any unauthorised distribution or use of this text may be a direct infringement of the author's and publisher's rights, and those responsible may be liable in law accordingly\n\nISBN 978\u20130\u2013571\u201326780\u20134\n","meta":{"redpajama_set_name":"RedPajamaBook"}}
+{"text":" \nTHE SHAPE OF SPECTATORSHIP\n\nFILM AND CULTURE\n\nJohn Belton, Editor\nFILM AND CULTURE\n\n_A series of Columbia University Press_\n\nEdited by John Belton\n\nFor the list of titles in this series, see Series List.\nTHE\n\nSHAPE\n\nOF\n\nSPECTATORSHIP\n\nART, SCIENCE, AND EARLY CINEMA IN GERMANY\n\nSCOTT CURTIS\n\nCOLUMBIA UNIVERSITY PRESS\n\nNEW YORK\n\nColumbia University Press\n\n_Publishers Since 1893_\n\nNew York Chichester, West Sussex\n\ncup.columbia.edu\n\nCopyright \u00a9 2015 Columbia University Press\n\nAll rights reserved\n\nE-ISBN 978-0-231-50863-6\n\nLibrary of Congress Cataloging-in-Publication Data\n\nCurtis, Scott.\n\nThe shape of spectatorship : art, science, and early cinema in Germany \/ Scott Curtis.\n\npages cm. \u2014 (Film and culture)\n\nIncludes bibliographical references and index.\n\nISBN 978-0-231-13402-6 (cloth : alk. paper) \u2014 ISBN 978-0-231-13403-3 (pbk. : alk. paper) \u2014 ISBN 978-0-231-50863-6 (ebook)\n\n1. Motion pictures\u2014Germany\u2014History\u201420th century. 2. Motion picture audiences\u2014Germany\u2014History\u201420th century. 3. Motion pictures\u2014Aesthetics. 4. Motion pictures in science\u2014Germany. 5. Documentary films\u2014Germany\u2014History\u201420th century. I. Title.\n\nPN1993.5.G3C88 2015\n\n791.430943\u2014dc23\n\n2015010546\n\nA Columbia University Press E-book.\n\nCUP would be pleased to hear about your reading experience with this e-book at cup-ebook@columbia.edu.\n\nCover Design: Jordan Wannemacher\n\nCover Image: From Wilhelm Braune and Otto Fischer, \"Versuche am unbelasteten und belasteten Menschen,\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 21, no. 4 (1895): 151\u2013322\n\nReferences to websites (URLs) were accurate at the time of writing. Neither the author nor Columbia University Press is responsible for URLs that may have expired or changed since the manuscript was prepared.\nTo my parents\n\nCONTENTS\n\nList of Illustrations\n\nAcknowledgments\n\nINTRODUCTION\n\n1. SCIENCE'S CINEMATIC METHOD: MOTION PICTURES AND SCIENTIFIC RESEARCH\n\nEarly Scientific Filmmaking: An Overview\n\nBergson, Cinema, and Science\n\nThe Science of Work and the Work of Science\n\nBrownian Motion and \"the Space Between\"\n\nNerve Fibers, Tissue Cultures, and Motion Pictures\n\n2. BETWEEN OBSERVATION AND SPECTATORSHIP: MEDICINE, MOVIES, AND MASS CULTURE\n\nThe Multiple Functions of the Medical Film\n\nMotion Pictures and Medical Observation\n\nTime, Spectatorship, and the Will\n\n3. THE TASTE OF A NATION: EDUCATING THE SENSES AND SENSIBILITIES OF FILM SPECTATORS\n\nCinema and the Spirit of Reform\n\nChildren, Crowds, and the Education of Vision and Taste\n\n\"Cinematic Lesson Plans\" in Elementary and Adult Education\n\n4. THE PROBLEM WITH PASSIVITY: AESTHETIC CONTEMPLATION AND FILM SPECTATORSHIP\n\nAgency and Temporality in the Aesthetic Experience of Cinema\n\n_Einf\u00fchlung_ , Identity, and Embodied Vision\n\nThe Politics of Contemplation\n\nCONCLUSION: TOWARD A TACTILE HISTORIOGRAPHY\n\nNotes\n\nBibliography\n\nIndex\nLIST OF ILLUSTRATIONS\n\nFigure 1.1. | Braune and Fischer's military recruit in the experimental suit  \n---|---  \nFigure 1.2. | The subject at rest with the grid superimposed  \nFigure 1.3. | Braune and Fischer's camera placement  \nFigure 1.4. | The resulting chronophotograph  \nFigure 1.5. | Determination of the coordinates of a point _P_ from the projections of _P_ on two planes as seen from the two cameras  \nFigure 1.6. | Side and top views of the instrument used to measure coordinates  \nFigure 1.7. | Measurement of a coordinate  \nFigure 1.8. | A table of the coordinates derived from experiment 1  \nFigure 1.9. | The graph of the coordinates (view from the right side)  \nFigure 1.10. | The graph of the coordinates (view from above of different body parts)  \nFigure 1.11. | Left and back views of the tridimensional model representing the attitudes of the human body during walking  \nFigure 1.12. | Seddig's cinematic apparatus for measuring Brownian motion  \nFigure 1.13. | Seddig's photographic rendering of Brownian motion  \nFigure 1.14. | The irregular paths of Brownian motion  \nFigure 1.15. | Hermann Braus  \nFigure 1.16. | Ross Granville Harrison  \nFigure 1.17. | Harrison's sketches of the elongation of frog nerve fibers grown in culture  \nFigure 2.1. | Frames from Ludwig Braun's film of a dog's beating heart  \nFigure 2.2. | Groedel's serial cassette X-ray apparatus  \nFigure 2.3. | Max Nordau  \nFigure 2.4. | A hypnotist practicing his craft, France, circa 1900  \nFigure 3.1. | A typical storefront movie theater ( _Ladenkino_ ) from the pre\u2013World War I era  \nFigure 3.2. | _The Wanderings of Odysseus_ , touted to be a \"Reformfilm\"  \nFigure 3.3. | \"The Cultural Work of the Film Theater: Thoughts from the Year 1784 by Friedrich von Schiller\"  \nFigure 3.4. | The geometry of taste  \nFigure 3.5. | Children at Luisen-Kino in Berlin, circa 1910  \nFigure 3.6. | A page from Lemke's _Die kinematographische Unterrichtsstunde_ (The Cinematic Lesson Plan, 1911)  \nFigure 3.7. | Hermann H\u00e4fker  \nFigure 4.1. | Aesthetic experience as a series of interlocking dichotomies  \nFigure 4.2. | A prewar audience enjoying a night at the Union-Theater in Berlin, 1913  \nFigure 4.3. | Kammer-Lichtspiele Theater in Berlin, 1912\nACKNOWLEDGMENTS\n\nThis has been, to use an inappropriate medical metaphor, a long and difficult birth, but certainly not for lack of attendants or painkillers. After so many years of conceptions, reconceptions, and labor, however, it is hard to know who to thank or how far back to go, so I will simply begin to recite and offer my deepest apologies to anyone I inadvertently leave out. Various versions took shape in various locations, where I owe debts to the institutions and people who supported me and this project. The initial research was made possible by a stipend from the German Academic Exchange Service. Several people made my stay in Frankfurt more productive than it might have been. Heide Schl\u00fcpmann, Helmut Diederichs, and Martin Loiperdinger offered good advice and kind, if somewhat bewildered encouragement as I struggled to define my topic. Diederichs also provided the rare image of Hermann H\u00e4fker for chapter 3. Special thanks go to the late Eberhard Spiess for allowing me access to the periodicals and holdings of the Deutsches Institut f\u00fcr Filmkunde, and to Brigitte Capitain for her patience as I took advantage of this generosity. In Iowa City, I appreciate the attention David Depew, Kathleen Farrell, and Hanno Hardt gave to the first version of this project, while John Durham Peters and Dudley Andrew deserve special thanks for their guidance through the years. In Los Angeles, I am grateful to Linda Mehr and the late Robert Cushman of the Academy of Motion Picture Arts and Sciences' Margaret Herrick Library; I learned much from their example. Joe Adamson, Val Almendarez, Anne Coco, Steve Garland, Harry Garvin, Barbara Hall, Doug Johnson, Janet Lorenz, David Marsh, Howard Prouty, Lucia Schultz, Matt Severson, Warren Sherk, and all of my fellow librarians at the Herrick deserve thanks as well.\n\nIn Evanston, I owe thanks to the Department of Radio\/Television\/Film\u2014especially my colleagues Bill Bleich, Michelle Citron, Laura Kipnis, Chuck Kleinhans, Larry Lichty, Hamid Naficy, Eric Patrick, Jeff Sconce, Jacob Smith, Jacqueline Stewart, Deb Tolchinsky, and the rest of the faculty\u2014for having faith in me. Chairs Annette Barbier, Mimi White, Lynn Spigel, and David Tolchinsky were steadfast in their support. I thank Lynn Spigel, especially, for all she has done on my behalf (as far back as Los Angeles) as a mentor, model, and friend. Deans David Zarefsky and Barbara O'Keefe at the School of Communication offered resources in various forms, and I am grateful for their time, money, and patience. In Berlin, stays at the Max Planck Institute, thanks to Lorraine Daston, significantly sharpened my thinking about physics and observation, especially. In Weimar, Karl Sierek, Friedrich Balke, Daniel Eschk\u00f6tter, and the graduate students at Bauhaus-Universit\u00e4t welcomed me and gave me space and time to work. In Innsbruck, Mario Klarer, Gudrun Grabher, Christian Quendler, Erwin Feyersinger, Robert Tinkler, Cornelia Klecker, Johannes Mahlknecht, Monika Datterl, and Maria Meth likewise gave time, space, and warm companionship freely, as well as trips to mountain cabins. In Doha, the entire staff and faculty of Northwestern University in Qatar made me feel welcome, especially program directors Mary Dedinsky and Sandra Richards, and colleagues Greg Bergida, David Carr, Susan Dun, Elizabeth Hoffman and Bob Vance, Joe Khalil, Muqeem Khan, John Laprise, Jocelyn Mitchell, Sue Pak, Christina Paschyn and Alex Demianczuk, Barry Sexton, Bianca Simon, Anne and Adam Sobel, Allwyn Tellis, Tim Wilkerson, and Ann Woodworth. Deans Jim Schwoch, Jeremy Cohen, and Everette Dennis displayed an inordinate amount of trust and confidence in my abilities; Dean Dennis, especially, offered whatever it took\u2014and it took a lot\u2014to get it done, and I am deeply indebted to him.\n\nAlong the way, a number of people deserve commendation for having read or commented on various parts of this project in various forms. Chapter 1 benefited greatly from the insights of Nancy Anderson, Charlotte Bigg, Thomas Haakenson, Andreas Mayer, and others at the Max Planck Institute; Hannah Landecker kindly shared her research and insights on cell biology; Martin Carrier and Alfred Nordmann sharpened my thinking about atomistic physics; Richard Kremer and Ken Alder offered face-saving corrections from the historian of science's point of view; and Dan Morgan, Oliver Gaycken, and Frank Kessler were kind enough to read the chapter at various points and help me clarify the argument. Chapter 2 profited from the attention of Ken Alder and the Science in Human Culture Group at Northwestern, who prompted me to rethink it, while Nancy Anderson and Mike Dietrich provided time and space at Dartmouth College to rewrite it. Lisa Cartwright, Oliver Gaycken, Andreas Killen, Kirsten Ostherr, and Henning Schmidgen were at various points inspirational and instrumental in shaping this chapter; Lisa Cartwright was especially helpful at a key point in the process. Chapter 3 owes its life to Steve Wurtzler, Jennifer Barker, and John Belton, and I owe Frank Kessler and Sabine Lenk for keeping it alive. A much earlier, different version of chapter 4 was lucky to have the scrutiny of Ben Singer, David Bordwell, David Levin, and Marc Silberman. For its current form, I must thank Robin Curtis, Gertrud Koch, Dan Morgan, Inga Pollmann, and especially Kaveh Askari and Tony Kaes for all of their insight and encouragement. There are still others who provided valuable assistance along the way, including research assistants David Gurney, Dan Bashara, and Rebecca Barthel. John Carnwath kindly and expertly corrected my translations, rescuing me from many infelicities. Stefanie Harris, J\u00f6rg Schweinitz, and various anonymous readers offered important insights that prompted revisions and changes in argument. For their stalwart professional support and friendship over the years, I must offer my heartfelt thanks to Richard Abel, Rick Altman, Matthew Bernstein, Jane Gaines, Dilip Gaonkar, the late Miriam Hansen, Tom Levin, Charlie Musser, Jan Olssen, Patrice Petro, Lauren Rabinovitz, Eric Rentschler, Mark Sandberg, Vivian Sobchack, and Virginia Wexman.\n\nAll of the people named so far I count as my friends, but some friends deserve special mention for their unselfish and nonjudgmental acceptance of me and my book. Tom Gunning has been a friend and mentor for a very long time; more than anyone, he has shaped the contours of this ongoing investigation, usually without even knowing it. Tony Kaes has been a loyal fan and inspiration since I was a student. Both Tony and Tom have offered insightful, transformative commentary on several versions. Oliver Gaycken, Vinzenz Hediger, and Kirsten Ostherr are my fellow travelers on this interdisciplinary journey; I don't often take a step without consulting them. Oliver read every word I have given him and always came back for more. Greg Waller and Brenda Weber have always offered valuable moral support and close friendship at just the right times. Ken Alder, Joe Carli, Lisa Cuklanz, Tracy Davis, Doug Johnson, Charlie Keil, and Will Schmenner have all been steady, life-long friends on whom I have leaned especially heavily at times. All the graduate students who have attended my seminars deserve note for their role in shaping my thinking over the years, but I thank especially Dan Bashara, Catherine Clepper, Beth Corzo-Duchardt, Alla Gadassik, Leslie Ann Lewis, Jason Roberts, Jocelyn Szczepaniak-Gillece, Kati Sweaney, and Meredith Ward. For their continuing friendship, I thank Richard Abel, Charles Acland and Haidee Wasson, Dana Benelli, Joanne Bernardi, Bill Bleich, Jeremy Cohen and Catherine Jordan, Kelley Conway and Matthew Sweet, Mark Garrett Cooper and Heidi Rae Cooley, Don Crafton and Susan Ohmer, Nick Davis, Leslie Midkiff DeBauche, Nico de Klerk, Carol Donelan and Shannon Spahr, Nata\u0161a \u010eurovi\u010dov\u00e1, Dirk and Myrna Eitzen, Jen Fay, Andr\u00e9 Gaudreault, Philippe Gauthier, Frank Gray, Alison Griffiths and William Boddy, Barbara Hall and Val Almendarez, Sara Hall and Monty George, Stefanie Harris, Micaela Hester, Chris Horak, Laura Horak and Gunnar Iverson, Rembert Hueser, Jenn Horne and Jonathan Kahana, Christopher Hurless and Rachel Henriquez, Zara Kadkani and Axel Schmitt, Jim Lastra, Tom Levin, Melody Marcus, Caitlin McGrath, Christie Milliken, Priska Morrissey, Tania Munz, Bill Palik, Anna Parkinson, Jennifer Peterson, Sarah Projansky, Christian and Grace Quendler, Isabelle Raynauld, Mark Sandberg, Ben Singer, Blane Skowhede, Jake Smith and Freda Love Smith, Stefan Soldovieri, Matthew Solomon, Shelley Stamp, Bing Stickney, Claudia Swan, Steve Tremble, Alison Trope, Mark Williams and Mary Desjardins, Tami Williams, Michael and Julia Wilson, Pam Robertson Wojcik, Robb Wood and Hanaa Issa, Steve Wurtzler, Harvey Young, and Josh Yumibe.\n\nI should stress that this book would not have been published except for the efforts of John Belton and Jennifer Crewe, whose stubborn determination to wrest the manuscript from me finally overmatched my stubborn refusal to give it up. I also thank my editorial team at Columbia University Press: Ben Kolstad, Roy Thomas, Jennifer Jerome, Anne McCoy, and Kathryn Schell.\n\nOf course, my family also deserves a large share of credit, not the least for their good-natured and bemused acceptance of a project that seemed never to end. Cindy and Randy Lee have been unconditional in their love and acceptance; Brandon and Noelle Lee, Trevor, Josh, and Michael Lee all are great relatives to have. Grant and Donna Boyles deserve mention for their love, care, and hospitality. I know my grandmother, Louise, and my aunt, Dian, would have been proud. I thank the Pike and Kelly families for welcoming me into their close-knit web of love and kindness: Ken and Elnora Kelly, Clayton and Carol Pike, Kerry and Kelli Graf (and Kaitlyn and Kayla), and Kory Pike; Kevin and Meredith Pike, especially, are not just relatives, but friends, which is a rare thing to say. But I owe most to Kirsten Pike, who has been my anchor, sail, and compass since I met her; this book and I would not be the same without her years of love and encouragement. If I had another book in me, I would dedicate it to her. But I must dedicate this work to my parents, Ray and Pat Curtis, whose inexhaustible patience, support, and love made everything possible.\n\nThere have been publications of parts of this project along the way, but what lies in the pages ahead is usually significantly different from what came before. Even so, we should note that parts of chapter 1 originally appeared as \"Die kinematographische Methode. Das 'Bewegte Bild' und die Brownsche Bewegung,\" _montage\/AV: Zeitschrift f\u00fcr Theorie & Geschichte audiovisueller Kommunikation_ 14, no. 2 (2005): 23\u201343; and \"Science Lessons,\" _Film History_ 25, nos. 1\u20132 (2013): 45\u201354. Parts of chapter 2 originally appeared as \"Between Observation and Spectatorship: Medicine, Movies, and Mass Culture in Imperial Germany,\" in _Film 1900: Technology, Perception, Culture_ , edited by Annemone Ligensa and Klaus Kreimeier (New Barnet, U.K.: Libbey, 2009), 87\u201398; and \"Dissecting the Medical Training Film,\" in _Beyond the Screen: Institutions, Networks and Publics of Early Cinema_ , edited by Marta Braun, Charlie Keil, Rob King, Paul Moore, and Louis Pelletier (New Barnet, U.K.: Libbey, 2012), 161\u2013167. Parts of chapter 3 originally appeared as \"The Taste of a Nation: Training the Senses and Sensibility of Cinema Audiences in Imperial Germany,\" _Film History_ 6, no. 4 (Winter 1994): 445\u2013469. Parts of chapter 4 originally appeared as \"Einf\u00fchlung und die fr\u00fche deutsche Filmtheorie,\" in _Einf\u00fchlung. Zur Geschichte und Gegenwart eines \u00e4sthetischen Konzepts_ , edited by Robin Curtis and Gertrud Koch (Paderborn: Fink, 2009), 61\u201384.\nINTRODUCTION\n\n_Probably no contemporary invention has generated quite as much discussion in the daily press and in daily conversation as the cinema. Everywhere new theaters shoot up overnight like mushrooms. Our cities at night can no longer be imagined without the beaming portals of the movie houses. But it's not just the simple folk pushing themselves through these \"narrow gates of grace.\" The educated class, as well as science and the schools, the state, the city, and rural communities have all grasped the cultural significance of cinema and have taken a step closer to the establishment and utilization of their own film theaters. Who would look upon this burning question with indifference?_\n\nADOLF SELLMANN (1912)\n\nWhatever cinema is, it has always been many things to many people. Even in 1912, it was clear to a reformer such as Adolf Sellmann that a variety of interest groups and interested parties, from scientists to educators to town councils, were using motion picture technology. Each group recognized cinema's \"cultural significance\" and power or acknowledged its inevitability, but not every group agreed on how motion pictures should be used, either in the public sphere or, especially, within the boundaries of the group or discipline. Everybody had their reasons for using motion pictures, and those reasons often diverged. This book attempts to understand how various disciplines or communities used motion pictures. What did cinema mean to these groups? What were the criteria for the acceptance of motion pictures as a tool within a given discipline? What problems presented themselves such that motion pictures were considered a solution? This book explores these questions to discover the criteria for the legitimacy of a new media technology within the disciplines of science (specifically, human motion studies, physics, and biology), medicine, education, and aesthetics in Germany before World War I.\n\nThese disciplines correspond roughly to the familiar historical trajectory of early cinema, from its roots in scientific research to its early bids for acceptance as an art form. Taken together, they also represent the heterogeneity of early cinema, not only in terms of the many types of films available during this period but also with respect to the varied venues, audiences, and uses of the medium. Additionally, they typify relatively well-defined communities with strong, native traditions, where, outside of the entertainment industry, the liveliest discussion of motion pictures took place during cinema's early period. This listing obviously leaves out the entertainment industry, but questions of appropriation and legitimacy are less interesting in this area (at least to me), where the criterion for acceptance of film within that industry was clear, even tautological, in that the medium only had to prove its commercial viability and little else. So with its focus on the way groups used film for purposes other than entertainment, this study is aligned primarily with recent work in nontheatrical uses of film.\n\nHowever, even within the framework of \"useful cinema\" and the good work that has been done to define that area of film history, questions of appropriation and legitimacy are not often explicitly asked. While we know much about the use of motion pictures in the classroom in the 1920s, for example, we still know comparatively little about the state of pedagogical theory and practice at that time and why some groups within the discipline saw motion pictures as a partial solution to a variety of problems, and why others did not. We know little of their disciplinary agenda. Perhaps inevitably, we approach these questions as film historians, not as historians of education. Yet to understand fully any given appropriation, we must fully understand the agenda that shapes it. There is an intimate and complex relationship between any technology and the agenda that makes use of it. The technology is not merely applied to a problem; the problem presents itself in part because of the technology. What any scientist investigates, for example, is partly due to what the available technology makes available for investigation. Historians of science are very good at demonstrating the dialectical relationship between tools, theories, and representations, which shows us that we cannot take \"use\" for granted; the criteria for use of any given tool within a given discipline are not obvious. As different groups used media technologies for their different purposes, the nature of those appropriations changed, and in a significant way, so did the medium. \"What cinema is\" for one group was not necessarily the same as for another. Indeed, the nature of the appropriation often depended on what the agents _thought_ film was. So there was more to \"use\" than simply taking a camera, recording an event, and projecting it; the representational problems faced by any given discipline shaped its appropriation of (media) technology. Understanding those representational problems demands an intimate knowledge of the historical contexts and camps of that discipline.\n\nSo this project is not just about the encounter between other disciplines and film but the encounter between other disciplines and _film studies_. Specifically, each chapter stages a meeting between methods or approaches common to film studies and those of the history of science, the philosophy of medicine, the history of education, or the history of aesthetics. What does the result of such an assignation look like? What can we take away from such an encounter? Or, to put it another way, what can we reasonably expect from interdisciplinary research? Max Weber has his hand up: \"With every piece of work that strays into neighboring territory... we must resign ourselves to the realization that the best we can hope for is to provide the expert with useful _questions_ of the sort that he may not easily discover for himself from his own vantage point inside his discipline.\" \"Useful questions,\" however, are rarely presented as such; they are instead approaches or agendas that seem foreign at first, yet bear on our own. To formulate them as questions, we need to know the discipline well enough to recognize the pattern common to the approaches. So interdisciplinary research should be more than cherry-picking a few juicy quotations from an exciting discovery in another field; it must entail some significant level of immersion. What useful questions does the history of science, for example, provide film and media studies, and vice versa? As hinted above, one broad question could be: What is the relationship between technology and a disciplinary agenda? This ambitious question might be answered only after an accumulation of case studies, but it is useful for film and media studies, because it leads us to speculate about the tangible relationship between a representational technology and a community's conception of the object of study. Another question might be something like: What is the relationship between a technology and other elements of the experimental system? This question forces us away from our habitual focus on film and toward an understanding of film and media technologies as part of a larger experimental arrangement or as part of a technological group along the lines of what Germans call a _Medienverbund_ , or \"media ensemble.\" With the help of the history of science, we can see film in these contexts as an important but nevertheless interdependent part of an experimental system or a larger institutional project. Each disciplinary encounter in the following chapters results, I hope, in a different set of similarly useful questions.\n\nWhat can film historians bring to the table? We can bring different kinds of answers. Our training in close analysis heightens our sensitivity to formal relationships that rely on analogies and homologies as well as causal or empirical connections. This would be useful, because the acceptance or legitimacy of any given technology for a discipline hinges not simply on the tool's function for a particular task but also on how that technology fits into a larger disciplinary system. A wrench is useful because it fits the bolt; certain parts of the bolt and wrench have similar shapes. In fact, the formal features of each determine their use, or vice versa; there is no reason to use the wrench on the bolt, except for this formal relationship. Likewise, film as a tool must have fit the object, task, and system of which it was a part. This fit was variable, because any given technology as complex as motion pictures is not a single thing but multiply adaptable to various agendas, so the relationship between them might be successful or not. The relationship was shaped discursively, too: the convergence between a technology and a discipline depended on the successful appropriation of the technology but also on the successful preparation of the discipline or agenda for that technology. If wrench and bolt are designed together from the start, the mutual shaping of film and discipline happened over time and was just as discursive as practical. That is, as mentioned earlier, the fit between a research task and film often depends on what the agents _think film is_ , as well as the analogical relationship between a technology and a system (such as how the film frame isolates an object in a way similar to how experiments attempt to isolate objects and variables). Film and media studies is especially good at teasing out film-theoretical assumptions and finding formal connections. So a useful question could be: To what extent can the _formal_ features of a technology and its representational products explain the relationship between that technology and the research agenda? This is a useful question for historians of science, because the significance of formal features of their objects of study rarely takes precedence.\n\nMore than a book about Germany, science, aesthetics, or even cinema, then, this is a book about historiography. How should we approach the relationship between a (media) technology and the group or discipline that uses it? This book insists that the formal features of the technology matter. It argues that the disciplinary legitimacy of a technology or the successful appropriation of a technology by an expert group depends on _a correspondence between the logic of the discipline and the formal features of the technology_. In this case, filmic form refers to two manifestations of the image: the projected image and the frames on the celluloid filmstrip. The logic of the discipline refers to its problem-solving protocols, its investigatory methods, and its ideological (in terms of its discipline) assumptions. Medical logic, for example, refers to a method of arriving at a diagnosis, etiology, prognosis, and treatment of a disease. It is a way of thinking that provides for a (more or less) consistent and balanced understanding of what is peculiar to the individual case and what is generalizable from it. As we shall see in chapter 2, this logic relied on series of cases for training and context. We will also see that the ambidexterity of film, its ability to be useful in both its still and moving forms, corresponded in unique ways to this medical logic and observational practice. The goal of that chapter, then, is in part to sketch the correlation between a \"medical way of thinking,\" as philosopher of science Ludwik Fleck put it, and the specific formal features of film. Likewise, in some scientific disciplines, the ability of film to frame and isolate the object under study and film's temporal malleability (its ability to expand or compress time) were analogous to features of scientific experiments, such as the ability to isolate variables and extend observational duration. In others, film's temporal discontinuity, indicated by the gap between film frames, matched theoretical ideals about the physical world. For education and aesthetics, the logic of the discipline was less about solving problems than about describing goals or mental operations to attain those goals. In education, the consonance between the detail and duration of the moving image and the richness of the natural world created a homology between the (perceived) realism of the image and the goal of visual instruction, which was to teach students to recognize objects and generalize from them. Aesthetics describes the terms of artistic production and, especially pertinent to this project, the conditions of aesthetic experience; it describes the set of presumed cognitive and emotional operations that accompany aesthetic experience, along with their ideological significance. With regard to film, then, the formal features of the object and the accompanying experience were compared with the prevailing understanding of aesthetic experience. In this case, the pace of the moving image worked against ideals of free will and agency embedded in common conceptions of the aesthetic experience, while the ability of the moving image to encourage emotional projection corresponded to different theories of aesthetic experience popular at the time.\n\nFrame, gap, detail, duration, and pace: each discipline or group, then, saw something useful (or counterproductive, as the case may be) about filmic form but emphasized different features for different goals. In the early period, as motion picture technology emerged as a real possibility for various applications, individual researchers, teachers, reformers, or cultural pundits took it upon themselves to justify motion pictures as a tool or good object to their colleagues. They endeavored to demonstrate that motion pictures could indeed conform to the logic and practices of the discipline. This is why the early period of film's cultural dissemination is so productive for this kind of project: the reasons for appropriation\u2014what the champions thought film was\u2014are often clearly stated, before the use of motion pictures becomes naturalized and obvious, requiring no justification at all (which is the case today when scientists, for example, use moving images as a matter of course without discussion). This is also why this project is less concerned about individual films than the contours of the discussion about the use of film in general; close analysis of filmic style can tell us very little about the justifications mounted for film or the correlation between disciplinary logic, practice, and film form. Such rationalizations can tell us much, however, about the state of the discipline at the time. Whether the use of film addressed common representational problems within a discipline (as in the case of cell biology at the turn of the century) or the discipline faced larger philosophical problems that film seemed to exemplify (as in the case of fin de si\u00e8cle aesthetics), the engagement with motion pictures tells us quite a lot about the priorities and changes that faced any given discipline. Again, this is especially the case during film's early period, when groups latched onto this new technology because of a pressing need or because of its prevalence. In other words, the timing of these appropriations is not coincidental: each group needed motion pictures in some way\u2014even as a scapegoat, which was often the case. Within any given discipline, these maneuvers were far from unanimously approved; dissent was more common in the cultural than scientific spheres, but the debates sharpen our understanding of what was at stake for each group.\n\nWhat was at stake exactly? As a primarily visual technology, motion pictures presented an aid or a challenge to _expert modes of viewing_ , which were the most common manifestation of disciplinary logic and practice. Around what practices, ideals, and \"ways of thinking\" do disciplines coalesce? There are many answers, ranging from laboratory procedures to nationalist rhetoric. But a very common way to train new members of a discipline is to teach them what to look for and how to look for it. Ludwik Fleck insisted that \"one has first to learn to look in order to be able to see that which forms the basis of the given discipline,\" but I would further insist that \"learning to look\" _is_ what often forms the basis of a discipline. Whether it is medical observation or aesthetic contemplation, disciplines have ways of looking that are assumed; if you have to ask what it is or how to do it, you obviously are not part of that group. It is a badge of honor or a barrier to entry. Scientific and medical disciplines, for example, invested much time and energy into training recruits to their modes of viewing, to what Fleck called \"the directed readiness to certain perceptions.\" British surgeon Sir James Paget emphasized in 1887 that \"becoming scientific in our profession [requires] the training of the mind in the power and habit of accurately observing facts.... The main thing for progress and for self-improvement is accurate observation.\" Likewise, Swiss pedagogue Johann Heinrich Pestalozzi organized his entire, influential educational program around close examination; he believed that direct perception of the world was \" _the foundation of all knowledge_.\" And perhaps it goes without saying that aesthetic experience, as expounded by German philosophers since Immanuel Kant, relied heavily on a mode of viewing that was leisurely, probing, and attentive\u2014in a word, contemplative. Above all, these disciplinary modes of looking are more than individual, as Fleck notes: \"We look with our own eyes, but we see with the eyes of the collective body, we see the forms whose sense and range of permissible transpositions is created by the collective body.\" So expert modes of vision and disciplines orbit each other closely and inextricably, held together by ideological bonds.\n\nThis is not to say there is only one mode of viewing for each discipline. Indeed, there are two obvious examples of different modes of expert viewing that obtain regardless of discipline: the holistic, all-at-once, instant appraisal; and the roaming, penetrating, leisurely, detail-oriented contemplation of the object. These two modes can be found in a range of disciplines from scientific observation to medical observation to aesthetics. In art history, for example, Robert Vischer distinguished between _Sehen_ and _Schauen_ , two modes of viewing artworks, which can be translated as \"seeing\" (by which he meant the synthetic, intuitive, instant appraisal) and \"scanning\" (by which he meant analytic, detail-oriented contemplation). These correspond roughly to our \"glance\" and \"gaze,\" as long as they are not confused with their current alignment in media studies with distraction and contemplation. For Vischer and experts across disciplines, glance and gaze were complementary modes of viewing, both requiring expertise. In medicine, physicians in turn-of-the-century Germany who were troubled by the increasingly scientific approach to healing\u2014represented in their minds by the analytic, focused gaze\u2014advocated a more holistic view that took in the entire patient at glance rather than a penetrating gaze that examined only the localized disease in detail. Even if in this case these modes of viewing were taken up as flags symbolizing different professional stances, in practice medical training encompassed both modes. An expert both synthesizes and analyzes.\n\nIn fact, as Michel Foucault has noted about medical observation, these modes of expert viewing were more than merely visual\u2014they were also a set of logical operations. Specifically, I find that expert viewing is largely a process of _correlation_. What happens during expert observation? The expert sees the parts and the whole and finds patterns between them and correlates those patterns to expert knowledge of the disciplinary context. Scientific observation, for example, connotes an analytic gaze, but the most important aspect of scientific observation is the context the scientist brings to it; the researcher assimilates observed data into an existing framework of knowledge. What the scientific observer already knows frames what he or she observes, and he or she incorporates or juxtaposes new data with old and thereby generates new insights. Observation therefore implies _the production of knowledge through correlative insight_. This is true even with art, in which the lynchpin of post-Kantian conceptions of aesthetic experience is the \"free play of associations.\" As the expert gazes upon the artwork, the parts work together in pleasing ways to prompt in the viewer a correlation of textual and contextual patterns. As these associations come together, interlock, separate, and recombine with others, the viewer supposedly experiences a pleasant, even moving state of being between artwork, world, and his or her own subjectivity\u2014all of which has been known as aesthetic experience, which depends fundamentally on a particular kind of viewing that is leisurely, active, attentive, and _correlative_.\n\nEspecially with regard to aesthetic contemplation, expert viewing was not only a disciplinary practice or a logical operation, it was also an exhortation. That is, expert viewing involved a measure of training and hence status, so it also often implied a way of looking at the world or an ideological stance. Immanuel Kant's and Friedrich Schiller's philosophies, for example, gave aesthetic experience a prominent ethical and moral position, in that it was both a mode of engaging with art and a way of bridging an impasse between competing forms of knowledge (Kant) or between conflicting human impulses (Schiller). Schiller's system, especially, suggested that aesthetic experience could lead the way to a better social organization, if only we applied this mode of engaging with art to our way of engaging with the world. Arthur Schopenhauer was even more insistent: aesthetic experience, for him, was one of the few ways by which we could escape the dreary consequences of our everyday impulses, and therefore in his system it became the fundamental model for interacting with the world in general. In the twentieth century, the discussion about the relative merits of contemplation and distraction was, as we know, highly politically charged precisely because of their implications for how one should conduct oneself; recall, for example, Georg Luk\u00e1cs's dismissal of the contemplative life as unconscionable in the face of changes that called for political action and initiative. Even scientific observation became a global ideal as \"objectivity\" came to mean more than refraining from theoretical speculation. I am not suggesting that these systems work or that they are universal. I am arguing that the operations and stances required by these expert modes of viewing also functioned widely as rhetorical instruments, as presumptions about how one should engage with the world.\n\nHow did motion pictures fit into these traditions of expert viewing? That is exactly the question the present project explores. If my larger argument claims that the legitimacy or success of a media technology depended on a correspondence between its formal features and the logic of the discipline, then the more specific argument holds that a discipline's expert viewing ideals and practices exemplified its logic and that _a discipline_ ' _s successful appropriation of film hinged on its ability to accommodate the new technology to its mode of expert viewing_. To put it another way, the acceptance of film as an appropriate tool or good object within a discipline depended on an alignment between the formal features of the filmic image and the practices and ideals of that discipline's modes of expert viewing. The acceptance of film as a tool rested in part on the advocate's ability to adapt\u2014physically and\/or rhetorically\u2014motion pictures to established modes of viewing. If proponents could prove, either through example or argument, that the filmic image could accommodate or even amplify established methods of observation, then the community accepted the justification and elaborated on it in subsequent literature.\n\nFor example, when teachers needed to justify the educational use of motion pictures, they aligned its formal features to already established conceptions of expert viewing and visual instruction; in Germany, these conceptions were encapsulated by the term _Anschauungsunterricht_ , which translates roughly as \"visual means of instruction.\" Their justifications were persuasive to the extent that certain features of film could match or accommodate the methods of this means of instruction. When cultural pundits argued against the idea that film could be art, their arguments were less often about its mechanical reproduction of nature and more about the relationship between the projected moving image and the possibility of aesthetic contemplation. In each case, the question was, can film be used in a manner familiar to our way of viewing and our way of viewing the world? Motion pictures also famously offered a view _different_ from what individual viewers or disciplines were accustomed to. My argument about the correspondence between film and disciplinary logic accommodates this difference as well, because historically the tug-of-war between the familiar and the novel stretched across the literature in a given field; advocates of film technology came to it because it was new and different, yet understood it in terms of what had already come before. Indeed, any given experimental system or discipline moves forward in the same way: methods, technologies, and inscriptions generate new insights that are folded into the existing system, which is thereby subtly changed. In other words, the patterns of justification and dissemination of this new media technology, like that of knowledge itself, were incremental and correlative.\n\nThis implies that expert vision is not the only criterion by which disciplines judged motion picture technology. A survey of the literature in each of these fields reveals that the advantages of film for their projects were often expressed in two ways: in terms of inscription (what the camera can record) and reception (how the image can be viewed). Chapter 1, unlike the rest of the chapters, will focus on inscription. It will examine how researchers adapted motion pictures as an inscription technology to their objects of study, their theories, and their disciplinary needs. The following chapters will explore how experts adapted motion pictures to their different modes of viewing: medical observation, visual methods of instruction, and aesthetic contemplation.\n\nNeedless to say, expert viewing requires training that derives from and reinforces a community. Whether that community is a scientific discipline or an educated class of citizens or both, training to partake of the privileges of that group often came in the form of _visual_ training as well as education in the relevant literature or canon. Sometimes that training was explicit, as in _Anschauungsunterricht_ , which was specifically designed to teach children how to observe; this was not considered disciplinary training so much as one criterion for good citizenship and cultivation. Sometimes it was implicit, as in aesthetic contemplation (although the German tradition of _Bildbetrachtung_ , or \"image viewing,\" explicitly instilled principles of looking, art appreciation, and aesthetic contemplation). In any case, experts and their apprentices came together to form a community (disciplinary, class based, national) through shared values and practices, especially practices of viewing. These bonds were shaped not only by the training and values themselves but also by comparison with their opposite: the untrained, \"valueless\" spectator. Indeed, the values of a group were often not explicitly stated except through reference to what was unacceptable. At stake was the proper method of processing visual information to achieve the desired end, yet the idea of _proper_ viewing was articulated equally often, if not more often, via denunciations of _improper_ viewing. The most obvious example, described in chapter 2, would be the medical experts' description of film audiences as passive, agog, and addicted, whereas their own practices of film viewing emphasized activity, detachment, and control. Yet the latter model was rarely explicitly stated. Instead, we must surmise that \"proper\" viewing model from descriptions of their practice as well as their explicit denunciations of \"improper\" viewing.\n\nIt is hardly surprising that a community of privileged white men in imperial Germany would condescend to audiences of a perceived lower-class amusement such as the movies. Yet perhaps we miss something if we leave it at that. Our understanding of the discursive construction of film spectatorship too often ignores the obvious _duality_ of these constructions. If \"proper\" modes of viewing relied on \"improper\" modes as a useful foil, then the opposite is also true: our understanding of spectatorship is incomplete without an understanding of expert observation. Observation and spectatorship depended on each other (perhaps still) in a figure\/ground relationship wherein the dominance of any given term was often difficult to determine. Spectatorship and observation functioned as each other's \"negative space,\" the outline of one shaping the other by default. This book contends that the \"shape of spectatorship\" can be truly determined only by simultaneously examining the \"shape of observation.\" Observation and spectatorship function akin to stillness and movement or analysis and synthesis: they are oscillating, dynamic dichotomies that are not opposites so much as necessary halves of a hermeneutic process. In this case the hermeneutic is one of self-identity; as experts worked to define their position with regard to disciplinary viewing, \"spectatorship\" was something of a by-product of the work process. More precisely, the work of (explicitly or implicitly) defining spectatorship or observation resulted in a coproduction of the opposite term in a strikingly consistent logic of the supplement. So yes, we know how privileged white men characterized spectators, and we have known that since audiences were aligned with crowds in discussions of early theater. But if we want to know _why_ any given characterization of spectators took its particular form, then we should be aware of the modes of viewing preferred _by the experts writing the description_.\n\nIf theories of film spectatorship in the 1970s and 1980s were preoccupied with the ideological dominance of Hollywood, then the historiographic innovations of film studies during the 1980s and 1990s demonstrated that the Hollywood model was, of course, not always dominant in production and reception; the paradigm of early cinema was not simply Hollywood in nuce but a positive model in its own right: these viewers saw film differently, and early film production and exhibition was heterogeneous. This heterogeneity also implied multiple types of textual address and hence multiple kinds of spectator in terms of gender, race, ethnicity, and class. So if the transitional era codified and standardized filmic address, then it also in a way standardized the spectator; the individual spectator of 1970s film theory was, then, an abstraction generated by filmmaking practice of the Hollywood era, just as the spectator of 1990s film history is a product of textual analysis of early film, or the latest theory of film spectatorship might be a derivation from journalistic accounts of audience experience or of the theorist's own experience. Theories of film spectatorship derive from either filmic textual address or written textual address, and these derivations are separate from historical, empirical viewers and audiences. While contemporary written discussions of viewers and audiences may seem closer to the historical viewer, the written account also produces an abstract notion of spectatorship. My point is that we must take into account the role and interest of the expert in the abstraction. We need to recognize our own expertise and role in producing spectators, but we also need to acknowledge the _disciplinary_ context of historical experts as they wrote about observation and spectatorship. If histories of early film helped to establish the multiplicity of film spectatorship through the categories of gender, race, ethnicity, and class, I would like to add one more category, expert\/lay, which seems a powerful determinate of the character of written discussions of audiences and of disciplinary modes of viewing. Expert\/lay appears closely aligned with high\/low class differences, but I would argue that it is more specific, because educational or disciplinary training provides a traceable, historiographically clear path to the fount of class-based distinctions.\n\nYet as Walter Benjamin argued, the line between expert observer and lay spectator was often fuzzy, especially as film viewers adopted \"an attitude of expert appraisal.\" Benjamin described a mode of reception in which the reactions of the individual were simultaneously amplified and regulated by the reactions of the mass, thereby giving the spectator the pleasure of viewing but also the detachment of the expert. Similarly, chapter 2 describes an expert mode of reception that was also simultaneously distanced from and thrilled by the moving image. Hence the dichotomy between expert and lay is heuristic; most often it was very fluid in practice, but the descriptions of spectatorship often functioned to chart a perceived difference that was helpful for self-identity or class identity. The significance of such descriptions has implications for the \"modernity thesis\" debate, which pivots on the validity of generalizing Benjamin's claim\u2014that our \"mode of perception\" changes over historical periods\u2014to questions of film history and style. That is, can we claim that any film style of a particular era expressed the \"mode of perception\" of the time? This debate has often been preoccupied with details of film style during the early period, or with periodization, or with the question of whether cognitive structures really change, but too often the debate has overlooked the obvious: experts of the time described the cinematic experience as precisely an expression of the state of urban life and as a reflection of the modern mode of perception. Writing about film was a primary means by which modern problems (of representation, observation, education, aesthetic standards) were articulated. In other words, Benjamin's claim did not come out of a blue sky: writers in the 1910s and 1920s described film in exactly these terms, so trying to _explain_ those historical descriptions by constructing a homology between filmic style and modern urban experience\u2014without overgeneralizing to include all films at all times during this period\u2014is not only valid, but logical.\n\nSo descriptions of the cinematic experience in Germany before World War I often functioned as a means to articulate modern problems of representation, observation, education, or aesthetic standards. To describe the experience of attending a film screening was to work through questions associated with modern urban upheaval and transformation. It was so in many places during the early period, when cinema was not really \"cinema\" but a heterogeneous mix of screen practices and ideals\u2014one of many \"cultural series,\" in Andr\u00e9 Gaudreault's term. In Germany, however, the lines between expert and lay were perhaps more starkly drawn (hence more visible) than elsewhere. At the same time, the deeply ambivalent character of discourse during this time of transition, as Germany struggled to balance rapid modernization with traditional values, makes it an especially interesting case study for the mutual construction of expert observation and lay spectatorship. Historians of Wilhelmine Germany have long recognized the dichotomous yet strikingly fluid relationship between left and right political positions, progressive and reactionary attitudes toward the role of the state, male and female roles in public service, or middle- and working-class positions on the social hierarchy. The same ambivalence is evident in the reactions to new media forms such as motion pictures, which makes it an intriguing and challenging case from which we can perhaps infer broader principles about the mechanism of cultural legitimacy. Of course, arguments for and against the use of motion pictures in Germany at this time are similar, perhaps identical, to arguments for and against in other countries. Yet every agent marshals evidence and rhetorical weight from his or her native tradition in support of his or her argument; the Germans were no different. So this book straddles the line between a specialist study on early cinema or German culture and a broader investigation of the nature of expert appropriations of new technologies. The mixture of these two goals differentiates it sufficiently, I hope, from other works on the discourse on early cinema in Germany or elsewhere.\n\nThis project therefore takes up the dual challenge of describing _how_ experts cleared a cultural space for motion picture technology while explaining _why_ that work (rhetorical or practical) took the form it did. Both questions require not just primary research into specific appropriations but an understanding of the disciplinary expectations and needs faced by the experts. So each chapter will explore the _history_ of that discipline (such as biology) by also using the _method_ of a corresponding discipline (such as the history of science). Each chapter, then, is intended to be a hybrid between the methods of film studies and those of another discipline. Explaining why the work of appropriation took the form that it did also requires an understanding of the historical and cultural context of the experts, in this case imperial Germany. So each chapter also attempts to provide a social context, but because arguments for and against carried the weight of past traditions and values, a detailed philosophical context is sometimes required as well. For example, to understand fully the visceral reaction to film's perceived accelerated pace, we will need to examine that reaction's relationship to Schiller's temporal understanding of aesthetic experience, which was accepted by experts and the middle class implicitly. Over time, philosophical ideas sometimes spread and harden into ideological dogma.\n\nI should emphasize that this project focuses on the question of cultural legitimacy or the acceptance of a new media technology by a given discipline at a historical moment. It therefore concentrates on the _correspondence between_ film form and discipline, rather than the _impact_ of form _on_ that discipline. Each chapter charts the relationship between the material or form of cinema (which may include the celluloid image, the projected image, the apparatus, or the venue) and the logic of a discipline by demonstrating how experts conformed that material to their disciplinary agenda. Charting this relationship synchronically is, I believe, preliminary to surveying the diachronic impact of film on any given discipline's understanding of its object of study. That is, any determination of the influence of moving images on a discipline's agenda or conception of its object would require, in my opinion, an examination of the changes in the discipline over time. An accumulation of case studies on a particular topic in which motion pictures were vital would be necessary to determine the change in research questions and the extent to which moving images shaped those questions. The conclusion hints at what such a diachronic or longitudinal study might look like, but the chapters ahead focus instead on the criteria for acceptance (or refusal) of film within each expert group, rather than on any transformation in that group's thinking as a result of using film. I believe we need first to clarify the pertinent elements of film form and disciplinary logic and their potential points of contact before we can argue for the impact of film form on that logic, if any.\n\nChapter 1 examines the use of chronophotography and motion picture technology for scientific research in the fields of human motion studies, physics, and biology. After a survey of early scientific filmmaking, the chapter leverages Henri Bergson's critique of modern science's \"cinematographical thinking\" to underscore the philosophical affinity between film and scientific method. Given that \"science\" is just as varied as \"film,\" however, the chapter finds that different disciplines used motion picture technology for different reasons and in ways that emphasized film's different formal features. In these case studies, the filmic material consisted of the chronophotographic image, the projected image, and the technological apparatus, all of which were adjusted to conform to various elements and processes of scientific inquiry. Accordingly, the chapter examines how film became evidence in relation to the discipline's object of study, its theories, and its representational options. This chapter does not focus on observation but emphasizes experiment instead.\n\nChapter 2 continues this investigation of film for research purposes but extends the argument to the field of medicine. The chapter provides an overview of early medical filmmaking that emphasizes not the different uses by different specialties but instead the common functions governing the use of film technology, such as the use of film to explore, document, or educate. Any given film functioned multiply, depending on who was using it for what purpose at the time; this rubric allows us to glimpse research films in tandem with their other uses. The chapter then explores the relationship between the formal features of film technology and expert medical observation. The use of motion pictures as both still and moving images\u2014the way researchers described their viewing of and extraction of data from these images\u2014subtly expressed the researchers' mode of observation. In this case, the chapter explores the similarity between a \"medical way of thinking\"\u2014its disciplinary logic\u2014and how investigators took advantage of the unique features of film technology, especially the celluloid image and the projected image. It also underlines the ideological investment in that mode of viewing, which was a sign of training and disciplinary allegiance. That investment explains diagnoses of film spectators that accompanied the general medicalization of society during the Wilhelmine period. That physicians dismissed movies in their mass cultural incarnation but lauded their use in the laboratory is best explained through the dual, mutually dependent character of observation and spectatorship.\n\nChapter 2 functions as a pivot between the specialized, rarified use of motion pictures in the laboratory (which was more common than we might think) and the emergence of film in the public sphere. If physicians diagnosed the cultural ills of a nation, reformers of all sorts took to the streets to carry out the treatment. Chapter 3 surveys the film reform movement of the early period and its relationship to larger reform movements, especially those that emphasized a renewal of cultural priorities through education. In this case, then, the filmic material was the cinema venue itself as reformers and educators attempted to mold exhibitor and spectator habits to certain educational standards or aspirations. The art education movement was especially pertinent for efforts to \"ennoble\" cinema and its audiences through an aesthetic education. The history of German pedagogy, moreover, reveals a deep investment increasing over the nineteenth century in a mode of visual instruction known as _Anschauungsunterricht_ , which spread quickly across Europe and the United States, where it was known in English as \"the object lesson.\" Film reformers such as Hermann Lemke worked hard to fashion film screenings for educational use that would conform to this method of visual instruction and observational training. Reformers such as Hermann H\u00e4fker, on the other hand, created film presentations that attempted to conform to models of aesthetic contemplation, which were not incompatible with these educational and ideological goals.\n\nH\u00e4fker's efforts to create a film screening that would make any middle-class aesthete proud speak to the perceived importance of aesthetic values for unifying German culture and ameliorating its social ills. Chapter 4 argues that the explosion of writings about cinema around 1912, which Anton Kaes called the _Kino-Debatte_ , was not only about the danger cinema presented to literary values and territory, as has long been argued, but also about the relationship between the cinematic experience and aesthetic reception in general. How could film be considered an aesthetic object? What aspects of film viewing fit or not with established conceptions of aesthetic contemplation? It is something of a critical commonplace that the advent of mass reception, of which the filmic experience was emblematic, prompted a change in the standards of \"traditional aesthetics.\" This chapter examines the validity of that claim by demonstrating what was precisely at stake in aesthetic contemplation as it was usually understood, and what exactly about the cinematic experience presented a challenge. So this chapter divides the ideological components of aesthetic contemplation into four main areas\u2014issues of agency, identity, time, and space\u2014to show where the objections to and applause for film borrowed from \"traditional aesthetics\" and where they diverged. Furthermore, the chapter shows that professional aesthetics of the day\u2014as encapsulated in discussions of _Einf\u00fchlung_ , or emotional projection during the aesthetic experience\u2014was wrestling with some of the same issues that challenged traditional aesthetics, such as emotional projection, synesthesia, and movement, even while descriptions of the cinematic experience also touched upon these issues. The _Kino-Debatte_ , then, anticipated the theoretical proclamations of the 1920s and 1930s that declared contemplation inadequate for modern living. Chapter 4 explicates the precise nature of the change in preference from contemplation to distraction and suggests that the _Kino-Debatte_ was an important turning point for this transition. Chapter 4 therefore focuses on how film's projected image could be discursively managed to fit (or not) into the logic of aesthetic contemplation. Finally, the conclusion emphasizes the importance of disciplinary context and expert\/lay distinctions for histories of nontheatrical film and suggests a historiographic approach that highlights how experts \"handled\" motion picture images, technologies, and audiences\u2014and the impressions those efforts left on their own institutions and on the history of cinema.\n\nEach chapter functions more or less on its own, but together I hope they will add to the conversation about nontheatrical film, spectatorship, and interdisciplinary work in film and media studies. Indeed, each chapter demonstrates that the appropriation of motion pictures was a series of constant adjustments between writing and reading, or between inscription and reception. Looking at film in this way, we can begin to explore how cinema fit into a cultural space, the work that went into making it fit, and what that work tells us about the people and institutions who did the work. In other words, how experts managed to call film their own while using it to manage the scientific and social problems they faced is the subject of this book.\n\nSCIENCE'S CINEMATIC METHOD\n\nMOTION PICTURES AND SCIENTIFIC RESEARCH\n\nTime would flee, I subdue it.\n\n\u2014CHARLES CROS (1877)\n\nIn the early 1890s, after \u00c9tienne-Jules Marey's successes with chronophotography ensured the continued viability of his \"graphic method\" of recording motion, two German physiologists, Wilhelm Braune and Otto Fischer, set out to improve upon Marey's techniques for the study of human locomotion. No mere dilettantes, Braune and Fischer were already well known in their field for studies of the human center of gravity and other investigations into the fundamentals of human motion. From 1889 to 1904 they published a series of studies of human locomotion, culminating in their landmark work, _Der Gang des Menschen_ (The Human Gait). In their Leipzig laboratory, they dressed their experimental subject, a military recruit, in a black jersey and attached to him an elaborate mechanical scaffolding of incandescent tubes designed to illuminate his stride with short, intensely bright bursts of light (fig. 1.1). They then photographed the subject as he walked, as comfortably as could be expected, across the darkened room. As he walked, the tubes fired, producing a strobe effect that was recorded through the camera's open shutter. The resulting image\u2014a series of white lines across a black background\u2014became the basis for hundreds of calculations concerning the specific mechanics of human motion (fig. 1.4). Interested in the economy of the laboring body\u2014its most efficient conservation and expenditure of energy\u2014they hoped their scientific analysis of human movement would lead eventually to a more efficient and productive society (or, at least, a more efficient soldier). Likewise, their improvements over Marey's system of photographic measurement led to a much more analyzable and therefore \"productive\" chronophotographic image. After Braune and Fischer, photographs became even more efficient and productive tools of scientific research.\n\nIn Marburg in 1907, a young physicist named Max Seddig presented his dissertation on \"Measurement of the Temperature Dependency of Brownian Motion.\" In an attempt to clarify the arguments for and against the atomic\u2013kinetic model of heat, while also trying to provide experimental confirmation of Albert Einstein's theories of Brownian motion, Seddig fashioned a device that could record chronophotographic images of microscopic particles affected by molecular activity. Seddig's microscope\u2013cinematograph combination supplied an objective record of Brownian motion from which he could calculate the velocity of these particles. Yet it was not the objective record that Seddig emphasized and praised but the machine's ability to measure time intervals precisely. Alternatively, Heidelberg biologist Hermann Braus presented in 1911 the results of his application of a similar microscope\u2013cinematograph combination to record and explore the growth of a tissue culture of a frog's heart. Using time-lapse cinematography, Braus sought to demonstrate that the culture actually grew rather than merely survived outside the organism. Unlike Seddig, he was not so concerned with measurement but with the temporal record of the event. With his motion picture record Braus was able to challenge competing claims about the growth of nerve cells.\n\nThese three cases\u2014from human motion studies, physics, and biology, respectively\u2014represent a fair sampling of the scientific use of film for research purposes in Germany before 1914. From these case studies we can draw some preliminary conclusions about the practical and philosophical connections between film and science. We might also be tempted, of course, to make general theoretical claims about the nature of cinema's relationship to modern science and temporality. But if we are to understand film's role in modern scientific inquiry, we must temper expansive claims with more historically localized analyses of how film technology was actually _used_. What \"film\" meant to any given scientist depended very little on a theoretical conception of cinema in general; instead it depended primarily on how some specific incarnations of motion picture technology could be applied to the specific and historically contingent problems the scientist faced in his or her discipline. Hence any study of scientific research films must incorporate methods common to both the history of science and film history by investigating the manner in which research questions and media technology mutually influenced each other. What questions does a given discipline privilege at a given time? How do those questions shape\u2014indeed, how are they shaped by\u2014experimental design and available instruments? The appropriation of motion picture technology as a scientific research tool, its specific use within the laboratory, reveals the researcher's assumptions about that which the camera is designed to capture. And these assumptions vary as widely as the different uses of film. But rather than making broad claims about what \"science\" or \"cinema\" is, thereby concealing this variety under top-down theoretical categories, we should let individual experiments reveal their assumptions and make our generalizations from those, if necessary. Design and deployment are themselves implicit theoretical statements.\n\nIf the cinematograph were an especially flexible tool that could be adapted to a number of different needs, these needs existed in the first place because of changes in the sciences themselves at the turn of the century. Biologists interested in cell development, for example, searched for new modes of visualization that would allow researchers a clear view of movement in time to resolve some heated disputes about the nature of cell growth; the techniques of tissue culture, on one hand, and those of motion pictures, on the other, offered two kinds of solutions, as we shall see. Physicists, too, looked to cinematography as a tool for investigating previously invisible phenomena, such as the effects of molecular movement, a topic of debates about the behavior of atomic phenomena. Seddig's use of chronophotography and motion pictures reflects a common application of this technology in scientific research; while scientists admired cinematography's ability to capture fleeting phenomena, they prized most highly film's ability to decompose the event into discrete, regular units, which permitted measurement of its temporal and spatial components. Indeed, this particular use of motion picture technology, especially in the physical sciences, betrays an assumption about the event or phenomenon under study as itself discrete, divisible, determined by classical laws, regular, and\u2014just like the filmstrip\u2014reversible. Seddig's use of motion pictures therefore demonstrates at once the value of film for scientific research and his (and Einstein's) commitment to a particular understanding of the relationships between movement, time, and space\u2014an understanding that French philosopher Henri Bergson was criticizing at precisely this moment in _Creative Evolution_ , his 1907 landmark study of the philosophy of biology. In short, as scientific disciplines reconceptualized the nature of matter, time, space, and the organism, they seized upon tools that could visualize these phenomena in accordance with these new concepts.\n\nBut film has never been just a convenient device, patiently waiting on the shelf as the scientist thinks up a new use for it as a solution to a new problem. Its availability and its existence also generated questions. Investigators' interest in temporal phenomena was in part spurred by the arrival of a machine that could make the phenomena amenable for analysis. Furthermore, those scientific research programs that featured film technology were not only shaped by that technology, but their scientific method itself had certain features in common with the filmic apparatus. Scientific experiment shares with motion pictures an impulse to record immediately and directly, a willingness to manipulate time, and an inclination to isolate and quantify phenomena. There are good practical reasons for using motion picture technology, of course. But in the late nineteenth century there developed also a _philosophical_ affinity between science and film that went beyond mere convenience. Cinema and science have come to share a certain way of thinking, so the history of the application of motion pictures in science can offer us a valuable opportunity to explore the relationship between science and modernity. Bergson was the first to suggest that science, through its parsing of continuous movement into discontinuous moments, proceeds in a way analogous to cinematography. If we are to understand fully the implications of the appropriation of motion picture technology by the scientific community, we must maintain a balance between the theoretical and the practical by considering this philosophical affinity alongside the way investigators put film to work in the laboratory. Bergson's conception of science as inherently \"cinematic\" offers us a logical point of departure for such considerations. True enough, Bergson was not as popular in Germany as he was in France and the United States. But because I am interested in his thoughts on cinema and science in general\u2014and not in his thoughts (if any) on particular applications in Germany or elsewhere\u2014his historical impact on German culture is actually not relevant to my approach. Bergson's theoretical framework can help us answer the question \"What did cinema and science see in each other at this particular moment?\" The individual case studies can reveal _how_ film was used; reading Bergson alongside these cases can help to reveal _why_ film was used. This chapter, then, will survey the use of motion pictures in the three case studies mentioned above.\n\nLet me first reiterate the role of this chapter in the book's overall goals. As I mentioned in the introduction, the larger argument (the \"general theory,\" if I may) concerns the correspondence and mutual accommodation between the logic of a discipline\u2014its problem-solving patterns, its investigatory methods, its ideological assumptions\u2014and film's formal characteristics. This implies not just taking advantage of a medium-specific formal feature, such as temporal malleability (for example, time-lapse cinematography), to solve a representational problem, it also implies a functional or productive homology between this formal feature (for example, the linear regularity of time-lapse recording as a statistical sample of a larger unit of time) and a way of solving problems (the primacy of mathematics, for example) or of viewing the world (as naturally divisible into equal, regular units, for example). This match\u2014between the formal features of the representational technology and the investigatory presumptions\u2014matters, because it provides the researcher, community, or discipline with the reassuring sense that the tool will fit the task to which it is assigned. However, we must note that the match is ideally never perfect; otherwise there would be no new information. Experimental systems are designed to generate new questions, so there should be a dislocation or displacement between the more or less ideological assurance of this tool's \"worldview,\" so to speak, and the strangeness of the view it provides. Time-lapse cinematography can, for example, confirm an understanding of nature as regular and divisible, but it also offers a surprising, even thrilling new image that prompts new questions. The larger argument of this book is that the acceptance of any new (media) technology depends in part on this correspondence between some set of its formal features and the logic of the discipline. The specific argument (the \"special theory\") is that expert vision expresses this disciplinary logic especially well and that film's legitimacy within disciplines depended on its accommodation to expert modes of viewing.\n\nBut expert viewing is not the only way that disciplinary logic is expressed; the experimental system itself is also a manifestation of the discipline's problem-solving patterns and theoretical presumptions. As Gaston Bachelard and others have argued, instruments are \"theories materialized\": the design and deployment of experimental technology carries a preconception or preunderstanding of the phenomenon it is designed to isolate. If we extend this system to include chronophotography or motion picture technology, we can see how the work of accommodating their formal features already made a statement about the relationship between the system and the object of study. Indeed, the work of creating a legible image\u2014that is, understandable and acceptable to the discipline\u2014reveals this relationship between system and object quite clearly. Likewise, the theory guiding the experimental observations is an expression of disciplinary logic. If instruments are theories materialized, then, as Hans-J\u00f6rg Rheinberger has noted, theories are also \"machines idealized.\" Einstein's theory of Brownian motion, for example, made several \"shortcuts\"\u2014including the presumption of molecular two-dimensionality and velocity without direction, as we shall see\u2014to manage the object mathematically and accommodate the phenomenon to both experimental confirmation and a particular disciplinary understanding of nature. Einstein's mathematics, in other words, simultaneously became an \"instrument\" to guide observation _and_ a theoretical rendering of the experimental situation. To offer one more example, disciplinary logic can be expressed through the experimental system's representational options. Film is only one part of an experimental system, of course, and only one in a range of representational tools that includes writings, sketches, graphs, and photographs. Selecting film as part of this media ensemble already implied a certain set of questions or needs. Hermann Braus, for example, found that using film was an especially powerful means of engendering belief among colleagues, while at the same time expressing, through its formal features (such as the duration of the projected image and its temporally forward motion) theoretical presumptions about cell growth. In this case, film and the new technology of tissue culture\u2014also a representational tool\u2014combined to express a new direction in the discipline's visual needs and strategies.\n\nSo the correspondence and accommodation between experimental system or discipline and technology can happen in several ways, depending on the researcher's or discipline's goals and needs, which are locally and historically determined. This chapter will explore the \"general theory\" rather than the \"special theory\" of media technology's disciplinary legitimacy. Specifically, this chapter will focus on three ways in which experimental systems incorporated chronophotographic or motion picture technology, especially on the correspondence and mutual accommodation between a given set of formal characteristics (of photography and\/or film) and (1) an _object of study_ , (2) a _theory_ , and (3) the _representational options_ of an experimental system and discipline. Or, to put it another way, each of these three adaptations required a certain kind and amount of work to make the chronophotographic or filmic image into evidence. A close analysis of Braune and Fischer's method will show how they created evidence in relation to their object of study (in this case, the human body). The discussion of Seddig's experiment stresses the creation of evidence in relation to a theory (Einstein's theory of Brownian motion). And the section on Braus emphasizes the creation of evidence in relation to a set of representational options within a discipline (here, cell biology). Each example deals to some extent with all three adaptations, of course, because they are intertwined, but the emphasis changes. In general, I will demonstrate that while the scientific community readily accepted chronophotography and film as valid instruments, investigators still had to perform considerable labor to adapt these devices to their tasks and to transform the resulting images into acceptable scientific evidence. Motion pictures may have allowed scientists to manage time and movement, but researchers first needed to manage film's temporal rush and excessive detail. The nature of this work was shaped by its historical context. The subject of this chapter is therefore the way that investigators were continuously obliged to adapt as they juggled chronophotographic or motion picture technology, the needs of the individual experiment, the theories shaping the experiment, and the discipline's priorities shaping the representational choices.\n\nWhile observation is not an explicit focus of this chapter, it is inescapable. Braune and Fischer decomposed movement to train expert vision to phenomena that it might not see or have been able to see. Einstein's theory of Brownian motion focused experimenters' attention, showing them what to look for. For Braus, film was an observational tool that forced researchers to abandon previous theories and modes of analysis (which emphasized discontinuity) for an approach that emphasized synthesis and continuity. These case studies demonstrate that, as Ian Hacking has noted, experiment and observation are only heuristically separable. Nevertheless, this chapter emphasizes other ways, besides observation, that disciplines accommodated the formal features of film and chronophotography. Expert vision is the explicit focus of the following chapters.\n\nThe chapter has five sections. It starts with a general overview of scientific research films (as opposed to popular science films) before World War I, in which I outline various prominent applications as well as some hypotheses about how motion pictures fit with the rhetorical goals of scientific enterprise. (A majority of scientific applications of film during the early period were devoted to various medical fields, which I will explore separately in chapter 2.) The next section continues to explore why motion picture technology was intriguing to researchers; it focuses on Bergson's discussion in _Creative Evolution_ of the relationship between cinema and science. In the third section, I place Braune and Fischer's work on human locomotion in the context of both social modernity and the science of work. This section contains a detailed explication of Braune and Fischer's method to show exactly how the merging of apparatus, image, and object actually occurs. The fourth section relates Seddig's work to the general problems of atomistic physics at the turn of the century. In the fifth section, I sketch Braus's cinematic contribution within the context of fin de si\u00e8cle changes in the discipline of cell biology. The concluding paragraphs of this chapter compare the different cases directly to emphasize the mutual dependence between motion picture technology and emerging scientific agendas.\n\nEARLY SCIENTIFIC FILMMAKING: AN OVERVIEW\n\nScientists from a wide variety of disciplines\u2014including but not limited to botany, military engineering, meteorology, neurology, psychology, and medicine, as well as the three already mentioned\u2014were among the earliest users of motion picture technology. Most histories of research films start with Jules Janssen, Eadweard Muybridge, and \u00c9tienne-Jules Marey, who were important transitional figures between scientific photography and motion pictures. The initial adoption of moving images was relatively smooth because so much work had already gone into creating scientifically valid photographic images during the middle decades of the nineteenth century. The slow process by which still photography had been standardized\u2014setting norms for emulsions, exposure times, preparation techniques, image interpretation, and so on\u2014meant that the enthusiasm for photography often collided with rapidly evolving disciplinary requirements for scientific documentation. As Jennifer Tucker and others have shown, photography was not immediately and unconditionally accepted as a completely objective and scientific record. Photography's evidentiary status depended on its ability to meet several criteria of production and reception. Photographs had to withstand cross-examination by experts from any discipline to which they wished to testify; they were as subject to expert judgment as drawings or other illustrations. In Germany, for example, microphotographs were not generally accepted as proper evidence by the scientific community until respected bacteriologist Robert Koch's innovations and rhetorical efforts made \"reading\" photographs of bacteria a truly collective activity among an international group of scientists and physicians. Yet by the 1890s, the protocols for generating acceptable scientific photographs had been established in most disciplines, so the innovation of motion pictures was greeted enthusiastically, their way smoothed by Marey's renowned chronophotographs and graphic inscription methods.\n\nMarey is indeed the most important figure in any history of early scientific film, because his efforts and resources shaped the application of motion pictures to scientific experiment. Marey was intrigued by Muybridge's serial photography when he first encountered it in 1881 but ultimately disappointed in its scientific value: Muybridge's 24-camera, trip-wire method of recording was prone to mechanical inaccuracy and incapable of managing the exact time intervals required for careful research. So at the Coll\u00e8ge de France in the 1880s and 1890s Marey explored photographic and chronophotographic methods for visualizations of movement that accounted for distances and times more precisely. The Institut Marey was founded in 1901 to carry on his work; researchers such as Lucien Bull, Pierre Nogu\u00e8s, and Joachim-L\u00e9on Carvallo continued to explore the relationship between experiment and visualization, especially in the areas of slow and high-speed cinematography and X-ray cinematography. Marey's assistant at the Coll\u00e8ge de France, Charles \u00c9mile Fran\u00e7ois-Franck, continued his work on microcinematography in particular; collaborating with Lucienne Chevroton, Fred Vl\u00e8s, and others, Fran\u00e7ois-Franck published widely on the chronophotographic and cinematographic recording of microscopic and macroscopic movement. These two sites were also magnets for individual researchers searching for novel ways to make visible their objects of study; French biologist Antoine Pizon and Swiss biologist Julius Ries both worked with the team at the Institut Marey to capture visually the process of cell division, for example, while Fran\u00e7ois-Franck helped French physicist Victor Henri with his research into Brownian motion (I will have more to say about all of these examples later in the chapter).\n\nWhether scientific cinema grew out of team efforts focusing on new visualization techniques or out of the work of individual researchers focused on experimental problems that motion pictures might solve, both models had one thing in common: the need for resources. Needless to say, the early motion picture apparatus was expensive, often cumbersome, and usually difficult to adapt. Its use in scientific circles, therefore, was generally restricted to established researchers who had the necessary financial and technical resources at their disposal. Indeed, the distribution of resources largely dictated the spread of motion picture technology in the laboratory. Thanks to Marey's considerable political and scientific skill, France could boast at least two sites with the necessary budget and expertise to pursue such a program. Germany also had a university research infrastructure in place that allowed individual researchers to explore the use of motion picture technology, but no single site dominated. Aside from the many medical applications, which we will examine closely in the next chapter, we can count botanist Wilhelm Pfeffer's time-lapse chronophotography of plant growth at Leipzig University and Carl Cranz's high-speed studies of ballistics at the military academy in Berlin-Charlottenburg as notable intersections of science and film at the university level.\n\nResearch film also received a boost from manufacturers who recognized that lending their equipment and funds provided not only a measure of legitimacy and good press but entertainment for movie-going audiences as well. Path\u00e9 Fr\u00e9r\u00e8s, for example, funded the filmmaking of microcinematographer Jean Comandon and then distributed his films to theaters around the world. Occasionally a German manufacturer would lend a hand to researchers; film pioneer Oskar Messter, for example, had pretensions in this arena, and companies such as Ernemann were sometimes acknowledged as technical patrons. But even researchers who purchased the basic apparatus from a manufacturer such as Lumi\u00e8re or Ernemann were still obliged to make modifications to the equipment in their own laboratory setting. Carl Zeiss's optical laboratory in Jena, for example, seems to have been especially suited to this kind of work. Generally speaking, despite the involvement of some film manufacturers, these films were usually made by specialists to be shown to other specialists at scientific conferences or in the lecture hall.\n\nThis is not to say, however, that scientific films were limited exclusively to an elite audience. Scientific titles were often part of the program at the local movie theater. Companies such as Path\u00e9, Gaumont, and \u00c9clair in France or Urban in England, especially, produced their own series of scientific films for general audiences. German audiences would have been familiar with this genre from the titles imported by these companies. (Foreign manufacturers generally dominated the German exhibition market until the 1910s.) In addition, the German periodical _Film und Lichtbild_ (a German _Popular Science_ for film enthusiasts) acted as something of a clearinghouse and ad hoc distribution company by publishing lists of scientific films and offering discounts to its readers. There were also dedicated screenings occasionally, as well as theaters devoted to the genre, such as the Fata Morgana theater in Dresden, which opened in August 1912 and showed only scientific, industrial, and nonfiction films.\n\nBut what of the research films of a Seddig or a Braus? Did they ever find their way to the public? It is very difficult to say without a complete survey and correlation of all films made in the laboratory with those screened in public or semipublic venues\u2014a task that is likely impossible to complete. There are good reasons for both possible answers, yes and no. On one hand, these films were the result of considerable expense and effort, so investigators might have been reluctant to give them up for duplication. (Comandon and others like him were exceptions, because they made legitimate research films for hire.) Also, some scientists might have hesitated to cross the line between serious research and its popularization. Motion pictures already had acquired the yellow tinge of mass culture, so some investigators were probably reluctant to have their work completely jaundiced by a matinee showing at the local nickelodeon. Not that the scientific application of motion pictures encountered serious objection; while film histories often repeat legends of academic hostility to researchers using motion pictures in their experiments (usually limited to French medical films and the Doyen controversy, described in chapter 2), evidence of such protests is actually rare in comparison with the general enthusiasm displayed for the new technology. On the other hand, research films were the topic of a considerable number of screenings and discussions, and teachers, reformers, and even scientists encouraged these ventures into the popular realm as important efforts at public outreach. Furthermore, there were already a number of screenings of these films in the semipublic realm of the university lecture, so it would not have been too much of a leap to take the next step. And as manufacturers such as Messter and Ernemann lent their equipment and expertise to researchers, they may have asked for copies of the films, which might have been made available for rental in the manufacturer's catalog. It seems that the public screening of any given laboratory film depended on the resources and predilections of the individual researcher or the manufacturer; I have not yet found a consistent conduit in Germany between the scientific laboratory and the movie theater.\n\nBy and large, then, these films served primarily as a form of scientific evidence. Despite its mass culture connotations, its high cost, the high level of technical expertise required, and the often futile results, motion picture technology offered several tempting benefits to the researcher. Like still photography, the motion picture camera provided a mechanical, automatic, hence \"objective\" record, thereby adding substantial evidentiary weight to scientific claims. The photographic image, like other graphic inscription devices (such as the electrocardiograph), seemed to provide researchers with an unmediated and permanent record of any given phenomenon, one that could be stored and disseminated with ease. And because it could be projected and reproduced, the photographic image proved useful for demonstrations as well as experiments; indeed, a motion picture projector was just as likely to be found in a lecture hall as in a laboratory.\n\nUnlike still photography, however, the motion picture had the capacity to record events as they occurred over time. This singular feature offered several advantages. The camera itself could act as a mechanical, indefatigable prosthesis of the scientist's eye, a tireless observer of events that was able to catch the slightest change without the interruption of a blink. Furthermore, motion pictures offered the scientist the option of manipulating time by recording (or projecting) the film at different speeds. Slow-moving events could be sped up with time-lapse techniques; fast-moving phenomena could be slowed down through high-speed cinematography. As a result, temporal events invisible to our ordinary perception became \"visible\"; film became a kind of temporal microscope or telescope, bringing nature's aloof wonders closer to our level. Finally, as implied above, the motion picture camera could also act as a precise measuring tool. By controlling the rate at which the film passed through the camera as the phenomenon traveled a set distance, the scientist could then calculate the speed of the recorded movement. This ability was by far the most intriguing aspect of cinema's scientific potential, and researchers spent considerable energy perfecting it.\n\nMotion picture technology, then, was an especially flexible tool that could be adapted to a number of different tasks. In this respect, however, it is no different than any number of technical innovations adapted for scientific use, from perspective drawing to the computer. Successful adaptation depends on a variety of circumstances, but as sociologist of science Bruno Latour has argued, the most salient predictor of which technologies the scientific community will adopt is the extent to which the adoption will aid the community in rhetorical struggles. Latour maintains that technologies that serve as inscription devices, or \"writing and imaging procedures,\" function rhetorically in debates between authors and groups as tools that help \"in the mustering, the presentation, the increase, the effective alignment, or ensuring the fidelity of new allies.\" Struggles between scientists are the same as those between generals and politicians, Latour argues; those with the most allies win. Therefore, the essential characteristics of any inscription device\u2014the qualities that will ensure its success in the scientific arena\u2014have less to do with its ability to provide accurate inscription (visualization, writing) per se than whether those properties can be put to use in rhetorical struggles. Latour explains:\n\nIn other words, it is not _perception_ which is at stake in this problem of visualization and cognition. New inscriptions, and new ways of perceiving them, are the results of something deeper. If you wish to go out of _your_ way and come back heavily equipped so as to force others to go out of _their_ ways, the main problem to solve is that of _mobilization_. You have to go and to come back _with_ the \"things\" if your moves are not to be wasted. But the \"things\" have to be able to withstand the return trip without withering away. Further requirements: the \"things\" you gathered and displaced have to be presentable all at once to those you want to convince and who did not go there. In sum, you have to invent objects which have the properties of being _mobile_ but also _immutable_ , _presentable_ , _readable_ , and _combinable_ with one another.\n\nMotion picture technology had all of the qualities of this sort of immutable mobile, a good indicator of its success as a scientific instrument. The instrument itself was mobile, but more importantly, so were the films. \"Immutability,\" in Latour's sense, refers to the permanency of both the inscription process and the object or condition represented. Simply, the inscription must be relatively permanent, as films were, while providing a translation without any seeming corruption of the thing represented. The photographic image's necessary physical connection (via light and chemical processes) to the object represented served to guarantee that the object was relatively uncorrupted by the recording process. The films were meant to be projected, so they were of course presentable, but because they were also photographs, they could be presented in a wide variety of ways, as illustrations in journal articles or as lecture slides, for example. In this way, the films could also be combined with other technologies, such as print technology, but the apparatus itself could be combined with others as well, such as the microscope. The \"readability\" or legibility of the technology is the most contentious aspect of any innovation, because the interpretation of new forms of inscription always requires negotiation within a discipline. Experts and innovators haggle over the meaning of signs until standards of production and protocols of interpretation emerge. Generally speaking, however, motion pictures, like photography before them, were considered very legible for scientific purposes.\n\nBERGSON, CINEMA, AND SCIENCE\n\nWithout a doubt, motion pictures presented scientists with new analytic techniques that could manipulate time and space. However, at the same time, science's very mode of analysis was newly subject to debate. At least since the romantics, some German thinkers had come to associate science's empirical, experimental, and materialist method with a cold, mechanistic, and \"disenchanted\" view of the world. As Anne Harrington has argued, the rebellion against mechanistic, reductionist science often consisted of calls for \"wholeness\" in various forms, from Hans Driesch's biological vitalism to Gestalt psychology to Richard Wagner's _Gesamtkunstwerk_. This reaction was not limited to Germany, of course; perhaps the most prominent figurehead of this rebellion was Henri Bergson, whose philosophy\u2014or its popularization\u2014spread like wildfire through the parlors and lecture halls of Europe and the United States in the years before World War I. If film was still a relatively marginal research technology at this time, Bergson's _Creative Evolution_ brought it to the center of the debates about scientific method.\n\n_Creative Evolution_ , which appeared in 1907 and quickly became Bergson's most famous work, was concerned with evolutionary biology in the same manner that his earlier _Matter and Memory_ (1896) was concerned with psychology and his later _Duration and Simultaneity_ (1922) dealt with physics. In _Creative Evolution_ , he took the evolution of life as a fact, but expressed dissatisfaction with scientific explanations of it. According to Bergson, the mechanistic approach to biology perpetuates a common analytic mistake: it divides up organisms and living processes in order to understand their parts, but does not, because of this division, fully understand their total, living reality. Consequently, the analytic, mechanistic, reductionist approach cannot satisfactorily explain change or the creation of new forms or new solutions. Bergson was intent to bind the organism's \"living reality\" to the flow of time. That is, Bergson made the flow of time, which we all experience as incessant and forward moving, the model of life itself. The central concept here is his notion of _dur\u00e9e_ , or \"duration.\" Bergson insisted that we make a category mistake when we divide the continuity of lived experience into the discontinuity of a series of separate points. Bergson reversed the traditional or commonsensical view that change is a succession of states, a view that makes those states logically anterior to change. Bergson's view, instead, was that change is primary and that to analyze change by breaking it into a succession of states misrepresents the true reality of the world, which exists in constant flux, \"becoming,\" or _dur\u00e9e_. Organisms exist in time, in _dur\u00e9e_ , and cannot be separated from it without losing an understanding of their lived reality.\n\nThis same logic applies to the examination of motion. Bergson saw the universe in a constant state of flux; change and movement are the only constants, the only true reality. Matter, form, or solidity are only stable views of this essential instability. Unfortunately, our common, ordinary, analytic perception cannot grasp this flux; it can only extract determinate moments, which we then mistake for an accurate picture of reality. Even our body is not solid but \"is changing form at every moment; or rather, there is no form, since form is immobile and the reality is movement. What is real is the continual _change of_ form: _form is only a snapshot view of a transition_.... [Therefore,] our perception manages to solidify into discontinuous images the fluid continuity of the real\" (328, emphasis in original). It is not so much that this mode of perception is wrong, it is just that it is incomplete and should not be mistaken for a true understanding of the world. Bergson acknowledged that human beings cannot help but think this way, dividing the flow of the world and time into discrete intervals, but he insisted that this habit of thought should be overcome: \"to invert the habitual direction of the work of thought.\" That is, given that analysis or this way of viewing the world is more or less habitual and necessary, Bergson declared that to recognize _dur\u00e9e_ , \"The mind must do violence to itself, has to reverse the direction of the operation by which it habitually thinks, has perpetually to revise, or rather to recast, all its categories.\" Bergson's philosophy, then, was an attempt to save us from our logical errors of thought so that we could see the world in a different way. Specifically, he argued against two category mistakes: mistaking discontinuity for continuity and space for time.\n\nBergson used the example of motion pictures, which consist of a series of still images projected to imitate real movement, to illustrate the relationship between the continuity of _dur\u00e9e_ and the discontinuity of our ordinary, analytic perception. The mechanism of cinema was analogous to, even expressed, our ordinary perceptual process. This process, Bergson elaborated,\n\nconsists in extracting from all the movements peculiar to all the figures an impersonal movement abstract and simple, _movement in general_ , so to speak: we put this into the apparatus, and we reconstitute the individuality of each particular movement by combining this nameless movement with the personal attitudes. Such is the contrivance of the cinematograph. And such is also that of our knowledge.... Whether we would think becoming, or express it, or even perceive it, we hardly do anything else than set a kind of cinematograph inside us. We may therefore sum up what we have been saying in the conclusion that the _mechanism of our ordinary knowledge is of a cinematographical kind_. (332, emphasis in original)\n\nWhat does it mean to \"set a kind of cinematograph inside us\"? It means that when we try to think movement or change, we cannot help but to conceive it first as a series of individual states of being or \"snapshots\" of form. The accumulation of these \"snapshots\" provides us with a conception of movement, a conception as illusory as the movement of the cinematic image at twenty-four frames per second. Thinking of movement or change as a series of states\u2014rather than as a thing in itself, as Bergson urged us to do\u2014forces us to extrapolate movement from the series. With that extrapolation, movement becomes universal, ideal, or \"movement in general.\" We attempt to reconstitute the particularity of the movement by an effort of synthesis, which is what the accumulation of snapshots amounts to. Out of habit, our ordinary way of thinking is cinematic.\n\nIn the same way, modern science \"proceeds according to the cinematographical method,\" in that \"it is the essence of science to handle _signs_ , which it substitutes for the objects themselves\" (357). These signs \"denote a fixed aspect of the reality under an arrested form. To think movement, a constantly renewed effort of the mind is necessary. Signs are made to dispense us with this effort by substituting, for the moving continuity of things, an artificial reconstruction which is its equivalent in practice and has the advantage of being easily handled\" (ibid.). According to Bergson, science is not _really_ able to understand movement per se, movement as continual flux. Yes, it can understand it as the difference between the changes of two stable states, but it cannot grasp the essential dynamism of change. Instead, the scientific approach creates abstractions\u2014the geometric understanding of movement as a line or the mathematical understanding of movement as an equation\u2014to help us grasp that which is in constant motion. For Bergson, the normal scientific approach to movement proceeds by leaps from moment to moment, from arrangement to rearrangement; science may increase the number of moments it isolates \"but it always isolates moments.... It does not bear on the interval, but only on extremities\" (357\u2013358). To the extent that science followed this tendency to divide movement into stable units and to treat the interval as an enabling ellipsis and not as precisely the problem, modern science for Bergson was essentially cinematic.\n\nBergson characterized the history of science in these terms as well. Aristotelian science was also cinematic, but it differed from modern science in the way it broke up time. For the ancients, the time of the movement of a falling body, for example, had certain determinate periods, whose natural articulations, like puberty, presented moments when there occurred the natural release of a new form. For modern science, according to Bergson, time \"has no natural articulations. We can, we ought to, divide it as we please. All moments count. None of them has the right to set itself up as a moment that represents or dominates the others. And, consequently, we know a change only when we are able to determine what it is about at any one of its moments\" (360). Time, then, has become democratic in the modern age; there are no more privileged moments. This democratization marks the difference between the _qualitative_ description of the ancients and the _quantitative_ measurement of the moderns (361). Modern science works only with a view to measure, and it selects as its objects only those phenomena that can be measured. \"It retains only the events or systems of events that can be thus isolated without being made to undergo too profound a deformation, because only these lend themselves to the application of its method. Our physics dates from the day when it was known how to isolate such systems\" (371\u2013372). In the same way, motion picture technology divides temporal events evenly, into a neat twenty-four frames per second, for example. The precise and democratic division of time common to the cinematic apparatus mimics modern science's insistence that no moment be privileged over another.\n\nIn this way, Bergson outlined the deep affinity between cinema and science, one that at least partially explains their immediate mutual attraction. But if Bergson condemned our \"ordinary perception\" and therefore cinema (and scientific method) as incomplete\u2014thereby joining the rebellion against mechanistic science\u2014he nevertheless recognized that cinema had potential beyond its analytic character. In an interview from 1914, Bergson regarded motion pictures somewhat more sympathetically:\n\nSeveral years ago, I went to the cinema. I saw it at its origins. Obviously, this invention, a complement to instant photography, can suggest new ideas to a philosopher. It could be an aid to the synthesis of memory, or even of thought. If the circumference [of a circle] is composed of a series of points, memory is, like cinema, a series of images. Immobile, it is in a neutral state; in movement, it is life itself.\n\nBergson recognized that cinema had the capacity to provide both analysis and synthesis, that it had an inherently ambiguous character precisely because it is both still and moving. Indeed, Bergson's invocation of the cinematographic apparatus was never an outright condemnation of the cinema but instead a way of describing a tendency in our habitual, everyday way of thinking that science had codified into experimental method. If the point of his philosophical project was to \"reverse\" this way of thinking toward a truer, less alienating view of the world\u2014toward an embrace of \"intuition\"\u2014then there was also the possibility that cinema could somehow participate in that reversal. In other words, if modernity\u2014\"the alienating, blinding experience of the age of large-scale industrialism\" to which, Walter Benjamin claims, Bergson's work responded\u2014exacerbated this alienating perception, cinema's analytic character aligned it with that alienation, while its synthetic nature associated it with authentic experience. Cinema was both a social irritant and amelioration. In other words, the Bergsonian tension between continuity and discontinuity, between unity and fragmentation, was an expression of \"the alienating, blinding experience of the age of large-scale industrialism,\" but it was also a tension expressed in the very form of film itself. The aspect of cinema that made it a significant tool for scientific analysis existed alongside the aspect of cinema that made it useful for philosophical thinking.\n\nWhile Bergson's philosophy struck a chord throughout Europe and the United States, its resonance was muted in Germany, where the reception of his work was not quite as overwhelming. Nevertheless, many German thinkers struck notes in much the same key. Wilhelm Dilthey, for example, argued that positivist philosophy and science alone could not grasp the whole of life's rich variety: \"The basic conception of my philosophy is that up to now no one has put whole, full, and unmutilated experience at the basis of philosophizing, that is to say, the whole and full reality.\" According to Dilthey, this \"whole and full reality\" could not be fully understood using the \"mutilating\" knives of reductionist analysis and causal explanation. So he proposed his famous division between the physical sciences, which rely on causal explanation, and the human sciences, which derive their insights from empathetic or hermeneutic understanding: \"The basis of the human studies is not conceptualization but total awareness of a mental state and its reconstruction based on empathy.\" This recalls Bergson's conclusion about the incompleteness of the scientific enterprise:\n\nIt seems then that, parallel to this physics, a second kind of knowledge ought to have grown up, which could have retained what physics allowed to escape. On the flux itself of duration, science neither would nor could lay hold, bound as it was to the cinematographical method. This second kind of knowledge would have set the cinematographical method aside. It would have called upon the mind to renounce its most cherished habits. It is within becoming that it would have transported us by an effort of sympathy. (372)\n\nIn other words, both philosophers were interested in opposing the machine of scientific method with the wholeness offered by a certain intuitive, empathetic understanding. Cinema, via Bergson, stood squarely in the middle of these debates about scientific method in fin de si\u00e8cle Germany. Its inherently ambiguous character was reflected in the different ways scientists used it in their experiments and in the different ends to which it was employed. It is true that Bergson in _Creative Evolution_ emphasized only the analytic potential of motion picture frames\u2014that is, the use of celluloid as a \"mutilating\" dissection knife trained on living movement. Yet scientists in their actual use of film also effectively employed the _projected_ film as an end in itself, revealing what Bergson would later recognize (and Gilles Deleuze would still later describe) as cinema's and science's capacities for synthesis within a persuasive image of movement itself. In the rest of this chapter, I will outline some of these different means and ends by examining a series of case studies of the appropriation of cinematic technology in the science of work, in physics, and in cell biology.\n\nTHE SCIENCE OF WORK AND THE WORK OF SCIENCE\n\nTo illustrate his point that \"with immobility set beside immobility, even endlessly, we could never make movement,\" Bergson suggested this thought experiment:\n\nSuppose we wish to portray on a screen a living picture, such as the marching past of a regiment. There is one way in which it might first occur to us to do it. That would be to cut out jointed figures representing the soldiers, to give each of them the movement of marching, a movement varying from individual to individual although common to the human species, and to throw the whole on the screen. We should need to spend on this little game an enormous amount of work, and even then we should obtain but a very poor result: how could it, at its best, reproduce the suppleness and variety of life? (331)\n\nThis \"little game\" bears a remarkable similarity to the serious experiments of Braune and Fischer, who used the marches of a military recruit to extract principles of motion \"common to the human species\" from his \"individual movement.\" Their results, after \"an enormous amount of work,\" look very much like \"jointed figures,\" although Braune and Fischer would protest that their representations were not meant to convey \"the suppleness and variety of life\" but rather the universal laws that subtend it. Interested primarily in isolating moments in motion and reducing them to universal principles, they were unapologetic contributors to the mechanistic, positivist, analytic trend in the sciences. In this section, I will use their work as an example of the \"cinematographical method\" in science that Bergson interrogates. But I also want to demonstrate how their agenda fits into larger social concerns about modernity. So I will first survey the science of work and its promise to offer a way to manage social problems and class relations. The military heritage of this discipline leads to a consideration of the \"docile body\" in the science of work and scientific photography or cinematography. Then I will turn to the actual process by which scientific images are rendered, by reading their technique as exemplary of the transformation of the chronophotographic image into acceptable evidence.\n\nBefore the image could be accepted as evidence, the subject itself had to be made manageable. At each stage of Braune and Fischer's experiment, they had to make adjustments to the subject, the apparatus, and the means of analysis. The recruit's body, for example, had to be continually manipulated and adjusted to create a legible image. Indeed, just as adapting the subject's body to the apparatus, and vice versa, played a large role in creating and legitimating a scientific image, so adapting labor to the machine age was the primary goal of the science of work. Likewise, Braune and Fischer's intricate efforts to prepare the military recruit for his mission correspond nicely with the mathematical contortions required to translate the image into acceptable scientific data. Understanding the mechanics of the human body required its submission and disassembly on both sides of the equation, before and after the chronophotographic inscription. As we shall see, disassembly, or analysis, was the first step to rebuilding the body and society. Braune and Fischer used the apparatus (both the camera and its accompanying scholarly translations) to break down human movement into its constituent parts. But the painstaking work of analyzing each movement would be rewarded only if they could find a way to eliminate needless effort and bring all the elements back together again so that they worked more efficiently, all in the name of conserving energy and forestalling fatigue.\n\nAs Anson Rabinbach has persuasively demonstrated, the twin concepts of \"energy\" and \"fatigue\" were enormously powerful tropes for scientists and reformers of fin de si\u00e8cle Europe. New scientific models of energy consumption and conservation, along with the ubiquitous technologies and techniques of mass production that accompanied the second industrial revolution, led many scientists to think of the human body as a motor, a machine governed by the same laws of physics and chemistry as its man-made counterparts. The centuries-old battle between vitalism and mechanism heated up once again as this new mechanistic theory of the human motor encountered the critical vitalism, or _Lebensphilosophie_ , of Bergson, Driesch, and others. As the demands of adapting human labor to new industrial techniques grew, a new science\u2014the science of work\u2014adopted these mechanistic principles. The science of work studied the \"human motor\" in order to define its laws and track down the origin of fatigue.\n\nYet the trope of fatigue was more than a scientific mania of the age; it expressed a profound concern over decline and social disintegration. As the structural changes wrought by the Industrial Revolution rippled through society, there arose a tendency \"to locate the body as the site where social deformations and dislocations can be most easily observed\" (21). Metaphors of health and sickness were used to express national anxiety. Fatigue became more than a physical ailment, it became a _moral_ problem, a sign of weakness and absence of will. According to Rabinbach, \"In fatigue the physical horizon of the body's forces was identified with the moral horizon of the species; the moral infirmity of the population was directly proportional to the debilitating effects of fatigue.... [Furthermore,] fatigue represented the membrane between morally sacrosanct labor and the violent, irrational impulses that constantly threatened to disrupt social order\" (43). The tensions between human labor, capital, and the machine age demanded some sort of solution, and European scientists studying the nature of work believed that greater productivity was the key to social harmony. Investigations into the nature of fatigue were hopeful steps toward resolving class conflict scientifically, that is to say, \"neutrally.\"\n\nThe science of work, which emerged in the nineteenth century on the periphery of scientific study, compensated for this marginal status by becoming a self-consciously international phenomenon. The relatively small group of researchers interested in this topic around the 1880s had very little support, institutional or otherwise. Ernest Solvay, a Belgian chemist, who conceived of society as \"an enormous industrial enterprise dedicated to increasing overall productivity while encouraging social justice,\" endowed the Institut de Sociologie in Brussels as part of his plan to study the nature of energy and fatigue. Angelo Mosso of Turin, an Italian physiologist and educational reformer, invented the first efficient and accurate measure of fatigue, the ergograph, in 1884. But it was Hermann von Helmholtz of Germany and \u00c9tienne-Jules Marey of France, in particular, who gave direction and means to the fledgling science.\n\nHelmholtz's contributions to science are legion, but he is perhaps best known for his version of the first law of thermodynamics, or the law of conservation of energy, which holds that energy is neither created nor destroyed but simply transferred. His mathematical formulation of this fundamental law of physics provided science with its most substantive version yet, and it soon became more than a truism. This law's companion axiom, the second law of thermodynamics, which was formulated by other scientists, including Rudolf Clausius and William Thomson (Lord Kelvin), also gained credence beyond the world of physics. The second law explained the process of _entropy_ , which holds that although energy cannot be destroyed, it tends over time to be degraded from useful forms to uselessness. The best example of entropy is the phenomenon of heat transference: when a hot body is placed next to a cold one, the potentially useful energy of the former will transfer to the latter until both have equal, and less useful, temperatures. The relevance of these laws of conservation and entropy was soon extended from the study of molecules to that of the human body and even to society as a whole. Fatigue became the corporal analog of the second law of thermodynamics, and degeneration became its social equivalent (45\u201348). Scientists, reformers, and opinion makers of all sorts soon began to depict the nature and problems of society in terms of energy, attention, will, and utility, on one hand, and fatigue, degeneration, entropy, and uselessness, on the other. The laws of thermodynamics therefore provided the metaphors that motivated social plans for managing the conflicts and excesses of industrial society.\n\nOn the other hand, Marey provided the means by which scientists could study the laws of energy and entropy as they were played out through the human body. Marey, a physiologist who made lasting contributions to the fields of cardiology, physiological instrumentation, and aviation, as well as to the craft of photography and the science of work, provided not only the means for a minute analysis of the movements of the human body but the basis upon which these studies could be counted as legible and legitimate areas of inquiry. His transformation of ephemeral phenomena, such as movement, into scientifically acceptable (that is, legible by disciplinary standards) visual evidence through his \"graphic method\" prompted a flood of motion studies that overran journals in the 1890s. In this respect, Marey is an exemplary figure in late nineteenth-century European positivism. His graphic method signaled the ascendancy of the process-oriented approach in physiology that has dominated the field for the past 150 years. His focus on the disassembly and reformation (in the broadest possible sense) of the human body is representative of the general goal of positivistic, inductive, physical sciences: analyzing individual instances of natural phenomena and provisionally concluding from them an ideal model or set of laws. As Rabinbach demonstrates, Marey represents the forging of a crucial link between cultural and social modernity, between late nineteenth-century disruptions in the perception of time and space and the efforts to manage the contemporary social crises (84\u201388). His kymograph and chronophotographs codified, even embodied, the historical confluence of these forces, giving the science of work direction, means, and legitimacy.\n\nDesigned to calculate \"the mechanical work expended in different movements,\" Marey's chronophotographs were also a link between cultural and social modernity, providing the basis for both a new leisure activity and a science of labor. Marey intended his \"ergonomics\"\u2014or science of efficient movement\u2014to lead to greater productivity. The concept of \"training\" was very important for Marey's ergonomics and the science of work in general. Marey maintained that all animal locomotion is characterized by the transformation of abrupt and disjointed movements into consistent motion. (This is also an accurate description of the technological principle of cinema.) The central feature of all work\u2014whether of humans or machines\u2014is the transformation of irregular, inconsistent, and jarring shocks into regular and uniform activity. The body's own elasticity permits the suppression of shock into regular effort. Muscles, for example, act to turn abrupt movements into dynamic work. Marey believed that animal and human motors are naturally efficient yet capable of improvement. His chronophotography attempted to demonstrate the potential for greater economy to be attained from \"training\"\u2014essentially a program of scientific and bodily discipline. Through careful study of the movement of the body in various stages of a work process\u2014whether forging iron, for example, or pole-vaulting\u2014scientists could spot and correct inefficient movements, thereby showing the worker how he or she might expend the least amount of force and consequently accomplish the task with the least amount of fatigue.\n\nBraune and Fischer's studies of human motion extended this tradition. To \"investigate the influence of a relatively heavy load on gait,\" they asked their experimental subject to carry \"an army regulation knapsack, three full cartridge pouches and an 88 rifle in the 'shoulder-arms' position\" in a number of different experimental settings. These studies, ostensibly dealing \"only with the experimental determination of the process of movement, without considering the cause,\" were designed to recreate the process of labored movement in the hopes that these activities could be improved ergonomically. In this they shared the approach and goal of other practitioners of the physiological branch of the German science of work, or _Arbeitswissenschaft_ , such as Nathan Zuntz and Wilhelm Schumberg, whose _Studien zu einer Physiologie des Marsches_ \"surveyed all aspects of military drill\" in the hopes of pinpointing the causes and consequences of fatigue and march-related illnesses. The military orientation was a common motif in the science of work: Braune and Fischer's experiments were supported by the German High Ministry; Marey stressed the potential benefits of motion study for military training in his numerous appeals for support from the French government; Wilhelm Weichardt tested his infamous \"fatigue vaccine\" on the Austro-Hungarian army; and Mosso tested his ideas about the relationship of mental and physical fatigue on Italian soldiers.\n\nIt is not too surprising that European theories of efficiency, elasticity, and fatigue often took an explicitly military orientation, considering that a military motto might be \"efficiency through training.\" As a matter of fact, the science of work was the industrial application of forms of discipline first deployed in the military. The temporal decomposition and reconstitution of the human body through chronophotographic or cinematic means also fit well into such agendas. _Moving Picture World_ once noted, \"The United States Army has had [motion] pictures taken of a soldier going through the manual of arms. Thumb books with these pictures are made up and furnished to the recruit, who by looking carefully through them can easily trace every minute movement that goes to make up the completed action.\" In this example the recruit was expected to incorporate the lessons of the cinematic image in much the same way that he was expected to embody military ideology. The point is not so much that cinema and the science of work cooperated with the military but that the military application of motion pictures and of these scientific principles point to a coincidence of techniques that provide further insight into the relationship between science and cinema. Michel Foucault cast some light onto the larger intellectual history connecting the science of work and the state:\n\nThe great book of Man-the-Machine was written simultaneously on two registers: the anatomico-metaphysical register, of which Descartes wrote the first pages and which the physicians and philosophers continued, and the technico-political register, which was constituted by a whole set of regulations and by empirical and calculated methods relating to the army, the school and the hospital, for controlling and correcting the operations of the body. These two registers are quite distinct, since it was a question, on one hand, of submission and use, and on the other, of functioning and explanation: there was a useful body and an intelligible body.\n\nBut these registers, Foucault continues, overlap in the notion of \"'docility,' which joins the analyzable body to the manipulable body. A body is docile that may be subjected, used, transformed and improved.\" The scientist's efforts to survey the human body and the colonel's attempts to modify it both required that the body submit to a regimen of exercises. This may have included a series of measurements, tests, recordings, or it might have meant calisthenics in the morning and full-load drills in the afternoon. Either way, whether under the rubric of science or the state, docility, according to Foucault, \"implies an uninterrupted, constant coercion, supervising the processes of the activity rather than its result and it is exercised according to a codification that partitions as closely as possible time, space, movement.\"\n\nThis last sentence is certainly an apt description of Braune and Fischer's experimental procedure, especially in that their chronophotographs \"partition as closely as possible time, space, movement.\" Foucault called these methods \"disciplines,\" and we would include science among them, because its work is essentially that of domestication. We may, after Ian Hacking, divide the work of the scientist into two types: representing and intervening. Hacking equated this division, generally speaking, with the split between theory and experiment, even while acknowledging that the two are inseparable. Indeed, experiment is tightly bound with the process of representing, as sociologists of science have shown. But whether through representation or experimentation, any phenomenon to be studied must be \"tamed\" before it can become scientific data. In the attempt to analyze (or, more accurately, render analyzable) natural phenomena, the work of the scientist involves any number of phases, such as selecting, partitioning, measuring, or representing. It is impossible to present a phenomenon in its \"natural\" state; it must be rendered into material images, such as graphs, photographs, tables, charts, and diagrams\u2014representations that function as Bergson's \"snapshot\" of duration. Sociologist of science Michael Lynch, following Foucault, calls the product of these scientific procedures a \"docile object\":\n\nIt is an object that \"behaves\" in accordance with a programme of normalization. This does not mean that it fails to resist, or that its recalcitrance does not serve to adumbrate its objective news for science. It is to say that, when an object becomes observable, measurable and quantifiable, it has already become _civilized_ ; the disciplinary organization of civilization extends its subjection to the object in the very way it makes it knowable. The docile object provides the material template that variously supports or frustrates the operations performed on it. Its properties become observable-reportable in reference to the practices for revealing them. If the object was not compliant to such a programme, its attributed properties would be incompletely or \"unscientifically\" observable.\n\nThis same civilizing process applies equally to the human body in motion studies, the paramecium under the biologist's microscope, or the astronomer's optical pulsars. I would argue that it also applies to the apparatus created or transformed to view or inscribe these phenomena. Motion picture technology also underwent a certain domestication as its image was transformed into acceptable scientific evidence. For the cinematic image to function legitimately as scientific evidence, it must undergo a transformation, a _rendering_ that is common to all scientific practice and \"docile objects.\" The army's flip books, for example, were used to train the soldier's body, to corral its forces into a productive and useful activity. The books were part of the system of discipline. Yet the very creation of the flip books was also one way of making the cinematic image analyzable by controlling its size, speed, and impact. In the same way, Braune and Fischer took specific, formal steps to domesticate both the human body and their chronophotographic apparatus. The next section will describe these steps in some detail and discuss how they fit into the broader process of representing natural phenomena in science.\n\nBRAUNE AND FISCHER'S _THE HUMAN GAIT_\n\n_The Human Gait_ appeared as a series of papers published between 1895 and 1904 in the _Proceedings of the Royal Saxon Society for Sciences_ (Abhandlungen der k\u00f6niglich s\u00e4chsischen Gesellschaft der Wissenschaften). Although Braune died immediately after the experiments described in their first chapter (perhaps testifying to the exhausting nature of the work), Fischer honored his teacher by making him the first author, and both names have been associated with the studies ever since. Chapter 1 appeared in 1895 and chapters 2 through 6 appeared in 1899, 1900, 1901, 1903, and 1904. Sponsored by the German High Ministry of War, the essays were the first quantitative analysis of human locomotion, and their precision set a standard that is still frequently cited today. That an English translation appeared in 1987\u2014and is still considered an important text in the modern field of human motion studies\u2014testifies to their continued relevance in the science of human movement. In addition to formulating important axioms for this field, Braune and Fischer developed new techniques and instruments for analyzing images. In fact, they are recognized as the originators of analytic, close-range photogrammetry, the science of measurement from photographs.\n\nPrevious investigators of human motion, such as the Weber brothers, Marey, Muybridge, Carlet, and Hermann Vierordt, were primarily concerned with qualitative, two-dimensional motion studies. That is, their largely descriptive studies focused on motion through only two axes\u2014the horizontal and vertical lift of the human leg, for example. Braune and Fischer, on the other hand, realized that animal locomotion takes place along a _z_ -axis as well, that is, _sideways_. They strove to create, through experimentation and exact measurement, an ideal, three-dimensional model of human movement, something that had not been done up to that point. Several researchers had tried to represent the intricacies of human movement graphically, but their methods lacked the scientific rigor to which Braune and Fischer aspired. Braune and Fischer noted, for example, that the achievements of Muybridge, Ansch\u00fctz, and Londe \"are very important for artists, particularly those who depict people and animals in motion,\" but \"the use of photography as a scientific research tool and the improvement of cameras to this end are due, above all, to Marey.\"\n\nThe primary difference between studies \"important for artists\" and those that could be counted as \"scientific\" was measurement. Muybridge and Londe had in common the instinct for \"automatic writing,\" that is, some method of creating automatic, mechanically inscribed signs of movement. But such a sign alone would be useless if it could not be held up to ever stricter scientific protocols. Marey's single-camera setup represented considerable progress over Muybridge's series of cameras, simply because, for Muybridge's series method to be successful, \"the distances between axes of the cameras, standing side by side, had to correspond to the phases of movement and the different cameras had to be optically similar\" (6). This distance depended upon the velocity of the moving body, which could not be known beforehand and is different for each body and each type of movement among the various body parts. Therefore, precise comparison between the pictures was nearly impossible. Yet Marey's single-camera system had problems as well. \"If all the points of a human or animal body moved in one plane during walking or running, these series of pictures would represent not only a one-sided projection but also a true picture of the whole process of movement. However, movement in space has to be taken into account, whereby the centers of all joints describe double curves. Thus, the projection achieved by Marey's method is insufficient to describe completely the movement in space\" (8). In other words, while Marey's pictures provided information about the horizontal and vertical motions of the entire body during walking, they provided no insight into its sideways oscillations. Braune and Fischer's contribution, then, was a four-camera system that produced \"two-sided\" chronophotography, because \"two simultaneous photographic exposures of the same movement are sufficient to determine the movement in any direction\" (10).\n\nTwo-sided chronophotography had its own peculiar difficulties, however. It \"requires the cameras to be opened and shut at short intervals at precisely the same time. This requirement can only be achieved using a highly complicated mechanism. Therefore, to interrupt the exposure, we relied not on shutting the camera but on _altering the photographic object itself_ , so that it was possible to dispense with a particular mechanism for shutting the camera\" (10, emphasis added). From the beginning, then, Braune and Fischer confronted the dilemma of simultaneously regulating subject and apparatus. This was not an either\/or situation; the subject was not completely subdued in favor of an implacable apparatus and method. Rather, both were altered until their properties _merged_ in the representation. That is, features of the body that could be graphically enhanced (such as a limb's straight line) were coordinated with characteristics of the camera (such as the two-dimensional film plane) to create a usable image. This stage of their experiment, then, was crucial for adapting the body to the chronophotographic image and vice versa.\n\nBraune and Fischer's alterations included, first of all, highlighting the recruit's body with a series of strategically placed Geissler tubes\u2014long, thin, straight tubes filled with rarefied nitrogen that, when exposed to an electric current, become incandescent. The recruit wore a black jersey, similar to the one used by Marey, which \"provided a dark background for the tubes and permitted better attachment of the tubes to the body\" (12) (fig. 1.1). When fired by a regulated electrical circuit, the flashes of light provided an ideally manageable strobe effect. The black jersey offered not only a dark background for the tubes, it effectively erased all extraneous details\u2014meaning most of the body itself\u2014from the picture and presented only the most graphic qualities of the process under examination. Significantly, Braune and Fischer also took into account the points that were not illuminated (fig. 1.2).\n\nFIGURE 1.1. Braune and Fischer's military recruit in the experimental suit\n\nFIGURE 1.2. The subject at rest with the grid superimposed\n\nIn some places the Geissler tubes were surrounded by narrow rings of black Japan varnish; these places thereby appeared as short interruptions in the line of light in the pictures. They were located near the ends of the tubes and at the same level as the center of the joint. They therefore marked the corresponding positions of the joints as isolated points of light in the photographs. Similar black rings were also located at the places corresponding to the center of gravity of each segment of the body; the different centers of gravity appeared, then, as black dots on the white lines in the photograph (14).\n\nGenerally speaking, Braune and Fischer were \"marking\" the object in preparation for its representation, a process that is common in scientific rendering practices. In recounting how a \"natural\" space is rendered into a geometricized workplace, Lynch identified a number of themes or processes. Exploring how representations\u2014graphs, tables, diagrams, and photographs\u2014come to embody the \"natural object,\" he asks \"how science initially determines what is natural on the basis of what its graphic qualities disclose.\" \"Marking\" is a first step toward identifying and cultivating those graphic qualities. Lynch finds that marking occurs in two phases: \"labeling\" and \"upgrading visibility.\" In the first phase, the visibility of the object is initially consolidated by first-order techniques, for example, dyeing a cell so that its constituent parts show up under microscopic study. In the second, scientists mark instances that stand out as clear examples, which is a process of selective perception while the project is underway. Braune and Fischer's Geissler tubes served this dual purpose, then, by both enabling the visibility of the object and making visible only what already had been identified as significant.\n\nThe second step common to this rendering process, so closely related to the first as to be only theoretically distinct, is \"the constitution of graphic space.\" Here a \"'mathematical' space comes to dwell within the 'natural' terrain.\" Scientists mark the space of the experiment in such a way that it can be measured or formally structured. In the happy coincidence of anatomy and classification, or the way a specimen is appropriated so that its visible properties are brought together with the graphic qualities of the representational medium, natural objects are prepared for \"mathematicization.\" In other words, the surfaces of the natural object are brought in line with ideal, geometric properties that can be mathematically useful. The placement of the Geissler tubes illustrates nicely how the protomathematical properties of the human body merge with the graphic qualities of the chronophotographic image to become an intelligible, analyzable, measurable scientific representation.\n\nBraune and Fischer also constituted the graphic space by careful camera placement and superimposition of a coordinate grid. They placed two cameras across from each other and perpendicular to the _x_ -axis (the recruit's path) and two other cameras at 30-degree angles to the _x_ -axis (fig. 1.3). The two simultaneous exposures (of each side of the body) could therefore determine the position of any given point in three-dimensional space. To ensure that the points on the different exposures were registered exactly in relation to one another, Braune and Fischer created a grid that could be superimposed upon the image, so that the points of light could be matched to an established set of coordinates (see fig. 1.2).\n\nFIGURE 1.3. Braune and Fischer's camera placement\n\nTo enable us to draw the trajectories in a system of tridimensional co-ordinates, after photographing the phases of movement we photographed on the same plate a network of 1-cm squares printed on a glass plate covered with Japan varnish. In order to improve accuracy we built a large wooden frame on which a 1-m square table of co-ordinates could rotate about a vertical axis. This frame rested on four small screws for which four metal recesses were prepared in the floor. The frame and the table of co-ordinates could thus be brought into exactly the same position at any time. (16)\n\nSo the wooden frame held a one-meter-square glass grid that could rotate parallel to the plane of each of the four cameras. After the initial exposures of the recruit were finished, Braune and Fischer would cover the cameras, screw the frame into its predetermined spot on the _x_ -axis, and superimpose the grid onto each of the four photographic plates, thereby creating a \"graphic space.\"\n\nIt was also necessary that the different phases of movement be regulated _temporally_. To coordinate the firing of the strobe effect, Braune and Fischer connected the primary circuit of the induction coil to a large tuning fork (15). They determined that the fork vibrated at a frequency of twenty-six vibrations per second, which meant that between any two phases of movement approximately 0.04 seconds elapsed (16). The frequency of the fork did not matter so much as its regularity.\n\nConnecting the subject to an electrical source also regulated him in a more indirect way. Braune and Fischer sewed long strips of gutta-percha (a substance resembling rubber but containing more resin) into the jersey where the tubes were to be placed to protect the recruit from electric shock. \"Perhaps we were overconcerned with regard to the insulation and could have saved ourselves a great deal of work since it usually took us between 6 and 8 hours to dress the experimental subject. However, we thought that the subject would walk naturally if he knew that the electric current, of which so many people are afraid, would not come into contact with his body\" (14). Indeed, it would be surprising if the recruit actually did walk naturally. The eleven Geissler tubes were connected in series and powered by a large induction coil in the laboratory. Wires from the coil hung from the ceiling, were draped over a light wooden rod fixed across the shoulders of the recruit (see fig. 1.1), and connected to the circuit on the body. The recruit could therefore walk freely for about ten meters, \"the length of the room necessary for the experiment\" (14). Camera placement played a crucial role in the success of the experiment, so it was essential that the subject not waver from a specified path, which the constraints of the wires and the threat of electrical shock certainly ensured.\n\nFIGURE 1.4. The resulting chronophotograph\n\nClearly, then, by the time the exposures were made, the phenomenon of human movement had already been transformed from an unreadable, \"natural\" object into a regulated, mathematicized process. The photographic image's unruly detail had also been restrained in a number of ways: the strobe effect reduced the image to only its most graphic, mathematical components; the camera placement and the coordinate grid regulated the space of the experiment; the tuning fork and Geissler tubes marked the temporality of the images. The resulting chronophotographic image, however, was far from self-explanatory (fig. 1.4). No image is meaningful without another set of interpretive routines.\n\nIf they were to create a three-dimensional model from these images, Braune and Fischer needed to measure the distances between the points of light and darkness. Figure 1.5 can only hint at some of the spectacular trigonometric operations involved in determining three-dimensional coordinates from two photographs, operations that I will not gloss in any detail. Generally, however, they used triangulation, the basic principle of photogrammetry, to find the position of any point in space from the bearings of two fixed points a known distance apart. This required, of course, that they take measurements directly from the photographic plates. To accomplish this task, they created an instrument designed especially for this purpose (fig. 1.6).\n\nFIGURE 1.5. Determination of the coordinates of a point _P_ from the projections of _P_ on two planes as seen from the two cameras\n\nThe photographic plate was fixed upon a mobile ring and viewed through a microscope, which could slide along a track and bring any point on the plate into view. The microscope gave a view of both the image on the photographic plate and a ruler placed alongside the plate, thereby allowing easy measurement of the points on the plate (fig. 1.7).\n\nFIGURE 1.6. Side ( _A_ ) and top ( _B_ ) views of the instrument used to measure coordinates\n\nFIGURE 1.7. Measurement of a coordinate: The instrument trained on the photograph ( _A_ ) and the view through the instrument ( _B_ )\n\nThese measurements for all four views were then collected and, \"to avoid an accumulation of data\" (!) (43), tabulated for only nine points on the human body: the shoulder, elbow, wrist, hip, knee, ankle, center of gravity of foot, tip of foot, and the point on the head (fig. 1.8). This reduction of the recorded points to a workable number was the first step in transforming the wealth of data into an ideal, that is, a theoretical model. It should also be noted that the gaps in the tables represent coordinates that for some reason could not be determined (a forearm blocking a point on the hip, for example); likewise, question marks indicate indeterminate data. These statistics, although varying by individual experiments, were consistent over time. \"Thus, the results reported in the tables of co-ordinates are valid not only for an individual. They also represent general laws of the movements of the limbs in human gait\" (80). At once more than and less than a human body, the numbers provided the principal tool by which Braune and Fischer reconstructed human movement.\n\nFIGURE 1.8. A table of the coordinates derived from experiment 1\n\nThe second step in \"polishing\" the rough data involved plotting the coordinate numbers onto a graph, essentially recreating the recruit's movement on another grid (fig. 1.9). Idiosyncrasies were reduced by the use of straight lines and points. Comparing one of the photographs (see fig. 1.2) with the graph, we can see clearly how theoretical presuppositions informed the creation of the image. Yet Braune and Fischer maintained, \"It must be stressed that the lines drawn in the diagram do not represent the Geissler tubes used for the experiment but the long axes situated inside the limbs\" (81). Their coordinates also provided for a series of horizontal, or overhead, views of human movement. Figure 1.10 provides successive overhead views of the movements of the nine sections of the body. The views were separated, of course, to make them easier to read.\n\nFIGURE 1.9. The graph of the coordinates (view from the right side)\n\nEdmund Husserl, writing on \"The Origin of Geometry,\" outlined how material objects in the real world are identified with idealized geometric forms:\n\nFirst to be singled out from the thing-shapes are surfaces\u2014more or less \"smooth,\" more or less perfect surfaces; edges, more or less rough or fairly \"even\"; in other words, more or less pure lines, angles, more or less perfect points; then, again, among the lines, for example, straight lines are especially preferred, and among the surfaces the even surfaces; for example, for practical purposes boards limited by even surfaces, straight lines, and points are preferred, whereas totally or partially curved surfaces are undesirable for many kinds of practical interests. Thus the production of even surfaces and their perfection (polishing) always plays its role in praxis.\n\nFIGURE 1.10. The graph of the coordinates (view from above of different body parts)\n\nLynch suggests that we read Husserl's account \"as a description, not of a once-and-for-all historical movement from proto-science to science, but as an account of what scientists do every time they prepare a specimen for analysis in actual laboratory work.\" We can see this mathematization, this \"polishing,\" take place as Braune and Fischer smoothed out the rough edges of their data from table to graph. The body\/image became progressively less recalcitrant as the process continued. Like a student who erases the rough pencil sketches and calculations only after finishing the final graph in pen, so Braune and Fischer's polishing erased the contingent, individual body for a generalized, ideal one. It became an _eidetic_ image. _Eidos_ is the Greek word for \"idea\" or \"species,\" but Bergson elaborated on this definition:\n\nWe might, and perhaps we ought to, translate _eidos_ by \"view\" or rather by \"moment.\" For _eidos_ is the stable view taken of the instability of things: the _quality_ , which is a moment of becoming; the _form_ , which is a moment of evolution; the _essence_ , which is the mean form above and below which the other forms are arranged as alterations of the mean; finally, the intention or _mental design_ which presides over the action being accomplished, and which is nothing else, we said, than the _material design_ , traced out and contemplated beforehand, of the action accomplished. To reduce things to Ideas is therefore to resolve becoming into its principle moments, each of these being, moreover, by the hypothesis, screened from the laws of time and, as it were, plucked out of eternity. That is to say that we end in the philosophy of Ideas when we apply the cinematographical mechanism of the intellect to the analysis of the real.\n\nThe culmination of Braune and Fischer's experiment, the final eidetic image, the reduction of real movement to an Idea\u2014arrived at through an application of the cinematographical mechanism\u2014was their three-dimensional model of human movement (fig. 1.11). With this model, they attempted to reconstruct movement by means of a series of discontinuous images. Their final operation, then, was essentially cinematic.\n\nIn this case, science's successful appropriation of cinema or chronophotography seems to have depended upon the ability to analyze the image and derive translatable information from it. The indeterminate detail of the image needed to be regulated, interpreted, and translated into data that could be mathematicized, tabulated, graphed, and modeled. Quantitative frame analysis, then, has been the traditional means of extracting this information or of disciplining the image. Anthony Michaelis, in his 1955 survey of the history of the scientific applications of motion pictures, declared that \"only the quantitative use of cinematography, combined with frame analysis, has produced the maximum amount of research data of which the motion picture film is inherently capable.\" In other words, if the image were to be a fully productive member of the scientific community, it needed to be, like the worker, broken down and reconstituted.\n\nFIGURE 1.11. Left ( _A_ ) and back ( _B_ ) views of the tridimensional model representing the attitudes of the human body during walking\n\nBraune and Fischer's efforts confirm Bergson's idea of the cinematic nature of science. In their attempts to chart the movement of the human body over time, Braune and Fischer were not concerned with duration so much as with isolation of particular moments. (They were also very interested in training the expert eye; their three-dimensional model in this respect functions as a guide to what to look for when studying human movement.) Film's success as a scientific instrument depended above all on its malleability and on the ability of scientists to manage the excessive detail within the image by reconfiguring the apparatus and image through various methods of frame analysis. Braune and Fischer's example is instructive because it provides a relatively clear view of the work involved in making a malleable and legitimate image. They also provide a good example because their simultaneous work on the image, the apparatus, and the human body replayed certain themes significant to cinema's relation to science and especially the science of work: the domestication of the object and apparatus, the isolation of physiological functions, and the disassembly and theoretical re-formation of movement and process. Braune and Fischer's example suggests that chronophotography's legitimacy as a scientific instrument rested not so much on its ability to record movement through time as its ability to stop that movement\u2014that is, not so much its continuity as its _dis_ continuity. Above all, the ability to divide processes into ever smaller units appealed to the late nineteenth-century researcher.\n\nBROWNIAN MOTION AND \"THE SPACE BETWEEN\"\n\nBraune and Fischer's method depended upon finding the geometry inherent in their subject\u2014in this case, the human body\u2014and translating it into mathematical terms. Chronophotography was essential to this operation, because it could isolate the \"determinate moments\" (in Bergson's words) of the flux of movement so that the inherently geometric aspects of the body could be highlighted and studied. As Bergson argues,\n\nThere is an order approximately mathematical immanent in matter, an objective order, which our science approaches in proportion to its progress. For if matter is a relaxation of the inextensive into the extensive and, thereby, of liberty into necessity, it does not indeed wholly coincide with pure homogenous space, yet is constituted by the movement which leads to space, and is therefore on the way to geometry. It is true that laws of mathematical form will never apply to it completely. For that, it would have to be pure space and step out of duration.\n\nHere Bergson is building on his argument that matter, or more precisely form, is a \"snapshot view\" of reality in transition. Yes, everything is in continual flux, but this constant movement (hence contingency, which, for Bergson, implies liberty) sometimes, to our perception, \"relaxes\" into a more or less stable shape. So from the perspective of this moment\u2014the \"moment\" during which the chair I am sitting on appears to be and acts solid and stable, which is a longer or shorter moment, depending on one's viewpoint\u2014we can map and measure this form and make use of this momentary \"interruption\" in duration. We can therefore make use of the geometry inherent in form, or what Bergson calls the \"mathematical immanent in matter.\"\n\nBoth Ren\u00e9 Descartes and Isaac Newton recognized this \"mathematical immanent in matter.\" Descartes worked toward a mathematical physics when he argued that certain qualities (such as extension, shape, and motion) were more knowable, objective, or certain than other qualities (such as color, sound, heat, and cold). He posited that we could know these \"primary\" qualities through measurement and ground our knowledge in the more certain world of mathematics. He thereby mapped geometry onto the physical world more thoroughly than his predecessors and opened the way for Newton's mechanics as mathematical formulations of motion. Classical physics was therefore founded upon matching the material world to mathematics. But, as Bergson noted, the pure form and space of geometry never completely match the matter of the object under study, simply because the object exists in time; in math, the elements of the equation do not age and change. Braune and Fischer's method indicates the lengths to which scientists must go to reconcile the _duration_ of the object (however momentary) with the _space_ of geometry and thereby harness the \"mathematical immanent in matter.\" Hence Bergson's conclusion that \"there is something artificial in the mathematical form of a physical law\" (238).\n\nYet science succeeds. Bergson offered this explanation: \"One hypothesis only, therefore, remains plausible, namely, that the mathematical order is nothing positive, that it is the form toward which a certain _interruption_ tends of itself, and that materiality consists precisely in an interruption of this kind.\" In other words, Bergson recognized a homology between science's method of arresting time and the momentary interruption of duration that is materiality. Or, to put it another way, science's tendency to _spatialize_ time is analogous to the \"relaxation\" of duration into form and matter. In any case, according to Bergson, science's success in finding mathematical correspondences depends on a temporal interruption.\n\nMax Seddig's attempt to confirm Einstein's theory of Brownian motion is an interesting case study, because his cinematic and chronophotographic method corresponded so closely to important features of the theory. Einstein's theory, as I will explain later, emphasized a gap or interruption: an elision of the path of particles in favor of two independent but related points along that path. In other words, Einstein stressed the _displacement_ of particles rather than their actual path. Likewise, Seddig's experimental method focused on the interval _between_ filmic exposures, so that two separate exposures represented two independent but related points. Seddig's results captured more fully than most the specifics of Einstein's theory precisely because his method was able to accommodate this gap or displacement that previous researchers missed or ignored. Seddig harnessed the inherent discontinuity of the chronophotographic\/cinematographic method to create an experimental system that corresponded to Einstein's important displacement equation. He experimentally created a \"temporal interruption\" akin to what Einstein had created theoretically, thereby instantiating the spatialization of time and form that Bergson found to be central to science's success. More than opening up new realms of the visible or even confirming important theories, this use of motion pictures in physics also illuminates certain features and limits of the scientific method as it had been traditionally understood. Indeed, examining Seddig's experiments alongside Bergson's critique allows us to see the deep compatibility between this application of motion pictures and the state of the physical sciences at the turn of the century. This section will proceed in the following manner: after a general discussion of the stakes involved in the discussion of atomistic physics, I will explicate Einstein's theory of Brownian motion as a watershed moment that collected and focused scientific observation. A survey of the experimental attempts to capture or confirm this phenomenon will lead us to Seddig's case and a close analysis of the connection between his experimental system and Einstein's theory. Finally, we will return to Bergson in order to ruminate on the homology between film form and the physics of Einstein and Seddig.\n\nEinstein's theorization of the phenomenon of Brownian motion, for which he assumed the atomic\u2013kinetic theory of matter, is generally considered to be \"one of the fundamental pillars (or even the main one) supporting atomism in its victorious struggle against phenomenological physics in the early years of this century.\" Throughout the nineteenth century, two approaches to scientific explanation had been in competition in classical physics: the atomic\u2013kinetic theory of heat, on one hand, and on the other, a phenomenological thermodynamics, which assumed that all natural phenomena could be encompassed by the application of Newton's laws of mechanics, but which did not presume to have access to the ultimate constituents of matter. (This approach to physics is called \"phenomenological\" because it deals with the description and classification of phenomena while refusing to indulge in any claims about causation.) This approach allowed physicists to describe certain phenomena (such as heat transference) and derive laws (such as the first and second laws of thermodynamics) from observation without explaining _why_ matter and energy acted in this way. These laws of thermodynamics therefore functioned much like other laws of classical physics, such as Newton's law of gravity, in that they presumed that phenomena would obey these laws no matter what their size or situation. These laws, in other words, were generally regarded as both scalable and absolute.\n\nThe rival approach to explaining thermal phenomena was the atomic\u2013kinetic theory of matter, which started with specific assumptions about the constituents of matter, that is, \"that it was discrete, molecular, ultimately atomic, and that heat was a 'concealed' form of motion associated with the molecules of a substance.\" This theory had the advantage of actually trying to _explain_ the nature of heat, but it had the disadvantage of being unobservable. While atomic theories of matter date back to the Greeks, many eminent nineteenth-century scientists, such as Ernst Mach and Wilhelm Ostwald, resisted building theories of physics on the supposed behavior of matter consisting of particles so small that they were invisible. If advocates of the kinetic theory were to succeed, they would need to deduce the existence of these particles from their effects. Einstein and others theorized that these effects could indeed be deduced mathematically from the random fluctuations in an observable system, such as a container full of a certain kind of gas. These random fluctuations would not occur, by definition, if the absolute laws of thermodynamics held. In other words, the proof of the real existence of molecules was tied to proving that classical thermodynamics was true only in a statistical\u2014not absolute\u2014sense. And at the turn of the century, the atomic theory of matter and its statistical correlation were far from universally accepted. The theory and confirmation of Brownian motion was a crucial victory in this struggle.\n\nBrownian motion, the irregular motion of microscopic particles suspended in fluid, had been known long before Scottish botanist Robert Brown described it in 1828, when he turned his microscope to cytoplasmic granules extracted from pollen. His achievement was \"to show that the motion could not be attributed to any supposed vitality of the particles themselves, since all kinds of inorganic as well as organic substances behaved similarly.\" This meant that the cause of the movement had to be external to the particles themselves. There were a variety of attempts to explain this phenomenon, but it was not until the close of the century that French physicist Louis-Georges Gouy suggested that Brownian motion constituted a clear demonstration of the existence of molecules in continuous, albeit random movement. However, Gouy did not work out any mathematical theory that could lead to experimental confirmation. This was Einstein's contribution in 1905. Not that previous scientists hadn't tried. But the attempts to clarify the phenomenon experimentally were hindered by the lack of agreement among the observations. Researchers simply could not agree on the principal features of Brownian motion. Not only were the movements too quick and irregular to submit themselves to steady observation, but researchers could not concur on even such basic presumptions as whether the movements were dependent on the temperature of the fluid. This tangle of confusion indicates, to Roberto Maiocchi at least, \"how difficult it is to make a meaningful and conclusive scientific 'observation' and, as a result, how any inductivist conception, which claims to start from an empirical base in order to then construct theories of some importance, is unsustainable.\"\n\nPhotography and cinematography offered at least the possibility of creating an \"objective\" record that could be compared with previous and current descriptions of Brownian motion. But again, without sufficient technical means and a theory to guide them, this hope was fool's gold. Take, for example, the case of Austrian scientist Felix Exner, who attempted \"to measure the size of the particles and their speed\" via direct photographic exposure. Unfortunately, the photographic plates at the time were not sensitive enough to register the small amount of light coming through the microscope lens. Undaunted, he observed the movements and traced them _manually_ onto a blackened photographic emulsion, projected these traces onto a screen, measured the distances between the points, and divided by the observation time. \"Of course, the values are not very exact,\" he admitted. Indeed. Exner was interested in establishing the numerical relationship between the temperature of the fluid and the velocity of the suspended particles. His article was the first attempt to quantify features of Brownian motion that previously had been described only qualitatively. Even though his results were the most accurate studies to date, given his method, it is not entirely surprising that they were not decisive. But he did spotlight the need for systematic observation and measurements and the role photography could play in this process.\n\nBut previous observation had very little impact on Einstein's formulation of his theory. The jumble of conflicting observations was of limited use. Einstein was not interested primarily in generating a theoretical explanation of a puzzling phenomenon. Instead, he saw Brownian motion as an observable system that could conceivably resolve the debate between phenomenological thermodynamics and the atomic\u2013kinetic model. Specifically, he derived equations governing motion based on the assumption of random molecular movement and then argued that their confirmation would demonstrate the existence of molecules and also show that the laws of thermodynamics did not apply _absolutely_ to particles of molecular dimensions. In his 1905 paper on the topic, Einstein indicated both the inadequacy of previous observations and the stakes involved for future observations:\n\nIn this paper it will be shown that according to the molecular-kinetic theory of heat, bodies of microscopically visible size suspended in a liquid will perform movements of such magnitude that they can be easily observed in a microscope, on account of the molecular motions of heat. It is possible that the movements to be discussed here are identical with the so-called \"Brownian molecular motion\"; however, the information available to me regarding the latter is so lacking in precision, that I can form no judgment in the matter.\n\nIf the movement discussed here can actually be observed (together with the laws relating to it that one would expect to find), then classical thermodynamics can no longer be looked upon as applicable with precision to bodies even of dimensions distinguishable in a microscope: an exact determination of actual atomic dimensions is then possible. On the other hand, had the prediction of this movement proved to be incorrect, a weighty argument would be provided against the molecular-kinetic conception of heat.\n\nEinstein tackled the big problems; his equations would either prove or disprove the atomic theory of matter.\n\nBecause he did not invoke previous studies or conduct experiments of his own, Einstein's vision of Brownian motion was not empirically based; instead, \"Brownian motion\" was for him a system that he had mathematically pared down, even created, which could then be observed and measured. Einstein did not so much _describe_ Brownian motion as _manage_ it mathematically, thereby providing researchers with the theoretical guidance they needed, giving them mathematical pointers so that they would know what to look for. As Maiocchi notes, \"Only when Einstein had constructed, _independently_ of the experimental accounts, a sufficiently articulated theory, did the experimenters know _what had to be observed_ and only after this theoretical clarification did the observations turn out to be conclusive.\" Einstein's theory, more than any technology, corralled subsequent observations into a stable of usable data.\n\nOf course, before they could be so guided, the researchers first had to _understand_ Einstein's work, and this was far from a foregone conclusion. As Mary Jo Nye remarks, Einstein's 1905 and 1906 papers included mathematical derivations that \"were certainly beyond the ken of even the more precocious experimentalist.\" The novelty of Einstein's equations lies in his emphasis on the _displacement_ of the particles rather than their actual path or velocity. As noted earlier, Einstein was interested in proving the real existence of molecules and also in demonstrating that thermodynamics was true only statistically and not absolutely. These two goals were intimately related, in that any kinetic theory of molecules had to incorporate a thorough understanding of statistical mechanics. The observed properties of a gas, for example, depend on the _average behavior_ of its molecules. But in any system that is sufficiently random\u2014and the model of gas as a system of molecules assumes this randomness\u2014there will be fluctuations from this average behavior. If such fluctuations were large enough, then they would put into doubt the stability of the measured properties and thereby raise questions about classical thermodynamics. In Brownian motion, Einstein had found a system of observable fluctuations that could prove the existence of molecules.\n\nThese fluctuations were expressed in his equations as the mean square displacement (\u03bb _x_ )2 of a particle in any given direction in any given time interval. Einstein realized that particle velocities were so great that they would never be observed directly and therefore could not be measured accurately. So his solution was to factor the displacement of the particles. Maiocchi explains:\n\nWhile previously the attempt had always been made to estimate the length of the trajectory actually traversed by a particle, Einstein's theory deals with the _displacement_ effected in a given time, i.e., the intervening distance between the points of departure and arrival, _independently of the path followed_. This is a change of radical importance because it changes completely _the object of the observation_ : it is no longer a matter of trying to measure the velocity of the Brownian movements (obtained by dividing the length actually traversed during the observation time by the time itself), but of a different quantity.\n\nThis solved the problem that had bedeviled researchers in their previous attempts to describe the phenomenon, as Stephen Brush argues:\n\nEinstein showed that there is no possibility of observing this velocity.... Hence any attempt to measure the \"instantaneous\" velocity of particles in Brownian movement will give erratic and meaningless results. It is for just this reason that all the efforts of the experimentalists... had failed to lead to any definite conclusion about the average speeds of suspended particles. They were simply measuring the wrong thing until Einstein pointed out that only the ratio of mean square displacement to time could be expected to have any theoretical significance.\n\nPrevious experiments, such as Exner's in 1900, had focused incorrectly on the _path_ of the particles. Since the invention of the ultramicroscope in 1903, researchers had turned to Brownian motion with renewed interest, further spurred by Einstein's paper in 1905. Some followed Exner's lead in measuring the actual paths, not catching Einstein's insight. French physicist Victor Henri's results, for example, published in 1908, are notable in that he too used cinematography to record particle movement and to trace particles' actual paths. His results did not agree with Einstein's theories and momentarily cast doubt on Einstein's equations. But it soon became clear that experimental error negated Henri's results. The winner in the Brownian motion sweepstakes was Henri's countryman Jean Perrin, who ran a series of experiments that were also published in 1908. Initially, Perrin paid little attention to Einstein's papers, but when he examined them more closely after 1908, he realized that his experiments confirmed Einstein's predictions. Further work by Perrin produced the most persuasive experimental confirmation of Einstein's theory; after that, virtually no one in the scientific community doubted the existence of atoms, and in 1926 Perrin won the Nobel Prize for his efforts.\n\nWhich brings us to Seddig. Even though Seddig achieved results comparable with those of Perrin, they were, according to Nye, \"neither as accurate nor as convincing.\" While Seddig is therefore little more than a footnote in the traditional accounts of this moment in the history of science, he did discover an important homology between an element of his experimental method (cinematography and chronophotography) and an aspect of the new theory of physics (displacement). Like Exner, Seddig was interested in verifying the dependence of particle velocity on the temperature of the fluid. And like Exner, Henri, and others, Seddig first attempted to record the actual paths of the particles via photographic exposure through an ultramicroscope. He hoped that with long exposures of the particles, the traces left on the photographic plates would correspond to the paths:\n\nIt seemed obvious to attempt to photograph the moving particles, which show up in the ultramicroscope as luminous points, on a stationary plate for a certain exposure time (around one second). The luminous, moving points should then sketch black lines on the plate, which should correspond to the horizontal path covered during this time. The lengths of the resulting curves obtained at _different_ temperatures but during _identical_ intervals should then stand in the reciprocal relation predicted by the theory.\n\nBut again, as with Exner, this method failed due to the weak light from the ultramicroscope and the relatively insensitive photographic emulsions available at the time.\n\nHowever, after this failure, Seddig employed a regular microscope, which provided stronger light and therefore better exposures, and an attached cinematograph. Again, like Henri, he tried to record the movements of the particles cinematographically. As he notes in his first presentation of his work, \"A cinematic method with a precision camera gave some results that were also sufficiently precise.\" While his modified cinematic apparatus seemed to achieve a greater degree of exactitude, he was eventually dissatisfied with the instability of the cinematic image in his efforts to trace the paths. So his final results were determined with the use of an ultramicroscope and a series of multiply exposed photographic plates (fig. 1.12). But while experimenting with cinematography, Seddig came upon the solution to a vexing problem. He had noticed that the strong light required to illuminate the particles also heated the suspending fluid, generating a temperature increase that could not be predicted and that therefore jeopardized his data. He minimized this problem by substituting an intermittent light source for a continuous one. The lamp fired two successive flashes one-tenth of a second apart, while the camera was rigged with a system to measure exactly the time interval between the flashes (fig. 1.13). The light source moreover was projected through a series of cooling and polarizing filters and reflected off a mirror into the microscope, which was attached to the camera. The camera was attached to an electrical circuit so that with each exposure the circuit would open and close, creating an electrical spark that was graphically recorded on a rotating drum covered with blackened paper. The graphic record served as a control for the frame rate so that the interval between exposures could be measured precisely.\n\nFIGURE 1.12. Seddig's photographic rendering of Brownian motion\n\nFIGURE 1.13. Seddig's cinematic apparatus for measuring Brownian motion\n\nFIGURE 1.14. The irregular paths of Brownian motion\n\nWith this setup, Seddig could record the movement of any given particle, but not completely: the intermittent flash recorded only two points along that highly irregular path (for an example, see fig. 1.14)\u2014say, point _A_ 1 and point _A_ 2. The path of the particle between these two points, which occurred _between_ the frames, left no trace and was therefore experimentally elided. All that could be measured was the straight line between the two points\u2014 _which was exactly the empirical translation of Einstein_ ' _s displacement equation_. In other words, Seddig had fashioned an experimental method that corresponded to (and therefore partially confirmed) Einstein's theory. Einstein praised Seddig's efforts, even if he was a bit befuddled by Seddig's method: \"I have read Seddig's paper. He has done it very well. I cannot quite make head or tail of his descriptions of the results.\"\n\nWhy was \"displacement\" so crucial to Einstein's theory? Because it worked around one of the major objections to the kinetic theory: the disparity between theoretical velocity and observational velocity at the molecular level. As Polish physicist Maryan Smoluchowski pointed out, experimental observation of particle velocity would never correspond to the theory, because theoretical velocity was simply not measurable by observational procedures: \"What we see is only the mean position of the particle, driven 10\u201320 times a second, each time in a different direction, by that velocity. Its center will describe an unpredictable zig-zag path made up of straight lines _much shorter in length than the size of the particle_. Its displacement becomes visible only when the geometric sum of these lines is raised to an appreciable value.\" Jean Perrin put it in another way:\n\nThe apparent mean speed of a grain during a given time varies _in the wildest way_ in magnitude and direction, and does not tend to a limit as the time taken for an observation decreases, as may easily be shown by noting, in the camera lucida, the positions occupied by a grain from minute to minute, and then every five seconds, or, better still, by photographing them every twentieth of a second, as has been done by Victor Henri, Comandon, and de Broglie when kinematographing the movement.\n\nThat is, the particle, buffeted randomly by molecules, constantly moves ever so slightly in a variety of directions\u2014these zigzags, smaller than the particle itself, cannot be observed and therefore measured or empirically confirmed. With the reduction of the equation to a question of displacement _without direction_ , velocity (speed + direction) was no longer an issue.\n\nThus the gap. Einstein's theory elided the actual path of the particles over time, erasing it from the equation, thereby creating a theoretical _interruption_ (\u00e0 la Bergson) that was matched by Seddig's cinematic interruption. By focusing on the dark space _between_ the frames\u2014or, chronophotographically, between exposures\u2014Seddig's method mimicked Einstein's theoretical attempt to erase the space between two points along a particle's unpredictable path. The gap between exposures was long enough to allow Seddig to measure the displacement of the particle from point _A_ 1 to point _A_ 2. If it had been too small a gap, Seddig would have traced (erroneously) the path of the particle, as Henri did. Seddig therefore found in his apparatus a specific use that serendipitously suited his needs.\n\nSeddig's application of photography, chronophotography, and motion pictures to the problem of Brownian motion represents a particularly resourceful partnership between film and physics. We could leave it at that and still note with satisfaction the especially neat fit of technology and theory that this application evinces. Seddig's techniques matched certain elements of Einstein's equations and while lacking the precision and expertise of Perrin's experiments, still counted as partial confirmation. But this correspondence of technology and theory also testifies to Bergson's insight into the deeper connection between motion pictures and science. If science's success in finding mathematical correspondences takes advantage of what might be considered a \"temporal interruption\" in the constant flux of becoming\u2014a \"temporal interruption\" we call \"form\"\u2014by finding the mathematical, atemporal line of geometry in that form, then the scientific use of motion picture technology often matched this process by creating its own temporal interruption\u2014the space between frames, for example\u2014and finding mathematical correspondences to the resulting form. The temporal character of motion was therefore reduced to a series of static, discrete, two-dimensional images; time and motion were then \"understood\" by the measurement of differences between them. Motion pictures, in this application, reinforced the tendency to mistake the category of space for the category of time.\n\nFurthermore, the spatialization of time allows us to conceive of time as reversible. Our felt experience of time is that it is irreversible, but our experience of space is that we can pursue it in any direction. So when we represent the flow of time as displacement along a homogenous axis, \"Nothing prevents us in this abstract representation,\" as French physicist Louis de Broglie remarked, \"from supposing that we may reverse the course of time, contrary to the most certain property of real duration.\" Likewise, a mathematical equation also implies this equivalence and reversibility. As Suzanne Guerlac explains, \"In geometrical terms, if we depict a movement from left to right, we can reformulate it as passing from right to left. In algebraic terms, equations are commutable.\" But when we conceive physical processes as reversible, we might fail to consider certain essential properties of real time, namely its directionality. Bergson, of course, argued for a dynamic ontology of irreversible time so that important properties of duration do not escape our understanding.\n\nBy erasing the direction of the particle\u2014whether the movement is from point _A_ 1 to point _A_ 2 or from point _A_ 2 to point _A_ 1 is irrelevant for the purposes of the theory\u2014Einstein's equations implied a temporal reversibility that was also mimicked by Seddig's use of photographic and motion picture technology. In more than one way, the scientific use of film (especially in the physical sciences) was mathematical; in Seddig's hands, film functioned mathematically. How was this accomplished? Three aspects of this kind of application\u2014the use of a flash, the use of the frame, and the form of the celluloid strip\u2014combined to create a formal, almost geometric support for the mathematical rendering of the phenomenon. Seddig used his flash as an intermittent light source that would not raise fluid temperature as much as constant illumination. But the flash also immobilized the particle by reducing exposure time to an instant and the spatial fullness of the phenomenon to a two-dimensional position. The flash focused both time and space to the sharpness of a point. Furthermore, the regularity of the flash created a series of equivalent points\u2014 _A_ 1, _A_ 2, _A_ 3...\u2014each of which corresponded to a single frame. The frame, in other words, determined the boundaries of the event, both spatially, in that the event took place within the field of the frame, and temporally, in that the frame represented a unit of time. So the flash and the frame reduced, limited, and focused the fullness of the event to a series of separable, measurable points\u2014which is to say that the technology accommodated a mathematical understanding of the phenomenon.\n\nWhile the photographic series is not strictly commutable\u2014one cannot rearrange the order of the units and still obtain the same result\u2014it is reversible, in that [ _A_ 1, _A_ 2, _A_ 3...] is mathematically the same as [... _A_ 3, _A_ 2, _A_ 1]. In this respect, the celluloid strip itself functioned as a line, as a trajectory of completed motion. Bergson distrusted the representation of movement as a line, because such a rendering confuses movement as we experience it\u2014that is, as something indivisible\u2014with our imaginative recreation of that movement as a series of stages or points. More precisely, it confuses movement with the line traversed; it conflates what is happening right now with what just happened. The path is past, while movement exists in the moment. We may divide the path into points, but these points have no reality outside of the line drawn. Bergson argued that the difference is simple: \"At a stage we _halt_ , whereas at these points the moving body _passes_.\" We therefore mistake immobility for mobility, discontinuity for continuity, and past for present. Representing motion as a line, especially one that predicts future motion, is an illusion, which rises from the conception that \"motion, _once completed_ , has deposited along its course an immobile trajectory on which one may count as many immobilities as one wishes. From this, one concludes that motion deposits at each instant a position with which it coincides.\" In truth, however, no such deposit occurs, because movement itself does not halt; otherwise it would not be movement. But motion pictures reinforce this illusion, not so much through the projected image, which, as Gilles Deleuze pointed out, _is_ movement, but through the celluloid strip of individual frames. What is an exposed film frame except a \"deposit\" of a position coinciding with movement? And just like a line, the series of immobile instants on the celluloid strip is completely reversible. Bergson might therefore have argued that cinematic exposure itself is a category mistake.\n\nNERVE FIBERS, TISSUE CULTURES, AND MOTION PICTURES\n\nToward the end of his dissertation, Seddig remarked on the close similarity of Brownian motion and cell movement, calling for further investigation into the relation between temperature fluctuation and \"biological motion\" that might lead to an understanding of the deeper \"causes of movement itself.\" Even if this particular research program never really caught on, in a way it points to the interesting history of cooperation between biologists and physicists during the early days of microcinematography. The precise date of the first moving pictures taken through a microscope has not been recorded, but Marey devotes a chapter to the technique in his 1894 book, _Le mouvement_. Marey's experiments were continued by various disciples, most of them working at the Institut Marey in France. Lucien Bull, for example, is known best for his high-speed cinematography of fast-moving objects, which was an important technique in physics for research on surface tension phenomena. In 1903 he helped biology Professor Antoine Pizon microcinematically record the multiplication of a colony of _Botryllus_ , or sea squirts. Another disciple of Marey and a pioneer in the field, Charles Fran\u00e7ois-Franck, adapted Marey's apparatus and made chronophotographic plates of a variety of biological phenomena from 1902 to 1908. Lucienne Chevroton, who worked at the Coll\u00e8ge de France alongside Fran\u00e7ois-Franck, similarly added a motion picture camera to a microscope apparatus in 1909 to record microcinematographic investigations of a sea urchin egg. Fran\u00e7ois-Franck and Chevroton offered their apparatus to their colleague Victor Henri, a physicist at the Coll\u00e8ge de France, who used it to record Brownian motion, as we saw in the previous section. He, in turn, may have introduced it to Jean Comandon, who during the early years became the most successful microcinematographer of cell movement. Lucien Bull also helped outfit Swiss biologist Julius Ries with a Lumi\u00e8re microscope\u2013cinematograph combination for his research on the fertilization of sea urchin eggs. As early as 1900, with the help of Charles Gaumont, French physicist Henri B\u00e9nard used cinematography to study dynamic, convective systems\u2014specifically, the spontaneous formation of inorganic matter into hexagonal shapes in a heated liquid\u2014and went so far to comment on the analogy between this type of motion and biological phenomena. In Germany, physicist Henry Siedentopf, who ran Carl Zeiss's optical laboratory in Jena and collaborated with Richard Zsigmondy on the invention of the ultramicroscope in 1903, actively used motion picture techniques to investigate crystallography. Siedentopf also helped Heidelberg biologist Hermann Braus with his cinematic investigations of tissue culture, thereby continuing this trend of cinematographic collaboration between biology and physics.\n\nThis cooperation between physicists and biologists interested in adapting motion picture technology is not surprising, given that investigators in both fields had set out to study motion as a way of answering difficult questions about causality. Brownian motion, as we have seen, functioned as an observable system that could provide clues to the constituents of matter itself. Biologists similarly searched for observable, temporally continuous systems in which the analysis of motion could resolve previously intractable disputes about the processes governing the organism. Braus, a morphologist and experimenter, used motion pictures for precisely this purpose. His case is interesting because it illustrates the close formal relation between cinema and biology at the turn of the century; motion picture technology not only helped solve evidentiary problems in biology, but the introduction of cinematic means into experimental technique matched broader changes in the discipline itself. Furthermore, Braus's use of motion pictures demonstrates especially dramatically cinema's _rhetorical_ power in science; Braus altered his viewpoint in a dispute because of what he witnessed in a filmic record. This section will place Braus's cinematic contribution in the context of certain broader debates and experimental trends in the discipline of biology.\n\nThe dispute in question\u2014between Braus (fig. 1.15) and American experimenter Ross Granville Harrison (fig. 1.16)\u2014concerned the development of nerve fibers. Braus and Harrison were not the only figures in the debate, which had been brewing since the mid-nineteenth century. The problem was this: while it was clear that nerve fibers grew, it was not clear _how_ they grew. Did a fiber grow from one cell or from a chain of cells? How was the connection between fibers and tissues established? Did the nerve grow from the center to the periphery, or was the connection already there in the embryo? The disputants offered at least three theories. Many believed that the formation of the nerve required the participation of many cells, not just one. This was known as the \"multicellular\" theory of nerve development. Others, such as Braus, maintained that nerve fibers had already been present in embryonic form and that as the organism developed and cells grew and multiplied, the nerves stretched across them, making bridges of nerve fibers that differentiated as they became functional. This was the \"protoplasmic bridge\" theory. Still others, such as Harrison, argued that nerve fibers grew outward from a single nerve cell into the interstices between other cells. This became known as the \"outgrowth\" theory, and most biologists would come to agree, but not until around 1930, that this theory was the most accurate and persuasive depiction of nerve fiber development (fig. 1.17).\n\nFIGURE 1.15. Hermann Braus\n\nCourtesy of the National Library of Medicine\n\nFIGURE 1.16. Ross Granville Harrison\n\nCourtesy of the National Library of Medicine\n\nFIGURE 1.17. Harrison's sketches of the elongation of frog nerve fibers grown in culture\n\nFrom Ross G. Harrison, \"The Outgrowth of the Nerve Fiber as a Mode of Protoplasmic Movement,\" _Journal of Experimental Zoology_ 9, no. 4 (December 1910): 787\u2013846\n\nThere were a number of reasons why investigators in the early twentieth century could not agree on this topic, including prevailing concepts of the structure of the nervous system. But a large part of the problem derived from contemporary methods of observation. As Harrison complained in 1906, \"Prior to the year 1904 all attempts to solve these problems were based on observations made upon successive stages of normal embryos. When one compares the careful analyses of their observations, as given by various authors, one cannot but be convinced of the futility of trying by this method to satisfy everyone that any particular view is correct.\" That is, researchers attempted to observe the growth of nerve fibers by slicing, staining, and mounting successive stages of an embryo, which were then viewed through a microscope. This technique had long been dominant in biology and developmental embryology. Crucial questions about the origin, direction, and character of growth had to be inferred from what was effectively a series of still images.\n\nBecause investigators basically had to speculate about any movement taking place between images, disputes over interpretation were common. Among the most famously frustrating was the debate between Santiago Ram\u00f3n y Cajal, who subscribed to the outgrowth theory, and Hans Held, who held onto the protoplasmic bridge theory. Susan Billings explains, \"Cajal claimed that _Plasmodesmen_ were artifacts of Held's preparative method, while Held, in turn, thought perhaps pale staining or alcohol fixation prevented Cajal from seeing them. The impression one might get from reading their papers is that Cajal and Held were discussing vastly different material.\" Yet when Cajal visited Held's laboratory in Germany, he said that he \"had the pleasure of examining Held's excellent preparations. Just as we expected, they are very successful, and to our great surprise, they show very much the same picture as ours.\" As with Brownian motion, researchers were unable to agree on what they saw in what were nearly identical preparations. (Indeed, this is another example of the influence of theory on observation.)\n\nThe histological technique of slicing, solidifying, staining according to type, and then mounting the preparation on a microscope slide had been the foundation of cell research up through the nineteenth century. It served biology well; as Hannah Landecker notes, \"the technique of staining, by suspending the cell in time, freed the experimenter from the temporal exigencies of a living subject.\" Precisely because the subject did not move, the researcher could examine the preparation at his or her leisure. \"Cellular movement, or lack thereof,\" Landecker continues, \"was a matter of inference when using static representations, the hardened moments preserved in histological specimens. It was about inferring what was happening in the spaces between the sequential slices of preserved moments.\" Indeed, perhaps because this method was so dominant, movement itself was not important to biology's research questions\u2014one could even argue that biologists' questions were often tailored to their technique. However, once questions of movement and growth could not be answered, new techniques were required; alternatively, we could argue that once the new techniques were available, new questions came to the foreground. Physicists had solved this problem of observation and interpretation in the case of Brownian motion by emphasizing the gap or discontinuity, thereby excising from the theory that which was not observable. Biologists could not do this, however, because their ostensible object of study was continuity itself. So they had to develop techniques that would allow them to study cell movement directly.\n\nHarrison believed that \"the only hope of settling these problems definitely lies, therefore, in experimentation.\" In 1905, Braus approached these questions experimentally by transplanting limbs of very young tadpoles to various areas of other tadpoles' bodies. He then examined the nerves in the transplanted limbs, finding evidence in favor of the protoplasmic bridge theory. Harrison repeated these experiments and came to completely different conclusions. In fact, Harrison argued, \"The same facts [in Braus] may be interpreted quite readily, if not more so, in accordance with outgrowth theory. The experiments do not approach the problem directly enough to determine questions of histogenesis [the origin of the nerve fiber], and there are too many loopholes left to permit a rigid proof.\" What was needed, apparently, was a means of isolating the nerve cell itself so that the growth of the nerve fiber could be observed directly. In fact, this is exactly what Harrison did in a now-famous experiment that he first described in a June 1907 announcement:\n\nThe immediate object of the following experiments was to obtain a method by which the end of a growing nerve could be brought under direct observation while alive.... The method employed was to isolate pieces of embryonic tissue, known to give rise to nerve fibers.... The pieces were taken from frog embryos about 3 mm. long.... After carefully dissecting it out, the piece of tissue is removed by a fine pipette to a cover slip upon which is a drop of lymph freshly drawn from one of the lymph sacs of an adult frog. The lymph clots very quickly, holding the tissue in a fixed position. The cover slip is then inverted over a hollow slide and the rim sealed with paraffin. When reasonable aseptic precautions are taken, tissues will live under these conditions for a week and in some cases specimens have been kept alive for nearly four weeks. Such specimens may be readily observed from day to day under highly magnifying powers.\n\nBy successfully maintaining a tissue specimen outside of the body (known as \"in vitro,\" whereas the state of tissue inside the body is known as \"in vivo\"), Harrison initiated the technique of \"tissue culture,\" a biological method whereby fragments of tissue from an organism are transferred to an artificial environment in which they can continue to survive and function in some form. With the cell population thereby isolated, the researcher could better examine and manipulate cell behavior. The histologist could observe the growth of the cell directly under the microscope and even slow down or accelerate that growth by manipulating the environment. Investigators from all quarters immediately took notice. Within the next few years, the literature on tissue culture exploded; its most famous experiments were those conducted by Alexis Carrel and Montrose Burrows at the Rockefeller Institute, where they managed to keep the culture from a chicken embryo alive for decades.\n\nThis method also presented a major break with traditional methods of visualization in biology. Not only did the in vitro experimental technique offer a different way of isolating the cells, but this form of representation also replaced the discontinuous form of representation then common to biology (for example, sequential slides, illustrations of stages of development, still photographs) with temporal continuity (movement, growth). Tissue culture allowed the researcher to observe movement in time; there still remained the challenge of representing this movement, of course, given the traditional means (slides at conferences or illustrations in texts and journals). Harrison's experiment also showed fairly conclusively that the nerve fiber grew from a single cell. It demonstrated that the fiber could and did grow without the help of other cells and that nerve growth did not require the presence of preformed protoplasmic bridges. Even so, biologists such as Hans Held challenged Harrison's results for years afterward, maintaining that the nerve fiber grew on its own in vitro but grew with the aid of bridges in vivo.\n\nSo in September 1911, Braus's demonstration before the Society of German Natural Scientists and Physicians spoke to both the interest in the new technique and the enduring controversy about nerve fiber development. His demonstration also addressed new challenges in representation in biology, in that he used motion pictures to depict these two issues. At the start of the talk, Braus discussed the still-new technique of tissue culture as Harrison and Carrel had developed it to that point. He then noted, \"I thought that many of you would be interested in seeing the phenomena of growth and movement in these cultures, if not in a live culture, at least via cinematographic images. I have films that were made in order to study the more detailed movements in the cultures of my many preparations, in which the transformations were too fast or too slow to be observed in the objects themselves.\" This offering to his colleagues at the conference deserves further examination, because it indicates two ways in which film had a crucial role in the experiment.\n\nFirst, Braus used film to _communicate_ the results of his work. In this respect, the medium of motion pictures functioned as one of any number of forms of communication that describe or depict experimental procedures so that the (expert) reader can judge the validity and value of the project. Some have called this expert communication and evaluation \"virtual witnessing\"; in the modern era, when there is no way to inspect or replicate every experiment personally, the significance of such reports for the dissemination and acceptance of new knowledge cannot be underestimated. In this early example, film promoted \"virtual witnessing\" in an important new way, not only because film made the cultures present to the expert witnesses in a more than virtual manner but because it was portable and repeatable in a way that live cultures were not.\n\nSecond, Braus used film as a _substitute_ for his object of study (the tissue from a frog's heart). He made the films to study movements that were \"too fast or too slow to be observed in the objects themselves\"; his films were therefore the object of his analysis. In fact, the capabilities of motion pictures so closely matched the capabilities of tissue culture that they became an experiential substitute for the technique. That is, the projected image moves and can thereby present movement or growth, but film was also, like tissue culture, fragmentary and easily malleable, both temporally and spatially. If the histologist could isolate, dissect, transplant, graft, and retard or accelerate the growth of cells, then the cinematographer could match these actions perfectly by framing, cutting, splicing, editing together, and slowing down or speeding up the film itself. (Of course, motion pictures were quantitatively different than tissue cultures because of the ease with which images could be multiplied or accelerated.) In this respect, Braus's use of film became what we might call a \"virtual experiment\"\u2014manipulation of the film could isolate and analyze aspects of the tissue that could also be manipulated, isolated, and analyzed in the technique of tissue culture itself.\n\nBraus then described the star attraction of his films: a beating heart extracted from a frog embryo and maintained in culture for days. That achievement was in itself noteworthy, but Braus was interested in proving something more: \"As far as I can tell, observations about the _growth_ of organs in vitro are not addressed in the literature.\" That is, he wanted to demonstrate that specimens actually grew in culture, they did not merely survive. So he used staining techniques and time-lapse cinematography: \"To prove that the culture, whose pulsation I've shown, is actually growing, I have filmed isolated, stained cells from the culture at greater magnifications. The exposures occurred, on average, every 10 minutes over the course of 10 hours. Here in the projection they have been compressed into just a few minutes, so that we can see the movements of the stained cells unfold rapidly, although in reality they are very slow.\" The stains allowed Braus to distinguish the movement of the cells and their changing form as they grew and multiplied. His films thereby functioned not just as a substitute for the culture, but as a new form of evidence.\n\nThis is a particularly potent example of the power of cinema to present evidence forcefully, because the cinematic record of his experiments with tissue culture caused Braus to reverse his position on the origin of nerve fibers.\n\nTo supplement this, I am showing images of growing nerves, in which\u2014just as with the mesoderm cells\u2014many of these growth phenomena can be observed under the microscope _directly_. The nerves are of particular interest because of the _in vitro_ heart movements. At the time of extraction and the end of the experiment, the heart structure had no ganglia cells.... But from our experiences with nerves grown _in vitro_ , we can rule out the claim that actual nerves originate in any other way than from the central neuroblasts. I want to discuss here objections that could be raised against this conclusion, reached by Harrison based on his preparations, which prompted me to verify his findings. This investigation has persuaded me that Harrison is basically correct.\n\nHaving witnessed these processes for himself through his filmic record, he was able to declare that \"only the isolation of neuroblasts [the nerve cells] gives total certainty; the nerve fiber grows out of the neuroblast like a mold grows out of an isolated spore.\" Braus went on to defend Harrison against the objections of other colleagues in the field, with whom he previously agreed.\n\nSo Braus also used film as a _confirmation_ of Harrison's theory. Braus's motion pictures were more than a demonstration; they _enacted_ the technique and Harrison's theory of growth. Harrison posited the nerve cell fiber as a plant-like \"outgrowth\" rather than a differentiated element of a multiplying mass. He argued that nerve fiber growth moved forward incrementally, outwardly, and linearly. Likewise, Braus's time-lapse technique in this instance\u2014with its statistical sampling of time through regular exposures and its amplification of the forward push of incremental growth\u2014enacted and insisted on an incremental, regular, mathematical, and resolutely _linear_ theory of growth. In other words, this set of techniques (the frame's isolation of the tissue, the temporal manipulation of growth, and the magnification and projection of the image) enacted, amplified, and confirmed Harrison's theory of growth and technique of tissue culture with incomparable rhetorical power. While cell growth, when illustrated by sequential images (such as slides), could have been seen as reversible and thus disputable when direction was at issue, the temporal continuity of the cinematic record _pushed_ the observer (including Braus) to a particular conclusion\u2014that the growth of the nerve fiber happens in _this_ way. If other scientific uses of cinema try to stop time, this application stressed duration and the teleology of growth. As a means of visualization and as an experimental tool, this application of motion pictures precisely matched broader changes in experimental technique and representation in biology at the turn of the century.\n\nAs we can see, motion picture technology presented itself as a potential research instrument to biologists involved in tissue culture, but film became a powerful tool for researchers because the research program in biology had changed to accommodate it. That is, Braus came to film not only because it solved a particular problem but because the problem itself was shaped by changes in conceptions of growth and time that motion picture technology could articulate (indeed, that the moving image might have prompted). Of course, the practical benefits outlined at the beginning of this chapter provided compelling reasons to use cinematography. In Braus's case, it offered the opportunity to record his experiments and then to present some of his findings to a wide audience. The photographic or filmic image also projected the impression of objectivity and thereby provided a potent rhetorical weapon in his arguments with the conclusions of other biologists. And film's ability to manipulate time allowed Braus to present quickly evidence that would have taken hours to observe directly. But beyond these obvious advantages, we could argue that the growth Braus documented was researchable only _because of_ cinema. The motion picture camera\u2014because it could alter the scale of cellular time through slow-motion or time-lapse cinematography\u2014could capture events that were not otherwise visible. These events were not merely difficult to observe without the camera, in a real sense they did not exist without it. They became, as Walter Benjamin noted, our \"optical unconscious,\" revealed to us by cinematography in the same way that our psychic unconscious is revealed by\u2014 _and comes into being by virtue of_ \u2014psychoanalysis.\n\nBut there is more to the success of cinema in science and its relatively rapid appropriation than even these good (practical) reasons. Not only were filmic techniques increasingly available, they were also adaptable to various scientific enterprises. If Seddig focused on the space between the frames, on cinema's _analytic_ ability, Braus similarly used the time between exposures to his advantage. But for Braus the image of the object was paramount. The images made little sense analytically; instead, Braus concentrated on their _synthesis_. For Braus the gap was crucial for the construction of the film, but the ellipsis was elided in favor of a temporal continuity or biological teleology. Growth is not reversible. This is not to say that film's analytic abilities were confined to (or more suitable for) the physical sciences, while its synthetic nature was best suited to the life sciences. The variety of applications in a range of disciplines makes any such generalization rash. In fact, as Latour might argue, cinema's adaptability was no greater or less than that of any technology\u2014from the microscope to the computer. Nor was Braus's \"synthetic\" use of cinema any less scientific than Seddig's \"analytic\" emphasis. Braus was doing more than \"simply\" recording a phenomenon and sharing it with others, even though he did not explicitly extract quantitative data from the images. Braus's use of time-lapse cinematography recorded only certain, regularly paced moments over the continuity of an event, thereby creating, I would argue, _a statistical sampling_ of the temporal dimension of the phenomenon. We can therefore see Braus's particular appropriation of motion picture technology as a statistical application of cinematography very much in keeping with the contemporary adoption of statistical thinking for the study of systems in both physics and biology. Indeed, part of the persuasiveness of these records could be found in their \"objective,\" mathematical construction of a representative sample.\n\nBergson, however, articulated the limits of analysis with respect to dynamic processes, and an extended comparison of the use of motion pictures in physics and biology would expose the paradox of analysis and synthesis as a problem of comprehending continuity through discontinuity. Thinking about Bergson and motion pictures together, in other words, highlights fundamental assumptions in certain scientific approaches about the nature of time and movement. Seddig's use of motion picture technology to study Brownian motion certainly reveals a commitment to interruption, reversibility, and immobility in the study of dynamic systems\u2014a commitment that was obviously present in Einstein's equations as well. The ambidexterity of motion picture technology\u2014its dual nature as an instrument of both still and moving images\u2014not only explains its success in science, but its varied and subtle permutations in scientific application can themselves be understood as a form of commentary on scientific theory and method. How researchers used motion picture technology in their experiments tells us much about their disciplinary and philosophical investments.\n\nAbove all, I want to stress the mutually reinforcing relationship between science and cinema. The problems and solutions that the technology and the research program presented to each other were entwined and dependent. For Braus and others, motion picture technology fit quite well with other elements of this experimental ensemble, in part because certain formal features\u2014its temporal malleability; its ability to frame and isolate; the forward, teleological motion of its projected image; and so on\u2014seemed to match similar features of the technique of tissue culture. There are other, broader homologies as well. The technique of tissue culture extracted life, reproduced it technologically, and prolonged it, allowing researchers to artificially accelerate or slow life down. And what is cinema except a technology that extracts bodies from their natural time and space, reproducing them mechanically, reanimating them repeatedly (faster or slower as needed) long after the host has expired? The idea that tissues, organs, and life itself are separable from the body\u2014and, by implication, that death is foreign to the organism\u2014is, like cinema, an especially modern notion. So we could say that cinema's singular facility with time, space, and life made it an ideal instrument to capture and confront these concepts in modern science. But we need not go so far. In fact, such expansive analogies tempt us to think of cinema and science as single entities, when the point of these case studies has been to demonstrate the opposite: that film is not monolithic and that science, like other broad umbrellas, contains many different disciplinary agendas, each of which has adopted some aspects of film technology while ignoring others. That so many different researchers found something to use in motion pictures indeed indicates a special relationship between scientists and film. Braune and Fischer's deconstruction and reconstruction of the human body, adapting it to modern labor systems; Einstein's and Seddig's flexible notions of time expressed in the invisible, random, and reversible world of molecular physics; Harrison's and Braus's conceptions of life as separable, temporally malleable, and technologically reproducible\u2014in each case, these scientists turned to motion pictures not merely to observe and record phenomena, but because in cinema they found a kindred spirit, an amiable partner that shared their vision, that could look at the world as they did. The cinematograph, then, was not just a handy tool. In more ways than Bergson imagined, the form and application of motion pictures articulated science's modern agendas.\n\nIn this chapter, we have examined the merger or accommodation of film form and objects, theories, and disciplinary agendas. In each case, film proved its worth because of this correspondence\u2014not exclusively, of course, because the amount of work necessary to adapt film to these aspects of scientific endeavor is prodigious and varied. But we can credit the researchers for recognizing the homology between film and experiment in the first place. The next chapter will examine medical research films and the correspondence between film form and _observational_ practices or ideals.\n\nBETWEEN OBSERVATION AND SPECTATORSHIP\n\nMEDICINE, MOVIES, AND MASS CULTURE\n\nStillness is the unattainable value.\n\n\u2014PAUL VAL\u00c9RY (1932)\n\nFrom around 1904 to 1914, motion pictures endured a difficult and very public transition between their good standing as a scientific tool and their growing notoriety as an instrument of mass culture. During the early period in Germany especially, there was a strong contrast between the enthusiasm for motion pictures as a scientific or pedagogical tool and the simultaneous condemnation of its public, commercial incarnation. Physicians in particular wrote many editorials and studies depicting the threat that motion pictures posed to the psychic, physical, and moral health of the nation. To a certain extent, this is unsurprising: the German complaints about cinema follow more or less the same pattern that we can find in the United Kingdom, France, Russia, and the United States at this time. There seems to be no reason to think that the discussions in the scientific community and those in the public sphere were related or exceptional. But if we look closely, we find that the scientific and public debates about cinema were in fact closely related: the reasons scientists and physicians accepted film as a scientific instrument were rooted in the same logic that prompted them and others to reject cinema's public manifestation. This logic concerned, to put it too simply, the perceived difference between _observation_ and _spectatorship_. That is, participants in both discussions seemed to agree on the advantages and dangers of the moving image, but many of these advantages and dangers appear to stem from _different ways of viewing the image_.\n\nIf the previous chapter focused primarily on the negotiation between object and apparatus\u2014such as Braune and Fischer's adjustments to their recruit, to their working space, and to their camera to create a scientifically productive image\u2014this chapter emphasizes the relationship between researcher and image. How the researcher _studies_ the image may be just as significant as how he or she produces it. Indeed, \"observation\" is a woefully underused key to understanding medical practice and identity. Many physicians felt that their professional reputation and livelihood depended on carefully cultivated observational skills. No less a personage than the eminent British surgeon Sir James Paget emphasized in 1887 that \"becoming scientific in our profession [requires] the training of the mind in the power and habit of accurately observing facts.... The main thing for progress and for self-improvement is accurate observation.\" As Paget indicates, part of this investment in observation stemmed from the contemporary adoption of scientific principles in medical practice, which some contested, but which ultimately bolstered the field's legitimacy. As physicians identified with scientists, \"observation\" became an important common link. But it is not simply a borrowed scientific practice, it is also key to understanding modern medical logic. As Michel Foucault and others have shown, modern medicine's understanding and approach to disease is inextricably tethered to specific habits of perception. The process of observation was not simply preliminary to diagnosis but actually rehearsed the logic by which medicine grasped its object.\n\nWe can see this process especially clearly when physicians encountered the cinematic image. How they used motion pictures and what they said about them can reveal the subtleties and complexities of their observational practice. As I have argued elsewhere, modern medicine's reliance on the correlation between life and death, or between movement and stillness, if you will, makes motion pictures a privileged representational technology for exploring medical hermeneutics. In their attempt to understand and locate pathology, physicians find in the corpse lesions that might be blamed for death and then compare these to signs in the living body that might be harbingers of disease. In the stilled corpse, researchers are able to grasp some elements of disease that are elusive in life. Similarly, physicians take advantage of the duality of motion pictures by going back and forth between the stilled and the moving image to grasp elusive properties of the recorded object or event. Exploring this process in detail reveals not only the doctor's cultural investment in observational practice but also the foundations of modern medical logic. Examining the medical community's reaction to movies, on the other hand, also reveals this investment in expert observation and their characterization of its opposite\u2014spectatorship\u2014as a pathological condition.\n\nNearly from the beginning, motion pictures were enlisted as an aid to medical research and education. German doctors, in particular, were captivated by the potential of the cinematic image. Certainly, the application of motion picture technology to medicine was not qualitatively different in Germany than in any of the other Western, industrialized countries. Germany, however, did enjoy a quantitative distinction: between 1900 and 1920, Germans wrote far more and far more frequently about medical cinematography than their counterparts in the United Kingdom, France, the United States, and the rest of the world. One could say that Germans wrote more about everything, but Germany's leadership in certain specialties that made extensive use of cinematography, notably radiology and neurology, helps validate this claim. It could also be argued that in the late nineteenth and early twentieth centuries, Germany's institutional research infrastructure was among the best in the world, which allowed its scientists to incorporate new instruments and agendas more quickly than, for example, researchers in the United States at this time. Of course, the number of journal articles praising cinema's potential as a new medical technology should not imply that the general practitioner put it to use on a daily basis. On the contrary, given the expense, skill, and patience required to operate a motion picture camera\u2014much less one adapted for medical use\u2014the average doctor never came in contact with one or even considered the possibility. During this period, the medical application of cinema remained confined primarily to university research laboratories and some medical school lecture halls, with a few privileged physicians leading the way. Still, German researchers enthusiastically applied motion pictures to a range of specialties from ophthalmology to gynecology.\n\nWhat did cinematography promise the intrepid researcher? This physician waxes poetic about microcinematography, but his claims are representative of all medical and scientific uses of motion pictures:\n\nMicrocinematography captures [ _fixiert_ ] what a scholar has witnessed and researched in his quiet laboratory, so that he can then demonstrate his collected research to a larger group, be it at a conference, before a medical society, or in an auditorium of students. But microcinematography not only documents movement processes, it is also a helpful tool for research itself. It captures every phase of movement and thereby allows the researcher to examine each image at length and to study the temporal and spatial relationships of the movements carefully and calmly. Microcinematography does not require hasty progress, as living movement often does; it allows the scholar the time he needs to grasp all worthwhile detail and lets him determine when to move to the next image.\n\nCompressed in this paragraph are most of the advantages typically cited in the early literature on the scientific application of motion picture technology. To recap briefly: first, the cinematic image, like the magic lantern slide, was projectable, and therefore could be shared among colleagues and students. This made it ideal for educational purposes, which required an efficient dissemination of information, but also for rhetorical purposes; the scientific film, like all scientific illustration, existed to present evidence meant to persuade others. The size and spectacle of the cinematic image had a powerful persuasive effect in itself, but part of that power came from the photographic character of the image. This speaks to the second advantage, its ability to \"capture\" or to document moving phenomena. Because its photographic base allowed it to record the variety and randomness of the natural world, the motion picture had an evidentiary status that was very useful for presentations and publications, because it could therefore act as a substitute for the living patient. This also meant that it could function as an object of study itself; it \"provides the researcher the opportunity to examine each image at length.\" This is the third advantage: the spatial and temporal malleability of the motion picture extended the senses and allowed the researcher to explore new domains previously unavailable. In addition, the individual frames of the film effectively managed the data, especially the informational complexity of any kind of movement. Indeed, the most noteworthy aspect of this excerpt is its enthusiasm for the researcher's ability to control the rate of movement of the motion picture. The author lauds the temporal control that came with the moving image and emphasizes the slow pace of scientific study, the almost contemplative stance before the image: \"in his quiet laboratory... to examine each image at length... carefully and calmly... the time he needs.\" The rest of this chapter will explore these advantages in detail.\n\nBut for now, this testimonial will stand in contrast to other descriptions of film common in the medical community\u2014those that decried cinema as a threat to public health. As medicine's prestige grew, physicians expanded their expertise to broader social questions and offered their prescriptions for a healthy nation. So alongside the enthusiasm for the scientific potential of motion pictures there was a separate yet related discourse that was not so enthusiastic. Between 1904 and 1914, reformers and physicians in Germany consistently viewed the new medium of motion pictures with suspicion. They not only protested the dank, unventilated storefronts in which many motion pictures were then being screened, but also felt that the motion pictures themselves presented a serious danger to public safety. The sensational subject matter of many of the movies threatened to corrupt the taste of the nation. But a particularly insidious form delivered this content, as this doctor attests:\n\nThe effect of this sensational subject matter is heightened by the _temporal concentration of events_. The cinema concentrates the sensations of a detective story or thick trashy novel into 10 or 15 minutes. The resulting psychological effect is thus completely different. When reading, we can pause at will, critically reflect on the text, relieve ourselves of the internal pressure through contemplation, digest the scary parts. In the cinema, such excitation of the passions is intensified and multiplied by the rapid succession of vivid images that passes before our eyes. There is no time for reflection or release, no time to find an inner balance. These spine-chilling and grotesque \"dramas\" distress the nervous systems of young and sensitive persons to the point of suffering, but without providing the spectator any of the means with which he usually defends himself against attacks on his nervous system: quiet _contemplation_ , _intellectual assimilation_ , and sober criticism are not possible.\n\nThe contrast with the previous quotation is instructive. If cinema provided the _researcher_ the opportunity \"to examine each image at length and to study the temporal and spatial relationships of the movements carefully and leisurely,\" it did not afford _the average moviegoer_ that same luxury\u2014\"quiet _contemplation_ , _intellectual assimilation_ , and sober criticism are not possible.\" The difference between the (boring) research film and the (exciting) film drama perhaps played a role in this characterization, but even more important for the purposes of this chapter was cinema's temporal rush, its insistent and impatient movement forward, which this doctor felt could have a lulling, even hypnotic effect. Those without sufficient training or education were susceptible to cinema's lurid charms. But the educated, professional class apparently had the tools to manage this onslaught. What were these tools? Most obviously, researchers, unlike the average moviegoing audience, were in control of the image and its projection\u2014they could literally manipulate their films however they pleased. But this ability to control the rate of projection went hand in hand with a mode of viewing that was very much part of a nineteenth-century researcher's training and identity, which distinguished itself from another mode of viewing attributed to the lay public, especially the cinema audience.\n\nExpert training depends on developing a set of skills, but it also distinguishes itself from those who lack the skills. In the medical profession, this dynamic determined the nature of the doctor\u2013patient relationship, or the nature of internal debates about midwives and quacks, for example; the medical profession relied on the constant maintenance of that hazy line between \"normal\" and \"abnormal,\" or between \"healthy\" and \"diseased\" in the broadest sense. Medical observation similarly depended on its opposite\u2014in this case, spectatorship. The debates about motion pictures are an excellent example of this dynamic. Comparing these two discourses allows us to see quite clearly the criteria for cinema's legitimacy. By understanding what German doctors considered to be the _proper_ mode of observation, we come to understand more fully what they thought was an _improper_ way of viewing images, and vice versa. We also see that this proper mode was not simply the result of disciplinary training. The \"objectivity\" of the scientific eye arose not merely out of professionalization but also in contrast to the \"subjectivity\" of the untrained other. That is, disciplinary modes of viewing relied on _class_ distinctions, as well as professional training.\n\nSo to appreciate the medical community's diagnosis of film spectatorship as passive, weak-willed self-abandonment to the flow of images\u2014a characterization that was not uniquely made by doctors, but which is best understood with reference to them\u2014we must take into account its investment in observational practice, especially as it relates to experiment and temporality. Physicians and their educated brethren distinguished themselves from the layperson via their viewing practices: by incorporating film into experimental method, for example, they observed and contemplated the image at a leisurely, disinterested pace, while ordinary spectators were swept up and seduced by it. These observational practices were therefore more than cultural capital, more than a sign of expert learning and status\u2014they were a badge of honor that also served as a shield against the encroachment of mass culture and the rush of modern life. How one approached cinema's temporality, it seems, determined whether one was an observer or a spectator.\n\nThe rest of this chapter consists of three sections. Because the early history of medical filmmaking is not yet common knowledge in film studies, the first section provides an introductory survey of the early use of motion picture technology in medical research and training, focusing on such work in Germany. This section will examine the multiple functions of film in the medical context by focusing on the needs that motion pictures served in the international medical community. (This discussion will focus on medical research and training films\u2014films by physicians for physicians\u2014rather than medical films intended to educate the general public.) The second section explores how the use of motion pictures corresponded to established principles of observation in medicine. It will also demonstrate the professional investment in this particular mode of viewing. While the first section emphasizes film and the history of medicine, this section focuses on film and the _philosophy_ of medicine. It therefore investigates the use of film in relation to medical logic. Finally, the last section takes a closer look at the medical editorials about the dangers of cinema, especially its hypnotic power, in order to illuminate the moral, ethical, and class dimensions of medical observation and cinematic spectatorship.\n\nTHE MULTIPLE FUNCTIONS OF THE MEDICAL FILM\n\nMost histories of research films recount individual cases within various disciplines or fields (just as I did in chapter 1), but it would be worthwhile at this point to step back and sketch broader patterns of use across specialties. Describing these patterns in terms of \"function\" has the advantage of accounting for the full life of a film beyond the immediate task. As we have seen, motion pictures functioned in various ways for researchers; they extended the senses, of course, but they also documented and displayed data. Even though a researcher made a film for a specific purpose\u2014to examine the mechanics of an organ, for example\u2014that same film also functioned as a record of the case and as an educational aid; it was a rare medical film indeed that had only one use. I would argue that there are three main functions of the research film: _exploratory_ , _documentary_ , and _educational_. These broad functions could be applied to much nonfiction filmmaking as well, but as a triad they are especially applicable to research films of all sorts, medical or scientific. Generally speaking, research films are made to explore unknown domains or relationships; to document and display results; and to train students, teachers, or, sometimes, the general public. These functions are by no means mutually exclusive; any film can have multiple, even simultaneous, functions. In fact, understanding how a film functions necessarily entails pinpointing who is using the film, when, and for what purpose.\n\nWe can group various cases in the history of the medical film around function, a retrospective critical category, but also around specific, historical needs that motion pictures satisfied for medical research. For example, as we have seen in physiology and biology, researchers at the turn of the century were increasingly interested in process and function over structure and morphology; the discovery of X-rays only heightened the prevailing interest in the mechanics of organ function, for instance. Motion pictures also proved to be a valuable technology for visualizing, documenting, and measuring the functions of the human body. Likewise, just as photography was an important method of documenting cases, motion pictures also proved indispensable for recording, for example, pathological movement. Such documents served as entries in an archive of images that medical professionals recognized to be vital to the health of their discipline. Physicians established image archives in hospitals and other research centers in Europe and the United States, and calls for similar motion picture archives of medical films were common at this time. Physicians recognized the importance of these films for medical education as well. While the profession was very self-conscious of the modernization of medical education that had taken place since the Enlightenment\u2014replacing the medieval, scholastic model of book learning with an emphasis on hands-on training and observation\u2014the practice of live demonstration of patient symptoms in medical lectures was logistically and ethically troublesome. Motion pictures offered a way out of this dilemma and presented a novel solution to issues in medical training. The rhetoric surrounding the use of film as an educational tool mirrored the broader discourse on pedagogical applications of cinema, but the medical training film illustrates especially clearly the contemporary emphasis on efficiency and modernization. So this section presents a brief survey of early medical filmmaking using the three functions as a preliminary heuristic, but it will also chart film's meanings in the context of specific issues (organ function, image archives, live demonstration, and efficiency) important to the medical profession at this historical moment.\n\nTHE EXPLORATORY FUNCTION\n\nOf course, the motion picture camera can be used to explore facets of the natural world below or beyond the threshold of human perception, such as the extremely small (via microcinematography) or the extremely slow (via time-lapse cinematography). The various means by which motion picture cameras can manipulate time and space have been very important to research films. For many medical professionals exploring the rhythms and movements of the human body, this ability to manipulate time also contributed to its use as an _experimental_ instrument, more than simply as an observational aid or recording device. Since the mid-nineteenth century, branches of the medical profession in the Western world had incorporated a scientific ethos into their research and practice. Even though this trend was often contested, by the beginning of the twentieth century, the role of experiment in medical research was firm, if not universal. The experimental use of motion pictures certainly had precedent in the chronophotographic research in physiology of Marey or of Braune and Fischer, but also in the increasingly frequent use of photography in medicine. How can the motion picture camera function as an experimental tool? For that matter, what does an experiment do? To answer these questions, let us consider a fairly well-known example in the history of medical filmmaking: Austrian cardiologist Ludwig Braun's use of a motion picture camera in 1897 to record the beating heart of a dog. A specialist in cardiac dynamics and mechanics, Braun employed frame-by-frame analysis to gauge changes in the size and position of the heart as it beat by measuring the shape and displacement of shadows and other markings on his filmed images. From these short strips he was able to extract conclusive information about, for example, the nature of the apex beat (the lowermost and outermost prominent cardiac pulsation) (fig. 2.1).\n\nFIGURE 2.1. Frames from Ludwig Braun's film of a dog's beating heart\n\nBraun used the camera in a way that instruments are often used in scientific experiments, that is, to test a theory, to measure constants, or to explore new domains. A scientific experiment is usually designed first to isolate and stabilize variables (such as temperature, air pressure, etc.) surrounding an event or phenomenon. An experiment is also designed to be consistently repeatable, not only so that other researchers can check the results for themselves, but, more importantly, so that the event itself can be scrutinized closely. And an experiment rarely stands alone\u2014usually it is one of a series of experiments, so repeatability is crucial for the success of the series as well. Time itself cannot be manipulated, of course, but an experiment allows the duration of an event to be significantly extended through repeated viewings. These three features of an experiment\u2014isolation, stabilization, and repeatability\u2014also allow the researcher to register quantifiable changes in the environment under study. A thermometer is an example of an experimental instrument that isolates and quantifies one particular variable, temperature. Other instruments are used to control or to create an (often artificial) environment for the experiment; the air pump, which creates a useful vacuum, is a good example. Film does not control or alter an environment to a great degree, but it can create something of a _new_ environment, which acts as a record of or substitute for the event. Through its framing and focus, for example, film isolated and stabilized the phenomenon for closer study; a beating heart is difficult to examine, so the filmed record of the heart became a \"working object\" that could be examined repeatedly. The motion picture was the best kind of repeatable experiment: if the record could function as a substitute, it could be endlessly repeated without the work involved in setting up the actual experiment again and again. In this way, as noted in the previous chapter, the motion picture functioned as a \"virtual experiment.\" Researchers also employed motion pictures to register and measure changes by using the constant frame size and frame rate of the film to calculate changes in time and space. Braun used the displacement between frames to measure the size and position of the heart. Finally, the success or legitimacy of an experiment is enhanced if its results can be somehow inscribed (via a published report or visual demonstration) and easily presented or exchanged. In the absence of the actual experiment, the researcher's report of the experiment allows readers a kind of \"virtual witnessing,\" as Steven Shapin calls it. Film, of course, facilitates this \"witnessing\" in an extremely persuasive way. All these features contributed to the motion picture camera's success as an experimental instrument.\n\nBraun's work also exemplified long-standing medical attention to the mechanics of organ function, which was only intensified with the discovery of X-rays and the development of motion pictures. As we know, the discovery of X-rays generated incredible enthusiasm\u2014in 1896 alone there were more than 1,000 articles published on the phenomenon. Much of this excitement came from the medical community, which immediately recognized benefits; engineers and experimenters sought to combine X-rays and cinematography. In 1897, Scottish physician John Macintyre used a cinematograph to take several X-ray exposures of a frog's leg on motion picture film to create the first moving X-ray image. Others continued to work on the combination to visualize organ function. For example, as early as 1903, Dutch physician P. H. Eykman used X-ray cinematography to study the swallowing mechanism in humans. Similarly, between 1903 and 1906, Joachim-L\u00e9on Carvallo at the Institut Marey made a series of X-ray films of swallowing and digestion in small animals. Starting around 1909, the German radiological team of K\u00e4stle, Rieder, and Rosenthal made X-ray films of joint, lung, and peristaltic movements. And American Lewis Gregory Cole likewise made films of gastric phenomena. But the combination was always plagued by difficulties: the low X-ray-tube power and slow emulsions of the early days resulted in weak exposures or overlong exposure times, on one hand, or the mechanical problems involved in capturing the image, either directly with large sheets of X-ray film (known as the \"direct method\") or indirectly through fluoroscopic screens (the \"indirect method\"), led to unstable images, on the other. Franz Groedel was the acknowledged leader in X-ray cinematography; between 1909 and 1915 he worked tirelessly to develop a viable direct method, including a mechanism consisting of a series of falling cassettes (fig. 2.2). His commitment to the idea of moving X-ray images never wavered: \"In spite of [the] present inadequate apparatus,\" he said in 1915, \"important questions have already been solved, or the solution thereof brought nearer.\"\n\nFIGURE 2.2. Groedel's serial cassette X-ray apparatus\n\nMany physicians also hoped that cinematography, and especially X-ray cinematography, could be used diagnostically, that is, as a way of searching for clues that aid the identification of a disease or injury, usually via the correlation of visible symptoms with known disease entities or classifications. But even as Groedel and others lauded its potential, the actual diagnostic value of X-rays and X-ray cinematography remained in doubt for many years. In 1912, one prominent leader in radiology said of X-ray cinematography, \"Of that, I don't expect too much. It will be didactically valuable, explain some processes, but it will not be able to serve diagnostic purposes in every instance.\" Nevertheless, researchers and engineers put considerable energy into crafting X-ray images that moved. French radiologist Andr\u00e9 Lomon collaborated in 1911 with scientific filmmaker Jean Comandon, known best for his masterful microcinematography, to produce moderately successful results with indirect X-ray cinematography. They worked on this approach up to the outbreak of World War I, which \"relegated all roentgen cinematographs to storerooms and museums.\" Groedel, Lomon, and others continued to work on the technical problems after the war, but it was not until the 1930s that the direct method proved regularly feasible, and it took until the 1950s for the indirect method to have daily clinical potential. Despite the inherent difficulties, X-ray cinematography promised at the very least the possibility of viewing hidden processes and movements that would help in understanding organ function and perhaps in diagnosing illness and injury. Indeed, what distinguished the exploratory function, whether manifested in experiment or diagnosis, was the possibility of _discovery_ \u2014the image presented a view that revealed something hidden from human perception. Yet to see something hidden, researchers often limited some variables and expanded others, a process made more convenient through motion picture technology, which could isolate or enlarge spatially (through lens or framing choices) or temporally (through recording or projection speed choices), or, as in the case of X-ray cinematography, film could be combined with other technologies that offered new ways of seeing. So film's exploratory function emphasized discovery, but it also underlined and depended upon film technology's formal analog to the functions of experimental restraint and modularity.\n\nTHE DOCUMENTARY FUNCTION\n\nIf the exploratory function charts new domains and reveals hidden spaces in order to help to understand processes or to identify disease, at some point the domain has been identified and the diagnosis agreed upon. At that point, film serves as an illustration of that which is already known; it becomes equivalent to the statement \"Here is an example of X.\" This is the documentary function of medical cinema. For anything to function as a document, it must have some evidentiary value; the evidential authority of the motion picture derives primarily from its photographic base, which has a well-known rhetorical power. Such is the nature of this image that it served meaningfully as a substitute for the object filmed, especially if the phenomenon was rare or difficult to reproduce in demonstration. This would explain the popularity of motion pictures among neurologists at the turn of the century, who used film to document examples of pathological movement associated with neurological disorders. For example, in 1897, neurologist Paul Schuster utilized the resources of Berlin clinics to create a series of short films of patients with a variety of neurological diseases, including Parkinson's, multiple sclerosis, myoclonus, hemichorea, and ataxia. These single-shot films, between three and ten seconds each, emphasized pathological movement and were designed primarily to illustrate Schuster's lectures without having to rely on live demonstrations of the patients. Schuster also hoped that these films and others like it would someday form an archive of material for medical educators. There were many other physicians and neurologists in Europe and the United States who saw the same potential: Gheorghe Marinescu in Bucharest, Paul Richer at La Salpetri\u00e8re in Paris, Walter Chase in Boston, Arthur Van Gehuchten in Louvain, Emil Kraepelin in Munich, Hans Hennes in Bonn, Osvaldo Polimanti in Naples and Perugia, and Theodore Weisenburg in Philadelphia\u2014all used motion pictures to document nervous disorders.\n\nThese films were especially helpful in medical demonstrations, for which the use of live patients was always troublesome. While the live demonstration was a major advance in medical education\u2014a huge step from the centuries-long, scholastic tradition of learning medicine only from ancient texts\u2014it had its own set of challenges. According to an American student taking classes at the Allgemeines Krankenhaus in Vienna in 1865, each professor was provided with a lecture room near his ward: \"At the time of lecture this room is filled in with 'specimens' in the shape of men and women who are transferred from the other wards for the occasion. These patients are looked upon and spoken of as 'material' for the medical instruction and as such are submitted to examination by the students without much reference to any feelings which they as men and women may have on the subject.\" Patients did not submit gladly, apparently. In another letter, the student draws a sketch of his routine at the hospital, which includes \"scolding and pitching into the patients for coming late (wh. they always do in Vienna).\" Motion pictures promised an alternative, as an obviously frustrated Hans Hennes noted in 1910:\n\nHow often does it happen to the professor that a patient fails during a lecture, that a manic suddenly changes his mood, a catatonic suddenly fails to perform his stereotyped movements. Although he executed his pathological movements without disturbance on the ward, the changed environment of the lecture hall has the effect of not letting him produce his peculiarities\u2014so that he does not display precisely what the professor wanted him to demonstrate. Other patients show their interesting oddities \"maliciously,\" only when there are no lectures, continuing education courses, and so on. Such occurrences, which are frequently disturbing to the clinical lecturer, are almost completely corrected by the cinematograph. The person doing the filming is in a position to wait calmly for the _best possible moment_ to make the recording. Once the filming is done, the pictures are available for reproduction at any moment. Film is always \"in the mood.\" There are no failures.\n\nHennes seemed not to be bothered by any ethical dilemmas; his concerns were purely practical. Motion pictures might have accidentally solved some ethical predicaments, but as Lisa Cartwright has demonstrated, filming patients often led to others. Film technology, in this respect, followed a pattern in medical history in which new instruments create a psychic and physical distance between doctor and patient and thereby further tip the power dynamic in the physician's favor. Physicians were already pleased to substitute photographs and slides for patients in lecture, if not to alleviate the obvious ethical concerns, then at least to present all the students with a larger, projected view. Schuster intended his films to substitute for live demonstrations as well: \"We wanted to use recordings of typical movement complexes to allow students to see the theoretically oriented lecture illustrated with motion pictures, regardless of the clinic's available material [i.e., patients].\"\n\nNeurologists such as Schuster also found motion pictures especially exciting, because the records could be used for further analysis of pathological movement. (This dual use is an example of the function of the film changing according to who is looking at it, students or researchers.) It was also not uncommon to use photography and motion pictures to document intervention outcomes. Orthopedic surgeons, for example, used motion pictures to film \"before and after\" records of corrective surgeries. These films were presented at congresses to persuade other physicians of a given diagnosis and therapy, or they were presented to students as examples of a particular surgical strategy. Even Comandon's motion pictures of microorganisms could be classified under this general rubric of \"demonstration.\"\n\nMedical films, like medical photographs, also appealed to researchers and educators because they could be part of a disciplinary archive of images. Hospitals such as the Saint-Louis in Paris, Bellevue in New York, and the Charit\u00e9 in Berlin established photographic departments for just this purpose. A report from Bellevue in 1869 indicates that a photographic archive and department could be a magnet for the discipline: \"Members of the medical profession begin to visit the Department periodically, for the purpose of obtaining such photographs as pertain to each one's more especial class of investigation. Many interesting cases of skin disease, fractures, and results of important surgical operations have been fully illustrated by series of photographs, which give opportunity for comparison and study not offered by any other means.\" Motion pictures promised the same opportunity for comparison \"fully illustrated by a series of photographs,\" so to speak. Calls for an archive of film records were common. In his important essay on the use of motion pictures in neurology, Hennes saw the immediate value of a permanent storehouse of medical films: \"If one were to collect the most interesting cases in this way with universal participation and support, a cinematic archive, analogous to the phonographic one, could be created, the lasting value of which could certainly not be denied.\" Franz Goerke went beyond medical cinema in his call for an archive:\n\nIs it not therefore obvious that we should take up the idea of creating a central collection place, a state archive for the deposit of motion pictures and so save them from destruction? Just as museums are collection places for works in the fine arts, just as the developments in the sciences have their place in city and state collections, just as libraries are protecting the intellectual achievements of mankind, and just as documents and government papers are kept in state archives, so equally important is a state archive for motion pictures, which will allow researchers in future with the help of other sources and sharply drawn conclusions to reconstruct a visual image of former times.\n\nFor researchers of all sorts, not just physicians, the idea of motion pictures as documents led inevitably to the impulse to collect and store them. As we shall see, this impulse was not exclusive but was especially pertinent to medical thinking. Indeed, of all the possible hopes the medical community had for photography and film, the dream of a universal and portable archive of cases was the most persistent.\n\nTHE EDUCATIONAL FUNCTION\n\nYet showing films was not as easy as it might seem. Despite the obvious suitability of motion pictures for demonstration or reference, film was simply not used as often as magic lantern slides in the lecture hall during the early period. Most universities lacked projection equipment, finding it expensive, cumbersome, and difficult to master. The films themselves, despite dreams of a ready archive, lacked reliable distribution outlets; other than the lists of films available from the major producers (Path\u00e9, Gaumont, Eclipse, Urban), there was no systematic information about available films on medical topics. The large firms generally distributed only the films they produced, so researchers such as Hennes had few options for distributing their films; procuring copies of someone else's usually involved a personal connection of some sort. Or, often, the films just did not exist anymore. Goerke complains, \"For a learned institute it is nearly impossible to obtain a film for an important learned lecture: we know of the film's existence, but in the meantime it has become a victim of destruction or oblivion.\" This is why the calls for an archive were so common: educators just wanted a reliable source of scientific and medical cinema. So if the use of film as an experimental tool or as a document attracted the interest of just a few dedicated (and historiographically visible) pioneers\u2014usually attached to well-funded research institutions in centers such as Berlin and Paris\u2014we cannot say the same for the clinical or educational applications of medical film, which were scarce, geographically scattered, and sparsely documented.\n\nStill, most medical filmmakers at this time cite as their motivation a desire to improve teaching. In fin de si\u00e8cle Paris, Eug\u00e8ne Louis Doyen, a maverick surgeon known for his innovative techniques and disdain for the academy, wrote, \"It has been with the object of completing our means of teaching the art of surgery that I have been led to study and employ the cinematograph.\" Doyen hired two cameramen to film his surgeries. Three of these films\u2014depicting a hysterectomy and a craniectomy\u2014were first presented at the July 1898 meeting of the British Medical Association in Edinburgh, where they were enthusiastically received. Despite this successful screening in the United Kingdom, the French Academy of Medicine refused to show Doyen's films to the membership; he had already run afoul of traditional medicine with his unusual (some would say risky) techniques. His colleagues were not about to let him have a forum. Doyen eventually made dozens of films, including one depicting his surgical separation of conjoined twins Radica and Doodica in 1902. During this time, one of his cameramen, Ambroise-Fran\u00e7ois Parnaland, tried to sell his prints of Doyen's films to Path\u00e9 and other companies, but it is not clear whether they were actually widely screened to the public at this early date. In any case, Doyen's controversial techniques and the graphic quality of the films further deteriorated his relationship with academic medicine. Doyen insisted that his films were didactically valuable; they were meant primarily to illustrate and publicize Doyen's tools and techniques, but they were also intended to serve as training films for surgeons.\n\nIn Germany, the watershed moment came at a February 1910 demonstration of \"Film in the Service of Medicine,\" in which the \"service\" was primarily educational. Representatives from the Berlin medical establishment were so tightly packed into the Kaiserin-Friedrich-Haus that the organizers had to turn people away. Among the films shown were two surgical films made by Doyen, the first featuring famed German surgeon Ernst von Bergmann and the second focused on Doyen himself. The principle organizer, Robert Kutner from Berlin, also showed his film demonstrating CPR techniques, which drew praise for their potentially wide appeal. Other films included James Fr\u00e4nkel's results of corrective orthopedic surgery, Braun's heart film, X-ray films by Carvallo and others, and Karl Reicher's microcinematographic records of single-celled organisms. While it was not the first time that films such as these had been shown in Germany, the event received much attention from the national and international medical community and helped to focus awareness on the educational potential of medical film. In a later speech, Kutner sang cinema's praises:\n\nAnd how convenient, how effortless!... The evidentiary power of [cinema] is more persuasive than that of any other document, exceeding even the most vivid description.... The motion picture projector demonstrates its most spectacular educational applications in auditorium demonstrations of microscopic or macroscopic images of movement. In a normal lecture-room demonstration of movement, especially that of small objects (think, for example, of a frog's beating heart), only a small part of the audience really sees anything, while everyone present can observe a film presentation equally well. Without the assistance of the motion picture projector, almost all X-ray motion pictures and certainly all motion pictures taken from a microscope could only be shown to a small group or even just to one person at a time.\n\nKutner described the pedagogical advantages of motion pictures in a language common to advocates of the educational film at the time, a rhetoric found in Germany but also elsewhere in Europe and in the United States. First, the rhetoric emphasized the \"vividness\" of the motion picture, usually in opposition to \"description\" or, more pointedly, book learning. What exactly is this \"vividness\"? The clarity, texture, and abundant detail of the photographic image combine with projected movement to give the image a _presence_ unlike any previous representational form. Its level of detail allows the photographic image to reproduce patterns of texture and variation, hence to represent the structure and randomness of the natural world, while the movement of the image presents this world in real time in a particularly striking way. The object \"lives\" onscreen. This is perhaps obvious, but it is all to say that \"vividness\" referred to the sense of presence that the moving image evokes. For early advocates of educational film, it was as if the thing itself were there in the room, available to direct perception. Film thereby functioned as an object lesson, an acceptable substitute for the thing itself (in chapter 3 I will discuss the educational implications of motion pictures as an \"object lesson\" more thoroughly).\n\nThis permitted Doyen to extol the virtues of the motion picture over books and corpses. Complaining about the inadequacy of \"surgery of the dead\"\u2014the practice of rehearsing operative techniques on cadavers\u2014to confer a sense of the surgical experience, Doyen asked, \"Do our books fill the gap thus left? Certainly not. The most detailed descriptions, the best diagrams or photographs of the various steps of an operation are inadequate.... It is not sufficient to follow the operation, as it were, secondhand; rather, the author of the technique, the master himself, must be seen at work. The surgeon is judged by his work, and no text-books, however well-illustrated, can sufficiently express his personality.\" In motion pictures, on the other hand, Doyen found a perfect medium to express vividly the personality of \"the master himself.\" Movies were not \"secondhand\"; they allowed Doyen to be \"present\" to the students. Especially noteworthy is Doyen's concept of \"personality.\" Doyen was not publicity shy, by any means, but he was not concerned here with conveying via a medical film his charisma and good looks, or not only those things. Primarily, his films were meant to present his technique and his tools. More specifically, they demonstrated how Doyen held himself and how he moved to accomplish his task. Film provided, better than any other previous medium, a demonstration of the actual movements required in surgery. Doyen's \"personality\" is his \"posture\" or \"attitude\"\u2014his _embodied_ technique. To convey that \"personality\" was to presume that the student would copy it, that while the student watched the film, a kind of kinesthetic empathy took place whereby the movements seen were somehow felt or incorporated into the student's own body. This is the mimetic presumption of most training films, it seems; many training films expect that we will copy their movements and that the student will take on the \"personality\" or \"attitude\" of the master.\n\nSecond, Kutner emphasized the _efficiency_ of the moving image for the pedagogical task. \"Efficiency\" was a mantra repeated by all sorts of social engineers in Europe and the United States around 1900; the medical community also chanted its name, especially but not exclusively in the United States. Efficiency was a key concept in transforming the turn-of-the-century hospital from \"a well of sorrow and charity\" into a \"workplace for the production of health.\" In the United States, for example, from around 1900 to 1920, health officials were increasingly dissatisfied with the duplication of services, the lack of coordination of units, and the general low level of effectiveness in patient care among clinics, dispensaries, and hospitals nationwide. \"Efficiency\" became an institutional logic to promote standardization of facilities, services, and administration. In fact, in the United States at least, efficiency was the rubric through which the modern hospital adopted business practices to establish itself as a desirable place for treatment and to attract paying patients. _Modern Hospital_ , the organ of the American Hospital Association, devoted itself to promoting economy and efficiency in hospital management, while the American College of Surgeons was established initially to focus on the standardization of tools and techniques within surgical practice.\n\nKutner's praise of film also relied on this concept of efficiency. Here he refers, of course, to economies of scale: the simple claim that more people could see a large projected image than could see a small image. As medical school enrollments grew steadily toward the turn of the century, this claim gained traction\u2014lecturers used projected images more and more from the 1870s onward. But Kutner had in mind another form of efficiency; when he wrote \"how convenient, how effortless!,\" he was probably not referring to the motion picture apparatus, which was definitely not convenient and effortless. Instead, he was referring to the efficiency of the image itself. It had a \"persuasive evidentiary power beyond that of any other document, beyond even the most vivid description.\" For Kutner and others, that power came naturally to the image, especially to the photographic image. It worked quickly and effortlessly on the spectator. When Doyen insisted that \"with the cinematograph we can make hundreds of people follow in one minute what a whole lecture could not make clear to a limited number of students,\" he made a similar claim about cinema's efficiency: not simply about numbers of students but about the immediacy of the image versus the indirectness of the spoken word. If the image was direct, instantaneous, vivid, and penetrating, then the written or spoken description was aloof, dull, and circuitous. In a way, Kutner's and Doyen's preferences echo a bias common in modern medical education. The nineteenth century continued a long transformation in medical education (and education in general) that emphasized direct perception of the objects of study over their mediated presentation in books. The discussion of film as an educational tool made this bias even more explicit. The direct perception of objects was seen as a much more effective and efficient mode of learning. The image was perceived as efficient because it could affect the viewer immediately, like a drug, or a blow to the head, whereas the word must be read or spoken and then processed cognitively, all of which takes time. The image was understood as physical and immediate, while the word was intellectual.\n\nPrecisely these perceived qualities of the filmic image\u2014its presence, its directness, its immediacy, and its ability to prompt mimesis\u2014made it an especially efficient training tool in the eyes of physicians and others. Protected by the twin firewalls of expertise and closed community, medical professionals could extol the virtues of film for educational goals. Once films such as these escaped these protections, however, they had the potential to become scandalous, as Doyen's example demonstrates. Furthermore, these cinematic properties _themselves_ were condemned as part of a system of commercial amusements. As we shall see, the objection to cinematic spectatorship revolved around precisely the same constellation of properties; the difference was not only the type of films (fiction versus nonfiction) but also the protections in place for regulating cinematic projection. Under the watchful eye of the educator, film's immediacy could be mediated, paradoxically, by the written or spoken word. The educator provided an expert framework, such as a lecture and projection control, that corralled and attenuated cinema's more vivid effects. In the movie theater, however, the content and its immediacy were left unchecked, which often led, these experts complained, to mimesis of a criminal sort. The paradox of the rhetoric of the educational film was that even though the moving image exemplified the very image of efficiency, that efficiency cut both ways, for good and for ill. The difference between these two visions of film was also the difference between observation and spectatorship, which is the subject of the following sections.\n\nMOTION PICTURES AND MEDICAL OBSERVATION\n\nThe exploratory, documentary, and educational functions of the medical film give some indication of how motion pictures were and are used by the medical community. But they do not, by themselves, present a clear picture of how motion pictures fit established, disciplinary observational models, nor do they explain the shrill and regular objections to movies in the theaters. The next section will examine those protests in detail, but the argument of this section is that to understand those complaints, we must first understand the researcher's investment in a particular mode of viewing related to medical and scientific observational practices. The condemnation of \"movies\" in the public sphere and the praise of \"motion pictures\" in the scientific context have in common a stance about the proper way to process visual information. This section outlines that position by exploring how researchers processed data in the motion pictures they used for medical purposes. On one hand, as we have already seen, medical researchers found the ability to analyze movement frame by frame to be an enormous advantage of motion pictures. On the other hand, the uses for motion pictures were not exclusively analytic\u2014the moving image itself was also very important for medical purposes; the efforts to perfect cineradiography are a good example of this application. In either case, however, the patterns of use reveal a need to regulate the insistent temporal push of the moving image. The patterns also correspond to certain common features of medical observation, especially the importance of the series to medical logic and the claim that observational accuracy depends on the observer's willingness to be methodical and leisurely.\n\nIn other words, I argue for a homology between observation and film; more precisely, there are certain features of film\u2014namely, its repeatability, its photographic detail, and its ambidexterity between movement and stillness\u2014that correspond to features of observational practice emphasized in the medical literature. Moreover, certain aspects of observation, such as correlation and description, could be easily adapted to film technology. Selected observational practices appear to have found purchase in the cinematic image. This section will chart these correspondences.\n\nMy approach to the argument will be unorthodox, however. Rather than survey the history of medical observation and compare it with early uses of motion pictures in medicine, I will take one statement as representative of all. In an 1897 presentation to the Society of German Natural Scientists and Physicians (Gesellschaft deutscher Naturforscher und \u00c4rzte), Ludwig Braun listed six characteristics of cinematography most important for his work. Braun provided, at the very beginning of the medical community's interest in the technology, an articulate and representative example of the discourse on medical film. Taken collectively, all six theses recognized and applauded the exploratory, documentary, and educational functions of motion pictures in the medical arena. The film provided a \"good, detailed\" document that could be used as a working object or substitute for the heart itself. The nature of the medium allowed frame-by-frame exploration of the movement of the heart. And the film could be presented in an educational setting, where everyone could see the movement clearly. Throughout the history of medical film, these have been the primary advantages that advocates have listed in favor of using this technology. Braun's list can therefore function synecdochially for the larger discourse on scientific and medical film. But it also demonstrates quite clearly the elements of medical observation most germane to his use of film. So in what follows, I examine each of his theses as a pretext to an explanation of medical logic, the relationship between analysis and synthesis in medical observation, and the temporality of the medical gaze.\n\n1. _The cinematograph delivers a long series of chronophotographic images. Each individual image has the virtues of a good, detailed photograph. The film can record the movements of a normal-colored, physiologically moving heart just as well as those of a heart that has been purposefully manipulated in various ways that serve the specific experiment._\n\nAt first, the last sentence appears out of place; its discussion of film's ability to record a manipulated organ equally as well as one that has not been altered seems both obvious and odd, given the emphasis on the photographic character of the medium in the previous sentences. But it is precisely the history of the use of photography in medicine to this point that prompts the statement. Despite the popular conception of photography as a reliable document, in 1897 there was a wide gap between what one saw in an organ and what one got in a photograph. The extreme variability in film emulsions, for instance, along with equal variability in photographic expertise meant that \"standardization was not one of photography's strong points\" in the nineteenth and early twentieth centuries. For example, until the introduction of orthochromatic and panchromatic photographic emulsions in 1884 and 1905, respectively, the reddish-yellow color of anatomical specimens came out too dark in photographs unless special precautions were taken. So if a researcher were interested in the color or texture of an organ, some sort of manipulation was required to create a good photograph. Braun emphasized here, however, that when using motion pictures, movement is the object of study, so one does not _need_ to make these adjustments, although that option is still available. For the purposes of recording a \"physiologically _moving_ heart,\" film's problems with color and texture were less relevant, thereby giving motion pictures a hidden advantage over the \"good, detailed photograph.\"\n\nNevertheless, we cannot ignore the importance of the \"good, detailed photograph\" in Braun's scheme. The photographic character of the image was the foundation upon which Braun built his argument for motion pictures. But the most important aspect of the image was its _repeatability_ : that \"each individual image\" could be examined as a photograph and that there were a series of them available for that purpose. This statement is one of the clearest indications that, as Lisa Cartwright has noted, medical researchers at this time used film primarily as series photography. But why was series photography interesting for physicians? How did it fit their disciplinary needs? Series photography presented, in a representational form, what we might call the \"statistical\" aspect of medical logic. Ludwik Fleck, in a 1927 article on \"Some Specific Features of the Medical Way of Thinking,\" argued that the fundamental cognitive task of medicine is to find a law for irregular phenomena. Medicine attends to the human body, which is a wildly variable and stubbornly individual object. Finding some sort of consistency in this variability, especially when focusing on atypical, morbid phenomena associated with disease, requires the examination of many, many cases. The clinic was the prototypical site where this observation occurred; physicians used \"statistical juxtaposition and comparison of many such phenomena\" to arrive at an \"ideal\" picture of the disease. Hence, \"the role of statistics in medicine is immense. It is only numerous, very numerous observations that eliminate the individual character of the morbid element\" and allow a regular \"law\" to emerge, to the extent that this is possible. Medical logic relies on series of cases found in clinics to come to an understanding of the \"facts\" or symptoms of a particular disease. Through the repetition of these facts and their variation, the researcher begins to see the pattern that becomes the ideal picture of the disease.\n\nCertainly, observing a patient in a clinic is much different than looking at a photograph or a film. The physical presence of the patient calls for not just a visual examination but palpation, percussion, auscultation, and other physical diagnostic techniques, not to mention the active questioning of the patient, none of which can be done to a photograph. But as Bernike Pasveer explained, part of the process of making images diagnostically viable involved encoding the images with information gathered by physical examination. That is, a successful X-ray image of a patient with pulmonary tuberculosis was able to show information (such as position of the lesions) that one could render or confirm from auscultation, for example. Learning to read the image required correlating sight and sound. The image thereby became a visual analog of the sounds and sensations obtained by physical examination. At that point, the photograph became a legitimate working object and could be compared with _other_ photographs. Photographs could be compared like cases, but the photo could even become the basis of comparison of these cases, in that the image eventually standardized a vocabulary and a way of looking at the disease (and the patient).\n\nSo series photography acted as a formal analog to the series of cases in a clinic, without having either the clinic or the actual cases present. Photography functioned \"statistically\" by presenting a series of images of different cases to be compared, or different views of the same case, either spatially (for example, different angles of view) or temporally (for example, tracking the stages of a disease as it develops). However, it is very important to emphasize that at this point in the development of medical cinema, the diagnostic value of photography, series photography, or cinematography was very limited. (Even the diagnostic value of X-ray images at this time was restricted.) Usually the image functioned as a document of an already established diagnosis or as the presentation of a question for others to answer. But the image itself did very little work in the detection required to establish an etiology of disease. Yes, the archive of images did help in the recognition of _patterns_ of disease\u2014a series of photographs might declare, \"Here is the course smallpox takes over nine days,\" for example. But the cause of the disease remained hidden to the camera. In this respect, the value of series photography and motion pictures for Braun and others was more experimental than diagnostic; the images functioned to control variables, to manipulate time, and to measure phenomena more than they detected causes or invisible relationships. It was only with the development of radiography and cineradiography that this possibility was broached, but the true diagnostic potential of the moving medical image was still decades away.\n\nSeries photography was much different than still photography in that the series was capable of depicting the duration and transformation of disease. But series photography and Braun's use of cinematography were similar to still photography in one clinic-like respect: they all functioned as an aid to _correlation_ , by which I mean establishing a mutual or reciprocal relationship between objects or events, such as correlating lesions in a cadaver to disease elements in a living patient. The clinic, as a collection of similar cases in a single location, functioned as an archive of the common symptoms and development of a disease. Series photography worked in that way as well. Photography's ability to isolate, frame, and repeat similar cases was a powerful aid in the standardization and multiplication of observational views. In addition, the arrangement of photographs in a series allowed not only their sequential organization but also their _simultaneous_ presentation. Georges Didi-Huberman has argued that Charcot's arrangement of his patients into living tableaux functioned like tables of data by organizing their signs into simultaneous events. Photography allowed this same organization, and much more easily. In series photography, the sequence was important, because it suggested a causal order or chronology, but the simultaneous display of images was arguably equally essential to the process of comparison and correlation. Series photography, as a research tool that allowed both sequential analysis and simultaneous display, thereby succinctly articulated the ideals of medical observation and logic. Braun's use of motion pictures as series photography also matched this logic, especially in the way he compared the images, as the next theses demonstrate.\n\n2. _The study of the resulting\u2014especially the enlarged\u2014images allows the analysis of movement, the recognition of every intermediate state and every phase of the process, and with that, a more exact assessment of the resulting transitional steps than was possible before._\n\n3. _The individual photograms are very uniform. If one lays two successive images on top of each other, those parts that remain motionless match perfectly, while the moving parts show positions that correspond to the differences in their movement. From this, one can perceive the spatial displacements, judge them better than before, and to a certain extent measure them and calculate the speed of the displacement in space from the time of exposure and the number of exposed frames._\n\nClearly, medical observation is not simply passive looking. Observation calls on more than vision alone\u2014it requires all of the senses combined with an extensive linguistic and logical apparatus. Foucault described \"the clinical gaze\" as a perceptual act sustained by logical operations, and correlation is one of its primary logical maneuvers. Medical photography and film functioned in many ways, but they operated primarily, I argue, as an aid to correlation. In fact, with his detailed description in thesis three of how he compares individual images, Braun offered an example of how correlation works. He laid two images on top of each other, aligning their similarities; from this alignment, the salient differences emerged. Likewise in the clinic, the repetition of cases over time created a cumulative, collective \"image\" of the disease, in which the more or less constant aspects of the entity were stabilized and the variations were set aside for further study, then reintegrated into the \"picture\" of the malady. Medical photography abstracted and accelerated this process; the number of photos of a particular affliction accumulated and created something of a \"virtual\" clinic, an archive of cases to be aligned and compared. Series photography worked the same way on a smaller scale by providing images to align and compare so that variations in the single case could be correlated. Cinematography worked on an even smaller scale: it created images not just of a single case but of a single moment. But the point here is that one of the key features of motion pictures in this mode was not necessarily the depiction of movement but _the generation of images for comparison_. That is, for Braun's minute research on the mechanics of the heart, ever more images were needed to make meaningful correlations. Cinema was useful in this case not because it depicted movement but because it functioned as an \"instant archive\" of images for comparison.\n\nBraun was mostly interested in measurement; he was working toward a precise tabulation of the dynamics of the heart. So his second thesis emphasized cinema's ability to aid the \"analysis of movement.\" The comparison of images that he outlined was designed, like Braune and Fischer's apparatus discussed in chapter 1, to extract data for the measurement of movements. The paradox, of course, is that his analysis of movement entailed, as we learned from Bergson in chapter 1, the comparison and measurement of different states of rest. When Braun emphasized \"the analysis of movement,\" he focused on various stages of motion or \"transitional steps.\" Those intermediate stages, however, were actually states of rest, or at least they functioned that way as still images. The analysis of movement was therefore a carefully designed comparison of various states of stillness. Just as paradoxical is the equation of \"intermediate states\" of movement with the film frame. That is, if analysis implied the \"recognition of every intermediate state,\" then it is not going too far to suggest that those states, those \"resulting transitional steps,\" to some extent resulted from the process of analysis itself. As the heart beat, the film frames divided and presented its natural articulations. The smallest possible \"step\" was between two frames; another step might have been between several. But in every case, the frame was the measure of the transition; the \"event\" was equated with the temporal boundaries implied by the spatial boundaries of the frame. Chronophotography, for example, was essentially a record of successive phases of motion: in deciding the time between exposures, the researcher already conceded that X amount of time would equal a single \"phase.\" This is as clear an indication as any that the technology the researcher used helped to shape his or her conception of the object. Braun's understanding of movement depended at least partly on his understanding of motion pictures. Indeed, it also appears at first glance that Braun's understanding of movement and continuity depended entirely on a method based on stillness and discontinuity, \u00e0 la Bergson. This, however, was not always the case, as the next three theses indicate.\n\n4. _The cinematographic shot can be used to present to the observer any movement of the heart synthetically, at will, and even decelerated to a rather great extent without impairing the clarity of the images._\n\nWhen Braun wrote that cinematography can present \"any movement of the heart synthetically,\" he meant it can show actual movement not the decomposed movement of analysis. If analysis breaks down movement, synthesis is the reconstitution of movement, the projection of a moving image. We could argue that the relationship between film's still images and their movement in projection corresponds to the relationship between analysis and synthesis in scientific method. In that tradition, synthesis functioned as an important check on analysis; analysis decomposed a phenomenon into its constituent parts, and synthesis reconstituted those parts to verify that the analysis was correct. Synthesis appears therefore to be only a supplement to the more valuable labor of analysis. This was how Marey used cinematography: as a way of training expert vision by decomposing movement and building it slowly back up through synthesis, which taught the viewer what to look for when the movement was encountered again. If he was dismissive of the moving image, having no interest in it other than as a verification of his analytic results, historians of the scientific film have often taken that dismissal to be definitive. But that ignores the real scientific value\u2014not to mention the pedagogical value\u2014of the moving image. Many other researchers, including Braun, have noted the importance of the moving image for understanding their objects of study. Thesis four gives an indication of that value, which in this case was primarily correlative. As we saw in the second and third theses, motion picture technology generated an \"instant archive\" of images for comparison; in frame-by-frame analysis, this comparison was used most often for measurement: the images were used to track spatial displacement. Braun used moving images not for measurement but for correlation of a different sort. The temporal malleability of film\u2014it could be \"decelerated to a rather great extent\"\u2014created a new set of views for comparison. That is, just as the proliferation of images generated many _spatial_ displacements to be compared, so the variability of projection speed generated many _temporal_ \"views\" to be evaluated. Different projection speeds revealed different aspects of the movement, which were collected and compared for a better understanding of the whole. This is a rather common procedure in the scientific appraisal of research films, which demonstrates that while frame-by-frame analysis may be the best way to derive quantitative data from film, it is clearly not the only kind of information that researchers get from motion pictures. Synthesis was not merely a supplement to analysis, but an equal partner.\n\n5. _The reproduction of movement in slow motion grants the observer more time to recognize significant features, to evaluate precisely the elements of movement, to determine whether or not all the parts of the heart start moving at the same time, and to compare the contraction process of the various parts of the heart, especially of both ventricles._\n\nIn thesis 5, Braun goes on to describe exactly how slow-motion projection of the images helped his project. It allowed him to recognize, compare, evaluate, and decide on various features of the heart's actions. How did it do this? The ability to manipulate the speed of the presentation gave him \"more time\" ( _l\u00e4ngere Zeitr\u00e4ume_ ). This is arguably the key feature of scientific and medical filmmaking, the reason any researcher chooses motion pictures over other representational technologies. To comprehend\u2014from the Latin \"to grasp\"\u2014any complex action requires that we slow it down and bring it within reach of our senses and limited processing abilities. Motion picture technology allowed that to happen. It gave the researcher \"more time\" to contemplate the details of the actions under study, especially when he or she exercised his or her exclusive privilege to control the pace of projection. In scientific filmmaking, as in experiment, the inexorable forward push of time and of the motion picture can be manipulated, slowed, stopped, accelerated. This was also true for narrative or any other kind of filmmaking, but the difference rested not in the technique but in the ability of the scientist to control the image, an ability not available to the spectator in the audience. I will have more to say later in the chapter about how this scientific approach to time becomes normative when applied to movies in the theater, but for now we can simply note the importance Braun and others give to cinema's ability to make \"more time.\"\n\nThis ability to control or to extend the time for examination of the event was a crucial advantage for researchers. Before exploring its manifestations in the medical research film, I want to point out how this ability related to established practices of medical observation. I am especially interested in the temporality of observation, which is addressed in historical discussions about proper method in medical observation, in Foucault's discussion of \"the clinical gaze,\" and in Michael Hau's concept of \"the holistic gaze.\" First, method: throughout the nineteenth century, physicians insisted on the difference between careful and careless observation, which depended on the ability to diligently apply a prescribed method. Textbooks at the time sought to outline this method for students and junior practitioners. British physiologist Thomas Laycock wrote one such text, in which he made it clear: \"The foundation of medical experience is observation of disease, and the requisites to successful observation are minuteness and accuracy. The clinical student must therefore make up his mind to be sedulously minute and carefully accurate in investigating the cases under his notice.\" German pathologist Rudolph Virchow described in detail his method of performing autopsies. Autopsies conducted in a haphazard way promoted interpretive error, he argued, so he \"drew particular attention to the necessity of insisting\u2014in autopsies for medico-legal purposes, as in everything else now\u2014upon completeness of examination and exactness of method, both in the investigation and in note-taking, so that it might be decided subsequently, though not in anticipation, whether there was any significance or importance in what was observed, or whether it was accidental and unessential.\" Virchow was particularly careful to describe exactly what should be observed in an autopsy and the order in which observations should be made. Method therefore precluded haste. An observation could be made quickly, to be sure, but over and over physicians warned their students against the dangers of hasty observation. Instead, observation was to be practiced carefully, leisurely, with an eye to detail (at least until one was trained).\n\nIndeed, detail itself prohibited hasty observation; if one properly attended to and assimilated the details, it took time to do so. Foucault argued that \"the clinical gaze\" emerged from precisely this intersection of vision, detail, and language. With the nineteenth century, according to Foucault, \"Rational discourse is based less on the geometry of light than on the insistent, impenetrable density of the object.\" That is, if Western thought since Descartes equated truth with light and light with an abstract ideal, then during the nineteenth century \"the solidity, obscurity, the density of things closed in upon themselves, have powers of truth that they owe not to light, but to _the slowness of the gaze_ that passes over them, around them, and gradually into them, bringing them nothing more than its own light.\" The clinical gaze cast its own light, searching across the dense landscape of information in the clinic and separating the essential from the inessential. So for Foucault, this careful gaze was inseparable from the process of analysis. Foucault characterized the analytic aspect of medical perception as a process of simultaneously recognizing, separating, naming, and acting upon some disease element. The clinical gaze was a process of seeing, thinking, and naming, which therefore became a deeply contemplative maneuver that took its time. German surgeon and professor Theodor Billroth emphasized the imaginative element in this process: \"Solitary, meditative observation is the first step in the poetry of research, in the formation of scientific phantasies, the reality of which we then test with the tools of logic, mathematics, physics and chemistry. Our tests will be the more successful the better we have learned to handle these tools. The diseased organism, the patient, must be observed in just this way, thoughtfully, and in a state of mental solitude and meditation.\" The abundance of detail, the richness of the information\u2014what Foucault calls \"the plentitude of concrete things\"\u2014was at least partly the reason for this emphasis on a contemplative, solitary, slow, and steady gaze.\n\nBut physicians characterized medical observation not only in these terms. Michael Hau has described medical observation as an active, gestalt-like process of holistic apprehension; he argues that many German physicians at the turn of the century objected to the analytic, overly scientific approach to observation that had become fashionable in the medical community. Threatened by the new technologies and methods common to laboratory science, many physicians took refuge in an explicitly artistic approach to observation, emphasizing their \"ability to 'see' and to synthesize seemingly fragmented characteristics of a human body into an aesthetic, coherent whole.\" By making proclamations about the relationship between health and beauty, these physicians stressed an aesthetic approach to the whole body to differentiate themselves from the more specialized and decidedly more fragmented approach of, say, bacteriologists. If the \"holistic\" gaze was more immediate and intuitive, that might distinguish it from the contemplative and correlative gaze described earlier, but it could also describe the brand of medical perception Foucault calls \"the glance.\" Foucault explains the difference between the clinical, analytic gaze and the more synthetic glance: \"The gaze implies an open field, and its essential activity is of the successive order of reading.... The glance, on the other hand, does not scan a field: it strikes at one point, which is central or decisive; the gaze is endlessly modulated, the glance goes straight to its object. The glance chooses a line that instantly distinguishes the essential.\" This form of the expert eye is similar to what Lorraine Daston describes as \"all-at-onceness\" in scientific observation: an intuitive, immediate understanding of the relationships presented by an image or by phenomenon. This distinction between types of medical perception is useful for our purposes, because it highlights the duration or temporality of observation: the difference between the searching, contemplative gaze of the explorer and the immediate, intuitive glance of the connoisseur. With regard to motion pictures, Braun found the first mode of observation especially valuable, because it corresponded with film's ability to make \"more time\" to recognize, compare, evaluate, and decide. But neither Marey nor Braun dismissed the glance or the synthesis of images entirely.\n\n6. _Finally, the cinematograph can project single images to a large auditorium, and can also present animated images as \"living photography\" by showing the entire chain of images synthetically._\n\nWhile they perhaps do not immediately correspond to the experience of motion pictures, Foucault's \"gaze\" and \"glance\" remind us that medical (and scientific) observation was not only analytic but also encompassed a more synthetic, holistic, or intuitive element. Likewise, Braun's use of motion pictures both as still photographs and as moving images should stress the equal value of both for medical cinema. The individual frame was very helpful for measurement or correlation, while the projected image had important rhetorical, educational, and even documentary uses. It makes little sense to separate them; we should, in fact, consider the relationship between the still image and the moving image as a vital dialectic for medical understanding. Physicians went back and forth between the two forms of the medical film in a productive, yet prescribed way that preserved and proved their expertise. Indeed, Braun's sixth thesis is all about expertise, however obliquely. Motion pictures could be used in the auditorium, where they could instruct and dazzle the audience with the latest in medical knowledge and technology. Imagine Braun (or others) giving a medical lecture on heart function, in which he has control of the film image. Braun could present his material one frame at a time, as a slide show, carefully delineating for his audience the precise difference between one phase and another. He could take as much time as he needed, with the image still projected, to describe the salient features of the organ's mechanism. But Braun could also capitalize on the rhetorical power of motion pictures by turning, with a flourish, the single image into \"living photography.\" This provided the medical students with another kind of information, while at the same time powerfully persuading them of Braun's argument. But even the spectacular aspect of the moving image was attenuated and contained by two barriers mentioned earlier in this chapter: expertise (\"we know what we are looking for in the image\") and closed community (\"this knowledge is exclusive to us\"). That is, the seriousness of purpose implied by the specialized educational setting contained the full power of the moving image. Even so, presenting \"the animated image as 'living photography'\" implied an almost Doyen-like commitment to showmanship, mastery, and self-promotion. Through the alternation of still and moving images, of analytic and synthetic approaches, surgeon became magician and back again, experts both.\n\nIt is also worthwhile to note the cultural capital invested in the gaze and the glance. In textbooks and treatises on medical observation, authors stressed that the expert eye did not come easily: \"Aspiring physicians will never be able to develop an eye for diagnostics from books. That can only be learned through careful observation and constant contact with his patients.\" Here the author emphasized a truism of medical education in the nineteenth century: direct observation was preferable to, even precluded, the scholastic, bookish approach that ruled medicine for so long. But he also confirmed several aspects of the clinical gaze: it was contemplative, it relied on the accumulation of cases, it was learned only through vigorous training. On the other hand, another author described the glance: \"It is that piercing gaze, which unconsciously separates the essential from the unessential and penetrates to the bottom of things with ease and certainty, without requiring difficult research, which is easily misled by irrelevancies that are easily apparent, while important things that elude perception are overlooked.\" The glance was decisive, confident, second nature. Like the gaze, the glance was acquired only after long training and experience. While Foucault characterized the distinction between the gaze and the glance as a historical progression from one mode of observation to another, and Hau described it as a cultural or disciplinary differentiation, I would suggest that the duo, like stillness and movement, constituted a set of alternatives or a dialectic, each always part of the larger category called observation. But the character of the two does not ultimately concern us here, nor even their relationship to each other, as much as their function as badges of professional expertise. The cultural investment in these modes of observation is palpable, as indicated by the numerous texts on observational method or by the number of debates about the specificity of clinical observation, especially when threatened by new technology (the sphygmomanometer, for example). Observation, in whatever form, was central to the German physician's professional and class identity. The explorer and the connoisseur may have had different skills, but they invariably came from the same place and were quite comfortable in each other's company (or even in the same skin).\n\nThis exercise in textual analysis has been designed to highlight the three-way correspondence between certain formal features of motion pictures, particular techniques used to examine them, and specific principles of observation. The way Braun used individual film frames, for example, illustrates and corresponds to the principle of correlation in observation. These correspondences demonstrate that the acceptance and legitimacy of motion pictures in science and medicine depended not merely on the availability of the technology but also on its ability to conform to established disciplinary imperatives. At this historical moment in the development of Western medicine, motion picture technology partly addressed a number of representational and observational issues, including the increased demand for rigorous observation in medicine and the rising interest in the documentation and analysis of complex movements. (So it is perhaps not surprising that the number of research projects on pathological movement, for example, increased as film was accepted as a tool for studying it.) Beyond the question of legitimacy, these correspondences also demonstrate, I hope, that observation is a complex operation and that we can learn much about it by studying how film is used. Specifically, it is clear that for nineteenth- and twentieth-century scientists, medical researchers, and scholars, \"observation\" was considered to be primarily _a self-disciplinary method of ordering thought_. As a means of protecting the community against the chaos of irrational and biased subjectivity, observation was closely aligned with experiment as a universally adopted method of enforcing professional standards. Observation was a prescribed method of organizing information through such means as stabilizing and isolating variables; measuring, describing, and classifying objects and events; and arranging and correlating these items or events to arrive at verifiable conclusions. This process is what it meant for physicians to be \"scientific\" as Paget so forcefully insisted: \"In our calling careful practice and scientific study should be inseparable\" (which also emphasizes the futility of separating experiment and observation).\n\nFilm worked as a research instrument to the extent that it could conform to or inform this method. In particular, film was an aid to correlation. The motion picture camera was a tool for generating images or views for comparison, either comparison of the individual frames or comparison of different temporal \"views\" via slow-motion or time-lapse cinematography. Alongside this feature, film was praised as something that gives the user \"more time,\" either by storing material for convenient retrieval, by reproducing and repeating events, or by offering variable filming and projection speeds. One expert applauded film's utility in the medical classroom:\n\nCinematography could help with that. The images can be shot with all the time in the world, using specimens that have been prepared with the utmost care, and are then permanently available. Instead of the uncertain outcomes of fleeting experiments, the serial images can be shown repeatedly, which not only makes a greater impression on students, but by presenting the frames individually, can also provide an analysis of the phenomenon.\n\nMotion pictures gave more time to the researcher and the student alike\u2014more time to prepare, more time to analyze and comprehend. Especially important was the flexibility of film with regard to the dialectic between analysis and synthesis; if researchers have always had analysis and synthesis, only with motion pictures did they have that choice in the same representational system. Alternating back and forth between the still image and the moving image, researchers took advantage of cinema's ambidexterity to present an \"impressive\" yet scientific picture of the phenomenon. Yet this ambidexterity was not merely an advantage of film\u2014it signaled a hermeneutic strategy that in part became standard procedure because of it. The cognitive gains that resulted from going back and forth between the still and moving image\u2014which have been known ever since a mental image could be sketched\u2014were codified into experimental and observational method with the advent of motion pictures. But more to the point, the scientific legitimacy of this alternation\u2014that stillness and movement could be _equated with_ analysis and synthesis\u2014was to some extent due to the availability of motion pictures as a research tool that could offer both options. So film was not simply a useful tool for understanding complex events: the use of film actually made manifest a mode of understanding. The ambidexterity of film operationalized a way of thinking and made it visible to the community in both images and practice. The motion picture camera was not just an instrument, but a theoretical approach.\n\nBut I want to return to the researcher's obvious glee, which the quotation above by K. W. Wolf-Czapek vividly demonstrates, in being able to manage the flow of time by controlling motion pictures. Wolf-Czapek's cheerleading betrays a certain emotional investment in this potential, as if it were crucial to the lecturer's pedagogical success or the physician's identity as a researcher. Controlling the passage of time almost became the very definition of \"scientific\" because of all it implied about careful preparation, leisurely observation, accurate description, and deliberate correlation. But as we saw with Braun earlier, the giddiness that the researcher felt toward the moving image was not only about the control of time, but the submission to it as well. Or more precisely, the moving image was thrilling in itself: its invitation to mimesis, its temporal rush, its presence. Controlling the moving image completely would also make it less spectacular, less effective as a persuasive representation. So the researcher had to negotiate this duality by deciding how and when to use the motion picture in its two forms. Various techniques in the laboratory and the lecture hall\u2014such as frame-by-frame analysis, slow-motion and time-lapse cinematography, varying projection speeds, looping a single shot so that it repeats, or tracing an image to abstract and manage its detail\u2014help to manage the thrill and temporality of the moving image (even if these techniques come with their own sensory pleasure). But even when the image was projected in all its glory as a thrilling example of \"living photography,\" the conventions of the lecture attenuate that potential. And it goes without saying that a film about pathological movement did not have the same immersive potential as a feature narrative. Physicians recognized the full impact of the motion picture, to be sure, but they did their best to manage that impact in the laboratory, the classroom, and the professional meeting. Was it simply a generic demand? That is, was it simply the case that any representational form would have the same restrictions? Other media did have similar boundaries of decorum and professionalization. But cinema was a special case unrelated to its content\u2014it _moved_ , and this meant that there was a little bit of carnival even in the most staid presentation or most boring research film. With the possibility of this kind of presentation, what signified \"scientific\" or \"professional\" depended especially _on how one managed the passage of time_. If scientific experiment always implied some control of the temporal variable, with motion pictures that control became explicit and essential to scientific identity, especially in the face of its mass culture incarnation. Hence the medical community's swift and vicious reaction to \"the movies.\"\n\nTIME, SPECTATORSHIP, AND THE WILL\n\nMovies were only one of many ailments of modernity, of course, and in the scheme of things, they took up no more editorial space\u2014probably considerably less\u2014than complaints about trashy literature, modern art, lurid advertising, trolleys, trade unions, or other by-products of industrialized life in the metropolis. But with their forceful entry into public consciousness, starting around 1907, motion pictures touched a very particular nerve among the educated elite. The previous section exposed one tendril of that nerve: the professional and emotional investment in methodical, leisurely, observational practice attached to scientific method. The pace of motion pictures threatened that practice; more precisely, for most commentators the (perceived) quickness of movies endangered a cultural tradition of learning, expertise, and status that invested heavily in studious attention, methodical description, and logical reasoning. Modernity jeopardized that investment in a variety of ways, but motion pictures best represented the problem of social acceleration: movies, like change itself, moved too quickly for comfort. They therefore put attention and reason at risk and, in that way, exemplified a problem of national importance. In their editorials about movies, physicians discussed the problem of cinema's pace in the context of this investment in attention and reasoning. They also addressed the objectionable subject matter of many films and the unhygienic conditions of many movie theaters. Physicians joined reformers in their protest against the number of \"trashy films\" ( _Schundfilme_ ) that exposed themselves to a dazed public in seedy storefront cinemas ( _Ladenkinos_ ). This final section will examine some of these complaints about the physical and psychic or moral threat of motion pictures. I want to provide a survey of the variety of complaints, but I also want to emphasize _pace_ as the primary source of nagging discomfort with movies felt by doctors and other members of the educated middle-class ( _Bildungsb\u00fcrgertum_ ). This section, then, serves as a segue between the focus on film as a research instrument in this and the previous chapter and the exploration of film as a social tool and phenomenon in the following chapters. Let us start this examination, however, with a brief check of credentials: How did German physicians position themselves as experts on movies, of all things?\n\nAs late as the 1850s, academically trained physicians in Germany were not considered experts of much. Their therapies were, by and large, no more effective than those available to the lay public; barber-surgeons, midwives, and folk medicine presented plenty of competition; and a physician's social standing was usually lower than the upper classes he served, which meant that medical advice was routinely questioned and rejected. Claudia Huerkamp has demonstrated that the turnaround was a result of the Prussian state bureaucracy's policy to take control of all guild organizations, to standardize medical education, to license medical practice, to thereby suppress folk alternatives, and to prioritize hygienic practices among the population. These policy decisions effectively eliminated the competition and, especially through the establishment of health insurance in the 1880s, expanded the clientele for academic physicians, particularly into the working classes. As doctors took on more patients from the working classes, their social standing was above the majority of their patients, thereby reversing the previous patient\u2013doctor power dynamic. In addition, the state directives promoted a general \"medicalization\" of society, in which academic concepts of health and disease were adopted by society at large; social problems that were not previously seen as medical problems, such as alcoholism, were gradually treated as diseases. Furthermore, the academic medical community stabilized and enforced professional standards, especially by adopting scientific methods and techniques, which led to new approaches to diagnostics, anesthetics, and surgery, for example. As their expertise and their association with the craft of science grew, so did their authority. So state policies, the expansion of clientele, the growing professionalization of medical ranks, and the association with scientific principles, all combined not only to create a medicalization of society but to place academically trained physicians in position as its chief diagnosticians.\n\nBy the 1890s this authority began to consolidate, and by 1914 it seemed perfectly natural to view many social problems within a medical framework and equally natural that physicians were qualified to comment on them. Doctors examined and prescribed for the body politic as well as for the individual. They saw themselves not merely as professionals or experts, but as _Kulturtr\u00e4ger_ (bearers of culture) who had a responsibility to support, carry on, and protect the nation's heritage. This intervention into the social sphere manifested itself in two ways: more doctors became involved in social medicine or public health, and more doctors wrote cultural critiques from a medical standpoint. For Munich psychiatrist Emil Kraepelin, it was simply a matter of applying one's skills in diagnosis, prophylactics, and therapy to the growing need for public medicine. Such public health issues as alcoholism, tuberculosis, and sexually transmitted diseases increasingly occupied the expertise of academically trained physicians in Germany at the turn of the century. While Germany lacked a national ministry of health until after World War I, individual doctors were very much involved in national campaigns. Kraepelin, for example, was committed to the temperance movement of the day, a typical example of the growing medicalization of social issues in industrialized nations. Likewise, motion pictures were swept up in this trend as certain aspects of the cinematic experience came to be regarded as a public health threat.\n\nKraepelin's prot\u00e9g\u00e9, Robert Gaupp, also directed his skills toward public health issues, such as child psychology and welfare, but he combined his specialized training with his cultural capital and status as a University of T\u00fcbingen professor to bring less obviously medical issues, such as motion pictures and pulp fiction ( _Schundliteratur_ ), under the rubric of medicine. Perhaps the most famous example of the medical diagnosis of culture, however, is Max Nordau's _Degeneration_ (1892). With chapters titled \"The Symptoms,\" \"Diagnosis,\" \"Etiology,\" and \"Prognosis,\" and various case studies of modernist endeavor, from the symbolists to Leo Tolstoy, Henrik Ibsen, Richard Wagner, and Friedrich Nietzsche, _Degeneration_ argued that cultural products of the day displayed classic symptoms of degeneration, decadence, and hysteria, which were \"the consequences of the excessive organic wear and tear suffered by the nations through the immense demands on their activity, and through the rank growth of large towns.\" Drawing in part on the contemporary discourse of \"nervousness,\" Nordau (fig. 2.3) found that the excesses of modernity had physical consequences, the symptoms of which could be read in cultural products. Just as Italian psychiatrist Cesare Lombroso theorized that social deviance was based on biological retrogression\u2014criminality, he surmised, must be due to some primitive remainder or reversal in the normal progression of the species\u2014so Nordau argued that the cultural avant-garde was not progressive and forward-thinking but instead atavistic and regressive, displaying traits common to deviants and hysterics. Using medical norms to evaluate literature and the arts, Nordau combined physiological theories of social deviance, Darwinian evolutionary models, and liberal ideals of ordered progress and rationality into an old-fashioned, curmudgeonly disapproval of modernism. Nordau's work was extremely visible\u2014it was one of Europe's ten best-selling books of the 1890s\u2014but certainly not unique. Darwinian theories of heredity and degeneration; medical or psychological investigations into hysteria, nervousness, and hypnotism; social ferment due to the rise of social democracy, industrialization, and modernization; all combined to create an uneasy mood of cultural pessimism among the educated middle classes of Wilhelmine Germany, which the commentaries of Gaupp and Nordau exemplify.\n\nSo the metaphorical extension of \"health\" and \"disease\" to all aspects of social life became more than a metaphor for doctors and their readers in imperial Germany. Likewise, the moral and physical threats that motion pictures and other cultural forms presented were also very real for these commentators. One physician's complaint about the dangers of representing the erotic and sexual in film presents a common stance:\n\nThe arrival of the cinematograph brought much damage in its wake.... While the danger, which undermines serious moral principles and hence the moral foundations of a man, must be fought, it is, of course, only a threat to the masses who don't think for themselves, for the minors, the young and easily impressionable, who are not used to having their own opinion about things or probing things with critical method. And because these masses make up the majority of our nation, this public, easily accessible sexual fare, which is apt to damage the sexual apparatus [ _sexuellen Tractus_ ], represents a serious _sexual danger_.\n\nFIGURE 2.3. Max Nordau\n\nOf course, the idea that sexually arousing images or narratives might damage one's moral fiber was not new. But this complaint is especially representative and remarkable for its insistence that \"critical method\" ( _kritischer Methode_ ) was the difference between the protected and the unprotected. Without it, apparently the masses were at modernity's mercy. But it is not simply that the masses lacked the tools to defend themselves; again and again, medical and lay pundits claimed that movies corroded \"critical method\" itself.\n\nWe find this idea even in editorials that outlined the mere physical threat that motion pictures presented to the average viewer. A Dr. Paul Schenk, for instance, discussed the flicker effect of early cinema, warning teachers who hoped to use motion pictures for educational purposes:\n\nFrom the standpoint of visual hygiene, the usefulness of cinema as an educational tool remains dubious. Modern man systematically ruins his eyes. We suffer from an excess of visual stimuli.... The much-maligned \"flicker\" of the cinematic image is a malaise that presently, and probably forever, deprives the cinema of the claim to be a \"hygienic\" means of instruction.... This impression is further strengthened by the unnaturally fast changes of scenery. Our eyes cling intently to the screen, where, in addition to the flicker of the images, a change of scenery takes place almost every minute within a time period that lasts 8 to 12 or up to 15 minutes.\n\nCinema's educational utility, according to Schenk, was hampered by the threat its \"flicker\" presented to student health. On one hand, this flicker supposedly damaged the eyes; there was apparently a physical connection between the flicker and nerve damage of some sort. On the other hand, the _pace_ of the film intensified this damage. In fact, this physical damage acted as a metaphor or outward symptom of a deeper, psychic damage caused by the temporal push of cinema. Reformers or _Kinogegner_ (enemies of the cinema) also took up this diagnosis in a general medicalization of cinema in the discourse of the time. Albert Hellwig, not a doctor but one of Germany's most prolific _Kinogegner_ , relied on medical terminology and cited an Italian doctor's discussion of cinema's assault on the sensitive mind: \"Of a group of neurasthenics, d'Abundo observed that frequenting cinematographic presentations brought about all sorts of ailments, especially insomnia. It was not so much the influence of the contents of the presentation, but rather an effect of the rapid, constantly moving action and attendant flickering.\" Another nonphysician, O. G\u00f6tze, put a finer point on it: \"The hasty tempo of the images, and especially the Leipziger-medley-like program [i.e., one resembling a mixed vegetable dish], seduce the child into superficial _viewing and observation_ habits. Even the images that could offer intellectual enrichment go by so quickly that it is impossible to form clear impressions.\" With this observation, G\u00f6tze articulated the position of the medical community and reformers with regard to the movies. Medically speaking, the cramped, oppressive atmosphere of the theaters, together with the flicker effect, presented a threat to physical health, while the immoral content of the films and their delivery by means of a relentless temporality presented psychological dangers. Cinema's quick pace threatened reason itself.\n\nThis complaint about tempo corresponded to a broader contemporary discussion about modernity and \"social acceleration.\" We could trace this feeling that \"the world is moving faster\" back to the eighteenth century, of course, but objections piled up toward the mid-nineteenth, especially at the fin de si\u00e8cle. Nordau complained about a lot, but close examination of _Degeneration_ reveals that the root cause of the state of things was, for him, the heightened pace of modern life:\n\nIts own new discoveries and progress have taken civilized humanity by surprise. It has had no time to adapt itself to its changed conditions of life. We know that our organs acquire by exercise an ever greater functional capacity, that they develop by their own activity, and can respond to nearly every demand made upon them; but only under one condition\u2014that this occurs gradually, that time be allowed them. If they are obliged to fulfill, without transition, a multiple of their usual task, they soon give out entirely.... To speak without metaphor, statistics indicate in what measure the sum of work of civilized humanity has increased during the half-century. It had not quite grown to this increased effort. It grew fatigued and exhausted, and this fatigue and exhaustion showed themselves in the first generation, under the form of acquired hysteria; in the second, as hereditary hysteria.\n\nSo, according to Nordau, the human body was not able to keep up with the increased demands and pace of modern life and grew fatigued, which led to a corrosion of the nerves and, subsequently, hysteria and degeneration, which showed itself in daily life and in modern art. \"All the symptoms enumerated are the consequences of states of fatigue and exhaustion, and these, again, are the effect of contemporary civilization, of the vertigo and whirl of our frenzied life, the vastly increased number of sense impressions and organic reactions, and therefore of perceptions, judgments, and motor impulses, which at present are forced into a given unity of time,\" writes Nordau. Too much information compressed into too little time was Nordau's recipe for decline.\n\nNordau's antidote for sensory overload was not withdrawal but \"attention.\" Attention was the savior, the critical faculty that brought order to chaos:\n\nThus we see it is only through attention that the faculty of association becomes a property advantageous to the organism, and attention is nothing but the faculty of the will to determine the emergence, degree of clearness, duration and extinction of presentations in consciousness.... Culture and command over the powers of nature are solely the result of attention; all errors, all superstition, the consequence of defective attention. False ideas of the connection between phenomena arise through defective observation of them, and will be rectified by a more exact observation. Now, to observe means nothing else than to convey deliberately determined sense-impressions to the brain, and thereby raise a group of presentations to such clearness and intensity that it can acquire preponderance in consciousness, arouse through association its allied memory-images, and suppress such as are incompatible with itself. Observation, which lies at the root of all progress, is thus the adaptation through attention of the sense-organs and their centers of perception to a presentation or group of presentations predominating in consciousness.\n\nAs Jonathan Crary has demonstrated, Nordau's views here represented a vast literature in experimental psychology and social commentary focused on attention. Crary has covered this ground well, but Nordau's remarks nevertheless bring up two issues especially salient to this chapter. First, we should note the absolute centrality of attention for Nordau's Enlightenment project: \"Culture and command over the powers of nature are solely the result of attention; all errors, all superstition, the consequence of defective attention.\" Attention was not simply the ability to focus, it was the motor of human progress. The stakes were high, precisely because Nordau, as others before and after, tied attention to human volition. \"Attention is nothing but the faculty of the will,\" which meant that human action relied almost entirely on attention in Nordau's scheme. Attention, as choice, expressed free will; to focus is to choose. But more than that, attention, as Crary points out, is also inhibition; it is the suppression of stimuli and the ordering of information. It is not simply choice that makes us human, according to Nordau, but the kind of choices we make for the good of progress. Attention, then, had a powerful ethical charge. Without the proper exercise of attention, according to Nordau and this tradition, we would be automatons and decadents, abandoning our future to the caprice of chance, fate, or worse, the flow of modernity.\n\nWhat did the proper exercise of attention look like? For Nordau, it looked exactly like \"observation,\" which brings us to the second issue: observation as the practical manifestation of attention and volition. According to Nordau, attention determined \"the emergence, degree of clearness, duration and extinction\" of stimuli, thus controlling the flow of information to consciousness. Attention was an important gatekeeper against the rush of modern life; significantly, it could control the _duration_ of the presentation. Attention, or its equivalent in observational practice, controlled the rate at which consciousness is exposed to stimuli, just as a scientific experiment isolates and controls variables and the rate at which the researcher receives information. The difference between overstimulation and an unacceptably quick pace is sometimes difficult to distinguish, but if we think of observation and experiment as a set of practices that manage the flow of data, then it really does not matter. In fact, attention itself, as Crary emphasizes, was a notoriously slippery concept; not only did it bleed easily into distraction, but psychologists more or less failed to locate or define it, except as an \"imprecise way of designating the relative capacity of a subject to selectively isolate certain contents of a sensory field at the expense of others in the interests of maintaining an orderly and productive world.\" Attention still received significant play, but precisely because of its ambiguity it makes good sense now to focus instead on observation. In the context of the hailstorm of modern life, attention functioned for Nordau and others as a \"stimulus shield,\" as well as a metaphor for bourgeois values of order, rationality, and discipline. But most of all, it functioned as an abbreviation for a set of observational practices derived, at least in part, from scientific method. These practices, which I outlined in the previous section, could be learned; the medical student or the young scientist was constantly required to adopt and perfect this mode of viewing. Once incorporated, this training became second nature, of course, but more importantly, as we see in Nordau and others, it carried an intense moral responsibility: \"Observation... lies at the root of all progress.\" For Nordau and the _Bildungsb\u00fcrgertum_ , that which threatened these practices, or risked the possibility of passing these practices to another generation, attacked the very foundation of human endeavor.\n\nSo it is easy to see why physicians and other members of the educated elite greeted cinema's temporal rush, emblematic of the incessant pace of modern life, with something less than enthusiasm. Stuck in a theater seat, with no way to slow or stop its continuously unfurling movement, they could only helplessly watch film corrode their (and their children's) \"critical faculties.\" We have already seen how psychiatrist Robert Gaupp, along with his colleague at T\u00fcbingen, art historian Konrad Lange, condemned \"the temporal concentration\" of cinema: \"In the cinema... excitation of the passions is intensified and multiplied by the rapid succession of vivid images that passes before our eyes. There is no time for reflection or release, no time to find an inner balance... quiet _contemplation_ , _intellectual assimilation_ , and sober criticism are not possible.\" Others echoed his medical opinion that the rush of images was too much for the average viewer: \"All eyes greedily fix on the living picture as it flits by. And the mind must hurriedly link together the connections between images that were only briefly glimpsed.... In the theater I must _mentally exert myself_ more throughout [than in the cinema]. I've got to pay attention to the words and thoughts, which often appear in a very terse, sharply focused form. Such a tight line of thought is not necessary in the movies.\" For some commentators, cinema's rush of images threatened reason itself:\n\nThe mere habituation to the darting, convulsive, twitching images of the flickering screen slowly and surely corrodes man's mental and, ultimately, moral strength. First, one gets used to switching quickly and abruptly from one impression to the next; one loses the slow continuity of the succession of ideas, the ability to grasp, which is a precondition for all sound judgment. Second, one becomes accustomed to yielding to and unthinkingly following its random string of images and ideas; one no longer misses the logical structure of an overarching thought, which is indeed what binds individual impressions together into what is generally referred to as \"thought.\"... Third, as a result of the rapid passing of images, one gets used to absorbing only approximations of an impression; one does not grasp the image clearly and consciously, in all its details.\n\nFor these writers, cinema's threat was ultimately a question of mental hygiene: methodical observation as taught by the scientific method was one good practice, akin to flossing, that would help insure the health of the psychic mechanism. It was only one of many good observational practices\u2014aesthetic contemplation was another\u2014that reflected a well-ordered mind. Motion pictures, on the other hand, encouraged sloppy habits of thought. Instead of the active self-control that these hygienic practices implied, motion pictures suggested disrepair and self-abandonment.\n\nWe can see these implications especially clearly in the characterization of cinema as a hypnotic agent. The ground between cinema and hypnotism has been well tilled, but I return to it because hypnotism provides not only a succinct and well-known model of spectatorship, but because, surprisingly enough, it also offers a model of observation. That is, if we consider the diagnostic value of hypnotism, or its function as an experimental tool, the double-edged value of this trope will become especially clear. So let us first briefly explore the discussion of hypnosis as a metaphor for film spectatorship. As Stefan Andriopolous has shown, the comparison of cinema with hypnosis tapped into contemporary debates about hypnotic crime that flourished during the initial heyday of hypnosis, between 1885 and 1900. In the clinical literature, researchers such as Hippolyte Bernheim in France and Auguste Forel in Switzerland wondered whether the power the hypnotist had over the subject could extend to forcing the hypnotized person to commit crimes against his or her will. To investigate this phenomenon, they even staged fake crimes, in which the hypnotized subject was asked to shoot or stab another person with blank bullets or wooden daggers. If the results were inconclusive, the \"belief in the possibility of perfectly camouflaged suggestions produced the paranoia that there might be an unlimited number of unknown hypnotic crimes.\" The idea that an agent could plant a suggestion in a hypnotized subject to be carried out later excited the already overwrought imaginations of many.\n\nFor many physicians and reformers, cinema acted as a hypnotist, sending impressionable subjects to the streets with powerful, posthypnotic suggestions to commit crimes of all varieties. Or it functioned as a \"trigger\" for latent psychopathies. One physician cited a 1911 case in Frankfurt, in which a twelve-year-old boy stole a purse. When interrogated, the boy confessed, \"I admit to having carried out the purse-snatching: I by chance ended up in a crowd where Frau K. stood with her purse. I had once seen the presentation of a purse-snatching in a movie theater. I was thereby compelled to try something like that, too.\" This criminal sounds rather literate for a twelve-year-old, but nevertheless the cinematic depiction of a crime apparently presented a suggestion too powerful to resist. The doctor commented, \"The effect of the dramatic, moving images on the child's psyche must have been especially strong because the purse-snatching took place on a crowded street in the middle of Frankfurt.\" The medical literature on cinema during this time was littered with such cases in which a youngster who has committed a crime admits to visiting the movie theater at least once a week or even to learning the technique at the movies. Apparently, like Drs. Caligari and Mabuse, cinema invisibly enlisted an army of impressionable youth to do its bidding.\n\nHow was this accomplished? Gaupp explained the connection between cinema and hypnotism:\n\nAdd to this the well-known psychological fact that, when hearing or reading about exciting events, few people have sufficient imagination to visualize the events graphically before the mind's eye. Cinema, however, brings everything right before our eyes in embodied form, and does so under psychological conditions that are conducive to deep and often lasting suggestive effect: the darkened room, the monotonous sound, the forcefulness of exciting scenes following each other beat by beat lull every critical faculty to sleep in impressionable souls, and thus, not infrequently the content of the drama becomes a fateful suggestion for the complaisant youthful mind. We know that _all suggestions adhere more strongly when the critical faculties sleep_.\n\nGaupp was not the first or the last to note the structural similarity between cinema and hypnosis; as Rae Beth Gordon notes, critics from Ycham to Raymond Bellour have been fascinated by the parallels: the darkened room and the luminous screen of the theater mimic or encourage the intense focus and narrowed consciousness of the hypnotized subject; the monotonous noise of projection and the \"scenes following each other beat by beat\" are like the lulling rhythms of the hypnotist. Everyone from Hugo M\u00fcnsterberg to Jean Epstein has remarked on film's suggestive power. Noteworthy, however, is the image that emerges from this varied commentary of the spectator as suggestible, childlike, and feminized\u2014a characterization that was fairly commonplace, especially in social psychology. The idea of \"suggestibility,\" especially, was a lynchpin that connected the trope of \"the child\" to other groups, namely women and crowds. That is, reformers and physicians made a conceptual leap from children to crowds (and to the cinema audience) via the idea of suggestibility, which had become an important concept in crowd psychology. For example, Gustave Le Bon, the most well-known popularizer of nineteenth-century crowd psychology, characterized the masses as \"an enraged child.\" Furthermore, according to Le Bon,\n\nIt will be remarked that among the special characteristics of crowds there are several\u2014such as impulsiveness, irritability, incapacity to reason, the absence of judgment and of the critical spirit, the exaggeration of the sentiments, and others besides\u2014which are almost always observed in beings belonging to inferior forms of evolution\u2014in women, savages, and children, for instance.\n\nDarwin's evolutionary scheme provided a quasi-scientific basis for comparing crowds to children, but \"suggestibility\" was even more significant for this comparison. Le Bon devoted a chapter to \"the suggestibility and credulity of crowds,\" arguing that the crowd is \"perpetually hovering on the borderland of unconsciousness, readily yielding to all suggestions,\" a mental state most commonly found in women, children, and the hypnotized subject.\n\nVia Charcot, hypnotism had already come to be associated with female hysteria. Under his supervision, female patients (and even some male patients) fell into a deep trance and, through the power of suggestion, disassociated themselves from their bodies, allowing Charcot to practically sculpt them into various positions. Likewise, in Le Bon's crowd psychology, this pliability and impressionability was a distinctly feminine trait. Those susceptible to suggestion\u2014or more precisely, those unable to withstand suggestion\u2014were feminized, namely women, children, savages, crowds, and weak-willed men (fig. 2.4). This is also why hypnosis was such a powerful trope for characterizing the cinema audience: for Gaupp and others, the viewer was in a state of self-abandonment and pliability, swept away by the moment like a hysteric or hypnotized subject. Film viewers\u2014whether actual children or merely uneducated and therefore childlike\u2014lacked the will to distance themselves from the psychological and physiological effects of suggestion. Precisely this lack of will was at issue; _willenlos_ is the word Gaupp uses to describe impressionable youth. His diagnosis described both pathology and morality; the lack of will was a moral weakness that manifested itself in mental impressionability and physical mimesis. Those who _could_ resist were, of course, male experts. Against the feminized subject, physicians such as Charcot, Le Bon, and Gaupp pitted the moral strength of the masculine professional.\n\nFIGURE 2.4. A hypnotist practicing his craft, France, circa 1900\n\nThis image of the film spectator is a familiar picture. But what of the hypnotist himself? That is, if hypnotism were a valuable trope for describing the spectator, could it be equally valuable to describe the observer? What properties of hypnotism expressed the hypnotist's stake in the process, especially with regard to his mode of viewing? What was the medical purpose of hypnosis? Psychoanalysis and the medical community abandoned serious work in hypnosis early in the twentieth century for a variety of reasons; not only was it becoming too much of a sensational sideshow, but the power dynamic it implied was indefensible within humanist inquiry. Nevertheless, before this crisis and in its modern form, hypnosis held therapeutic potential, because it allowed the investigator to probe deeply into the patient's psyche. It had the effect of anesthesia\u2014it allowed the physician to explore the psyche without the interference of a \"live\" consciousness. It was considered a form of \"psychic dissection.\" In this respect, it _suspended time_. More precisely, it functioned _experimentally_ : like an experiment, it controlled variables, stabilized the environment, and provided enough time to examine. Berlin psychiatrist Albert Moll declared, \"Hypnotism is a mine for the psychological investigator, for hypnosis is nothing but a mental state. When we think that psychologists have always used dreams so much in their investigations of mental life, and that experiments can be better made in hypnosis than in ordinary sleep, _because it can be regulated at pleasure_ , we cannot deny the value of hypnosis to psychology.\" One prominent French psychiatrist put it this way:\n\nThrough numerous examples, I will try to show that with hypnotic processes we may practice, if I may express it in this way, an actual _moral vivisection_ (if the reader is not too frightened by the word) and witness with our own eyes and _make_ function the intellectual mechanism just as the physiologist sees and _makes_ function the organic machine.\n\nWhen did a physiologist \"make\" a body function like a machine? In the course of an experiment, of course. Similarly, hypnotism was a process that stabilized the subject; isolated important variables; and allowed the time to inventory, describe, and classify those variables in a scientific way. Like a filmstrip, the subject's consciousness under hypnotism could be paused, rewound, slowed down, or inspected bit by bit. It gave the researcher time to explore, to accumulate data, to contemplate the details, and to correlate their patterns. In this way, the comparison between cinema and hypnosis goes further than what we find in the discourse itself: it is not just that cinema was a hypnotist or even that hypnotism was a constant theme in the films themselves, but that hypnotism and cinema were functionally equivalent in the eyes of the researcher. Both were experimental apparatuses that had a flexible, controllable regulatory mechanism in the hands of the scientist, and both had, in relation to the layperson, a potentially dangerous pace precisely because it could not be controlled.\n\nHence, proper observational method and experimental control functioned as mental prophylactics against the sweep of time. \"Will\" was the name they often gave to the power to resist, but if \"will\" ever existed beyond the realm of bourgeois fantasy, it existed as ingrained, scientific, observational method. For example, hypnosis was often characterized as an extreme \"narrowing\" of consciousness with an attenuation of peripheral awareness; the paradox, of course, being that those most capable of intense focus and attention were most easily hypnotized. Scientific observation, on the other hand, was emphatically _not_ considered a \"narrowing\" of consciousness; this was not what Nordau or others meant when they lauded \"attention.\" Indeed, given the emphasis on logical operations such as correlation, observation was nothing if not an \"expansion\" of awareness; the researcher constantly mentally compared the object under study to other objects in past and present experience. So the prevailing view of observation held that logical operations attached to a viewing protocol\u2014the finest aspect of \"attention\" as the educated elite understood it\u2014were the best defense against slipping into a dangerous immersion.\n\nThe masculine, medical professional was distinguished not only by his will, but by his training in scientific observation. Indeed, this training was his primary defense against the onslaught and seductive power of (moving) images. The scientific appropriation of motion pictures depended on the ability of the researcher to halt this onslaught, to forestall its assault and to read _slowly_. It must be so: if the researcher were to correlate the data, he had to be able to control its flow. Ultimately, then, the negative comparison between cinema and hypnosis was about mastery: mastery not only of the hypnotized subject but of the ability to hypnotize. Hypnotism and motion pictures were perfectly acceptable scientific tools, as long as they were in the qualified hands of (male) professionals. Hypnotism, for example, was condoned (and still is) as a legitimate procedure for psychic exploration. Likewise, physicians in Germany used motion pictures to help diagnose or visualize disease. Like motion pictures, hypnosis was used to isolate, stabilize, and present\u2014in this case, psychic or somatic trauma. Hypnosis was a tool for acquiring the distance and time necessary to observe and to diagnose. Physicians used motion pictures to record the human body, but then they slowed the images down or stopped them to examine the phenomenon frame by frame, also giving them the time and distance they needed to master the event. In the same way, hypnosis allows access to different temporal registers. In both cases, the scientific use of hypnosis or motion pictures implied a mastery of time and of the human body. Film's popular incarnation, however, implied a lack of mastery of both.\n\nIn his essay \"What Is Enlightenment?\" (1784), Immanuel Kant made a compelling argument for self-mastery: \"Enlightenment is man's release from his self-incurred tutelage  _selbstverschuldete Unm\u00fcndigkeit_ ]. Tutelage is man's inability to make use of his understanding without direction from another. It is self-incurred when its cause lies not in lack of understanding but in lack of resolution and courage to use it without direction from another.\" For Kant, true freedom meant being able to think (and speak) for oneself. Those who did not or could not were condemned to political and social infancy. \"Tutelage\" is an important term, in this respect; literally translated, _Unm\u00fcndigkeit_ , means \"minority\" in the sense of not yet being \"of age.\" ( _M\u00fcndigkeit_ means \"majority,\" as in \"the age at which full civil rights are accorded.\") Kant basically argued that those who refused to assume this responsibility to think for themselves were politically no more than children. So in this essay he gave license to society's \"enlightened\" to lead the others. Hence the importance of education, especially _Bildung_ (self-cultivation), for attaining this state of political and social responsibility. The emphasis we have seen so far on observation and attention reveals that _educating the eye_ was absolutely central to discharging this ethical duty. Self-cultivation was not limited to training in literature, music, or history; training _vision_ was also very much a part of this process. Scientific or medical observation was only one element of a range of viewing practices that could be considered _Bildung_ -worthy. Pedagogical practices of visual education and practices of aesthetic contemplation, which the next two chapters respectively explore, were two others. Their opposite number\u2014what we call \"spectatorship\"\u2014can be fully appreciated only by examining the infantilization of the \"unenlightened,\" which is the task of [chapter 3.\n\nTHE TASTE OF A NATION\n\nEDUCATING THE SENSES AND SENSIBILITIES OF FILM SPECTATORS\n\nI step in; intermission has just begun. An oppressive, damp draft hits me, even though the doors are open. The spacious room (500 capacity) is filled to the last seat with children. There is an incredible ruckus: running, yelling, shrieking, laughing, chatting. Boys scuffle. Orange peels and empty bon-bon boxes fly through the air. The floor is studded with candy wrappers. Along the windowsills and radiators young toughs roughhouse. Girls and boys sit together, densely packed. Fourteen-year-old boys and girls with hot, excited faces tease each other in unchildlike ways.... Children of all ages, even two- and three-year-olds, sit there with glistening cheeks. Women walk among them selling sweets. Many children sneak candy and drink soda [ _Brause_ ]; young boys smoke furtively.\n\nThen the movie begins.\n\n\u2014A SCHOOLTEACHER FROM BREMEN (1913)\n\nFor the cinema reformers of imperial Germany, this was a scene from hell. This is what German _Kultur_ had come to, what modernity had wrought: children melting and spoiled like day-old candy on the floor of a movie theater. Like Professor Rath of _Der Blaue Engel_ (The Blue Angel, 1930), who follows his students into a seedy nightclub and is initially shocked by the sexuality and degeneracy within, this schoolteacher from Bremen walked into a matinee and was horrified by what she saw. Her emphasis on the corporeality of the scene, on the _body_ of the audience, so to speak\u2014fighting, eating, sweating, and awakening sexuality\u2014attests to the perception that cinema presented a grave danger to the children's emotional and moral fitness and especially their physical health. Given the traditional bourgeois association of sensuality with the lowly masses, this scene also represented a threat to the well-being of the nation, of the body politic. As this chapter will demonstrate, \"children\" and \"the masses\" were often interchangeable concepts. Indeed, while cinema was often portrayed as a gaping Moloch devouring innocent children in some pagan ritual, the children depicted in this passage are far from sacrificial lambs. They present something of a veiled threat to the narrator, as if she had entered a strange, chaotic culture. In the contemporary literature on children and cinema, scenes like this one provoked twin paternal urges: to protect and to control.\n\n\"Women walk among them selling sweets.\" Along with the concern for sensuality (and its tacit partner, capitalism), the numerous references to sweets stand out in this description. Implicit was the assumption that cinema was spoiling the \"taste\" of the children for financial gain. Reformers complained constantly about the \"tastelessness\" of both the theater atmosphere and the films themselves. Figuratively speaking, the concession candy that ate at the children's teeth was also rotting their aesthetic sensibilities. Konrad Lange, a noted art historian at the University of T\u00fcbingen and a ferocious enemy of film, put it more bluntly: \"If one were to judge the artistic understanding of our good, middle-class citizens, one would have to say that their taste is rotten to the core. They have a morbid taste for the slick, the effeminate, the sentimental, and the sugary. They display a demoralizing aversion to the healthy dark bread of true art.\" Lange made the relationship between taste, class, and the body as explicit as possible. Taste, as the simultaneous expression of individual judgment and social distinction, serves as a connection between the private and the public spheres. It is, as Pierre Bourdieu notes, \"a class culture turned into nature, that is, _embodied_.\" It therefore provides a link between individual consumption and a national agenda. Protective of the masculine ideals at the heart of that agenda, Lange condemned the feminization of culture accompanying the onslaught of modernity; he was not alone: most middle-class males of the Western world seemed to share his concerns.\n\nLange's solution, like that of many teachers and educators involved in cinema reform, stressed the education of children and adults. The abiding faith in the ability of education to overcome social ills and promote social progress was a fundamental plank in the platform of many fin de si\u00e8cle movements, from the socialists to the progressives. But the tradition of _aesthetic_ education, with its promise to harmonize the senses and sharpen judgment, offered a quintessentially German solution. By pointing the way to a \"true\" and \"pure\" aesthetic experience, aesthetic education also pledged to counteract the corrupting influence of the cinema. While many reformers, such as Lange, steadfastly refused to be seduced by cinema's charms, some flirted with this particular Lola, courting her in hopes of making her an honest woman by giving her an aesthetic education (or an education in aesthetics). Even while the film industry used the reformers for its own ends, it revealed the contradictions of their ideology. Just as Professor Rath's affair with Lola reveals the indefensibility of his position\u2014a teacher who, ultimately, does not have the best interests of his students at heart\u2014so, too, the reformers' involvement with film shows that there were larger issues at stake than the health of the children.\n\nEven if Professor Rath portrays the hypocrisy of his class, I see him as a distinctly modern character caught between\u2014and profoundly ambivalent about\u2014the solemn textbooks of his classroom and the cunning spectacle of the nightclub, or more broadly, between _Kultur_ and _Zivilisation_. As Norbert Elias and others have noted, these terms played an important function in German self-identity; by the beginning of the twentieth century, they formed a contrasting pair. If _Kultur_ implied \"inwardness, depth of feeling, immersion in books, development of the individual personality,\" or the general cultivation of one's inner life, then _Zivilisation_ implied \"superficiality, ceremony, formal conversation.\" Or, as Raymond Geuss puts it, _Zivilisation_ \"has a mildly pejorative connotation and was used to refer to the external trappings, artifacts, and amenities of an industrially highly advanced society and also to the overly formalistic and calculating habits and attitudes that were thought to be characteristic of such societies.\" By the 1910s and 1920s, _Zivilisation_ came to be associated with the primary burdens of industrialized society: capitalism and technology, or the utilitarian, material, even decadent world of commercial interests. By contrast, _Kultur_ was associated with not only inner, spiritual values but a specifically German configuration of ethical and moral ideals. Professor Rath is caught between his allegiance to learning and teaching the subtleties of the German cultural heritage, to cultivating his soul and those of his students to become good Germans, on one hand, and his attraction to the superficial, voyeuristic pleasures of a crass milieu that combines the worst of sensuality and capitalism, on the other. His downward spiral, of course, begins with his choice between them.\n\nGerman educators interested in reforming film and its theaters felt they faced the same dilemma. But unlike Rath, they did not approach it as an either\/or choice. Instead, the appropriation of motion pictures for educational purposes was a negotiation between aesthetic and moral values, on one hand, and modern technology or commercial interests, on the other. Such negotiations necessarily implied that commercial cinema was not viewed negatively across the board. Just like the reform movements in general, film reform was nothing if not multiple, consisting of many different voices expressing the full range between progressive optimism and cultural pessimism. Yet a picture emerges of a well-meaning but deeply ambivalent group committed to preserving traditional ideals while modernizing the social curriculum. Motion pictures, in this case, were for many not merely a problem to be solved but perhaps also a solution in themselves; if they could be reformed, or \"ennobled\" as reformers often put it, then they might serve as a means to reconcile _Zivilisation_ and _Kultur_ , a goal shared by most of the reformers of imperial Germany, no matter what their chosen object of improvement. The educational use of motion picture technology reveals this attempt at reconciliation, especially in the way that films were made to fit established pedagogical practices. As reformers pressed film into an educational mold, their work also highlighted the ideological and practical contours of that framework.\n\nSpecifically, the use of motion pictures in education followed the contours of a pedagogical trend or approach known as _Anschauungsunterricht_ , or \"visually based means of instruction.\" Popularized in the nineteenth century, this method exemplified the reformist impulse in education, in that it attempted to counter the perceived overrationalization and rote memorization of the traditional pedagogical approach with a self-consciously modern strategy that emphasized the immediate visual perception of things themselves, as opposed to their description in books. Known as \"the object lesson\" in English-speaking countries, the approach asked teachers to present an object, such as a seashell, and ask the students a series of simple questions about it. The questions were designed to lead the students from concrete description to a higher, abstract understanding of the object in relation to its environment and to other things. Many claimed that motion pictures could function as outstanding representations of objects that could not be brought into the classroom, if and only if film could be made to conform to this and other methods of visual training. This chapter, then, continues some of the concerns traced in the previous chapter, especially the investment of the expert class in specific methods of processing visual information. At stake here is not merely the type of viewing, such as medical or scientific observation, but the culture's interest in _what counts as learning. Anschauungsunterricht_ , in this respect, was not just one more pedagogical method but a way of acquiring _Kultur_ ; it was a strong component of _Bildung_ , or the process of self-cultivation. What counted as learning, in this case, was a way of forging _vision_ and _thought_ into _taste_ ; if \"taste\" were the sign of cultivation, then \"vision\" was the means by which taste was trained.\n\nThis chapter argues that the goal of reform was not merely to align film and its theaters with standards of taste and morality but to conform motion pictures to specific modes of viewing; _Anschauungsunterricht_ serves as an obvious and convenient method of training vision to which reformers and educators adapted motion pictures and their projection. While _Anschauungsunterricht_ was explicitly a method of training observation in school-age children, the ideal pertained to adults as well, as we see in all sorts of adult education programs at the time, especially those concerned with aesthetic education. Hence, the image of spectatorship or of ordinary movie audiences at this time must be understood in contrast to this attempt to assimilate motion pictures into a larger ideological arena, signaled most strongly by the desire to train audiences to a particular mode of viewing associated with _Anschauungsunterricht_ and aesthetic education.\n\nSo this chapter charts the work involved in fitting motion pictures to educational and reformist agendas; it also surveys the cultural and ideological landscape on which these efforts took place. Three sections alternate between practical efforts and historical and theoretical context. The first section discusses the general contours of reform in Wilhelmine Germany before outlining some of the concrete steps reformers took to protect child audiences from the hazards of cinema. Censorship, taxes, and child protection laws were accompanied by attempts to create an alternative film system by controlling means of production, distribution, and exhibition. I will also describe reformers' efforts to persuade production companies and theater owners to support reform films and exhibition values, which led to the creation of reform theaters and community cinemas. The second section examines some of the guiding principles or assumptions behind these efforts, including _Anschauungsunterricht_ , aesthetic education, and the discourse on \"the child\" and \"the masses.\" The urge to protect children from the \"degeneracy\" of mass entertainments went hand in hand with the desire to educate the general public. Both concerns drew life from child and crowd psychology popularized at the turn of the century. Reformers saw aesthetic education and _Anschauungsunterricht_ , entwined in theory and practice, as potential solutions to the problematic picture of spectators the experts painted. The third section looks closely at the work of two reformers who set out to create alternative, educational, and edifying exhibition spaces in response to the perceptions and problems outlined in the previous section. Educator and reformer Hermann Lemke articulated principles for the educational use of film for school-age children, especially at commercial theaters that arranged special screenings for elementary schools. Another reformer, Hermann H\u00e4fker, hoped to offer educational or edifying film presentations for adults. H\u00e4fker's attempts to create \"model presentations\" ( _Mustervorstellungen_ ) exemplify the use of film as an instrument of ideological solidarity. Increasingly worried about the \"bad taste\" of mass entertainments, H\u00e4fker enlisted film in a program of aesthetic education designed to raise the sensibility of the people to a unified, national level. Taking his cue from a long tradition of aesthetic education dating back to Schiller, as well as the art education movement then taking place, H\u00e4fker wanted to use motion pictures to train the tastes of the nation. Finally, this section takes a longer look at Schiller's ideas about aesthetic education to lay bare Germany's long-standing ideological investment in the relationships between vision, taste, pedagogy, and nation.\n\nCINEMA AND THE SPIRIT OF REFORM\n\nIn their desire to make a change for the better, the men and women involved in film reform were part of a much larger set of movements sweeping the industrialized world around the turn of the century. As increased industrialization and urbanization brought one social upheaval after another, \"reform\" expressed the mood of the times in a variety of ways throughout Europe and the United States. In the United Kingdom, constitutional reforms swept through Parliament as groups demanded suffrage throughout the last third of the century. In the United States, the agrarian Populist movement of the 1890s and the Progressive movement of the 1900s reflected a broad impulse toward criticism and change. Progressivism, in particular, captured the spirit of reform through its outrage over the excesses of capitalism, its faith in progress, and its interventionist policies. During the 1880s, the pressures of industrialization and democracy prompted the French parliament to create the only state-run, compulsory, secular primary school system in the world. The growing confrontations between the forces of labor and capital also prodded republican politicians to campaign for social legislation, such as regulation of working conditions, to ensure social peace.\n\nGermany, in particular, was deluged by swelling transformations in the public sphere provoked by rapidly changing demographics. The Industrial Revolution and national unification came relatively late to Germany and accelerated very quickly. The resulting discord between the classes and between rural and urban lifestyles seemed especially acute. During the high tide of these changes, which occurred from around 1890 to 1920, the concept of \"reform\" as an expression of the sense of transition and as a plan for managing it took on special significance for self-understanding. Germany's groundbreaking legislation providing for compulsory insurance for workers' sickness, workplace accidents, and retirement pensions became an influential model for the United Kingdom, the United States, and France. These measures, dealing in some form with physical conditions and consequences of the workplace, illustrate the strong connection between class and somatic issues in reform movements during the late nineteenth century. Reform manifested itself in everything from _Jugendstil_ decor, to a new, more \"natural\" style in women's clothing ( _Reformkleidung_ ), to nutrition reform ( _Ern\u00e4hrungsreform_ ). \"Reform\" implied a battle against tradition, against perceived cultural and social stagnation; as such, it provided a plan for the formation of new, more \"authentic\" concepts for living. In fact, the connections between the reform movements and the more general tradition of _Kulturkritik_ are very strong; from Jean-Jacques Rousseau and Johann Heinrich Pestalozzi in the eighteenth century to Friedrich Nietzsche and Paul de Lagarde in the nineteenth, the critique of society paved the way for a general re-evaluation of values in the twentieth.\n\nVery often, the critique of society focused on the educational system. Education reform was among the first movements to sweep across Germany. The kaiser himself had set the agenda on a December morning in 1890 while addressing a congress of educators in Berlin; Wilhelm II claimed he grasped \"the spirit of an expiring century\" with his calls for school reform. In answering the question of how the German schools of the nineteenth century could be reshaped to meet the needs of the twentieth, the kaiser echoed sentiments that had been expressed throughout Europe during the often rocky transition from the Victorian age to the modern. Specifically, he voiced his fears that the _Gymnasium_ (high school) failed to train its students adequately for the requirements of Germany's rapid industrialization. Second, he complained about the \"excess of mental work\" in the schools, arguing that such \"overburdening\" was threatening the physical health of Germany's youth. Finally, he insisted that German schools devote more time and energy to fostering specifically national values, thus turning away from the traditional emphasis on the classics: \"We must make German the basis of the _Gymnasium_ ; we should raise young Germans, not young Greeks and Romans.\"\n\nThe kaiser's concerns about \"the modern,\" \"the healthy,\" and \"the national\" reflected and reinforced similar fixations of the European elite. Like them, he found the educational system to be both the problem and the solution to perceived crises. By inadequately preparing the nation's children for the demands of the future, the system risked irrelevance, according to reformers of the time. Swift reform promised both a brighter future and a greater measure of control over the rapid changes taking place. Among the different examples of education reform were the country boarding schools ( _Landerziehungsheime_ ), which were experimental schools located in the countryside as an explicit rejection of urban culture. Their emphasis on physical education echoed the hopes of the youth movement for a spiritually renewing combination of countryside, fresh air, and _Volk_. Likewise, work schools ( _Arbeitsschule_ ) hoped to renew the creative (and ethical, hence political) spirit through manual labor, such as gardening, and handicrafts, such as wood sculpting or leatherwork. The art education movement ( _Kunsterziehungsbewegung_ ) similarly stressed the creative capacities of children, advocating renewal through art and education of the aesthetic sensibility.\n\nFilm reformers shared the kaiser's interest in \"the modern,\" \"the healthy,\" and \"the national.\" At the center of their concerns lay motion pictures, which they also found to be both scourge and cure. An emblem of modernity, cinema represented a plague, especially toxic to children, and proper education of the public was the only hope to halt the epidemic. At the same time, cinema was emerging as the most powerful instrument of mass education and therefore potentially provided the surest treatment for whatever ills modernity had spread. Before treatment could begin, however, commercial interests had to be persuaded to participate in this remedy. Moreover, film needed a stamp of legitimacy to have any authority in this rescue mission. Film reform was the process by which these goals were attempted, if not completely achieved. It shared roots, objectives, and ideology with other reform movements of the day, especially, and not surprisingly, educational reform.\n\nIt does seem rather unusual that the head of the German empire, almost by definition the representative of a conservative status quo, would come out so strongly in favor of reform. A mixture of progressive reforms and reactionary politics indicated the ambivalent attitude of the bourgeoisie toward the troubling issues of the day. All reform movements revealed, in one way or another, the fundamental irony of the kaiser's position. The calls for clothing reform in Germany exemplified this contradiction. In his 1901 book, _The Culture of the Female Body as a Foundation for Women_ ' _s Clothing_ , Paul Schultze-Naumberg made an extensive study of the debilitating physical effects resulting from methods of forcing the female form into an aesthetic ideal. In a graphic and impassioned plea to eliminate the corset, in particular, Schultze-Naumberg demonstrated how its use eventually caused deformation of the muscles, bones, and internal organs. He called for a more functional clothing in keeping with \"a new concept of corporeality.\" While consistent with similar efforts by women's movements to liberate themselves from the pressures of social constraints, Schultze-Naumberg's \"new concept\" of more \"natural\" bodies included only those from healthy German stock. Carl Heinrich Stratz, another strong advocate of clothing reform, took a similar approach in his book _Women_ ' _s Clothing and Its Natural Development_ , grounding his arguments for the elimination of the corset on the conclusion that it threatened the racial superiority of European women. Schultze-Naumberg and Stratz, whose worries about the integrity of the Fatherland were cloaked in concerns for the health of women, are excellent examples of the fusion of progressive goals of more liberal movements with reactionary, nationalist politics.\n\nClearly, Schultze-Naumberg and Stratz shared with their fellow guardians of culture a rather paternalistic attitude toward the role of women in the changing public sphere of modernizing Germany. Women in general, and female sexuality in particular, were often targets of public disapproval about modern life, as in Stratz's work above, or in the complaints about Asta Nielsen's \"unladylike\" behavior in her films, or in the concerns about the number of women in cinema audiences. For some, these complaints are signs of a deeper anxiety about the increasing role of women in public life in Germany; the women's movement or the youth movement in their various manifestations are often cited as prime motivators in the perceived decline of male cultural authority. According to this version, the women's movement exemplified a menace that appeared to surround German intellectuals; actions such as the youth movements seemed to threaten paternal credibility even in the home. Faced with such massive structural changes, the cultural elite embraced reform as a way of coping with, and controlling, modernization. While it is true that most voices that survive in print are male, we must not forget that women were not simply the objects but the agents of reform. In fact, late nineteenth-century imperial Germany saw a proliferation of volunteer organizations\u2014instigating reform, helping the poor, and exchanging information\u2014that provided structure to a society in transition. Women entered the public sphere not only as consumers but also through volunteer organizations that functioned in tandem with, even as a substitute for, the state and provided steady and urgent pressure for changes in social policy. So social policy in imperial Germany was not simply a top-down affair instigated by male experts but grew out of women's culture. Likewise, while Germany's \"crisis in culture\" might have been acutely felt by male intellectuals as a decline of male authority, this perception must be tempered by the knowledge that the social mobility that women and workers gained from modernization also benefited males and the middle class not only through the greater economic power that came with a consumer society but the political power that came with the alignment of state policy with middle-class goals\u2014an alignment that occurred partly as a result of the intervention of women's and volunteer organizations. So while the hyperbole of public debate sometimes provides an easy example of male, middle-class \"anxiety,\" we should not take it entirely at face value.\n\nThe _Schund_ campaigns of late nineteenth-century Germany are a great example of the battle between modern consumer culture and the goals of middle-class volunteer organizations. _Schund_ is usually translated as \"trash\" or \"rubbish,\" but reformers of the late nineteenth century applied it to serialized novels and pamphlets purveyed by colporteurs or kiosks, especially literature perceived to lack any redeeming value yet not obscene. While the serialized novel emerged in the mid-nineteenth century, the market grew exponentially thereafter; by 1890 it was so popular that _Schundliteratur_ accounted for fully two-thirds of all German literary sales. By the time of the Weimar Republic, _Schund_ referred in general to any thin, mass-market paperback novel sold by the millions at kiosks and stationery shops.\n\nEducators and school administrators were the first to launch campaigns against this form of entertainment; according to Corey Ross, \"the roots of the campaign against _Schund_ can indeed be traced back to the efforts of elementary school teachers in the 1870s and 1880s to influence the reading habits of their pupils by drawing up lists of recommended titles.\" Teachers continued to be the primary force behind these campaigns, even as they comprised a diverse group of interested parties, including temperance groups, religious associations, women's commissions, leagues against public vice, and local police forces. Yet, as Kara Ritzheimer has argued, the rhetoric of reform unified these groups, despite their diverse goals and methods. Ritzheimer paraphrases, for example, a professor who warned that\n\nchildren who read excessively were likely to develop a lust for reading ( _Lesewut_ ) that might \"effeminize the body,\" \"cause the senses to lose their acuteness,\" \"weaken the memory,\" \"over-excite the imagination,\" and \"destroy a will to pay attention to serious matters.\" Furthermore, this \"reading lust\" was capable of breeding indolence ( _Schlaffheit_ ), indifference ( _Gleichg\u00fcltigkeit_ ), absentmindedness ( _Zerfahrenheit_ ), mental laziness ( _Denkfaulheit_ ), extravagance ( _\u00dcberspanntheit_ ), and slack behavior when it came to work and play ( _Unlust zu Arbeit und Spiel sich einstellen_ ).\n\nThis rhetoric of sensuality, addiction, feminization, and weakness was a portrait of media effects that transformed apparently normal children into the worst caricature of the lower class. Beyond the vocabulary of media effects, these groups also promoted _children_ as the primary focus of their efforts, thereby unifying their protectionist rhetoric around a class-neutral issue, rather than waging a campaign in the name of _adults_ , which could barely evade paternalistic connotations, if not all-out class warfare. In any case, the rhetoric was incredibly pliable, in that it was applied equally vociferously to books and motion pictures.\n\nCinema reform not only patterned itself after the educational reform movements, the instigators were often hardened veterans of the anti- _Schund_ campaigns. Karl Brunner, for example, was a _Gymnasium_ professor and a leading anti- _Schund_ campaigner who eventually became the head film censor in Berlin. The Hamburg commission discussed later started as a response to _Schund_. Most of the reformers were teachers and educators, so their close ties to the education reform groups of the period were also formative. Hermann Lemke, a _Gymnasium_ professor from Storkow and one of the founders of _Kinoreform_ , was well connected to the Society for the Dissemination of Popular Education (Gesellschaft zur Verbreitung von Volksbildung), the leading educational organization in Germany. Hermann H\u00e4fker, the most articulate representative of film reform and arguably Germany's first film theorist, was a journalist and writer who was also close to the leaders of the art education movement. Konrad Lange, one of the leading voices of the art education movement, taught art history at the University of T\u00fcbingen.\n\nDespite their similar backgrounds, these reformers were not all of one mind. The disparate views and priorities of all involved, as well as the absence of a central organization or platform, make it difficult even to characterize _Kinoreform_ as a movement. Scattered around mostly northern and small-town Germany, the representatives worked primarily at the local level, trying to coordinate national efforts through friendly trade journals, such as _Der Kinematograph_ (1907\u20131935) out of D\u00fcsseldorf and especially _Bild und Film_ (1912\u20131914) out of M\u00f6nchen-Gladbach. The birth of trade magazines devoted exclusively to film coincides with the birth of the reform movement in 1907; during its earliest years, _Der Kinematograph_ acted as a willing partner in _Kinoreform_. The range of viewpoints in its pages, and in the other magazines that followed shortly thereafter, testifies to the difficulty the reformers had in choosing the most effective course of action.\n\nIf they did not agree on methods, their approaches at least reflected the experience and infrastructure already gained in the work against _Schund_. Basically, the efforts of the film reform movement overall can be divided into \"negative\" and \"positive\" solutions, in the parlance of the day: those that emphasized regulation, taxes, police enforcement, and censorship, and those that offered alternatives to the objectionable fare; this chapter will focus on the latter, \"positive\" approach. We can also divide these approaches further based on the object of reform. _Filmreform_ , for example, expressed an emphasis on the _content_ of films, with an accompanying strategy that focused on production. _Kinoreform_ , on the other hand, expressed an emphasis on the _space_ of film exhibition, and with it a strategy to clean up and \"uplift\" film theaters (fig. 3.1). _Filmreformers_ such as Brunner and Lange spent their energy devising negative methods to control film production and reception, while Lemke and H\u00e4fker developed positive strategies for both _Filmreform_ and _Kinoreform_.\n\nFIGURE 3.1. A typical storefront movie theater ( _Ladenkino_ ) from the pre\u2013World War I era\n\nCourtesy Deutsches Institut f\u00fcr Filmkunde, Wiesbaden\n\nYet these reformers had a set of common objectives framed in ways similar to those in the _Schund_ campaigns. First and foremost, they felt compelled to protect children from what they perceived to be the dangerous effects of cinema. This was first explicitly stated in 1907, when a teacher's group in Hamburg, the Society of Friends of the Schools and Instruction for the Fatherland (Gesellschaft der Freunde des vaterl\u00e4ndischen Schul- und Erziehungswesens), formed a commission to study the effects of cinema on schoolchildren. Its conclusions were predictable and familiar: both the films themselves and the theaters produced physical and moral side effects in school-age children. The combination of the \"flicker effect\" and the lack of adequate ventilation in theaters caused \"eyestrain, nausea, and vomiting,\" according to the commission. Emphasizing the connection between the body and ethical judgment, the symptoms were a sign of a deeper moral sickness, manifested in school by \"apathy for learning, carelessness, and a tendency to daydream.\" Jurist Albert Hellwig, certainly the most prolific reformer who advocated the negative approach, echoed these concerns in 1914, when he argued that \"a promotion of a certain superficiality and inattentiveness, as well as a retardation of concentration and aesthetic cultivation\" could be counted among the psychological dangers to young moviegoers\u2014a diagnosis familiar from the discourse examined in the previous chapter.\n\nSecond, the reformers made it clear from the very beginning that they hoped to use cinema for educational purposes. In this and many other ways, the German reformers were very similar to their American counterparts, who also took it upon themselves to \"uplift\" both the theaters and the films for the good of the masses. The Hamburg commission concluded its study with the recommendation that\n\nTechnically and thematically impeccable cinematic presentations can be an outstanding instrument for education and entertainment. Pedagogically and artistically minded groups must advocate for better, nobler uses of the cinematograph by encouraging the big corporations in this industry to present good, child-oriented productions in special screenings for children.\n\nHermann Lemke answered this call to arms independently in the summer of 1907, when he persuaded a Friedenau cinema theater owner to host Germany's first \"reform theater,\" which was likely merely a \"film reform\" night or series at a commercial theater, given that it did not last very long in this form. Lemke gave the opening address, making the goals of cinema reform clear to the mostly middle-class audience of teachers, press, and community leaders. He charged that the current state of cinema had caused the aesthetic sensibilities of the people to regress. Calling on the combined power of educators and the press, he maintained that \"when the taste of the people is so backward, it's the duty of the intellectual [ _geistigen_ ] leaders to influence them and put their aesthetic taste back on the right track.\" Cleaning up the cinema theaters was the first order of business in this project, making it one of the earliest examples of _Kinoreform_. Lemke applauded the improvements the theater owner had already made:\n\nThis reformation is already apparent in the way this auditorium has been given a worthy interior decoration. Gone is the small, narrow room where everyone is crammed and squeezed together; in its place we find a larger, airier hall... so that the patron's sense that he is in a second-rate establishment vanishes all on its own. Good ventilation has been provided in order to reduce health risks.\n\nLemke's concerns demonstrate how closely \"taste\" and \"respectability\" were tied to \"the body,\" and especially the body of \"the masses.\" He was preaching to the converted, however. _Der Kinematograph_ later reported, \"it seems that the middle class is more interested than the working class in the direction the reform theater is taking. While the seats in the third section show hardly any patrons, the first section (50 cents admission) is mostly sold out.\" Still, Lemke was sufficiently encouraged to organize a Cinema Reform Association the following autumn. Represented by teachers, members of the press, theater owners, and production companies, the association was one of many throughout Germany that hoped to coordinate efforts from these quarters toward their educational goals. Indeed, composed of businessmen, teachers, council members, and theater owners in the community, local _Kino-Kommissions_ such as this were the primary means through which reformers organized their efforts, disseminated their results, and created larger networks. Lemke's society received contributions from such firms as the German branches of Eclipse and Gaumont. While cleaning up the _Kinos_ , the reformers turned their attention to the films themselves.\n\nEnjoying an easy fraternity with producers during the early years, film reformers hoped to capitalize upon their good relations with the motion picture companies to increase the number and availability of reform-type productions. In a particularly idealistic move indicative of the \"positive\" approach, Lemke in 1908 suggested that his reform association act as a \"Film-Idea-Central,\" a clearinghouse of sorts for reform-minded scripts. Members of the association could submit ideas for scenarios, and the society would negotiate with the studios on the writers' behalf. Lemke explained, \"Because we're in constant contact with the manufacturers, such an exchange will be relatively easy to arrange, particularly because we know what is being demanded. We would provide distribution free of charge and only require that those who use it become members. In this way, we may be able to bring the film companies to the cutting edge of this moment and also have a productive influence internationally.\" Unfortunately, while the members of the movement might have held some early enthusiasm for this plan, the film companies themselves apparently did not take to it; the idea never went beyond the initial stages, and no further mention is made of the Film-Idea-Central in the trade press or reform publications.\n\nThe failure of the Film-Idea-Central and the film reform theater in Friedenau established something of a frustrating pattern for the reformers. Film companies and exhibitors expressed early interest in reform projects, even going so far as to sponsor events, but eventually refused more meaningful and lasting support. The end of 1908 saw the opening of Germany's first film trade show\/exhibition at Berlin's Zoological Gardens. Jointly sponsored by Lemke's reform party and the leading film companies at the time, and with the rather obvious motto of Refining the Cinema ( _Veredelung des Kinos_ ), it was nonetheless heavily criticized even by friendly periodicals for its lack of organization. Exhibitors, manufacturers, and production companies declined the reformers' help for the next exhibit in 1912. Likewise, when Lemke and H\u00e4fker attempted to muster support for their special exhibitions, the film companies were initially supportive but lost interest fairly quickly. Realizing that domestic companies could not or would not produce sufficient numbers and variety of educational films, H\u00e4fker went so far as visiting foreign film companies, such as the Charles Urban Trading Company in London and Eclipse in Paris, to find suitable nature films for his exhibitions. Lemke even went to England and wrote film treatments to jump-start some sort of interest in his program. Very early on, it was quite clear that the production companies were cautious about backing the reformers and their schemes.\n\nThis did not mean, however, that the film companies wanted nothing to do with the reform movement. They were certainly willing to use the reform movement for their own ends; despite their difficulties, the reformers were still a legitimating presence\u2014they were, after all, educators, clergy, journalists, and otherwise pillars of their respective communities. Film companies were eager to cash in on this allegiance. Advertising trumpeted this relationship, even if the reformers had nothing to do with the making of the film. A 1912 Italian film distributed by the German company PAGU, _Die Irrfahrten des Odysseus_ (The Wanderings of Odysseus), was rather disingenuously labeled a \"Reformfilm\" and carried this blurb: \"From a special press screening, which was attended by the most respected Berlin literary figures and art critics, came the unanimous decision: 'This film signals the long-awaited reform of cinema'\" (fig. 3.2). Aware of the potential directions cinema could take, the film companies initially went along with the reformers, especially if they could be used as a selling point. But as soon as it became apparent that the vast majority of the viewing public was more interested in narrative entertainment free from \"ennobling\" connotations, the companies brushed off the reform societies' efforts to influence the product directly.\n\nThe extreme positions of some reformers did little to help the overall cause with the production companies. Lange and Brunner, for example, were steadfast in refusing film any legitimacy whatsoever as a medium of entertainment. Their regular denunciations of \"film drama\" ( _Kinodrama_ ) merely antagonized an industry leaning heavily toward narrative films. This prejudice against narrative films often disguised stronger rhetoric against international domination of the German film market. \"In the international film drama, the wildest passions of all nations come together for a gruesome rendezvous,\" clergyman Paul Samuleit charged. Likewise, their complaints about capitalist interests tainting cinema's potential were thinly veiled laments about the presence of _foreign_ capital. Some reformers, such as H\u00e4fker and Lange, dismissed film drama because of aesthetic concerns. It did not offend their sensibilities because of sloppy production qualities, although these did attract attention. Rather, the filmed drama betrayed what they saw to be cinema's primary mission: to record movement and \"real life.\" The argument for filmic realism, of course, coincided with their desire to use cinema for educational purposes, as we will see in the next section. As Sabine Hake notes, they did not dismiss the possibility of story elements in their educational films, but the excesses of the \"trashy film\" so contradicted their stated ideals that many rallied against film drama altogether, for both political and aesthetic reasons.\n\nFIGURE 3.2. _The Wanderings of Odysseus_ (L'Odissea, Italy, Milo, 1911), touted to be a \"Reformfilm\"\n\nLemke hoped to reform the cinema through example, stressing cooperation with and from the industry, and to rally schools together to create a distribution system. Others were not so willing to rely on this teamwork. One faction of the reform movement, led by Albert Hellwig and Karl Brunner, saw censorship and regulation (the \"negative\" approach) as the best way to combat the onslaught of _Schundfilme_. Both Hellwig and Brunner advocated a series of legal restrictions on the cinema, including censorship, entertainment taxes ( _Lustbarkeitsteuer_ ), poster censorship, safety regulations, and child protection laws ( _Kinderschutz_ ). Authorities tried to maintain some control over child audiences (and, consequently, theaters) by restricting their visits to specific hours of the day, regulating the length of the matinees, and requiring that they be accompanied by an adult, that police should have unlimited access to the theater during the matinees, that the day's program must be given prior approval, or that a \"suitable pause\" separate the films.\n\nReformers recognized early on the importance of establishing a distribution network for their educational films. For this task, Lemke and his circle enlisted the help of the Society for the Dissemination of Adult Education (Gesellschaft zur Verbreitung von Volksbildung, hereafter referred to as the GVV). An umbrella organization for more than 8,000 local education groups, clubs, associations, and societies, it was a formidable partner in _Kinoreform. Bildungs-Verein_ , the house publication, had a circulation of 13,000\u2014many times that of any film trade magazine. Yet the GVV leadership was hesitant about cinema's importance as an educational tool. Even though Johannes Tews, the director of the GVV and editor of _Bildungs-Verein_ , had attended the opening of Lemke's Friedenau reform theater, he still considered cinema to be of minor significance. The GVV resisted involvement with cinema until 1912, when it established a film distribution center of 180 films in 16 categories, from history of the Fatherland to educational films on biology.\n\nThe reformers found a more willing and beneficial partner in the Lichtbilderei, established in 1909 as a foundation of the Association for Catholic Germany (Volksverein f\u00fcr das katholische Deutschland). The Lichtbilderei was Germany's largest educational film institute before World War I, with an extensive catalog of titles. It began as a rental source for magic lantern slides, which could be used for public lectures, but started collecting films as well after 1911. By the end of 1912, it reportedly had around 900 titles and was collecting more at about 30 films per week, and by 1913 offered 400 slide series and 1,400 film titles. The Lichtbilderei was not limited to providing films for schools, churches, and clubs; it also provided programming for many commercial theaters. Approximately 40 weekly theaters and 50 to 60 Sunday _Kinos_ showed Lichtbilderei films regularly. The Lichtbilderei was also involved in the distribution of more commercial dramas, actually acquiring \"monopoly\" rights over such established hits as _Quo Vadis?_ (Italy, 1913), _Giovanna d_ ' _Arco_ (The Maid from Orleans, 1913), and _Tirol in Waffen_ (Tirol in Arms, 1914). From 1912 to 1915, the Lichtbilderei was something of an organizational center for the cinema reform movement. Its stock of films gave life to the community cinemas and private _Reformkinos_ , and its publications\u2014the periodical _Bild und Film_ (Image and Film) and the series of books from the association's Volksvereins publishing company\u2014were the principal forum for the discussion of _Kinoreform_ issues after 1912.\n\nIn 1912, the GVV, in association with the Lichtbilderei, established the funds for two educationally oriented _Wanderkino_. These traveling cinemas toured from town to town, playing for four to six weeks in each place, in an effort to offset the influence of commercial cinemas and unify aesthetic and educational standards across the nation. Showing between nine and eleven films an evening, accompanied by lectures concerning such topics as \"A Modern Factory,\" the enterprise was basically modeled after the GVV's successful _Wandertheater_ and public lecture series. Between the fall of 1912 and the outbreak of war, the _Wanderkinos_ offered a total of 1,279 such evenings.\n\nReformers had most success with their exhibition experiments. In addition to the reform theaters and _Wanderkinos_ already mentioned, a number of communities established their own public cinemas. The first was founded in the town of Eickel at a cost to the community of 14,000 German marks. Others opened soon afterward, in such towns as Altona, Wiesbaden, Osterfeld, Frankfurt (Oder), Gleiwitz, and Stettin. These cinemas became the center of local reform activity and provided the precedent for the state-run cinemas of modern Germany, which continue to illustrate the relation between taste and nation. The proclamations of the early _kommunale Kinos_ articulated this relationship and the goals of the reform movement in general:\n\nTo oppose, for aesthetic, cultural, and patriotic reasons, the trash that is generally offered in the private theaters; to replace it with films of scientific, entertaining, and educational value; to exert, in association with established institutions with similar principles, a gradual influence on the film market, which is currently almost entirely dependent on foreign countries, and thereby keep here the millions that are flowing out of the country. Finally, to enlist cinema in the service of youth organizations and colleges by providing suitable presentations.\n\nTo the modern observer, the cinema reformers of imperial Germany might seem a bit quixotic. Tilting their lances to such impassive windmills as capitalism and narrative, they only reluctantly and belatedly conceded that they were charging against the wind of public opinion. As the movies became more popular and an evening's entertainment began to look less and less like a lecture series, instead relying more heavily on _Kinodrama_ , the reformers began to look more and more irrelevant. Their own Dulcinea\u2014the children of the nation\u2014seemed oblivious to their activities. Even those sympathetic to their cause, like this reviewer of a book on cinema and theater reform, found their efforts somewhat naive.\n\n[The author] is certainly entitled to his opinion in this terribly serious matter. However, he will surely also understand the skepticism of those who do not believe at all in the \"ennobling\" of films towards a literary bent [ _literarischen Seite_ ], because they see completely heterogeneous things being forced into an unhappy marriage. The idea that benevolent corporations will free the theaters of commercial interests altogether is too pretty to be given much credence.\n\nOthers were not so kind. Speaking on behalf of the industry in 1911, a trade journal editor pointedly replied to the reformers, \"We ourselves know what we need, and we don't need tutelage.\" One theater owner from the 1920s remembered them as \"sanctimonious folks and hypocrites, morality sleuths in male and female guise.\" Film histories, until recently, have been equally dismissive. Siegfried Kracauer charged simply that, with their zealous efforts to defend the literary canon of the nation, \"they yielded to the truly German desire to serve the established powers.\" Even if a bit condescending, Kracauer was not far off the mark. While the proclamations of the various _Kinoreformers_ embraced a wide range of opinion, they never strayed far from the status quo. As Sabine Hake noted, \"In sharp contrast to the intellectuals, the reformers aligned themselves openly with the existing power structure.\" We must not, however, underestimate the reformers' contribution to German culture. In trying to sway what Kracauer called \"the salutary indifference of the masses,\" the reformers succeeded in dominating the discourse on cinema in the years before World War I. In addition to the permanent impression they left on German film culture, German mass communication research owes them an especially heavy debt: their focus on media effects had a lasting influence on the vocabulary and goals of modern mass media studies in Germany.\n\nUltimately, of course, cinema reform was not completely successful. The reformers failed to meet their stated goals and, considering the extreme position of many reformers, this is perhaps all for the best. World War I abruptly changed the nation's priorities, and even though the calls for reform were heard again through the Weimar years, the urgency of the moment had passed. In 1913, lances heavy with disappointment, the movement clearly appeared to be running out of breath. Sighed Lemke, \"I was always hoping that someone would take over the chairmanship from me, assist me, and further expand the [Cinema Reform] Association, but no one was willing to do so and the result was that the Association remained in its infancy [ _in seinen Kinderschuhen stecken blieb_ , or literally, \"stuck in its children's shoes\"].\"\n\nCHILDREN, CROWDS, AND THE EDUCATION OF VISION AND TASTE\n\nLemke's metaphor was apt, because it reveals the extent to which the reformers thought about the cinema (and themselves) through the metaphor of \"the child.\" Because they were educators and teachers, this is perhaps to be expected. It is noteworthy, however, that they applied this trope to adult audiences. References to their audiences as \"children,\" especially in connection with mention of \"the masses,\" are scattered throughout the discourse. One reformer, looking for the underlying causes of cinematic drama's continued popularity, maintained that \"just as much blame belongs to the audience, the people [ _das Volk_ ], this 'big child,' whose alarmingly spoiled taste craves for cinema's dramatic trash and silly comedies, practically forcing the theater owners to present them with aesthetic and moral duds week after week.\" Even Georg Luk\u00e1cs thought about cinema spectatorship in terms of children: \"In the 'cinema' we should forget these heights [of great drama] and become irresponsible. The _child_ in every individual is set free and becomes lord of the psyche of the spectator.\" Whether Luk\u00e1cs's \"inner child\" was inherently good or evil depends upon one's viewpoint, of course, and there were many to choose from at this time.\n\nThis section will survey some of the prevailing assumptions about child psychology and pedagogy to clarify the underlying ideological presumptions about child and adult moviegoers. \"Suggestibility\" was the common denominator linking children and crowds; studies of children were even the source of theories about social psychology. So this section will demonstrate how the portrait of (film) spectatorship usually derived from expert analyses of children and crowds. At the same time, I will show that symptoms of this spectatorship were problems to be solved by training in observational methods, specifically aesthetic education exemplified by the kind of programs promoted by Hamburg museum director Alfred Lichtwark, who was the driving force behind the art education movement of the time. His approach was popular and well known to film reformers\u2014especially because its nationalist flavor appealed to the taste of many experts of the day\u2014but it was also familiar because it exemplified the observational approach to general education known as _Anschauungsunterricht_. Looking closely at this approach or trend in pedagogy reveals it was a self-conscious response to the perceived disorder and quickened pace of modern life; observational training was a way of ordering thought that countered pace and disorder by emphasizing \"dwelling\" and correlation. This section thereby connects psychology, reform movements, and pedagogy to explain the ideological and practical emphasis on expert modes of viewing as a solution to the multiple problems spectatorship posed to film reform.\n\nChild psychology of the period provides a partial explanation for the equation of children and the masses. Swedish author Ellen Key's _Century of the Child_ , an enormously popular children's rights manifesto published originally in 1900, advocated a reassessment of the prevailing view that children were inherently evil. Summing up a trend in child psychology that emphasized the creative nature of the child, it called for new teaching methods to correspond to the new century, leaving behind the authoritarian methods of the old school and reassessing pedagogy \"from the child outward\" ( _vom Kinde aus_ ). If adult society, utilitarianism, and the demands of the \"real world\" had determined the standards of pedagogy before, now attention turned to the child's needs and inner nature. Whereas the old pedagogy might have emphasized uniformity, now the child could expect to be treated as \"the measure of itself.\" As Key insisted, \"instruction should only cultivate the child's own individual nature,\" which Key and others assumed to be creative, good, and even wise.\n\nFreud, however, was less optimistic about the life of the child. His essay on \"Infantile Sexuality,\" published in his 1905 _Three Essays on Sexuality_ , painted a darker picture of childhood as a \"hothouse of nascent psychopathology.\" His explanation of the importance of the child's body\u2014describing the oral, anal, and phallic stages\u2014on mental development was groundbreaking. Its lasting contribution is manifold, but most immediately it underlined the influence of childhood development on adult mental life. While there is little indication that Freud's theories were wholeheartedly accepted by garden-variety reformers, the new child psychology of both Key and Freud provides a clue to the urgency reformers felt when they argued for aesthetic cultivation and against the influence of sexually charged _Kinodramen_.\n\nDespite Sigmund Freud's seminal contributions, Darwin's evolutionary theories of child development still had a strong grip on the public imagination during this period. In particular, Darwin argued that child development recapitulated the mental evolution of the species. Accordingly, the maturing child was expected to exhibit mental characteristics of subhuman species. In _The Descent of Man_ , Darwin observed, \"We daily see these faculties developing in every infant; and we may trace a perfect gradation from the mind of an utter idiot, lower than that of an animal low in the scale, to the mind of a Newton.\" Discussions of crowd psychology latched onto this teleological comparison between children and primitive mentalities. Gustave Le Bon, the most well known popularizer of nineteenth-century crowd psychology, characterized the masses as \"an enraged child.\" Furthermore, according to Le Bon,\n\nIt will be remarked that among the special characteristics of crowds there are several\u2014such as impulsiveness, irritability, incapacity to reason, the absence of judgment and of the critical spirit, the exaggeration of the sentiments, and others besides\u2014which are almost always observed in beings belonging to inferior forms of evolution\u2014in women, savages, and children, for instance.\n\nDarwin's evolutionary scheme provided a quasi-scientific basis for comparing crowds with children, but even more significant for this comparison was the concept of \"suggestibility.\" Le Bon devoted a chapter to \"the suggestibility and credulity of crowds,\" arguing that the crowd is \"perpetually hovering on the borderland of unconsciousness, readily yielding to all suggestions\" (21), a mental state most commonly found in women and children (29). Most serious psychologists of his time dismissed Le Bon's rather crude arguments, but the metaphorical connection between children and the masses was still quite powerful for researchers. In fact, one could argue that social psychology has its roots in child study. Alfred Binet, a disciple of La Salp\u00eatri\u00e8re's Charcot and one of the founders of experimental social psychology, used the observational opportunities provided by public school classrooms to test his evolving theories of suggestibility. His conclusions about children and suggestibility worked their way into his formative studies of social behavior, which had a profound impact on the direction of modern social psychology.\n\nReformers borrowed the concept of \"suggestibility\" as they described the cinema audiences and their scopophilia or _Schaulust_. The Hamburg commission noted this condition in their report, complaining that\n\nmany cinema presentations endanger children morally as well. Let's assume, for example, that a young boy with a tendency towards thievery were to see crimes presented with elegance and brilliant success. Wouldn't that arouse his imitative instinct? A young girl could easily learn how to get easy money and enjoy an apparently carefree and, in her eyes, wonderful life by selling her honor. When she needs to earn a living later in life, she might ask herself: \"why work at a sewing machine for 10 pfennigs an hour, why work at a factory for 10 marks a week?\"\n\nWhy, indeed? These remarks prefigure persistent themes in the discourse on cinema during this period, especially the preoccupations with suggestibility, crime, and female sexuality. Emilie Altenloh, author of the first sociological study of cinema, even found parts of this equation in the nature of female spectatorship:\n\nThe female sex, of which it is generally said that it always purely and emotionally absorbs an impression in its entirety, must be particularly receptive to filmic presentation. By contrast, it seems very difficult for people who are highly developed intellectually to project themselves into the sequences of events, which are often strung together haphazardly. On various occasions people who were used to grasping things on a purely intellectual level said that it was extremely hard for them to comprehend the logic of a movie plot.\n\nAltenloh equated holistic or synthetic approaches to the image with female spectatorship, and analytic approaches with expert or educated observation. She suggested that, on one hand, this open or holistic approach to the image enabled an empathetic projection that is unavailable to those who approach the film analytically. On the other hand, this empathetic mode of viewing left the spectator vulnerable to suggestion, and in this step she equated feminine and childlike modes of spectatorship. She further maintained that, in the absence of a strong family life or education, cinema held a mesmerizing influence on its young patrons, especially young male workers: \"It is undeniable that the cinema's lack of all higher interests has a certain influence on the entire way of thinking and living for these unstable people [ _ungefestigen Menschen_ ],\" she concluded. \"From the lives of outlaws, the morals of Apaches, and the fearlessness of heroes in cowboy films they take a philosophy of life that forces them into trajectories similar to that of their celebrated idols.\"\n\nAlbert Hellwig also wrote often on the suggestive power of cinema and its dangers for the criminally inclined or morally weak. In one article, he described a neurasthenic woman's response to a night at the movies. In the film, a postal clerk dreams that he is attacked by robbers: \"there appear a series of threatening faces and ghostlike hands, which reach out to others in their sleep.\" This made such an impression on the young lady that she began to see hallucinations of these hands day and night. \"The rather intelligent lady was initially fully aware that it was merely a hallucination, a product of her imagination. She was nonetheless quite upset because she saw this group of gigantic hands appear suddenly and in a variety of circumstances.\" Hellwig implied that the cause of the woman's hallucinations was a combination of cinema's suggestive power and the woman's pathological condition, neurasthenia, a vague, yet debilitating nervous condition in vogue during this time. It left its victims incapable of work and inflicted upon them a dazzling array of symptoms, including headaches, the fear of responsibility, graying hair, and insomnia. According to Anson Rabinbach, \"neurasthenics were identifiable by their impoverished energy and by the excessive intrusion of modern urban society on their physical and mental organization.\" It was a form of mental fatigue that left its victims unable to resist the stimuli of the modern world; it was characterized, in short, as a weakness of the will, as moral exhaustion.\n\nThe combination of pathology and morality is significant, because the concept of \"moral weakness\" metaphorically connected judgment and physical strength. The reformers' focus on both the unhealthy atmosphere of the nickelodeons and suggestive power of film reveals an underlying concern for both the bodies of the audiences and their moral judgment. This concern manifested itself as a problem of \"taste\"\u2014taste lies between the realms of sensuality and reason. As with the question of the nature of the child, reformers were divided over the nature of the masses, especially their judgment. Against those who argued that the masses were not ready for reform, that they were not interested in what interested the educated classes, Hermann Lemke argued, \"I for one cannot imagine that the general population has such bad taste; and even if the people were not yet mature enough for it [cinema's reform], they would have to be educated. But one must never indulge their lowest instincts\u2014that is harmful to the community and must be prevented.\" Hellwig was less willing to entertain the idea that the masses were inherently good: \"It is the bad taste of the audience that ultimately makes the trashy film.\" The solution to this problem of taste and, by extension, the crisis of moral judgment, was aesthetic education.\n\nSince Schiller, aesthetic education has offered a solution to the twin problems of sensuality and suggestibility. That is, Schiller suggested the category of the aesthetic as a medium between alienated Nature and Reason. In an alienated world, the aesthetic provided Schiller with hope for reintegration and, hence, social harmony. The aesthetic category acted as a corridor between raw nature and a higher morality. \"In a word,\" Schiller wrote, \"there is no other way of making sensuous man rational except by first making him aesthetic.\" The reformers were very interested in making \"sensuous man rational.\" Schiller's importance for the reformist agenda is illustrated by an editorial in the trade periodical, _Lichtbild-B\u00fchne_ (fig. 3.3). The headline reads, \"The Cultural Work of the Cinema Theater: Thoughts from the Year 1784, by Friedrich von Schiller.\" The essay invoked Schiller's \"The Stage Considered as a Moral Institution\" to argue that cinema could function in the same manner. The aesthetic, however, was a precondition to the moral, and cinema must first go through that transformation. An illustration from a 1918 reform pamphlet illustrates the axiomatic nature of this relationship between the aesthetic and the moral (fig. 3.4). The upper-left sphere represents \"entertainment with immoral effect\" and \"morally unobjectionable entertainment,\" while the upper-right sphere signifies \"art\" and \"non-art.\" A transubstantiation occurs when the rather plain problems of morality (left) and aesthetics (right) are superimposed to reveal the nature and proportion of \"art,\" \"trash,\" and _Kitsch_. We could say that this new sphere represents the issue of \"taste.\"\n\nFIGURE 3.3. \"The Cultural Work of the Film Theater: Thoughts from the Year 1784 by Friedrich von Schiller\"\n\nFIGURE 3.4. The geometry of taste: the superimposition of morality (\"entertainment with immoral effect\" [ _Unmoralisch wirkende Unterhaltung_ ] and \"morally unobjectionable entertainment\" [ _Moralisch einwandfrei Unterhaltung_ ]) and aesthetics (\"art\" [ _Kunst_ ] and \"non-art\" [ _Unkunst_ ]) reveals the nature of taste (\"art\" [ _Kunst_ ], \"trash\" [ _Schund_ ], and _Kitsch_.)\n\nSchiller represents the beginning of a long tradition of aesthetic education in Germany, one that eventually became grafted onto questions of nationalism. The most famous, or infamous, example of this development was August Julius Langbehn's _Rembrandt as Educator_ , first published anonymously and with enormous success in 1890. Like Schiller and de Lagarde before him, Langbehn reacted against the perceived excessive rationalization of the Enlightenment. The preoccupation with systemization, objectivity, and book learning had, in his opinion, brought about \"the decline of the spiritual life of the German people\" (7). Specialization, he complained, precluded exercise of creative power: \"one thirsts for synthesis\" in overeducated Germany, he wrote, and so \"one turns to art!\" (8). The German people could be rescued from this \"systematic, scholarly, cultured barbarism\" by \"going back to their original source of power, their individualism\" (9). Furthermore, if individualism was the root of all art, and he claimed it was, and if education should correspond to the nature of its students, then art education would be the most effective and natural form of instruction. Rembrandt, even though he was Dutch, was for Langbehn \"the most individual of all German artists\" (11). \"The scholar is characteristically international, the artist national,\" he wrote, underlining the difference between science and art, word and image (11). The goal of art education, as Langbehn saw it, was to effect a spiritual regeneration of the German people by reacquainting them with their own inner nature as it was exemplified by the masterpieces of national art. But if this program sounds reasonable, most of Langbehn's essay was noxiously antimodern, antiliberal, and even anti-Semitic, which unfortunately struck a chord among the reading public and resounded throughout German culture of the time. He advocated approaches to aesthetic education rooted in national (as opposed to international\/foreign) sources, but he understood the local and national in biological, racial, and ethnic terms. Progressive agendas had no place in his conception of aesthetic education.\n\nAlfred Lichtwark, generally recognized to be the driving force behind the art education movement, also valued local artistic traditions in his address to the 1901 art education conference in Dresden. \"Our education still lacks a _firm national foundation_ ,\" he declared. The basis for a national culture, as with Langbehn, could be found in German art. \"Up to now,\" Lichtwark said, \"the schools have not considered it their task to acquaint youth not only with the names, but the works of the great artists who express the German character\" (104). And he blamed this lack of attention to \"national art\" for the lack of \"formative power\" in German culture. But Lichtwark's idea of _Heimat_ was not rooted in blood and soil, like Langbehn's, but rather in the historical and cultural environment in which talent and cultural forms develop; Lichtwark's more historically contingent conception of _Heimat_ therefore led to a more liberal and modernist understanding of the relationship between local and national culture. Yet like Langbehn, Lichtwark held that \"the challenge of art education\" was inseparable from \"a moral renewal of our life\" (99). This hope was certainly not limited to Lichtwark; most representatives of the art education movement held it as their ultimate goal.\n\nBut Konrad Lange was cautious of such sanguine hopes, asking at that same conference whether \"with ' _Kunsterziehung_ ' [art education] we've actually found the magic word to solve all social questions.\" If he seemed less concerned about the spiritual state of the people, he was, like Lichtwark, still very anxious about the state of German art. He acknowledged that \"we actually have masters of the first order in all the areas of the fine arts, men who are living proof that the creative German spirit is not yet dead,\" but claimed that this was not enough. For this relative good health to survive, it needed good soil in which to grow. \"And this soil can only be the people's understanding of art.\" Worried that the elements of modern urban life could undermine children's sense of culture, Lange advocated leading them to art to maintain a sense of artistic tradition, to bring \"the artistic education of our youth... in closer connection to the living, creative art of the present.\"\n\nThe education of taste was also very important to Lichtwark. To his contemporaries, he was even better known as the director of the Hamburg _Kunsthalle_ , which came into international prominence during his tenure. There he was instrumental in organizing groundbreaking exhibits of amateur and artistic photography, promoting local artists, and rediscovering forgotten local talents, such as the romantic painter Philipp Otto Runge. \"We do not want a museum that simply stands and waits,\" proclaimed Lichtwark upon assuming the directorship of the Hamburg _Kunsthalle_ in 1886. \"Rather, we want an institution that actually works for the aesthetic education of our population.\" Aesthetic education, for Lichtwark, was about individual self-cultivation, of course, but it also had value for the community. Summarizing Lichtwark, Eckard Schaar writes, \"Artistic cultivation is not an innate ability... but rather a participation in a national collective property that, carried by the spirit of the people, influences the soul of the individual.\" Lichtwark envisioned his museum as an educational center for the artistic life of the region that would focus and direct this process of individual and communal aesthetic development. It would be a clearinghouse of taste, wherein exhibits of art from around the world would help raise the sensibilities of the general public and teach new techniques to local artists. For instance, in his introduction to the first exhibit of amateur photography in 1893, Lichtwark stated that the show's goal was to \"raise the artistic taste of the public and stir interest\" in the new art. The development of a national art depended upon the aesthetic education of both the public and the artists\u2014his museum would take up that task.\n\nLichtwark's influence on actual educational practices came through one of his most popular books, _Exercises in the Contemplation of Art Works_. The drills consisted of Aristotelian question-and-answer sessions between teacher and student, demonstrating by example how the child's inherent aesthetic taste could be cultivated and guided to acceptable standards. The student would gaze upon a painting and answer the teacher's questions about its form and content until the work's meaning would reveal itself to the child. For this process to be successful, Lichtwark stressed the importance of extended contemplation of single artworks in a quiet, conducive environment. Consistent with the _vom Kinde aus_ philosophy mentioned earlier, this method of aesthetic training soon gained wide favor among German art educators. Lichtwark's system also confronted the important issue of national taste.\n\nThe typical modern German has a weakness in the area of aesthetic education. He lacks an external refinement and solidity of form as well as an inner connection to the visual arts. He has no desire for artistic pleasures that require an education of the eye and heart. His eyes see poorly and his soul not at all. For the preservation of our nation as well as our national economy, these inadequacies must be forcefully redressed.\n\nLichtwark designed his _Exercises_ to provide a training program for children and others who were \"aesthetically weak.\" By teaching youngsters how to look, gaze, and, ultimately, _see_ , Lichtwark was following a set of presumptions common to aesthetic education: train the eye and the heart follows. For the art education reformers of imperial Germany, then, educating public taste was a project in nation building. Simply, education _through_ art was a way of building a distinctly national art, while education _to_ art was designed to build consensus and therefore national unity, as well as maintain traditional standards and methods of evaluation. These two directions were and are common for all art education programs from Friedrich Schiller to John Dewey.\n\nLichtwark's approach caught on. A number of books in the following years staged an encounter between an imaginary viewer, a painting, and a helpful questioner. _Bildbetrachtung_ (\"image viewing\" or \"image contemplation\") became a common method of teaching art appreciation, which emphasized the contemplative, especially the spiritual side of aesthetic education and experience. Schoolteacher Heinrich Wolgast echoed the views of many in Hamburg and elsewhere who stressed the connection between vision and spiritual development through disciplined viewing exercises: \"the child who conquers the world with these more sensitive organs will reap greater rewards than one with untrained eyes,\" he wrote, and \"intellectual understanding of the world based on a deeper grasp of its appearances will guarantee an improved preparedness for life.\" Reformers such as Wolgast hoped that training the observational acuity of children and adults would result not just in spiritual but ultimately national renewal; the art education movement is therefore a good example of the explicit, parallel investment in visual education and national goals that we see consistently in discussions of positive film reform. Lichtwark's program enjoyed national visibility and increasing popularity, especially among educators interested in current ideas about visual education. The parallels were not lost on film reformers such as Lemke, Sellmann, and H\u00e4fker, who had similar educational aspirations for film.\n\nYet these pedagogical impulses sprang from a broader trend in visual education that suffused nineteenth-century pedagogy, for which Wolgast's formulation could have easily served as the motto. This trend, known generally as _Anschauungsunterricht_ (\"observational instruction\" or \"visual means of instruction\" or simply \"visual education\"), started with the educational principles of Swiss pedagogue Johann Heinrich Pestalozzi (1746\u20131827), who believed that _Anschauung_ , or \"sense impression\" was \" _the foundation of all knowledge_.\" _Anschauung_ is a difficult word even for Germans: in Kant's _Critiques_ it is usually translated as \"intuition,\" but it also means \"sense perception,\" which are two very different things. In German dictionaries the two meanings of the word, one cognitive, one perceptual, sit side by side, matching the epistemological and phenomenological (or the knowable and visible) sides of the experiential coin. Yet _Anschauugsunterricht_ strove to develop the child's innate sense of form (in the Kantian sense) through observational exercises focused on the visible world, so it thereby embraced _Anschauung_ 's seemingly opposed connotations. It is perhaps best understood as a process through which the pupil attains an appreciation of both the detail of the individual object and its place in a larger system, whether philosophical, taxonomic, or social. Pestalozzi's program was notable for its then-radical approach to education. Rather than a top-down, authoritarian, deductive, and speculative method that demanded the student listen to the lecturer and recite back what was said or read in books, Pestalozzi advocated a bottom-up, democratic, inductive, and empirical approach to learning that asked the teacher to interact with the student\u2014similar to the process in Lichtwark's _Exercises_ \u2014and trust that the student could come to a higher understanding of the material through careful observation of the object in front of him or her. It countered mere book learning and rote memorization with a program that emphasized the pedagogical power of natural objects themselves.\n\nBut it was not merely about bringing the child to an object; the method was primarily about teaching the child how to _observe_. Yet as we saw in the previous chapter, observation is above all a complex logical operation, as Clive Ashwin notes about Pestalozzi's _ABC der Anschauung_ (1803): \"Its content was designed to activate and exercise the child's faculties, first by distinguishing separate entities and isolating them within the perceptual manifold; secondly by enabling the child to observe and note their peculiarities of form; and thirdly by associating each form with its correct name. This integration of number, form, and word led Pestalozzi to put the _ABC der Anschauung_ at the centre of his educational scheme.\" In practice, this involved a specific, step-by-step process. The teacher and the student would look at an object, such as a seashell, and the child would be asked to describe what he or she saw and be encouraged to point out individual characteristics of the object (\"distinguishing separate entities and isolating them\"). A series of questions asked by the teacher would bring the student to understand the basic form of the object, sometimes by drawing the object or its geometrical shape (\"enabling the child to observe and note their peculiarities of form\"). Naming each element and its form would be an important part of the exercise (\"associating each form with its correct name\"). Equally as important, the student would come to an understanding of the salient elements of the object (such as the seashell's function as both protective covering and housing) and its relationship to other such objects or abstract principles. Through this \"object lesson,\" the child would learn from direct perception but would also learn the method for correct observation \"in which essential qualities of the object are distinguished from the accidental ones.\"\n\nPestalozzi perceived this process as a solution to the problem of rote memorization and excessive book learning, but also to the problem of society's perceived acceleration. He sought to advance learning through direct, visual encounters as opposed to verbal or written encounters, but he also implicitly designed this approach to _decelerate_ the learning experience. The entry on Pestalozzi in the _Encyclopedia of Philosophy_ summarizes the goal: \"According to Pestalozzi, it is the curse of modern civilization that its hasty and primarily verbal education does not give man enough time for the process of _Anschauung_ , a term perhaps best translated as 'internalized apperception,' or as dwelling on the meaning and challenge of an impression.\" Written and spoken information were too abstract for Pestalozzi, but the spoken lecture also expressed the \"hasty\" pace of modern life. The object lesson allowed the child to dwell on the salient features and relationships, or on _details_ and their _correlation_ to principle and form. Indeed, Pestalozzi's method embodied the modern educator's contested relationship to detail, which on one hand exemplified the confusion, ornamentation, and overstimulation that many saw as the problem with modernity's visual field. Melanie Keene, in her dissertation on the object lesson in England, discusses the example of palaeontologist Gideon Algernon Mantell, whose distaste for the crowds and hyperstimuli of the 1851 Great Exhibition in London fortified his belief in the value of attention to single, small objects as doorways to general principles. Mantell's motivation for adopting Pestalozzi's pedagogical principles was common; in the profusion of detail, the object lesson commanded _time to dwell_ on the form underlying the confusion.\n\nYet on the other hand, this very detail functioned as the object's ground of authenticity. Not just any object could serve as the focus of a lesson. Preferably, the object came from \"nature,\" a term which for Pestalozzi was synonomous with all that is genuine, authentic, and free from artificiality. A useful contrast in this scheme is the drawing, which was often an important _part_ of the lesson\u2014students learned about form by drawing shapes\u2014but which was explicitly eschewed as the _basis_ of a lesson. That is, students could draw an object and thereby learn about its form, but they could not use a drawing as the lesson's object itself. Pestalozzi was unhappy with rote memorization of words, but he was \"equally dissatisfied with the use of pictures as a substitute for the direct experience of objects.\" The difference, of course, is in the details, which in this method signified the randomness, contingency, and authenticity of nature; \"authentic\" meant \"free from human influence,\" or \"that which resists us\" (one German word for \"object\" is _Gegenstand_ , or \"that which stands against\"). Pestalozzi and his disciples complained about the overabundance of stimuli and detail in the modern world\u2014almost invariably associated with the man-made world and its objects\u2014but they clung to the detail of nature as the ground for their method.\n\nThe _Encyclopedia of Philosophy_ 's entry on Pestalozzi translates _Anschauung_ as \"internalized apperception,\" which Christopher Ritter helpfully unpacks as \"self-directed understanding through which newly observed qualities of an object are related to past experience.\" The editor and translator of Johann Herbart's 1804 elaboration of Pestalozzi's method agreed that apperception was key: \"He [Herbart] teaches that the chief object of instruction is to secure the reaction of the mind upon what is offered to sense-perception. We must understand what we see. We must explain it by what we know already.\" Looking at _Anschauungsunterricht_ in this way, it is clear that Pestalozzi's method signaled a distinctly modern moment when self-identity was aligned with observation, or more precisely, with the logical operation most closely associated with the practice of observation: _correlation_ , which we explored in the previous chapter. To relate newly observed qualities to past experience and then to order those qualities and experience into a hierarchy and network of relationships\u2014this was what modern educators and experts defined as _understanding_. And this is what they demanded from their students as they trained their vision to be expert. Observation and correlation ruled as all fields committed to visualization and new forms of seeing developed across the disciplines. Herbert Spencer, who popularized Pestalozzi's method in England, exclaimed, \"Of new practices that have grown up during the decline of these old ones, the most important is the systematic culture of observation.\" So educators versed in _Anschauungsunterricht_ understood observation as a twofold operation: to _dwell_ on authentic, natural detail and to _correlate_ that newly observed detail to past experience with form and function. The object lesson was therefore a reaction to the perceived _quickness_ and _disorder_ of modern life.\n\nPestalozzi and his followers soon and quickly disseminated his method throughout Europe, where it found fertile ground. The commitment to visualization in the sciences encouraged visual instruction in education, which was the logical extension of Pestalozzi's approach. And obviously, given its emphasis on nature, _Anschauungsunterricht_ was perceived to be an especially effective method for teaching natural history and science. Science educators, like nearly all those interested in visual education whatever their discipline or national origin, expressed the belief that images worked _quickly_ and _efficiently_ on the viewer's mind. Chapter 2 explored this assumption about the presence and power of images; briefly, the trope presumed a homology between image, idea, and the structure of the human mind. Because of this presumed structural similarity, images were thought to leave a stronger impression than verbal or written descriptions. German physiologist Carl Jacobj put it best:\n\nSymbolically descriptive word images [ _Wortbilder_ ] of concepts can be replaced by the simultaneously created visual image [ _Anschauungsbild_ ] that represents the factual object of observation immediately and in all its details, so that it [the visual image] is imprinted in a faster, stronger, and more sustainable way on the conscious mind and, as a consequence, on memory.\n\nEven if the image worked as a fine substitute for a wordy description, Jacobj indicated that no educator ever left images to work on their own. Indeed, without proper guidance, images were almost always considered anathema to proper understanding. So the second major trope or guideline in visual education, beyond the vividness and efficiency of images, was that the real power remained with the instructor's words. But this relationship to words was fraught, as we have seen: a discomfort with words as the basis of a lesson but a recognition that words needed to frame a lesson. As Ashwin notes, \"In its most fundamental sense, then, _Anschauung_ meant something like 'direct and correct observation': observation which was closely _associated with_ language to the extent that the child was given the correct verbal equivalent at the point of making the observation, but which was not _mediated by_ language.\" These dicta remained unchanged as motion picture technology became an option for visual education, as we will see in the next section.\n\n\"CINEMATIC LESSON PLANS\" IN ELEMENTARY AND ADULT EDUCATION\n\nYet this tidy portrait of Pestalozzi and _Anschauungsunterricht_ should not deceive us into envisioning nineteenth-century pedagogy as an orderly sequence of inheritances. Over the course of the century, these principles were not only diffused so broadly that some did not even know their origin (if they could be said to have one), but there were so many interpreters, objections, and tweaks to theories of educational praxis that average reformers could be forgiven if they were sometimes confused about which practice belonged to which theory. For example, the art education movement positioned itself against what it perceived to be the excessive emphasis on science and mathematics in the approach of Herbart and his disciples, even as it adopted his method for ordering the lesson plan and even though Herbart himself argued against excessive rationalism in education, too. Nor should we be confused about the degree of impact these theories actually had in the authoritarian, state-run classrooms of imperial Germany\u2014which is to say, hardly at all. We should, however, recognize that all of these reform efforts hoped to provide _balance_ and _order_ to the educational experience, even if the understanding of proper balance and order was constantly in dispute. So when faced with the educational potential of film, which hardly anyone disputed, and the chaotic application of such potential, which everyone noted, advocates such as Lemke and H\u00e4fker drew upon the trends in pedagogical theory at the time in order to place film into an orderly and recognizable system of practice. This section will discuss precisely how this was accomplished with regard to elementary education (Lemke) and adult education (H\u00e4fker).\n\nFilms in the elementary school classroom were very exceptional, if not unheard of, during the early period in Germany. The apparatus was cumbersome and difficult for the untrained, but the expense was prohibitive as well. Because hardly any elementary schools could afford motion picture technology, the only solution, if film were to be a modern addition to their visual instruction agenda, was to take students to the local theaters. These excursions would generally take place during special screening times set aside during classroom hours, when the children and teachers could attend a matinee showing of a program of films deemed suitable by teachers, reformers, and school administrators. Most teachers and reformers, however, balked at the idea, having already decided that the movie theater was inherently corrupting, no matter what the content. Even so, commercial theaters were undaunted, especially after decrees in 1910 and 1912 in Breslau and Prussia, which restricted entry for children to film theaters except for special children's matinees. Most provinces eventually adopted these laws, leaving theaters without an important part of the market and prompting many exhibitors to cooperate more readily with schools and educators. So a proprietor of a film theater in Berlin, in a pamphlet promoting the educational use of (his) commercial theaters, assessed the situation, stressing the importance of film for visual instruction:\n\nIt is hardly necessary to mention the value of cinematic presentations for visual instruction [ _Anschauungsunterricht_ ]. For some time there have been efforts everywhere to use this important element in teaching. Institutions of higher education, which have the necessary resources, have their own screening rooms equipped with cinematographic equipment. Similar plans have repeatedly been made for elementary schools, but they have not yet been carried out, mainly because of high costs.\n\nThe word _Anschauungsunterricht_ was commonly used in connection to film's potential place in the curriculum. It was especially popular with regard to teaching natural history, for which motion pictures seemed to enjoy a preternatural inclination. Yet almost as often as educators suggested that motion pictures could function well as an object lesson, they questioned the \"motion\" part of \"motion pictures,\" as in this declaration by a teacher named R\u00fcswald: \"Based on my experience, I would only support the use of film in teaching in the following circumstance: namely, that slides and film are used simultaneously. This demand is based on the facts that the impressions of the cinematographic image are too quick and thus too superficial, and that now more than ever a calm, quiet, measured dwelling on a single subject matter of education is bitterly needed.\" This equation of movement, haste, and superficiality was common, and it was certainly consistent with the principles outlined in the previous section (we will see more of it in chapter 4). But educator Adolf Sellmann would have none of it:\n\nThis objection is unjustified. Does the observation of movements in nature make one superficial? Not at all. On the contrary, this kind of observation can and must lead in many cases to an especially acute attention and thoroughness. If I want to grasp the action precisely, I must look closely, observe keenly, and turn focused attention to the moving process. Motion processes observed in nature can imprint themselves deeply on the soul, so that the observed motion becomes an inner experience. This can, of course, also be the case with motion that is observed on film. If I have focused on it with all my attention and therefore with all my soul, the impression lasts longer. _The living picture can often have a longer-lasting impact than the still picture._ Why should observed movement only fleetingly be remembered? It surely depends entirely on the mind [ _Seele_ ] that looks at it.\n\nSellmann and other advocates argued that movement actually _sharpened_ the attention and that film's ability to replicate that movement\u2014and, crucially, to reveal hidden aspects of it\u2014gave it a privileged role among the _Medienverbund_ (media ensemble) of early visual instruction. Educators argued back and forth about the role of filmed movement in the classroom, but their discussion could be distilled to a question that was rarely articulated: What is the role of observed movement in understanding the natural world? Or better: What does it mean to understand movement? Fundamentally, the split in camps corresponded to a choice between discontinuity and continuity, with teachers such as R\u00fcswald advocating the use of slides (and film, in his case, although some argued for slides only), while Lemke, Sellmann, and others supported the use of _motion_ pictures. Lobbyists for film saw themselves on the side of modern pedagogy, while critics of film defended against unnecessary and potentially dangerous technology.\n\nYet the primary justification for the use of film as an object lesson resided not necessarily in its movement but in the ability of the photographic image to replicate in its detail the randomness, variety, and disorder of the natural world. Educators signaled this by consistently calling film's image \"faithful to nature\" ( _Naturgetreu_ ), which was a term often used by scientists and researchers when justifying their faith in the photographic image. Of course, not every aspect of the photograph is perfectly faithful to nature, but in these discussions, _Naturgetreu_ referred most often to the level of detail that allowed the photographic image to reproduce patterns of texture and variation. Physician Richard Kretz wrote, \"Photography is perfectly faithful to nature [ _Naturgetreu_ ], that is, the images reproduce... all forms and proportions, the distribution of light and shadow in a completely correct manner.\" Later, a geography instructor declared,\n\nThe cinematograph offers an excellent substitute for student hikes and field trips. It leads the student not only through the wider area of his home province, but also through the most distant latitudes. He gets to know countries and peoples through his own observation [ _aus eigener Anschauung_ ]. The most accurate description of a landscape, the most in-depth, vivid [ _anschaulichste_ ] description of life and the activity in it, the most detailed painting cannot replace what film, with true fidelity to nature [ _Naturgetreu_ ], parades before the eyes of the students.\n\nThis \"fidelity\" referred not to color or depth or emphasis or any of the aspects of nature and observation that many complained photography could not represent well. Instead, it referred to the same qualities that brought scientific curiosity to bear on nature in the first place: the abundant variations on patterns of similarity and difference found in the forms and random textures of natural phenomena. Because photography could replicate these forms and textures with such detail, it could act as a substitute or a representation of the object of study. Like \"vividness\" ( _Anschaulichkeit_ ), _Naturgetreu_ referred to the advantage of images over words. But _Naturgetreu_ was finally a stronger justification for film's role in the object lesson. Even if some disagreed on the role of movement in films seen in the classroom, _Naturgetreu_ was a description of film that nearly all could agree upon, especially given the importance of nature's details to the principles of _Anschauungsunterricht_ , as we have seen.\n\nNevertheless, if the image itself was more or less pedagogically controversial, educators objected, often rightly, to the lack of films specifically made for their curricula or the lack of rigor in most special school presentations at commercial theaters. Many complained that the _wissenschaftliche_ (\"scientific\" or \"academic\") presentations programmed by commercial exhibitors were often nothing more than inoffensive nonfiction titles randomly strung together. _Reformkinos_ , or theaters especially procured in order to offer more edifying screenings, were sometimes a solution to this problem, but they required financial and institutional support not often forthcoming. In his 1909 pamphlet, _Kinematographie und Schule_ , Georg Victor Mendel argued for creating standing theaters devoted to educational films for schoolchildren, a goal later achieved in the Urania theaters and the _kommunales Kinos_ discussed earlier. Ernemann, the Dresden-based equipment manufacturer, dedicated in 1909 its standing exhibition space to educational or scientific screenings for children three afternoons per week. Also in Dresden in 1910, civil engineer August Kade funded twice-yearly educational and scientific screenings\u2014with live musical and song accompaniment\u2014in a city-owned exhibition space, which was dubbed the Kosmographia \"scientific theater.\" Overall, at least eight reform-oriented theaters or screening spaces opened in Germany between 1909 and 1914, all of which were touted as alternatives to educational programs at commercial theaters. Even with these efforts, commercial cinemas remained the most widely available option for elementary school educational film screenings (fig. 3.5).\n\nFIGURE 3.5. Children at Luisen-Kino in Berlin, circa 1910\n\nYet the problem of curricular integration remained. Educators complained that the film programs at these theaters were well intentioned, but offered far too much far too quickly:\n\nIt is precisely the broad scope of the programs, which are all condensed into one to two hours, that must stir the most concern from a pedagogical point of view, as students do not arrive at calm observation or reflection, and none of the images can leave a lasting impression on them.... Not until projectors and film are cheaper and each school has its own cinematograph, or a special device can be attached to the projection apparatus that occasionally allows cinematographic images to be shown, which the teacher can present himself and explain as the lesson requires\u2014only then can film have a profitable application in the school.\n\nFrom the typical teacher's point of view, school screenings at commercial theaters fundamentally lacked the _control_ they required: motion pictures moved at a pace that could not be controlled, but also the program itself was out of their hands. According to this teacher, film had a future in the classroom only if that control\u2014in a literally hands-on manner\u2014could be assured. Lemke, whose efforts represented the most serious attempt to accommodate films and schools, took up the difficult challenge to integrate motion pictures into the grade-school learning experience. He not only had to take into account these complaints but also to assuage implicit anxieties about the role of the teacher in a technologically mediated classroom; admittedly, his and other utopian proclamations about the coming ascendancy of visual media and the subsequent decline of lectures did not help matters in that regard.\n\nEven if teachers such as Lemke assumed that film's fidelity to nature (its _Naturgetreu_ qualities) allowed it to adequately substitute for the object, that was only half of _Anschauungsunterricht_ , or the \"object lesson\"; film needed to be integrated into the lesson plan as well. Lemke attacked this problem by following the principles for guided apperception that developed in the late nineteenth century, especially after a new generation of pedagogues in the 1880s further refined Herbart's interpretation of Pestalozzi. As a concession to those against commercial screenings for children, Lemke argued for a distinction between special screenings for schoolchildren ( _Sch\u00fclervorstellungen_ ) and screenings that incorporated methods unique to the classroom, which he dubbed _Schulvorstellungen_ ; Lemke thereby put the film program in the teacher's control. He advocated that teachers understand the available films, actively work with the exhibitor to curate the programs, and then incorporate the screenings into lesson plans according to accepted Herbartian principles. Lemke also emphasized discussion sessions before and after film screenings in the commercial theaters. This plan required much more preparation: preselection of the films, discussion among the faculty about modifying or accommodating the program to the existing curriculum, and teacher training in using the films and leading discussion. For a brief time, Lemke even held intensive teacher-training seminars on educational film issues and techniques.\n\nIn fact, Lemke's suggestions for organizing film into a lesson plan followed Herbartian principles step by step. One of Herbart's enduring legacies in pedagogy is a five-step program for guiding apperception within a lesson:\n\n1. preparation ( _Vorbereitung_ )\n\n2. presentation ( _Darbietung_ )\n\n3. association ( _Verkn\u00fcpfung_ )\n\n4. generalization ( _Zusammenfassung_ )\n\n5. application ( _Anwendung_ )\n\nThrough these reform-minded and theoretical steps, teachers would introduce new knowledge of an object to a student by first reminding the student of already known material (preparation); then presenting the new material, repeating as necessary (presentation); encouraging associations between what is known already and what is new speculating on abstract principles linking the two objects (generalization); and finally thinking about how to apply this knowledge to new objects. In his own plan for the educational use of film, which he outlined in detail in _Die kinematographische Unterrichtsstunde_ (The Cinematic Lesson Plan, 1911), Lemke followed these steps closely by explaining how film could be deployed through each (see fig. 3.6).\n\nFIGURE 3.6. A page from Lemke's _Die kinematographische Unterrichtsstunde_ (The Cinematic Lesson Plan, 1911), in which he provides a Herbartian lesson plan for a specific film\n\nHe also recommended specific films and groupings of films that followed the idea of apperception: introducing new objects and concepts through abstract connections to the familiar. In this way, Lemke and others accommodated film to principles of _Anschauungsunterricht_ that were already widely accepted by reform-minded teachers: first, by connecting the specific features of the cinematic image to the visual emphasis in that tradition ( _Naturgetreu_ and the _object_ lesson), then by demonstrating that film could be incorporated into the curriculum in a familiar way (apperception, film, and the object _lesson_ ). With these efforts, and his own journal devoted to educational film and slide material ( _Die Lichtbildkunst in Schule, Wissenschaft und Volksleben_ , Storkow 1912\u20131914), Lemke was on the leading edge of the educational use of film in Europe and the United States. For an excellent example of the use of film for adult edification, however, we must turn to Hermann H\u00e4fker (fig. 3.7).\n\nAfter the failure or, at best, limited success of the attempts to create an alternative production and distribution system, reformers realized that focusing on exhibition held the most promise for fulfillment of their program. So like Lemke, H\u00e4fker looked to existing commercial theaters to establish an alternative exhibition venue to create a suitable educational or edifying environment. Miriam Hansen has argued that the peculiarities of early cinema exhibition presented the structural possibility of an alternative public sphere. The variety format; the sense of theatrical space; the combination of lectures, live music, sound effects, and so on; and the uneven development of modes of production, distribution, and exhibition\u2014all contributed to \"overlapping types of public sphere, of 'nonsynchronous' layers of cultural organization.\" Between the \"fissures of institutional development,\" alternative modes of reception and experience could emerge. The reformers, of course, hoped to \"synchronize\" these layers, not only by coordinating the modes of production, distribution, and exhibition, but also by integrating the various cultural spheres that commercial cinema was already grafting upon itself: literature, science, the tradition of the lecture series, and art.\n\nFIGURE 3.7. Hermann H\u00e4fker\n\nHermann H\u00e4fker's \"model presentations\" ( _Mustervorstellungen_ ) are the best example of the reformist exhibition program aimed at adults. Some have called him Germany's first film theorist\u2014he was certainly one of the very first to write regularly about the cinema. He began the century working as a writer, journalist, and translator for a number of periodicals, covering a range of topics, from Shakespeare's sonnets to his own bicycle tour of Finland. He was one of the first writers for _Der Kinematograph_ and a spirited contributor to and editor of _Bild und Film_ , eventually writing three books on film for the Volksvereins publishing company. His _Bild und Wort_ (Image and Word) society film exhibitions were prototypes for many \"model presentations\" that reformers tried to implement on a regular basis around Germany. His 1913 book, _Kino und Kunst_ (Cinema and Art), was an elaborate justification of the artistic potential of cinema and an extension of his earlier work in the reform journals. In this monograph, he describes his attempts to create aesthetically pleasing and educationally effective cinema programs. As we have seen, H\u00e4fker was not alone in these attempts, but he was unique in providing theoretical justifications of his programs.\n\nLike Lange and the other reformers, H\u00e4fker was concerned with the aesthetic sensibility of the masses and the influence of bad taste. His comments about taste were directed particularly to the contemporary state of film exhibition. Of the nickelodeons of the 1910s, H\u00e4fker noted, \"the educated circles have been repulsed by the tastelessness of the programs.\" Further, \"it's not the What of the program, but the How of the presentation that makes the impression.\" Of course, he certainly did not withhold complaints about the \"sensational\" films the producers presented to the audience. But unlike many of his contemporaries, such as Willi Warstat, who felt that censorship was the proper solution, H\u00e4fker continued to express his concern for the \"tasteless\" exhibition. This tastelessness referred, most generally, to the intrusion of modern life's hectic pace into the auditorium, where spectators were assaulted with a \"breathless chase of one number after another, accompanied by intertitles, the uninterrupted noise of the projector, the lights, etc.\" In this regard, H\u00e4fker's goals were consonant with those of _Anschauungsunterricht_. H\u00e4fker demanded an exhibition that avoided the exciting and the extraordinary and instead tried to establish \"a quiet and natural mood.\" He advised exhibitors to program their films in accordance with classical aesthetic principles, building tension and then release by alternating comedies with dramas and \"scenes from the life of nature and simple people.\" The exhibitor should also refrain from putting all the films on one reel, allowing instead a short pause between them so that \"the spectator's eyes would receive their necessary recovery time and the nerves a moment to relax.\"\n\nThis last bit of advice points to a range of literature dealing with visual fatigue and the motion pictures. In this discussion, the equation of cinema with modernity was most explicit. H\u00e4fker expressed the concerns of the day quite well when he complained, \"image and form, word and sound, color and line... rain like a hailstorm on the nerves of modern man, especially in, but not limited to, the city.\" Cinema came to epitomize this hailstorm. Some of the first articles written on cinema in Germany were medical papers on the harmful results of the \"flicker\" effect. Other medical investigations dealt with the threat of eyestrain in the film theaters. Nearly all reformers or opponents of cinema criticized its threat to public health and vision, as we saw in chapter 2. And as we saw in chapter 1, this outcry represented the larger preoccupation with fatigue that characterized debates coming out of the late nineteenth century. As Anson Rabinbach has shown, the trope of fatigue was more than a scientific mania of the age; it expressed a profound anxiety of decline and social disintegration. In the medical, scientific, and even literary study of fatigue, there was \"a tendency to equate the psychological with the physical and to locate the body as the site where social deformations and dislocations can be most easily observed.\" In other words, metaphors of health and sickness were used to express national anxiety. Fatigue, together with neurasthenia, was more than a physical ailment\u2014it was also perceived as a _moral_ disorder, a sign of weakness and the absence of will. Neurasthenia, mentioned before in connection to cinema's suggestive power, was the most typical metaphor for the delicate condition of the national psyche.\n\nH\u00e4fker viewed modernity as a series of \"shocks\"; he sought a haven to which he could escape the hailstorm of modernity. He just wanted to rest for a while, to give his nerves time to recuperate, and he wanted to make cinema such a haven. But cinema would never be this sanctuary, he said in 1908, \"so long as the corresponding sense of illusion is missing and the correct mood is lacking.\" There is so much in the modern world to disturb this mood, but treating film as an art form, especially exhibiting films \"tastefully,\" could slow this flood of \"the much-too-much\" ( _eine Eind\u00e4mmung des Vielzuvielen_ ). He planned to do just this with his \"model presentations.\" In 1910 he presented to the Image and Word association in Dresden a model program that was to be the prototype for other cities. The selection consisted mostly of nature films, but plans for further exhibitions included travelogues, scientific films, and _actualit\u00e9s_. Originally, he intended to continue the exhibitions in coordination with local schools, but the project fizzled due to lack of readily available films for continuous programming.\n\nThe 1910 presentation, entitled \"Spectacles of the Earth,\" highlighted H\u00e4fker's preferred form, the nature film: \"The first part showed high mountains and deserts; the second part concerned ethnological subjects (Laplanders, Chinese, Arabs, Indians, cannibals, etc.). The third part dealt with 'The Thousand Games of Water' (Victoria Falls, Niagara Falls, storms on the coast, surfs, rapids, geysers, underwater volcanoes from New Zealand).\" The films were accompanied by lectures, slides, music, and nature sound effects, all of which H\u00e4fker tried to orchestrate into a _Gesamtkunstwerk_ of Wagnerian proportions. The presentation began with a lecture of what to expect, what to look for, and \"in which sense to take it.\" It would then alternate films with slides and lectures, carefully presenting each. H\u00e4fker provides a detailed\u2014and obviously quite proud\u2014description of the final section of the program:\n\nThen it became dark once again. You could hear the sound of water, and as the curtain parted, you could see an actual waterfall, etc. At the end of this section there was a beautiful image\u2014one of the few that were also artistically impeccable. [\"Trip on the Avon River in New Zealand\"]. The spectators didn't know at first exactly where they were, when, as if by magic an invisible, delicate music sounded, completely in rhythm and harmony, as if made for the image (and, of course, purposefully arranged), accompanying the scene to its conclusion. As the lights came up in the auditorium in front of the closed curtain, the loud applause was not only for all that had been seen up to that point, but for the last scene and the genuine musical enjoyment that accompanied it. The proscenium seemed a magical realm, a mysterious land of light, life, and music.\n\nH\u00e4fker's further descriptions show the pains he took to assure a proper environment and mood. He reports that three men worked the slides to guarantee precise timing; curtains hung all around the auditorium to dampen the sound; colored stage lights splashed the proscenium as the audience seated themselves (59\u201360).\n\nThese preparations certainly have many precedents in traditions of theatrical and orchestral performance, and the format is adapted from the long tradition of lecturing in performance halls. Like other reformers, H\u00e4fker insisted that focusing on the viewing environment was the first step toward cinema's eventual aesthetic respectability. But H\u00e4fker set himself apart from his contemporaries with his claim that the entire cinematic apparatus\u2014image, light, music, sound effects, lectures\u2014could be used in combination for the artistic presentation of film, calling this Wagnerian use of cinema _Kinetographie_. H\u00e4fker's efforts to guarantee the proper conditions show his concern was primarily with the spectator's relation to the film. The conditions of reception were vitally important to his program and his conception of the function of art. The full effect of the \"total presentation\" ( _Gesamt-vorf\u00fchrung_ )\u2014here illustrated by the audience's reported confusion\/illusion that they were in New Zealand\u2014required the spectators' complete and undistracted attention. It required, in short, their _contemplating_ the film as they would an artwork. He hoped that he could educate audiences to this way of viewing films.\n\nH\u00e4fker took his cue from Lichtwark's _Exercises in the Contemplation of Art Works_ , which provided the foundation for the training of taste and vision, a way of viewing art that H\u00e4fker transferred to film. This way of viewing was certainly not unique, having immediate precedent in the German tradition of _Bildbetrachtung_ , as exemplified by Lichtwark's exercises. His presentations did not simply provide an environment conducive to the passive reception of art; they set out to actually train the audience's vision. Through the lectures, H\u00e4fker guided the audience to what was important and \"in which sense to take it\"\u2014that sense being, primarily, vision. But he did not want to stop there: \"In order to draw attention to especially interesting images, perhaps one should _occasionally_ employ little _signal lights_. They could be colored incandescent lamps placed around the screen that light up shortly before surprising scenes or scenes that are difficult to see\" (57\u201358). These visual cues would reinforce his verbal guidance, perhaps eventually creating some sort of physiological response. (Apparently, H\u00e4fker did not consider that the lights might have been a distraction.) There was also a moral dimension to this way of looking. In H\u00e4fker's discussion of approaches to art, contemplation was exemplary of a certain economy of energy, in that focused attention on the artwork is a way of exercising the will against the excessive stimuli of modernity. If neurasthenia was a type of mental fatigue caused by the difficulties of dealing every day with modern life, art provided not only a haven of unity and harmony in a distracted and disorganized world, it also offered an opportunity to train the attention and exercise the taste. Art and the artistic presentation of film were workouts for the mind; museums and film theaters could be mental health clubs.\n\nLemke's and H\u00e4fker's education of vision and taste exemplifies the strikingly ambivalent tone of contemporary reform movements, in that they were both nationalistic and progressive, both protective of traditional values and open to modern innovations. Yet that combination of tradition and modernity was and is common for all early adopters of new (media) technology who try to incorporate their new tool into an established disciplinary method, or especially an established mode of expert viewing. In their case, their technophilia or excitement over the new medium prompted them to find within it some potential for compromise or appeal to their less excited colleagues. It is hardly coincidental that the problems most cited about film and its exhibition\u2014its quick tempo, excessive detail, and jumbled programs\u2014corresponded to some of the most likely complaints about modernity in general, namely, quick pace and disorder. Lemke's and H\u00e4fker's plans, anxiously aware of these charges, attempted to contain them by enveloping film within a protective casing of order, dwelling, and observational method, whether called _Anschauungsunterricht_ or contemplation. Whether motion pictures, per se, could be incorporated into \"traditional aesthetics\" is the question of the next chapter. But before we move to that topic, let us briefly review the ideological connection between vision, taste, morality, and education.\n\nSchiller, unlike most philosophers, was not suspicious of the senses, least of all of the sense of sight. According to Schiller, knowledge of the physical world passes through the senses and is therefore contingent on them, but vision provides the opportunity to transcend the physical world and enter the aesthetic on the way to the moral realm. The key to this journey is \"contemplation.\" Schiller declared, \"As long as man, in that first physical state, is merely a passive recipient of the world of sense... he is still completely One with that world.... Only when, at the aesthetic stage, he puts it outside himself, or _contemplates_ it, does his personality differentiate itself from it.\" Upon entering the aesthetic, the subject renounces his or her passions and creates the possibility of becoming a _moral_ being. Contemplation is the exercise through which this process begins. The very act of perception, the very apparatus of vision is both inextricably implicated in the sensual world and ironically outside of it. \"From the moment a man _sees_ an object, he is no longer in a merely physical state,\" Schiller noted. That is, while exercise of the other senses testifies to one's _proximity_ to the natural world, vision offers the opportunity for _distance_. The aesthetic of contemplation, exemplified by what Benjamin called the \"aura\" of an artwork, is based on distance. The aesthetic of distraction, again illustrated by Benjamin's discussion of cinema and architecture's tactile qualities, is based on proximity. Schiller again: \"If desire seizes directly upon its object, contemplation removes its object to a distance, and makes it into a true and inalienable possession by putting it beyond the reach of passion.\"\n\nHence the whole concept of subjectivity\u2014becoming a knowing subject by objectifying and therefore \"possessing\" Nature\u2014is dependent upon the refusal of passion and sensuality. Once \"outside\" this sphere, the moral becomes possible. For Schiller, the renunciation of Nature is not a goal in itself as much as a necessary step toward the fulfillment of humanity's moral potential. Like the act of vision, always in the physical world while simultaneously having the potential to transcend it, humanity balances on the fine line between the sensual and the moral. Schiller called this line \"the aesthetic.\"\n\nTaste, like vision, is both embedded in Nature and somehow removed from it. Even more than vision, taste implies participation in the social world. An artwork affects the individual, but the exercise of aesthetic judgment implies universality. When we find ourselves agreeing that something is beautiful or sublime, we are exercising a unique and precious form of intersubjectivity based on our recognition of shared capacities for aesthetic experience. This is what Kant meant when he, following Vico, called taste a _sensus communis_ \u2014a communal sense.\n\nThe concept of taste provides the ideal illustration of the relation between aesthetics and ideology. While society could impose moral behavior on its subjects by appealing only to Reason, it is more efficient to employ the emotions in this task. As the medium between Nature and Reason, the aesthetic allows this operation. Schiller explained:\n\nThe ethical State can merely make it (morally) necessary, by subjecting the individual will to the general; the aesthetic State alone can make it real, because it consummates the will of the whole through the nature of the individual. Though it may be his needs which drive man into society, and reason which implants within him the principles of social behavior, beauty alone can confer upon him a _social character_. Taste alone brings harmony into society, because it fosters harmony in the individual.\n\nFaced with a society that they felt was becoming more alienated and fractured, reformers latched onto the promise of harmony and unity offered by the aesthetic realm. Lichtwark and H\u00e4fker focused on vision to effect a renewal in taste. Their exercises in the contemplation of artworks were, like Lemke's Herbartian ordering of the \"cinematic lesson plan,\" attempts to ward off the distractions of modernity, prophylactics against the \"much-too-much.\" If spectatorship had been characterized as an addiction that lulled audiences into an impressionable somnambulism, H\u00e4fker, Lemke, and other reformers hoped to counteract this state by inscribing cinema into an aesthetic of contemplation and reflection. The audience's vision required _training_ so that mental and physical fatigue would not set in; it was a way of \"pumping up\" moral weaklings. While H\u00e4fker's _Gesamtkunstwerkeffekt_ would provide the illusion necessary for the aesthetic experience, it was not intended to lull the audience into distractedness. Rather, it provided access to the _sensus communis_ through a _disinterested_ , distanced aesthetic experience. Nature films were both safely asexual and reminders of potential harmony. Yet the use of nature films is ironic; the reformers' emphasis on vision and distance and disavowal of _Kinodrama_ and \"sensational\" films amounted to a refusal of sensuality and corporeality\u2014in short, a refusal of Nature. Training audiences to conform to certain rules of observation\u2014an ascetic education of their vision\u2014was part of an ideology that combined educational practices and Kantian aesthetics to establish some sense of social order.\n\nThis legitimation strategy\u2014anesthetizing\/aestheticizing cinema and its audiences\u2014was a response to modernity's perceived assault on the body and the body politic, often exemplified by cinema's \"flicker.\" H\u00e4fker and others felt that training the aesthetic sensibility could fend off the \"shocks\" of modern life. The combined concepts of \"the child\" and \"taste\" served as a fulcrum for the reformers, allowing them to \"uplift\" the motion pictures and incorporate cinema into their ideology. As Germany's _Kinoreformers_ attempted to redeem and legitimate cinema as Art, they recognized within it the potential for recovering a lost utopia of unity and, ultimately, a means for social control. Yet even by 1912, presenting contemplation as a solution to modernity's ills seemed slightly old-fashioned, as we shall see.\n\nTHE PROBLEM WITH PASSIVITY\n\nAESTHETIC CONTEMPLATION AND FILM SPECTATORSHIP\n\nBut the difficulties which photography caused for traditional aesthetics were child's play compared to those presented by film.\n\n\u2014WALTER BENJAMIN (1936)\n\nAs cinema's bandwagon\u2014already heavy with reformers and trade journal reporters\u2014rolled toward World War I, literary intellectuals, pundits, and other belletrists climbed aboard (sometimes climbing down again after their thousand words) just to see what the ride was like. Judging from the sharp spike in the number of film essays written between 1912 and 1914, it was apparently de rigueur to offer a learned editorial on the way modern life and culture found expression through this new phenomenon. Just as a range of opinions comprised the reformist discourse explored in chapter 3, so the tone of this collection of articles, which Anton Kaes felicitously dubbed the _Kino-Debatte_ , extended from peevish outrage and haughty condescension to diplomatic concession or even roguish delight in the new medium. This expansion of the discourse in Germany corresponded to cinema's more visible public profile at this time, due to a stronger domestic film industry; the production of longer and more emotionally involving story films; the rise of film stars, such as Asta Nielsen and Henny Porten; the emergence of picture palaces and the successful \"embourgeoisement\" of the cinematic experience; and, most tellingly, the development of the _Autorenfilm_ , a short-lived strategy that attached literary and theatrical luminaries to industry projects. Perhaps because those weighing in\u2014including Ernst Bloch, Max Brod, Alfred D\u00f6blin, Georg Luk\u00e1cs, Kurt Pinthus, Walter Serner, and others\u2014were or were to become such prominent names in the German literary tradition, this part of the conversation about film has received the most attention in secondary surveys of the period. We should be quick to note, however, that reformers and trade journal writers did not disappear during this time\u2014on the contrary, they exerted a clear influence on the direction of the discussion, even if negatively\u2014but the sheer number of new voices in the mix has tended to shift our attention from the pedagogical and commercial sections of the debate.\n\nThis chapter will be no different in that respect. It will, however, back away from the scholarly emphasis on the relation between literature and film. While depictions of the _Kino-Debatte_ have been as varied as the debate itself, scholars usually focus on the battle between image and word in the contemporary discussion of cinema's relationship to German culture. It is indeed hard to ignore the incessant complaints about the supposed decline in literacy attributed to the consumption of sensational and superficial images rather than great literature or the many declarations that cinema could never be considered genuine art as long as it lacked words to express the depths of the human soul. Anton Kaes was absolutely correct when he noted that the tension between old and new \"found expression in a vigorous discourse about the relationship between literature and cinema,\" or, simply, that the debate about cinema was \"a debate about the literature of the time.\" While theories of film started to disengage themselves from literary or theatrical models by the 1920s, before World War I, cinema and its champions felt the need to justify themselves in terms of literature. For Sabine Hake, literature was \"the primary reference point\" for writers coming to terms with modernity through their essays on cinema. Peter Jelavich emphasized the attempts to conform film to \"traditional bourgeois aesthetics, which demanded clarity of authorial voice and rootedness in the written word.\" Helmut Diederichs similarly charted the ways in which this group divided the ground between literature and film. Stefanie Harris has demonstrated, on the other hand, how cinema's unique form shaped the literary work of such writers as Kurt Pinthus. As Heinz-B. Heller usefully pointed out, these were _literary_ intellectuals after all, so it comes as no surprise that their response to film would be from a position firmly grounded in their chosen medium.\n\nFocusing so closely on early cinema's relationship to literary form and turf, however, unintentionally narrows our understanding of cinematic experience to that particular relationship, leaving relatively unexplored the question of film and aesthetic reception in general. The above quotation from Benjamin\u2014his point that the challenge that photography presented to traditional aesthetics was \"child's play\" compared with film\u2014summarizes a fairly common conception: that film was emblematic of a change in aesthetic standards at the fin de si\u00e8cle as mass reception and distraction replaced individual contemplation as the dominant or most appropriate mode of aesthetic reception. Indeed, in film and media studies this account is more or less taken on faith in Benjamin's word alone. I have no argument with the general outlines of this story, but even his well-known \"Work of Art\" essay compresses the events considerably. So we have a dual historiographical problem, as I see it: the emphasis on the literary misses a large swath of the cinematic experience, specifically the relationship between viewer and image, while the leap from contemplation to distraction in film history and theory is too often taken for granted without spelling out the character of \"traditional aesthetics\" and the transition to whatever replaced it. This chapter argues that a close, renewed examination of the _Kino-Debatte_ is essential to solving both historiographical problems.\n\nPrevious chapters were concerned with the criteria for film's legitimacy within any given discipline and the adaptability of motion picture technology to an expert mode of viewing as a major factor in establishing that legitimacy; this chapter will explore film's legitimacy within the realm of aesthetics and its degree of adaptability to the expert mode of viewing known as aesthetic contemplation. It presumes that, following a trend in German aesthetics since Kant, the question of aesthetic value hinged on _reception_ more than form. That is, the major statements on aesthetics in the long nineteenth century\u2014especially those of Kant, Schiller, Schopenhauer, and others, but excluding those within the Hegelian tradition, which was concerned with how meaning inhered in form rather than how we experienced it\u2014were concerned primarily with the role of aesthetics as a way of being in the world, more than whether any particular form was more artistic than another. They were concerned with the function of art within a moral, ethical, social, and philosophical system. The value and legitimacy of art in this system depended primarily on the singularity of the experience it aroused. The exact nature of that experience has been a topic of constant exploration since these statements, but the significance of that experience for the system in general\u2014whether philosophical, moral, or political\u2014cannot be underestimated. Form prompts experience, to be sure, but these and other statements emphasized the universal character of the aesthetic experience rather than the variety of experiences created by various forms. More often than not, evaluations of any given form, such as music, rested on the ability of that form to catapult the reader\/listener\/viewer into a particular kind of aesthetic experience. Better experience usually equaled more hallowed form.\n\nSo to understand fully the relationship between early cinema and aesthetics, we must focus on the relationship between the cinematic experience and aesthetic experience as it was understood, or between watching movies and the expert mode of viewing called aesthetic contemplation. This chapter asks the following questions: to what extent did the cinematic experience, as these authors described it, conform to their understanding of aesthetic experience? Specifically, how did the experience of film align with their understanding of aesthetic contemplation as an implicitly expert mode of viewing? To what extent did the descriptions of cinematic experience participate in changes to expert conceptions of this mode of viewing?\n\nTo answer these questions requires first understanding what the authors presumed about aesthetic experience. The trouble with aesthetic experience, of course, as countless analytic philosophers have complained, is its notoriously slippery surface. It is very difficult to define logically, or even on an individual basis. Fortunately, for the purposes of this project we need not come to a philosophically rigorous conclusion; instead, we need only outline what the authors _thought_ aesthetic experience was. Even that is elusive, because any given writer borrowed ideas or presumptions, often haphazardly, from a long and varied aesthetic tradition. Indeed, we might find it more historiographically productive to think of descriptions of aesthetic experience (rather than aesthetic experience per se) as statements often expressing competing values. If one writer latched onto Kant's idea of disinterest and detachment as the point of aesthetic experience, another might champion, after Schopenhauer, the idea of losing oneself in the artwork. If one author saw repose as the fundamental criterion of any aesthetic encounter, another might have emphasized the value of the free play of associations while engaged with a work of art. My point is that these key ideas or terms\u2014detachment, loss of self, repose, free play, and others\u2014describing any aesthetic experience (not just cinematic) _functioned within a system of implicit dichotomies_. Let me explain.\n\nIf we gather common ideas about the nature of aesthetic experience in the major statements of Kant, Schiller, Schopenhauer, and others within the tradition of nineteenth-century German aesthetics, it appears that aesthetic contemplation entailed (1) _detachment or disinterest_ , meaning that aesthetic judgment is without desire, passion, or self-interest; (2) _repose_ , in that the object is lingered over without haste, giving enough time for the free play of the imagination; (3) _activity_ , meaning that the mind is alive with associations and correlations; and (4) _loss of self_ , in that the contemplation of the object can lead to either a transcendental immersion into the object (Schopenhauer) or a moment of awareness of one's participation in a larger community (Kant). Under this category we can also subsume discussions about the role of the body in aesthetic experience; for some, aesthetic contemplation was a purely mental operation; others looked to the body as a model for understanding aesthetic pleasure, while avoiding the purely sensual. Some terms are mutually reinforcing (disinterest and participation in a community, for example), while others are contradictory (detachment and immersion seem to be opposite ideas).\n\nBut we can arrange these terms\u2014detachment, repose, activity, and loss of self\u2014into spheres or larger categories that also contain their opposites. These categories are: _space_ (distance\/proximity or detachment\/immersion), _identity_ (loss of self\/self-awareness), _time_ (repose\/haste), and _agency_ (activity\/passivity or free will\/determination) (see fig. 4.1). This arrangement shows especially clearly the _ideological_ implications of these terms and of the descriptions of aesthetic experience they evoked. That is, to give aesthetic experience its moral or philosophical weight within a larger system\u2014the pivotal role of Kant's _Critique of Judgment_ with regard to his other _Critiques_ comes to mind\u2014the terms writers and philosophers used to describe aesthetic experience were rhetorically linked to fundamental ideological values (or ideologically inflected categories of experience) such as agency and identity. Understanding the rhetorical role these terms played allows us to glimpse the stakes of any historical debate regarding aesthetic experience. Pairing the terms with their opposites also underlines their mutual dependence in those historical debates.\n\nFIGURE 4.1. Aesthetic experience as a series of interlocking dichotomies\n\nThis scheme also allows us to escape the analytic philosophers' futile attempts to logically reconcile descriptions, an evasion much more in keeping with the sharpest understandings of aesthetic experience as indeterminate. Schiller argued, for example, that the aesthetic state is a _mediating_ moment \"midway between matter and form, passivity and activity,\" wherein indeterminacy is the preferred state of being. Accepting the indeterminacy of the aesthetic state should turn us away from pat definitions and toward an understanding of aesthetic contemplation as dichotomous, fluid, oscillating, or unfixed. (Again, I want to stress that questions about what the aesthetic state actually is and even whether it exists are irrelevant when considering historical descriptions of it.) For example, descriptions of contemplation almost always preferred repose over haste\u2014thereby presuming that both the object and the subject were more or less in a state of stillness\u2014but the quick, gestalt-like expert glance over the artwork as a whole was not ruled out as part of the process. However, detachment was almost a universal feature of these descriptions, so its opposites, self-interest or passion, were hardly ever included as viable elements of the experience. On the other hand, descriptions of the aesthetic experience often oscillated between sides of a dichotomy: claims that emotional detachment or psychic distance were crucial to aesthetic experience (Kant, Schiller) must be balanced or reconciled with descriptions that emphasized emotional projection or immersion into the artwork (Schopenhauer, Adolf Hildebrand). Likewise, the relationship between active and passive stances in aesthetic experience is often unclear from the descriptions; mastery alternates with surrender (as in many descriptions of the experience of the sublime), while focused attention alternates with the free play of associations or loss of self in the experience. This approach, then, matches a theoretical understanding of aesthetic experience as indeterminate to a historiographical scheme that does not try to resolve contradictions but allows them to stand to highlight their ideological function.\n\nObviously these categories overlap; questions of agency and identity are indeed hard to separate. But that is precisely the point: the overlapping conceptual categories strengthen the ideological weight of any given description. So this scheme might be useful for an investigation of _any_ historical description of aesthetic experience; it is definitely useful to understanding the early debates about motion pictures. To summarize, viewing the historical claims for aesthetic experience in terms of a series of dichotomies, rather than as a series of firm characteristics, has three advantages. First, we avoid the futile philosophical debate about what the aesthetic experience actually is and replace it with the recognition that historical descriptions of aesthetic contemplation\u2014what writers and aestheticians _thought_ it was\u2014have been varied and contradictory, but not infinitely so. Second, by subsuming the dichotomies under the general categories of identity and agency, we emphasize that most philosophers (in the neo-Kantian tradition) recognized aesthetics as a moral category that mediated between opposite poles of their choice. For example, the Kantian\/Schillerian tradition of putting Art between Reason and Sensuality extends in spirit to at least Theodor Adorno, if not also Jacques Ranci\u00e8re and other contemporary philosophers who hope to find for Art a place apart and some emancipatory potential. Seeing the aesthetic experience as a series of unstable dichotomies recognizes not only Art's socially mediating function but also that historical descriptions of aesthetic experience emphasized its productive indeterminacy.\n\nFinally, with regard to this specific project, these dichotomies allow us to see more clearly how the discussion of motion pictures was ultimately an _aesthetic_ debate, in the sense that these essays on cinema may be about the status of literature, the content of the films, or whatever, but the core issue was the larger ideological problem of _the moral significance of the aesthetic experience_. These writers obviously cared about the decline in reading skills, about the loss of ability to concentrate, the superficiality of the mass audience, the novelty of the moving image, and many other things that may or may not have indicated the decline of civilization. But, at bottom, all these complaints can be traced to the _character_ of the aesthetic experience, which apparently mattered most to these writers. \"Film presentations are generally rejected from an artistic perspective,\" wrote Emilie Altenloh about the taste of the upper classes. While Altenloh undoubtedly refers to judgments of artistic _form_ (\"von _k\u00fcnsterlischen_ Gesichtspunkten\"), this chapter agues that complaints or judgments of form were ultimately in the service of a better aesthetic experience. Modernity was therefore an affront to their aesthetic sensibilities and values, which were rooted in a philosophical tradition that privileged a particular, expert mode of viewing a stationary image or object. The extent to which cinema challenged or could be accommodated to these values is the question of this chapter.\n\nTo burrow further, we could argue that these dichotomies grew out a more fundamental, historical division between active and passive viewers. As we have seen, film spectators were often characterized as hypnotized by the cinematic image, even addicted to it, and captivated by the ease with which it entered the stream of consciousness, while cinema's fragmented temporality, quick pace, and motley form matched the habits and psyche of the modern city dweller, who was at once impatient and distracted, unable to concentrate on a single, main idea. Hence movies were perceived as effortless, whereas real art required work; movies were a jumble, whereas real art had a distinct form; movie spectators needed their next film like a drug, while real art encouraged a detached observer; movies moved by far too quickly, disallowing the continuous, leisurely attention required of real art. If these sets seem familiar, it is partly because they have been repeated over the centuries whenever audiences are described; theater audiences were probably the first victims of such characterizations. Ranci\u00e8re's explanation of the paradox of spectatorship sheds some light on this issue:\n\nThere is no theatre without a spectator.... But according to the accusers, being a spectator is a bad thing for two reasons. First, viewing is the opposite of knowing: the spectator is held before an appearance in a state of ignorance about the process of production of this appearance and about the reality it conceals. Second, it is the opposite of acting: the spectator remains in her seat, passive. To be a spectator is to be separated from both the capacity to know and the power to act.\n\nThe idea of aesthetic contemplation from Kant and Schiller to early twentieth-century professional aestheticians looks like a deliberate attempt to counter the problem of passivity in spectatorship by endowing the viewer with both a protocol and a philosophical justification that connect aesthetic enjoyment to reasoning and knowledge production (\"the capacity to know\"), on one hand, and political maturity or citizenship (\"the power to act\") on the other. Endowing aesthetic contemplation with agency and linking it to individual or civic identity gave this mode of viewing the moral weight it needed to counter a mode of viewing perceived to be common to the passive, irresponsible crowd. The contemporary discussion of the cinematic experience could not avoid confronting this weighty connection between agency and contemplation\u2014often to film's disadvantage.\n\nPrecisely because contemplation had long been endowed with high moral import\u2014with values such as reason and citizenship\u2014any changes to the conditions of contemplation put these values at risk, especially as mass reception became more common with new urban demographics and forms. If cinema were not at the heart of these transformations, for many essayists it was a worst-case scenario. At the same time, however, the discussion about cinema was not merely a reactionary defense of Enlightenment-era ideals; it was a collective attempt to find some common ground, to ask (often implicitly) whether contemplation was the proper way to engage with these new forms, and if not, what was? In fact, Benjamin's own position on the relative value of contemplation and distraction was not limited to his words in the \"Work of Art\" essay. As Carolin Duttlinger has shown, Benjamin's conception of attention ( _Aufmerksamkeit_ ) played a central and complex role from his earliest essays, and if he advocated distraction ( _Zerstreuung_ ) as the perceptual stance most appropriate toward modernity in the \"Work of Art\" essay, that was only one part of a long conversation he had with himself about the relationship between a mode of viewing and one's position toward and within modern life. Ultimately, this conversation was about the relationship between \"a way of being with images,\" in Dudley Andrew's ingenious phrase, and a way of being in the world. For Benjamin as well as the writers of the _Kino-Debatte_ decades earlier, the two stances were one and the same.\n\nSo this chapter will explore the status of aesthetic reception in the context of discussions about cinematic spectatorship. The first section will trace the broad themes in the _Kino-Debatte_ about movies, modernity, and spectatorship in relation to the rhetorical or ideological categories of time and agency. Through an examination of Schiller and Schopenhauer, it will stress the philosophical and subsequently ideological connection between temporality, indeterminacy, and aesthetic experience, and how this constellation persisted in the descriptions of early cinema. The second section will inspect discussions of aesthetic experience in professional aesthetics of the time, especially around the concept of _Einf\u00fchlung_ , or \"feeling into\" the artwork. These discussions about the relationship of emotions and the body to aesthetic experience were attempting to come to grips with, on one hand, unresolved ideas about reception in nineteenth-century aesthetics, and on the other, modern aesthetic reception characterized by turn of the century democratic access to art. This section will demonstrate that discussions of the cinematic experience also pressed on some of the same troubling issues\u2014such as emotional projection, immersion and detachment, and visual pleasure\u2014regarding the nature of aesthetic contemplation in the modern world that professional aestheticians discussed. This section explores the categories of space and identity. The third section focuses on Georg Luk\u00e1cs's famous early essay on film, \"Thoughts Toward an Aesthetic of the Cinema\" (1913), to demonstrate the nascent political critique of contemplation that was to be such a central feature of his and other theorists' stance after World War I. This final section will also return to Benjamin, Siegfried Kracauer, and the discussion of contemplation and distraction to realign our history of these theories in light of the _Kino-Debatte_ 's discursive construction of spectatorship.\n\nAGENCY AND TEMPORALITY IN THE AESTHETIC EXPERIENCE OF CINEMA\n\nThe scheme outlined above could apply to any historical description of aesthetic experience after Kant, but this section will recast some familiar themes in the _Kino-Debatte_ in terms of the roles time and agency played in contemporary understandings of aesthetic experience. Let us begin with a relatively obvious case: motion pictures as an emblem of social acceleration. This was a bright, visible thread in early debates about cinema all over the world. Basically, commentators complained that the incessant tempo of motion pictures\u2014its relentless push forward and its quick change from one view to the next\u2014exemplified the problems of modern social acceleration. Educator Georg Kleib\u00f6mer discussed cinema's pace at length in his 1909 essay, \"Cinema and Schoolchildren.\" He opened his attack by describing the Great Lisbon Earthquake's profound effect on young Johann Wolfgang von Goethe, and then wondered whether the eruption of Mount Pel\u00e9e in 1902 had the same effect:\n\nAnd let us search for just one child for whom this natural event had an indelible significance. How is it that we all well know that this search will be in vain? Because in our time all the world's grand strokes of fate are immediately known to us in every detail; because one harrowing incident displaces the other before it can be \"worked over\" by our mind. Now even the most tragic events only touch the surface of the soul.\n\nSuperficiality abounds, according to Kleib\u00f6mer, because we do not have enough time to absorb the emotional significance of events before we are compelled to move to the next one. \"The moral education of a child,\" he instructed, \"should include a _few_ events that give him an understanding of certain emotional values,\" but \"above all, such emotions must not confront other feelings in close temporal proximity.\" And in the cinema? \"In rapid succession jest and seriousness alternate on the screen, since variety must reign.... So first _horror_ , _fear_ in the highest degree, then _compassion_ increased to the utmost.\" He therefore criticized \"the danger that this rapid traversal of the entire scale of feelings poses for the truth and depth of children's emotions.\" Kleib\u00f6mer saw filmic form as a synecdoche for the rapidity with which modern information was conveyed; he assumed that because of that rapidity, children could not absorb the significance of events, and this was the root cause of their perceived superficiality (as opposed to, perhaps, the possibility that they were six-year-olds not named Goethe).\n\nKleib\u00f6mer was not alone in his opinion; writers of all sorts, not just cranky reformers, connected movies and modernity in this way. Austrian author Karl Hans Strobl similarly argued, \"The cinema is one of the most perfect expressions of our time. Its hasty, erratic tempo corresponds to the nervousness of our lives; the anxious flickering, the whisking away of its scenes is the extreme opposite of a measured, regular stride, of confident persistence.\" In the pages of _Der Kinematograph_ a commentator noted matter of factly, \"And so the cabaret, with its indiscriminate, democratic disposition, appeals best to the nervous haste and variety of our time, which of course is not say that the theater is doomed. But now the cinema, the projection house, successfully competes with the cabaret for the affection of the people just as the latter surpassed the legitimate stage.\" Right-wing editor of _Der Kunstwart_ and proto-Nazi Wilhelm Stapel was even more explicit:\n\nBecause of the rush of images, you get used to absorbing only an approximation of the impression; you do not get a clear and conscious understanding of the image in its details. Therefore only the coarse, surprising, sensational impressions stick. The sense for the intimate, the precise, the delicate is lost. The patrons of the cinema \"think\" only in garish, vague ideas. Any image that lights up their mind's eye takes up all of their attention; they no longer mull and reconsider it, they no longer indulge in the particularities and the reasons.\n\nDevotees of the connection between early cinema and modernity will find this theme familiar. Noteworthy, however, is not simply this well-known correspondence but the strong implication that cinema's tempo interfered with the usual aesthetic experience of viewing images. Stapel was very clear in this regard: the spectator does not \"get a clear and conscious understanding of the image in its details.... they no longer mull and reconsider it.\" If one is to understand the image, one must attend to the details slowly. Now this could just be a commonsensical caution that one cannot simultaneously do anything hastily and well, but it was not usually put in those terms. Instead, the complaint was specifically about the superficiality that results from processing (or being forced to process) visual imagery too rapidly. What was the connection between viewing tempo and shallowness?\n\nTo appreciate the moral implications of this equation, we must return to Schiller and Schopenhauer, who based their philosophies on a common understanding of time. The importance of temporality for Schiller's aesthetics is not only rarely discussed, it is key to understanding later ideological objections to film. So the following will explicate their aesthetic schemes while emphasizing temporality and indeterminacy, which will clarify certain aspects of the debates about cinema. Even if these early nineteenth-century philosophers were in many ways considered obsolete by the early twentieth century, their ideas about the moral and cultural importance of aesthetic experience\u2014and the relationship between that significance and the passing of time\u2014were widely and implicitly assumed. Schiller, especially, presumed that everyday, sensuous existence is necessarily tied to the inexorable succession of moments, while abstract thought and reason\u2014or the Platonic idea\u2014exist outside this temporal course in a timeless, static, changeless existence. Schiller called our animal nature the \"sense drive\" and the spiritual or intellectual side of our existence the \"form drive.\" He described the sense drive:\n\nSince everything that exists in time exists as a succession, the very fact of something existing at all means that everything else is excluded.... when man is sensible of the present, the whole infinitude of his possible determinations is confined to this single mode of being. Wherever, therefore, this drive functions exclusively, we inevitably find the highest degree of limitation. Man in this state is nothing but a unit of quantity, an occupied moment of time\u2014or rather, he is not at all, for his Personality is suspended as long as he is ruled by sensation, and swept along by the flux of time.\n\nHere and elsewhere, Schiller defined his understanding of the different facets of human existence in terms of their relationship to time. In the sense drive, one is nothing but \"an occupied moment of time\"; by being occupied by (in terms of both concerned with and controlled by) sensuous existence\u2014ruled only by needs and desires of the moment\u2014one is \"swept along by the flux of time.\" This conception of temporality, for Schiller and others, implied a limit on human potential: \"Wherever, therefore, this drive functions exclusively, we inevitably find the highest degree of limitation.\" The form drive, however, \"embraces the whole sequence of time, which is as much as to say: it annuls time and annuls change. It wants the real to be necessary and eternal, and the eternal to be necessary and real\" (81). Ideas aspire to timelessness, being outside of the succession of events and therefore outside of perception. Reason is timeless, in Schiller's scheme, but also ethereal. So Schiller invented another motive force to harmonize, balance, or counter the other two:\n\nFor as long as he only feels, his Person, or his absolute existence, remains a mystery to him; and as long as he only thinks, his existence in time, or his Condition, does likewise. Should there, however, be cases in which he were to have this twofold experience _simultaneously_ , in which he were to be at once conscious of his freedom and sensible of his existence, were, at one and the same time, to feel himself matter and to know himself as mind, then he would in such cases, and in such cases only, have a complete intuition of his human nature. (95, emphasis in original)\n\nFor Schiller, the desiring, physical side and the spiritual, intellectual side of our nature have their limitations, but when we are able to experience both at the same time we are without limitations. That is, if our lopsided, specialized existence is determined by our physical desires (that is, as matter without form), or by our intellectual aspirations (that is, form without matter), then the third way escapes determination entirely; it is indeterminate. Schiller called this third mode of existence \"the play drive,\" which is exemplified by aesthetic experience. It is a moment of indeterminacy, of oscillation between Sense and Reason, or between physical necessity and law. That moment of indeterminacy holds the greatest human potential, when free will is exercised most productively in the service of \"a complete intuition of [our] human nature.\" Schiller concluded:\n\nOur psyche passes, then, from sensation to thought via a middle disposition in which sense and reason are both active _at the same time_. Precisely for this reason, however, they cancel each other out as determining forces, and bring about a negation by means of an opposition. This middle disposition, in which the psyche is subject neither to physical nor to moral constraint, and yet is active in both these ways, pre-eminently deserves to be called a free disposition... we must call this condition of real and active determinability the _aesthetic_. (141, emphasis in original)\n\nThe exercise of free will, then, is the goal of aesthetic experience. But not entirely: the goal is to be \"sensible of existence\" without being determined by it; to be aware of all aspects of our nature while they are held in abeyance. Aesthetic experience is a balancing act between physical necessity and moral obligation, actively taking part in both sides of the human condition without letting either determine us. Schiller thereby gave the aesthetic experience a strong ethical charge; it serves as the moment when we are most fully human, overly determined by neither of our natures: \"For, to mince matters no longer, man only plays when he is in the fullest sense of the word a human being, and _he is only fully a human being when he plays_ \" (107, emphasis in original). For Schiller, aesthetic experience was the grandest kind of play, which brings our fullest human potential into relief.\n\nEspecially interesting and rarely noted, however, is the emphasis Schiller placed on the temporal dimension of this experience. The sense drive is \"swept along by the flux of time,\" the formal drive \"annuls time,\" but the play drive is repeatedly described as an experience of simultaneity: \"a middle disposition in which sense and reason are both active _at the same time._ \" The importance of simultaneity in his scheme marks Schiller's move as a very modern gesture; both simultaneous and indeterminate (\"this condition of real and active determinability [ _Bestimmbarkeit_ ]\"), his category feels very modern indeed. It is not too great a leap to argue that simultaneity creates this indeterminacy; we are both in and outside of time, which puts us in a position of being in neither one drive nor the other, but both states at the same time. This clarifies his formulation of the play drive: \"the play-drive, therefore, would be directed towards annulling time _within time_ , reconciling with absolute being and change with identity\" (97, emphasis in original). The annulment of time experienced in the formal drive is reconciled with the experience of time within the sense drive, thereby resulting in the \"annulling time _within time_.\" Whatever one makes of the validity of his interpretation, there can be no doubt that Schiller's conception of the aesthetic experience was ultimately temporal.\n\nSchopenhauer also took pains to underline the temporality of the experience, even if it was different from Schiller's: \"It is the state where, simultaneously and inseparably, the perceived individual thing is raised to the idea of its species, and the knowing individual to the pure subject of will-less knowing, and now the two, as such, no longer stand in the stream of time and of all other relations.\" Both subject and object are raised to a transcendent state in Schopenhauer's conception of contemplation, during which the object becomes an idea and the subject loses itself and its desire in the process, thereby also finding temporary relief from the misery of longing and striving. The emancipatory potential of aesthetic experience derives from its ability to provide a momentary escape from time; this is different from Schiller's more moral and community-oriented aesthetics, but it affirms rather than cancels Schiller's conception of the aesthetic as a primarily temporal experience. For both philosophers, and in the most common conceptions of aesthetic experience in the long nineteenth century, human limitation was tied to \"the flux of time,\" and its emancipatory potential was linked to the escape from that flux or being able to maintain a balance between competing temporalities.\n\nThis review of Schiller's and Schopenhauer's philosophies is necessary, because it reminds us of the _ethical or moral mission_ they assigned to aesthetic experience, thereby giving it an ideological weight that it carried through the nineteenth and twentieth centuries. It also establishes _the importance of time_ within traditional aesthetics, especially the notion of repose in the aesthetic stance. Finally, it emphasizes that Schiller and others calibrated their conception of contemplation to highlight its _indeterminacy_ , or more precisely, the importance of the indeterminate quality of the aesthetic experience as an emblem of human liberty. All of these features of aesthetic experience\u2014especially the focus on experience, rather than artistic form\u2014spread and took root in everyday understandings of aesthetic issues that we see in the _Kino-Debatte_ and other, early discussions about viewing motion pictures. Again, the goal is to demonstrate that these debates were largely discussions about aesthetic (as opposed to narrowly literary) experience, especially this model of contemplation, to which modern spectatorship was detrimentally compared. Indeed, the unfavorable comparisons functioned primarily as a defense of contemplation's ideological value. We could dismiss the comparisons out of hand as ideologically compromised, but we would lose the deeply ambivalent and complex character of aesthetic reception. Contemplation was not yet dead; whether the values associated with it actually ever died is still open to question.\n\nThose values were primarily ethical in imperial Germany. Modernity, it appears, was too pushy; it shoved its spectators along, giving them no pause for reflection, which was an offense to values of repose. So when writers such as Gaupp, Sellmann, Stapel, Strobl, or others noted the potential effect of the fast pace of movies and modernity, they were not simply complaining about the vapid superficiality of \"kids these days\"; they were defending a very long tradition that insisted on leisurely approaches to the image. To allow oneself to succumb to the \"flux of time\" while attending to an image\u2014or to let the image impose such limitations\u2014was an abdication of the duty to self-cultivation, of the obligation to become fully human. Contemplation was depicted as a moment of escape, but also a moment of self-awareness; the aesthetic experience had a special place in state and cultural self-conceptions in Germany, which the righteous defended vigorously. But because of its pivotal role between emancipation, civic duty, and self-awareness, contemplation represented a complex, ambivalent stance toward the world that was not so easily dismissed, as Benjamin recognized (and which I will discuss at greater length later in the chapter).\n\nAnother good example within the discussion concerns the fragmentary, disjointed quality authors attributed to modern life and to cinema. If movies and modernity were too pushy and quick, they were also disjunctive: the cinema \"is short, rapid, even encrypted, and it stops for nothing. There is something compact, precise, even military about it. This fits very well with our age, an age of extracts.\" These comments on form were often framed as an analogy between movies, modernity, and the mind of its spectators; modern life was disordered, as were movies and the thought processes of its audience. This homology\u2014a discursive construction of film's spectator\u2014was usually an implicit defense of aesthetic ideals of detachment and effort. Theater critic Hermann Kienzl's brief commentary is exemplary:\n\nThe psychology behind the triumph of cinema is urban psychology. Not only because the metropolis is the natural focal point from which all social life radiates, but especially because the metropolitan soul\u2014this inquisitive and inscrutable soul perpetually on the run, stumbling from one fleeting impression to the next\u2014is really the soul of the cinema! Indeed, some city dwellers even lack the stamina and concentration for mental and emotional absorption\u2014and probably also the time, especially in Berlin, a metropolis agitated by work-fever. The same trivial drive for relaxation that leads city dwellers to the operetta or farce after work to replenish their exhausted energies leads them likewise to seek the effortless pleasures offered by movie theaters.\n\nHere we have the usual stereotypes about the metropolis and its inhabitants: they are driven by passion, desire, necessity; their actions are hurried and jumbled. Needless to say, these were also transgressions against the aesthetic values of detachment and repose. Kienzl pretended to offer merely a description of the situation, but it was an implicit defense of the principles outlined earlier.\n\nThere is also the implication, well before Hugo M\u00fcnsterberg, that movies were popular and effective because they somehow mimicked the structure of the modern mind, or at least the mind of its audience. For many commentators, the link between movies, modernity, and mind made film \"the most psychological representation in our time.\" The \"effortless pleasures\" of the cinema replenished the modern soul in a way\u2014a trivial way, they would say\u2014that other entertainments could not. Most writers disparaged movies and the mind of the masses in a dual dismissal, but others saw it differently:\n\nOnly film technology permits the rapid sequence of images that roughly corresponds to our own imaginative faculty and in some measure imitates its jerky unpredictability [ _Sprunghaftigkeit_ ]. Part of the fatigue to which we finally fall prey while watching theatrical works of art results not from the noble effort of aesthetic enjoyment, but rather from the exertion in adapting to the plodding, affected movement of life on stage. Spared this effort in the cinema, one is free to devote a considerably more uninhibited commitment to the illusion.\n\nWhereas Kienzl and others assumed that the mind of an intellectual is ordered and disciplined, Lou Andreas-Salom\u00e9 concluded, after spending a year with Sigmund Freud and Viktor Tausk, that the mind is no such thing. When Kienzl remarked that \"city dwellers lack the stamina and concentration for mental and emotional absorption,\" he compared the stalwart literary mind with the flighty, disordered, lazy mind of the moviegoer. But Andreas-Salom\u00e9 acknowledged that movies moved quickly and disjunctively, which matched her understanding of the \"imaginative faculty.\" This put both in a favorable, or at least neutral, light. Indeed, her sympathetic view of cinema's aesthetic potential depended on this structural similarity; the speed at which we think\u2014slowed down considerably by theater\u2014was matched only by cinema. Likewise, she saw in early cinema's unpredictability\u2014we can imagine that to early, novice audiences the continual surprise at the next view might have given them the impression that film narrative or editing was capricious (which it sometimes was)\u2014a disconnectedness that fit her understanding of human thought. Actually, the primary difference between her idea of mind and Kienzl's was that she refused to create two models divided by class, one for elite intellectuals and one for the mass audience. Her model assumed that, when it comes to thinking, we are all a hot mess, trained or not. M\u00fcnsterberg also developed a single model, but presumed that all our imaginative faculties are well ordered. In any case, many authors assumed a structural similarity between movies and mind, but their aesthetic judgment depended on whose mind movies resembled.\n\nBut Kienzl and Andreas-Salom\u00e9 shared one assumption: true art required effort and movies were effortless. The \"noble effort of aesthetic enjoyment\" was a persistent trope in these and other discussions about the difference between high and low cultural forms, in which high art was usually differentiated by its complexity or difficulty and low art by its simplicity. For Andreas-Salom\u00e9, cinema's effortlessness was a result of its structural similarity with the mind, especially with regard to its motion and pace. It had also something to do with the age-old comparison of images and ideas, a comparison common to the _Kino-Debatte_ , too, as when Hermann Duenschmann, apparently a big fan of Gustave Le Bon, compared the film audience with a crowd: \"The crowd thinks only in images and can only be influenced through images that act suggestively on their imagination.\" This implied that the learned think in words, but the visual status of ideas has always been ambiguous. At least since Descartes, many philosophers have defined \"ideas\" as images, not words. Descartes consistently made the analogy that ideas are \"like portraits drawn from Nature.\" Or Locke, in his _Essay Concerning Human Understanding_ , wrote that ideas are \"Pictures drawn in our Minds,\" or that the \"Idea is just like that Picture, which the Painter makes of the visible Appearances joyned together.\" Now an important caveat here is that Descartes and Locke were thinking not of concepts, but of sensory ideas\u2014the images that our brain creates from the sensory information gathered by normal perception. But the leap to concepts is not too hard to make and has been made since Plato, perhaps. The point is that one support for this notion that films were \"effortless\" was the isomorphism between images and the way the mind is thought to work. (This was also a support for the assumption within discussion about educational media that visual instruction is more efficient than instruction with other kinds of materials.)\n\nYet images per se cannot be easier to comprehend; otherwise the aesthetic contemplation of paintings would have no pedestal upon which to place itself. So there must have been something other than the similarity between images and mind that supported the claim that movies were effortless. Ulrich Rauscher gives a clue: \"I fear the cinema has one disadvantage for the audience: because it tells its story so comfortably, because it takes over the operations of sense-making itself, the cinema, which could foster the creativity of our literati, will bring about a general laziness of the public's imagination.\" Film is effortless, because it \"takes over the operations of sense-making itself\" ( _weil er die Versinnlichung der Vorg\u00e4nge selber \u00fcbernimmt_ ) for the audience, leaving nothing for the imagination to work on. The \"noble effort\" of aesthetic enjoyment was perceived as the free play of associations that accompanied a productively ambiguous artwork; the imagination filled in interpretive gaps with its own associations, thereby bringing subject and object together as the hermeneutic circle between part, whole, and subject tightened. Just as photography was not considered legitimate aesthetic material because of its mechanical nature and its excessive detail\u2014which left nothing to the imagination, presumably\u2014so, too, motion pictures were dismissed because their detail and their narrative patterns left the viewer nothing to do but watch. There was more to it than that, however, because these same movies were disdained for their chaotic, confusing stories, which doubtless would leave much to the imagination. The problem, then, was that the succession of images was not merely obvious, but _preordained_. Whether the images were confusing or crystal clear, they were always _someone else_ ' _s_ images, and if we combine this concept with the \"images = ideas\" formula, we have a series of views _imposed_ upon the viewer in a preordained temporal sequence, which does not happen with paintings or literary images. Hence the loss of free play, or more importantly, _free will_. The cinematic experience, in this view, was not _indeterminate_ , but rather _determined_. (Benjamin quotes Georges Duhamel: \"I can no longer think what I want to think. My thoughts have been replaced by moving images.\") By showing us where to look, editing and cinematic narrative forced these writers to look there; this was for many patrons a step too far.\n\nThose patrons would presumably never allow themselves to enjoy cinema's flow of images. But the inadequacy of this rigid decree\u2014that the flux of time jeopardizes agency and thereby imposes limits on human potential\u2014for modern life becomes apparent in the _Kino-Debatte_ (and elsewhere) as well, especially in discussions that emphasized _alertness_ and _somnambulism_. Essays often invoked these tropes when describing the tolls of modern life. Recalling Georg Simmel's vivid portrait of \"The Metropolis and Mental Life\" (1905) or Freud's idea of the \"stimulus shield\" (1920), these essays also assumed that life in the big city dulled the senses and made one blas\u00e9, requiring ever greater thrills to be satisfied. For many writers, cinema was just the ticket: \"But every day and every hour cinema restores to our pampered senses, which in the heyday of technology have forgotten how to be astonished, the feeling of Pygmalion's enchantment with Galatea.\" Anticipating film theory of the 1920s, this author and others claimed that cinema renewed vision by offering a point of view we otherwise could not have. For writers such as Rauscher, cinema's preordained succession of images was oppressive and imprisoning, whereas for this writer, it was liberating to see with another's eyes. Max Brod, too, was rejuvenated by a visit to the cinema:\n\nThe vividness with which so much happens [in the film] has finally shaken me out of my semi-somnolent state. Now on the way home I become an inventor, imagining new images for the Biograph: a pursuit in which, instead of automobiles, locomotives or trolleys, two ships, a cruiser and a pirate ship, race against each other over the wide surface of the sea, the gap between them narrowing amid the most furious shooting.\n\nOr this early editorial: \"I believe that through the cinema we have only now learned to see. We have been awakened to the pleasure of watching [ _Schauen_ ].... Reality appears much more clearly before us, and the interest is twice as great. We can almost let our mind fall asleep and reap with our eyes what the soul desires.\" If these writers were drifting through their modern lives, semi-awake to its challenges and blas\u00e9 to its pleasures, cinema roused them from this aesthetic slumber to give them a new outlook. Here, then, a trip to the cinema did exactly what any aesthetic experience should do: renew our perception. This idea, however, was relatively new in aesthetics and already expressed the waning dominance of the Schillerian model.\n\nIndeed, that aesthetic contemplation was undergoing a transformation is evident in essays that evoked alertness and somnambulism in terms of modern life. Strobl both admired and was uneasy with social acceleration and its effect on our perception:\n\nBut wherever the streets of world commerce run, wherever the billions cross paths, wherever goods are converted into money and money into power\u2014at these hubs of human and commercial relations a sustained readiness is necessary. Constant presence of mind has replaced the old contemplativeness that let one's mind wander, because we no longer need to have it at hand.\n\nAccording to Strobl, a leisurely approach to the traffic of images or to the city was simply not viable. One required instead a \"presence of mind\" that was very much embedded in the present but not necessarily \"swept away by the flux of time.\" Alertness, as we will see in our discussion of Benjamin, was considered a more modern response to time and succession than repose, yet was no less complex in its relationship to time.\n\nAesthetic experience, as discussed by Kant, Schiller, and Schopenhauer in the early nineteenth century, was a complex and contradictory structure that was nonetheless required to bear a particularly heavy ethical load. If it collapses logically with the barest nudge, it was still home to a family of values related to agency and identity. As we have seen, early discussions about film were often couched in terms of agency, especially with regard to the spectator's relation to the passing of time. Complaints about cinema's incessant temporal push and its preordained succession of imagery continued at least one line of thinking about aesthetic experience, which insisted that the viewer's stance before the image was also a stance before the world, one that recognized our limits but also strove to glimpse human potential in an ephemeral moment of transcendence. Aesthetic contemplation was understood as an exercise of free will, so to surrender oneself completely to the image or to the flow of time was an abdication of duty; indeterminacy meant liberty, but the rush of preordained images meant constraint. The temporal complexity of Schiller's and Schopenhauer's visions of aesthetic experience was therefore at odds with conceptions of cinematic temporality as constraining or controlling. But with statements attesting to the importance of alertness in modern life or positing that seeing the world from another viewpoint could reawaken the senses, the discussion began to question the utility of a leisurely aesthetic stance in favor of an approach to images\u2014and the world\u2014that was not merely cognitively active and unhurried, but _reactive_ and _quick_. So in the _Kino-Debatte_ we can begin to see the \"difficulties\" new forms might have posed for what Benjamin called \"traditional aesthetics.\"\n\n_EINF\u00dcHLUNG_ , IDENTITY, AND EMBODIED VISION\n\nBut an understanding of aesthetic contemplation as inherently tied to leisure and free will\u2014or, basically, the cultured gentleman's prerogative\u2014is only partially correct. We must also consider the relationship between aesthetic experience and identity or subjectivity. One might think that the gentleman's prerogative also presumed a stable, unified self, which would be confirmed in aesthetic experience. Kant's theory of the beautiful, for example, held that pleasure arose from the harmony between the artwork and our cognitive faculties, which assumed a resonance between them that was stable and stabilizing. His theory of the sublime, however, maintained that its pleasure derived from the oscillation between the physical threat of nature's awesome power or infinitude and our cognitive framing of that boundlessness. Faced with this power, the threat of physical annihilation presented a literal loss of self, while the mastery implied by cognitively framing such power was self-affirming. So the existence of that particular kind of aesthetic experience, especially given the importance of the sublime in aesthetic theory, meant that aesthetic experience per se was never inherently stabilizing.\n\nThis split between body and mind, or between loss of self (whether through physical danger or an experience of transcendence) and self-awareness (whether cognitive or corporeal), was exacerbated by the structure of Schopenhauer's system. On one hand, Schopenhauer insisted that a loss of self was absolutely necessary to any experience calling itself aesthetic, if it were to have any mollifying effect at all. As Schopenhauer described it, the viewer attends to the object and gradually loses self-awareness as the associations and Ideal become prominent. Likewise, his description of aesthetic contemplation emphasized the purely visual and cognitive\u2014as opposed to corporeal\u2014experience of viewing. For him, the aesthetic gaze divorced itself from the world, from the flux of time, and even from the artwork itself, the specifics of which did not figure largely in his understanding of aesthetic experience. Indeed, Kant, Schiller, Schelling, Schopenhauer, and Hegel all ignored the specific features of any given artwork in favor of using the object as a prompt for abstract thought and for mounting ever more complex philosophical systems. Johann Friedrich Herbart continued this approach in his formalist aesthetics, which \"proposed a simplified theory of form, one that defined aesthetics essentially as the science of elementary relations to lines, tones, planes, colors, ideas, and so on,\" without reference to the specter of \"content\" or \"meaning.\" Similarly, \"the perfect aesthetic frame of mind for Herbart was a state of absolute indifference, that 'quiet seriousness' that lies 'between depression and excitation.'\" In this tradition, their understanding of the gaze matched their understanding of the object: neither emphasized detail or materiality; both could be characterized as disembodied or ephemeral.\n\nOn the other hand, Schopenhauer also simultaneously pursued another approach, which he did not see as contradictory, but complementary. Part of the second volume of _The World as Will and Representation_ speculated on the _physiology_ of the perceptual act, an approach that aesthetically minded scientists such as Hermann von Helmholtz, Gustav Fechner, and Wilhelm Wundt pursued further in their experiments on the nature of (aesthetic) perception. Their findings, however, indicated that the human body was a transient, variable, and unstable field upon which any subjectivity rested at its own risk. That is, if a stable and unified subjectivity previously depended on a happy conception of the body as similarly stable and unified, experimental physiology and psychology of the mid-nineteenth century invalidated that guarantee. As Jonathan Crary has argued, the fleeting nature of the human body left barely any solid ground for subjectivity.\n\nMany aestheticians during the late nineteenth century looked for an approach to questions about aesthetic perception that would navigate between the Scylla of abstract, disembodied philosophizing about art and the Charybdis of fragmenting, \"scientistic\" approaches to the aesthetic experience. They were less interested in philosophical or physiological questions about how we perceive form and space than in the psychological problem of how we take delight in the specific characteristics of form and space. Robert Vischer's seminal 1873 dissertation, _On the Optical Sense of Form_ , coined the term _Einf\u00fchlung_ , or \"feeling into,\" as an answer to this question. Vischer meant it to explain \"the symbolism of form,\" or how spatial form has meaning for us: the subject \"unconsciously projects its own bodily form\u2014and with this also the soul\u2014into the form of the object\" (92). But _Einf\u00fchlung_ eventually came to mean emotional projection in general, and then, with Edward B. Titchener's translation of the term as \"empathy,\" it transformed into a broad psychological concept. In aesthetics, however, _Einf\u00fchlung_ was discussed in terms of several problems, including a renewed interest in analyzing the specific formal and material features of artworks and the experience they prompted; describing the emotional content of the aesthetic experience in terms of projection of self in relation to distinct forms; and understanding the role of the body as a measure of both art and aesthetic experience, especially in terms of aesthetic pleasure. That is, writers taking up the idea of _Einf\u00fchlung_ explained aesthetic pleasure as a resonance between the structure of the body and the structure of the artwork, thereby explicitly acknowledging the embodied nature of perception.\n\nThis brief history is necessary, because it clarifies \"traditional aesthetics\" and its \"challenges,\" which were twofold: (1) new artistic _forms_ , such as photography, fit uncomfortably in the canon of fine or even applied art; and (2) new aesthetic _experiences_ , especially mass reception. The question of form is outside this chapter's purview, but we can address the nature of mass reception, which differs from individual contemplation in at least two pertinent ways: (1) the emotions of the audience amplify those of the individual, thereby making emotional projection into the work much more prominent; and (2) because of this amplification, and the awareness of being in an audience, the sense of embodiment is more pronounced as well. We could therefore view the discussion of _Einf\u00fchlung_ in the late nineteenth and early twentieth centuries as an exploration of conflicting dichotomies nascent in \"traditional aesthetics\" (such as the relations between mind and body, or between free will and destabilizing aesthetic experience), while also attempting to reconcile the contradictions inherent in these grand aesthetic systems with changes in class demographics as more and more people gained access to and were interested in art. Specifically, I would argue that _Einf\u00fchlung_ aesthetics could be seen as an attempt to reconcile issues especially pertinent to mass reception (embodiment, emotional projection) with individual contemplation and its ideological implications. While mass reception was not explicitly acknowledged in discussions of _Einf\u00fchlung_ , the theories pave the way for a deeper understanding of the role of emotion and corporeality in aesthetic experience. Indeed, many contemporary theorists have looked back to _Einf\u00fchlung_ aesthetics as a possible framework for new theories of embodied spectatorship.\n\nThe dichotomies circling around the category of identity were less fraught ideologically than those concerning agency. Whereas no one argued seriously for \"determination\" against \"free will\" (although \"surrender\" might have been an option), philosophers were able to describe aesthetic experience in terms of \"loss of self\" just as persuasively as \"self-awareness.\" The issue of embodied perception, however, brushed against the almost universal requirement of disinterest, in that any hint of sensuality immediately lowered the status of aesthetic experience to mere pleasure. Like paper and scissors, physicality always lost against reason in this ideological game. But because the ideological stakes were generally not as high in this category, this section will not focus on those stakes; instead it will emphasize descriptions of aesthetic experience. Specifically, in an attempt to understand precisely the nature of film's \"challenge\" to \"traditional aesthetics,\" this section will draw upon theories of _Einf\u00fchlung_ , which were particularly adept at describing emotional projection and embodied perception in aesthetic experience, two issues also present in the debates about cinema. In fact, if the legitimacy of cinema as an aesthetic form in part hinged on the answers to these questions, it was also true that these two issues\u2014which I want to stress were present in \"traditional aesthetics\" from the beginning, if we look closely\u2014mounted the strongest theoretical and practical challenge to individual contemplation as it was generally understood. Emotional projection and embodied perception threatened to change the questions about aesthetic legitimacy itself. If _Einf\u00fchlung_ aesthetics tried to reconcile individual contemplation with features of aesthetic experience that were also prominent in mass reception, then the presence of a form such as cinema pressed harder on those questions.\n\nThis is not to say, however, that early discussions of cinema participated directly in debates within professional aesthetics or that professional aestheticians participated in the _Kino-Debatte_ ; to my knowledge no writings on film during this period in Germany ever mentioned the term _Einf\u00fchlung_. But the early discussions of film brought up the same issues. To be sure, salons crowded with paintings and people had already prompted much complaint as mass reception elbowed its way into the art world and demanded a seat at the table. But cinema was not merely one of many emblems of modernity; the nature of the experience itself\u2014the feeling of movement, synesthesia, emotional projection, and spatial depth\u2014coincided precisely with issues in aesthetic reception that professional aesthetics hoped to pin down. We can therefore read the _Kino-Debatte_ as part of an extended conversation in German culture about the nature of aesthetic experience. The debates were not just a series of complaints about film encroaching on literary form and territory; they were also a very sophisticated debate about the nature of aesthetic reception, about what it meant, in a broad sense, to contemplate an image.\n\nAs an opening example, consider this quotation from a 1912 article describing an _actualit\u00e9_ that featured Wilhelm II on one of his hunting trips:\n\nThe Kaiser sits motionlessly\u2014only the image twitches slightly here and there, flickering and spotting up as if something were boiling under the projected surface. Suddenly, he raises his rifle and my ear hears the gunshot without its being fired. The audience silently rejoices over their Kaiser's fine shot; a wave-like movement goes through the room. The mountain goat rolls.\n\nThis otherwise incidental passage is exemplary of the early debates about film in its focus on audience reaction. This writer described, for example, a process of emotional projection: the audience understood the representation by projecting itself into the scene of the hunt. They rejoiced _with_ the kaiser as well as _over_ his excellent aim. The writer also emphasized the _physical_ response of the audience, demonstrating the importance of the body for this process of projection. The \"wave-like movement\" that ripples through the room was an effect of this projection. But here we should note what is different about the cinematic experience over the solitary enjoyment of a painting. This writer described the mutual amplification of audience emotion (which Benjamin later theorized in his \"Work of Art\" essay) that is inherent to the cinematic experience and that manifested itself here as a physical reaction. There is a reciprocity between spectator and screen, just as _Einf\u00fchlung_ aesthetics described the mutual relationship between viewer and artwork, but here that feeling of resonance was amplified by the number of people in the room all feeling it at once (fig. 4.2).\n\nFIGURE 4.2. A prewar audience enjoying a night at the Union-Theater in Berlin, 1913\n\nNote, too, that if _Einf\u00fchlung_ aestheticians thought about that resonance between painting or sculpture and viewer in terms of \"movement,\" here that term is no longer metaphorical but strikingly literal: the movement of the image, of the kaiser, and of the audience were all working in concert to provide the basis for emotional projection. If before\u2014as I will explain later\u2014the idea of \"movement\" in aesthetics was limited to what we might term \"inner movement,\" or emotional resonance, now that same reciprocity depended on a _physical_ sensation of movement. Furthermore, just as writers on _Einf\u00fchlung_ argued that our senses were not strictly delimited and that stimuli for one sense could also stimulate another, so we see here a similar recognition of the confusion of the senses. In noting that he \"hears the gunshot,\" the writer emphasized a _synesthetic_ response; he recognized that the film experience is not merely visual, but that vision itself is embodied and connected to the other senses. (This \"gunshot\" could have been a sound effect, of course, but we should also consider that it could have been psychosomatic, an effect of deep immersion.)\n\nThis speaks to another theme in _Einf\u00fchlung_ aesthetics: the idea of contemplation as the ground for aesthetic experience. Our usual assumption that the spectator is held in continuous rapt attention is troubled by this passage, for it also emphasizes the _materiality_ of the experience: \"the image twitches slightly here and there, flickering and spotting up as if something were boiling under the projected surface.\" The film experience seemed to require the audience, like the motionless kaiser and the rolling mountain goat, to hold opposite attitudes\u2014stasis and movement, contemplation and distraction\u2014at the same time. At once immersed in the illusion while blithely noting the twitching image, the audience alternated between attention and detachment. Already, the cinematic experience highlighted contradictions at the heart of contemplation. The rest of this section, then, will explore the issues of emotional projection, synesthesia, and movement as they relate to identity and embodied perception in both _Einf\u00fchlung_ aesthetics and early discussions of cinema in the _Kino-Debatte_.\n\nEmotional projection was a key issue in aesthetics during this period. An essay from the debates on film will help explicate this topic. Alfred Polgar felt that attending the cinema was a sensual experience. In his 1911 essay, \"Drama in the Film Theaters,\" he described seeing pretty girls on the screen; they smiled at him and he felt that he should smile back, even wait for them outside the theater once the show was over. He knows it is silly to think of such things, but there is something strangely compelling in the way the film takes on a life of its own, the way it \"smiles back at me.\" This could be the sigh of another lonely film guy, or the first glimmer of a phenomenology of film, in which the film itself has a subjectivity that engages with the spectator, such as that suggested by Vivian Sobchack. But it also hints at the connection between emotional projection, _Einf\u00fchlung_ , and identity.\n\nPolgar's emotional investment in the girls on the screen recalls one of the central concepts of Vischer's dissertation and of this trend in German aesthetics: the imputation of one's inner life to an inanimate object, which Vischer termed _Einf\u00fchlung_. Vischer wrote:\n\nWe have seen how the perception of a pleasing form evokes a pleasurable sensation and how such an image symbolically relates to the idea of our own bodies\u2014or conversely, how the imagination seeks to experience itself through the image. We thus have a wonderful ability to project and incorporate our own physical form into an objective form.... What can that form be other than the form of a content identical with it? It is therefore our own personality that we project into it. (104)\n\nAccording to Vischer, we project our emotions and sense of our body (such as our orientation in space) onto an inanimate object, just as we project these aspects of ourselves onto other people to understand their expressions. To us, the work of art itself is permeated by human sensations. In fact, subjectivity is present not only in our attitude toward the work but also within the work itself. \"There is also a will _within_ the image\" (114), Vischer wrote, meaning that we project our experience on to the relation _between_ the parts of the work as well. Vischer's concept of _Einf\u00fchlung_ described how the projection of human feelings onto inanimate objects plays a role in the creation, shaping, and reception of artworks.\n\nLikewise, Adolf Hildebrand's _The Problem of Form in the Fine Arts_ (1893)\u2014which had already gone through nine editions by 1914\u2014was an incredibly popular exploration of the problems posed by sensual perception. He was especially interested in legibility\u2014how we can understand the relation between changing appearances in people, nature, and art. Hilde-brand explained:\n\nNature in its movements and transformations produces changes in its appearance, which we interpret as the visible signs of those processes. We perceive the signs and imagine the process; we participate in it, so to speak, perform along with it, and accept the internal activity as the cause of the external appearance. Just as the child learns to understand laughter and tears by joining the process and is able to feel, through muscular activity that he himself calls forth, the inner cause of the pleasure or pain, so does all gestural expression and all movement on the part of others become for us a comprehensible expression of internal processes, a comprehensible language.\n\nHildebrand extended this analogy to inanimate objects and representations as well. We understand images by imputing them with a \"story\" of their processes, similar to our own; our own bodily sensations and experiences enliven the images and make them comprehensible to us. We understand art through a process of empathy. Taken together, the theories of Vischer and Hildebrand helped to establish _Einf\u00fchlung_ as a popular explanation of the aesthetic experience.\n\nPolgar also made a comparison between film and music to further his thesis about the power of the cinematic image. Music, he wrote, has the power to \"move\" us; it moves our \"inner humanity\" in a way quite mysterious. It could also provoke a synesthetic response: \"a powerful suite of images, color, feeling, and idea.\" Film, Polgar argued, functions in the same way: it is \"a similar fertilization of all the other senses through the stimulation of an optical sense.\" This idea of synesthesia was important to _Einf\u00fchlung_ theorists, because it helped to describe what actually happens during aesthetic reception, but also because it indicated the centrality of the body as a measure of aesthetic response. _Einf\u00fchlung_ theory postulated that the harmonious correlation of form to the physical or sensory structure of the viewer was the key to aesthetic pleasure. That is, according to the theories, if the object has a structure similar to our body, it is pleasing to us; if not, it strikes us as unpleasant. By extension, the symmetry of the body accounts for the tendency toward symmetry in art. A sympathetic response to this harmony could take place on a number of levels. There is, for example, in terms of symmetry, the physical correspondence of the structure of sense organs and various objects (a horizontal line matches the horizontality of our eyes, for instance). In terms of regularity, the formal arrangement in series matches the rhythmic function of our organs. In other words, \"the body, in effect, imposes an 'organic norm' in viewing the world, according to which regularity, symmetry, and proportion induce pleasing sensations by emulating the normal human body.\"\n\nBut this \"viewing\" is not merely visual. Vischer noted that sometimes \"a visual stimulus is experienced not so much with our eyes as with a different sense in another part of our body\" (98). Sunglasses could have the effect of \"cooling\" the skin, while \"loud\" colors might offend our auditory nerves. \"For in the body there is, strictly speaking, no such process as localization.\" The embodiment of vision meant that \"the whole body is involved; the entire physical being is moved\" (99). On one hand, this embodiment implied the possibility\u2014even necessity\u2014of synesthesia, at least at some level. The senses resonate with one another; the boundaries between them are not simply blurred, but porous, causing a stimulus to one sense to spark a response in the others. On the other hand, Vischer also argued that this resonance between the senses is the model for the relation between the arts: \"These reflexes or reciprocal vibrations of the senses are the physical cause of the unity of the arts\" (99). While this parallel between the structure of the body and the relation between the arts was just a footnote to his argument, it points to an ambiguity in Vischer's book and in subsequent discussions of _Einf\u00fchlung_ , as we will see: the question of embodied spectatorship did not always imply physical participation in the act of viewing; instead, it often implied shared forms and orientations between the artwork and the human body per se. That is, the body was more often a _model for_ art than a _participant in_ art. Vischer's evocation of synesthesia functioned ambiguously by pointing both to embodied spectatorship and to the body as an idealized model.\n\nHowever, Ernst Bloch's 1914 essay, \"Melody in the Cinema, or Immanent and Transcendental Music,\" pressed harder on this issue via the example of silent film accompanied by music. A musician by training, Bloch placed greater emphasis on the redemptive and sensual nature of sound. But he argued that, while it was exclusively visual, the film image gained another dimension when combined with the proper music. Indeed, his article was a plea for music that was appropriate for the film and not simply random notes played to cover the sound of the projector. He found synesthetic possibilities in film music, which could present a complete sensual experience initiated by the ear, not the eye.\n\nAs visitors to the cinema we must initially rely exclusively upon our eyes. Now, the sense of touch conveys the impression of reality most immediately; in front of the film screen, however, we must renounce everything\u2014pressure, warmth, scent, sound, and the feeling of being sensually encompassed [ _sinnliches Mittendarinsein_ ]\u2014that gives the appearance of things its fully \"real\" character. The skin, the nose, ears, and all other senses are switched off, while the eyes are overwhelmed. Only an optical impression of black and white is excerpted from the real world, and since this impression is presented in the most confusing momentary motion and without any stylization, it produces the uncanny impression of a solar eclipse, a silent and sensuously deprived reality that is heightened only in its speed and its concentration, but without departing from our world aesthetically and ideally. But now the ear takes on an unusual function: it fills in as the replacement for all the other senses. Because the rustling, rubbing, and noisy collision of objects, because above all, human voices (which are themselves ringing with emotion) can blend seamlessly into [musical] tones\u2014indeed, precisely because there is nowhere a natural or manufactured sound in the world that competes with music\u2014this art form [film music] is able to reflect the colorfulness of lived reality and achieve at one stroke a sensuous totality that never makes us aware of our individual senses.\n\nLike many writers during this period, Bloch presented film as a lack: in front of the screen we must \"renounce everything\" that gives the world its \"fully 'real' character,\" such as \"pressure, warmth, scent, sound.\" We may disagree with his assessment of the cinematic experience\u2014we might rightly wonder where he found such a cool, odorless, silent, and spacious film theater\u2014but we can also see this as a condemnation of the poverty of any form that relies on vision alone. In this case, Bloch argued that film's silent, black-and-white world presented only an attenuated version of the world, a meager representation that could not satisfy the need for sensuality until it was matched with music. Then, as if music were color itself, this world came alive, and the filmic representation was able to regain the \"colorfulness of lived reality.\" It seems at first glance that music was the sole hero here, rescuing bored spectators everywhere from the paucity of filmed reality. But it was precisely the _combination_ of film and music that created this embodied experience. Bloch called film \"a silent and sensuously deprived reality that is heightened only in its speed and its concentration,\" but it was exactly that speed and concentration that allowed a \"sensuous totality,\" in that film and music are both temporal forms. It was their combination (as in opera, for example) that swept the spectator into a powerful representation of \"the colorfulness of lived reality.\"\n\nBut film is not like opera; \"its speed and its concentration\" are like no other forms. What did he mean by \"concentration\" ( _Konzentration_ )? On one hand, he referred to the temporal density of filmic representation: its ability to create temporal ellipses through editing and to pack more events into a limited amount of time\u2014film as a precipitate of time. On the other hand, it recalled the concentration of the film viewer, the spectator's absorption and immersion in the film, the kind of contemplation that almost allowed the viewer to forget \"pressure, warmth, scent, sound,\" and so on. Both were required for this new form of embodied spectatorship. Almost despite himself, Bloch argued for a synesthetic experience unique to cinema. In so doing, he extended further the issue that Vischer and others explored: the role of the body in the aesthetic experience. Bloch's description of film and music's \"sensuous totality\" ( _Gesamtsinnlichkeit_ ) certainly pushed synesthetic principles of artistic formation and reception much further than what we find in most theories of _Einf\u00fchlung_.\n\nBloch's description of film and music and Polgar's case of the pretty girls bring up an interesting question in relation to this theory of aesthetic response. Generally speaking, Vischer, Hildebrand, and other theorists of _Einf\u00fchlung_ limited their discussion to _static_ forms, such as painting, sculpture, and architecture. We cannot blame them for not including cinema in their investigations, but even theater and dance or other temporal forms were rarely, if ever, evoked. Music featured prominently, but only because it is abstract; these writers were working on the problem of legibility\u2014how is it that we understand something that is not like us?\u2014so music's abstractness, not its temporality, was intriguing to them. Polgar's pretty girls were just a representation, certainly\u2014black and white, silent, two-dimensional\u2014but they _moved_ , and the moving image's precise role in _Einf\u00fchlung_ theory was still indeterminate. To what extent could _Einf\u00fchlung_ aesthetics accommodate a temporal form such as moving pictures? Vischer, Hildebrand, and others frequently discussed \"movement,\" but we should first understand what they meant by this term.\n\nMovement is implied by the very term _Einf\u00fchlung_ , which translates literally as \"feeling into.\" Yet it also goes without saying that the combination of \"feeling\" and \"into\"\u2014that is, the comparison of emotion and space\u2014must be metaphorical. Nevertheless, the emphasis in _Einf\u00fchlung_ aesthetics on physiology and on spectator response left open the possibility that \"movement\" during the aesthetic experience might be more than merely metaphorical. This was indeed the case for both Vischer and Hildebrand, who discussed physical movement of the spectator in terms of the movement of the eye over the object before it. But this activity, as Vischer explained, is largely confined to the eye: \"We achieve this muscular activity, by moving the eye while looking at the object: that is, by scanning [ _Schauen_ ]\" (94). Hildebrand agreed; both theorists distinguished seeing ( _Sehen_ ) from scanning ( _Schauen_ ) and likened the latter to touch as a more sensual, tactile appropriation of the image. Vischer allowed that there might be a more visceral or holistic physical response to art when he ventured that \"mental stimuli can bring about motor stimuli in the lower organs, and vice versa. The whole body is involved; the entire physical being is moved\" (99). Ultimately, however, this response was not what he had in mind when discussing the aesthetic experience. Instead, both he and Hildebrand stressed the importance of the imagination as a mediator between raw stimuli and a purely aesthetic response. \"I might imagine myself,\" Vischer wrote, \"moving along the line of a range of hills guided by kinesthetic imagination (be it direct or mediated by the reflex stimuli of sensitized nerves). In the same way, fleeting clouds might carry me far away. This is no longer seeing [ _Sehen_ ] but a _watching_ [ _Zusehen_ ]: the forms appear to move, but only _we_ move in the imagination\" (101, emphasis in original). In other words, our projection into the work\u2014and our physical response to it\u2014depends on an act of imagination, ensuring the aesthetic experience remains within the domain of the \"higher\" functions. The movement of the eye initiates the process, but in our response any \"movement\" is to be construed as virtual, not real.\n\nIf _Einf\u00fchlung_ was a combination of emotional projection and (a limited form of) embodied spectatorship, the work of architectural historian August Schmarsow is especially appropriate. His inaugural lecture from 1893, \"The Essence of Architectural Creation,\" outlined a theory of architecture as \"the creatress of space\": \"Our sense of space [ _Raumgef\u00fchl_ ] and spatial imagination [ _Raumphantasie_ ] press toward spatial creation [ _Raumgestaltung_ ]; they seek their satisfaction in art. We call this art architecture; in plain words, it is the _creatress of space_ [ _Raumgestalterin_ ].\" Schmarsow argued that we understand architectural form by projecting ourselves into it\u2014by correlating our bodily axis, for example, to the vertical lines of a building. For Schmarsow, this was a process of spatial creation ( _Raumgestaltung_ ), closely linked to proprioception, our sense of spatial orientation, or what he called \"the intuited form of space\": \"The intuited form [ _Anschauungsform_ ] of space... consists of the residues of sensory experience to which the muscular sensations of our body, the sensitivity of our skin, and the structure of our body all contribute\" (286). Our bodily activity creates or contributes to a physical \"memory\" of sensation, which serves as the basis for a physiological understanding of architectural form. Like Vischer and Hildebrand, Schmarsow found the imagination central to this process: \"It is an act of free aesthetic contemplation when, with the aid of our imagination, we transport ourselves from the exterior that we see before us into the center of the interior space; when, by inquiring into its axial system, we strive to open up a remote organism to the analogous feeling within ourselves\" (293). This \"striving\" to open up the interior of a building by means of our already existing sense of space is to Schmarsow a creative process, and \"movement\" was vital to it.\n\nAs in the work of Vischer and Hildebrand, this \"movement\" seemed virtual or imagined, detached from real muscular tension, but in Schmarsow's essays something more seems to be at play: \"We cannot express its relation to ourselves in any way other than by imagining that we are in motion, measuring the length, width, and depth, or by attributing to the static lines, surfaces, and volumes the movement that our eyes and our kinesthetic sensations suggest to us, even though we survey the dimensions while standing still\" (291). Here Schmarsow hinted that our \"kinesthetic sensations\" play a part, that in the appropriation of architectural form there is a productive tension between stillness and movement in the body at the moment of apperception. Indeed, architecture may be the \"creatress of space,\" but Schmarsow emphasized the creative aspect of spectatorship as well:\n\nJust as, in response to external events, shared emotional feelings intensify in their rise and fall into moods or press in their growth toward blissful delight or convulsive pain in order to move farther out and fill the immediate surroundings with the vibrations of the inner life and to influence those surroundings, if only through the fleeting sound of the human voice\u2014so, too, the purely imagined impressions and their integration or combination into three-dimensional visual forms involuntarily project themselves into the world outside and develop further into a sensorially perceptible reality. (292)\n\nSchmarsow argued that _emotion_ was key to understanding space and that this emotion projected outward to create a larger reality. Furthermore, when he wrote that \"the spatial construct is, so to speak, an emanation of the human being present, a projection from within the subject\" (289), Schmarsow suggested not simply that architectural space was a mutual construction of architect and viewer (\u00e0 la Kant), but that the apperception of architecture involved a simultaneous construction of lived space, that the architectural representation of space was not merely imagined but felt emotionally and physically\u2014and that this emotional and physical \"movement,\" in a real way, _creates_ the space represented. (In this respect, Schmarsow's ideas might be useful for thinking about our bodily and emotional engagement with cinematic space.)\n\nOn the other end of the spectrum, however, we find Munich psychologist Theodor Lipps, regarded as the chief proponent and theorist of _Einf\u00fchlung_ aesthetics. Between 1890 and 1914, he was a prolific\u2014not to say incontinent\u2014explicator of _Einf\u00fchlung_ , both in aesthetic terms (emotional projection of ourselves into art) and psychological terms (as the fundamental way we understand others). On the question of movement, Lipps was contradictory. In a typically confusing passage, Lipps defined his terms: \"Finally, one could figuratively say that 'activity' is the inner breath or heartbeat, or more generally, it is the inner motion. But 'motion' here is not meant as a simple event within me, but rather the fact that I move. Of course, this 'motion' has nothing to do with space.\" In postulating that \"I move\" but that \"this 'motion' has nothing to do with space,\" Lipps hoped to have it both ways, succinctly expressing the tantalizing tension in the definition of movement in _Einf\u00fchlung_ aesthetics. Yet, in the end, Lipps was fairly clear that this movement was limited to \"some inner, striving motion\" ( _der inneren strebenden Bewegung_ ). Indeed, we might assume (or hope) that kinesthetic affect is an important aspect of the aesthetic experience, but Lipps was adamant that if we _feel_ any bodily response, if we are aware of our bodies in any way, then we are no longer in the realm of the aesthetic proper. Real bodily movement or physical response may be mimicry or erotic pleasure, according to Lipps, but at the point that it is physical, it ceases to be aesthetic. He stated it flatly:\n\nBut the pleasure which I feel while I am paying attention to my physical states or the processes in my physical organs cannot be identical, neither wholly nor in part, with the joy which I feel when I do _not_ pay attention to the processes in my physical organs, but devote my whole attention to the aesthetic object. In short, _A_ cannot equal non- _A_.... _Einf\u00fchlung_ means, not a sensation in one's _body_ , but feeling something, namely, _oneself_ , into the aesthetic object.... Actually, the sensations of my own bodily state are only present in aesthetic contemplation in order to be entirely absent to me.\n\nThis, of course, meant that any sexual feeling or arousal could not be part of the aesthetic experience:\n\nThe sexual has nothing, absolutely nothing, to do with the aesthetic. Those who employ it to explain aesthetic feeling know as little of the meaning of beauty and aesthetic contemplation as those who warn against \"nudity\" in art because they fear that even chaste nudity will threaten morality: first their own morality, then that of others to whom they ascribe their own crudity.\n\nIn sum, Lipps insisted that the movement implied by _Einf\u00fchlung_ was metaphorical and must remain so if it were to be aesthetic; he firmly stood by a strict, conservative delimitation of art and body that required the terms of aesthetic pleasure to be divorced from bodily response.\n\nThe cinematic experience, however, brings us back to the question. As we become immersed in the cinematic image, we might, as Lipps demanded, forget about our bodies, but this very forgetfulness might result in involuntary movement, such as gripping the arm of the chair (or the person next to you) during a car chase or moving slightly side to side during a well-choreographed fight sequence. It might even result in erotic pleasure. During the time that Lipps wrote about _Einf\u00fchlung_ , these pleasures in the cinema would not have even been included in the category of \"the aesthetic,\" but the character of the moving image\u2014among other things\u2014may have forced a reconsideration of the definition of aesthetic pleasure. We can see this renegotiation start to take place with Walter Serner's 1913 essay, \"Kino und Schaulust,\" which is one of the first to suggest the sexual dimension of visual pleasure. Serner's discussion of the link between cinematic movement and bodily reaction implicitly challenged the division between visual stimuli and visceral response that we have seen in traditional and _Einf\u00fchlung_ aesthetics. I am not suggesting that Serner was actively intervening in this discussion; indeed, his attitude toward cinema was ambivalent at best. But his essay indicates that what remains virtual and latent in _Einf\u00fchlung_ aesthetics\u2014movement in the image and in the spectator\u2014was even at the time visceral and literal in the experience of cinema.\n\nSerner was explicit that the _Schaulust_ he described was not the polite, distanced gaze of the aesthete: \"Not the harmless kind, for which everything is only movement or only color or both, but a frightening desire that is no less powerful than the very deepest kind. It is a desire that boils the blood and makes it rage, until an unfathomably powerful excitement common to all desire races through the flesh.\" This \"unfathomably powerful excitement\" was, of course, sexual desire. Serner was at pains to point out that this brand of sexual desire was not merely erotic but was tinged as well with the thirst for violence. Cinema appealed to our basest instincts, our darkest needs. But it was not simply the content of the films that made this appeal; it was cinematic movement itself:\n\nIt was neither the quick victory over something that was dying nor an astonishing profitability that with one stroke put this [cinema] into place from the outset. It was the exciting adventurousness of a tiger hunt, of a daring mountain run, of a death-defying automobile ride; it was the breathtaking pursuit of a wounded, bleeding thug over the dizzyingly high rooftops of New York; it was the eerie district filled with misery, sickness, and crime, and the entire horrific detective genre with murder and violence, Browning and Navajo; and it was all the bloody, blazing images of fire and death, of horror and terror, on which all eyes sate themselves after long privation. A gaze, it was, that had rhythm and life and was desire.\n\nThe tiger hunt, the mountain ride, the chase\u2014these were the components of cinema as much as the bloody pictures of fire and death. Serner named the _thrill_ of cinema as an explicitly visceral reaction in which the spectator was swept away by the movement of the image. The eye was no longer detached from the body but connected \"through the sheer endless rush, which is all the more welcome, since it knows that movement is the greatest component of the desire to look and its innermost essence.\" _Einf\u00fchlung_ aesthetics saw the body as a model for art, as a category for understanding, but mostly not as an active participant in the aesthetic experience, except for the initial roaming gaze. And there is no indication that Serner or anyone else at this moment viewed what he described as aesthetic. But as the filmic image and cinematic experience _became_ associated with Art through other means (for example, the immersive form of film narrative, movie palaces, and so on), this residual experience of movement and visceral response could not be logically left behind, even if it were attenuated in practice or, as Tom Gunning argues, took its place in lower forms of cinema. In any case, after cinema, aesthetics could no longer logically justify the well-mannered separation between contemplation and physical response.\n\nTHE POLITICS OF CONTEMPLATION\n\nWhatever their differences regarding the role of the entire body in aesthetic response, all writers on _Einf\u00fchlung_ (indeed, most writers on aesthetics in general) assumed that contemplation was absolutely central to the aesthetic experience. Recall that Schmarsow declared, \"It is an act of _free aesthetic contemplation_ when, with the aid of our imagination, we transport ourselves from the exterior that we see before us into the center of the interior space\" (293, emphasis added). Likewise, Lipps consistently invoked contemplation as the prerequisite to, even the ground for, _Einf\u00fchlung_ : \"The contemplation of the observed movement awakens the tendency to a corresponding inner response [ _Selbstbet\u00e4tigung_ ]; and by corresponding we mean that which would be connected with the execution of such a movement if I were to perform it. And this tendency is at the same time _actualized_ in the act of contemplation.\" Lipps argued that whatever movement we might feel when encountering a work (be it a painting or dance or another form), we do not actually move, nor do we need to move. Instead, the impulse toward imitation of that movement in the work is resolved or dissipated by the act of contemplation itself. In Lipps's version, the contemplative act transmutes _motion_ into _emotion_. Contemplation thereby allows emotional projection to take place, undisturbed by the intrusion of physical sensation. It is a surrender to the object and to the process: \"the aesthetic experience is the way that I feel moved when I engage in aesthetic contemplation, when I surrender myself completely to the representation,\" wrote Lipps. In this respect, _Einf\u00fchlung_ aesthetics grafted common notions of aesthetic contemplation onto the process of _Einf\u00fchlung_. We could even say that the theory of _Einf\u00fchlung_ was constructed to explain what happens during the contemplation of artworks, but without disturbing the moral and ideological significance of the operation. That is, the meaning of \"contemplation\" in _Einf\u00fchlung_ aesthetics was not markedly different from what we might find in aesthetics generally, even if elements of _Einf\u00fchlung_ theory tread close to an expansion of that meaning.\n\nCould this model of contemplation accommodate the cinematic experience? An initial answer might be no, it could not, especially given the standard history, which dictates that contemplation faded and distraction took its place. The evidence in this chapter elaborates that history. The first section demonstrated that objections to cinema's rush of images were grounded not simply in contemplation's leisurely approach but also in the historically strong tension between the flux of time and free will. That is, writers were not just _unaccustomed to_ the rush of imagery but felt _determined by_ the choice and flow of imagery that seemed to push them along ever more impatiently through the present, giving them a strong, lived sense of time's passage. Meanwhile, other writers reveled in the sense of presence and the present that motion pictures gave them, which sharpened their senses and put them on alert, as if caught on foot in midday traffic. Likewise, the second section sketched the trouble with contemplation from another angle as aesthetics tried to accommodate aspects of the aesthetic experience that were nascent in early theories but never fully accounted for, especially emotional projection and embodied perception. As these features became more prominent in the theories, it was more difficult to graft a common conception of contemplation (as largely inattentive to the body) onto this model. The cinematic experience, however, was varied. As we saw in chapter 3, reformers such as H\u00e4fker worked hard to fit film presentations into the ideologically dominant model of contemplative experience. We could also argue, after Charles Musser, that some film genres, such as landscape films, were designed from the start to provide for a contemplative gaze.\n\nWe could further contend, after Miriam Hansen and Heide Schl\u00fcpmann, that cinema's public sphere had already created an alternative to detached aesthetic contemplation. They argue persuasively that the usual interpretation of these early discussions of film audiences in terms of class misreads to a certain extent the masculine anxieties of the moment, which should more properly be classified in terms of gender and sexuality. The presence of women in the public sphere in general and in the movie theater specifically, according to Hansen and Schl\u00fcpmann, indicates not only a change in gender dynamics in pre\u2013World War I Germany, but also a change in aesthetic modes of reception\u2014specifically, a shift from detached aesthetic contemplation to a more immersive, emotionally involved mode of viewing that has often been characterized as female. Their point is that, if we are going to look to the debates as evidence of a shift in aesthetic reception, we would do well to consider that most interlocutors were male and that many in the audience were female; contemplation was an ideological tool in service of not just class-based but also gender-based goals.\n\nHansen and Schl\u00fcpmann provide an important corrective and an avenue of research that has been enormously productive. I would only indicate that, as chapter 3 demonstrated, children also presented an important model of spectatorship. In fact, this book argues that gender, class, and age were only some of the categories through which spectatorship was discursively constructed, and that our understanding of this process is best grasped via the larger division between _expert_ and _lay_ viewers. An expert could very well have an immersive, emotionally involved reaction to a film or a painting, as in Diderot's famous description of one of his encounters with a painting: \"I was motionless, my eyes wandered without fixing themselves on any object, my arms fell to my sides, my mouth opened.... I shall not tell you how long my enchantment lasted. The immobility of being, the solitude of a place, its profound silence, all suspend time; time no longer exists, nothing measures it, man becomes as if eternal.\" Apart from the final venture into metaphysics (\"man becomes as if eternal\"), this description recalls numerous reports of ordinary moviegoers, female and otherwise. But because it was written by an expert, it is free of the damning taint of passivity, which characterized all spectators\u2014young and old, female and male. Diderot was absorbed by the image, but he wandered through it of his own accord. Passivity, not necessarily immersion or emotion, was the feminized trait that characterized lay spectators of all sorts.\n\nYet class does matter; if contemplation held most aesthetic theories in a tight ideological grip, it did so in service of implicit class privilege and agency. After World War I, however, when class barriers were subject to attack, the reaction to contemplation took on a more overtly political tone. While nineteenth-century philosophers might have viewed contemplation (and themselves) as active, revolutionaries saw contemplation (and the philosophers) as bourgeois and passive in favor of the status quo. This critique of contemplation was voiced most vociferously by the Dadaists, who vowed to no longer assume the role of mere \"passive spectator [ _passiver Beschauer_ ] of this comical world, of an aesthete like Oscar Wilde,\" for, as Richard Huelsenbeck warned at the time, \"to sit in a chair for a single moment is to risk one's life.\" For the Dadaists, contemplation was irredeemably tied to passivity. They condemned the class dimensions of contemplation as a bourgeois appropriation of art for art's sake that ignored the revolutionary potential of the aesthetic experience. Early discussions of cinema reversed this complaint. Various _Kinogegner_ rejected motion pictures for their perceived association with the working classes and further argued that the nature of the filmic medium precluded the possibility of contemplation, which they defended as if protecting class boundaries. Either way, it seemed, motion pictures were not usually connected to contemplation. If they were, as in H\u00e4fker's case, it was in defense of film, contemplation, and middle-class values. In fact, the extent to which film was considered suitable for either contemplation or a distracted mode of viewing depended precisely on the class alignments of any given writer.\n\nAfter World War I, then, the passive and active connotations of contemplation and distraction traded places. The rehabilitation of distraction began, at least in film theory, with Siegfried Kracauer's 1926 essay, \"The Cult of Distraction,\" in which he described film presentations in Berlin's lush movie palaces (fig 4.3). For Kracauer, these inherently fragmented, varied, and artistically incoherent presentations (which included live revues, advertisements, films, lighting schemes, music, and other spectacular elements) structurally matched \"the disorder of society\" and, hence, had the potential to present the working classes with a true picture of \"the uncontrolled anarchy of our world\" (327). That picture could also function as an image of the mass audience or masses ( _die Masse_ ) of and for itself; that is, for Kracauer, if the mass audience could recognize itself in an image of itself, it would be a step closer to political consciousness: \"Here, in pure externality, the audience encounters itself; its own reality is revealed in the fragmented sequence of splendid sense impressions. Were this reality to remain hidden from the viewers, they could neither attack nor change it; its disclosure in distraction is therefore of _moral_ significance\" (326, emphasis in original). But Kracauer complained that picture palaces in 1926 papered over this fragmentation with \"the glue of sentimentality\" or an \"artistic\" unity, thereby hiding the true picture of alienation behind a surface sheen. They functioned in this case, for Kracauer, as distraction _from_ , whereas he advocated distraction _for_ political awakening. Although never named in this essay, contemplation was implicitly no more than a phantom appendage of an \"idealist culture that haunts us today only as a specter\" (327).\n\nFIGURE 4.3. Kammer-Lichtspiele Theater in Berlin, 1912\n\nBenjamin also gave distraction an explicitly political valence. In his 1929 \"Surrealism\" essay, for example, he discussed the \"transformation of a highly contemplative attitude into revolutionary opposition.\" But Benjamin's understanding of the nature of that political charge differed from Kracauer's. His \"Work of Art\" essay (1935\u20131939) remains the definitive sign of the rising political stock of distraction and the devalued currency of contemplation between the wars. This essay has received more than its share of commentary, so I only want to underline certain keys differences between Benjamin and Kracauer. For Kracauer, distraction functioned as an image of the masses for the masses; the homology between leisure and labor, between the disjunctive form of entertainment and the alienated character of work and modern, urban life was something that the audience _could see_ or picture through their experience in the picture palace. Indeed, Kracauer assumed that, once they grasped this picture, the mass audience would _reflect_ on it and come to an understanding of its political situation. So the image that distraction presented would be structurally no different than any image the audience would contemplate and reflect on.\n\nBenjamin, on the other hand, argued that modernity and technological reproducibility had destroyed the cult value of images, so he searched for an alternative mode of thinking that would not rely on images per se, or even vision. Distraction, for him, was this revolutionary mode that struck at the heart of knowledge as usual. For Benjamin, contemplation comes at knowledge through the encounter with\u2014or reliance on\u2014a transcendental Ideal (recall Schopenhauer's understanding of aesthetic experience as loss of oneself and a nirvana-like communion with the Ideal). It is a top-down approach that already presumes form, like a Kantian a priori. That \"already-given\" rigs the game of knowledge production, stacking the deck against experience. Contemplation, for Benjamin, succumbed to the tyranny of forms of knowledge already given. Distraction, however, represented another way of thinking that worked from the bottom up; it takes the world and experience for what they are, not what they are presumed to be: \"A person who concentrates before a work of art is absorbed by it.... By contrast, the distracted masses absorb the work of art into themselves.\" The tables have turned: contemplation is now passive, in that the viewer is ruled by the rules and form of the work of art. Distraction, on the other hand, is now active, in that the masses control the work and care not for its form or the usual ways of appropriating it. Benjamin evoked touch and habit\u2014senses other than vision\u2014to describe this mode; distraction was a famously tactile form of knowledge: \"The values of distraction should be defined with regard to film, just as the values of catharsis are defined with regard to tragedy... Distraction, like catharsis, should be conceived as a _physiological_ phenomenon.\" Like Bergson, he hoped to turn us away from our common, ordinary perception that actually keeps us chained to the status quo.\n\nGeorg Luk\u00e1cs also offered a thorough and trenchant analysis of the class dimensions of contemplation. From his early _Theory of the Novel_ (1916) to _History and Class Consciousness_ (1923), Luk\u00e1cs presented a strong critique of the _vita contemplativa_. For Luk\u00e1cs, contemplation was not merely a mode of aesthetic viewing, but a general reaction to the alienating character of modern life that must be surmounted for any transformation of the world to take place. It was a \"pattern of consciousness in which man contemplates from a position of formal freedom his own integration in a system of alien compulsions and confuses this formal 'freedom' of his contemplation with an authentic freedom.\" Contemplation, for Luk\u00e1cs, was simultaneously tied to both transcendence and complacency. In the preface to the 1967 edition of _History and Class Consciousness_ , Luk\u00e1cs wrote, \"Above all, I was absolutely convinced of one thing: that the purely contemplative nature of bourgeois thought had to be radically overcome.\" Even in _Theory of the Novel_ , Luk\u00e1cs organized his argument around two modes: contemplation and action. Specifically, he found two broad trends in the history of the novel, \"abstract idealism, which concentrates on pure action, and Romanticism, which interiorizes action and reduces it to contemplation.\" Novels such as _Don Quixote_ (1605) emphasized action without reflection, while novels such as Novalis's _Heinrich von Ofterdingen_ (1802) expressed the inward turn of romanticism, away from the disappointment and failures of modern alienation. Luk\u00e1cs explained:\n\nIn Romanticism, the literary nature of the _a priori_ status of the soul vis-\u00e0-vis reality becomes conscious: the self, cut off from transcendence, recognizes itself as the source of the ideal reality, and, as a necessary consequence, as the only material worthy of self-realization. Life becomes a work of literature; but, as a result, man becomes the author of his own life and at the same time the observer of that life as a created work of art.\n\nLuk\u00e1cs set up agency\/identity dichotomies similar to Benjamin's: the paths to identity or self-realization were twofold, either through transcendence or self-awareness. If one denied the possibility of transcendence, then only a materialist, humanist approach would be possible, which was what Luk\u00e1cs advocated. Likewise, once this path was chosen, agency returned and one could be an active \"author\" of one's life rather than a passive \"observer,\" which Luk\u00e1cs condemned. In _Theory of the Novel_ , he advocated humanism (exemplified by Goethe's _Wilhelm Meister_ ) as a middle ground between action and contemplation, but in his later, more explicitly Marxist works, Luk\u00e1cs focused primarily on a radical critique of contemplation.\n\nBut the first rumblings of this dissatisfaction could be felt before World War I, especially in Luk\u00e1cs's 1913 essay, \"Thoughts Toward an Aesthetic of the Cinema\" (\"Gedanken zu einer Aesthetik des 'Kino'\"). This brief but provocative article for the _Frankfurter Zeitung_ \u2014it was chosen for publication over Bloch's piece on film and music\u2014has been given insightful consideration by a number of scholars over the years, but it is worth another look, because it both addresses the most insistent themes of the _Kino-Debatte_ (the distinction between theater and cinema, word and image) and looks past them to anticipate themes in film theory of the 1920s. While it does not speak to most issues common in _Einf\u00fchlung_ aesthetics (for example, emotional projection, synesthesia, or embodied spectatorship), it offers in nascent form a critique of contemplation that Luk\u00e1cs developed in his later work; in this way, it articulates cinema's implicit challenge to \"traditional aesthetics\" that will come out more forcefully in Luk\u00e1cs's later work and the writings of Benjamin, Kracauer, and others.\n\nLuk\u00e1cs did this through what at first appears to be a standard comparison of theater and cinema, a strategy common to much early writing on film. The difference between theater and film, according to Luk\u00e1cs, is the difference between presence and absence, specifically, the presence of the actor on stage. But it is not the gestures or words of the actor that give theater its effect, nor the action of the drama. Instead, it is \"the power with which a person, the living will of a person, without mediation and without restraining direction, streams forth onto an equally living mass or multitude\" (13). The very _presence_ of the actor\u2014the force of his or her personal will, his or her drive toward the fulfillment of his or her being, his or her destiny\u2014is the essence of theater. This living, breathing essence of another person is inherently transitory\u2014tied to the now of the moment, to the _present_ \u2014but its very ephemerality is \"not a deplorable weakness, but rather a productive limit, the necessary correlate and most observable expression of destiny in drama\" (13\u201314). That is, the presence of the actor in the transitory moment shared by everyone in the theater is the most visible expression of the enactment of becoming that is drama itself. This sense of becoming, of the lived moment, is available to us all merely through the act of living intensely, but \"this so-called 'life' never attains an intensity that could raise everything up to the sphere of destiny\" (14). Drama, in other words, frames and concentrates this sense of destiny and fate for us: \"Throughout the presentation of the drama this metaphysical feeling grows in immediacy and perceptibility: out of the deepest truth of people and their position in the universe grows a self-evident reality\" (14). The stage, by virtue of its emphasis on _presence_ and _the moment_ , becomes a vehicle for the profound truths of human existence. Actors are an instantiation of these metaphysical truths in that they focus the force of living, or the direction of fate, into an ephemeral here-and-now shared by the actor and the audience; in fact, the mere presence of a great actor is \"already and without any great drama destiny, divine service, tragedy, mystery\" because of his or her unique ability to be \"absolutely present\" (14).\n\nCinema, on the other hand, does not share this emphasis on \"presence\" or \"the present.\" Motion pictures lack this \"presence\" not because of any inherent defect, but simply because in films \"there are only movements and actions of people\u2014but _no people_ \" (14, emphasis in original). The people on the screen are not present\u2014nor do they exist in the present\u2014so they have no presence. Even so, the representations in cinema are \"no less organic and alive than those images of the stage\" (14). They are simply different: \"they maintain a life of a completely different kind. In a word, they become _fantastic_ \" (14, emphasis in original). They present \"a new aspect\" of life: \"one without the present, a life without fate, without reasons, without motives, a life without measure or order, without essence or value, a life without soul, of pure surface, a life with which the innermost of our soul does not want to coincide.... The world of the 'cinema' is thus a world without background or perspective, without any difference in weight or quality, as only the present gives things fate and weight, light and lightness\" (14). At first glance, this judgment sounds very much like it might have come from a _Kinoreformer_ , like a condemnation of the cinema as \"soulless\" and suitable for only the basest amusements that require no thought or reflection. But Luk\u00e1cs was after something else here. He saw in cinema the same utopian potential that one could find in fantasy: \"' _Everything is possible_ ': this is the worldview of the 'cinema'\" (15, emphasis in original). Cinema was not just the poor cousin of the stage; it offered a completely different set of possibilities.\n\nThe basis for this difference lies in their formal properties as well as their dissimilar relationship to time and the word. The stage is populated with living, breathing people; drama focuses on exactly that moment of their shared presence with the audience. Motion pictures, however, do not picture people, but representations of people. Nevertheless, \"not only in their technique, but also in their effect, cinematic images\" are \"equal in their essence to nature\" (14). The \"technique\" Luk\u00e1cs speaks of here is the photographic quality of the cinematic image, while its \"effect\" is the illusion of movement that the apparatus creates. This movement, this temporal flow, also distinguishes cinema from theater. The stage has a paradoxical relation to time: \"it is the flow of grand moments, something internally deep at rest, almost 'arrested,' something become eternal, as a direct result of the painfully strong 'present'\" (14). Theater's emphasis on presence and the present gives it, oddly, a sense of stillness. The essence of cinema, on the other hand, \"is movement in itself, an eternal variability, the never-resting change of things\" (15). These different concepts of time, according to Luk\u00e1cs, correspond to the fundamental difference between the stage and cinema: \"The one is purely metaphysical, distancing itself from all that is empirically alive; the other is so strongly unmetaphysical, so exclusively empirically alive, that through this sheer extremity of its nature another entirely different metaphysics arises\" (15). One is fate, thought, stillness, presence, metaphysics. The other is fantasy, surface, movement, absence, action. But, as Tom Levin has noted in his reading of this essay, the intriguing aspect of cinema is that it is both \"empirical\" and \"fantastic.\" That is, its photographic image grounds it in reality, in the everyday, while its temporality\u2014its rush of images, as well as their arbitrary relation to laws of physics and causality\u2014frees it of necessity and allows it flights of fantasy. As Luk\u00e1cs declares, \"The fundamental law of connection for the stage and the theater is inexorable necessity; for the 'cinema' it is unlimited possibility\" (15). The uniqueness of cinema lies not simply in its ability to portray fantastic worlds but, as Levin argued, in its dialectical relation between the real and the possible. \"Everything is true and real, is equally true and equally real\" in the cinema, according to Luk\u00e1cs. The combination of reality and possibility and the _equality_ of the two even gives cinema a utopian dimension.\n\nBut I would also suggest that, in Luk\u00e1cs's essay, cinema provided a critical dimension alongside its utopian potential. In his comparison of stage and film in terms of \"metaphysics\" and \"soullessness,\" there was, I think, a critique of contemplation that emerged more forcefully in his later work, especially _History and Class Consciousness_ (1923). Consistently throughout the essay, Luk\u00e1cs distinguished the \"soul\" of the theater (its stillness, its metaphysical depth, its emphasis on destiny and the human condition, its presence) with the \"soullessness\" of the cinema (its constant movement, its emphasis on surface, its metaphysical \"lightness,\" its fantastic quality, its absence). In this distinction, Luk\u00e1cs was not alone. Many other writers of this period also called cinema \"soulless.\" Franz Pfemfert, editor of _Die Aktion_ , bemoaned in 1911 cinema's role in the \"soullessness\" of modern society. For Pfemfert, the naked reality presented by the cinematic image\u2014which he blamed for the _lack_ of fantasy in the filmic world\u2014could best be utilized for educational purposes. But Kurt Pinthus, introducing his 1913 _Kinobuch_ , a collection of scenarios written specifically for cinema, warned his readers, \"Always remember: this is not high art, not 'soulful' art, not theatrical art. We write only pieces for cinema, not for theater.\"\n\nHere we find what Luk\u00e1cs meant by \"soul.\" Pinthus wrote, \"not 'soulful' art, not theatrical art,\" as if they are one and the same. Pinthus and Luk\u00e1cs referred, of course, to German inwardness ( _Innerlichkeit_ ), that national tendency toward contemplation and reflection (and the demand for it in art). In his consistent opposition of \"action\" (cinema) to \"soul\" (theater), Luk\u00e1cs indicated that cinema offered an alternative to this cul-de-sac of reflection. His distinction between cinema and theater corresponds to the dichotomy between abstract idealism and romantic inwardness found in his _Theory of the Novel_ , which in turn was the foundation for his critique of contemplation in _History and Class Consciousness_. The latter book, especially, condemned as bankrupt the alienated, contemplative character, in favor of political or radical action. His discussion of cinema's \"soullessness,\" then, was not a condemnation, not even a slight in favor of the theater, but the articulation of a new possibility, a new form that more closely corresponded to the practical needs of the people. Cinema's emphasis on surface and lightness was an opportunity to escape from the suffocating and futile tradition of _Innerlichkeit_ , a formulation remarkably similar to those of Kracauer and Benjamin. Luk\u00e1cs thereby subtly pointed to the emergence of a new aesthetic, which Kracauer and Benjamin later articulated as \"distraction.\" So when Luk\u00e1cs declared that, with cinema, \"Man has lost his _soul_ ; in return, however, he gains his _body_ \" (16), he celebrated the potential for a new aesthetic standard that incorporated physiological, even tactile, responses to cinema, thereby pointing the way for film theorists of the 1920s and beyond.\n\n\"What does all this mean? It means that real presentation banishes contemplation,\" Benjamin announced, only slightly ironically, in 1930. It certainly seems that way, with Luk\u00e1cs proclaiming that our soul has been replaced by our body and Benjamin theorizing a physiological and tactile model of knowledge. Yet as Carolin Duttlinger has shown, Benjamin's own conception of contemplation and distraction was much more complex and dialectical than the version even the \"Work of Art\" essay provides. In his essays from the early 1920s, Benjamin saw in religious contemplation a presence of mind that allowed for both absorption and alertness, which was different from what he saw in the secular, solipsistic immersion of the modern age. Indeed, he and Bertolt Brecht agreed that modern contemplation and distraction were two sides of the same coin: both precluded critical engagement and response and therefore impeded political action. If his discussion of distraction in the \"Work of Art\" essay reversed this view, it is only because he dialectically leavened distraction with both habit ( _Gewohnheit_ ) and alertness or presence of mind ( _Geistesgegenwart_ ). Yet later, in some of his historical works, Benjamin's conception of modern contemplation again adds _Geistesgegenwart_ as the essential ingredient. So Duttlinger concludes that Benjamin's writings consistently reject \"solipsistic contemplation in favor of a more flexible, perpetually alert presence of mind.\"\n\nThe balance between immersion and detachment has been historically difficult to find, not only because it is challenged with every new art form, but because it is, as Benjamin's work so clearly shows, a _moral and political_ stance as well as an aesthetic stance, so finding the balance also implies finding a proper stance toward the historical moment. This is why the task continues to fascinate, and why contemplation, no matter what opprobrium it receives, will never die. For example, as Jocelyn Szczepaniak-Gillece has shown, contemplation and immersion made a comeback in film theory in the 1930s through the 1950s, as modernist architects attempted to design movie theaters that would create an ideal, immersive viewing experience and hence an ideal spectator, who was contemplative yet alert and responsive. Indeed, the change in ideas about contemplation seems to respond to Benjamin and Brecht's complaints that the inherently individualist nature of contemplation separates the viewer from the collective, thereby inhibiting communication, solidarity, and political action. These architect-theorists, such as Benjamin Schlanger and Frederick Kiesler, hoped to induce an almost Schopenhauerian disembodied viewing experience, while also making that experience resolutely communal. Like Benjamin, they accepted and worked with cinema's collectivity. This is the primary difference between the writers of the _Kino-Debatte_ and later theorists: for the most part, the earlier writers were unwilling to relinquish the historically strong connection between contemplation and individual viewing. Mass reception, for them, held little aesthetic potential (and the fear of social democracy often shut down any political potential). Yet we have seen glimmers of this possibility in various individual essays across the debates; if they often argued individually against film's aesthetic potential, as a group the essays helped clear the way. If _single_ writers clung to models of individual, expert viewing, the _collection_ of essays put mass reception on the film-theoretical agenda. Later shifts in film theory were therefore anticipated collectively by the film essays that appeared in the years before World War I.\nCONCLUSION\n\nTOWARD A TACTILE HISTORIOGRAPHY\n\nOf course, after World War I everything changed. On 1 July 1918, the newly established state film production company, UFA, created a _Kulturabteilung_ , or cultural division, to make and distribute a regular series of educational films. The leader of this division, Ernst Krieger, had been a major in the imperial army and had served alongside Alexander Grau, the wartime minister of culture. The war had offered a number of lessons, but in this case it had persuaded Krieger, Grau, and others beyond a doubt of the value of motion pictures as a means of mass education and indoctrination\u2014which after the war could be directed toward peacetime social issues. Krieger hired a number of doctors, scientists, and educators fresh from the front to make educational films encompassing titles from _The Alps_ (1918) to _Marital Hygiene_ (1922). The division also funded scientific research films, thereby giving the state's seal of approval and support to scientific and medical cinematography. The number of universities and institutes using motion pictures to document and to explore increased dramatically after the war, as film found a secure place in laboratories and lecture halls. Hermann H\u00e4fker's dream of a state-run, educational cinema had come true, even if\u2014in UFA's case, at least\u2014it had to make room for fictional films as well. Indeed, cinema's aesthetic pretensions received a boost after 26 February 1920, when _The Cabinet of Dr. Caligari_ opened in Berlin to much hype and wide acclaim. Its remarkable mise-en-sc\u00e8ne, acting, and narrative framework struck a chord with all audiences, even those beyond Germany's borders; its self-consciously artistic presentation drew from a distinctly German style, marking it immediately as \"Art.\" _Caligari_ became a lightning rod of debate about the nature of film, even while people lined up at the box office. Ushering in German cinema's golden era, _Caligari_ gave cinematic form some measure of aesthetic legitimacy. Meanwhile, the credibility of film in terms of aesthetic reception received more attention and validation with new theories (Rudolf Arnheim, Bal\u00e0zs, Benjamin, Kracauer) that acknowledged its significance for the transformation of aesthetic standards.\n\nSo in terms of disciplinary legitimacy, cinema had much less to worry about after the war. Debates continued about its proper function in society or how its artistic potential could be best realized, but they were not nearly as heated as in the prewar years. German society seemed to have adjusted itself to cinema, and cinema to it. In addition to prompting UFA's role as state-sanctioned protector of educational (and propaganda) film, the war had also cut off the supply of imported films, thereby allowing domestic production companies to flourish; the complaint about foreign films could no longer fuel debates, and the supply of \"quality\" German films also rose. The international success of _Caligari_ awakened the establishment to the artistic and economic potential of film, giving it a stamp of legitimacy that before had been only grudgingly offered at home. Firmly entrenched in German culture by the 1920s, cinema spent considerably less time defending its right to exist.\n\nOn the other hand, little has changed. We are still as preoccupied as ever\u2014perhaps even more\u2014with social acceleration and the dangers of distraction. If motion pictures exemplified the quick, relentless pace of modernity and the social consequences of inattentiveness, their secure place in mass culture did nothing to calm nervousness about their perceived effects. In fact, those concerns have doubled or tripled in our new century, predictably, as the emergence of new media forms seems to underline the lack of control we have over the pace of change and the technologies that come with it. The perceived effects have not changed, only the media to which they are attached. The rhetorical patterns we have seen over the course of this investigation have remained remarkably steady over the century; with each new media form we have a new round of experts decrying the infantilization of its audience while appropriating it for their own disciplinary projects. The difference between expert observers and lay spectators, as we have seen, resides in the amount of control one can wield over the technology and its product. The audience is infantilized precisely because it is perceived to surrender to the technology without maintaining a proper distance or detachment from it; the expert is able to use the same technology because he or she is able to master the medium\u2014which testifies to his or her own self-mastery as well. This dichotomy between resistance and surrender seems at first indistinguishable from middle-class ideals of self-mastery or restraint. These class ideals definitely reinforce the disciplinary distinctions, but because experts have the technology _in their hands_ , often literally exercising their control in this way, their disciplinary alignment is a more historiographically proximate explanation of their ideological stance. Hence the importance of examining these rhetorical patterns, which still persist in our media landscape today, in terms of _disciplines_ as well as class.\n\nExpert\/lay distinctions are perhaps just as intractable as class distinctions, and they can be just as blunt. Yet by implicating education, training, and disciplinary communities, they offer an obvious and clear point of entry for historical investigation. Indeed, this project has argued that a full description of the rhetorical dichotomies common to debates about media\u2014which would allow us to see their porousness and mutual dependence\u2014requires an acknowledgment of the heterogeneity of film technologies, products, and experiences. Taken as a whole, the expressions of film's potential or dangers are dynamic and conflicted\u2014a result of the many-sided quality of the products or technologies the historical agents encounter on the ground. Any given agent might be intransigent in this or that essay or speech, but the collection of voices within a discipline or community are, like the object itself, heterogeneous. The heterogeneity of object and audience is, in my opinion, best (but not exclusively) expressed through the specific institutional appropriations of film in the nontheatrical context, and if we want to understand these applications, it seems apparent that we need to understand the disciplinary agendas at play and their relationship to the application. I am convinced that the historical interplay between these heterogeneous agendas and objects holds more insight into \"what cinema is\" than our own top-down theoretical proclamations.\n\nSo the way that experts _handled_ the technology, both literally and figuratively, fills out the history of nontheatrical uses of media such as film. Production, distribution, and exhibition histories are traditionally important paths of inquiry for film and media studies, but we can also add descriptions of how experts have studied, manipulated, and adapted the image to their own ends, which reveals presumptions and expectations about their audience, their own expertise, and the role of film in their discipline. Experts have emphasized different aspects of film form in their use or study of the image: the still image is good for some tasks, the moving image is appropriate for other approaches; the detail of an image is an intriguing landscape open to discovery for some, while others (or even the same) gravitated toward the usefully abstract animated image. We are certainly aware of all these various facets of film, but we could focus more on patterns of use and their relationship to historically persistent rhetorical patterns. Investigating those varied uses also allows us to see the precise relationship between film form and disciplinary agendas. Moreover, the variety of approaches or entry points for expert analysis of the image also testifies to the ambidexterity of film, or its usefulness as both a still and moving image, among its other, varied manifestations. By emphasizing film's variety of forms and ways of \"handling\" it, film's malleability prompts what we might call a _tactile historiography_.\n\nIndeed, it was precisely film's adaptability that appealed to experts, that brought them to the idea that they could appropriate it, transform it, and make it their own. These experts sculpted cinema's _dispositif_ \u2014the technology, the image, the audience, and the relationship between all three\u2014in markedly different ways. The scientific disciplines had the most rigid framework within which motion pictures could fit; the technology was used primarily to control duration and to aid correlation. From the aesthetic debates, we can see the negotiation between the cinematic experience\u2014as varied as it was\u2014and ideals of individual contemplation. In any case, the extent to which film could be shaped according to preexisting standards and practices determined its acceptability. What was this shaping, exactly? For researchers, it involved a very literal hands-on tinkering with the technology to adapt it to standards of evidence and imaging. It also involved, as we have seen, tinkering with the object of study to adapt it to the representational technology. For someone like Lemke or H\u00e4fker, on the other hand, it sometimes meant tinkering with the technology or the screening venue, but more often corralling funders, exhibitors, town councils, and other members of the community and driving them toward a common goal. Persuading these groups required a similar negotiation between the film experience or _dispositif_ and established, often disciplinary agendas or ideals (\"modernizing education,\" for example). For essayists and aestheticians, it meant thinking through how some aspect of film form could fit established or emerging ideals. To press film into service, then, meant shaping it to an existing framework of institutional resources, policies, practices, and ideals.\n\nSo tactile historiography is sensitive to the historical impressions left on cinema (the filmic \"material\" that includes the technology, the image, and the audience). These impressions are of at least two types. On one side, we have the institutional or disciplinary framework to which film is made to fit; this is a historically specific but more or less stable configuration of disciplinary ideals, established practices, rules, policies, norms, and conventions. In education, for example, limited funding meant that elementary schools would not include film projection equipment in the classroom until the 1920s or so. So in the early 1910s educational screenings were held in commercial film theaters, which of course shaped the educational experience, sometimes objectionably. Yet teachers undoubtedly managed these educational screenings in such a way that the norms of the classroom were imported to the theater, so the use of film in this context expressed a combination of two institutional frameworks or two sets of institutional or disciplinary norms: commercial and educational.\n\nOn the other side, historical agents such as Lemke, who worked to make film fit into educational norms, left on these experiences the impression of their specific sculpting, tinkering, or negotiating. In this case, Lemke's specific adjustments in part consisted of his arrangement of the films and discussion into a program designed around Herbartian principles. A discursive capitulation to disciplinary norms in education, this move shaped the experience in a local, perhaps fleeting way, in that the audience might have been only vaguely aware of this justification, so its impression was likely not durable. Still, the effort itself led to the acceptance of film as a tool that _could_ be managed, as opposed to the earlier conception of film as totally unsuitable. This shift did indeed leave a lasting impression on the practices of educational film in Germany. Norms change, as do specific, local adjustments to them, which then incrementally extend or change the norms (or not). This historiographic approach sees film as an adaptable material, the form of which at any given historical moment expresses these norms and adjustments.\n\nYet if form matters, _how_ does it matter? How can an understanding of film form contribute to an understanding of broader historical trends, or vice versa? Usually film historians illustrate this relationship through close analyses of individual films, the best of which present analogies between textual and historical structures, as in Tom Gunning's essay on D. W. Griffith's _The Lonedale Operator_ (1909), which demonstrates the homology between the mise-en-sc\u00e8ne, the editing patterns of the film, and the emergence of new gender roles and modern forms of transportation and communication. In this approach, the historian illuminates film style and historical moment simultaneously\u2014each expresses the other. I advocate, in addition to this method, an approach that similarly demonstrates the mutually expressive relationship between disciplinary practice, ideals, and agendas, on one hand, and film form broadly speaking, on the other, which could include the specific quality of the image, the structure of the technology at the time, or the experience itself. The particular style of, say, a research film could be pertinent, but the way the technology is used in a research setting might reveal more substantial and specific patterns that help to explain the medium's use in that particular laboratory at that particular time. The style of the research film could also express these patterns of use, so that is an area worthy of further investigation, but this project has stressed the experts' own understanding of film form expressed through their patterns of use.\n\nWe must acknowledge, however, that all of these aspects of film form\u2014style, technology, image, experience, and so on\u2014are unstable and subjective; they change shape with the historical moment, of course, but what counts most as \"form\" depends on what the experts take it to be. This historiographic approach acknowledges that disciplines may see film differently than we do. Ludwig Braun, for example, saw motion pictures as an extension of serial photography; he therefore understood the technology as an image generator that could be best used to examine minute differences in the rhythmic function of rapidly moving objects such as the heart. In this case, Braun took film form to be a series of slightly different exposures, and the specific photographic quality of the image, for example, counted very little for his task. (If the technology could have generated successive line drawings of the heart just as easily, he would have been thrilled.) He took what he needed and left the rest. This unspoken selection or emphasis usually follows disciplinary logic, so this understanding of form\u2014of what film is\u2014also expresses that logic; form and discipline illuminate each other. Braun understood film to be \"incremental exposures,\" which expressed the serial logic in scientific and especially medical thinking at the time. The analogy between \"incremental exposure\" and \"series\" has explanatory power, because it demonstrates\u2014more powerfully than, for example, just listing advantages and disadvantages\u2014 _why a tool would be useful_. If the form of a wrench is crucial to understanding why it works on a bolt, the relationship between film and any given discipline is not so straightforward; analogies and correspondences can help us see how film fit an agenda.\n\nSo this historiographic strategy differs from the usual, first, in its emphasis on patterns of use rather than style. Style is important and can be helpful, but patterns of use apply to adjustments to the technology, the circulation of images, the multiple function of any given film, or the role of moving images in a selection of representational options\u2014a partial list at best. Patterns of use provide many more points of contact with the organization's goals or the discipline's logic than style alone. An analysis of style helps us understand, for example, the conventions within a given genre, which is good, but it limits us to the genre, when patterns of use take us into the heart of the discipline itself. Second, this approach emphasizes the notion of film form that arises from patterns of use. These patterns of use make sense only against a background of disciplinary problems and solutions. So the third way that this approach differs from the norm is in its emphasis on the disciplinary (as opposed to economic, technological, biographical, or aesthetic) context. This requires, as I have noted, some significant immersion into those contexts, but we would be rewarded with a deeper understanding of why this or that media technology mattered to the discipline. Having this insight into a discipline also helps us, as film historians, better argue the significance of film for that discipline. If we are determined to venture into the realm of the nontheatrical film, and we are convinced of its importance for this or that task, then we cannot back away from the possibility that film had some impact on the way an organization, community, or even discipline understood its agenda, its object, and itself. Having expertise in the discipline helps make the case for the significance of moving images on collective \"thought styles,\" in Ludwik Fleck's phrase, of which disciplinary logic is one expression.\n\nThis project has emphasized the correspondence between material and discipline. If we are interested in the mechanism of cultural legitimacy\u2014the process by which a new technology or form comes to be accepted by a group or discipline\u2014then this approach makes sense: any new form is not only understood in terms of what came before, but it must play by the rules set out by the discipline. The rules of evidence or legibility in the physical sciences, for example, are to a certain extent malleable or accommodating to new technologies, but if the technology does not offer representational solutions at least somewhat familiar with more or less agreed-upon conventions, it will not be considered useful. But if we are to explain not just legitimacy and acceptance but also the extent to which the technology _transforms_ the discipline, then we must also consider the _strangeness_ or difference the new form brings to the endeavor. This strangeness evokes wonder, as we have seen in the revelationist film theory of Bal\u00e1zs, Epstein, or Benjamin, or in the descriptions of researchers as they gaze into the microscopic sublime. This wonder may propel agents to replicate the experiments or experiences; it may reveal new vistas that shift the discipline's horizon of experience. In other words, the strangeness of the experience of film might have acted as an engine of change in a given discipline. Cell biology provides a good example; descriptions of cell \"behavior\"\u2014and the conclusions about cell life that rested upon such observations\u2014could not have existed without the temporal manipulations afforded by time-lapse cinematography. That is, the descriptions and therefore understanding of cell behavior depended on the technology's ability to match one timescale\u2014that of the cells\u2014to another, our human perception. Cell biologists really saw life through film's eye, and it changed what they thought life was; it changed their \"thought style.\" The strangeness of film transformed biological conceptions of life, even if only slightly.\n\nYet these and other transformations are incremental, not revolutionary. Hans-J\u00f6rg Rheinberger's idea of \"differential reproduction\" in experimental method is pertinent here; experiments are designed to be reproducible, to be sure, but that is not so important as the slight difference that each experiment affords. The difference between what is known and what is unexpected is usually only slight, but the gap between them is the real goal and product of experimental practice, because that is the space where new knowledge is produced. Likewise, the use of a new media technology as a tool is almost always experimental, and the difference between current understanding and the new view it provides is sometimes a surprisingly productive opening. The use of this tool is an attempt to solve a problem, and we can see over the course of the disciplinary appropriations of this tool a series of linked solutions to a common problem\u2014like a series of experiments. Looking at the history of the disciplinary uses of film helps us to understand its role in a larger series of linked solutions and to see film's incremental effect on the problem itself. It is as if Braun's method of comparing minute differences in his incremental exposures and then projecting them to get a sense of the whole in its duration could be applied to historiography: we note the slight differences in applications of film technology in a discipline, but we are able to see the cumulative effect only by running the series through the flip book of history, so to speak. Or, to apply a biological metaphor, film technology could be the mutant gene in the disciplinary organism, the true effects of which we notice only after a generation or two.\n\nSo this project has envisioned film's _dispositif_ \u2014its technology, its image, its audience\u2014as material in a grand, cross-disciplinary series of experiments. Experts shaped this \"material,\" adjusted or trimmed it to fit a set of needs, but the crucial difference that film provided to these disciplines came from its resistance to these efforts. The material was not infinitely malleable. The technology could be made to do only so much, the image was only so informative, the audience only so agreeable. We see this resistance in the strangeness of the view to the experts, but also in its inappropriate fit; some disciplines, such as geology, found little use for it. We see it also in the excess or residue, such as pleasure or thrill, which spills sloppily over the disciplinary framework; pleasure continues to be difficult to fit even into aesthetics. Expert vision wrestles with this remainder, even as it depends on it. Aesthetic contemplation, for example, is at once surrender and mastery; the oscillation between them relies on the irreducibly material, sensual, and singular character of the artwork, which is the basis for immersion and pleasure, but also the ground of its resistance to rationalization and abstraction\u2014both of which are also part of the aesthetic experience. Similarly, the filmic _dispositif_ as \"material\" partly resists expert efforts to sculpt it; this is especially true when it comes to the audience, which has been subject to much rationalization and abstraction. The \"shape of spectatorship\" in this regard was not just the negative outline or boundary with expert vision but the product of expert modeling. The form spectatorship has taken depended on the discursive and practical molding of experts but also on the resistance of the audience and cinema itself to this kneading and on the spectators' willingness to take the experiment to places beyond which they were prodded.\nNOTES\n\nINTRODUCTION\n\n 1. Adolf Sellmann, \"Das Geheimnis des Kinos,\" _Bild und Film_ 1, nos. 3\u20134 (1912): 65\u201367, here 65. All translations are my own unless otherwise noted.\n\n 2. The scholarship on \"useful film\" and educational film is growing. See, e.g., the special issues on \"Gebrauchsfilm\" in _montage\/AV: Zeitschrift f\u00fcr Theorie & Geschichte audiovisueller Kommunikation_ 14, no. 2 (2005), and no. 3 (2006); Vinzenz Hediger and Patrick Vonderau, eds., _Films That Work: Industrial Film and the Productivity of Media_ (Amsterdam: Amsterdam University Press, 2009); Charles R. Acland and Haidee Wasson, eds., _Useful Cinema_ (Durham, N.C.: Duke University Press, 2011); and Devin Orgeron, Marsha Orgeron, and Dan Streible, eds., _Learning with the Lights Out_ (New York: Oxford University Press, 2012).\n\n 3. Max Weber, \"Science as a Vocation,\" in _The Vocation Lectures_ , ed. and with an introduction by David Owen and Tracy B. Strong, trans. Rodney Livingstone (Indianapolis: Hackett, 2004), 1\u201331, here 7 (emphasis in original). This and the following paragraph have been adapted from my essay, \"Science Lessons,\" _Film History_ 25, nos. 1\u20132 (2013): 45\u201354.\n\n 4. See Norbert Bolz, _Am Ende der Gutenberg-Galaxis. Die neuen Kommunikationsverh\u00e4ltnisse_ (Munich: Fink, 1993), 115, or chap. 3; or Thomas Elsaesser, \"Die Stadt von Morgen: Filme zum Bauen und Wohnen in der Weimarer Republik,\" in _Geschichte des dokumentarischen Film in Deutschland, Band 2: Weimarer Republik (1918\u20131933)_ , ed. Klaus Kreimeier, Antje Ehmann, and Jeanpaul Goergen (Stuttgart: Reclam-Verlag, 2005), 381\u2013410; as well as Vinzenz Hediger and Patrick Vonderau, \"Record, Rhetoric, Rationalization: Industrial Organization and Film,\" in Hediger and Vonderau, _Films That Work_ , 35\u201349; and Petr Szczepanik, \"Modernism, Industry, Film: A Network of Media in the Ba\u0165a Corporation and the Town of Zl\u00edn in the 1930s,\" in Hediger and Vonderau, _Films That Work_ , 349\u2013376.\n\n 5. On experimental systems, see Hans-J\u00f6rg Rheinberger, _Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube_ (Stanford, Calif.: Stanford University Press, 1997).\n\n 6. Ludwik Fleck, \"Some Specific Features of the Medical Way of Thinking [1927],\" in _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle (Dordrecht, Netherlands, and Boston: Reidel, 1986), 39\u201346.\n\n 7. Ludwik Fleck, \"Scientific Observation and Perception in General [1935],\" in Cohen and Schnelle, _Cognition and Fact_ , 59\u201378, here 60.\n\n 8. Fleck, \"Scientific Observation and Perception in General [1935],\" 61.\n\n 9. Sir James Paget, \"An Address on the Utility of Scientific Work in Practice,\" _British Medical Journal_ (15 October 1887): 811\u2013814, here 811.\n\n 10. Johann Heinrich Pestalozzi, _How Gertrude Teaches Her Children_ [1801], ed. Ebenezer Cooke, trans. Lucy E. Holland and Frances C. Turner, 2d ed. (Syracuse, N.Y.: Bardeen, 1898), tenth letter, 220 (emphasis in original).\n\n 11. Ludwik Fleck, \"To Look, To See, To Know [1947],\" in Cohen and Schnelle, _Cognition and Fact_ , 129\u2013151, here 137.\n\n 12. Robert Vischer, _On the Optical Sense of Form_ [1873], in _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou (Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994), 89\u2013124, esp. 93\u201395. In art history, see Norman Bryson's discussion of the terms in _Vision and Painting: The Logic of the Gaze_ (New Haven: Yale University Press, 1983). For the use of \"gaze\" and \"glance\" in media studies, see John Ellis, _Visible Fictions: Cinema, Television, Video_ (London: Routledge, 1982); and Timothy Corrigan, _A Cinema Without Walls: Movies and Culture After Vietnam_ (New Brunswick, N.J.: Rutgers University Press, 1991).\n\n 13. Michael Hau, \"The Holistic Gaze in German Medicine, 1890\u20131930,\" _Bulletin of the History of Medicine_ 74, no. 3 (Fall 2000): 495\u2013524.\n\n 14. Michel Foucault, _The Birth of the Clinic: An Archaeology of Medical Perception_ , trans. A. M. Sheridan Smith (New York: Vintage, 1973), 109.\n\n 15. See William Egginton, \"Intimacy and Anonymity, or How the Audience Became a Crowd,\" in _Crowds_ , ed. Jeffrey T. Schnapp and Matthew Tiews (Stanford, Calif.: Stanford University Press, 2006), 97\u2013110.\n\n 16. \"The Work of Art in the Age of Its Technological Reproducibility (Second Version),\" in _Walter Benjamin: Selected Writings_ , vol. 3, _1935\u20131938_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott, Howard Eiland, and others (Cambridge, Mass.: Belknap, 2002), 101\u2013136, here 116.\n\n 17. Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Second Version),\" 104.\n\n 18. Representative interventions in the debate about the role of modernity in a history of film style include the collection _Cinema and the Invention of Modern Life_ , ed. Leo Charney and Vanessa Schwartz (Berkeley: University of California Press, 1995); David Bordwell, _On the History of Film Style_ (Cambridge, Mass.: Harvard University Press, 1997), 141\u201346; Noel Carroll, \"Modernity and the Plasticity of Perception,\" _Journal of Aesthetics and Art Criticism_ 59, no. 1 (Winter 2001): 11\u201318; Ben Singer, _Melodrama and Modernity: Early Sensational Cinema and Its Contexts_ (New York: Columbia University Press, 2001); Charlie Keil, \"'To Here from Modernity': Style, Historiography, and Transitional Cinema,\" in _American Cinema_ ' _s Transitional Era: Audiences, Institutions, Practices_ , ed. Charlie Keil and Shelley Stamp (Berkeley: University of California Press, 2004), 51\u201365; Tom Gunning, \"Modernity and Cinema: A Culture of Shocks and Flows,\" in _Cinema and Modernity_ , ed. Murray Pomerance (New Brunswick, N.J.: Rutgers University Press, 2006), 297\u2013315; and Frank Kessler, \"Viewing Change, Changing Views: The 'History of Vision' Debate,\" in _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier (New Barnet, U.K.: Libbey, 2009), 23\u201335.\n\n 19. Among many other surveys discussed in chap. 4, see esp. Sabine Hake, _The Cinema_ ' _s Third Machine: Writing on Film in Germany, 1907\u20131933_ (Lincoln: University of Nebraska Press, 1993).\n\n 20. Andr\u00e9 Gaudreault, _Film and Attraction: From Kinematography to Cinema_ , trans. Timothy Barnard (Urbana: University of Illinois Press, 2011).\n\n 21. See, e.g., Kevin Repp, _Reformers, Critics, and the Paths of German Modernity: Anti-Politics and the Search for Alternatives, 1890\u20131914_ (Cambridge, Mass.: Harvard University Press, 2000); Andrew Lees, _Cities, Sin, and Social Reform in Imperial Germany_ (Ann Arbor: University of Michigan Press, 2002); and Dennis Sweeney, \"Reconsidering the Modernity Paradigm: Reform Movements, the Social and the State in Wilhelmine Germany,\" _Social History_ 31, no. 4 (November 2006): 405\u2013434.\n\n 22. Ben Singer names this ambivalence \"ambimodernity\" and brings it to the attention of the film studies community in \"The Ambimodernity of Early Cinema: Problems and Paradoxes in the Film-and-Modernity Discourse,\" in Ligensa and Kreimeier, _Film 1900_ , 37\u201351.\n\n1. SCIENCE'S CINEMATIC METHOD\n\n 1. Charles Cros, \"Inscription,\" in _Oeuvres completes_ , ed. Louis Forestier and Pascal Pia (Paris: Pauvert, 1964), 135\u2013136.\n\n 2. On the graphic method, see Merriley Borell, \"Extending the Senses: The Graphic Method,\" _Medical Heritage_ 2, no. 2 (March\/April 1986): 114\u2013121; Robert G. Frank Jr., \"The Telltale Heart: Physiological Instruments, Graphic Methods, and Clinical Hopes, 1854\u20131914,\" in _The Investigative Enterprise: Experimental Physiology in Nineteenth-Century Medicine_ , ed. William Coleman and Frederic L. Holmes (Berkeley: University of California Press, 1988), 211\u2013290; Soraya de Chadarevian, \"Graphical Method and Discipline: Self-Recording in Nineteenth-Century Physiology,\" _Studies in History and Philosophy of Science_ 24, no. 2 (June 1993): 267\u2013291; and Robert M. Brain, \"Representation on the Line: Graphic Recording Instruments and Scientific Modernism,\" in _From Energy to Information: Representation in Science and Technology, Art, and Literature_ , ed. Bruce Clarke and Linda Dalrymple Henderson (Stanford, Calif.: Stanford University Press, 2002), 155\u2013177.\n\n 3. Wilhelm Braune and Otto Fischer, _The Human Gait_ , trans. Paul Maquet and Ronald Furlong (Berlin and New York: Springer, 1987). In addition to _The Human Gait_ , Braune and Fischer's human motion studies included _Das Gesetz der Bewegungen an der Basis der mittleren Finger und im Handgelenk des Menschen_ (Leipzig, 1887); _\u00dcber den Schwerpunkt des menschlichen K\u00f6rpers mit R\u00fccksicht auf die Ausr\u00fcstung des deutschen Infanteristen_ (Leipzig, 1889), translated as _On the Centre of Gravity of the Human Body as Related to the Equipment of the German Infantry Soldier_ by Paul Maquet and Ronald Furlong (Berlin and New York: Springer, 1985); _Bestimmung der Tr\u00e4gheitsmomente des menschlichen Koerpers und seiner Glieder_ (Leipzig, 1892), translated as _Determination of the Moments of Inertia of the Human Body and Its Limbs_ by Paul Maquet and Ronald Furlong (Berlin and New York: Springer, 1988); and Fischer's _Theoretische Grundlagen f\u00fcr eine Mechanik der lebenden K\u00f6rper_ (Leipzig, 1906).\n\n 4. Max Seddig, \"Ueber Abh\u00e4ngigkeit der Brown'schen Molekularbewegung von der Temperatur,\" _Sitzungsberichte der Gesellschaft zur Bef\u00f6rderung der gesammten Naturwissenschaften zu Marburg_ 18 (1907): 182\u2013188; Seddig, \"\u00dcber die Messung der Temperaturabh\u00e4ngigkeit der Brownschen Molekularbewegung,\" _Physikalische Zeitschrift_ 9, no. 14 (15 July 1908): 465\u2013468; Seddig, \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown'schen Molekularbewegung,\" Habilitationsschrift, Akademie in Frankfurt a. M., 1909; Seddig, \"Exacte Messung des Zeitintervalles bei kinematographischen Aufnahmen,\" _Jahrbuch f\u00fcr Photographie und Reproduktionstechnik_ 26 (1912): 654\u2013657; and Seddig, \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown-Zsigmondyschen Bewegung,\" _Zeitschrift f\u00fcr Anorganische Chemie_ 73\u201374 (1912): 360\u2013384.\n\n 5. Hermann Braus, \"Mikro-Kino-Projektionen von in vitro gez\u00fcchteten Organanlagen,\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 83, part 2 (1911): 472\u2013475.\n\n 6. For a fascinating account of cinema's relationship to time and science, see Mary Ann Doane, _The Emergence of Cinematic Time: Modernity, Contingency, the Archive_ (Cambridge, Mass.: Harvard University Press, 2002).\n\n 7. Examinations of the relationship between illustrative materials and the agendas of scientists are increasingly popular in the history of science; a good, representative example is Martin Kemp, \"Temples of the Body and Temples of the Cosmos: Vision and Visualization in the Vesalian and Copernican Revolutions,\" in _Picturing Knowledge: Historical and Philosophical Problems Concerning the Use of Art in Science_ , ed. Brian S. Baigrie (Toronto and Buffalo, N.Y.: University of Toronto Press, 1996), 40\u201384. Two especially influential collections are Michael Lynch and Steve Woolgar, eds., _Representation in Scientific Practice_ (Cambridge, Mass.: MIT Press, 1990); and Caroline A. Jones and Peter Galison, eds., _Picturing Science, Producing Art_ (New York: Routledge, 1998). Catelijne Coopmans, Janet Vertesi, Michael E. Lynch, and Steve Woolgar, eds., _Representation in Scientific Practice Revisited_ (Cambridge, Mass.: MIT Press, 2014) is an excellent recent collection.\n\n 8. For more on the relationship between motion pictures and scientific experiment, see the section on \"The Multiple Functions of the Medical Film\" in chap. 2 in this volume.\n\n 9. On experimental systems and \"dislocation\" or \"differential reproduction,\" see Hans-J\u00f6rg Rheinberger, _Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube_ (Stanford, Calif.: Stanford University Press, 1997), esp. chap. 5. Generally speaking, experimental systems are designed to produce incremental differences, which ultimately produce new inquiries and systems. But the balance between old and new is not symmetrical; my theory of disciplinary appropriation accommodates both the _correspondence_ between disciplinary ideals and film and the _difference_ between them, but the history I tell in this book favors the former. For more on the latter, see the conclusion in this volume.\n\n 10. Gaston Bachelard, _The New Scientific Spirit_ , trans. Arthur Goldhammer (Boston: Beacon, 1984), 13. See also Davis Baird, _Thing Knowledge: A Philosophy of Scientific Instruments_ (Berkeley: University of California Press, 2004).\n\n 11. Rheinberger, _Toward a History of Epistemic Things_ , 21.\n\n 12. Ian Hacking, _Representing and Intervening: Introductory Topics in the Philosophy of Natural Science_ (Cambridge: Cambridge University Press, 1983).\n\n 13. For surveys of scientific uses of photography and film, see Karl Wilhelm Wolf-Czapek, ed., _Angewandte Photographie in Wissenschaft und Technik_ (Berlin: Union Deutsche Verlagsgesellschaft, 1911); Martin Weiser, _Medizinische Kinematographie_ (Dresden and Leipzig: Steinkopff, 1919); F. Paul Liesegang, _Wissenschaftliche Kinematographie_ (D\u00fcsseldorf: Liesegang, 1920); Anthony R. Michaelis, _Research Films in Biology, Anthropology, Psychology, and Medicine_ (New York: Academic, 1955); Virgilio Tosi, _Cinema Before Cinema: The Origins of Scientific Cinematography_ , trans. Sergio Angelini (London: British Universities Film & Video Council, 2005); Timothy Boon, _Films of Fact: A History of Science in Documentary Films and Television_ (New York: Wallflower, 2008); and Kelly Wilder, _Photography and Science_ (London: Reaktion, 2009).\n\n 14. The literature on this transition between chronophotography and film is vast, but one place to start is Deac Rossell, _Living Pictures: The Origins of the Movies_ (Albany: State University of New York Press, 1998). On Janssen, see esp. the work of Jimena Canales, who describes the history of Janssen's photographic revolver in the context of emerging cinematographic forms of representation in the following: \"Photogenic Venus: The 'Cinematographic Turn' in Science and Its Alternatives,\" _Isis_ 93 (2002): 585\u2013613; \"Sensational Differences: The Case of the Transit of Venus,\" _Cahiers Fran\u00e7ois Vi\u00e8te_ 1, nos. 11\/12 (September 2007): 15\u201340; _A Tenth of a Second: A History_ (Chicago: University of Chicago Press, 2009), 87\u2013115; and \"Desired Machines: Cinema and the World in Its Own Image,\" _Science in Context_ 24, no. 3 (September 2011): 329\u2013359.\n\n 15. Jennifer Tucker, _Nature Exposed: Photography as Eyewitness in Victorian Science_ (Baltimore, Md.: Johns Hopkins University Press, 2005); or Tucker, \"Photography as Witness, Detective, and Imposter: Visual Representation in Victorian Science,\" in _Victorian Science in Context_ , ed. Bernard Lightman (Chicago: University of Chicago Press, 1997), 378\u2013408; see also Joel Snyder, \" _Res Ipsa Loquitur_ ,\" in _Things That Talk: Object Lessons from Art and Science_ , ed. Lorraine Daston (New York: Zone, 2004), 195\u2013221; as well as Wilder, _Photography and Science_.\n\n 16. An excellent discussion of one scientist's dissatisfaction with photography can be found in Sarah de Rijcke, \"Drawing Into Abstraction. Practices of Observation and Visualisation in the Work of Santiago Ram\u00f3n y Cajal.\" _Interdisciplinary Science Reviews_ 33, no. 4 (2008): 287\u2013311. Ram\u00f3n y Cajal found that photography could not capture the three-dimensionality of nerve cells as well as drawings.\n\n 17. Thomas Schlich, \"'Wichtiger als der Gegenstand selbst': Die Bedeutung des fotografischen Bildes in der Begr\u00fcndung der bakteriologischen Krankheitsauffassung durch Robert Koch,\" in _Neue Wege in der Seuchengeschichte_ , ed. Martin Dinges and Thomas Schlich (Stuttgart: Steiner, 1995), 143\u2013174. See also Olaf Breidbach, \"Representation of the Microcosm: The Claim for Objectivity in 19th Century Scientific Microphotography,\" _Journal of the History of Biology_ 35 (2002): 221\u2013250.\n\n 18. On the similarities between the graphic method and cinema, see Lisa Cartwright, \"'Experiments of Destruction': Cinematic Inscriptions of Physiology,\" _Representations_ 40 (Fall 1992): 129\u2013152; and Cartwright, _Screening the Body: Tracing Medicine_ ' _s Visual Culture_ (Minneapolis: University of Minnesota Press, 1995).\n\n 19. On Germany's research infrastructure, see, e.g., Margit Sz\u00f6ll\u00f6si-Janze, \"Science and Social Space: Transformations in the Institutions of _Wissenschaft_ from the Wilhelmine Empire to the Weimar Republic,\" _Minerva_ 43 (2005): 339\u2013360.\n\n 20. Carl Cranz, \"\u00dcber einen ballistischen Kinematographen,\" _Deutscher Mechaniker-Zeitung_ 18 (15 September 1909): 173\u2013177. See also P. W. W. Fuller, \"Carl Cranz, His Contemporaries, and High-Speed Photography,\" _Proceedings of SPIE_ , no. 5580, 26th International Congress on High-Speed Photography and Photonics (25 March 2005): 250\u2013260; and Wilhelm Pfeffer, \"Die Anwendung des Projectionsapparates zur Demonstration von Lebensvorg\u00e4ngen,\" _Jahrb\u00fccher wissenschaftliche Botanik_ 35 (1900): 711\u2013745. On Pfeffer, see esp. Oliver Gaycken, \"'The Swarming of Life': Moving Images, Education, and Views Through the Microscope,\" _Science in Context_ 24, no. 3 (September 2011): 361\u2013380; and Gaycken, \"The Secret Life of Plants: Visualizing Vegetative Movement, 1880\u20131903,\" _Early Popular Visual Culture_ 10, no. 1 (2012): 51\u201369.\n\n 21. The best overview of Comandon's life and work is B\u00e9atrice de Pastre and Thierry Lefebvre, eds., _Filmer la science, comprendre la vie: Le cinema de Jean Comandon_ (Paris: Centre national de la cin\u00e9matographie, 2012).\n\n 22. On Messter, see Christian Ilgner and Dietmar Linke, \"Filmtechnik: Vom Malteserkreuz zum Panzerkino,\" in _Oskar Messter: Filmpioneer der Kaiserzeit_ , ed. Martin Loiperdinger (Basel and Frankfurt: Stroemfeld\/Roter Stern, 1994), 93\u2013134, esp. 128\u2013134; as well as Frank Kessler, Sabine Lenk, and Martin Loiperdinger, eds., _Oskar Messter_ \u2014 _Erfinder und Gesch\u00e4ftsmann_ , KINtop Schriften 3 (Basel and Frankfurt: Stoemfeld\/Roter Stern, 1994). On Ernemann, see, e.g., the nod to the manufacturer in Hans Hennes,\"Die Kinematographie der Bewegungsst\u00f6rungen,\" _Die Umschau_ 15, no. 29 (1911): 605\u2013606; as well as Hanns G\u00fcnther, \"Mikrokinematographische Aufnahmeapparate,\" _Film and Lichtbild_ 1, no. 1 (1912): 4\u20136; 1, no. 2 (1912): 13\u201314.\n\n 23. On popular scientific cinema, see Thierry Lefebvre, \"The Scientia Production (1911\u20131914): Scientific Popularization Through Pictures,\" _Griffithiana_ no. 47 (May 1993): 137\u2013153; Oliver Gaycken, \"'A Drama Unites Them in a Fight to the Death': Some Remarks on the Flourishing of a Cinema of Scientific Vernacularization in France, 1909\u20131914,\" _Historical Journal of Film, Radio and Television_ 22, no. 3 (2002): 353\u2013374; and Gaycken, _Devices of Curiosity: Early Cinema and Popular Science_ (Oxford and New York: Oxford University Press, 2015). For an excellent account of popular science films in the United Kingdom, see Boon, _Films of Fact_. On the entwinement of scientific experiment, projection, popular science, and Victorian physics, see Simon Schaffer, \"Transport Phenomena: Space and Visibility in Victorian Physics,\" _Early Popular Visual Culture_ 10, no. 1 (February 2012): 71\u201391. On popularization in science in general, see Roger Cooter and Stephen Pumfrey, \"Separate Spheres and Public Places: Reflections on the History of Science Popularization and Science in Popular Culture,\" _History of Science_ 32, no. 97 (1994): 237\u2013267.\n\n 24. Readers were asked to write the editor for details; see \"Verzeichnis wissenschaftlich und technisch wertvoller Films,\" _Film und Lichtbild_ 1, no. 2 (1912): 16; or \"An unsere Abonnenten!\" _Film und Lichtbild_ 2, no. 5 (1913): 84.\n\n 25. For reports of screenings, see \"Wissenschaft und Lichtspiele,\" _Bild und Film_ 1, no. 2 (1912): 49\u201350; \"Kino und Wissenschaft,\" _Bild und Film_ 1, no. 2 (1912): 55; W. Thielemann, \"Kinematographie und biologische Forschung,\" _Bild und Film_ 3, no. 7 (1913\/1914): 171\u2013172; and \"Wissenschaftliche Abende,\" _Film und Lichtbild_ 1, no. 3 (1912): 30\u201331.\n\n 26. \"Ein neues wissenschaftliches Kino,\" _Film und Lichtbild_ 1, no. 5 (1912): 62. It is possible that the Fata Morgana was the only one of its kind.\n\n 27. See Thierry Lefebvre, _La chair et le celluloid: Le cin\u00e9ma chirurgical du Docteur Doyen_ (Brionne: Jean Doyen \u00e9diteur, 2004), for a discussion of the controversy surrounding the theft and possible unauthorized exhibition of Parisian doctor Eug\u00e8ne-Louis Doyen's surgical films in the early 1900s. See also chap. 2 in this volume.\n\n 28. For example, Wilhelm Richter, a Berlin teacher and school reformer, often wrote on scientific cinema in the popular press, cheering all efforts to bring new views to public perception. See \"Der Kinematograph als naturwissenschaftliches Anschauungsmittel,\" _Naturwissenschaftliche Wochenschrift_ 12, no. 52 (28 December 1913): 817\u2013820.\n\n 29. On the use of film in university teaching, see Franz Bergmann, \"Der Kinematograph im Hochschulunterricht,\" _Bild und Film_ 2, no. 2 (1912\/1913): 48; Wilhelm Richter, \"Hochschulkinematographie,\" _Bild und Film_ 2, nos. 11\/12 (1912\/1913): 253\u2013257; and L. Segmiller, \"Das Skizzieren nach Lichtbildern bei Tageslicht und k\u00fcnstlicher Beleuchtung,\" _Film und Lichtbild_ 1, no. 4 (1912): 35\u201339.\n\n 30. One survey of the contemporary use of microcinematography notes, \"Films of the latter [Ernst Sommerfeldt] are available commercially from Ernemann and depict crystallographic phenomenon,\" so apparently whether a research film made it into theaters depended on both the researcher's willingness and the manufacturer's evaluation of its popular appeal. On this example, see Ernst Sommerfeldt, \"\u00dcber fl\u00fcssige und scheinbar lebende Kristalle; mit kinematographischen Projektionen,\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 79, part 2 (1907): 202. The survey of microcinematography is Engelhard Wychgram, \"Aus optischen und mechanischen Werkst\u00e4tten IV,\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und f\u00fcr mikroskopishe Technik_ 28 (1911): 337\u2013361, esp. 351\u2013361.\n\n 31. On \"mechanical objectivity,\" see Lorraine Daston and Peter Galison, \"The Image of Objectivity,\" _Representations_ 40 (Fall 1992): 81\u2013128; and Daston and Galison, _Objectivity_ (New York: Zone, 2007).\n\n 32. On the difference between instruments for experimentation and for demonstration, see Thomas L. Hankins and Robert J. Silverman, \"The Magic Lantern and the Art of Demonstration,\" in _Instruments and the Imagination_ (Princeton, N.J.: Princeton University Press, 1995), 37\u201371.\n\n 33. Bruno Latour, \"Visualization and Cognition: Thinking with Eyes and Hands,\" _Knowledge and Society: Studies in the Sociology of Culture Past and Present_ 6 (1986): 1\u201340, here 5. Brian Winston has also considered Latour's work in relation to film: \"The Documentary Film as Scientific Inscription,\" in _Theorizing Documentary_ , ed. Michael Renov (London and New York: Routledge, 1993), 37\u201357.\n\n 34. Latour, \"Visualization and Cognition,\" 7 (emphasis in original).\n\n 35. See esp. Nicolas Rasmussen, _Picture Control: The Electron Microscope and the Transformation of Biology in America, 1940\u20131960_ (Stanford, Calif.: Stanford University Press, 1997); Bernike Pasveer, \"Knowledge of Shadows: The Introduction of X-Ray Images in Medicine,\" _Sociology of Health and Illness_ 11, no. 4 (December 1989): 361\u2013381; and Edward Yoxen, \"Seeing with Sound: A Study of the Development of Medical Images,\" in _The Social Construction of Technological Systems: New Directions in the Sociology and History of Technology_ , ed. Wiebe E. Bijker, Thomas P. Hughes, and Trevor J. Pinch (Cambridge, Mass.: MIT Press, 1987), 281\u2013303.\n\n 36. Anne Harrington, _Reenchanted Science: Holism in German Culture from Wilhelm II to Hitler_ (Princeton, N.J.: Princeton University Press, 1996). For an account of earlier debates about mechanistic approaches in science, see Peter Hanns Reill, _Vitalizing Nature in the Enlightenment_ (Berkeley: University of California Press, 2005).\n\n 37. Henri Bergson, _Creative Evolution_ , trans. Arthur Mitchell (New York: Random House, 1944). Hereafter cited parenthetically. Bergson indicates in the opening footnote of chap. 4 that he began thinking about science and film during his 1902\u20131903 course on the \"History of the Idea of Time\" at the Coll\u00e8ge de France.\n\n 38. The mechanistic approach to biology was represented in Germany by such \"biophysicists\" as Hermann von Helmholtz and Emil Du Bois-Reymond, who hoped to demonstrate that life operated under the same physical and chemical laws as other phenomena. See Helmholtz's _\u00dcber die Erhaltung der Kraft_ (Berlin: Reimer, 1847) for a comparison of muscles and mechanics, or Du Bois-Reymond's _Untersuchungen \u00fcber thierische Elektricit\u00e4t_ (Berlin: Reimer, 1848\u20131884) for an exploration of life's basic physical forces.\n\n 39. Henri Bergson, _An Introduction to Metaphysics_ , trans. T. E. Hulme (New York: Macmillan, 1955), 52.\n\n 40. Bergson, _Metaphysics_ , 51.\n\n 41. At this point we should note the influence of Bergson's philosophy on film theory from the work of Jean Epstein to Gilles Deleuze. For Epstein, see representative selections in _French Film Theory and Criticism_ , vols. 1 and 2, ed. Richard Abel (Princeton, N.J.: Princeton University Press, 1988); for Deleuze, see _Cinema 1: The Movement-Image_ and _Cinema 2: The Time-Image_ , trans. Hugh Tomlinson and Barbara Habberjam (Minneapolis: University of Minnesota, 1986 and 1989).\n\n 42. Michel Georges-Michel, \"Henri Bergson nous parle au cin\u00e9ma,\" _Le Journal_ (20 February 1914): 7; translated by Louis-Georges Schwartz as \"Henri Bergson Talks to Us About Cinema,\" _Cinema Journal_ 50, no. 3 (Spring 2011): 79\u201382, here 81. See also Frank Kessler's German translation and discussion of this article: \"Henri Bergson und die Kinematographie,\" _KINtop_ 12 (2003): 12\u201316.\n\n 43. I want to thank Paula Amad, whose doctoral dissertation \"Archiving the Everyday: A Topos in French Film History, 1895\u20131931\" (University of Chicago, 2002) led me in this direction. See also Amad's _Counter-Archive: Film, The Everyday, and Albert Kahn's_ Archives de la Plan\u00e8te (New York: Columbia University Press, 2010). For more on Bergson's understanding of movement, see Jimena Canales, \"Movement Before Cinematography: The High-Speed Qualities of Sentiment,\" _Journal of Visual Culture_ 5, no. 3 (December 2006): 275\u2013294.\n\n 44. Walter Benjamin, \"On Some Motifs in Baudelaire,\" in _Walter Benjamin: Selected Writings_ , vol. 4, _1938\u20131940_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott and others (Cambridge, Mass.: Belknap, 2003), 313\u2013355, here 314.\n\n 45. This is not to say that Bergson had no effect in Germany. Georg Simmel, e.g., was impressed and influenced by Bergson. See Gregor Fitzi, _Soziale Erfahrung und Lebensphilosophie. Georg Simmels Beziehung zu Henri Bergson_ (Konstanz, Germany: UVK Verlagsgesellschaft, 2002). For Bergson's reception by such thinkers as Max Scheler, Roman Ingarden, Hans Driesch, Max Horkeimer, and others, see Rudolf W. Meyer, \"Bergson in Deutschland. Unter besonderer Ber\u00fccksichtigung seiner Zeitauffassung,\" in _Studien zum Zeitproblem in der Philosophie des 20. Jahrhunderts_ , ed. Rudolf W. Meyer et al. (Munich: Alber, 1982), 10\u201364; and G\u00fcnther Pflug, \"Die Bergson-Rezeption in Deutschland,\" _Zeitschrift f\u00fcr philosophische Forschung_ 45, no. 2 (April\u2013June 1991): 257\u2013266.\n\n 46. Cited in Michael Ermarth, _Wilhelm Dilthey: The Critique of Historical Reason_ (Chicago: University of Chicago Press, 1978), 24.\n\n 47. Harrington, _Reenchanted Science_ , 27.\n\n 48. Wilhelm Dilthey, \"The Construction of the Historical World in the Human Studies,\" in _Selected Writings_ , ed., trans., and with an introduction by H. P. Rickman (Cambridge: Cambridge University Press, 1976), 168\u2013245, here 181.\n\n 49. My discussion of the science of work is indebted to Anson Rabinbach, _The Human Motor: Energy, Fatigue, and the Origins of Modernity_ (Berkeley and Los Angeles: University of California Press, 1990). Hereafter cited parenthetically. Closely related to this idea of fatigue was the modern notion of \"nervousness\": see Andreas Killen, _Berlin Electropolis: Shock, Nerves, and German Modernity_ (Berkeley: University of California Press, 2006).\n\n 50. In addition to Harrington's _Reenchanted Science_ , a good overview of the debate, from the vitalist's point of view, is Frederick Burwick and Paul Douglass, eds., _The Crisis in Modernism: Bergson and the Vitalist Controversy_ (Cambridge: Cambridge University Press, 1992). With regard to cinema and vitalism, see also Inga Pollmann, \"Cinematic Vitalism: Theories of Life and the Moving Image\" (PhD diss., University of Chicago, 2011).\n\n 51. Rabinbach, _Human Motor_ , 181; see also Ernest Solvay, _Notes sur le productivisme et le comptabilisme_ (Brussels: Misch and Thron, 1900) 2: 323. On the institute, see Daniel Warnotte, _Ernest Solvay et l_ ' _Institut de Sociologie: Contribution \u00e0 l_ ' _histoire de l_ ' _\u00e9nerg\u00e9tique sociale_ (Brussels: Bruylant, 1946).\n\n 52. Rabinbach, _Human Motor_ , 134; see also Angelo Mosso, _Fatigue_ , trans. Margaret Drummond and W. B. Drummond (New York: Putnam, 1904); and Karen J. Fleckenstein, \"The Mosso Plethysmograph in 19th-Century Physiology,\" _Medical Instrumentation_ 18, no. 6 (November\u2013December 1984): 330\u2013331.\n\n 53. See Leo Koenigsberger, _Hermann von Helmholtz_ (Braunschweig, Germany: Viewig, 1911); as well as David Cahan, ed., _Hermann von Helmholtz and the Foundations of Nineteenth-Century Science_ (Berkeley and Los Angeles: University of California Press, 1993).\n\n 54. See especially Greg Myers, \"Nineteenth-Century Popularizations of Thermodynamics and the Rhetoric of Social Prophecy,\" in _Energy & Entropy: Science and Culture in Victorian Britain_, ed. Patrick Brantlinger (Bloomington: Indiana University Press, 1988), 307\u2013338; Ed Block Jr., \"T. H. Huxley's Rhetoric and the Popularization of Victorian Scientific Ideas, 1854\u20131874,\" in Brantlinger, _Energy & Entropy_, 205\u2013228; and _Degeneration: The Dark Side of Progress_ , ed. J. Edward Chamberlin and Sander L. Gilman (New York: Columbia University Press, 1985). The concept of degeneration will receive much more attention in chap. 2 of this volume.\n\n 55. Recent studies of Marey include Marta Braun, _Picturing Time: The Work of \u00c9tienne-Jules Marey_ (Chicago: University of Chicago Press, 1992); and Francois Dagognet, _\u00c9tienne-Jules Marey: A Passion for the Trace_ , trans. Robert Galeta with Jeanine Herman (New York: Zone, 1992). See also Marey's _La methode graphique dans les sciences \u00e9xperimentales et principlement en physiologie et en m\u00e9decine_ (Paris: Masson, 1885); _Movement_ , trans. Eric Pritchard (London: Heinemann, 1895); and _La chronophotographie_ (Paris: Gauthier-Villars, 1899).\n\n 56. \u00c9tienne-Jules Marey, \"\u00c9tudes sur la marche de l'homme,\" _Revue Militaire de M\u00e9decine et de Chirurgie_ 1 (1880): 244\u201346, cited in Rabinbach, _Human Motor_ , 116.\n\n 57. For the culmination of this approach, see Frederick Winslow Taylor, _The Principles of Scientific Management_ (1911; New York: Norton, 1947); and Frank B. Gilbreth, _Motion Study: A Method for Increasing the Efficiency of the Workman_ (New York: Van Nostrand, 1911). On the use of motion pictures in this program, see Richard Lindstrom, \"'They All Believe They Are Undiscovered Mary Pickfords': Workers, Photography, and Scientific Management,\" _Technology and Culture_ 41, no. 4 (2000): 725\u2013751; Ram\u00f3n Reichert, \"Der Arbeitstudienfilm: Eine verborgene Geschichte des Stummfilms,\" _medien & zeit: Kommunikation in Vergangenheit und Gegenwart_ 5 (2002): 46\u201357; Philipp Sarasin, \"Die Rationalisierung des K\u00f6rpers: \u00dcber 'Scientific Management' und 'biologische Rationalisierung'\" in _Geschichtswissenschaft und Diskursanalyse_ (Frankfurt: Suhrkamp, 2003), 61\u201399; Elspeth H. Brown, _The Corporate Eye: Photography and the Rationalization of American Commercial Culture, 1884\u20131929_ (Baltimore: Johns Hopkins University Press, 2005); Florian Hoof, \"'The One Best Way': Bildgebende Verfahren der \u00d6konomie und die Innovation der Managementtheorie Nach 1860,\" _montage\/AV: Zeitschrift f\u00fcr Theorie und Geschichte audiovisueller Kommunikation_ 15, no. 1 (2006): 123\u2013138; and Scott Curtis, \"Images of Efficiency: The Films of Frank B. Gilbreth,\" in _Films That Work: Industrial Film and the Productivity of Media_ , ed. Vinzenz Hediger and Patrick Vonderau (Amsterdam: Amsterdam University Press, 2009), 85\u201399.\n\n 58. This tradition goes back as far as 1836, with the publication of the work of Wilhelm Weber, who with his brothers Ernst and Eduard investigated human motion using a variety of innovative visual and graphic technologies. See _Mechanik der menschlichen Gehwerkzeuge: Eine anatomisch-physiologische Untersuchung_ , in _Wilhelm Weber_ ' _s Werke_ , vol. 6, ed. Der k\u00f6niglichen Gesellschaft der Wissenschaften zu G\u00f6ttingen (Berlin: Springer, 1894), 1\u2013305. (Wilhelm Braune and Otto Fischer were closely involved in the selection and editing of Weber's works.) For a comprehensive history of human motion studies, see Andreas Mayer, _Wissenschaft vom Gehen. Die Erforschung der Bewegung im 19. Jahrhundert_ (Frankfurt: Fischer, 2013).\n\n 59. Braune and Fischer, _The Human Gait_ , 18.\n\n 60. Braune and Fischer, _The Human Gait_ , 4.\n\n 61. Rabinbach, _Human Motor_ , 189; see also Nathan Zuntz and Wilhelm Schumberg, _Studien zu einer Physiologie des Marsches_ (Berlin: Hirschwald, 1901).\n\n 62. Rabinbach, _Human Motor_ , 104; see also \u00c9tienne-Jules Marey, _Animal Mechanism: A Treatise on Terrestrial and Aerial Locomotion_ (New York: Appleton, 1874).\n\n 63. Rabinbach, _Human Motor_ , 144; see also Wilhelm Weichardt, \"Erm\u00fcdungsbek\u00e4mpfung durch Antikenotoxin,\" _Deutsche milit\u00e4r\u00e4rztliche Zeitschrift_ 42, no. 1 (5 January 1913): 12\u201313.\n\n 64. Rabinbach, _Human Motor_ , 135; see also Mosso, _Fatigue_ , 121.\n\n 65. \"Novel Uses for Moving Pictures,\" _Moving Picture World_ 1, no. 3 (23 March 1907): 39\u201340.\n\n 66. Michel Foucault, _Discipline and Punish_ , trans. Alan Sheridan (New York: Vintage, 1979), 136.\n\n 67. Foucault, _Discipline and Punish_ , 137.\n\n 68. Ian Hacking, _Representing and Intervening: Introductory Topics in the Philosophy of Natural Science_ (Cambridge: Cambridge University Press, 1983).\n\n 69. Michael Lynch, \"Discipline and the Material Form of Images: An Analysis of Scientific Visuality,\" _Social Studies of Science_ 15, no. 1 (February 1985): 43\u201344 (emphasis in original). Daston and Galison, in their work on objectivity (cited in note 31), call it a \"working object,\" which has become the more common term in the history and sociology of science.\n\n 70. Some of the most notable early sociological studies of the scientific process include Harold Garfinkel, Michael Lynch, and Eric Livingston, \"The Work of a Discovering Science Construed with Materials from the Optically Discovered Pulsar,\" _Philosophy of the Social Sciences_ 11, no. 2 (June 1981): 131\u2013158; Karin Knorr-Cetina, _The Manufacture of Knowledge: An Essay on the Constructivist and Contextual Nature of Science_ (Oxford: Pergamon, 1981); Bruno Latour and Steve Woolgar, _Laboratory Life: The Social Construction of Scientific Facts_ (London and Beverly Hills, Calif.: Sage, 1979); and Latour, _Science in Action_ (Cambridge, Mass.: Harvard University Press, 1987).\n\n 71. Chapter 1 appeared as \"Versuche am unbelasteten und belasteten Menschen,\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 21, no. 4 (1895): 151\u2013322; chap. 2 as \"Die Bewegung des Gesamtschwerpunktes und die \u00e4u\u00dferen Kr\u00e4fte\" in 25, no. 1 (1899): 1\u2013130; chap. 3 as \"Betrachtungen \u00fcber die weiteren Ziele der Untersuchung und \u00dcberblick \u00fcber die Bewegungen der unteren Extremit\u00e4ten\" in 26, no. 3 (1900): 85\u2013170; chap. 4 as \"\u00dcber die Bewegung des Fu\u00dfes und die auf denselben einwirkenden Kr\u00e4fte\" in 26, no. 7 (1901): 467\u2013556; chap. 5 as \"Die Kinematik des Beinschwingens\" in 28, no. 5 (1903): 319\u2013418; and chap. 6 as \"\u00dcber den Einflu\u00df der Schwere und der Muskeln auf die Schwingungsbewegung des Beins\" in 28, no. 7 (1904): 531\u2013617.\n\n 72. Herman J. Wohlring, review of _The Human Gait_ , _Human Movement Science_ 8, no. 1 (February 1989): 79\u201383. Aerial photography, the counterpart to close-range photogrammetry, also developed from military applications. See Teodor J. Blachut and Rudolf Burkhardt, _Historical Development of Photogrammetric Methods and Instruments_ (Falls Church, Va.: American Society for Photogrammetry and Remote Sensing, 1989); and Paul Virilio, _War and Cinema: The Logistics of Perception_ , trans. Patrick Camiller (London: Verso, 1989).\n\n 73. Eadweard Muybridge, _Animal Locomotion: An Electro-Photographic Investigation of Consecutive Phases of Animal Movement_ (Philadelphia: University of Pennsylvania, 1887); or _Muybridge_ ' _s Complete Human and Animal Locomotion_ (New York: Dover, 1979); and Albert Londe, _Photographie m\u00e9dicale_ (Paris: Gauthiers-Villars, 1893). There also seems to have been surprisingly little crossover between Braune and Fischer's work and that of other German chronophotographers, such as Ottomar Ansch\u00fctz and Ernst Kohlrausch, but that is primarily a disciplinary issue; as the quotation above indicates, Braune and Fischer saw Ansch\u00fctz's decision to pursue the popular potential of his devices, rather than any research applications, as a markedly different path. A potentially interesting overlap might be the German Ministry of War, with which Ansch\u00fctz worked in 1884, but more research needs to be done here. On Ansch\u00fctz, see Deac Rossell, _Faszination der Bewegung: Ottomar Ansch\u00fctz zwischen Photographie und Kino_ , KINtop Schriften 6 (Frankfurt: Stroemfeld\/Roter Stern, 2001).\n\n 74. Braune and Fischer, _The Human Gait_ , 6. Hereafter cited parenthetically.\n\n 75. Lynch, \"Discipline and the Material Form of Images,\" 43.\n\n 76. Lynch, \"Discipline and the Material Form of Images,\" 42.\n\n 77. Edmund Husserl, _The Crisis of European Sciences and Transcendental Phenomenology_ , trans. David Carr (Evanston, Ill.: Northwestern University Press, 1970), 376.\n\n 78. Michael Lynch, \"The Externalized Retina: Selection and Mathematization in the Visual Documentation of Objects in the Life Sciences,\" in _Representation in Scientific Practice_ , ed. Michael Lynch and Steve Woolgar (Cambridge, Mass.: MIT Press, 1990), 153\u2013186, here 170.\n\n 79. Bergson, _Creative Evolution_ , 342 (emphasis in original).\n\n 80. Michaelis, _Research Films in Biology, Anthropology, Psychology, and Medicine_ , 371.\n\n 81. Bergson, _Creative Evolution_ , 238.\n\n 82. Jill Vance Buroker, \"Descartes on Sensible Qualities,\" _Journal of the History of Philosophy_ 29, no. 4 (October 1991): 585\u2013611.\n\n 83. Bergson, _Creative Evolution_ , 240 (emphasis in original).\n\n 84. An earlier, shorter version of this section appeared as \"Die kinematographische Methode. Das 'Bewegte Bild' und die Brownsche Bewegung,\" _montage\/AV: Zeitschrift f\u00fcr Theorie & Geschichte audiovisueller Kommunikation_ 14, no. 2 (2005): 23\u201343.\n\n 85. Roberto Maiocchi, \"The Case of Brownian Motion,\" _British Journal for the History of Science_ 23 (September 1990): 257\u2013283, here 257. See also Stephen G. Brush, \"A History of Random Processes: I. Brownian Movement from Brown to Perrin,\" _Archive for History of Exact Sciences_ 5, no. 1 (1968): 1\u201336; Mary Jo Nye, _Molecular Reality: A Perspective on the Scientific Work of Jean Perrin_ (New York: American Elsevier, 1972); Milton Kerker, \"Brownian Movement and Molecular Reality Prior to 1900,\" _Journal of Chemical Education_ 51, no. 12 (December 1974): 764\u2013768; Peter Clark, \"Atomism Versus Thermodynamics,\" in _Method and Appraisal in the Physical Sciences_ , ed. Colin Howson (Cambridge: Cambridge University Press, 1976), 41\u2013106; Brush, _Statistical Physics and the Atomic Theory of Matter from Boyle and Newton to Landau and Onsager_ (Princeton, N.J.: Princeton University Press, 1983), 79\u2013104; and John Stachel, \"Einstein on Brownian Motion,\" in _The Collected Papers of Albert Einstein_ , vol. 2, _The Swiss Years: Writings, 1900\u20131909_ , ed. John Stachel (Princeton, N.J.: Princeton University Press, 1989), 206\u2013222. On the modern implications and theoretical offspring of Brownian motion, see Erwin Frey and Klaus Kroy, \"Brownian Motion: A Paradigm of Soft Matter and Biological Physics,\" _Annalen der Physik_ 14, nos. 1\u20133 (February 2005): 20\u201350.\n\n 86. Clark, \"Atomism Versus Thermodynamics,\" 42.\n\n 87. For Mach's objections to the kinetic theory, see Ernst Mach, _Die Principien der W\u00e4rmlehre: Historisch-kritisch Entwickelt_ (Leipzig: Barth, 1896), 428\u2013429; for a discussion of Mach and the historical context of the debate, see Stephen G. Brush, _The Kind of Motion We Call Heat: A History of the Kinetic Theory of Gases in the 19th Century_ (Amsterdam and New York: North-Holland and American Elsevier, 1976), 274\u2013299; for more on Mach, see John T. Blackmore, _Ernst Mach: His Work, Life, and Influence_ (Berkeley: University of California Press, 1972). For Ostwald and other anti-atomists, see Mary Jo Nye, _Molecular Reality_ ; and Nye, \"The Nineteenth-Century Atomic Debates and the Dilemma of an 'Indifferent Hypothesis,'\" _Studies in History and Philosophy of Science_ 7, no. 3 (1976): 245\u2013268.\n\n 88. Brush, _Statistical Physics_ , 97.\n\n 89. Louis-Georges Gouy, \"Le mouvement brownien et les mouvements mol\u00e9culaires,\" _Revue g\u00e9n\u00e9rale des sciences pures et appliqu\u00e9es_ 6, no. 1 (15 January 1895): 1\u20137.\n\n 90. Maiocchi, \"The Case of Brownian Motion,\" 260.\n\n 91. Felix M. Exner, \"Notiz zu Brown's Molecularbewegung,\" _Annalen der Physik_ 2, no. 8 (1900): 843\u2013847.\n\n 92. Exner, \"Notiz,\" 844\u2013845.\n\n 93. Albert Einstein, \"On the Movement of Small Particles Suspended in a Stationary Liquid Demanded by the Molecular-Kinetic Theory of Heat,\" in _Investigations on the Theory of the Brownian Movement_ , ed. R. F\u00fcrth, trans. A. D. Cowper (New York: Dover, 1956), 1\u201318, here 1\u20132.\n\n 94. The best explanation of Einstein's use of Brownian motion as a statistical system is Martin J. Klein, \"Fluctuations and Statistical Physics in Einstein's Early Work,\" in _Albert Einstein: Historical and Cultural Perspectives_ , ed. Gerald Holton and Yehuda Elkana (Princeton, N.J.: Princeton University Press, 1982), 39\u201358. Klein remarks, \"Einstein had _invented_ the Brownian motion. To say anything less, to describe this paper in the usual way, that is, as his _explanation_ of the Brownian motion, is to undervalue it\" (47, emphasis in original). See also J\u00fcrgen Renn, \"Einstein's Invention of Brownian Motion,\" _Annalen der Physik_ 14, supplement (2005): 23\u201337.\n\n 95. Maiocchi, \"The Case of Brownian Motion,\" 260 (emphasis in original).\n\n 96. Nye, _Molecular Reality_ , 111.\n\n 97. Significantly, Brownian motion described an observable system in liquid, whereas up to this point discussions of random systems and fluctuations had focused only on gases. That is, the kinetic theory to this point applied only to gases, not liquids or solids. By removing that particular restriction in his focus on Brownian motion, Einstein also raised the stakes of the debate over the existence of atoms.\n\n 98. Einstein further simplified the system by limiting his mathematical derivations to two dimensions, thereby only taking into account the horizontal displacement of particles. In this way, Einstein's theory corresponds to experimental practice in that the field of observation corresponds to the flat, two-dimensional field of the microscope.\n\n 99. Maiocchi, \"The Case of Brownian Motion,\" 263\u2013264 (emphasis in original).\n\n. Brush, \"A History of Random Processes,\" 22\u201323.\n\n. On the invention and use of the ultramicroscope, see David Cahan, \"The Zeiss Werke and the Ultramicroscope: The Creation of a Scientific Instrument in Context,\" in _Scientific Credibility and Technical Standards in 19th and Early 20th Century German and Britain_ , ed. Jed Z. Buchwald (Dordrecht, Netherlands: Kluwer Academic, 1996), 67\u2013115.\n\n. Victor Henri, \"\u00c9tudes cin\u00e9matographique des mouvements browniens,\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 146 (18 May 1908): 1024\u20131026; and Henri, \"Influence du milieu sur les mouvements browniens,\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 147 (6 July 1908): 62\u201365. For a later use of motion pictures to record the distribution of particles in a gaseous system, which builds upon Seddig's and Henri's work, see Richard Lorenz and W. Eitel, \"\u00dcber die \u00f6rtliche Verteilung von Rauchteilchen,\" _Zeitschrift f\u00fcr anorganische Chemie_ 87, no. 1 (12 May 1914): 357\u2013374.\n\n. For a critique of Henri's results, see Aim\u00e9 Cotton, \"Recherches r\u00e9centes sur les mouvements browniens,\" _La Revue du Mois_ 5 (10 June 1908): 737\u2013741.\n\n. The most influential paper of many is Jean Perrin, \"Mouvement brownien et mol\u00e9cules,\" _Annales de chimie et de physique_ 18 (September 1909): 1\u2013114, translated by F. Soddy as \"Brownian Movement and Molecular Reality,\" in _The Question of the Atom_ , ed. Mary Jo Nye (Los Angeles, Calif.: Tomash, 1984), 507\u2013601. The best commentary on Perrin is Mary Jo Nye, _Molecular Reality_. For Perrin's own discussion of Henri, Seddig, and others, see _Atoms_ , trans. D. L. Hammick, 2d English ed. rev. (London: Constable, 1923), 109\u2013133. On Perrin's visualization techniques, see Charlotte Bigg, \"Evident Atoms: Visuality in Jean Perrin's Brownian Motion Research,\" _Studies in History and Philosophy of Science_ 39 (2008): 312\u2013322. See also Bigg, \"A Visual History of Jean Perrin's Brownian Motion Curves,\" in _Histories of Scientific Observation_ , ed. Lorraine Daston and Elizabeth Lunbeck (Chicago: University of Chicago Press, 2011), 156\u2013180.\n\n. Nye, _Molecular Reality_ , 97.\n\n. Seddig, \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown'schen Molekularbewegung,\" 12 (emphasis in original).\n\n. Seddig, \"Ueber Abh\u00e4ngigkeit,\" 185.\n\n. Marey came upon this solution as early as 1891. See Braun, _Picturing Time_ , 166.\n\n. Einstein to Jakob Laub, 30 July 1908, quoted in Stachel, \"Einstein on Brownian Motion,\" 220. We should also note that in 1907 there was another attempt to verify Einstein's theories: Theodor Svedberg, _Studien zur Lehre von der Kolloiden L\u00f6sungen_ (Uppsala: Akademische Buchdruckerei Edv. Berling, 1907). Einstein was not so kind: \"The errors in Svedberg's method of observation and also in his theoretical treatment became clear to me at once. I wrote a minor correction at the time, which only addressed the worst, as I couldn't bring myself to detract from Mr. S's great pleasure in his work.\" (Einstein to Jean Perrin, 11 November 1909, quoted in Stachel, \"Einstein on Brownian Motion,\" 220).\n\n. Maryan Smoluchowski, \"Essai d'une th\u00e8orie cin\u00e8tique du movement brownien et des milieux troubles,\" _Krakau Anzeiger_ 7 (1906): 585\u2013586, quoted in Maiocchi, \"The Case of Brownian Motion,\" 264.\n\n. Jean Perrin, _Les atoms_ , 4th ed. rev. (Paris: Librarie F\u00e9lix Alcan, 1914), 157; _Atoms_ , 2d English ed. rev., 109\u2013110 (emphasis in original). On Jean Comandon's Brownian motion films, see Jean Comandon with Albert Dastre, \"Cin\u00e9matographie, \u00e0 l'ultra-microscope, de microbes vivants et des particules mobiles,\" _Comptes rendus hebdomadaires des s\u00e9ances de l'Acad\u00e9mie des Sciences_ 149 (22 November 1909): 938\u2013941. Perrin showed the films of Henri and Comandon to the Soci\u00e9t\u00e9 des Amis de l'Universit\u00e9 de Paris in 1911: Jean Perrin, \"La realit\u00e9 des molecules,\" _Revue Scientifique_ 49, no. 2 (1911): 774\u2013784, quoted in Nye, _Molecular Reality_ , 153. See also Hannah Landecker, \"Cellular Features: Microcinematography and Film Theory,\" _Critical Inquiry_ 31, no. 4 (2005): 903\u2013937.\n\n. On the debate in the 1920s between Bergson and Einstein regarding this category mistake, see Jimena Canales, \"Einstein, Bergson, and the Experiment That Failed: Intellectual Cooperation at the League of Nations,\" _Modern Language Notes_ 120, no. 5 (2006): 1168\u20131191.\n\n. Louis de Broglie, \"The Concepts of Contemporary Physics and Bergson's Ideas on Time and Motion,\" in _Bergson and the Evolution of Physics_ , ed. and trans. P. A. Y. Gunter (Knoxville: University of Tennessee Press, 1969), 45\u201362, here 49.\n\n. Suzanne Guerlac, _Thinking in Time: An Introduction to Henri Bergson_ (Ithaca, N.Y.: Cornell University Press, 2006), 19.\n\n. Bergson was not alone in this stance; even Nobel Prize\u2013winning Belgian physicist and chemist Ilya Prigogine argued that physics should account for the irreversibility of time. See, e.g., _From Being to Becoming: Time and Complexity in the Physical Sciences_ (San Francisco: Freeman, 1980); and with Isabelle Stengers, _Order Out of Chaos: Man_ ' _s New Dialogue with Nature_ (Boulder, Colo.: New Science Library, 1984); and _The End of Certainty: Time, Chaos, and the New Laws of Nature_ (New York: Free Press, 1997). For an introduction to this contradiction between time in physics and time as it is experienced, see David Z. Albert, _Time and Chance_ (Cambridge, Mass.: Harvard University Press, 2000).\n\n. For different interpretations of the instant and the point in photography and cinema, see Thierry de Duve, \"Time Exposure and Snapshot: The Photograph as Paradox,\" _October_ 5 (Summer 1978): 113\u2013125; and Doane, _The Emergence of Cinematic Time_ , 208\u2013230.\n\n. Henri Bergson, _Matter and Memory_ , trans. Nancy Margaret Paul and W. Scott Palmer (Cambridge, Mass.: Zone, 1988), 247 (emphasis in original).\n\n. Bergson, _Matter and Memory_ , 250 (emphasis in original).\n\n. Deleuze, _Cinema 1_.\n\n. Seddig, \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown'schen Molekularbewegung,\" 36\u201337.\n\n. Marey, _Movement_.\n\n. _Lucien Bull_ (Brussels: Hayez, 1967), 11.\n\n. For a representative sampling, see Charles Fran\u00e7ois-Franck, \"La chronophotographie simultan\u00e9e du coeur et des courbes cardiographiques chez les mammif\u00e8res,\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 54 (8 November 1902): 1193\u20131197; \"Note sur quelques points de technique relatifs \u00e0 la photographie et \u00e0 la chronophotographie avec le magn\u00e9sium \u00e0 deflagration lente,\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 55 (5 December 1903): 1538\u20131540; \"\u00c9tudes graphiques et photographiques de m\u00e9canique respiratoire compar\u00e9e,\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 61 (28 July 1906): 174\u2013176; and \"D\u00e9monstrations de microphotographie instantan\u00e9e et de chronomicrophotographie,\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 62 (25 May 1907): 964\u2013967. Although many of his citations are incorrect, Thierry Lefebvre, \"Contribution \u00e0 l'histoire de la microcin\u00e9matographie: De Fran\u00e7ois-Franck \u00e0 Comandon,\" _1895_ 14 (June 1993): 35\u201343, is an essential introduction.\n\n. L. Chevroton and F. Vl\u00e8s, \"La cin\u00e9matique de la segmentation de l'oeuf et la chronophotographie du d\u00e9veloppement de l'Oursin,\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 149 (8 November 1909): 806\u2013809.\n\n. Henri, \"\u00c9tudes cin\u00e9matographique des mouvements browniens.\"\n\n. Isabelle do O'Gomes, \"L'oeuvre de Jean Comandon,\" in _Le cin\u00e9ma et la science_ , ed. Alexis Martinet (Paris: CNRS \u00c9ditions, 1994), 78\u201385.\n\n. Julius Ries, \"Kinematographie der Befruchtung und Zellteilung,\" _Archiv f\u00fcr Mikroskopische Anatomie und Entwicklungsgeschichte_ 74 (1909): 1\u201331.\n\n. Henri B\u00e9nard, \"Les tourbillons cellulaires dans une nappe liquide. II. Proc\u00e9d\u00e9s m\u00e9caniques et optiques d'examens, lois num\u00e9riques des ph\u00e9nom\u00e8nes,\" _Revue g\u00e9n\u00e9rale des sciences pures et appliqu\u00e9es_ 11 (1900): 1309\u20131328; and B\u00e9nard, \"Formation de centres de giration \u00e0 l'arri\u00e8re d'un obstacle en movement,\" _Comptes rendus de l_ ' _Acad\u00e9mie des Sciences_ 147 (1908): 839\u2013842. See also Jos\u00e9 Eduardo Wesfreid, \"Scientific Biography of Henri B\u00e9nard (1874\u20131939),\" in _Dynamics of Spatio-Temporal Cellular Structures: Henri B\u00e9nard Centenary Review_ , ed. Innocent Mutabazi, Jos\u00e9 Eduardo Wesfreid, and \u00c9tienne Guyon (New York: Springer, 2006), 9\u201340; and David Aubin, \"'The Memory of Life Itself': B\u00e9nard's Cells and the Cinematography of Self-Organization,\" _Studies in History and Philosophy of Science_ 39, no. 3 (2008): 359\u2013369.\n\n. H. Siedentopf and E. Sommerfeldt, \"\u00dcber die Anfertigung kinematographischer Mikrophotographien der Kristallisationserscheinungen,\" _Zeitschrift f\u00fcr Elektrochemie_ 13, no. 24 (14 June 1907): 325\u2013326; and Siedentopf, \"\u00dcber ultramikroskopische Abbildung,\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und mikroskopische Technik_ 26 (1909): 391\u2013410.\n\n. A contemporary overview of Braus's career can be found in Walther Vogt's eulogy, \"Hermann Braus,\" _M\u00fcnchener medizinische Wochenschrift_ 72, no. 8 (20 February 1925): 304\u2013305. An assessment of his legacy in morphology is in Lynn K. Nyhart, \"Learning from History: Morphology's Challenges in Germany ca. 1900,\" _Journal of Morphology_ 252 (April 2002): 2\u201314.\n\n. A shorter version of this section appeared as \"Science Lessons,\" _Film History_ 25, nos. 1\u20132 (2013): 45\u201354.\n\n. My presentation of these theories and the history of this debate is indebted to Susan M. Billings's excellent survey, \"Concepts of Nerve Fiber Development, 1839\u20131930,\" _Journal of the History of Biology_ 4, no. 2 (Fall 1971): 275\u2013305.\n\n. Ross Granville Harrison, \"Further Experiments on the Development of Peripheral Nerves,\" _American Journal of Anatomy_ 5, no. 2 (31 May 1906): 121\u2013131. A brief overview of Harrison's career can be found in J. S. Nicholas, \"Ross Granville Harrison, 1870\u20131959,\" _Yale Journal of Biology and Medicine_ 32, no. 6 (June 1960): 407\u2013412.\n\n. Peter J. Taylor and Ann S. Blum, \"Pictorial Representation in Biology,\" _Biology and Philosophy_ 6, no. 2 (April 1991): 125\u2013134; and Nick Hopwood, \"Producing Development: The Anatomy of Human Embryos and the Norms of Wilhelm His,\" _Bulletin of the History of Medicine_ 74 (2000): 29\u201379.\n\n. Billings, \"Concepts,\" 293\u2013294.\n\n. Santiago Ram\u00f3n y Cajal, \"New Observations on the Development of Neuroblasts, with Comments on the Neurogenetic Hypothesis of Hensen-Held (1908),\" in _Studies on Vertebrate Neurogenesis_ , trans. Lloyd Guth (Springfield, Ill.: Thomas, 1960), 71\u201376, quoted in Billings, \"Concepts,\" 294. See also De Rijcke, \"Drawing Into Abstraction.\"\n\n. Hannah Landecker, \"New Times for Biology: Nerve Cultures and the Advent of Cellular Life in Vitro,\" _Studies in the History and Philosophy of Biological and Biomedical Sciences_ 33, no. 4 (2002): 667\u2013694, here 672.\n\n. Landecker, \"New Times,\" 673.\n\n. Landecker examines this formulation with regard to the rise of microcinematography in \"Creeping, Drinking, Dying: The Cinematic Portal and the Microscopic World of the Twentieth-Century Cell,\" _Science in Context_ 24, no. 3 (September 2011): 381\u2013416; and \"The Life of Movement: From Microcinematography to Live-Cell Imaging,\" _Journal of Visual Culture_ 11, no. 3 (December 2012): 378\u2013399.\n\n. Harrison, \"Further Experiments,\" 121.\n\n. Hermann Braus, \"Experimentelle Beitr\u00e4ge zur Frage nach der Entwickelung peripherer Nerven,\" _Anatomischer Anzeiger_ 26, nos. 17\/18 (1 April 1905): 433\u2013479.\n\n. Ross Granville Harrison, \"Experiments in Transplanting Limbs and Their Bearing Upon the Problems of the Development of Nerves,\" _Journal of Experimental Zoology_ 4, no. 2 (June 1907): 239\u2013281, here 241.\n\n. Ross Granville Harrison, \"Observations on the Living Developing Nerve Fiber,\" _Anatomical Record_ 1, no. 5 (1 June 1907): 116\u2013118, here 116. Harrison describes the method and its results more fully in \"The Outgrowth of the Nerve Fiber as a Mode of Protoplasmic Movement,\" _Journal of Experimental Zoology_ 9, no. 4 (December 1910): 787\u2013846.\n\n. Leo Loeb is known to have accomplished in vivo tissue culture as early as 1897. See Lewis Philip Rubin, \"Leo Loeb's Role in the Development of Tissue Culture,\" _Clio Medica_ 12, no. 1 (1977): 33\u201356. See also H. M. Carleton, \"Tissue Culture: A Critical Summary,\" _British Journal of Experimental Biology_ 1, no. 1 (October 1923): 131\u2013151.\n\n. On this experiment, see the announcements by Alexis Carrel and Montrose T. Burrows, including \"Cultivation of Adult Tissues and Organs Outside of the Body,\" _Journal of the American Medical Association_ 55, no. 16 (15 October 1910): 1379\u20131381; \"Cultivation of Sarcoma Outside of the Body: A Second Note,\" _Journal of the American Medical Association_ 55, no. 18 (29 October 1910): 1554; \"Human Sarcoma Cultivated Outside of the Body: A Third Note,\" _Journal of the American Medical Association_ 55, no. 20 (12 November 1910): 1732; and \"Cultivation of Tissues in Vitro and Its Technique,\" _Journal of Experimental Medicine_ 13, no. 3 (1 March 1911): 387\u2013396.\n\n. The definitive argument for this break in biological representation, to which I am indebted, is Hannah Landecker, _Culturing Life: How Cells Became Technologies_ (Cambridge, Mass.: Harvard University Press, 2007); see also her \"Technologies of Living Substance: Tissue Culture and Cellular Life in Twentieth Century Biomedicine\" (PhD diss., Massachusetts Institute of Technology, 1999).\n\n. This shift in the discipline's attention was not just a result of representational issues but also to a certain extent due to philosophical discomfort as a result of Bergson's critiques. For a good example of a biologist wrestling with these issues, albeit much later, see P. Lecomte du No\u00fcy, _Biological Time_ , with a foreword by Alexis Carrel (New York: Macmillan, 1937).\n\n. Billings, \"Concepts,\" 301\u2013302.\n\n. Braus, \"Mikro-Kino-Projektionen\"; this was reprinted in slightly revised form in _Wiener medizinische Wochenschrift_ 61, no. 44 (1911): 2809\u20132812.\n\n. Braus, \"Mikro-Kino-Projektionen,\" 472\u2013473.\n\n. Steven Shapin, \"Pump and Circumstance: Robert Boyle's Literary Technology,\" _Social Studies of Science_ 14, no. 4 (November 1984): 481\u2013520. See also Steven Shapin and Simon Schaffer, _Leviathan and the Air-Pump: Hobbes, Boyle, and the Experimental Life_ (Princeton, N.J.: Princeton University Press, 1985).\n\n. Braus, \"Mikro-Kino-Projektionen,\" 473.\n\n. Braus, \"Mikro-Kino-Projektionen,\" 474 (emphasis in original).\n\n. Braus, \"Mikro-Kino-Projektionen,\" 474.\n\n. Walter Benjamin, \"Little History of Photography,\" in _Walter Benjamin: Selected Writings_ , vol. 2, _1927\u20131934_ , ed. Michael W. Jennings, Howard Eiland, and Gary Smith, trans. Rodney Livingstone and others (Cambridge, Mass.: Belknap, 1999), 507\u2013530, here 512.\n\n. For another example of the statistical use of film frames, see Scott Curtis, \"'Tangible as Tissue': Arnold Gesell, Infant Behavior, and Film Analysis,\" _Science in Context_ 24, no. 3 (September 2011): 417\u2013442.\n\n2. BETWEEN OBSERVATION AND SPECTATORSHIP\n\n 1. Paul Val\u00e9ry, _Id\u00e9e Fixe_ , trans. David Paul (New York: Pantheon, 1965), 22.\n\n 2. For similar debates, see Michael Chanan, _The Dream That Kicks: The Prehistory and Early Years of Cinema in Britain_ (London and Boston: Routledge & Kegan Paul, 1980); Richard Abel, _The Cin\u00e9 Goes to Town: French Cinema, 1896\u20131914_ (Berkeley: University of California Press, 1994); Yuri Tsivian, _Early Cinema in Russia and Its Cultural Reception_ , ed. Richard Taylor, trans. Alan Bodger (London and New York: Routledge, 1994); Lee Grieveson, _Policing Cinema: Movies and Censorship in Early-Twentieth-Century America_ (Berkeley: University of California Press, 2004).\n\n 3. Sir James Paget, \"An Address on the Utility of Scientific Work in Practice,\" _British Medical Journal_ (15 October 1887): 811\u2013814, here 811.\n\n 4. On physicians' adoption of scientific method, see W. F. Bynum, _Science and the Practice of Medicine in the Nineteenth Century_ (Cambridge: Cambridge University Press, 1994); on the resistance of doctors to science, see John Harley Warner, \"Ideals of Science and Their Discontents in Late Nineteenth-Century American Medicine,\" _Isis_ 82, no. 3 (September 1991): 454\u2013478.\n\n 5. Michel Foucault, _The Birth of the Clinic: An Archaeology of Medical Perception_ , trans. A. M. Sheridan Smith (New York: Vintage, 1973); Michael Hau, \"The Holistic Gaze in German Medicine, 1890\u20131930,\" _Bulletin of the History of Medicine_ 74, no. 3 (Fall 2000): 495\u2013524.\n\n 6. Scott Curtis, \"Still\/Moving: Digital Imaging and Medical Hermeneutics,\" in _Memory Bytes: History, Technology, and Digital Culture_ , ed. Lauren Rabinovitz and Abraham Geil (Durham, N.C.: Duke University Press, 2004), 218\u2013254.\n\n 7. Like chapter 1, this chapter will focus on research films, but for more on the early medical education film in Germany, see Waldemar Schweisheimer, _Die Bedeutung des Films f\u00fcr soziale Hygiene und Medizin_ (Munich: M\u00fcller, 1920). On early American medical education films, see Martin Pernick, _The Black Stork: Eugenics and the Death of_ \" _Defective_ \" _Babies in American Medicine and Motion Pictures Since 1915_ (New York: Oxford University Press, 1999); and esp. Kirsten Ostherr's valuable studies, _Cinematic Prophylaxis: Globalization and Contagion in the Discourse of World Health_ (Durham, N.C.: Duke University Press, 2005); and _Medical Visions: Producing the Patient Through Film, Television, and Imaging Technologies_ (New York: Oxford University Press, 2013).\n\n 8. The most comprehensive survey of early literature on medical research films remains Anthony R. Michaelis, _Research Films in Biology, Anthropology, Psychology, and Medicine_ (New York: Academic, 1955). Specific statistics about Germany's output compared with other countries can be found on p. 326. See also Adolf Nichtenhauser, \"History of Motion Pictures in Medicine\" (unpublished manuscript, MS C 380, History of Medicine Division, National Library of Medicine, Bethesda, Md.). The definitive contemporary treatment of the subject is Lisa Cartwright, _Screening the Body: Tracing Medicine_ ' _s Visual Culture_ (Minneapolis: University of Minnesota Press, 1995).\n\n 9. A particularly vivid indication of the rise of German radiology is the change in the rosters of editorial board advisors of the major radiological journals. _The Archives of the Roentgen Ray_ (London), e.g., had no one from Germany on its board in 1907, but it had seven members from Germany and Austria by 1913.\n\n 10. On Germany's research infrastructure, see, e.g., Margit Sz\u00f6ll\u00f6si-Janze, \"Science and Social Space: Transformations in the Institutions of _Wissenschaft_ from the Wilhelmine Empire to the Weimar Republic,\" _Minerva_ 43 (2005): 339\u2013360.\n\n 11. The best survey of the early literature in Germany is Martin Weiser, _Medizinische Kinematographie_ (Dresden and Leipzig: Theodor Steinkopff, 1919). See also Karl Wilhelm Wolf-Czapek, _Die Kinematographie: Wesen, Entstehung und Ziele des lebenden Bildes_ (Berlin: Union Deutsche Verlagsgesellschaft, 1908; 2d enl. ed., 1911); and relevant sections in Wolf-Czapek, ed., _Angewandte Photographie in Wissenschaft und Technik_ (Berlin: Union Deutsche Verlagsgesellschaft, 1911); Hans Lehmann, _Die Kinematographie: Ihren Grundlagen und ihre Anwendungen_ (Leipzig: Teubner, 1911); Oswald Polimanti, \"Der Kinematograph in der biologischen und medizinischen Wissenschaft,\" _Naturwissenschaftliche Wochenschrift_ 10, no. 49 (3 December 1911): 769\u2013774; and Polimanti, \"Die Anwendung der Kinematographie in den Naturwissenschaften, der Medizin und im Unterricht,\" in _Wissenschaftliche Kinematographie_ , by Franz Paul Liesegang, with Karl Kieser and Oswald Polimanti (D\u00fcsseldorf: Liesegang, 1920), 257\u2013310.\n\n 12. Weiser, _Medizinische Kinematographie_ , 62.\n\n 13. Robert A. Nye, _Crime, Madness, and Politics in Modern France: The Medical Concept of National Decline_ (Princeton, N.J.: Princeton University Press, 1984). See esp. his final chapter on the United Kingdom and Germany.\n\n 14. Robert Gaupp, \"Der Kinematograph vom medizinischen und psychologischen Standpunkt,\" in _Der Kinematograph als Volkunterhaltungsmittel_ , by Robert Gaupp and Konrad Lange (Munich: D\u00fcrer-Bund-Flugschrift zur Ausdruckskultur 100, 1912), 9 (emphasis in original).\n\n 15. The definitive statement on the role of experiment in medicine is Claude Bernard, _Introduction \u00e0 l_ ' _\u00e9tude de la m\u00e9decine exp\u00e9rimentale_ (Paris: Bailli\u00e8re, 1865), translated as _An Introduction to the Study of Experimental Medicine_ by Henry Copley Greene (New York: Macmillan, 1927). See also Bynum, _Science and the Practice of Medicine in the Nineteenth Century_ ; and Warner, \"Ideals of Science.\"\n\n 16. For overviews of the origins of medical photography, see Alison Gernsheim, \"Medical Photography in the Nineteenth Century,\" _Medical and Biological Illustration_ (London) 11, no. 2 (April 1961): 85\u201392; Renata Taurek, _Die Bedeutung der Photographie f\u00fcr die medizinische Abbildung im 19. Jahrhundert_ (Cologne: Hansen, 1980); Daniel M. Fox and Christopher Lawrence, _Photographing Medicine: Images and Power in Britain and America Since 1840_ (New York: Greenwood, 1988); Andreas-Holger Maehle, \"The Search for Objective Communication: Medical Photography in the Nineteenth Century,\" in _Non-verbal Communication in Science Prior to 1900_ , ed. Renato G. Mazzolini (Florence: Olschki, 1993), 563\u2013586; Monique Sicard, Robert Pujade, and Daniel Wallach, _\u00c0 corps et \u00e0 raison: Photographies m\u00e9dicales, 1840\u20131920_ (Paris: Marval, 1995); and Gunnar Schmidt, _Anamorphotische K\u00f6rper: Medizinische Bilder vom Menschen im 19. Jahrhundert_ (Cologne: B\u00f6hlau, 2001). On the impact of photography on medical practice, see Stanley Joel Reiser, _Medicine and the Reign of Technology_ (Cambridge: Cambridge University Press, 1978).\n\n 17. Ludwig Braun, _\u00dcber Herzbewegung und Herzstoss_ (Jena: Fischer, 1898). On Braun, see Nichtenhauser, \"History of Motion Pictures in Medicine,\" 35\u201338; Michaelis, _Research Films_ , 133; Cartwright, _Screening the Body_ , 20\u201324; and Peter Geimer, \"Living and Non-living Pictures,\" in _Undead: Relations Between the Living and the Lifeless_ , ed. Peter Geimer (Berlin: Max-Planck-Institut f\u00fcr Wissenschaftsgeschichte, 2003), 39\u201351. For other early attempts to film the action of the heart, see \u00c9tienne-Jules Marey, _Movement_ , trans. Eric Pritchard (London: Heinemann, 1895), chap. 16; and Charles Fran\u00e7ois-Franck, \"La chronophotographie simultan\u00e9e du coeur et des courbes cardiographiques chez les mammif\u00e8res,\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 54 (8 November 1902): 1193\u20131197. For other uses of film in experimental physiology, see, e.g., Martin Philippson, _L'autonomie et la centralisation dans le syst\u00e8me nerveux des animaux: \u00e9tude de physiologie exp\u00e9rimentale et compare_ (Brussels: Falk, 1905); and esp. the work of Oswald Polimanti, \"\u00dcber Ataxie cerebralen und cerebellaren Ursprungs,\" _Archiv f\u00fcr Physiologie_ (1909): 123\u2013134; and \"Zur Physiologie der Stirnlappen,\" _Archiv f\u00fcr Physiologie_ (1912): 337\u2013342.\n\n 18. On the nature of scientific experiment, see Hans-J\u00f6rg Rheinberger, _Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube_ (Stanford, Calif.: Stanford University Press, 1997); Ian Hacking, _Representing and Intervening: Introductory Topics in the Philosphy of Natural Science_ (Cambridge: Cambridge University Press, 1983); and Hans Radder, esp. _The Material Realization of Science_ (Assen and Maastricht: Van Gorcum, 1988), 59\u201369; _In and About the World: Philosophical Studies of Science and Technology_ (Albany: State University of New York, 1996), 11\u201320; and his edited volume, _The Philosophy of Scientific Experimentation_ (Pittsburgh, Pa.: University of Pittsburgh Press, 2003). See also Theodore Arabatzis, \"Experiment,\" in _The Routledge Companion to Philosophy of Science_ , ed. Stathis Psillos and Martin Curd (London and New York: Routledge, 2008), 159\u2013170.\n\n 19. On the notion of \"working objects,\" see Lorraine Daston and Peter Galison, \"The Image of Objectivity,\" _Representations_ 40 (Fall 1992): 81\u2013128; and _Objectivity_ (New York: Zone, 2007).\n\n 20. Steven Shapin, \"Pump and Circumstance: Robert Boyle's Literary Technology,\" _Social Studies of Science_ 14, no. 4 (November 1984): 481\u2013520. See also Steven Shapin and Simon Schaffer, _Leviathan and the Air-Pump: Hobbes, Boyle, and the Experimental Life_ (Princeton, N.J.: Princeton University Press, 1985).\n\n 21. See the bibliography in Otto Glasser, _Wilhelm Conrad Roentgen and the Early History of the Roentgen Rays_ (Springfield, Ill.: Thomas, 1934).\n\n 22. Excellent reviews of the subject include Hans A. Jarre, \"Roentgen Cinematography,\" in _The Science of Radiology_ , ed. Otto Glasser (Springfield, Ill.: Thomas, 1933), 198\u2013209; and L. J. Ramsey, \"Early Cineradiography and Cinefluorography,\" _History of Photography_ 7, no. 4 (October\u2013December 1983): 311\u2013322. See also Monika Dommann, _Durchsicht, Einsicht, Vorsicht: Eine Geschichte der R\u00f6ntgenstrahlen, 1896_ \u2013 _1963_ (Z\u00fcrich: Chronos, 2003). For early cinema and the X-ray, see Cartwright, _Screening the Body_ ; and Solveig J\u00fclich, \"Seeing in the Dark: Early X-Ray Imaging and Cinema,\" in _Moving Images: From Edison to the Webcam_ , ed. John Fullerton and Astrid S\u00f6derberg Widding (Sydney: Libbey, 2000), 47\u201358.\n\n 23. John Macintyre, \"X-Ray Records for the Cinematograph,\" _Archives of Skiagraphy_ 1, no. 2 (April 1897): 37; and the report of his screening for \"Ladies Night\" of the Royal College of Surgeons of England in \"The Royal Society Conversazione,\" _Lancet_ 149 (19 June 1897): 1706.\n\n 24. P. H. Eykman, \"Der Schlingact, dargestellt nach Bewegungsphotographien mittelst R\u00f6ntgen-Strahlen,\" _Pfl\u00fcger_ ' _s Archiv f\u00fcr die gesammte Physiologie des Menschen und der Thiere_ 99 (1903): 513\u2013571.\n\n 25. Virgilio Tosi, _Cinema Before Cinema: The Origins of Scientific Cinematography_ , trans. Sergio Angelini (London: British Universities Film & Video Council, 2005), 170.\n\n 26. C. K\u00e4stle, H. Rieder, and J. Rosenthal, \"Ueber kinematographisch aufgenommene R\u00f6ntgenogramme (Bio-R\u00f6ntgenographie) der inneren Organe des Menschen,\" _M\u00fcnchener medizinsiche Wochenschrift_ 56, no. 6 (9 February 1909): 280\u2013283; \"The Bioroentgenography of the Internal Organs,\" _Archives of the Roentgen Ray_ 15, no. 1 (June 1910): 3\u201312.\n\n 27. Lewis Gregory Cole, \"The Gastric Motor Phenomena Demonstrated with the Projecting Kinetoscope,\" _American Quarterly of Roentgenology_ 3, no. 4 (1912): 1\u201311.\n\n 28. Franz M. Groedel, \"The Present State of Roentgen Cinematography and Its Results as to the Study of the Movements of the Inner Organs of the Human Body,\" _Interstate Medical Journal_ 22 (March 1915): 281\u2013290, here 290. Of his numerous texts on the topic, see esp. \"Roentgen Cinematography and Its Importance in Medicine,\" _British Medical Journal_ (24 April 1909): 1003; and his three-part series \"Die Technik der R\u00f6ntgenkinematographie,\" _Deutsche medizinsiche Wochenschrift_ 35 (11 March 1909): 434\u2013435; 39 (6 February 1913): 270\u2013271; and 39 (24 April 1913): 798\u2013799. See also W. Bruce Fye, \"Franz M. Groedel,\" _Clinical Cardiology_ 23, no. 2 (February 2000): 133\u2013134.\n\n 29. On diagnosis, see Carlo Ginzburg, \"Clues: Roots of an Evidential Paradigm,\" in _Clues, Myths, and the Historical Method_ , trans. John and Ann C. Tedeschi (Baltimore: Johns Hopkins University Press, 1989), 96\u2013125; or Caroline Whitbeck, \"What Is Diagnosis? Some Critical Reflections,\" _Metamedicine_ 2, no. 3 (October 1981): 319\u2013329.\n\n 30. On doubts about the clinical application of X-rays, see Andrew Warwick, \"X-Rays as Evidence in German Orthopedic Surgery, 1895\u20131900,\" _Isis_ 96, no. 1 (March 2005): 1\u201324.\n\n 31. Friedrich Dessauer, _Die neuesten Fortschritte in der R\u00f6ntgenphotographie_ (Leipzig: Nemnich Verlag, 1912), 15. For a more optimistic view, see Carl Bruegel, \"Bewegungsvorg\u00e4nge am pathologischen Magen auf Grund r\u00f6ngenkinematographischer Untersuchungen,\" _M\u00fcnchener medizinische Wochenschfrift_ 60, no. 4 (28 January 1913): 179\u2013181.\n\n 32. Andr\u00e9 Lomon and Jean Comandon, \"Radiocin\u00e9matographie par la photographie des \u00e9crans intensificateurs,\" _La Presse M\u00e9dicale_ 35 (3 May 1911): 359.\n\n 33. Jarre, \"Roentgen Cinematography,\" 202.\n\n 34. For a more complete survey, see K. Podoll and J. L\u00fcning, \"Geschichte des wissenschaftlichen Films in der Nervenheilkunde in Deutschland 1895\u20131929,\" _Fortschritte der Neurologie, Psychiatrie_ 66 (1998): 122\u2013132; and Genevi\u00e8ve Aubert \"From Photography to Cinematography: Recording Movement and Gait in a Neurological Context,\" _Journal of the History of the Neurosciences_ 11, no. 3 (2002): 255\u2013264. See also Juliet Clare Wagner, \"Twisted Bodies, Broken Minds: Film and Neuropsychiatry in the First World War\" (PhD diss., Harvard University, 2009).\n\n 35. Paul Schuster, \"Vorf\u00fchrung pathologischer Bewegungscomplexe mittelst des Kinematographen und Erl\u00e4uterung derselben,\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 69, part 1 (1898): 196\u2013199. For more on Schuster and his context, see Bernd Holdorff, \"Die privaten Polikliniken f\u00fcr Nervenkranke vor und nach 1900\" and \"Zwischen Hirnforschung, Neuropsychiatrie und Emanzipation zur klinischen Neurologie bis 1933,\" in _Geschichte der Neurologie in Berlin_ , ed. Bernd Holdorff and Rolf Winau (Berlin and New York: de Gruyter, 2001), 127\u2013137, and 157\u2013174.\n\n 36. Gheorghe Marinescu, \"Les troubles de la marche dans l'h\u00e9mipl\u00e9gie organique \u00e9tudi\u00e9s \u00e0 l'aide du cin\u00e9matographe,\" _La semaine M\u00e9dicale_ (1899): 225\u2013228; see also Alexandru C. Barboi, Christopher G. Goetz, and Radu Musetoiu, \"The Origins of Scientific Cinematography and Early Medical Applications,\" _Neurology_ 62 (June 2004): 2082\u20132086.\n\n 37. See Albert Londe's report of his work with Richer in Albert Londe, _Notice sur les titres et travaux scientifiques_ (Paris: Masson, 1911).\n\n 38. Walter Greenough Chase, \"The Use of the Biograph in Medicine,\" _Boston Medical and Surgical Journal_ 153, no. 21 (23 November 1905): 571\u2013572. See also Cartwright, _Screening the Body_ , chap. 3.\n\n 39. Arthur Van Gehuchten, \"Coup de couteau dans la moelle lombaire. Essai de physiologie pathologique,\" _Le N\u00e9vraxe_ 9 (1907): 208\u2013232. See also Genevi\u00e8ve Aubert, \"Arthur Van Gehuchten Takes Neurology to the Movies,\" _Neurology_ 59 (November 2002): 1612\u20131618.\n\n 40. Emil Kraepelin, \"Demonstration von Kinematogrammen,\" _Centralblatt f\u00fcr Nervenheilkunde und Psychiatrie_ 32 (1909): 689.\n\n 41. Hans Hennes, \"Die Kinematographie im Dienste der Neurologie und Psychiatrie, nebst Beschreibung einiger selteneren Bewegungsst\u00f6rungen,\" _Medizinische Klinik_ 6, no. 51 (18 December 1910): 2010\u20132014.\n\n 42. See Polimanti titles cited in note 11.\n\n 43. T. H. Weisenburg, \"Moving Picture Illustrations in Medicine, with Special Reference to Nervous and Mental Diseases,\" _Journal of the American Medical Association_ 59, no. 26 (28 December 1912): 2310\u20132312.\n\n 44. This section on live demonstration draws from my essay, \"Photography and Medical Observation,\" in _The Educated Eye: Visual Pedagogy in the Life Sciences_ , ed. Nancy Anderson and Michael R. Dietrich (Hanover, N.H.: Dartmouth College Press, 2012), 68\u201393.\n\n 45. Clare [Blake] to Dear Pater, Vienna, 9 November [1865], Clarence John Blake Papers, Francis A. Countway Library of Medicine, Harvard University, quoted in John Harley Warner, _Against the Spirit of System: The French Impulse in Nineteenth-Century American Medicine_ (Princeton, N.J.: Princeton University Press, 1998), 304.\n\n 46. Clare to Sister Agnes, Vienna, 29 March 1869, Clarence John Blake Papers, Francis A. Countway Library of Medicine, Harvard University, quoted in Warner, _Against the Spirit of System_ , 311.\n\n 47. Hennes, \"Die Kinematographie im Dienste der Neurologie,\" 2012, quoted in Friedrich A. Kittler, _Gramophone, Film, Typewriter_ , trans. and with an introduction by Geoffrey Winthrop-Young and Michael Wutz (Stanford, Calif.: Stanford University Press, 1999), 145 (emphasis in original).\n\n 48. See Cartwright, _Screening the Body_ , chap. 3: \"An Etiology of the Neurological Gaze.\"\n\n 49. See Reiser, _Medicine and the Reign of Technology_. See also Joel D. Howell, _Technology in the Hospital: Transforming Patient Care in the Early Twentieth Century_ (Baltimore: Johns Hopkins University Press, 1995).\n\n 50. For discussion of the projection of images in medical education, see A. Wassermann, \"Die medizinische Fakult\u00e4t,\" in _Die Universit\u00e4ten im Deutschen Reich_ , ed. W. Lexis (Berlin: Asher, 1904), 146\u2013147; or Erwin Christeller, \"Die Bedeutung der Photographie f\u00fcr den pathologisch-anatomischen Unterricht und die pathologisch-anatomische Forschung,\" _Berliner klinische Wochenschrift_ 55, no. 17 (29 April 1918): 399\u2013401; for broader overviews, see Sigmund Theodor Stein, _Das Licht im Dienste wissenschaftlicher Forschung: Handbuch der Anwendung des Lichtes und der Photographie in der Natur- und Heilkunde_ (Leipzig: Spamer, 1877); Sigmund Theodor Stein, _Die optische Projektionskunst im dienste der exakten Wissenschaften: ein Lehr- und Hilfsbuch zur unterst\u00fctzung des naturwissenschaftlichen Unterrichts_ (Halle an der Saale, Germany: Knapp, 1887); see also Henning Schmidgen, \"Pictures, Preparations, and Living Processes: The Production of Immediate Visual Perception ( _Anschauung_ ) in Late-19th-Century Physiology,\" _Journal of the History of Biology_ 37, no. 3 (October 2004): 477\u2013513.\n\n 51. Schuster, \"Vorf\u00fchrung pathologischer Bewegungscomplexe,\" 196\u2013197.\n\n 52. See, e.g., Albert E. Stein, \"Ueber medizinisch-photographische und -kinematographische Aufnahmen,\" _Deutsche medizinische Wochenschrift_ 38 (20 June 1912): 1184\u20131186. For early reviews of the use of photography and cinematography for the study of pathological movement, with implications for therapy, see Ernst Jendrassik, \"Klinische Beitr\u00e4ge zum Studium der normalen und pathologischen Gangarten,\" _Deutsche Archiv f\u00fcr klinische Medizin_ 70 (1901): 81\u2013132; and James Fr\u00e4nkel, \"Kinematographische Untersuchung des normalen Ganges und einiger Gangst\u00f6rungen,\" _Zeitschrift f\u00fcr orthop\u00e4dische Chirurgie_ 20 (1908): 617\u2013646.\n\n 53. An example of a German report on Comandon's work that stresses the importance of his films as both exploratory and documentary is \"Die Kinematographie des Unsichtbaren,\" _Prometheus_ 21, no. 1054 (5 January 1910): 218\u2013220.\n\n 54. J. Frey, \"Report of the Photographic Department of Bellevue Hospital for the Year 1869,\" in _Tenth Annual Report of the Commissioners of Public Charities and Correction of the City of New York for the Year 1869_ (Albany: van Benthuysen, 1870), 85. www.artandmedicine.com\/ogm\/1869.html.\n\n 55. Hennes, \"Die Kinematographie im Dienste der Neurologie und Psychiatrie,\" 2014. For other calls for a central agency to handle medical or scientific films, see Robert Kutner, \"Die Bedeutung der Kinematographie f\u00fcr medizinische Forschung und Unterricht sowie f\u00fcr die volkshygienische Belehrung,\" _Zeitschrift f\u00fcr \u00c4rztliche Fortbildung_ 8, no. 8 (15 April 1911): 249\u2013251; or Weiser, _Medizinische Kinematographie_ , 4. This call was eventually answered after World War I with the formation of the Kulturfilm section of UFA. See Universum-Film A. G., _Das medizinische Filmarchiv bei der Kulturabteilung der Universum-Film A.G._ (Berlin: Gahl, 1919).\n\n 56. Franz Goerke, \"Proposal for Establishing an Archive for Moving Pictures (1912),\" trans. Cecilie L. French and Daniel J. Leab, _Historical Journal of Film, Radio and Television_ 16, no. 1 (March 1996): 9\u201312, here 10. Originally published as \"Vorschlag zur Einrichtung eines Archives f\u00fcr Kino-films\" in _Der Deutsche Kaiser im Film. Zum 25 j\u00e4hrigen Regierungs-Jubil\u00e4um Seiner Majest\u00e4t des Deutschen Kaisers K\u00f6nigs von Preu\u00dfen Wilhelm II_ , ed. Paul Klebinder (Berlin: Klebinder, 1912), 63\u201368.\n\n 57. Goerke, \"Proposal for Establishing an Archive,\" 9\u201310.\n\n 58. Eug\u00e8ne Doyen, \"Le cin\u00e9matograph et l'enseignement de la chirurgie,\" _Revue critique de m\u00e9decine et de chirurgie_ 1, no. 1 (15 August 1899): 1\u20136; partially translated as \"The Cinematograph and the Teaching of Surgery,\" _British Gyn\u00e6cological Journal_ 15 (1899): 579\u2013586, here 581. On Doyen, see Robert Didier, _Le Docteur Doyen: Chirurgien de la Belle \u00c9poque_ (Paris: Librairie Maloine, 1962); and esp. the work of Thierry Lefebvre, including \"Le cas \u00e9trange du Dr Doyen, 1859\u20131916,\" _Archives_ 29 (February 1990): 1\u201312; \"Le Dr Doyen, un pr\u00e9curseur,\" in _Le cin\u00e9ma et la science_ , ed. Alexis Martinet (Paris: CNRS \u00c9ditions, 1994), 70\u201377; \"Die Trennung der Siamesischen Zwillinge Doodica und Radica durch Dr. Doyen,\" _KINtop 6_ (1997): 97\u2013101; and _La chair et le celluloid: Le cin\u00e9ma chirurgical du Docteur Doyen_ (Brionne: Jean Doyen \u00e9diteur, 2004).\n\n 59. Although the films were not mentioned in the _British Medical Journal_ 's proceedings of the July 1898 meeting, there is a letter to the editor that remarks on the strong impression they made: G. P. Coldstream, \"The Cinematoscope as an Aid in Teaching,\" _British Medical Journal_ (3 September 1898): 658. On Doyen's films, see Thierry Lefebvre, \"La collection des films du Dr Doyen,\" _1895_ 17 (December 1994): 100\u2013114; and Tiago Baptista, \"' _Il faut voir le ma\u00eetre_ ': A Recent Restoration of Surgical Films by E.-L.Doyen (1859\u20131916),\" _Journal of Film Preservation_ 70 (November 2005): 42\u201350. After 1907, French film manufacturer Eclipse distributed Doyen's films throughout Europe (see Lefebvre, _La chair et le celluloid_ , 67\u201376).\n\n 60. Doyen, \"Le cin\u00e9matograph et l'enseignement de la chirurgie,\" 3.\n\n 61. For a catalog of Doyen's films to that point, see Eug\u00e8ne Louis Doyen, _L'enseignement de la technique op\u00e9ratoire par les projections anim\u00e9es_ (Paris: Soci\u00e9t\u00e9 g\u00e9n\u00e9rale des cin\u00e9matographes Eclipse, ca. 1911).\n\n 62. For Doyen's detailed discussion of the medical case, the surgical technique, and its ethical aftermath, see Eug\u00e8ne Louis Doyen, _La cas des xiphopages hindoues Radica et Doodica_ (Paris: Bourse de Commerce, 1904). This pamphlet also reprints a heated exchange of letters between Doyen and a Dr. Legrain, who accused Doyen of besmirching the profession with the film. See also Lefebvre, \"Die Trennung der Siamesischen Zwillinge.\" On the Parnaland incident, see Lefebvre, _La chair et le celluloid_ , 39\u201359.\n\n 63. The concern about spectacle in medicine was more pronounced in France\u2014probably due to the Doyen controversy\u2014but there were grumblings from England and Germany as well, especially with regard to Doyen's films. See \"A Surgical Showman,\" _British Medical Journal_ (19 January 1907): 163, which reports from Germany and elsewhere about screenings of Doyen films that left doctors disgusted; at one of his demonstrations, \"it is said that he was hissed at Brussels.\"\n\n 64. See the report in \"Verwandte Gebiete,\" _Zentralblatt f\u00fcr R\u00f6ntgenstrahlen, Radium und verwandte Gebiete_ 1, no. 2 (1910): 78\u201380.\n\n 65. Kutner was a proponent of medical photography in general, having taken some of the earliest images from a cytoscope with famed urologist Max Nitze. See Max Nitze, _Kystophotographischer Atlas_ (Wiesbaden: Bergmann, 1894). See also Harry W. Herr, \"Max Nitze, the Cystoscope and Urology,\" _Journal of Urology_ 176, no. 4 (October 2006): 1313\u20131316. Kutner was also a strong advocate of continuing education for physicians, having founded the journal _Zeitschrift f\u00fcr \u00c4rztliche Fortbildung_.\n\n 66. See also James Fr\u00e4nkel, \"Kinematographische Demonstration,\" _Verhandlungen der freien Vereinigung der Chirurgen Berlins_ 20, part 1 (1907): 12\u201313.\n\n 67. Karl Reicher, \"Kinematographie in der Neurologie,\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 79, part 2 (1907): 235\u2013236.\n\n 68. See \"Special Correspondence: Berlin,\" _British Medical Journal_ (5 March 1910): 598, for another report of the evening. In England, Dr. William Stirling was Kutner's counterpart as a champion of the educational value of medical films. See reports of Stirling's presentations of a variety of films in medicine and biology in \"Medical News,\" _Lancet_ 177 (27 May 1911): 1470; and _Lancet_ 182 (11 October 1913): 1083\u20131084.\n\n 69. Kutner, \"Die Bedeutung der Kinematographie,\" 250.\n\n 70. Alongside this discourse of \"seeing as\" (film as an extension of direct perception and the moving image as a substitute for the thing itself) there was another discourse of \"seeing differently\" (film as a technology for representing things in ways the naked eye could not perceive). Educators regarded both features of cinema to be pedagogically useful, while film theorists such as Jean Epstein and B\u00e9la Bal\u00e0zs prioritized the latter. This difference also corresponds to the \"documentary\" and \"exploratory\" functions outlined so far.\n\n 71. Doyen, \"The Cinematograph and the Teaching of Surgery,\" 580\u2013581 (translation modified).\n\n 72. A good overview is Jennifer Karns Alexander, _The Mantra of Efficiency: From Waterwheel to Social Control_ (Baltimore: Johns Hopkins University Press, 2008). See also Evelyn Cobley, _Modernism and the Culture of Efficiency: Ideology and Fiction_ (Toronto and Buffalo, N.Y.: University of Toronto Press, 2009).\n\n 73. Paul Starr, _The Social Transformation of American Medicine_ (New York: Basic, 1982), 146.\n\n 74. George Rosen, \"The Efficiency Criterion in Medical Care, 1900\u20131920,\" _Bulletin of the History of Medicine_ 50, no. 1 (Spring 1976): 28\u201344; Margarete Arndt and Barbara Bigelow, \"Toward the Creation of an Institutional Logic for the Management of Hospitals: Efficiency in the Early Nineteen Hundreds,\" _Medical Care Research and Review_ 63, no. 3 (June 2006): 369\u2013394.\n\n 75. See the issue devoted to the \"Conference on Hospital Standardization,\" _Bulletin of the American College of Surgeons_ 3, no. 1 (1917); or Frank B. Gilbreth, \"Scientific Management in the Hospital,\" _Modern Hospital_ 3 (1914): 321\u2013324.\n\n 76. My essay on \"Photography and Medical Observation\" (see note 44) likewise examines correspondences between formal features of still photography and practices\/ideals of medical observation.\n\n 77. Ludwig Braun, \"Ueber den Werth des Kinematographen f\u00fcr die Erkenntniss der Herzmechanik,\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 69, part 1 (1898): 185\u2013186. This list is included and elaborated in his _\u00dcber Herzbewegung und Herzstoss_ (Jena: Fischer, 1898).\n\n 78. Kelly Wilder, _Photography and Science_ (London: Reaktion, 2009), 23.\n\n 79. Gernsheim, \"Medical Photography in the Nineteenth Century,\" 87.\n\n 80. Cartwright, _Screening the Body_ , 38, 48.\n\n 81. Ludwik Fleck, \"Some Specific Features of the Medical Way of Thinking [1927],\" in _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle (Dordrecht, Netherlands, and Boston: Reidel, 1986), 39\u201346, here 39\u201340. Foucault also discusses the importance of the series in medical thinking in _The Birth of the Clinic_ , 97. On the importance of comparing pathological states to find some sense of consistency, see Georges Canguilhem, _The Normal and the Pathological_ , trans. Carolyn R. Fawcett in collaboration with Robert S. Cohen (New York: Zone, 1991), 51. On medical logic, see Friedrich Oesterlen, _Medical Logic_ , trans. G. Whitley (London: Sydenham Society, 1855); Frederick P. Gay, \"Medical Logic,\" _Bulletin of the History of Medicine_ 7 (1939): 6\u201327; Lester S. King, \"Medical Logic,\" _Journal of the History of Medicine and Allied Sciences_ 33, no. 3 (July 1978): 377\u2013385; and King, _Medical Thinking: A Historical Preface_ (Princeton, N.J.: Princeton University Press, 1982).\n\n 82. Bernike Pasveer, \"Representing or Mediating: A History and Philosophy of X-Ray Images in Medicine,\" in _Visual Cultures of Science: Rethinking Representational Practices in Knowledge Building and Science Communication_ , ed. Luc Pauwels (Lebanon, N.H.: Dartmouth College Press\/University Press of New England, 2006), 41\u201362.\n\n 83. For an example of the use of series photography to track the development of smallpox in a patient over several days, see Samuel A. Powers, _Variola: A Series of Twenty-One Heliotype Plates Illustrating the Progressive Stages of the Eruption_ (Boston: Samuel A. Powers, 1882). My thanks to Mark Rowland for pointing me to this text. For examples of different angles of a patient during the same session, see Albert Londe's photographs of female patients documented in the journal _Nouvelle iconographie de la Salp\u00eatri\u00e8re_ (Paris: Masson, 1888\u20131918).\n\n 84. For an interesting discussion of X-ray cinematography's diagnostic value, see the exchange between Drs. Fr\u00e4nkel, von Bergmann, Levy-Dorn, Albu, and Kutner in Albert Fr\u00e4nkel, \"R\u00f6ntgendiagnosen und R\u00f6ntgenfehldiagnosen beim Magenkarzinom; diagnostischer Fortschritt durch R\u00f6ntgenkinographie.\" _Zentralblatt f\u00fcr R\u00f6ntgenstrahlen, Radium und verwandte Gebiete_ 3, no. 4 (1912): 149\u2013150.\n\n 85. Warwick, \"X-Rays as Evidence in German Orthopedic Surgery, 1895\u20131900.\"\n\n 86. Photographs were often used as illustrations exchanged between attendees of a lecture. An example, picked more or less at random, is Adolf Magnus-Levy, \"Ueber Organ-Therapie beim endemischen Kretinismus,\" _Verhandlungen der Berliner medicinischen Gesellschaft_ 34, part 2 (1903): 350\u2013357. See esp. the discussion of this presentation on 22 July 1903 in part 1, pp. 246\u2013249. No photos are published with the paper, but they discuss the photographs that were passed around among the audience. Many such uses of photographs can be found in the _Verhandlungen_ and similar proceedings.\n\n 87. Cartwright, _Screening the Body_ , 36.\n\n 88. Georges Didi-Huberman, _Invention of Hysteria: Charcot and the Photographic Iconography of the Salp\u00eatri\u00e8re_ , trans. Alisa Hartz (Cambridge, Mass.: MIT Press, 2003), 24\u201325.\n\n 89. For more on this topic, see Scott Curtis, \"Photography and Medical Observation.\"\n\n 90. Foucault, _Birth of the Clinic_ , 109.\n\n 91. Foucault, _Birth of the Clinic_ , esp. 107\u2013123.\n\n 92. Neurologists such as Schuster needed movement to recognize the disorder\u2014which would otherwise be lost in the individual frames\u2014so they consistently slowed (but did not stop) the image in projection. See Schuster, \"Vorf\u00fchrung pathologischer Bewegungskomplexe.\"\n\n 93. Many researchers emphasized this feature of motion picture technology, but for an extended discussion of the applications of film's temporal malleability, with interesting responses from the expert audience, see the report of Herr v. Gr\u00fctzner's presentation at the T\u00fcbingen Society of Medical and Natural Scientists: \"Medizinisch-Naturwissenschaftlicher Verein T\u00fcbingen,\" _M\u00fcnchener medizinische Wochenschrift_ 56, no. 3 (19 January 1909): 154\u2013155.\n\n 94. Thomas Laycock, _Lectures on the Principles and Methods of Medical Observation and Research_ (Philadelphia: Blanchard and Lea, 1857), 64.\n\n 95. Rudolph Virchow, _Post-mortem Examinations_ , trans. T. P. Smith, 3d ed. (Philadelphia: Blakiston, 1895), 12.\n\n 96. Foucault, _Birth of the Clinic_ , xiv.\n\n 97. Foucault, _Birth of the Clinic_ , xiii (emphasis added).\n\n 98. Foucault, _Birth of the Clinic_ , esp. 107\u2013122.\n\n 99. Theodor Billroth, _The Medical Sciences in the German Universities: A Study in the History of Civilization_ , trans. William H. Welch (New York: Macmillan, 1924), 52\u201353. Originally published as _\u00dcber das Lehren und Lernen de medicinischen Wissenschaften an den Universit\u00e4ten der deutschen Nation nebst allgemeinen Bemerkungen \u00fcber Universit\u00e4ten; eine culturhistorische Studie_ (Vienna: Gerold, 1876).\n\n. Foucault, _Birth of the Clinic_ , xiii.\n\n. Hau, \"The Holistic Gaze in German Medicine, 1890\u20131930,\" 503. For more on the resistance of clinicians to laboratory methods in medicine, see Russell C. Maulitz, \"Physician Versus Bacteriologist: The Ideology of Science in Clinical Medicine,\" in _The Therapeutic Revolution: Essays in the Social History of American Medicine_ , ed. Morris J. Vogel and Charles E. Rosenberg (Philadelphia: University of Pennsylvania Press, 1979), 91\u2013107.\n\n. Foucault, _Birth of the Clinic_ , 121.\n\n. Lorraine Daston, \"On Scientific Observation,\" _Isis_ 99, no. 1 (2008): 97\u2013110. See also Lorraine Daston and Elizabeth Lunbeck, eds., _Histories of Scientific Observation_ (Chicago: University of Chicago Press, 2011).\n\n. There are perhaps some connections here to be made between the glance of the connoisseur and theories of cinephilia. See Christian Keathley, _Cinephilia and History; or, The Wind in the Trees_ (Bloomington: Indiana University Press, 2006).\n\n. Again, see Curtis, \"Still\/Moving: Digital Imaging and Medical Hermeneutics,\" for more on the relationship between film and medical hermeneutics.\n\n. Which should remind us of similar patterns of showmanship in early entertainment film described by Tom Gunning in \"An Aesthetic of Astonishment: Early Film and the (In)Credulous Spectator,\" _Art and Text_ 34 (Spring 1989): 31\u201345.\n\n. My thanks to Christian Quendler for stating this so succinctly for me.\n\n. Erwin Risak, _Der klinische Blick_ , 7th and 8th eds. (Vienna: Springer, 1943), 4.\n\n. Carl F. Flemming, _Pathologie und Therapie der Psychosen_ (Berlin: Hirschwald, 1859), 281\u2013282.\n\n. For a good overview of the relationship between clinical observation and scientific methods, see Kenneth D. Keele, _The Evolution of Clinical Methods in Medicine_ (London: Pitman, 1963). On the debates about clinical observation and the sphygmomanometer specifically, see Jeremy Booth, \"A Short History of Blood Pressure Measurement,\" _Proceedings of the Royal Society of Medicine_ 70, no. 11 (November 1977): 793\u2013799; Reiser, _Medicine and the Reign of Technology_ , 101\u2013106; and Hughes Evans, \"Losing Touch: The Controversy Over the Introduction of Blood Pressure Instruments Into Medicine,\" _Technology and Culture_ 34, no. 4 (October 1993): 784\u2013807.\n\n. Paget, \"An Address on the Utility of Scientific Work in Practice,\" 811.\n\n. Karl Wilhelm Wolf-Czapek, \"Die Kinematographie im medizinische Unterricht,\" _Jahrbuch f\u00fcr Photographie und Reproduktionstechnik_ 22 (1908): 58\u201359, here 58.\n\n. A close review of the primary literature on scientific film shows that analysis and synthesis were always considered two sides of the same coin. See \u00c9tienne-Jules Marey, _Movement_ , trans. Eric Pritchard (London: Heinemann, 1895), esp. chap. 18: \"Synthetic Reconstruction of the Elements of an Analyzed Movement.\" See also Hannah Landecker, \"Microcinematography and the History of Science and Film,\" _Isis_ 97, no. 1 (2006): 121\u2013132; and Oliver Gaycken, \"'The Swarming of Life': Moving Images, Education, and Views Through the Microscope,\" _Science in Context_ 24, no. 3 (September 2011): 361\u2013380.\n\n. On the theoretical implications of experimental apparatuses, see Davis Baird, _Thing Knowledge: A Philosophy of Scientific Instruments_ (Berkeley: University of California Press, 2004).\n\n. More could be said about the relationship between control of and submission to or pleasure in the scientific image. A good place to start would be Anne Secord, \"Botany on a Plate: Pleasure and the Power of Pictures in Promoting Early Nineteenth-Century Scientific Knowledge,\" _Isis_ 93, no. 1 (March 2002): 28\u201357.\n\n. Claudia Huerkamp, _Der Aufstieg der Arzte im 19. Jahrhundert: Vom gelehrten Stand zum professionellen Experten: Das Beispiel Preu\u00dfens_ (G\u00f6ttingen: Vandenhoeck & Ruprecht, 1985). See also Ute Frevert, _Krankheit als politisches Problem 1770\u20131880. Soziale Unterschichten in Preu\u00dfen zwischen medizinischer Polizei und staatlicher Sozialversicherung_ (G\u00f6ttingen: Vandenhoeck & Ruprecht, 1984).\n\n. Huerkamp stresses this medicalization of culture in her essay, \"The Making of the Modern Medical Profession, 1800\u20131914: Prussian Doctors in the Nineteenth Century,\" in _German Professions, 1800\u20131950_ , ed. Geoffrey Cocks and Konrad H. Jarausch (New York and Oxford: Oxford University Press, 1990), 66\u201384.\n\n. On the social prestige and authority of physicians, see Alfons Labisch, _Homo Hygienicus: Gesundheit und Medizin in der Neuzeit_ (Frankfurt and New York: Campus, 1992); Michael H. Kater, \"Professionalization and Socialization of Physicians in Wilhelmine and Weimar Germany,\" _Journal of Contemporary History_ 20 (1985): 677\u2013701; Paul Weindling, \"Bourgeois Values, Doctors and the State: The Professionalization of Medicine in Germany 1848\u20131933,\" in _The German Bourgeoisie_ , ed. David Blackbourn and Richard J. Evans (London and New York: Routledge, 1991), 198\u2013223; Charles E. McClelland, \"Modern German Doctors: A Failure of Professionalization?\" in _Medicine and Modernity: Public Health and Medical Care in Nineteenth- and Twentieth-Century Germany_ , ed. Manfred Berg and Geoffrey Cocks (Cambridge and New York: Cambridge University Press, 1997), 81\u201397. For a discussion of this phenomenon from the viewpoint of medical ethics, see Robert M. Veatch, \"Generalization of Expertise,\" _Hastings Center Studies_ 1, no. 2 (1973): 29\u201340.\n\n. On scientists, especially, as _Kulturtr\u00e4ger_ , see Fritz K. Ringer, _The Decline of the German Mandarins: The German Academic Community, 1890\u20131933_ (Cambridge, Mass.: Harvard University Press, 1969), 6; and Russell McCormmach, \"On Academic Scientists in Wilhelmian Germany,\" in _Science and Its Public: The Changing Relationship_ , ed. Gerald Horton and William A. Blanpied (Dordrecht, Netherlands, and Boston: Reidel, 1976), 157\u2013171.\n\n. Paul Weindling, \"Public Health in Germany,\" in _The History of Public Health and the Modern State_ , ed. Dorothy Porter (Amsterdam and Atlanta, Ga.: Rodopi, 1994), 119\u2013131. See also Weindling, _Health, Race, and German Politics Between National Unification and Nazism, 1870\u20131945_ (Cambridge and New York: Cambridge University Press, 1989).\n\n. Eric J. Engstrom, \"Emil Kraepelin: Psychiatry and Public Affairs in Wilhelmine Germany,\" _History of Psychiatry_ 2, no. 6 (June 1991): 111\u2013132. See also Engstrom's _Clinical Psychiatry in Imperial Germany: A History of Psychiatric Practice_ (Ithaca, N.Y.: Cornell University Press, 2003); and Emil Kraepelin, _Memoirs_ , ed. H. Hippius, G. Peters, D. Ploog in collaboration with P. Hoff and A. Kreuter, trans. Cheryl Wooding-Deane (Berlin and New York: Springer-Verlag, 1987). Kraepelin was also swept up by the degeneration craze described below: see Emil Kraepelin, \"Zur Entartungsfrage,\" _Zentralblatt f\u00fcr Nervenheilkunde und Psychiatrie_ 31 (1908): 745\u2013751, translated as \"On the Question of Degeneration,\" _History of Psychiatry_ 18, no. 3 (2007): 399\u2013404.\n\n. According to Andreas Killen, as early as 1912 the Reich Health Office started to collect materials documenting the educational benefits of scientific films and the health risks of commercial cinema. Andreas Killen, \"Psychiatry, Cinema, and Urban Youth in Early-Twentieth-Century Germany,\" _Harvard Review of Psychiatry_ 14, no. 1 (2006): 38\u201343.\n\n. Robert Gaupp, _Psychologie des Kindes_ (Leipzig: Teubner, 1910), and \"Das Pathologische in Kunst und Literatur,\" _Deutsche Revue_ 36, no. 2 (April 1911): 11\u201323. For an especially explicit statement of the physician's duty to society, see Gaupp, \"Der Arzt als Erzieher seines Volkes,\" _Medicinisches Correspondenz-Blatt_ 89, no. 32 (9 August 1919): 295\u2013296. On Gaupp, see William Mayer, \"Robert Gaupp,\" _American Journal of Psychiatry_ 108, no. 10 (April 1952): 724\u2013725.\n\n. Max Nordau, _Degeneration_ (London: Appleton, 1895), 43. Originally published as _Entartung_ , 2 vols. (Berlin: Duncker, 1892\u20131893). Nordau, an Austro-Hungarian physician living in Paris, was a prolific writer of fiction and cultural criticism as well as a foreign correspondent for German-language newspapers in Berlin, Vienna, and Budapest.\n\n. For contemporary discussions of nervousness, see, e.g., Wilhelm Heinrich Erb, _Ueber die wachsende Nervosit\u00e4t unserer Zeit_ (Heidelberg: Universit\u00e4ts Buchdruckerei von J. H\u00f6rning, 1893); Auguste Forel, _Hygiene der Nerven und des Geistes im gesunden und kranken Zustande_ (Stuttgart: Moritz, 1903); and Robert Gaupp, \"Die Nervosit\u00e4t unserer Zeit im Lichte der Wissenschaft,\" _Medicinisches Correspondenz-Blatt_ 77, no. 31 (3 August 1907): 633\u2013639. On nervousness and modernity in Germany, see Joachim Radkau, _Das Zeitalter der Nervosit\u00e4t: Deutschland zwischen Bismarck und Hitler_ (Munich: Hanser, 1998); Andreas Killen, _Berlin Electropolis: Shock, Nerves, and German Modernity_ (Berkeley: University of California Press, 2006); Michael Cowan, _Cult of the Will: Nervousness and German Modernity_ (University Park: Pennsylvania State University Press, 2008).\n\n. On Lombroso and Nordau, see Charles Bernheimer, \"Decadent Diagnostics,\" in _Decadent Subjects: The Idea of Decadence in Art, Literature, Philosophy, and Culture of the_ Fin de Si\u00e8cle _in Europe_ , ed. T. Jefferson Kline and Naomi Schor (Baltimore: Johns Hopkins University Press, 2002), 139\u2013162. For more on Nordau, see George L. Mosse, \"Max Nordau and His Degeneration,\" in Max Nordau, _Degeneration_ (New York: Fertig, 1968), xiii\u2013xxxvi; Thomas Anz, \"Gesundheit, Krankheit und literarische Norm: Max Nordaus 'Entartung' als Paradigma pathologisierender Kunstkritik,\" in _Gesund oder Krank?: Medizin, Moral und \u00c4sthetik in der deutschen Gegenwartsliteratur_ (Stuttgart: Metzler, 1989), 33\u201352; Hans-Peter S\u00f6der, \"Disease and Health as Contexts of Modernity: Max Nordau as a Critic of Fin-de-Si\u00e8cle Modernism,\" _German Studies Review_ 14, no. 3 (October 1991): 473\u2013487; Christoph Schulte, _Psychopathologie des Fin de Si\u00e8cle: Der Kulturkritiker, Arzt und Zionist Max Nordau_ (Frankfurt: Fischer Taschenbuch, 1997); C\u00e9line Kaiser, _Rhetorik der Entartung: Max Nordau und die Sprache der Verletzung_ (Bielefeld, Germany: Transcript, 2007).\n\n. To be fair, even those friendly to modern art and culture often saw it in similar, especially primitivist terms. See Doris Kaufmann, \"'Pushing the Limits of Understanding': The Discourse on Primitivism in German _Kulturwissenschaften_ , 1880\u20131930,\" _Studies in History and Philosophy of Science_ 39 (2008): 434\u2013443.\n\n. S\u00f6der, \"Disease and Health as Contexts of Modernity,\" 474.\n\n. Surveys of cultural pessimism in Germany include Fritz Stern, _The Politics of Cultural Despair: A Study in the Rise of the Germanic Ideology_ (Berkeley: University of California Press, 1961); and Ringer, _The Decline of the German Mandarins_.\n\n. Ike Spier, \"Die sexuelle Gefahr des Kinos,\" _Die neue Generation_ 8 (1912): 192\u2013198, here 192 (emphasis in original). I will discuss the battle against _Schundfilms_ (\"trash films\") in chap. 3, but for representative works, see Albert Hellwig, _Schundfilms: Ihr Wesen, ihre Gefahren und ihre Bek\u00e4mpfung_ (Halle an der Saale, Germany: Waisenhaus, 1911); Max Grempe, \"Gegen die Frauenverbl\u00f6dung im Kino,\" _Gleichheit_ 23, no. 5 (1912): 70\u201372; Malwine Rennert, \"Die Zaung\u00e4ste des Lebens im Kino,\" _Bild und Film_ 4, no. 11 (1914\/1915): 217\u2013218; and, for a reasonable rebuttal, Joseph Landau, \"Mechanisierte Unsterblichkeit,\" in _Der Deutsche Kaiser im Film. Zum 25j\u00e4hrigen Regierungs-Jubil\u00e4um Seiner Majest\u00e4t des Deutschen Kaisers K\u00f6nigs von Preu\u00dfen Wilhelm II_ , ed. Paul Klebinder (Berlin: Klebinder, 1912), 18\u201322.\n\n. Paul Schenk, \"Der Kinematograph und die Schule,\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 14, no. 15 (1 August 1908): 312\u2013313. For an entertaining \"experiment\" in which a writer submits three men to hours of continuous, flickering projection with predictable results, see Naldo Felke, \"Die Gesundheitssch\u00e4dlichkeit des Kinos,\" _Die Umschau_ 17, no. 1 (1 January 1913): 254\u2013255.\n\n. A survey of the (mostly French) discussion of \"flicker\" in early cinema can be found in Thierry Lefebvre, \"Flimmerndes Licht: Zur Geschichte der Filmwahrnehmung im fr\u00fchen Kino,\" _KINtop_ 5 (1996): 71\u201380.\n\n. For excellent surveys of this trend, see Killen, \"Psychiatry, Cinema, and Urban Youth in Early-Twentieth-Century Germany\"; and Killen, \"The Scene of the Crime: Psychiatric Discourses on the Film Audience in Early Twentieth Century Germany,\" in _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier (New Barnet, U.K.: Libbey, 2009) 99\u2013111.\n\n. Albert Hellwig, \"\u00dcber die sch\u00e4dliche Suggestivkraft kinematographischer Vorf\u00fchrung,\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 20, no. 6 (15 March 1914): 122; I will discuss Hellwig's work in more depth in chap. 3, but for a taste of his reliance on medical terminology and audiences, see Hellwig, \"Zur Psychologie kinematographischer Vorf\u00fchrungen,\" _Zeitschrift f\u00fcr Psychotherapie und medizinische Psychologie_ 6 (1916): 88\u2013120; and \"Hypnotismus und Kinematograph,\" _Zeitschrift f\u00fcr Psychotherapie und medizinische Psychologie_ 6 (1916): 310\u2013315.\n\n. O. G\u00f6tze, \"Jugendpsyche und Kinematograph,\" _Zeitschrift f\u00fcr Kinderforschung_ 16 (1911): 418 (emphasis in original).\n\n. Thierry Lefebvre quotes a similar, French objection from 1913 to film's temporal malleability: \"The cinema, with its rapid unfolding, its somewhat brutal speed of images which follow one another, distort the slow and progressive work of nature. Here is a film showing a seed which suddenly sprouts, becomes stem, flower, fruit all in just a couple of seconds. Nature does not do this; nature 'does not jump,' as told to us by the old philosophy.\" \"The Scientia Production (1911\u20131914): Scientific Popularization Through Pictures,\" _Griffithiana_ no. 47 (May 1993): 137\u2013153, here 145.\n\n. A good survey is Hartmut Rosa and William E. Scheuerman, eds., _High-Speed Society: Social Acceleration, Power, and Modernity_ (University Park: Pennsylvania State University Press, 2009).\n\n. Nordau, _Degeneration_ , 40. For more on Nordau's critique of the speed of modernity, see G\u00fcnther A. H\u00f6fler, \"La naissance de la 'nervosit\u00e9' issue de l'esprit de la modernit\u00e9 technologique. D\u00e9g\u00e9n\u00e9rescence et nomadisation chez Max Nordau et Adolph Wahrmund,\" in _Max Nordau (1849\u20131923): Critique de la D\u00e9g\u00e9n\u00e9rescence, M\u00e9diateur Franco-Allemand_ , _P\u00e8re Fondateur du Sionisme_ , ed. Delphine Bechtel, Dominique Bourel, and Jacques Le Rider (Paris: Cerf, 1996), 149\u2013160.\n\n. Nordau, _Degeneration_ , 42.\n\n. Nordau, _Degeneration_ , 55.\n\n. Jonathan Crary, _Suspensions of Perception: Attention, Spectacle, and Modern Culture_ (Cambridge, Mass.: MIT Press, 1999).\n\n. Crary, _Suspensions of Perception_ , 25. See also Friedrich Nietzsche's discussion of attention and the will to mastery in _Beyond Good and Evil_ , sect. 19, in _Basic Writings of Nietzsche_ , trans. Walter Kaufmann (New York: Modern Library, 1966), 215\u2013217.\n\n. Crary, _Suspensions of Perception_ , 17. If \"attention\" were a difficult concept to define, at least it remained ideologically productive through the twentieth century; the same cannot be said for \"will\" or \"volition,\" which lost currency after around 1900. See G. E. Berrios and M. Gili, \"Will and Its Disorders: A Conceptual History,\" _History of Psychiatry_ 6 (1995): 87\u2013104.\n\n. Gaupp, \"Der Kinematograph vom medizinischen und psychologischen Standpunkt,\" 9 (emphasis in original).\n\n. Adolf Sellmann, \"Das Geheimnis des Kinos,\" _Bild und Film_ 1, nos. 3\u20134 (1912): 65\u201367, here 66 (emphasis in original).\n\n. Wilhelm Stapel, \"Der homo cinematicus,\" _Deutsches Volkstum_ 21 (October 1919): 319\u2013320, here 319.\n\n. See the articles by Andreas Killen (notes 122 and 133 above), as well as Stefan Andriopoulos, _Possessed: Hypnotic Crimes, Corporate Fiction, and the Invention of Cinema_ (Chicago: University of Chicago Press, 2008); and Rae Beth Gordon, _Why the French Love Jerry Lewis: From Cabaret to Early Cinema_ (Stanford, Calif.: Stanford University Press, 2001).\n\n. Stefan Andriopoulos, \"Spellbound in Darkness: Hypnosis as an Allegory of Early Cinema,\" _Germanic Review_ 77, no. 2 (Spring 2002): 102\u2013116, here 103.\n\n. Leopold Laquer, \"\u00dcber die Sch\u00e4dlichkeit kinematographischer Veranstaltungen f\u00fcr die Psyche des Kindesalters,\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 27, no. 11 (1 June 1911): 221.\n\n. Laquer, \"\u00dcber die Sch\u00e4dlichkeit kinematographischer Veranstaltungen,\" 222.\n\n. The most famous of these was the case of the Borbacher Knabenmord, in which a young man accused of killing a little boy recounted the films he saw leading up to the crime. This case was a touchstone for medical and reformist literature on cinema through the 1920s. See Killen, \"The Scene of the Crime,\" 104; and Hellwig, \"\u00dcber die sch\u00e4dliche Suggestivkraft kinematographischer Vorf\u00fchrung.\"\n\n. Gaupp, \"Der Kinematograph vom medizinischen und psychologischen Standpunkt,\" 9 (emphasis in original).\n\n. Gordon, _Why the French Love Jerry Lewis_ , 128.\n\n. Gustave Le Bon, _The Crowd: A Study of the Popular Mind_ (Atlanta, Ga.: Cherokee, 1982), 16. Originally published as _Psychologie des foules_ (Paris: Alcan, 1895). Translated and published in German as _Psychologie der Massen_ (Leipzig: Klinkhardt, 1908).\n\n. Le Bon, _The Crowd_ , 21. For a version of this argument applied to film audiences, see Hermann Duenschmann, \"Kinematograph und Psychologie der Volksmenge. Eine sozialpolitische Studie,\" _Konservative Monatsschrift_ 69, no. 9 (June 1912): 920\u2013930.\n\n. Le Bon, _The Crowd_ , 29.\n\n. Didi-Huberman, _Invention of Hysteria_.\n\n. L\u00e9on Chertok and Isabelle Stengers, _A Critique of Psychoanalytic Reason: Hypnosis as a Scientific Problem from Lavoisier to Lacan_ , trans. Martha Noel Evans (Stanford, Calif.: Stanford University Press, 1992). For a detailed study of the historical relationship between hypnosis and psychoanalysis, see Andreas Mayer, _Sites of the Unconscious: Hypnosis and the Emergence of the Psychoanalytical Setting_ (Chicago: University of Chicago Press, 2013).\n\n. Albert Moll, _Hypnotism_ (London: Scott, 1890), 333 (emphasis added). Originally published as _Der Hypnotismus_ (Berlin: Fischer's Medicinische, 1889). To be fair, it should be noted that this particular application of hypnosis is relatively uncommon during the nineteenth and early twentieth centuries, when the therapeutic technique is used overwhelmingly for somatic ailments. For an example, see W. P. Carr, \"Suggestion as Used and Misused in Curing Disease,\" in _Hypnotism and Hypnotic Suggestion_ , ed. E. Virgil Neal and Charles S. Clark (Rochester: New York State Publishing, 1900), 5\u201317. For more on the experimental and therapeutic uses of hypnosis, see Mayer, _Sites of the Unconscious_.\n\n. [Henri-\u00c9tienne] Beaunis, \"L'exp\u00e9rimentation en psychologie par le somnambulisme provoqu\u00e9,\" _Revue philosophique_ 10, no. 7 (1885): 2 (emphasis in original).\n\n. Immanuel Kant, \"What Is Enlightenment?,\" in _German Aesthetic and Literary Criticism_ , ed. David Simpson (Cambridge: Cambridge University Press, 1984), 29\u201334, here 30.\n\n3. THE TASTE OF A NATION\n\n 1. \"Die Bremer Lehrerinnen und die Kinogefahr,\" _Die Lehrerin_ 30 (1913): 156, quoted in Albert Hellwig, _Kind und Kino_ (Langensalza: Beyer, 1914), 71.\n\n 2. Stephen Kern discusses the bourgeois attitude toward sexuality in _Anatomy and Destiny: A Cultural History of the Human Body_ (Indianapolis: Bobbs-Merrill, 1975). Cf. Peter Gay, _The Education of the Senses_ , vol. 1 (New York: Oxford University Press, 1984); and Michel Foucault, _The History of Sexuality_ , trans. Robert Hurley (New York: Vintage, 1990).\n\n 3. Konrad Lange, _Die k\u00fcnstlerische Erziehung der deutschen Jugend_ (Darmstadt: Bergstrae\u00dfer, 1893), 12.\n\n 4. Pierre Bourdieu, _Distinction_ (Cambridge, Mass.: Harvard University Press, 1984), 190 (emphasis in original).\n\n 5. A more complete treatment of this theme can be found in Patrice Petro, _Joyless Streets: Women and Melodramatic Representation in Weimar Germany_ (Princeton, N.J.: Princeton University Press, 1989). Ann Douglas discusses a similar concern in the United States in _The Feminization of American Culture_ (New York: Knopf, 1977).\n\n 6. See Josef Chytry, _The Aesthetic State: A Quest in Modern German Thought_ (Berkeley and Los Angeles: University of California Press, 1989).\n\n 7. Norbert Elias, _The Civilizing Process_ , vol. 1, _The History of Manners_ , trans. Edmund Jephcott (New York: Pantheon, 1978), 19. For other discussions of _Kultur_ and _Zivilisation_ in the German context, see Fritz Ringer, _The Decline of the German Mandarins: The German Academic Community, 1890\u20131933_ (Cambridge, Mass.: Harvard University Press, 1969); and Jeffrey Herf, _Reactionary Modernism: Technology, Culture, and Politics in Weimar and the Third Reich_ (Cambridge and New York: Cambridge University Press, 1984). See also J\u00f6rg Fisch, \"Zivilisation, Kultur,\" in _Geschichtliche Grundbegriffe. Historisches Lexikon zur politisch-sozialen Sprache in Deutschland_ , ed. Otto Brunner, Werner Conze, and Reinhardt Koselleck (Stuttgart: Klett, 1992), 7: 679\u2013774.\n\n 8. Raymond Geuss, \"Kultur, Bildung, Geist,\" _History and Theory_ 35, no. 2 (May 1996): 151\u2013164, here 153.\n\n 9. Another dichotomy, Ferdinand T\u00f6nnies's _Gemeinschaft_ (community) and _Gesellschaft_ (society), struck a similarly antimodernist tone. Contrasting the unity of the small, rural community, which he felt was disappearing in the modern industrial transformation, with the alienation and fragmentation of the metropolis, T\u00f6nnies was perhaps more elegiac than staunchly antimodernist. Still, the tendency to describe or criticize modern bourgeois society through reference to a precapitalist past was described by Georg Luk\u00e1cs as \"romantic anti-capitalism,\" capturing the deeply ambivalent, contradictory character of nineteenth-century reactions to industrialization. See T\u00f6nnies, _Gemeinschaft und Gesellschaft_ (Leipzig: Fues, 1887), translated by Charles Loomis as _Community and Society_ (East Lansing: Michigan State University Press, 1957); Georg Luk\u00e1cs, \"\u00dcber den Dostojewski-Nachlass,\" _Moskauer Rundschau_ 17 (22 March 1931): 4; Robert Sayre and Michael L\u00f6wy, \"Figures of Romantic Anti-capitalism,\" _New German Critique_ 32 (Spring\/Summer 1984): 42\u201392. For a good, general overview of Germany's ambivalent reaction to modernity, see Kenneth D. Barkin, \"The Crisis of Modernity, 1887\u20131902,\" in _Imagining Modern German Culture, 1889\u20131910_ , ed. Fran\u00e7oise Forster-Hahn (Washington, D.C.: National Gallery of Art, 1996), 19\u201335.\n\n 10. Dennis Sweeney, \"Reconsidering the Modernity Paradigm: Reform Movements, the Social and the State in Wilhelmine Germany,\" _Social History_ 31, no. 4 (November 2006): 405\u2013434, here 406. See also Kevin Repp, _Reformers, Critics, and the Paths of German Modernity: Anti-Politics and the Search for Alternatives, 1890\u20131914_ (Cambridge, Mass.: Harvard University Press, 2000); and Andrew Lees, _Cities, Sin, and Social Reform in Imperial Germany_ (Ann Arbor: University of Michigan Press, 2002).\n\n 11. Repp, _Reformers, Critics, and the Paths of German Modernity_ , 278.\n\n 12. This chapter began life as \"The Taste of a Nation: Training the Senses and Sensibility of Cinema Audiences in Imperial Germany,\" _Film History_ 6, no. 4 (Winter 1994): 445\u2013469.\n\n 13. On reform movements in general, see Norman Rich, _The Age of Nationalism and Reform, 1850\u20131890_ (New York: Norton, 1970), 103\u2013122; Maureen A. Flanagan, _America Reformed: Progressives and Progressivisms, 1890s_ \u2013 _1920s_ (New York: Oxford University Press, 2007); or Judith F. Stone, _The Search for Social Peace: Reform Legislation in France, 1890\u20131914_ (Albany: State University of New York Press, 1985).\n\n 14. See, e.g., such general surveys as Hans-Ulrich Wehler, _The German Empire, 1871\u20131918_ (Leamington Spa, U.K.: Berg, 1985); or David Blackbourn, _History of Germany, 1780\u20131918: The Long Nineteenth Century_ , 2d ed. (Malden, Mass.: Blackwell, 2003). See also studies of German urbanization, such as J\u00fcrgen Reulecke, _Geschichte der Urbanisierung in Deutschland_ (Frankfurt: Suhrkamp, 1985); or surveys of European modernity, such as Andrew Lees and Lynn Lees, eds., _The Urbanization of European Society in the Nineteenth Century_ (Lexington, Mass.: Heath, 1976); and Hans J\u00fcrgen Teuteberg, ed., _Urbanisierung im 19. und 20. Jahrhundert: historische und geographische Aspekte_ (Cologne: B\u00f6hlau, 1983).\n\n 15. On reform in Germany, in addition to Repp ( _Reformers, Critics, and the Paths of German Modernity_ ) and Lees and Lees ( _The Urbanization of European Society_ ), see R\u00fcdiger vom Bruch, _Wissenschaft, Politik und \u00f6ffentliche Meinung: Gelehrtenpolitik im Wilhelminischen Deutschland, 1890\u20131914_ (Husum, Germany: Matthiesen, 1980); J\u00fcrgen Reulecke, _Sozialer Frieden durch soziale Reform: Der Centralverein f\u00fcr das Wohl der Arbeitenden Klassen in der Fr\u00fchindustrialisierung_ (Wuppertal: Hammer, 1983); and vom Bruch, ed., _Weder Kommunismus noch Kapitalismus: B\u00fcrgerliche Sozialreform in Deutschland vom Vorm\u00e4rz bis zur \u00c4ra Adenauer_ (Munich: Beck, 1985). On educational reform in particular, see Christa Berg, ed., _Handbuch der deutschen Bildungsgeschichte_ (Munich: Beck, 1991); and Wolfgang Scheibe, _Die Reformp\u00e4dagogische Bewegung, 1900\u20131932: Eine einf\u00fchrende Darstellung_ , 9th ed. (Weinheim and Basel: Beltz, 1984). Some have rightly argued that, despite the implications of the concept of \"reform,\" we should be careful not to view the educational or social theories and practices that came out of this period as complete breaks with tradition. See J\u00fcrgen Oelkers, _Reformp\u00e4dagogik. Eine kritische Dogmengeschichte_ (Weinheim and Munich: Juventa, 1989).\n\n 16. A good survey of late nineteenth-century _Kulturkritik_ is David L. Gross, \" _Kultur_ and Its Discontents: The Origins of a 'Critique of Everyday Life' in Germany, 1880\u20131925,\" in _Essays on Culture and Society in Modern Germany_ , ed. Gary D. Stark and Bede Karl Lackner (College Station: Texas A&M University Press, 1982), 70\u201397.\n\n 17. _Verhandlung \u00fcber Fragen des h\u00f6heren Unterrichts, Berlin 4. bis 17. Dezember 1890_ (Berlin, 1891), 770, quoted in James C. Albisetti, _Secondary School Reform in Imperial Germany_ (Princeton, N.J.: Princeton University Press, 1983), 4. For an especially compelling discussion of the debates about the value of Greek ideals in imperial Germany, see Suzanne L. Marchand, _Down from Olympus: Archaeology and Philhellenism in Germany, 1750\u20131970_ (Princeton, N.J.: Princeton University Press, 1996).\n\n 18. See, e.g., Wilhelm Frei, _Landerziehungsheime: Darstellung und Kritik einer modernen Reformschule_ (Leipzig: Klinkhardt, 1902); or Herbert Bauer, _Zur Theorie und Praxis der ersten deutschen Landerziehungsheime: Erfahrungen zur Internats- und Ganztagserziehung aus den Hermann-Lietz-Schulen_ (Berlin: Volk und Wissen, 1961).\n\n 19. See Georg Kerschensteiner, \"Begriff der Arbeitsschule,\" in _Die deutsche Reformp\u00e4dagogik_ , ed. Wilhelm Flitner and Gerhard Kudritzki (D\u00fcsseldorf and Munich: K\u00fcpper, 1961), 222\u2013238.\n\n 20. Essays expressing the themes of \"the modern,\" \"the healthy,\" and \"the national\" might be, respectively: Hermann Kienzl, \"Theater und Kinematograph,\" _Der Strom_ 1, no. 7 (October 1911): 219\u2013221; Robert Gaupp, \"Die Gefahren des Kino,\" _S\u00fcddeutsche Monatshefte_ 9, no. 9 (1911\/1912): 363\u2013366; and Albert Hellwig, \"Kinematograph und Zeitgeschichte,\" _Die Grenzboten_ 72, no. 39 (1913): 612\u2013620. These and other representative essays can be found in J\u00f6rg Schweinitz, ed., _Prolog vor dem Film_ : _Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ (Leipzig: Reclam, 1992). Essays from this period are also collected in Anton Kaes, ed., _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_ (T\u00fcbingen: Niemeyer, 1978); and Fritz G\u00fcttinger, ed., _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ (Frankfurt: Deutsches Filmmuseum, 1984). Among the numerous commentaries, see especially Kaes's introduction, revised and translated as \"Literary Intellectuals and the Cinema: Charting a Controversy (1909\u20131929),\" _New German Critique_ 40 (Winter 1987): 7\u201334; Heide Schl\u00fcpmann, _Unheimlichkeit des Blicks: Das Drama des fr\u00fchen deutschen Kinos_ (Frankfurt: Stroemfeld\/Roter Stern, 1990), 189\u2013243; and Sabine Hake, _The Cinema_ ' _s Third Machine: Writing on Film in Germany, 1907\u20131933_ (Lincoln: University of Nebraska Press, 1993), 27\u201342.\n\n 21. Repp ( _Reformers, Critics, and the Paths of German Modernity_ ) and Sweeney (\"Reconsidering the Modernity Paradigm\") emphasize imperial Germany's diversity of approaches to the problems of modernity, as well as the deep ambivalence toward the modern that most elites felt. On the other hand, for histories of ideas that stress the retrograde elements of German society and the reactionary responses that, for some, foreshadow National Socialism, see Fritz Stern, _The Politics of Cultural Despair: A Study in the Rise of the Germanic Ideology_ (Berkeley and Los Angeles: University of California Press, 1961); or George L. Mosse, _The Crisis of German Ideology: Intellectual Origins of the Third Reich_ (New York: Grosset & Dunlap, 1964).\n\n 22. Paul Schultze-Naumberg, _Die Kultur des weiblichen K\u00f6rpers als Grundlage der Frauenkleidung_ , quoted in Kern, _Anatomy_ , 15. Schultze-Naumberg shifted easily from advocating \"natural clothing\" to supporting art fashioned after natural bodies; during the Third Reich he was an architect of the campaign against \"degenerate\" art. See Kern, _Anatomy_ , 223\u2013226.\n\n 23. Carl Heinrich Stratz, _Die Frauenkleidung und ihre nat\u00fcrliche Entwicklung_ (Stuttgart: Enke, 1900).\n\n 24. See Schl\u00fcpmann, _Unheimlichkeit_ , 8\u201325; Beth Irwin Lewis, \" _Lustmord_ : Inside the Windows of the Metropolis,\" in _Berlin: Culture and Metropolis_ , ed. Charles W. Haxthausen and Heidrun Suhr (Minneapolis: University of Minnesota Press, 1990), 111\u2013140; and the essays included in J. Edward Chamberlin and Sander L. Gilman, eds., _Degeneration: The Dark Side of Progress_ (New York: Columbia University Press, 1985).\n\n 25. Along with Kaes (\"Literary Intellectuals and the Cinema\"), Schl\u00fcpmann ( _Unheimlichkeit_ ), and Hake ( _Third Machine_ ), see Miriam Hansen, \"Early Silent Cinema: Whose Public Sphere?,\" _New German Critique_ 29 (Spring\/Summer 1983): 147\u2013184. On women and reform in Germany, see Christoph Sach\u00dfe, _M\u00fctterlichkeit als Beruf: Sozialarbeit, Sozialreform und Frauenbewegung, 1871\u20131929_ (Frankfurt: Suhrkamp, 1986); and Ann Taylor Allen, _Feminism and Motherhood in Germany, 1800\u20131914_ (New Brunswick, N.J.: Rutgers University Press, 1991).\n\n 26. Susanne Asche, \"F\u00fcrsorge, Partizipation und Gleichberechtigung\u2014die Leistungen der Karlsruherinnen f\u00fcr die Entwicklung zur Gro\u00dfstadt (1859\u20131914),\" in _Karlsruher Frauen, 1715\u20131945: Eine Stadtgeschichte_ (Karlsruhe: Badenia, 1992), 171\u2013256.\n\n 27. Eventually exemplified by Oswald Spengler's _The Decline of the West_ , trans. Charles Francis Atkinson (New York: Knopf, 1926\u201328). There are numerous commentaries, but see, e.g., Ringer, _The Decline of the German Mandarins_ ; Klaus Vondung, \"Zur Lage der Gebildeten in der wilhelminischen Zeit,\" in _Das wilhelminische Bildungsb\u00fcrgertum Zur Sozialgeschichte seiner Ideen_ , ed. Klaus Vondung (G\u00f6ttingen: Vandenhoeck & Ruprecht, 1976), 20\u201333; or Charles E. McClelland, \"The Wise Man's Burden: The Role of Academicians in Imperial German Culture,\" in Stark and Lackner, _Essays on Culture and Society in Modern Germany_ , 45\u201369. An excellent expression of the perceived loss of cultural authority of experts in an age of mass culture and mediocre scientists is Ernst Meumann, \"Wilhelm Wundt. Zu seinem achtzigsten Geburtstag,\" _Deutsche Rundschau_ 38, no. 11 (August 1912): 193\u2013224.\n\n 28. On the vital role of feminist activists in shaping the direction of reform movements and the public sphere in general, see Allen, _Feminism and Motherhood in Germany, 1800\u20131914_ ; and Kathleen Canning, _Languages of Labor and Gender: Female Factory Work in Germany, 1850\u20131914_ (Ithaca, N.Y.: Cornell University Press, 1996).\n\n 29. Mirjam Storim, \"'Einer, der besser ist, als sein Ruf': Kolportageroman und Kolportagebuchhandel um 1900 und die Haltung der Buchbranche,\" in _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , ed. Kaspar Maase and Wolfgang Kaschuba (Cologne: B\u00f6hlau, 2001), 252\u2013282, here 255\u2013256. See also Rudolf Schenda, _Die Lesestoffe der kleinen Leute: Studien zur popul\u00e4ren Literatur im 19. und 20. Jahrhundert_ (Munich: Beck, 1976).\n\n 30. Luke Springman, \"Poisoned Hearts, Diseased Minds, and American Pimps: The Language of Censorship in the _Schund und Schmutz_ Debates,\" _German Quarterly_ 68, no. 4 (Autumn 1995): 408\u2013429, here 413.\n\n 31. Corey Ross, _Media and the Making of Modern Germany: Mass Communications, Society, and Politics from the Empire to the Third Reich_ (New York: Oxford University Press, 2008), 66.\n\n 32. Kaspar Maase, \"Krisenbewu\u00dftsein und Reformorientierung: Zum Deutungshorizont der Gegener der modernen Popul\u00e4rk\u00fcnste 1880\u20131918,\" in Maase and Kaschuba, _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , 290\u2013342. See also his \"Struggling About 'Filth and Trash': Educationalists and Children's Culture in Germany Before the First World War,\" _Paedagogica Historica_ 34, no. 1 (1998): 8\u201328.\n\n 33. Professor Dr. Fri\u00df Johannesson, \"Das Lesen der Jugend au\u00dferhalb der Schule,\" _Die Hochwacht_ no. 2 (November 1911), quoted in Kara L. Ritzheimer, \"Protecting Youth from 'Trash': Anti- _Schund_ Campaigns in Baden, 1900\u20131933,\" PhD diss. (State University of New York\u2013Binghamton, 2007), 25.\n\n 34. Class warfare, however, was not unknown in these campaigns, especially given the historical coincidence of the rise of mass entertainment and the rise of the Social Democratic Party of Germany (SPD), which many (such as Karl Brunner) saw as uncoincidental and fought both with equal vigor. For more, see Ross, _Media and the Making of Modern Germany_.\n\n 35. A fine articulation of Brunner's position with regard to film (and the SPD) is _Der Kinematograph von heute_ \u2014 _eine Volksgefahr_ (Berlin: Vaterl\u00e4ndischen Schriftenverbandes, 1913).\n\n 36. Other trade periodicals included _Der deutsche Lichtspiel-Theater-Besitzer_ (Berlin, 1909\u20131914), _Erste Internationale Film-Zeitung_ (Berlin, 1908\u20131920), _Film und Lichtbild_ (Stuttgart, 1912\u20131914), and _Die Lichtbild-B\u00fchne_ (Berlin, 1908\u20131940). Helmut H. Diederichs provides a more complete survey of the trade press in his _Anf\u00e4nge deutscher Filmkritik_ (Stuttgart: Fischer, 1986). On _Der Kinematograph_ in particular, see Thomas Schorr, \"Die Film- und Kinoreformbewegung und die Deutsche Filmwirtschaft. Eine Analyse des Fachblatts _Der Kinematograph_ (1907\u20131935) unter p\u00e4dagogischen und publizistischen Aspekten,\" PhD diss. (Universit\u00e4t der Bundeswehr, Munich, 1990). Hake also discusses the trade press in _Third Machine_ , 3\u201326.\n\n 37. C. H. Dannmeyer, _Bericht der Kommission f\u00fcr_ \" _Lebende Photographien_ \" (Hamburg: Kampen, 1907), 27\u201328.\n\n 38. Hellwig, _Kind und Kino_ , 22. Hellwig wrote much on _Schundfilms_ and censorship, including \"Die Beziehungen zwischen Schundliteratur, Schundfilms und Verbrechen,\" _Archiv f\u00fcr Kriminal-Anthropologie und Kriminalistik_ 51, no. 1 (24 January 1913): 1\u201332; \"Die ma\u00dfgebenden Grunds\u00e4tze f\u00fcr Verbote von Schundfilms nach geltendem und k\u00fcnstigem Rechte,\" _Verwaltungsarchiv_ 21 (1913): 405\u2013455; and _Die Filmzensur: Eine rechtsdogmatische und rechtpolitische Er\u00f6rterung_ (Berlin: Frankenstein, 1914).\n\n 39. See Eileen Bowser, _The Transformation of Cinema, 1907\u20131915_ (New York: Scribner, 1990), 37\u201352. See also J. A. Lindstrom, \"'Getting a Hold Deeper in the Life of the City': Chicago Nickelodeons, 1905\u20131914.\" PhD diss. (Northwestern University, 1998); Lee Grieveson, _Policing Cinema: Movies and Censorship in Early-Twentieth-Century America_ (Berkeley: University of California Press, 2004); and Jennifer Lynn Peterson, _Education in the School of Dreams: Travelogues and Early Nonfiction Film_ (Durham, N.C.: Duke University Press, 2013), esp. chap. 3.\n\n 40. Dannmeyer, _Bericht der Kommission_ , 39.\n\n 41. \"Die Er\u00f6ffnung des Reform-Kinematographentheater,\" _Der Kinematograph_ no. 32 (7 August 1907). _Der Kinematograph_ was not paginated. For more on the sometimes stuffy discussion of ventilation, see a translation of the American Society of Heating and Ventilation Engineers' \"Report of Committee on Standards for Ventilation Legislation for Motion Picture Show Places,\" in _Gesundheits-Ingenieur_ 36, no. 22 (31 May 1913): 409\u2013410; and a German response, Konrad Meier, \"Vorschriften \u00fcber L\u00fcftung von Kinotheatern,\" _Gesundheits-Ingenieur_ 36, no. 26 (28 June 1913): 483\u2013484.\n\n 42. \"Ein kurzer R\u00fcckblick auf die erste Woche des Reform-Kinematographen-Theaters,\" _Der Kinematograph_ no. 33 (14 August 1907).\n\n 43. \"Kinematographische Reformvereinigung,\" _Der Kinematograph_ no. 43 (23 October 1907).\n\n 44. For more on _Kino-Kommissions_ , see Sabine Lenk and Frank Kessler, \"The Institutionalization of Educational Cinema: The Case of the _Kinoreformbewegung_ in Germany,\" in _The Institutionalization of Educational Cinema: Educational Cinemas in North America and Europe in the 1910s and 1920s_ , ed. Marina Dahlquist and Joel Frykholm (Bloomington: Indiana University Press, forthcoming). See also Rudolf W. Kipp, _Bilddokumente zur Geschichte des Unterrichtsfilms_ (Gr\u00fcnwald, Germany: Institut f\u00fcr Film und Bild in Wissenschaft und Unterricht, 1975), 13\u201315.\n\n 45. \"Kinematographische Reformvereinigung.\" Oskar Kalbus, one of the driving forces behind UFA's _Kulturfilm_ division, later intimated that these donations were not uncontroversial: \"Although this association was soon sharply criticized because of the close relationship between Lemke and the French film industry, it can nevertheless take credit for having given the first important impetus for the introduction of film in schools.\" Kalbus, \"Abri\u00df einer Geschichte der deutschen Lehrfilmbewegung,\" in _Das Kulturfilmbuch_ , ed. Edgar Beyfu\u00df and Alexander Kossowsky (Berlin: Chryselius'scher, 1924), 1\u201313, here 3.\n\n 46. Hermann Lemke, \"Die Verwertung und Nutzbarmachung neuer Film-Ideen\u2014K\u00fcnstlerische Films,\" _Der Kinematograph_ no. 57 (29 January 1908).\n\n 47. Ludwig Brauner, \"Die Kino-Ausstellung in Berlin,\" _Der Kinematograph_ no. 104 (25 December 1908).\n\n 48. Indeed, by this time the relations between the exhibitors and the reformers and trade journals were downright hostile. See \"Die Kino-Austellung und 'Wir,'\" _Erste Internationale Film-Zeitung_ 6, no. 50 (14 December 1912): 52.\n\n 49. Hermann H\u00e4fker, \"Eine Reise an die Quellen der Kinematographie,\" _Der Kinematograph_ no. 163 (9 February 1910); and 172 (13 April 1910).\n\n 50. Hermann Lemke, \"Volkst\u00fcmliche Reisebeschreibungen,\" _Der Kinematograph_ no. 34 (21 August 1907).\n\n 51. _Der Kinematograph_ no. 258 (6 December 1911).\n\n 52. Paul Samuleit and Emil Borm, _Der Kinematograph als Volks- und Jugendbildungsmittel_ (Berlin: Gesellschaft f\u00fcr Verbreitung von Volksbildung, 1912), 23\u201324, quoted in Hake, _Third Machine_ , 36 (translation modified).\n\n 53. Hake, _Third Machine_ , 36\u201338.\n\n 54. Unwilling to rely just on production companies, by 1909 Lemke hoped to create a cost-sharing distribution cooperative among interested schools. See Hermann Lemke, _Praktische Forderungen f\u00fcr die Verwertung der Kinematographie im Unterricht_ (Friedenau: Schule und Technik, 1909). Georg Victor Mendel agreed and followed up with a plan to open a \"purely scientific [ _wissenschaftlichen_ ] theater\": Georg Victor Mendel, _Kinematographie und Schule: Plan zur Gr\u00fcndung eines rein wissenschaftlichen Theaters f\u00fcr Kinematographie und Projektion_ (Berlin: privately printed, 1909).\n\n 55. The legal discourse on cinema in Germany is far too vast to even attempt a survey here. Albert Hellwig's reviews are the best place to start, however: _Rechtsquellen des \u00f6ffentlichen Kinematographenrechts_ (M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1913); and _\u00d6ffentliches Lichtspielrecht_ (M. Gladbach: Volksvereins, 1921). Other contemporary surveys include Bruno May, _Das Recht des Kinematographen_ (Berlin: Falk, 1912); and Hans M\u00fcller-Sanders, \"Die Kinematographenzensur in Preu\u00dfen,\" PhD diss. (Badischen Ruprecht-Karls-Universit\u00e4t, Heidelberg, 1912). See also Gary D. Stark, \"Cinema, Society, and the State,\" in Stark and Lackner, _Essays on Culture and Society in Modern Germany_ , 122\u2013166; and Kaspar Maase, \"Massenkunst und Volkserziehung: Die Regulierung von Film und Kino im deutschen Kaiserreich,\" _Archiv f\u00fcr Sozialgeschichte_ 41 (2001): 39\u201377.\n\n 56. Hellwig, _\u00d6ffentliches Lichtspielrecht_ , 32\u201333. Not all regulations applied to the same theaters at the same time, of course. For an excellent case study of the variety of local tactics, see Amelie Duckwitz, Martin Loiperdinger, and Susanne Theisen, \"'Kampf dem Schundfilm!': Kinoreform and Jugendschutz in Trier,\" _KINtop_ 9 (2000): 53\u201363.\n\n 57. My presentation of the GVV is indebted to Schorr, \"Die Film- und Kinoreformbewegung,\" 81\u201394; and Horst Dr\u00e4ger, _Die Gesellschaft f\u00fcr Verbreitung von Volksbildung: Eine historisch-problemgeschichtliche Darstellung von 1871\u20131914_ (Stuttgart: Klett, 1975), 226\u2013237.\n\n 58. Dr\u00e4ger, _Gesellschaft f\u00fcr Verbreitung_ , 236.\n\n 59. Willi Warstat and Franz Bergmann, _Kino und Gemeinde_ (M. Gladbach: Volksvereins, 1913), 114\u2013116.\n\n 60. Heiner Schmitt, _Kirche und Film: Kirchliche Filmarbeit in Deutschland von ihren Anf\u00e4ngen bis 1945_ (Boppard: Boldt, 1979), 41. For more on the role of Catholicism in this sphere, see Margaret Stieg Dalton, _Catholicism, Popular Culture, and the Arts in Germany, 1880\u20131933_ (Notre Dame, Ind.: University of Notre Dame Press, 2005).\n\n 61. The \"monopoly\" system, established in Germany between 1910 and 1911, allowed distributors to acquire sole rights to a film and pass this exclusivity to cinema managers in the form of local exhibition rights. The theater owner's local monopoly enabled him to charge more and make, for the first time in Germany, a considerable profit. See Corinna M\u00fcller, _Fr\u00fche deutsche Kinematographie: formale, wirtschaftliche und kulturelle Entwicklungen 1907\u20131912_ (Stuttgart and Weimar: Metzler, 1994), 126\u2013158; as well as her essay, \"The Emergence of the Feature Film in Germany Between 1910 and 1911,\" in _Before Caligari: German Cinema, 1895\u20131920_ , ed. Paolo Cherchi Usai and Lorenzo Codelli (Madison: University of Wisconsin Press, 1990), 94\u2013113.\n\n 62. The best survey of the role of the _Lichtbilderei_ in the reform movement is Diederichs, _Anf\u00e4nge deutscher Filmkritik_ , 84\u201388.\n\n 63. Dr\u00e4ger, _Gesellschaft f\u00fcr Verbreitung_ , 234\u2013235.\n\n 64. Volker Schulze, \"Fr\u00fche kommunale Kinos und die Kinoreformbewegung in Deutschland bis zum Ende des ersten Weltkriegs,\" _Publizistik_ 22, no. 1 (January\u2013March 1977): 61\u201371.\n\n 65. Minutes from the meeting of the community representatives of Eickel, 14 May 1912 (archive of the City of Wanne-Eickel), quoted in Schulze, \"Fr\u00fche kommunale Kinos,\" 64.\n\n 66. Rudolf Pechel in _Literarischen Echo_ 16 (1913\/1914): 582, quoted in Ludwig Greve, Margot Pehle, and Heidi Westhoff, eds., _H\u00e4tte ich das Kino! Die Schriftsteller und der Stummfilm_ (Munich: K\u00f6sel, 1976), 68. Pechel reviewed Willy Rath's _Kino und B\u00fchne_ (M. Gladbach: Volksvereins, 1913).\n\n 67. Arthur Mellini, \"Die ganze Richtung passt uns nicht!\" _Lichtbild-B\u00fchne_ 5 (4 February 1911): 3\u20134, quoted in Karen J. Kenkel, \"The Nationalisation of the Mass Spectator in Early German Film,\" in _Celebrating 1895: The Centenary of Cinema_ , ed. John Fullerton (Sydney: Libbey, 1998), 155\u2013162, here 158.\n\n 68. Max Kullmann, \"Die Entwicklung des deutschen Lichtspieltheater,\" PhD diss. (University of Nuremberg, 1935), quoted in Hake, _Third Machine_ , 27. Kullmann quoted a film theater owner.\n\n 69. Siegfried Kracauer, _From Caligari to Hitler_ (Princeton, N.J.: Princeton University Press, 1947), 19.\n\n 70. Hake, _Third Machine_ , 28.\n\n 71. Helmut Kommer, _Fr\u00fcher Film und sp\u00e4te Folgen: Zur Geschichte der Film- und Fernseherziehung_ (Berlin: Basis, 1979).\n\n 72. Hermann Lemke, _Die Kinematographie der Gegenwart, Vergangenheit und Zukunft_ (Leipzig: Demme, 1911), 24.\n\n 73. Many scholars have stressed the connection between \"the masses\" and \"the feminine\" as an indication of the anxieties and spirit of the age. This line of reasoning is indeed extremely significant, but the connection between \"the masses\" and \"children\" (as a similarly charged rhetorical construction) deserves a closer look. On the masses as feminine, see esp. Susanna Barrows, _Distorting Mirrors: Visions of the Crowd in Late Nineteenth-Century France_ (New Haven: Yale University Press, 1981). For another, parallel examination of German reformers on cinema, children, and the masses, see Karen J. Kenkel, \"The Adult Children of Early Cinema,\" _Women in German Yearbook_ (2000): 137\u2013160.\n\n 74. Lorenz Pieper, \"Kino und Drama,\" _Bild und Film_ 1, no. 1 (1912): 5.\n\n 75. Georg Luk\u00e1cs, \"Thoughts Toward an Aesthetic of the Cinema,\" trans. Janelle Blankenship, _Polygraph_ 13 (2001): 13\u201318, here 16 (emphasis in original). Originally published as \"Gedanken zu einer Aesthetik des 'Kino,'\" _Frankfurter Zeitung_ 251 (10 September 1913): 1\u20132.\n\n 76. This phrase and _vom Kinde aus_ are attributed to Hamburg pedagogue Johannes Gl\u00e4ser, one of many who popularized and realized Key's suggestions. See Scheibe, _Die Reformp\u00e4dagogische Bewegung, 1900\u20131932_ , 65.\n\n 77. Ellen Key, \"Erziehung,\" in _Das Jahrhundert des Kindes_ (Berlin, 1905), in Flitner and Kudritzki, _Die deutsche Reformp\u00e4dagogik_ , 52\u201354, here 52.\n\n 78. Stephen Kern, \"Freud and the Emergence of Child Psychology, 1880\u20131910,\" PhD diss. (Columbia University, 1970), 264.\n\n 79. Charles Darwin, _The Descent of Man_ (London, 1871), quoted in Kern, \"Freud,\" 212.\n\n 80. The importance of \"primitivism\"\u2014of which Darwin was a prime but not uncommon example\u2014for this connection between children (or the feminine) and the masses cannot be underestimated. For its role in shaping turn of the century cultural agendas in Germany, see Doris Kaufmann, \"'Pushing the Limits of Understanding': The Discourse on Primitivism in German _Kulturwissenschaften_ , 1880\u20131930,\" _Studies in History and Philosophy of Science_ 39 (2008): 434\u2013443. For examinations in relation to cinema, see Assenka Oksiloff, _Picturing the Primitive: Visual Culture, Ethnography, and Early German Cinema_ (New York: Palgrave, 2001); and Beth Corzo-Duchardt, \"Primal Screen: Primitivism and American Silent Film Spectatorship,\" PhD diss. (Northwestern University, 2013).\n\n 81. Gustave Le Bon, _The Crowd: A Study of the Popular Mind_ (Atlanta, Ga.: Cherokee, 1982), 16. Hereafter cited parenthetically. Originally published as _Psychologie des foules_ (Paris: Alcan, 1895). Translated and published in German as _Psychologie der Massen_ (Leipzig: Klinkhardt, 1908). See also Robert A. Nye, _The Origins of Crowd Psychology: Gustave Le Bon and the Crisis of Mass Democracy in the Third Republic_ (London and Beverly Hills, Calif.: Sage, 1975)..\n\n 82. Erika Apfelbaum and Gregory R. McGuire, \"Models of Suggestive Influence and the Disqualification of the Social Crowd,\" in _Changing Conceptions of Crowd Mind and Behavior_ , ed. C. F. Graumann and S. Moscovici (New York and Berlin: Springer, 1986), 27\u201350.\n\n 83. See Walter Serner, \"Kino und Schaulust,\" in Schweinitz, _Prolog vor dem Film_ , 208\u2013214. Originally published in _Die Schaub\u00fchne_ 9, nos. 34\/35 (1913): 807\u2013811. Chapter 4 will discuss _Schaulust_ and this essay in more detail.\n\n 84. Dannmeyer, _Bericht der Kommission_ , 27\u201328.\n\n 85. Emilie Altenloh, _Zur Soziologie des Kino: Die Kino-Unternehmung und die sozialen Schichten ihrer Besucher_ (Jena: Diederichs, 1914), 91.\n\n 86. Altenloh, _Zur Soziologie des Kino_ , 65.\n\n 87. Albert Hellwig, \"\u00dcber die sch\u00e4dliche Suggestivkraft kinematographischer Vorf\u00fchrung,\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 20, no. 6 (15 March 1914): 122. Hellwig was reviewing and citing from an article by Italian psychiatrist Giuseppe d'Abundo, \"Sopra alcuni particolari effetti delle projezioni cinematografiche nei nevrotici,\" _Rivista Italiana di Neuropatologia, Psichiatria ed Elettroterapia_ 4, no. 10 (October 1911): 433\u2013442; for another German review, see \"Kinematograph als Krankheitsstifter,\" in _Fortschritte der Medizin_ 30 (1912): 302. For more on Italian uses of cinematography in the human sciences, see Silvio Alovisio, _L'occhio sensibile. Cinema e scienze della mente nell'Italia del primo Novecento. Con una antologia di testi d'epoca_ (Turin: Edizioni Kaplan, 2013).\n\n 88. Anson Rabinbach, _The Human Motor: Energy, Fatigue, and the Origins of Modernity_ (Berkeley and Los Angeles: University of California Press, 1990), 157.\n\n 89. Hermann Lemke, \"Die kinematographische Reformpartei, ihre Aufgaben und Ziele,\" _Der Kinematograph_ no. 42 (16 October 1907).\n\n 90. Albert Hellwig, _Schundfilms: Ihr Wesen, ihre Gefahren und ihre Bek\u00e4mpfung_ (Halle an der Saale, Germany: Waisenhaus, 1911), 33, quoted in Hake, _Third Machine_ , 39.\n\n 91. Friedrich Schiller, _On the Aesthetic Education of Man_ , trans. Elizabeth M. Wilkinson and L. A. Willoughby (Oxford: Oxford University Press, 1967), 161.\n\n 92. \"Die Kultur-Arbeit des Kinematographen-Theaters,\" _Die Lichtbild-B\u00fchne_ 2, no. 41 (4 February 1909).\n\n 93. Erwin Ackerknecht, _Das Lichtspiel im Dienste der Bildungspflege: Handbuch f\u00fcr Lichtspielreformer_ (Berlin: Weidmannsche, 1918), 66.\n\n 94. August Julius Langbehn, \"Rembrandt als Erzieher,\" in _Die Kunsterziehungsbe-wegung_ , ed. Hermann Lorenzen (Bad Heilbrunn, Germany: Klinkhardt, 1966), 7\u201317. Hereafter cited parenthetically. Stern's _The Politics of Cultural Despair_ provides the standard account of Langbehn's place in history.\n\n 95. For more on the reverberations of Langbehn's essay through Wilhelmine Germany, see Corona Hepp, _Avantgarde: Moderne Kunst, Kulturkritik und Reformbewegungen nach der Jahrhundertwende_ (Munich: Deutscher Taschenbuch, 1987).\n\n 96. Alfred Lichtwark, \"Der Deutsche der Zukunft,\" in Flitner and Kudritzki, _Die deutsche Reformp\u00e4dagogik_ , 99\u2013110, here 104 (emphasis in original). Hereafter cited parenthetically. Lichtwark and Langbehn were acquaintances; Lichtwark introduced Langbehn to the work of Rembrandt in 1887. See Gisela Wilkending, _Volksbildung und P\u00e4dagogik_ \" _vom Kinde aus_ \" _: Eine Untersuchung zur Geschichte der Literaturp\u00e4dagogik in den Anf\u00e4ngen der Kunsterziehungsbewegung_ (Weinheim, Germany: Beltz, 1980), 79\u201385. For more on Lichtwark, see Julius Gebhard, _Alfred Lichtwark und die Kunsterziehungsbewegung in Hamburg_ (Hamburg: Hoffmann und Campe, 1947); Hans Pr\u00e4ffcke, _Der Kunstbegriff Alfred Lichtwarks_ (Hildesheim, Z\u00fcrich, and New York: Olms, 1986); Carolyn Kay, _Art and the German Bourgeoisie: Alfred Lichtwark and Modern Painting in Hamburg, 1886\u20131914_ (Toronto and Buffalo, N.Y.: University of Toronto Press, 2002); but esp. Jennifer Jenkins, _Provincial Modernity: Local Culture and Liberal Politics in Fin-de-Si\u00e8cle Hamburg_ (Ithaca, N.Y.: Cornell University Press, 2003).\n\n 97. Jenkins, _Provincial Modernity_ , 76.\n\n 98. Konrad Lange, \"Das Wesen der k\u00fcnstlerischen Erziehung,\" in Lorenzen, _Kunsterziehungsbewegung_ , 21\u201326, here 22. For more on the art education movement, see Peter Joerissen, _Kunsterziehung und Kunstwissenschaft im wilhelminischen Deutschland, 1871\u20131918_ (Cologne and Vienna: B\u00f6hlau, 1979).\n\n 99. Lange, _Die k\u00fcnstlerische Erziehung der deutschen Jugend_ , 10.\n\n. Lange, \"Das Wesen der k\u00fcnstlerischen Erziehung,\" 26.\n\n. Alfred Lichtwark, \"Die Aufgaben der Kunsthalle: Antrittsrede den 9. December 1886,\" In _Drei Programme_ , 2d ed. (Berlin: Cassirer, 1902), 11\u201331, here 29.\n\n. Eckard Schaar, \"Zust\u00e4nde,\" in Alfred Lichtwark, _Erziehung des Auges: Ausgew\u00e4hlte Schriften_ , ed. Eckard Schaar (Frankfurt: Fischer, 1991), 8, quoted in Jenkins, _Provincial Modernity_ , 64.\n\n. Alfred Lichtwark, _Die Bedeutung der Amateur-Photographie_ (Halle an der Saale, Germany: Knapp, 1894), 1.\n\n. Alfred Lichtwark, \"Museen als Bildungsst\u00e4tten,\" _Der Deutsche der Zukunft_ (Berlin: Cassirer, 1905), 89\u2013107.\n\n. Alfred Lichtwark, _\u00dcbungen in der Betrachtung von Kunstwerken_ (Dresden: K\u00fchtmann, 1900), 17.\n\n. See, for instance, John Dewey, _Art as Experience_ (New York: Putnam, 1984).\n\n. See, e.g., Walter Geisel, _Wie ich mit meinen Jungens Kunstwerke betrachte_ (Gl\u00fcckstadt: Geisel, 1904); Paul Quensel, _Meisterbilder und Schule: Anregungen zu praktischen Versuchen_ (Munich: Kunstwart-Verl, 1905); Leipziger Lehrerverein, ed., _Bildbetrachtungen: Arbeiten aus der Abteilung f\u00fcr Kunstpflege des Leipziger Lehrervereins_ (Leipzig: Teubner, 1906); and Ulrich Diem, _Bildbetrachtung: Eine Wegleitung f\u00fcr Kunstfreunde_ (St. Gallen: Fehr'sche, 1919). _Bildbetrachtung_ was even more popular as a teaching method after World War II.\n\n. Heinrich Wolgast, \"Die Bedeutung der Kunst f\u00fcr die Erziehung,\" in Lorenzen, _Die Kunsterziehungsbewegung_ , 17\u201320, here 19.\n\n. For a European history of the movement, along with a clear explication of the principles of _Bild_ \\- _und Kunstbetrachtung_ , see Ludwig Praehauser, _Erfassen und Gestalten: Die Kunsterziehung als Pflege formender Kr\u00e4fte_ (Salzburg: M\u00fcller, 1950).\n\n. Pestalozzi, _How Gertrude Teaches Her Children_ [1801], ed. Ebenezer Cooke, trans. Lucy E. Holland and Frances C. Turner, 2d ed. (Syracuse, N.Y.: Bardeen, 1898), tenth letter, 220 (emphasis in original).\n\n. Clive Ashwin, \"Pestalozzi and the Origins of Pedagogical Drawing,\" _British Journal of Educational Studies_ 29, no. 2 (June 1981): 138\u2013151, here 146.\n\n. Keiichi Takaya, \"The Method of _Anschauung_ : From Johann H. Pestalozzi to Herbert Spencer,\" _Journal of Educational Thought_ 37, no. 1 (2003): 77\u201399, here 84.\n\n. Robert Ulich, \"Pestalozzi, Johann Heinrich,\" in _The Encyclopedia of Philosophy_ , vol. 6, ed. Paul Edwards (New York: Macmillan, 1967), 121\u2013122.\n\n. Melanie Judith Keene, \"Object Lessons: Sensory Science Education, 1830\u20131870,\" PhD diss. (University of Cambridge, 2008), 51\u201354.\n\n. Ulich, \"Pestalozzi, Johann Heinrich,\" 122.\n\n. Ashwin, \"Pestalozzi and the Origins of Pedagogical Drawing,\" 146.\n\n. Christopher Owen Ritter, \"Re-presenting Science: Visual and Didactic Practice in Nineteenth-Century Chemistry,\" PhD diss. (University of California, Berkeley), 2001, 128.\n\n. W. T. Harris, editor's preface to _Herbart_ ' _s ABC of Sense Perception and Minor Pedagogical Works_ , by Johann Friedrich Herbart, ed. and trans. William J. Eckoff (New York: Appleton, 1896), vii.\n\n. Herbert Spencer, _Education: Intellectual, Moral, and Physical_ [1861] (New York: Appleton, 1896), quoted in Takaya, \"The Method of _Anschauung_ ,\" 81.\n\n. The best survey of its dissemination in Germany is Gottlieb Gustav Deussing, \"Der Anschauungsunterricht in der deutschen Schule von Comenius bis zur Gegenwart,\" PhD diss. (Universit\u00e4t Jena, 1884).\n\n. On _Anschauungsunterricht_ in the natural sciences, see Massimiano Bucchi, \"Images of Science in the Classroom: Wallcharts and Science Education, 1850\u20131920,\" _British Journal for the History of Science_ 31, no. 2 (1998): 161\u2013184; Lynn K. Nyhart, \"Science, Art, and Authenticity in Natural History Displays,\" in _Models: The Third Dimension of Science_ , ed. Soraya de Chadarevian and Nick Hopwood (Stanford, Calif.: Stanford University Press, 2004), 307\u2013335; Nyhart, _Modern Nature: The Rise of the Biological Perspective in Germany_ (Chicago: University of Chicago Press, 2009), esp. chap. 5, \"The 'Living Community' in the Classroom\"; Henning Schmidgen, \"Pictures, Preparations, and Living Processes: The Production of Immediate Visual Perception ( _Anschauung_ ) in Late-19th-Century Physiology,\" _Journal of the History of Biology_ 37, no. 3 (October 2004): 477\u2013513; and Schmidgen, \"1900\u2014The Spectatorium: On Biology's Audiovisual Archive,\" _Grey Room_ 43 (2011): 42\u201365.\n\n. I have examined the trope of efficiency in visual education in \"The Efficiency of Images: Educational Effectiveness and the Modernity of Motion Pictures,\" in _The Visual Culture of Modernism_ , SPELL: Swiss Papers in English Language and Literature 26, ed. Deborah L. Madsen and Mario Klarer (T\u00fcbingen: Narr, 2011), 41\u201359; and \"Dissecting the Medical Training Film,\" in _Beyond the Screen: Institutions, Networks and Publics of Early Cinema_ , ed. Marta Braun et al. New Barnet, U.K.: Libbey, 2012), 161\u2013167.\n\n. Carl Jacobj, \"Anschauungsunterricht und Projektion,\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und mikroskopische Technik_ 36, no. 4 (1919): 273\u2013314, here 275, quoted in Schmidgen, \"1900\u2014The Spectatorium,\" 51.\n\n. Ashwin, \"Pestalozzi and the Origins of Pedagogical Drawing,\" 146 (emphasis in original).\n\n. For a contemporary assessment of the gap between reform ideals and actual practice, see I. L. Kandel, \"Germany,\" in _Comparative Education: Studies of the Educational Systems of Six Modern Nations_ , ed. Peter Sandiford (London and Toronto: Dent, 1918), 121\u2013130; or, to a lesser extent, the apologetic William S. Learned, _An American Teacher_ ' _s Year in a Prussian Gymnasium_ (New York: Educational Review, 1911).\n\n. \"Besuch kinematographischer Vorf\u00fchrung durch Sch\u00fcler h\u00f6herer Lehranstalten (Breslau 1910)\" and \"Besuch der Kinematographentheater durch Sch\u00fcler und Sch\u00fclerinnen sowie durch die Z\u00f6glinge der Seminare und Pr\u00e4parandenanstalten (Berlin 1912)\" in _Dokumente zur Geschichte der Schulfilmbewegung in Deutschland_ , ed. Fritz Terveen (Emsdetten: Lechte, 1959), 16\u201317.\n\n. Otfrid von Hanstein, _Kinematographie und Schule. Ein Vorschlag zur Reform des Anschauungs-Unterrichts_ (Berlin: Lichtspiele Mozartsaal, 1911), 3. Other major statements about the educational use of film before World War I include Samuleit and Borm, _Der Kinematograph als Volks- und Jugendbildungsmittel_ ; Adolf Sellmann, _Der Kinematograph als Volkserzieher?_ (Langensalza, Germany: Beyer, 1912); Friedrich Murawski, _Die Kinematographie und ihre Beziehungen zu Schule und Unterricht_ (Dresden: Bieyl and Kaemmerer, 1914); Sellmann, _Kino und Volksbildung_ (M. Gladbach: Volksvereins Verlag, 1914); and esp. Sellmann, _Kino und Schule_ (M. Gladbach: Volksvereins Verlag, 1914).\n\n. For a friendly assessment, see Wilhelm Richter, \"Der Kinematograph als naturwissenschaftliches Anschauungsmittel,\" _Naturwissenschaftliche Wochenschrift_ 12, no. 52 (28 December 1913): 817\u2013820.\n\n. K. R\u00fcswald, \"Der Film im Erdkundlichen und Naturwissenschaftlichen Unterricht,\" in Terveen, _Dokumente zur Geschichte der Schulfilmbewegung in Deutschland_ , 43\u201344. A fine summary of the arguments forwarded against educational uses of film can be found in H. Graupner, \"Unterrichtshygiene,\" in _Handbuch der deutschen Schulhygiene_ , ed. Hugo Selter (Dresden and Leipzig: Steinkopff, 1914), 174\u2013321, esp. his section on \"Kinematograph und Unterrichtshygiene,\" 302\u2013307.\n\n. Sellmann, _Kino und Schule_ , 15 (emphasis in original).\n\n. Richard Kretz, \"Die Anwendung der Photographie in der Medicin,\" _Wiener klinische Wochenschrift_ 7, no. 44 (1 November 1894): 832.\n\n. Paul Knospe, _Der Kinematograph im Dienste der Schule. Unter besonderer Ber\u00fccksichtigung des erdkundlichen Unterrichts_ (Halle an der Saale, Germany: Waisenhaus, 1913), 9. There are many more examples, but see also Bastian Schmid, \"Kinematographie und Schule,\" _Die Naturwissenschaften_ 1, no. 6 (7 February 1913): 145\u2013146.\n\n. Oskar Kalbus summarizes and quotes from such complaints in _Der Deutsche Lehrfilm in der Wissenschaft und im Unterricht_ (Berlin: Heymanns, 1922), 6.\n\n. Mendel, _Kinematographie und Schule_.\n\n. \"Zur Er\u00f6ffnung des Ernemann-Kino in Dresden,\" _Der Kinematograph_ no. 134 (21 July 1909). It was not unusual for film equipment manufacturers in Germany to have exhibition storefronts that were open to the public in a quasi-museum-like setting. See Deac Rossell, \"Beyond Messter: Aspects of Early Cinema in Berlin,\" _Film History_ 10 (1998): 52\u201369.\n\n. \"Die Dresdner 'Kosmographia,'\" _Bild und Film_ 1, no. 1 (1912): 19. See also Uli Jung, \"Film f\u00fcr Lehre und Bildung,\" in _Geschichte des dokumentarischen Films in Deutschland_ , vol. 1, ed. Uli Jung and Martin Loiperdinger (Stuttgart: Reclam, 2005), 333\u2013340.\n\n. Those venues were the Ernemann-Kino (1909) and the Kosmographia (1910) in Dresden; the Reform-Kino in Braunschweig (1910); the Reformtheater in Bremen (1911); the Gemeindekino in Eickel (1912); the Germania Saal in Hagen (1912); the Musterlichtbildb\u00fchne in Altona (1912); and the Urania in Stettin (1914).\n\n. Hermann Bredtmann, \"Kinematographie und Schule,\" _P\u00e4dagogisches Archiv_ 56, no. 3 (1914): 154\u2013163, here 161 and 163.\n\n. On Lemke, see Schorr, \"Die Film- und Kinoreformbewegung,\" 56ff; M\u00fcller, _Fr\u00fche deutsche Kinematographie_ , 71\u201376; and M\u00fcller, \"Der fr\u00fche Film, das fr\u00fche Kino und seine Gegner und Bef\u00fcrworter,\" in _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , ed. Kaspar Maase und Wolfgang Kaschuba (Cologne, Weimar, and Vienna: B\u00f6hlau, 2001), 62\u201391.\n\n. See the technophilia of Hermann Lemke's _Durch die Technik zur Schulreform. Zwei modern-technische Lehrmethoden und Veranschaulichungsmittel in der Schule der Zukunft_ (Leipzig: Demme, 1911), in which he predicts that the combination of films and phonographs will make teachers obsolete. See also Sellmann's vision of a \"complete revolution\" in teaching once projectors are universally installed; _Kino und Schule_ , 39.\n\n. Hermann Lemke, _Die kinematographische Unterrichtsstunde_ (Leipzig: Demme, 1911), 5.\n\n. M\u00fcller, \"Der fr\u00fche Film, das fr\u00fche Kino und seine Gegner und Bef\u00fcrworter,\" 75. Sellmann held similar workshops in Eickel, but they also did not last in the long run. See \"Bericht \u00fcber eine Besprechung der Kinokommssion des Westf\u00e4lischen Landgemeindetages anl\u00e4\u00dflich der Er\u00f6ffnungsfeier des Gemeindelichtspielhauses in Eickel,\" _Bild und Film_ 2, no. 3 (1912): 70\u201371. See also Lenk and Kessler, \"The Institutionalization of Educational Cinema.\"\n\n. Actually, Herbart was not this clear or consistent, so these steps are the result of refinements by later Herbartians such as Wilhelm Rein, esp. _P\u00e4dagogik im Grundri\u00df_ (1890), 4th ed. (Leipzig: G\u00f6schen, 1907), 109. For a helpful discussion of Herbart and Herbartianism, see Harold B. Dunkel, _Herbart and Education_ (New York: Random House, 1969).\n\n. Miriam Hansen, _Babel and Babylon: Spectatorship in American Silent Film_ (Cambridge, Mass.: Harvard University Press, 1991), 93.\n\n. Helmut H. Diederichs, \"Naturfilm als Gesamtkunstwerk: Hermann H\u00e4fker und sein 'Kinetographie'-Konzept,\" _Augenblick_ 8 (1990): 37\u201360. If H\u00e4fker is known at all to English-speaking readers, it is through Kracauer's characterization of him in _From Caligari to Hitler_ as the man who \"praised war as the salvation from the evils of peace\" (28). H\u00e4fker saw World War I mainly as an opportunity for the state to take control of cinema and put his plans into action. While there is no doubt that H\u00e4fker was conservative, nationalistic, and blind to the horrors of war, it would be unfair to depict him as a warmonger with the prefascist tendencies implied by Kracauer. H\u00e4fker earned a \"heart attack\" in a concentration camp for his resistance to the Nazi government. All biographical information comes from Diederichs's article and his entry on H\u00e4fker in _Cinegraph_ , ed. Hans-Michael Bock (Munich: edition text + kritik, 1984). My presentation of H\u00e4fker is indebted to these essays and my conversations with Diederichs.\n\n. Hermann H\u00e4fker, \"Zur Dramaturgie der Bilderspiele,\" _Der Kinematograph_ no. 32 (7 August 1907).\n\n. Hermann H\u00e4fker, \"Meisterspiele,\" _Der Kinematograph_ no. 56 (22 January 1908).\n\n. H\u00e4fker, \"Zur Dramaturgie der Bilderspiele.\"\n\n. Hermann H\u00e4fker, _Kino und Kunst_ (M. Gladbach: Volksvereins, 1913), 5.\n\n. See, for instance, Robert Gaupp and Konrad Lange, _Der Kinematograph als Volksunterhaltungsmittel_ (Munich: Callwey, 1912); R. Stigler, \"\u00dcber das Flimmern der Kinematographen,\" _Archiv f\u00fcr die gesamte Physiologie des Menschen und der Tiere_ (Bonn) 123 (1908): 224\u2013232; or Naldo Felke, \"Die Gesundheitssch\u00e4dlichkeit des Kinos,\" _Die Umschau_ 17, no. 1 (1 January 1913): 254\u2013255.\n\n. Rabinbach, _Human Motor_ , 21.\n\n. H\u00e4fker, \"Meisterspiele.\"\n\n. H\u00e4fker, _Kino und Kunst_ , 9. Hereafter cited parenthetically.\n\n. Ernst Schultze, _Der Kinematograph als Bildungsmittel_ (Halle an der Saale, Germany: Waisenhaus, 1911), 118.\n\n. Max Brethfeld, \"Neue Versuche, die Kinematographie f\u00fcr die Volksbildung und Jugenderziehung zu verwerten,\" _Neue Bahnen_ (1910): 422, quoted in Diederichs, \"Naturfilm,\" 41.\n\n. H\u00e4fker, _Kino und Kunst_ , 61. Hereafter cited parenthetically.\n\n. On the Urania lecture hall influence, see Gerhard Ebel and Otto L\u00fchrs, \"Urania\u2014eine Idee, eine Bewegung, eine Institution wird 100 Jahre alt,\" in _100 Jahre Urania: Wissenschaft heute f\u00fcr morgen_ (Berlin: Urania Berlin, 1988), 15\u201374. There are also structural similarities between H\u00e4fker's presentations and the presentation of early cinema's passion plays. See Charles Musser, _The Emergence of Cinema: The American Screen to 1907_ (New York: Scribner, 1990), 208\u2013218.\n\n. Jonathan Crary offers a concise overview of the attention psychologists paid to attention in his \"Unbinding Vision,\" _October_ 68 (Spring 1994): 21\u201344, and offers a more lengthy treatment in _Suspensions of Perception: Attention, Spectacle, and Modern Culture_ (Cambridge, Mass.: MIT Press, 1999). With regard to the perceived increase in sensual diversions, see, e.g., George M. Beard, _American Nervousness: Its Causes and Consequences_ (New York: Putnam, 1881); and Tom Lutz, _American Nervousness, 1903: An Anecdotal History_ (Ithaca, N.Y.: Cornell University Press, 1991).\n\n. See Martin Jay, _Downcast Eyes: The Denigration of Vision in Twentieth-Century French Thought_ (Berkeley and Los Angeles: University of California Press, 1993).\n\n. Schiller, _On the Aesthetic Education of Man_ , 183 (emphasis in original)\n\n. Walter Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Third Version),\" in _Walter Benjamin, Selected Writings_ , vol. 4, _1938\u20131940_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott and others (Cambridge, Mass.: Belknap, 2003), 251\u2013283.\n\n. Schiller, _On the Aesthetic Education of Man_ , 183.\n\n. Immanuel Kant, _Critique of Judgement_ , trans. James Creed Meredith (Oxford: Oxford University Press, 1952), 150\u2013154, \u00a740.\n\n. Schiller, _On the Aesthetic Education of Man_ , 215 (emphasis in original). For my discussion of aesthetics and ideology, I am indebted to Terry Eagleton, _The Ideology of the Aesthetic_ (Cambridge, Mass.: Blackwell, 1990).\n\n4. THE PROBLEM WITH PASSIVITY\n\n 1. Walter Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Second Version),\" in _Walter Benjamin: Selected Writings_ , vol. 3, _1935\u20131938_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott, Howard Eiland, and others (Cambridge, Mass.: Belknap, 2002), 101\u2013133, here 109.\n\n 2. For representative essays, see the following anthologies: Anton Kaes, ed., _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film, 1909\u20131929_ (T\u00fcbingen: Niemeyer, 1978); Ludwig Greve, Margot Pehle, and Heidi Westhoff, eds., _H\u00e4tte ich das Kino! Die Schriftsteller und der Stummfilm_ (Munich: K\u00f6sel, 1976); Fritz G\u00fcttinger, ed., _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ (Frankfurt: Deutsches Filmmuseum, 1984); J\u00f6rg Schweinitz, ed., _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ (Leipzig: Reclam, 1992); Helmut H. Diederichs, ed., _Geschichte der Filmtheorie: Kunsttheoretische Texte von M\u00e9li\u00e8s bis Arnheim_ (Frankfurt: Suhrkamp, 2004); and Richard W. McCormick and Alison Guenther-Pal, eds., _German Essays on Film_ (New York: Continuum, 2004).\n\n 3. The best overview of the early German film industry remains Corinna M\u00fcller, _Fr\u00fche deutsche Kinematographie: formale wirtschaftliche und kulturelle Entwicklungen, 1907\u20131912_ (Stuttgart and Weimar: Metzler, 1994). On early German cinema in general, see the following anthologies: Paolo Cherchi Usai and Lorenzo Codelli, eds., _Before Caligari: German Cinema, 1895\u20131920_ (Madison: University of Wisconsin Press, 1990); Thomas Elsaesser with Michael Wedel, eds., _A Second Life: German Cinema_ ' _s First Decades_ , (Amsterdam: Amsterdam University Press, 1996); and Thomas Elsaesser and Michael Wedel, eds., _Kino der Kaiserzeit: Zwischen Tradition und Moderne_ (Munich: edition text + kritik, 2002). On the _Autorenfilm_ , see esp. Helmut H. Diederichs, _Anf\u00e4nge deutscher Filmkritik_ (Stuttgart: Fischer, 1986); Diederichs, \"The Origins of the _Autorenfilm_ ,\" in Cherchi Usai and Codelli, _Before Caligari_ , 380\u2013401; and Leonardo Quaresima, \" _Dichter, Heraus!_ The _Autorenfilm_ and German Cinema of the 1910s,\" _Griffithiana_ 38\/39 (October 1990): 101\u2013120.\n\n 4. Anton Kaes, \"Literary Intellectuals and the Cinema: Charting a Controversy (1909\u20131929),\" _New German Critique_ 40 (Winter 1987): 7\u201334, here 7\u20138.\n\n 5. Sabine Hake, _The Cinema_ ' _s Third Machine: Writing on Film in Germany, 1907\u20131933_ (Lincoln: University of Nebraska Press, 1993), 63.\n\n 6. Peter Jelavich, \"'Am I Allowed to Amuse Myself Here?': The German Bourgeoisie Confronts Early Film,\" in _Germany at the Fin de Si\u00e8cle: Culture, Politics, and Ideas_ , ed. Suzanne Marchand and David Lindenfeld (Baton Rouge: Louisiana State University Press, 2004), 227\u2013249, here 247.\n\n 7. Helmut H. Diederichs, \"Kino und die Wortk\u00fcnste: Zur Diskussionen der deutschen literarischen Intelligenz 1910 bis 1915,\" _KINtop_ 13 (2004): 9\u201323. See also Diederichs, \"Fr\u00fchgeschicht deutscher Filmtheorie. Ihre Entstehung und Entwicklung bis zum Ersten Weltkrieg,\" unpublished Habilitationsschrift (J. W. Goethe-Universit\u00e4t Frankfurt am Main, 1996).\n\n 8. Stefanie Harris, _Mediating Modernity: German Literature and the_ \" _New_ \" _Media, 1895\u20131930_ (University Park: Pennsylvania State University Press, 2009).\n\n 9. Heinz-B. Heller, _Literarische Intelligenz und Film: Zu Ver\u00e4nderungen der \u00e4sthetischen Theorie und Praxis unter dem Eindruck des Films 1910\u20131930 in Deutschland_ (T\u00fcbingen: Niemeyer, 1985). See also Thomas Koebner, \"Der Film als neue Kunst: Reaktionen der literarischen Intelligenz: Zur Theorie des Stummfilms (1911\u20131924),\" in _Literaturwissenschaft-Medienwissenschaft_ , ed. Volker Canaris and Helmut Kreuzer (Heidelberg: Quelle und Meyer, 1977), 1\u201331. While they do not focus as much on the relationship between literature and film, Miriam Hansen and Heide Schl\u00fcpmann set the agenda for the study of this era in other ways, which will be discussed later in the chapter: Miriam Hansen, \"Early Silent Cinema: Whose Public Sphere?,\" _New German Critique_ 29 (Spring\/Summer 1983): 147\u2013184; Heide Schl\u00fcpmann, _Unheimlichkeit des Blicks: Das Drama des fr\u00fchen deutschen Kinos_ (Frankfurt: Stroemfeld\/Roter Stern, 1990).\n\n 10. This applies, of course, to descriptions of the _beautiful_ , rather than to those of the _sublime_ , which often emphasized an immediate overwhelming of the imagination. My thanks to Dan Morgan for reminding me of this distinction.\n\n 11. Friedrich Schiller, _On the Aesthetic Education of Man_ , trans. Elizabeth M. Wilkinson and L. A. Willoughby (Oxford: Oxford University Press, 1967), 123.\n\n 12. See, for example, Robert Vischer's description of the difference between _Sehen_ and _Schauen_ (\"seeing\" and \"scanning\") in _On the Optical Sense of Form_ (1873), in _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou (Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994), 89\u2013124, esp. 93\u201395. See also Adolf Hildebrand's elaboration of this idea in _The Problem of Form in the Fine Arts_ (1893), in Mallgrave and Ikonomou, _Empathy, Form, and Space_ , 227\u2013279, esp. 229\u2013232.\n\n 13. For a complete survey, see Steve Odin, _Artistic Detachment in Japan and the West: Psychic Distance in Comparative Aesthetics_ (Honolulu: University of Hawaii Press, 2001).\n\n 14. Emilie Altenloh, _Zur Soziologie des Kino: Die Kino-Unternehmung und die sozialen Schichten ihrer Besucher_ (Jena: Diederichs, 1914), 91.\n\n 15. William Egginton, \"Intimacy and Anonymity, or How the Audience Became a Crowd,\" in _Crowds_ , ed. Jeffrey T. Schnapp and Matthew Tiews (Stanford, Calif.: Stanford University Press, 2006), 97\u2013110.\n\n 16. Jacques Ranci\u00e8re, _The Emancipated Spectator_ , trans. Gregory Elliott (London and New York: Verso, 2009), 2.\n\n 17. Carolin Duttlinger, \"Between Contemplation and Distraction: Configurations of Attention in Walter Benjamin,\" _German Studies Review_ 30, no. 1 (February 2007): 33\u201354.\n\n 18. Dudley Andrew, \"Film and Society: Public Rituals and Private Space,\" in _Exhibition: The Film Reader_ , ed. Ina Rae Hark (New York: Routledge, 2001), 161\u2013172, here 165. Originally published in _East-West Film Journal_ 1, no. 1 (1986): 7\u201322.\n\n 19. A good introduction to the broader social problem, minus cinema, is Hartmut Rosa and William E. Scheuerman, eds., _High-Speed Society: Social Acceleration, Power, and Modernity_ (University Park: Pennsylvania State University Press, 2009).\n\n 20. Georg Kleib\u00f6mer, \"Kinematograph und Schuljugend,\" _Der Kinematograph,_ no. 124 (12 May 1909) (emphasis in original in this and subsequent quotations).\n\n 21. Karl Hans Strobl, \"Der Kinematograph,\" in _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ , ed. Fritz G\u00fcttinger (Frankfurt: Deutsches Filmmuseum, 1984), 50\u201354, here 52. Originally published in _Die Hilfe_ 17, no. 9 (2 March 1911).\n\n 22. Ph. Sommer, \"Zur Psychologie des Kinematographen,\" _Der Kinematograph_ no. 227 (3 May 1911).\n\n 23. Wilhelm Stapel, \"Der homo cinematicus,\" _Deutsches Volkstum_ 21 (October 1919): 319\u2013320, here 319.\n\n 24. Schiller, _On the Aesthetic Education of Man_ , 79. Hereafter cited parenthetically.\n\n 25. Arthur Schopenhauer, _The World as Will and Representation_ , trans. E. F. J. Payne (New York: Dover, 1966), vol. 1, \u00a738, p. 197.\n\n 26. Josef Chytry, _The Aesthetic State: A Quest in Modern German Thought_ (Berkeley and Los Angeles: University of California Press, 1989). See also David Aram Kaiser, _Romanticism, Aesthetics, and Nationalism_ (Cambridge: Cambridge University Press, 1999), esp. his chapter on \"Schiller's Aesthetic State.\"\n\n 27. Egon Friedell, \"Prolog vor dem Film,\" in Kaes, _Kino-Debatte_ , 42\u201347, here 43. Originally published in _Bl\u00e4tter des Deutschen Theaters_ 2 (1912): 509\u2013511.\n\n 28. Hermann Kienzl, \"Theater und Kinematograph,\" in Schweinitz, _Prolog vor dem Film_ , 230\u2013234, here 231. Originally published in _Der Strom_ 1, no. 7 (October 1911): 219\u2013221.\n\n 29. Walter Hasenclever, \"Der Kintopp als Erzieher: Eine Apologie,\" in Kaes, _Kino-Debatte_ , 47\u201349, here 48. Originally published in _Revolution_ 1, no. 4 (1 December 1913): n.p.\n\n 30. Lou Andreas-Salom\u00e9, _In der Schule bei Freud: Tagesbuch eines Jahres 1912\/1913_ , ed. Ernst Pfeiffer (Z\u00fcrich: Niehans, 1958), 102\u2013103.\n\n 31. Hermann Duenschmann, \"Kinematograph und Psychologie der Volksmenge. Eine sozialpolitische Studie,\" _Konservative Monatsschrift_ 69, no. 9 (June 1912): 920\u2013930, here 924.\n\n 32. Ren\u00e9 Descartes, \"The Search for Truth,\" _The Philosophical Writings of Descartes_ , trans. John Cottingham, Robert Stoothoff, and Dugald Murdoch, vol. 2 (Cambridge and New York: Cambridge University Press, 1984\u20131991), 400\u2013420, here 406.\n\n 33. John Locke, _An Essay Concerning Human Understanding_ , ed. and with an introduction by Peter H. Nidditch (Oxford: Oxford University Press, 1975), 152, book 2, chap. 10, sect. 5.\n\n 34. Locke, _Essay_ , 607, book 4, chap. 7, sect. 16.\n\n 35. Lex Newman, \"Ideas, Pictures, and the Directness of Perception in Descartes and Locke,\" _Philosophy Compass_ 4, no. 1 (2009): 134\u2013154.\n\n 36. Ulrich Rauscher, \"Die Kino-Ballade,\" in G\u00fcttinger, _Kein Tag ohne Kino_ , 143\u2013149, here 148. Originally published in _Der Kunstwart_ 26, no. 13 (1 April 1913): 1\u20136.\n\n 37. Georges Duhamel, _Sc\u00e8nes de la vie future_ (Paris: Mercure de France, 1930), 52, quoted in Walter Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Third Version),\" in _Walter Benjamin: Selected Writings_ , vol. 4, _1938\u20131940_ , trans. Edmund Jephcott and others, ed. Howard Eiland and Michael W. Jennings (Cambridge, Mass.: Belknap, 2003), 267.\n\n 38. Georg Simmel, \"The Metropolis and Mental Life,\" in _The Sociology of Georg Simmel_ , ed. and trans. Kurt H. Wolff (New York: Free Press, 1950), 409\u2013424; Sigmund Freud, _Beyond the Pleasure Principle_ , trans. James Strachey (London: Hogarth, 1950).\n\n 39. Joseph Landau, \"Mechanisierte Unsterblichkeit,\" in _Der Deutsche Kaiser im Film_ , ed. Paul Klebinder (Berlin: Klebinder, 1912), 18\u201322, here 20.\n\n 40. Max Brod, \"Kinematographentheater,\" in Kaes, _Kino-Debatte_ , 39\u201341, here 41. Originally published in _Die neue Rundschau_ 20, no. 2 (February 1909): 319\u2013320.\n\n 41. \"Neuland f\u00fcr Kinematographentheater,\" in Kaes, _Kino-Debatte_ , 41. Originally published in _Lichtbild-B\u00fchne_ 3 (September 1910): 3.\n\n 42. Strobl, \"Der Kinematograph,\" in _Kein Tag ohne Kino_ , 51.\n\n 43. Juliet Koss describes the gentleman art historian in terms of a unified, stable subjectivity in \"On the Limits of Empathy,\" _Art Bulletin_ 88, no. 1 (March 2006): 139\u2013157; see also Koss, _Modernism After Wagner_ (Minneapolis: University of Minnesota Press, 2010).\n\n 44. Harry Francis Mallgrave and Eleftherios Ikonomou, \"Introduction,\" _Empathy, Form, and Space_ , 10\u201311. Herbart's aesthetics were widely influential in the nineteenth century (but not as influential as his pedagogical ideas, as we saw in the previous chapter). See his 1813 _Lehrbuch zur Einleitung in die Philosophie_ , third edition (K\u00f6nigsberg: Unzer, 1834) or his 1831 _Kurze Encyklop\u00e4die der Philosophie_ (Hamburg: Voss, 1884). Robert Zimmermann later expanded Herbartian aesthetics into an equally influential comprehensive system devoted to the \"the science of form.\" See his _Allgemeine Aesthetik als Formwissenschaft_ (Vienna: Braum\u00fcller, 1865).\n\n 45. Many of Schopenhauer's arguments in \"Supplements to the Third Book\" take into account questions of perception and physiology, esp. chap. 30, \"On the Pure Subject of Knowing,\" and chap. 39, \"On the Metaphysics of Music.\" Schopenhauer, _The World as Will and Representation_ , vol. 2, pp. 367\u2013375 and 447\u2013457, respectively.\n\n 46. Looking for \"laws\" of beauty as a scientist might look for laws of nature, Fechner measured hundreds of paintings to find a statistical, scientific basis for the perfect format. See Gustav Fechner, _Vorschule der Aesthetik_ (Leipzig: Breitkopf & H\u00e4rtel, 1897 [1876]). Likewise, Wundt, the father of experimental psychology, tried to link pleasure in the perception of forms to the physiological structure of the eye by noting the ease with which the eye traced the contours of various forms. See Wilhelm Wundt, _Grundz\u00fcge der physiologischen Psychologie_ , 5th ed. (Leipzig: Engelmann, 1902\u20131903 [1874]), 1: 486ff. For a discussion of the implications of the work of these and other researchers for modern subjectivity, see Jonathan Crary, _Techniques of the Observer: On Vision and Modernity in the Nineteenth Century_ (Cambridge, Mass.: MIT Press, 1990). For more on the relationship between the sciences, especially physiology, and aesthetics, see Robert Michael Brain, \"The Pulse of Modernism: Experimental Physiology and Aesthetic Avant-Gardes Circa 1900,\" _Studies in History and Philosophy of Science_ 39, no. 3 (2008): 393\u2013417.\n\n 47. Mallgrave and Ikonomou, \"Introduction,\" 2.\n\n 48. Vischer, _On the Optical Sense of Form_ , 89\u2013124. Hereafter cited parenthetically.\n\n 49. Gustav Jahoda, \"Theodor Lipps and the Shift from 'Sympathy' to 'Empathy,'\" _Journal of the History of the Behavioral Sciences_ 4, no. 2 (2005): 151\u2013163; and Susan Lanzoni, \"Empathy in Translation: Movement and Image in the Psychological Laboratory,\" _Science in Context_ 25, no. 3 (September 2012): 301\u2013327.\n\n 50. The early \"formal-analytic\" approach to art, so important for the formation of the discipline of art history, is exemplified by Conrad Fiedler, _\u00dcber die Berurtheilung von Werken der bildenden Kunst_ (Leipzig: Hirzel, 1876). Translated by Henry Schaefer-Simmern and Fulmer Mood as _On Judging Works of Visual Art_ (Berkeley: University of California Press, 1949).\n\n 51. The _Zeitschrift f\u00fcr \u00c4sthetik und allgemeine Kunstwissenschaft_ (Journal for Aesthetics and Art History), which began in 1906 under the editorship of Max Dessoir, was the leading forum in Germany for discussions of _Einf\u00fchlung_ and its implications for aesthetics and reception.\n\n 52. These questions were not limited to Germany, as many ideas and approaches spread to the United Kingdom, France, and the United States during the nineteenth century. Representative English-language examples of psychological aesthetics include Herbert Spencer, _The Principles of Psychology_ , 2 vols. (London: Williams and Norgate, 1855); Grant Allen, _Physiological Aesthetics_ (London: King, 1877); James Sully, \"Pleasure of Visual Form,\" _Mind: A Quarterly Review of Psychology and Philosophy_ 5, no. 18 (April 1880): 181\u2013201; and Vernon Lee and C. Anstruther-Thomson, _Beauty and Ugliness and Other Studies in Psychological Aesthetics_ (London and New York: Lane, 1912). On Lee, see esp. Hilary Fraser, \"Women and the Ends of Art History: Vision and Corporeality in Nineteenth-Century Critical Discourse,\" _Victorian Studies_ 42, no. 1 (Autumn 1998): 77\u2013100. Applications of these ideas to film include Hugo M\u00fcnsterberg, _The Photoplay: A Psychological Study_ (London and New York: Appleton, 1916); Victor Oscar Freeburg, _Pictorial Beauty on the Screen_ (New York: Macmillan, 1923); and Frances Taylor Patterson, _Scenario and Screen_ (New York: Harcourt, Brace, 1928). Contemporary analyses of this trend in English-language (film) aesthetics include Laura Marcus, _The Tenth Muse: Writing About Cinema in the Modernist Period_ (Oxford and New York: Oxford University Press, 2007); Lynda Nead, _The Haunted Gallery: Painting, Photography, Film c. 1900_ (New Haven and London: Yale University Press, 2007); and Kaveh Askari, _Making Movies into Art: Picture Craft from the Magic Lantern to Early Hollywood_ (London: British Film Institute, 2014), esp. chap. 3.\n\n 53. Joseph Imorde, \"Einf\u00fchlung in der Kunstgeschichte,\" in _Einf\u00fchlung. Zur Geschichte und Gegenwart eines \u00e4sthetischen Konzepts_ , ed. Robin Curtis and Gertrud Koch (Paderborn, Germany: Fink, 2009), 127\u2013142.\n\n 54. In addition to my own essay on \"Einf\u00fchlung und fr\u00fche deutsche Filmtheorie,\" in Curtis and Koch, _Einf\u00fchlung. Zur Geschichte und Gegenwart eines \u00e4sthetischen Konzepts_ , 61\u201384, see Robin Curtis, \"Einf\u00fchlung and Abstraction in the Moving Image: Historical and Contemporary Reflections,\" _Science in Context_ 25, no. 3 (September 2012): 425\u2013446; and Robert Michael Brain, \"Self-Projection: Hugo M\u00fcnsterberg on Empathy and Oscillation in Cinema Spectatorship,\" in the same issue, pp. 329\u2013353.\n\n 55. Even when one of the preeminent names in aesthetic and _Einf\u00fchlung_ theory, Max Dessoir, was given the opportunity to discuss the phenomenon of film from a theoretical standpoint, he chose, rather uninterestingly, to extol the virtues of words for literature and denounce the lack of words in silent film. See \"Kino und Buchhandel,\" in Schweinitz, _Prolog vor dem Film_ , 284\u2013285.\n\n 56. Walter von Molo, \"Im Kino,\" in Schweinitz, _Prolog vor dem Film_ , 28\u201339, here 31. Originally published in _Velhagen & Klasings Monatshefte_ 26, no. 8 (April 1912): 618\u2013627.\n\n 57. Alfred Polgar, \"Das Drama im Kinematographen,\" in _Kein Tag ohne Kino_ , 56\u201361, here 60. Originally published in _Der Strom_ 1, no. 2 (May 1911).\n\n 58. Vivian Sobchack, _The Address of the Eye: A Phenomenology of Film Experience_ (Princeton, N.J.: Princeton University Press, 1992).\n\n 59. Hildebrand, _The Problem of Form in the Fine Arts_ , 261.\n\n 60. Polgar, \"Das Drama im Kinematographen,\" 59.\n\n 61. Mallgrave and Ikonomou, \"Introduction,\" 23.\n\n 62. Ernst Bloch, \"Die Melodie im Kino oder immanente und transzendentale Musik,\" in Schweinitz, _Prolog vor dem Film_ , 326\u2013334, here 328\u2013329. Originally published in _Die Argonauten_ 1, no. 2 (1914): 84\u201385. This translation, while largely my own, borrows some phrases from Ernst Bloch, \"On Music in the Cinema,\" in _Literary Essays_ , trans. Andrew Joron and others (Stanford, Calif.: Stanford University Press, 1998), 157\u2013158.\n\n 63. See also Hildebrand, _The Problem of Form in the Fine Arts_ , 229. Hildebrand further equates _Schauen_ with _Abtasten_ (\"probing\" or \"touching\").\n\n 64. See Hildebrand, _The Problem of Form in the Fine Arts_ , 261, for a concurring opinion: \"What we simply call the life of nature is actually the animation of nature through the imagination.\"\n\n 65. August Schmarsow, \"The Essence of Architectural Creation\" (1893), in Mallgrave and Ikonomou, _Empathy, Form, and Space_ , 281\u2013297, here 287 (emphasis in original). Hereafter cited parenthetically.\n\n 66. For a survey of his output, see Niels W. Bokhove and Karl Schuhmann, \"Bibliographie der Schriften von Theodor Lipps,\" _Zeitschrift f\u00fcr philosophische Forschung_ 45, no. 1 (January\u2013March 1991): 112\u2013130.\n\n 67. Theodor Lipps, \"Einf\u00fchlung und \u00e4sthetischer Genu\u00df,\" _Die Zukunft_ 54, no. 14 (20 January 1906): 100\u2013114, here 101.\n\n 68. Lipps, \"Einf\u00fchlung und \u00e4sthetischer Genu\u00df,\" 103.\n\n 69. Theodor Lipps, \"Einf\u00fchlung, innere Nachahmung, und Organempfindungen,\" _Archiv f\u00fcr die gesamte Psychologie_ 1, nos. 2\/3 (1903): 185\u2013204, here 201, 202, 203. Translated as \"Empathy, Inner Imitation, and Sense-Feeling,\" in _A Modern Book of Aesthetics_ , ed. Melvin M. Rader (New York: Holt, 1935), 291\u2013304, here 302\u2013303 (translation modified).\n\n 70. Theodor Lipps, _\u00c4sthetik. Psychologie des Sch\u00f6nen und der Kunst. Erster Teil: Grundlegung der \u00c4sthetik_ (Hamburg and Leipzig: Voss, 1903), 148.\n\n 71. Other writers on _Einf\u00fchlung_ explored the relation between empathy and bodily response, however tentatively. See Karl Groos, \"Das \u00e4sthetische Miterleben und die Empfindungen aus dem K\u00f6rperinnern,\" _Zeitschrift f\u00fcr \u00c4sthetik und allgemeine Kunstwissenschaft_ 4 (1909): 161\u2013182; Vernon Lee, \"Weiteres \u00fcber Einf\u00fchlung und \u00e4sthetisches Miterleben,\" _Zeitschrift f\u00fcr \u00c4sthetik und allgemeine Kunstwissenschaft_ 5 (1910): 145\u2013190; and later, Johannes Volkelt, _System der \u00c4sthetik, Erster Band: Grundlegung der \u00c4sthetik_ , 2d ed. (Munich: Beck, 1927), esp. 186\u2013201, in which he discusses motor sensations as way of facilitating _Einf\u00fchlung_. See also Christian G. Allesch, _Geschichte der psychologischen \u00c4sthetik_ (G\u00f6ttingen: Verlag f\u00fcr Psychologie, 1987) for a complete discussion of this topic.\n\n 72. _Schaulust_ has been translated as \"scopophilia\" by Freud's translators, but this rendering gives it a clinical connotation that neither Freud nor Serner intended. It literally means \"the desire to look\" or \"the sexual pleasure in looking,\" but \"voyeurism\" tends to narrow its meaning as well. Not having an adequate English word at hand, I will simply refer to the concept in German. (On the inadequacies of the standard translation of Freud, see Bruno Bettelheim, _Freud and Man_ ' _s Soul_ [New York: Knopf, 1983]).\n\n 73. Walter Serner, \"Kino und Schaulust,\" in Schweinitz, _Prolog vor dem Film_ , 208\u2013214, here 208. Originally published in _Die Schaub\u00fchne_ 9, nos. 34\/35 (1913): 807\u2013811.\n\n 74. Serner, \"Kino und Schaulust,\" 210.\n\n 75. Serner, \"Kino und Schaulust,\" 211.\n\n 76. Tom Gunning, \"The Cinema of Attractions: Early Film, Its Spectator and the Avant-Garde,\" in _Early Cinema: Space, Frame, Narrative_ , ed. Thomas Elsaesser with Adam Barker (London: British Film Institute, 1990), 56\u201362.\n\n 77. Lipps, \"Einf\u00fchlung, innere Nachahmung, und Organempfindungen,\" 195 (emphasis in original). Translated as \"Empathy, Inner Imitation, and Sense-Feeling,\" in Rader, _A Modern Book of Aesthetics_ , 300\u2013301 (translation modified).\n\n 78. Lipps, \"Einf\u00fchlung und \u00e4sthetischer Genu\u00df,\" 113.\n\n 79. Charles Musser, \"A Cinema of Contemplation, A Cinema of Discernment: Spectatorship, Intertextuality and Attractions in the 1890s,\" in _The Cinema of Attractions Reloaded_ , ed. Wanda Strauven (Amsterdam: Amsterdam University Press, 2006), 159\u2013179.\n\n 80. Miriam Hansen, \"Early Silent Cinema: Whose Public Sphere?,\" _New German Critique_ 29 (Spring\/Summer 1983): 147\u2013184; Heide Schl\u00fcpmann, _Unheimlichkeit des Blicks: Das Drama des fr\u00fchen deutschen Kinos_ (Frankfurt: Stroemfeld\/Roter Stern, 1990).\n\n 81. Diderot, _Salons_ , III, ed. Jean Seznec and Jean Adh\u00e9mar (Oxford, 1963), 134\u2013135, quoted in Michael Fried, _Absorption and Theatricality: Painting and Beholder in the Age of Diderot_ (Chicago: University of Chicago Press, 1980), 125.\n\n 82. Richard Huelsenbeck, _Wozu Dada. Texte 1916\u20131936_ (Giessen: Anabas, 1994), 35, quoted in David C. Durst, _Weimar Modernism: Philosophy, Politics, and Culture in Germany 1918\u20131933_ (Lanham, Md.: Lexington, 2004), 48. Durst's book was instrumental in crafting my argument about the politics of contemplation.\n\n 83. Siegfried Kracauer, \"Cult of Distraction: On Berlin's Picture Palaces,\" in _The Mass Ornament: Weimar Essays_ , trans. and ed. Thomas Y. Levin (Cambridge, Mass.: Harvard University Press, 1995), 323\u2013328. Hereafter cited parenthetically.\n\n 84. Walter Benjamin, \"Surrealism: The Last Snapshot of the European Intelligensia,\" in _Walter Benjamin: Selected Writings_ , vol. 2, _1927\u20131934_ , ed. Michael W. Jennings, Howard Eiland, and Gary Smith, trans. Rodney Livingstone and others (Cambridge, Mass.: Belknap, 1999), 207\u2013221, here 213.\n\n 85. Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Third Version)\"; and Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Second Version),\" 101\u2013136.\n\n 86. Benjamin, \"The Work of Art in the Age of Its Technological Reproducibility (Second Version),\" 119.\n\n 87. Benjamin, \"Theory of Distraction,\" in _The Work of Art in the Age of Its Technological Reproducibility, and Other Writings on Media_ , ed. Michael W. Jennings, Brigid Doherty, and Thomas Y. Levin, trans. Edmund Jephcott, Rodney Livingstone, Howard Eiland, and others (Cambridge, Mass.: Belknap, 2008), 56\u201357 (emphasis added).\n\n 88. Of the many commentaries on Benjamin and Kracauer on distraction, see esp. Miriam Bratu Hansen, _Cinema and Experience: Siegfried Kracauer, Walter Benjamin, and Theodor W. Adorno_ (Berkeley: University of California Press, 2012); and Paul North, _The Problem of Distraction_ (Stanford, Calif.: Stanford University Press, 2012).\n\n 89. The standard postwar discussion of this topic is Hannah Arendt, _The Human Condition_ (Chicago: University of Chicago Press, 1958).\n\n 90. Georg Luk\u00e1cs, _History and Class Consciousness_ , trans. Rodney Livingstone (Cambridge, Mass.: MIT Press, 1971), 319.\n\n 91. Luk\u00e1cs, \"Preface to the New Edition [1967],\" _History and Class Consciousness_ , xviii.\n\n 92. Georg Luk\u00e1cs, _The Theory of the Novel_ , trans. Anna Bostock (Cambridge, Mass.: MIT Press, 1971), 135.\n\n 93. Luk\u00e1cs, _The Theory of the Novel_ , 118.\n\n 94. Georg Luk\u00e1cs, \"Gedanken zu einer Aesthetik des 'Kino,'\" _Frankfurter Zeitung_ 251 (10 September 1913): 1\u20132. There is also an earlier version, \"Gedanken zu einer Aesthetik des 'Kino,'\" which appeared in the German-Hungarian journal _Pester Lloyd_ (16 April 1911): 44\u201346. I will be using Janelle Blankenship's excellent translation, \"Thoughts Toward an Aesthetic of the Cinema,\" _Polygraph_ 13 (2001): 13\u201318. Hereafter cited parenthetically.\n\n 95. See especially Tom Levin, \"From Dialectical to Normative Specificity: Reading Luk\u00e1cs on Film,\" _New German Critique_ 40 (Winter 1987): 35\u201361; and Janelle Blankenship, \"Futurist Fantasies: Luk\u00e1cs's Early Essay 'Thoughts Toward an Aesthetic of the Cinema,'\" _Polygraph_ 13 (2001): 21\u201336.\n\n 96. Janelle Blankenship notes that this formulation recalls Bergson's concept of _dur\u00e9e_ , which might have decisively shaped Luk\u00e1cs's later work. Blankenship \"Futurist Fantasies,\" 22.\n\n 97. Levin, \"From Dialectical to Normative Specificity,\" 35\u201361.\n\n 98. We must here note that this designation of cinema\u2014or any form for that matter\u2014as utopian is very provisional in Luk\u00e1cs's work. Luk\u00e1cs's early work sometimes endorsed the possibility of a glimpse in art of unalienated, authentic life and, at other times, foreclosed that possibility, depending on his mode of analysis, metaphysical or historical. See Gy\u00f6rgy M\u00e1rkus, \"Life and the Soul: The Young Luk\u00e1cs and the Problem of Culture,\" in _Luk\u00e1cs Revalued_ , ed. Agnes Heller (London: Blackwell, 1983), 1\u201326.\n\n 99. Franz Pfemfert, \"Kino als Erzieher,\" in Schweinitz, _Prolog_ , 165\u2013169. Originally published in _Die Aktion_ 1, no. 18 (19 June 1911): 560\u2013563.\n\n. Kurt Pinthus, _Das Kinobuch_ (Frankfurt: Fischer Taschenbuch, 1983), 27.\n\n. Benjamin, \"Garlanded Entrance: On the 'Sound Nerves' Exhibition at the Gesundheitshaus Kreuzberg,\" in Jennings, Doherty, and Levin, eds., _The Work of Art in the Age of Its Technological Reproducibility, and Other Writings on Media_ , 60\u201366, here 62.\n\n. Duttlinger, \"Between Contemplation and Distraction,\" 51.\n\n. Jocelyn Szczepaniak-Gillece, \"Machines for Seeing: Cinema, Architecture, and Mid-century American Spectatorship,\" PhD diss. (Northwestern University, 2013).\n\nCONCLUSION\n\n 1. For a history of the _Kulturfilm_ , see Oskar Kalbus, _Pionere des Kulturfilms: Ein Beitrag zur Geschichte des Kulturfilmschaffens in Deutschland_ (Karlsruhe: Neue Verlags-Gesellschaft, 1956).\n\n 2. For a clear explanation of _dispositif_ as a historiographical concept, see Frank Kessler, \"La cin\u00e9matographie comme dispositif (du) spectaculaire,\" _CiN\u00e9MAS_ 14, no. 1 (2003): 21\u201334; and \"The Cinema of Attractions as _Dispositif_ ,\" in _The Cinema of Attractions Reloaded_ , ed. Wanda Strauven (Amsterdam: Amsterdam University Press, 2006), 57\u201369.\n\n 3. Tom Gunning, \"Systematizing the Electric Message: Narrative Form, Gender, and Modernity in _The Lonedale Operator_ ,\" in _American Cinema_ ' _s Transitional Era: Audiences, Institutions, Practices_ , ed. Charlie Keil and Shelley Stamp (Berkeley: University of California Press, 2004), 15\u201350. See also Tom Gunning, \"Film History and Film Analysis: The Individual Film in the Course of Time,\" _Wide Angle_ 12, no. 3 (July 1990): 4\u201319.\n\n 4. Ludwik Fleck, _Genesis and Development of a Scientific Fact_ , ed. Thaddeus J. Trenn and Robert K. Merton, trans. Fred Bradley and Thaddeus J. Trenn (Chicago: University of Chicago Press, 1979); and _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle (Dordrecht, Netherlands, and Boston: Reidel, 1986).\n\n 5. Nicolas Rasmussen follows the development of a representational technology and its disciplinary conventions in _Picture Control: The Electron Microscope and the Transformation of Biology in America, 1940\u20131960_ (Stanford, Calif.: Stanford University Press, 1997).\n\n 6. See my \"Vergr\u00f6sserung und das mikroskopische Erhabene,\" _Zeitschrift f\u00fcr Medienwissenschaft_ 5 (2011): 96\u2013110. See also Hannah Landecker, \"Cellular Features: Microcinematography and Film Theory,\" _Critical Inquiry_ 31, no. 4 (2005): 903\u2013937.\n\n 7. Hannah Landecker, \"Creeping, Drinking, Dying: The Cinematic Portal and the Microscopic World of the Twentieth-Century Cell,\" _Science in Context_ 24, no. 3 (September 2011): 381\u2013416.\n\n 8. Hans-J\u00f6rg Rheinberger, _Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube_ (Stanford, Calif.: Stanford University Press, 1997).\n\n 9. These ideas about linked solutions and mutant genes I owe to George Kubler, _The Shape of Time: Remarks on the History of Things_ (New Haven: Yale University Press, 1962).\n\n 10. A particularly compelling statement of this dynamic is in Andreas Gailus, \"Of Beautiful and Dismembered Bodies: Art as Social Discipline in Schiller's _On the Aesthetic Education of Man_ ,\" in _Impure Reason: Dialectic of Enlightenment in Germany_ , ed. W. Daniel Wilson and Robert C. Holub (Detroit, Mich.: Wayne State University Press, 1993), 146\u2013165, here 158\u2013159.\nBIBLIOGRAPHY\n\nPRIMARY SOURCES\n\nAckerknecht, Erwin. _Das Lichtspiel im Dienste der Bildungspflege: Handbuch f\u00fcr Lichtspielreformer_. Berlin: Weidmannsche, 1918.\n\nAllen, Grant. _Physiological Aesthetics_. London: King, 1877.\n\nAltenloh, Emilie. _Zur Soziologie des Kino: Die Kino-Unternehmung und die sozialen Schichten ihrer Besucher_. Jena: Diederichs, 1914.\n\nAmerican Society of Heating and Ventilation Engineers. \"Report of Committee on Standards for Ventilation Legislation for Motion Picture Show Places.\" _Gesundheits-Ingenieur_ 36, no. 22 (31 May 1913): 409\u2013410.\n\nAndreas-Salom\u00e9, Lou. _In der Schule bei Freud: Tagesbuch eines Jahres 1912\/1913_. Edited by Ernst Pfeiffer. Z\u00fcrich: Niehans, 1958.\n\n\"An unsere Abonnenten!\" _Film und Lichtbild_ 2, no. 5 (1913): 84.\n\n\"A Surgical Showman.\" _British Medical Journal_ (19 January 1907): 163.\n\nBeard, George M. _American Nervousness: Its Causes and Consequences_. New York: Putnam, 1881.\n\nBeaunis, [Henri-\u00c9tienne]. \"L'exp\u00e9rimentation en psychologie par le somnambulisme provoqu\u00e9.\" _Revue philosophique_ 10, no. 7 (1885): 2.\n\nB\u00e9nard, Henri. \"Formation de centres de giration \u00e0 l'arri\u00e8re d'un obstacle en movement.\" _Comptes rendus de l_ ' _Acad\u00e9mie des Sciences_ 147 (1908): 839\u2013842.\n\n\u2014\u2014. \"Les tourbillons cellulaires dans une nappe liquide. II. Proc\u00e9d\u00e9s m\u00e9caniques et optiques d'examens, lois num\u00e9riques des ph\u00e9nom\u00e8nes.\" _Revue g\u00e9n\u00e9rale des sciences pures et appliqu\u00e9es_ 11 (1900): 1309\u20131328.\n\nBenjamin, Walter. \"Garlanded Entrance: On the 'Sound Nerves' Exhibition at the Gesundheitshaus Kreuzberg.\" In _The Work of Art in the Age of Its Technological Reproducibility, and Other Writings on Media_ , ed. Michael W. Jennings, Brigid Doherty, and Thomas Y. Levin, trans. Edmund Jephcott, Rodney Livingstone, Howard Eiland, and others, 60\u201366. Cambridge, Mass.: Belknap, 2008.\n\n\u2014\u2014. \"Little History of Photography.\" In _Walter Benjamin: Selected Writings_ , vol. 2, _1927\u20131934_ , ed. Michael W. Jennings, Howard Eiland, and Gary Smith, trans. Rodney Livingstone and others, 507\u2013530. Cambridge, Mass.: Belknap, 1999.\n\n\u2014\u2014. \"On Some Motifs in Baudelaire.\" In _Walter Benjamin: Selected Writings_ , vol. 4, _1938\u20131940_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott and others, 313\u2013355. Cambridge, Mass.: Belknap, 2003.\n\n\u2014\u2014. \"Surrealism: The Last Snapshot of the European Intelligensia.\" In _Walter Benjamin: Selected Writings_ , vol. 2, _1927\u20131934_ , ed. Michael W. Jennings, Howard Eiland, and Gary Smith, trans. Rodney Livingstone and others, 207\u2013221. Cambridge, Mass.: Belknap, 1999.\n\n\u2014\u2014. \"Theory of Distraction.\" In _The Work of Art in the Age of Its Technological Reproducibility, and Other Writings on Media_ , ed. Michael W. Jennings, Brigid Doherty, and Thomas Y. Levin, trans. Edmund Jephcott, Rodney Livingstone, Howard Eiland, and others, 56\u201357. Cambridge, Mass.: Belknap, 2008.\n\n\u2014\u2014. \"The Work of Art in the Age of Its Technological Reproducibility (Second Version).\" In _Walter Benjamin: Selected Writings_ , vol. 3, _1935\u20131938_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott, Howard Eiland, and others, 101\u2013133. Cambridge, Mass.: Belknap, 2002.\n\n\u2014\u2014. \"The Work of Art in the Age of Its Technological Reproducibility (Third Version).\" In _Walter Benjamin: Selected Writings_ , vol. 4, _1938\u20131940_ , ed. Howard Eiland and Michael W. Jennings, trans. Edmund Jephcott and Others, 251\u2013283. Cambridge, Mass.: Belknap, 2003.\n\nBergmann, Franz. \"Der Kinematograph im Hochschulunterricht.\" _Bild und Film_ 2, no. 2 (1912\/1913): 48.\n\nBergson, Henri. _Creative Evolution_. Translated by Arthur Mitchell. New York: Random House, 1944.\n\n\u2014\u2014. _An Introduction to Metaphysics_. Translated by T. E. Hulme. New York: Macmillan, 1955.\n\n\u2014\u2014. _Matter and Memory_. Translated by Nancy Margaret Paul and W. Scott Palmer. Cambridge, Mass.: Zone, 1988.\n\n\"Bericht \u00fcber eine Besprechung der Kinokommssion des Westf\u00e4lischen Landgemeindetages anl\u00e4\u00dflich der Er\u00f6ffnungsfeier des Gemeindelichtspielhauses in Eickel.\" _Bild und Film_ 2, no. 3 (1912): 70\u201371.\n\n\"Besuch kinematographischer Vorf\u00fchrung durch Sch\u00fcler h\u00f6herer Lehranstalten (Breslau 1910)\" and \"Besuch der Kinematographentheater durch Sch\u00fcler und Sch\u00fclerinnen sowie durch die Z\u00f6glinge der Seminare und Pr\u00e4parandenanstalten (Berlin 1912).\" In _Dokumente zur Geschichte der Schulfilmbewegung in Deutschland_ , ed. Fritz Terveen, 16\u201317. Emsdetten: Lechte, 1959.\n\nBillroth, Theodor. _The Medical Sciences in the German Universities: A Study in the History of Civilization_. Translated by William H. Welch. New York: Macmillan, 1924. Originally published as _\u00dcber das Lehren und Lernen de medicinischen Wissenschaften an den Universit\u00e4ten der deutschen Nation nebst allgemeinen Bemerkungen \u00fcber Universit\u00e4ten; eine culturhistorische Studie_ (Vienna: Gerold, 1876).\n\nBloch, Ernst. \"Die Melodie im Kino oder immanente und transzendentale Musik.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 326\u2013334. Leipzig: Reclam, 1992. Originally published in _Die Argonauten_ 1, no. 2 (1914): 84\u201385.\n\n\u2014\u2014. \"On Music in the Cinema.\" In _Literary Essays_ , trans. Andrew Joron and others, 157\u2013158. Stanford, Calif.: Stanford University Press, 1998.\n\nBraun, Ludwig. _\u00dcber Herzbewegung und Herzstoss._ Jena: Fischer, 1898.\n\n\u2014\u2014. \"Ueber den Werth des Kinematographen f\u00fcr die Erkenntniss der Herzmechanik.\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 69, part 1 (1898): 185\u2013186.\n\nBraune, Wilhelm, and Otto Fischer. \"Betrachtungen \u00fcber die weiteren Ziele der Untersuchung und \u00dcberblick \u00fcber die Bewegungen der unteren Extremit\u00e4ten.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 26, no. 3 (1900): 85\u2013170.\n\n\u2014\u2014. _Das Gesetz der Bewegungen an der Basis der mittleren Finger und im Handgelenk des Menschen_. Leipzig, 1887.\n\n\u2014\u2014. _Determination of the Moments of Inertia of the Human Body and Its Limbs_. Translated by Paul Macquet and Ronald Furlong. Berlin and New York: Springer, 1988. Originally published as _Bestimmung der Tr\u00e4gheitsmomente des menschlichen Koerpers und seiner Glieder_. Leipzig, 1892.\n\n\u2014\u2014. \"Die Bewegung des Gesamtschwerpunktes und die \u00e4u\u00dferen Kr\u00e4fte.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 25, no. 1 (1899): 1\u2013130.\n\n\u2014\u2014. \"Die Kinematik des Beinschwingens.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 28, no. 5 (1903): 319\u2013418.\n\n\u2014\u2014. _The Human Gait_ , trans. Paul Maquet and Ronald Furlong. Berlin and New York: Springer, 1987.\n\n\u2014\u2014. _On the Centre of Gravity of the Human Body as Related to the Equipment of the German Infantry Soldier_. Translated by Paul Macquet and Ronald Furlong. Berlin and New York: Springer, 1985. Originally published as _\u00dcber den Schwerpunkt des menschlichen K\u00f6rpers mit R\u00fccksicht auf die Ausr\u00fcstung des deutschen Infanteristen_. Leipzig, 1889.\n\n\u2014\u2014. \"\u00dcber den Einflu\u00df der Schwere und der Muskeln auf die Schwingungsbewegung des Beins.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 28, no. 7 (1904): 531\u2013617.\n\n\u2014\u2014. \"\u00dcber die Bewegung des Fu\u00dfes und die auf denselben einwirkenden Kr\u00e4fte.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 26, no. 7 (1901): 467\u2013556.\n\n\u2014\u2014. \"Versuche am unbelasteten und belasteten Menschen.\" _Abhandlungen der Mathematisch-Physischen Klasse der K\u00f6niglich S\u00e4chsischen Gesellschaft der Wissenschaften_ 21, no. 4 (1895): 151\u2013322.\n\nBrauner, Ludwig. \"Die Kino-Ausstellung in Berlin.\" _Der Kinematograph_ no. 104 (25 December 1908).\n\nBraus, Hermann. \"Experimentelle Beitr\u00e4ge zur Frage nach der Entwickelung peripherer Nerven.\" _Anatomischer Anzeiger_ 26, nos. 17\/18 (1 April 1905): 433\u2013479.\n\n\u2014\u2014. \"Mikro-Kino-Projektionen von in vitro gez\u00fcchteten Organanlagen.\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 83, part 2 (1911): 472\u2013475.\n\n\u2014\u2014. \"Mikro-Kino-Projektionen von in vitro gez\u00fcchteten Organanlagen.\" _Wiener medizinische Wochenschrift_ 61, no. 44 (1911): 2809\u20132812.\n\nBredtmann, Hermann. \"Kinematographie und Schule.\" _P\u00e4dagogisches Archiv_ 56, no. 3 (1914): 154\u2013163.\n\nBrod, Max. \"Kinematographentheater.\" In _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_ , ed. Anton Kaes, 39\u201341. T\u00fcbingen: Niemeyer, 1978. Originally published in _Die neue Rundschau_ 20, no. 2 (February 1909): 319\u2013320.\n\nBruegel, Carl. \"Bewegungsvorg\u00e4nge am pathologischen Magen auf Grund r\u00f6ngenkinematographischer Untersuchungen.\" _M\u00fcnchener medizinische Wochenschfrift_ 60, no. 4 (28 January 1913): 179\u2013181.\n\nBrunner, Karl. _Der Kinematograph von heute_ \u2014 _eine Volksgefahr_. Berlin: Vaterl\u00e4ndischen Schriftenverbandes, 1913.\n\nCarr, W. P. \"Suggestion as Used and Misused in Curing Disease.\" In _Hypnotism and Hypnotic Suggestion_ , ed. E. Virgil Neal and Charles S. Clark, 5\u201317. Rochester: New York State Publishing, 1900.\n\nCarrel, Alexis, and Montrose T. Burrows. \"Cultivation of Adult Tissues and Organs Outside of the Body.\" _Journal of the American Medical Association_ 55, no. 16 (15 October 1910): 1379\u20131381.\n\n\u2014\u2014. \"Cultivation of Sarcoma Outside of the Body: A Second Note.\" _Journal of the American Medical Association_ 55, no. 18 (29 October 1910): 1554.\n\n\u2014\u2014. \"Cultivation of Tissues in Vitro and Its Technique.\" _Journal of Experimental Medicine_ 13, no. 3 (1 March 1911) 387\u2013396.\n\n\u2014\u2014. \"Human Sarcoma Cultivated Outside of the Body: A Third Note.\" _Journal of the American Medical Association_ 55, no. 20 (12 November 1910): 1732.\n\nChase, Walter Greenough. \"The Use of the Biograph in Medicine.\" _Boston Medical and Surgical Journal_ 153, no. 21 (23 November 1905): 571\u2013572.\n\nChevroton, L., and F. Vl\u00e8s. \"La cin\u00e9matique de la segmentation de l'oeuf et la chronophotographie du d\u00e9veloppement de l'Oursin.\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 149 (8 November 1909): 806\u2013809.\n\nChristeller, Erwin. \"Die Bedeutung der Photographie f\u00fcr den pathologisch-anatomischen Unterricht und die pathologisch-anatomische Forschung.\" _Berliner klinische Wochenschrift_ 55, no. 17 (29 April 1918): 399\u2013401.\n\nColdstream, G. P. \"The Cinematoscope as an Aid in Teaching.\" _British Medical Journal_ (3 September 1898): 658.\n\nCole, Lewis Gregory. \"The Gastric Motor Phenomena Demonstrated with the Projecting Kinetoscope.\" _American Quarterly of Roentgenology_ 3, no. 4 (1912): 1\u201311.\n\nComandon, Jean with Albert Dastre. \"Cin\u00e9matographie, \u00e0 l'ultra-microscope, de microbes vivants et des particules mobiles.\" _Comptes rendus hebdomadaires des s\u00e9ances de l'Acad\u00e9mie des Sciences_ 149 (22 November 1909): 938\u2013941.\n\n\"Conference on Hospital Standardization.\" _Bulletin of the American College of Surgeons_ 3, no. 1 (1917).\n\nCotton, Aim\u00e9. \"Recherches r\u00e9centes sur les mouvements browniens.\" _La Revue du Mois_ 5 (10 June 1908): 737\u2013741.\n\nCranz, Carl. \"\u00dcber einen ballistischen Kinematographen.\" _Deutscher Mechaniker-Zeitung_ 18 (15 September 1909): 173\u2013177.\n\nCros, Charles. \"Inscription.\" In _Oeuvres completes_ , ed. Louis Forestier and Pascal Pia, 135\u2013136. Paris: Pauvert, 1964.\n\nd'Abundo, Giuseppe. \"Sopra alcuni particolari effetti delle projezioni cinematografiche nei nevrotici.\" _Rivista Italiana di Neuropatologia, Psichiatria ed Elettroterapia_ 4, no. 10 (October 1911): 433\u2013442.\n\nDannmeyer, C. H. _Bericht der Kommission f\u00fcr_ \" _Lebende Photographien._ \" Hamburg: Kampen, 1907.\n\nDescartes, Ren\u00e9. \"The Search for Truth.\" _The Philosophical Writings of Descartes_ , vol. 2. Translated by John Cottingham, Robert Stoothoff, and Dugald Murdoch, 400\u2013420. Cambridge and New York: Cambridge University Press, 1984.\n\nDessauer, Friedrich. _Die neuesten Fortschritte in der R\u00f6ntgenphotographie_. Leipzig: Nemnich, 1912.\n\nDewey, John. _Art as Experience_. New York: Putnam, 1984.\n\n\"Die Dresdner 'Kosmographia.'\" _Bild und Film_ 1, no. 1 (1912): 19.\n\n\"Die Er\u00f6ffnung des Reform-Kinematographentheater.\" _Der Kinematograph_ no. 32 (7 August 1907).\n\n\"Die Kinematographie des Unsichtbaren.\" _Prometheus_ 21, no. 1054 (5 January 1910): 218\u2013220.\n\n\"Die Kino-Austellung und 'Wir.'\" _Erste Internationale Film-Zeitung_ 6, no. 50 (14 December 1912): 52.\n\n\"Die Kultur-Arbeit des Kinematographen-Theaters.\" _Die Lichtbild-B\u00fchne_ 2, no. 41 (4 February 1909).\n\nDiem, Ulrich. _Bildbetrachtung: Eine Wegleitung f\u00fcr Kunstfreunde_. St. Gallen: Fehr'sche, 1919.\n\nDoyen, Eug\u00e8ne. \"Le cin\u00e9matograph et l'enseignement de la chirurgie.\" _Revue critique de m\u00e9decine et de chirurgie_ 1, no. 1 (15 August 1899): 1\u20136. Partially translated as \"The Cinematograph and the Teaching of Surgery,\" _British Gyn\u00e6cological Journal_ 15 (1899): 579\u2013586.\n\nDoyen, Eug\u00e8ne Louis. _La cas des xiphopages hindoues Radica et Doodica_. Paris: Bourse de Commerce, 1904.\n\n\u2014\u2014. _L'enseignement de la technique op\u00e9ratoire par les projections anim\u00e9es_. Paris: Soci\u00e9t\u00e9 g\u00e9n\u00e9rale des cin\u00e9matographes Eclipse, ca. 1911.\n\nDu Bois-Reymond, Emil. _Untersuchungen \u00fcber thierische Elektricit\u00e4t_. Berlin: Reimer, 1848\u20131884.\n\nDuenschmann, Hermann. \"Kinematograph und Psychologie der Volksmenge. Eine sozialpolitische Studie.\" _Konservative Monatsschrift_ 69, no. 9 (June 1912): 920\u2013930.\n\n\"Ein kurzer R\u00fcckblick auf die erste Woche des Reform-Kinematographen-Theaters.\" _Der Kinematograph_ no. 33 (14 August 1907).\n\n\"Ein neues wissenschaftliches Kino.\" _Film und Lichtbild_ 1, no. 5 (1912): 62.\n\nEinstein, Albert. \"On the Movement of Small Particles Suspended in a Stationary Liquid Demanded by the Molecular-Kinetic Theory of Heat.\" In _Investigations on the Theory of the Brownian Movement_ , ed. R. F\u00fcrth, trans. A. D. Cowper, 1\u201318. New York: Dover, 1956.\n\nErb, Wilhelm Heinrich. _Ueber die wachsende Nervosit\u00e4t unserer Zeit_. Heidelberg: Universit\u00e4ts Buchdruckerei von J. H\u00f6rning, 1893.\n\nExner, Felix M. \"Notiz zu Brown's Molecularbewegung.\" _Annalen der Physik_ 2, no. 8 (1900): 843\u2013847.\n\nEykman, P. H. \"Der Schlingact, dargestellt nach Bewegungsphotographien mittelst R\u00f6ntgen-Strahlen.\" _Pfl\u00fcger_ ' _s Archiv f\u00fcr die gesammte Physiologie des Menschen und der Thiere_ 99 (1903): 513\u2013571.\n\nFechner, Gustav. _Vorschule der Aesthetik_. Leipzig: Breitkopf & H\u00e4rtel, 1897.\n\nFelke, Naldo. \"Die Gesundheitssch\u00e4dlichkeit des Kinos.\" _Die Umschau_ 17, no. 1 (1 January 1913): 254\u2013255.\n\nFiedler, Conrad. _\u00dcber die Berurtheilung von Werken der bildenden Kunst_. Leipzig: Hirzel, 1876. Translated by Henry Schaefer-Simmern and Fulmer Mood as _On Judging Works of Visual Art_ (Berkeley: University of California Press, 1949).\n\nFischer, Otto. _Theoretische Grundlagen f\u00fcr eine Mechanik der lebenden K\u00f6rper_. Leipzig, 1906.\n\nFlemming, Carl F. _Pathologie und Therapie der Psychosen._ Berlin: Hirschwald, 1859.\n\nForel, Auguste. _Hygiene der Nerven und des Geistes im gesunden und kranken Zustande_. Stuttgart: Moritz, 1903.\n\nFran\u00e7ois-Franck, Charles. \"D\u00e9monstrations de microphotographie instantan\u00e9e et de chronomicrophotographie.\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 62 (25 May 1907): 964\u2013967.\n\n\u2014\u2014. \"\u00c9tudes graphiques et photographiques de m\u00e9canique respiratoire compar\u00e9e.\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 61 (28 July 1906): 174\u2013176.\n\n\u2014\u2014. \"La chronophotographie simultan\u00e9e du coeur et des courbes cardiographiques chez les mammif\u00e8res.\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 54 (8 November 1902): 1193\u20131197.\n\n\u2014\u2014. \"Note sur quelques points de technique relatifs \u00e0 la photographie et \u00e0 la chronophotographie avec le magn\u00e9sium \u00e0 deflagration lente.\" _Comptes rendus hebdomadaires des s\u00e9ances et m\u00e9moires de la Soci\u00e9t\u00e9 de Biologie_ 55 (5 December 1903): 1538\u20131540.\n\nFr\u00e4nkel, Albert. \"R\u00f6ntgendiagnosen und R\u00f6ntgenfehldiagnosen beim Magenkarzinom; diagnostischer Fortschritt durch R\u00f6ntgenkinographie.\" _Zentralblatt f\u00fcr R\u00f6ntgenstrahlen, Radium und verwandte Gebiete_ 3, no. 4 (1912): 149\u201350.\n\nFr\u00e4nkel, James. \"Kinematographische Demonstration.\" _Verhandlungen der freien Vereinigung der Chirurgen Berlins_ 20, part 1 (1907): 12\u201313.\n\n\u2014\u2014. \"Kinematographische Untersuchung des normalen Ganges und einiger Gangst\u00f6rungen.\" _Zeitschrift f\u00fcr orthop\u00e4dische Chirurgie_ 20 (1908): 617\u2013646.\n\nFreeburg, Victor Oscar. _Pictorial Beauty on the Screen_. New York: Macmillan, 1923.\n\nFrei, Wilhelm. _Landerziehungsheime: Darstellung und Kritik einer modernen Reformschule_. Leipzig: Klinkhardt, 1902.\n\nFreud, Sigmund. _Beyond the Pleasure Principle_. Translated by James Strachey. London: Hogarth, 1950.\n\nFrey, J. \"Report of the Photographic Department of Bellevue Hospital for the Year 1869.\" In _Tenth Annual Report of the Commissioners of Public Charities and Correction of the City of New York for the Year 1869_ , 85. Albany: van Benthuysen, 1870. www.artandmedicine.com\/ogm\/1869.html.\n\nFriedell, Egon. \"Prolog vor dem Film.\" In _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_ , ed. Anton Kaes, 42\u201347. T\u00fcbingen: Niemeyer, 1978. Originally published in _Bl\u00e4tter des Deutschen Theaters_ 2 (1912): 509\u2013511.\n\nGaupp, Robert. \"Das Pathologische in Kunst und Literatur.\" _Deutsche Revue_ 36, no. 2 (April 1911): 11\u201323.\n\n\u2014\u2014. \"Der Arzt als Erzieher seines Volkes.\" _Medicinisches Correspondenz-Blatt_ 89, no. 32 (9 August 1919): 295\u2013296.\n\n\u2014\u2014. \"Der Kinematograph vom medizinischen und psychologischen Standpunkt.\" In _Der Kinematograph als Volkunterhaltungsmittel_ , by Robert Gaupp and Konrad Lange, 1\u201312. Munich: D\u00fcrer-Bund-Flugschrift zur Ausdruckskultur 100, 1912.\n\n\u2014\u2014. \"Die Gefahren des Kino.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 64\u201369. Leipzig: Reclam, 1992. Originally published in _S\u00fcddeutsche Monatshefte_ 9, no. 9 (1911\/1912): 363\u2013366.\n\n\u2014\u2014. \"Die Nervosit\u00e4t unserer Zeit im Lichte der Wissenschaft.\" _Medicinisches Correspondenz-Blatt_ 77, no. 31 (3 August 1907): 633\u2013639.\n\n\u2014\u2014. _Psychologie des Kindes_. Leipzig: Teubner, 1910.\n\nGaupp, Robert, and Konrad Lange, _Der Kinematograph als Volksunterhaltungsmitte._ Munich: Callwey, 1912.\n\nGeisel, Walter. _Wie ich mit meinen Jungens Kunstwerke betrachte_. Gl\u00fcckstadt: Geisel, 1904.\n\nGeorges-Michel, Michel. \"Henri Bergson nous parle au cin\u00e9ma.\" _Le Journal_ (20 February 1914): 7. Translated by Louis-Georges Schwartz as \"Henri Bergson Talks to Us About Cinema.\" _Cinema Journal_ 50, no. 3 (Spring 2011): 79\u201382.\n\nGilbreth, Frank B. _Motion Study: A Method for Increasing the Efficiency of the Workman_. New York: Van Nostrand, 1911.\n\n\u2014\u2014. \"Scientific Management in the Hospital.\" _Modern Hospital_ 3 (1914): 321\u2013324.\n\nGoerke, Franz. \"Proposal for Establishing an Archive for Moving Pictures (1912).\" Translated by Cecilie L. French and Daniel J. Leab. _Historical Journal of Film, Radio and Television_ 16, no. 1 (March 1996): 9\u201312. Originally published as \"Vorschlag zur Einrichtung eines Archives f\u00fcr Kino-films,\" in _Der Deutsche Kaiser im Film: zum 25j\u00e4hrigen Regierungs-Jubil\u00e4um Seiner Majest\u00e4t des Deutschen Kaisers K\u00f6nigs von Preu\u00dfen Wilhelm II_ , ed. Paul Klebinder, 63\u201368 (Berlin: Klebinder, 1912).\n\nG\u00f6tze, O. \"Jugendpsyche und Kinematograph.\" _Zeitschrift f\u00fcr Kinderforschung_ 16 (1911): 418.\n\nGouy, Louis-Georges. \"Le mouvement brownien et les mouvements mol\u00e9culaires.\" _Revue g\u00e9n\u00e9rale des sciences pures et appliqu\u00e9es_ 6, no. 1 (15 January 1895): 1\u20137.\n\nGraupner, H. \"Unterrichtshygiene.\" In _Handbuch der deutschen Schulhygiene_ , ed. Hugo Selter, 174\u2013321. Dresden and Leipzig: Theodor Steinkopff, 1914.\n\nGrempe, Max. \"Gegen die Frauenverbl\u00f6dung im Kino.\" _Gleichheit_ 23, no. 5 (1912): 70\u201372.\n\nGroedel, Franz M. \"Die Technik der R\u00f6ntgenkinematographie.\" _Deutsche medizinsiche Wochenschrift_ 35 (11 March 1909): 434\u2013435.\n\n\u2014\u2014. \"Die Technik der R\u00f6ntgenkinematographie.\" _Deutsche medizinsiche Wochenschrift_ 39 (6 February 1913): 270\u2013271.\n\n\u2014\u2014. \"Die Technik der R\u00f6ntgenkinematographie.\" _Deutsche medizinsiche Wochenschrift_ 39 (24 April 1913): 798\u2013799.\n\n\u2014\u2014. \"The Present State of Roentgen Cinematography and Its Results as to the Study of the Movements of the Inner Organs of the Human Body.\" _Interstate Medical Journal_ 22 (March 1915): 281\u2013290.\n\n\u2014\u2014. \"Roentgen Cinematography and Its Importance in Medicine.\" _British Medical Journal_ (24 April 1909): 1003.\n\nGroos, Karl. \"Das \u00e4sthetische Miterleben und die Empfindungen aus dem K\u00f6rperinnern.\" _Zeitschrift f\u00fcr \u00c4sthetik und allgemeine Kunstwissenschaft_ 4 (1909): 161\u2013182.\n\nG\u00fcnther, Hanns. \"Mikrokinematographische Aufnahmeapparate.\" _Film and Lichtbild_ 1, no. 1 (1912): 4\u20136; 1, no. 2 (1912): 13\u201314.\n\nH\u00e4fker, Hermann. \"Eine Reise an die Quellen der Kinematographie.\" _Der Kinematograph_ no. 163 (9 February 1910); 172 (13 April 1910).\n\n\u2014\u2014. _Kino und Kunst_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1913.\n\n\u2014\u2014. \"Meisterspiele.\" _Der Kinematograph_ no. 56 (22 January 1908).\n\n\u2014\u2014. \"Zur Dramaturgie der Bilderspiele.\" _Der Kinematograph_ no. 32 (7 August 1907).\n\nHarris, W. T. Editor's preface to _Herbart_ ' _s ABC of Sense Perception and Minor Pedagogical Works_ , by Johann Friedrich Herbart. Translated and edited by William J. Eckoff, vii\u2013xi. New York: Appleton, 1896.\n\nHarrison, Ross Granville. \"Experiments in Transplanting Limbs and Their Bearing Upon the Problems of the Development of Nerves.\" _Journal of Experimental Zoology_ 4, no. 2 (June 1907): 239\u2013281.\n\n\u2014\u2014. \"Further Experiments on the Development of Peripheral Nerves.\" _American Journal of Anatomy_ 5, no. 2 (31 May 1906): 121\u2013131.\n\n\u2014\u2014. \"Observations on the Living Developing Nerve Fiber.\" _Anatomical Record_ 1, no. 5 (1 June 1907): 116\u2013118.\n\n\u2014\u2014. \"The Outgrowth of the Nerve Fiber as a Mode of Protoplasmic Movement.\" _Journal of Experimental Zoology_ 9, no. 4 (December 1910): 787\u2013846.\n\nHasenclever, Walter. \"Der Kintopp als Erzieher: Eine Apologie.\" In _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_ , ed. Anton Kaes, 47\u201349. T\u00fcbingen: Niemeyer, 1978. Originally published in _Revolution_ 1, no. 4 (1 December 1913): n.p.\n\nHellwig, Albert. \"Die Beziehungen zwischen Schundliteratur, Schundfilms und Verbrechen.\" _Archiv f\u00fcr Kriminal-Anthropologie und Kriminalistik_ 51, no. 1 (24 January 1913): 1\u201332.\n\n\u2014\u2014. \"Die ma\u00dfgebenden Grunds\u00e4tze f\u00fcr Verbote von Schundfilms nach geltendem und k\u00fcnstigem Rechte.\" _Verwaltungsarchiv_ 21 (1913): 405\u2013455.\n\n\u2014\u2014. _Die Filmzensur: Eine rechtsdogmatische und rechtpolitische Er\u00f6rterung_. Berlin: Frankenstein, 1914.\n\n\u2014\u2014. \"Hypnotismus und Kinematograph.\" _Zeitschrift f\u00fcr Psychotherapie und medizinische Psychologie_ 6 (1916): 310\u2013315.\n\n\u2014\u2014. _Kind und Kino_. Langensalza: Beyer, 1914.\n\n\u2014\u2014. \"Kinematograph und Zeitgeschichte.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 97\u2013109. Leipzig: Reclam, 1992. Originally published in _Die Grenzboten_ 72, no. 39 (1913): 612\u2013620.\n\n\u2014\u2014. _\u00d6ffentliches Lichtspielrecht_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1921.\n\n\u2014\u2014. _Rechtsquellen des \u00f6ffentlichen Kinematographenrechts_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1913.\n\n\u2014\u2014. _Schundfilms: Ihr Wesen, ihre Gefahren und ihre Bek\u00e4mpfung_. Halle an der Saale, Germany: Waisenhaus, 1911.\n\n\u2014\u2014. \"\u00dcber die sch\u00e4dliche Suggestivkraft kinematographischer Vorf\u00fchrung.\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 20, no. 6 (15 March 1914): 122.\n\n\u2014\u2014. \"Zur Psychologie kinematographischer Vorf\u00fchrungen.\" _Zeitschrift f\u00fcr Psychotherapie und medizinische Psychologie_ 6 (1916): 88\u2013120.\n\nHelmholtz, Hermann von. _\u00dcber die Erhaltung der Kraft_. Berlin: Reimer, 1847.\n\nHennes, Hans. \"Die Kinematographie der Bewegungsst\u00f6rungen.\" _Die Umschau_ 15, no. 29 (1911): 605\u2013606.\n\n\u2014\u2014. \"Die Kinematographie im Dienste der Neurologie und Psychiatrie, nebst Beschreibung einiger selteneren Bewegungsst\u00f6rungen.\" _Medizinische Klinik_ 6, no. 51 (18 December 1910): 2010\u20132014.\n\nHenri, Victor. \"\u00c9tudes cin\u00e9matographique des mouvements browniens.\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 146 (18 May 1908): 1024\u20131026.\n\n\u2014\u2014. \"Influence du milieu sur les mouvements browniens.\" _Comptes rendus hebdomadaires des s\u00e9ances de l_ ' _Academie des Sciences_ 147 (6 July 1908): 62\u201365.\n\nHerbart, Johann Friedrich. _Kurze Encyklop\u00e4die der Philosophie_. Hamburg: Voss, 1884.\n\n\u2014\u2014. _Lehrbuch zur Einleitung in die Philosophie_ , third edition (K\u00f6nigsberg: Unzer, 1834).\n\nHildebrand, Adolf. _The Problem of Form in the Fine Arts_ (1893). In _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou, 227\u2013279. Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994.\n\nJacobj, Carl. \"Anschauungsunterricht und Projektion.\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und mikroskopische Technik_ 36, no. 4 (1919): 273\u2013314.\n\nJendrassik, Ernst. \"Klinische Beitr\u00e4ge zum Studium der normalen und pathologischen Gangarten.\" _Deutsche Archiv f\u00fcr klinische Medizin_ 70 (1901): 81\u2013132.\n\nKalbus, Oskar. \"Abri\u00df einer Geschichte der deutschen Lehrfilmbewegung.\" In _Das Kulturfilmbuch_ , ed. Edgar Beyfu\u00df and Alexander Kossowsky, 1\u201313. Berlin: Chryselius'scher, 1924.\n\n\u2014\u2014. _Der Deutsche Lehrfilm in der Wissenschaft und im Unterricht_. Berlin: Heymanns, 1922.\n\nKandel, I. L. \"Germany.\" In _Comparative Education: Studies of the Educational Systems of Six Modern Nations_ , ed. Peter Sandiford, 121\u2013130. London and Toronto: Dent, 1918.\n\nKant, Immanuel. _Critique of Judgement_. Translated by James Creed Meredith. Oxford: Oxford University Press, 1952.\n\n\u2014\u2014. \"What Is Enlightenment?\" In _German Aesthetic and Literary Criticism_ , ed. David Simpson, 29\u201334. Cambridge: Cambridge University Press, 1984.\n\nK\u00e4stle, C., H. Rieder, and J. Rosenthal. \"The Bioroentgenography of the Internal Organs.\" _Archives of the Roentgen Ray_ 15, no. 1 (June 1910): 3\u201312.\n\n\u2014\u2014. \"Ueber kinematographisch aufgenommene R\u00f6ntgenogramme (Bio-R\u00f6ntgenographie) der inneren Organe des Menschen.\" _M\u00fcnchener medizinsiche Wochenschrift_ 56, no. 6 (9 February 1909): 280\u2013283.\n\nKey, Ellen. \"Erziehung,\" _Das Jahrhundert des Kindes_ (Berlin, 1905). In _Die deutsche Reformp\u00e4dagogik_ , ed. Wilhelm Flitner and Gerhard Kudritzki, 52\u201354. D\u00fcsseldorf and Munich: K\u00fcpper, 1961.\n\nKienzl, Hermann. \"Theater und Kinematograph.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 230\u2013234. Leipzig: Reclam, 1992. Originally published in _Der Strom_ 1, no. 7 (October 1911): 219\u2013221.\n\n\"Kinematograph als Krankheitsstifter.\" _Fortschritte der Medizin_ 30 (1912): 302.\n\n\"Kinematographische Reformvereinigung.\" _Der Kinematograph_ no. 43 (23 October 1907).\n\n\"Kino und Buchhandel.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 272\u2013289. Leipzig: Reclam, 1992.\n\n\"Kino und Wissenschaft.\" _Bild und Film_ 1, no. 2 (1912): 55.\n\nKleib\u00f6mer, Georg. \"Kinematograph und Schuljugend.\" _Der Kinematograph_ no. 124 (12 May 1909).\n\nKnospe, Paul. _Der Kinematograph im Dienste der Schule. Unter besonderer Ber\u00fccksichtigung des erdkundlichen Unterrichts_. Halle an der Saale, Germany: Waisenhaus, 1913.\n\nKracauer, Siegfried. \"Cult of Distraction: On Berlin's Picture Palaces.\" In _The Mass Ornament: Weimar Essays_ , trans. and ed. Thomas Y. Levin, 323\u2013328. Cambridge, Mass.: Harvard University Press, 1995.\n\nKraepelin, Emil. \"Demonstration von Kinematogrammen.\" _Centralblatt f\u00fcr Nervenheilkunde und Psychiatrie_ 32 (1909): 689.\n\n\u2014\u2014. _Memoirs_. Edited by H. Hippius, G. Peters, and D. Ploog in collaboration with P. Hoff and A. Kreuter. Translated by Cheryl Wooding-Deane. Berlin and New York: Springer-Verlag, 1987.\n\n\u2014\u2014. \"Zur Entartungsfrage.\" _Zentralblatt f\u00fcr Nervenheilkunde und Psychiatrie_ 31 (1908): 745\u2013751. Translated as \"On the Question of Degeneration,\" _History of Psychiatry_ 18, no. 3 (2007): 399\u2013404.\n\nKretz, Richard. \"Die Anwendung der Photographie in der Medicin.\" _Wiener klinische Wochenschrift_ 7, no. 44 (1 November 1894): 832.\n\nKutner, Robert. \"Die Bedeutung der Kinematographie f\u00fcr medizinische Forschung und Unterricht sowie f\u00fcr die volkshygienische Belehrung.\" _Zeitschrift f\u00fcr \u00c4rztliche Fortbildung_ 8, no. 8 (15 April 1911): 249\u2013251.\n\nLandau, Joseph. \"Mechanisierte Unsterblichkeit.\" In _Der Deutsche Kaiser im Film. Zum 25j\u00e4hrigen Regierungs-Jubil\u00e4um Seiner Majest\u00e4t des Deutschen Kaisers K\u00f6nigs von Preu\u00dfen Wilhelm II_ , ed. Paul Klebinder, 18\u201322. Berlin: Klebinder, 1912.\n\nLangbehn, August Julius. \"Rembrandt als Erzieher.\" In _Die Kunsterziehungsbewegung_ , ed. Hermann Lorenzen, 7\u201317. Bad Heilbrunn, Germany: Klinkhardt, 1966.\n\nLange, Konrad. \"Das Wesen der k\u00fcnstlerischen Erziehung.\" In _Die Kunsterziehungsbewegung_ , ed. Hermann Lorenzen, 21\u201326. Bad Heilbrunn, Germany: Klinkhardt, 1966.\n\n\u2014\u2014. _Die k\u00fcnstlerische Erziehung der deutschen Jugend_. Darmstadt: Bergstrae\u00dfer, 1893.\n\nLaquer, Leopold. \"\u00dcber die Sch\u00e4dlichkeit kinematographischer Veranstaltungen f\u00fcr die Psyche des Kindesalters.\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 27, no. 11 (1 June 1911): 221\u2013222.\n\nLaycock, Thomas. _Lectures on the Principles and Methods of Medical Observation and Research_. Philadelphia: Blanchard and Lea, 1857.\n\nLe Bon, Gustave. _The Crowd: A Study of the Popular Mind_. Atlanta, Ga.: Cherokee, 1982. Originally published as _Psychologie des foules_ (Paris: Alcan, 1895). Translated and published in German as _Psychologie der Massen_ (Leipzig: Klinkhardt, 1908).\n\nLearned, William S. _An American Teacher_ ' _s Year in a Prussian Gymnasium_. New York: Educational Review, 1911.\n\nLecomte du No\u00fcy, P[ierre]. _Biological Time_. With a foreword by Alexis Carrel. New York: Macmillan, 1937.\n\nLee, Vernon. \"Weiteres \u00fcber Einf\u00fchlung und \u00e4sthetisches Miterleben.\" _Zeitschrift f\u00fcr \u00c4sthetik und allgemeine Kunstwissenschaft_ 5 (1910): 145\u2013190.\n\n\u2014\u2014, and C. Anstruther-Thomson. _Beauty and Ugliness and Other Studies in Psychological Aesthetics_. London and New York: Lane, 1912.\n\nLehmann, Hans. _Die Kinematographie: ihren Grundlagen und ihre Anwendungen_. Leipzig: Teubner, 1911.\n\nLeipziger Lehrerverein, ed. _Bildbetrachtungen: Arbeiten aus der Abteilung f\u00fcr Kunst-pflege des Leipziger Lehrervereins_. Leipzig: Teubner, 1906.\n\nLemke, Hermann. _Die Kinematographie der Gegenwart, Vergangenheit und Zukunft_. Leipzig: Demme, 1911.\n\n\u2014\u2014. \"Die kinematographische Reformpartei, ihre Aufgaben und Ziele.\" _Der Kinematograph_ no. 42 (16 October 1907).\n\n\u2014\u2014. _Die kinematographische Unterrichtsstunde_. Leipzig: Demme, 1911.\n\n\u2014\u2014. \"Die Verwertung und Nutzbarmachung neuer Film-Ideen\u2014K\u00fcnstlerische Films.\" _Der Kinematograph_ no. 57 (29 January 1908).\n\n\u2014\u2014. _Durch die Technik zur Schulreform. Zwei modern-technische Lehrmethoden und Veranschaulichungsmittel in der Schule der Zukunft_. Leipzig: Demme, 1911.\n\n\u2014\u2014. _Praktische Forderungen f\u00fcr die Verwertung der Kinematographie im Unterricht._ Friedenau: Schule und Technik, 1909.\n\n\u2014\u2014. \"Volkst\u00fcmliche Reisebeschreibungen.\" _Der Kinematograph_ no. 34 (21 August 1907).\n\nLichtwark, Alfred. \"Der Deutsche der Zukunft.\" In _Die deutsche Reformp\u00e4dagogik_ , ed. Wilhelm Flitner and Gerhard Kudritzki, 99\u2013110. D\u00fcsseldorf and Munich: K\u00fcpper, 1961.\n\n\u2014\u2014. \"Die Aufgaben der Kunsthalle: Antrittsrede den 9. December 1886.\" In _Drei Programme_ , 2d ed., 11\u201331. Berlin: Cassirer, 1902.\n\n\u2014\u2014. _Die Bedeutung der Amateur-Photographie_. Halle an der Saale, Germany: Knapp, 1894.\n\n\u2014\u2014. \"Museen als Bildungsst\u00e4tten.\" _Der Deutsche der Zukunft_ , 89\u2013107. Berlin: Cassirer, 1905.\n\n\u2014\u2014. _\u00dcbungen in der Betrachtung von Kunstwerken_. Dresden: K\u00fchtmann, 1900.\n\nLiesegang, F. Paul. _Wissenschaftliche Kinematographie_. D\u00fcsseldorf: Liesegang, 1920.\n\nLipps, Theodor. _\u00c4sthetik. Psychologie des Sch\u00f6nen und der Kunst. Erster Teil: Grundlegung der \u00c4sthetik_. Hamburg and Leipzig: Voss, 1903.\n\n\u2014\u2014. \"Einf\u00fchlung, innere Nachahmung, und Organempfindungen.\" _Archiv f\u00fcr die gesamte Psychologie_ 1, nos. 2\/3 (1903): 185\u2013204. Translated as \"Empathy, Inner Imitation, and Sense-Feeling,\" in _A Modern Book of Aesthetics_ , ed. Melvin M. Rader, 291\u2013304 (New York: Holt, 1935).\n\n\u2014\u2014. \"Einf\u00fchlung und \u00e4sthetischer Genu\u00df.\" _Die Zukunft_ 54, no. 14 (20 January 1906): 100\u2013114.\n\nLocke, John. _An Essay Concerning Human Understanding_. Edited and with an introduction by Peter H. Nidditch. Oxford: Oxford University Press, 1975.\n\nLomon, Andr\u00e9, and Jean Comandon. \"Radiocin\u00e9matographie par la photographie des \u00e9crans intensificateurs.\" _La Presse M\u00e9dicale_ 35 (3 May 1911): 359.\n\nLonde, Albert. _Notice sur les titres et travaux scientifique_. Paris: Masson, 1911.\n\n\u2014\u2014. _Nouvelle iconographie de la Salp\u00eatri\u00e8re_. Paris: Masson, 1888\u20131918.\n\n\u2014\u2014. _Photographie m\u00e9dicale_. Paris: Gauthiers-Villars, 1893.\n\nLorenz, Richard, and W. Eitel. \"\u00dcber die \u00f6rtliche Verteilung von Rauchteilchen.\" _Zeitschrift f\u00fcr anorganische Chemie_ 87, no. 1 (12 May 1914): 357\u2013374.\n\nLuk\u00e1cs, Georg. _History and Class Consciousness_. Translated by Rodney Livingstone. Cambridge, Mass.: MIT Press, 1971.\n\n\u2014\u2014. _The Theory of the Novel_. Translated by Anna Bostock. Cambridge, Mass.: MIT Press, 1971.\n\n\u2014\u2014. \"Thoughts Toward an Aesthetic of the Cinema.\" Translated by Janelle Blankenship. _Polygraph_ 13 (2001): 13\u201318. Originally published as \"Gedanken zu einer Aesthetik des 'Kino,'\" _Frankfurter Zeitung_ 251 (10 September 1913): 1\u20132.\n\n\u2014\u2014. \"\u00dcber den Dostojewski-Nachlass.\" _Moskauer Rundschau_ 17 (22 March 1931): 4.\n\nMach, Ernst. _Die Principien der W\u00e4rmlehre: Historisch-kritisch Entwickelt_. Leipzig: Barth, 1896.\n\nMacintyre, John. \"X-Ray Records for the Cinematograph.\" _Archives of Skiagraphy_ 1, no. 2 (April 1897): 37.\n\nMagnus-Levy, Adolf. \"Ueber Organ-Therapie beim endemischen Kretinismus.\" _Ver-handlungen der Berliner medicinischen Gesellschaft_ 34, part 2 (1903): 350\u2013357; 34, part 1 (1903): 246\u2013249.\n\nMarey, \u00c9tienne-Jules. _Animal Mechanism: A Treatise on Terrestrial and Aerial Locomotion_. New York: Appleton, 1874.\n\n\u2014\u2014. \"\u00c9tudes sur la marche de l'homme.\" _Revue Militaire de M\u00e9decine et de Chirurgie_ 1 (1880): 244\u2013246.\n\n\u2014\u2014. _La chronophotographie_. Paris: Gauthier-Villars, 1899.\n\n\u2014\u2014. _La methode graphique dans les sciences \u00e9xperimentales et principlement en physiologie et en m\u00e9dicine_. Paris: Masson, 1885.\n\n\u2014\u2014. _Movement_. Translated by Eric Pritchard. London: Heinemann, 1895. Originally published as _Le movement_. Paris: Masson, 1894.\n\nMarinescu, Gheorghe. \"Les troubles de la marche dans l'h\u00e9mipl\u00e9gie organique \u00e9tudi\u00e9s \u00e0 l'aide du cin\u00e9matographe.\" _La semaine M\u00e9dicale_ (1899): 225\u2013228.\n\nMay, Bruno. _Das Recht des Kinematographen_. Berlin: Falk, 1912.\n\n\"Medical News.\" _Lancet_ 177 (27 May 1911): 1470.\n\n\"Medical News.\" _Lancet_ 182 (11 October 1913): 1083\u20131084.\n\n\"Medizinisch-Naturwissenschaftlicher Verein T\u00fcbingen.\" _M\u00fcnchener medizinische Wochenschrift_ 56, no. 3 (19 January 1909): 154\u2013155.\n\nMeier, Konrad. \"Vorschriften \u00fcber L\u00fcftung von Kinotheatern.\" _Gesundheits-Ingenieur_ 36, no. 26 (28 June 1913): 483\u2013484.\n\nMellini, Arthur. \"Die ganze Richtung passt uns nicht!\" _Lichtbild-B\u00fchne_ 5 (4 February 1911): 3\u20134.\n\nMendel, Georg Victor. _Kinematographie und Schule: Plan zur Gr\u00fcndung eines rein wissenschaftlichen Theaters f\u00fcr Kinematographie und Projektion_. Berlin: privately printed, 1909.\n\nMeumann, Ernst. \"Wilhelm Wundt. Zu seinem achtzigsten Geburtstag.\" _Deutsche Rundschau_ 38, no. 11 (August 1912): 193\u2013224.\n\nMoll, Albert. _Hypnotism_. London: Scott, 1890. Originally published as _Der Hypnotismus_ (Berlin: Fischer's Medicinische, 1889).\n\nMosso, Angelo. _Fatigue_. Translated by Margaret Drummond and W. B. Drummond. New York: Putnam, 1904.\n\nM\u00fcller-Sanders, Hans. \"Die Kinematographenzensur in Preu\u00dfen.\" PhD diss., Badischen Ruprecht-Karls-Universit\u00e4t, Heidelberg, 1912.\n\nM\u00fcnsterberg, Hugo. _The Photoplay: A Psychological Study_. London and New York: Appleton, 1916.\n\nMurawski, Friedrich. _Die Kinematographie und ihre Beziehungen zu Schule und Unterricht_. Dresden: Bieyl and Kaemmerer, 1914.\n\nMuybridge, Eadweard. _Animal Locomotion: An Electro-Photographic Investigation of Consecutive Phases of Animal Movement_. Philadelphia: University of Pennsylvania, 1887.\n\n\u2014\u2014. _Muybridge_ ' _s Complete Human and Animal Locomotion_. New York: Dover, 1979.\n\n\"Neuland f\u00fcr Kinematographentheater.\" In _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_ , ed. Anton Kaes, 41. T\u00fcbingen: Niemeyer, 1978. Originally published in _Lichtbild-B\u00fchne_ 3 (September 1910): 3.\n\nNietzsche, Friedrich. _Beyond Good and Evil_. In _Basic Writings of Nietzsche_ , trans. Walter Kaufmann, 181\u2013435. New York: Modern Library, 1966.\n\nNitze, Max. _Kystophotographischer Atlas_. Wiesbaden: Bergmann, 1894.\n\nNordau, Max. _Degeneration_. London: Appleton, 1895. Originally published as _Entartung_ , 2 vols. (Berlin: Duncker, 1892\u20131893).\n\n\"Novel Uses for Moving Pictures.\" _Moving Picture World_ 1, no. 3 (23 March 1907): 39\u201340.\n\nPaget, Sir James. \"An Address on the Utility of Scientific Work in Practice.\" _British Medical Journal_ (15 October 1887): 811\u2013814.\n\nPatterson, Frances Taylor. _Scenario and Screen_. New York: Harcourt, Brace, 1928.\n\nPerrin, Jean. \"La realit\u00e9 des molecules.\" _Revue Scientifique_ 49, no. 2 (1911): 774\u2013784.\n\n\u2014\u2014. _Les atoms_. 4th ed. rev. Paris: Librarie F\u00e9lix Alcan, 1914. Translated by D. L. Hammick as _Atoms_ , 2d English ed. rev. (London: Constable, 1923).\n\n\u2014\u2014. \"Mouvement brownien et mol\u00e9cules.\" _Annales de chimie et de physique_ 18 (September 1909): 1\u2013114. Translated by F. Soddy as \"Brownian Movement and Molecular Reality,\" in _The Question of the Atom_ , ed. Mary Jo Nye, 507\u2013601 (Los Angeles, Calif.: Tomash, 1984).\n\nPestalozzi, Johann Heinrich. _How Gertrude Teaches Her Children_. Edited by Ebenezer Cooke. Translated by Lucy E. Holland and Frances C. Turner. 2d ed. Syracuse, N.Y.: Bardeen, 1898. Originally published as _Wie Gertrud ihre Kinder lehrt, ein Versuch den M\u00fcttern Anleitung zu geben, ihre Kinder selbst zu unterrichten, in Briefen_ (Bern and Z\u00fcrich: Ge\u00dfner, 1801).\n\nPfeffer, Wilhelm. \"Die Anwendung des Projectionsapparates zur Demonstration von Lebensvorg\u00e4ngen.\" _Jahrb\u00fccher wissenschaftliche Botanik_ 35 (1900): 711\u2013745.\n\nPfemfert, Franz. \"Kino als Erzieher.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 165\u2013169. Leipzig: Reclam, 1992. Originally published in _Die Aktion_ 1, no. 18 (19 June 1911): 560\u2013563.\n\nPhilippson, Martin. _L'autonomie et la centralisation dans le syst\u00e8me nerveux des animaux: \u00e9tude de physiologie exp\u00e9rimentale et compare_. Brussels: Falk, 1905.\n\nPieper, Lorenz. \"Kino und Drama.\" _Bild und Film_ 1, no. 1 (1912): 5.\n\nPinthus, Kurt. _Das Kinobuch_. Frankfurt: Fischer Taschenbuch, 1983.\n\nPolgar, Alfred. \"Das Drama im Kinematographen.\" In _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ , ed. Fritz G\u00fcttinger, 56\u201361. Frankfurt: Deutsches Filmmuseum, 1984. Originally published in _Der Strom_ 1, no. 2 (May 1911).\n\nPolimanti, Oswald. \"Der Kinematograph in der biologischen und medizinischen Wissenschaft.\" _Naturwissenschaftliche Wochenschrift_ 10, no. 49 (3 December 1911): 769\u2013774.\n\n\u2014\u2014. \"Die Anwendung der Kinematographie in den Naturwissenschaften, der Medizin und im Unterricht.\" In _Wissenschaftliche Kinematographie_ , by Franz Paul Liesegang, with Karl Kieser and Oswald Polimanti, 257\u2013310. D\u00fcsseldorf: Liesegang, 1920.\n\n\u2014\u2014. \"\u00dcber Ataxie cerebralen und cerebellaren Ursprungs.\" _Archiv f\u00fcr Physiologie_ (1909): 123\u2013134.\n\n\u2014\u2014. \"Zur Physiologie der Stirnlappen.\" _Archiv f\u00fcr Physiologie_ (1912): 337\u2013342.\n\nPowers, Samuel A. _Variola: A Series of Twenty-One Heliotype Plates Illustrating the Progressive Stages of the Eruption_. Boston: Samuel A. Powers, 1882.\n\nQuensel, Paul. _Meisterbilder und Schule: Anregungen zu praktischen Versuchen_. Munich: Kunstwart-Verl, 1905.\n\nRam\u00f3n y Cajal, Santiago. \"New Observations on the Development of Neuroblasts, with Comments on the Neurogenetic Hypothesis of Hensen-Held [1908].\" In _Studies on Vertebrate Neurogenesis_ , trans. Lloyd Guth, 71\u201376. Springfield, Ill.: Thomas, 1960.\n\nRath, Willy. _Kino und B\u00fchne_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1913.\n\nRauscher, Ulrich. \"Die Kino-Ballade.\" In _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ , ed. Fritz G\u00fcttinger, 143\u2013149. Frankfurt: Deutsches Filmmuseum, 1984. Originally published in _Der Kunstwart_ 26, no. 13 (1 April 1913): 1\u20136.\n\nReicher, Karl. \"Kinematographie in der Neurologie.\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 79, part 2 (1907): 235\u2013236.\n\nRein, Wilhelm. _P\u00e4dagogik im Grundri\u00df_ (1890). 4th ed. Leipzig: G\u00f6schen, 1907.\n\nRennert, Malwine. \"Die Zaung\u00e4ste des Lebens im Kino.\" _Bild und Film_ 4, no. 11 (1914\/1915): 217\u2013218.\n\nRichter, Wilhelm. \"Der Kinematograph als naturwissenschaftliches Anschauungsmittel.\" _Naturwissenschaftliche Wochenschrift_ 12, no. 52 (28 December 1913): 817\u2013820.\n\n\u2014\u2014. \"Hochschulkinematographie.\" _Bild und Film_ 2, nos. 11\/12 (1912\/1913): 253\u2013257.\n\nRies, Julius. \"Kinematographie der Befruchtung und Zellteilung.\" _Archiv f\u00fcr Mikroskopische Anatomie und Entwicklungsgeschichte_ 74 (1909): 1\u201331.\n\nRisak, Erwin. _Der klinische Blick_. 7th and 8th eds. Vienna: Springer, 1943.\n\n\"The Royal Society Conversazione.\" _Lancet_ 149 (19 June 1897): 1706.\n\nR\u00fcswald, K. \"Der Film im Erdkundlichen und Naturwissenschaftlichen Unterricht.\" In _Dokumente zur Geschichte der Schulfilmbewegung in Deutschland_ , ed. Fritz Terveen, 43\u201344. Emsdetten, Germany: Lechte, 1959.\n\nSamuleit, Paul, and Emil Borm. _Der Kinematograph als Volks- und Jugendbildungsmittel_. Berlin: Gesellschaft f\u00fcr Verbreitung von Volksbildung, 1912.\n\nSchenk, Paul. \"Der Kinematograph und die Schule.\" _Aerztliche Sachverst\u00e4ndigen-Zeitung_ 14, no. 15 (1 August 1908): 312\u2013313.\n\nSchiller, Friedrich. _On the Aesthetic Education of Man_. Translated by Elizabeth M. Wilkinson and L. A. Willoughby. Oxford: Oxford University Press, 1967.\n\nSchmarsow, August. \"The Essence of Architectural Creation (1893).\" In _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou, 281\u2013297. Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994.\n\nSchmid, Bastian. \"Kinematographie und Schule.\" _Die Naturwissenschaften_ 1, no. 6 (7 February 1913): 145\u2013146.\n\nSchopenhauer, Arthur. \"On the Metaphysics of Music.\" In _The World as Will and Representation_. Volume 2. Translated by E. F. J. Payne, 447\u2013457. New York: Dover, 1966.\n\n\u2014\u2014. \"On the Pure Subject of Knowing.\" In _The World as Will and Representation_. Volume 2. Translated by E. F. J. Payne, 367\u2013375. New York: Dover, 1966.\n\n\u2014\u2014. _The World as Will and Representation_. Volume 1. Translated by E. F. J. Payne. New York: Dover, 1966.\n\nSchultze, Ernst. _Der Kinematograph als Bildungsmittel_. Halle an der Saale, Germany: Waisenhaus, 1911.\n\nSchuster, Paul. \"Vorf\u00fchrung pathologischer Bewegungscomplexe mittelst des Kinematographen und Erl\u00e4uterung derselben.\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 69, part 1 (1898): 196\u2013199.\n\nSchweisheimer, Waldemar. _Die Bedeutung des Films f\u00fcr soziale Hygiene und Medizin_. Munich: M\u00fcller, 1920.\n\nSeddig, Max. \"Exacte Messung des Zeitintervalles bei kinematographischen Aufnahmen.\" _Jahrbuch f\u00fcr Photographie und Reproduktionstechnik_ 26 (1912): 654\u2013657.\n\n\u2014\u2014. \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown'schen Molekularbewegung.\" Habilitationsschrift, Akademie in Frankfurt a. M., 1909.\n\n\u2014\u2014. \"Messung der Temperatur-Abh\u00e4ngigkeit der Brown-Zsigmondyschen Bewegung.\" _Zeitschrift f\u00fcr Anorganische Chemie_ 73\u201374 (1912): 360\u2013384.\n\n\u2014\u2014. \"\u00dcber die Messung der Temperaturabh\u00e4ngigkeit der Brownschen Molekularbewegung.\" _Physikalische Zeitschrift_ 9, no. 14 (15 July 1908): 465\u2013468.\n\n\u2014\u2014. \"Ueber Abh\u00e4ngigkeit der Brown'schen Molekularbewegung von der Temperatur.\" _Sitzungsberichte der Gesellschaft zur Bef\u00f6rderung der gesammten Naturwissenschaften zu Marburg_ 18 (1907): 182\u2013188.\n\nSegmiller, L. \"Das Skizzieren nach Lichtbildern bei Tageslicht und k\u00fcnstlicher Beleuchtung.\" _Film und Lichtbild_ 1, no. 4 (1912): 35\u201339.\n\nSellmann, Adolf. \"Das Geheimnis des Kinos.\" _Bild und Film_ 1, nos. 3\u20134 (1912): 65\u201367.\n\n\u2014\u2014. _Der Kinematograph als Volkserzieher?_ Langensalza, Germany: Beyer, 1912.\n\n\u2014\u2014. _Kino und Schule_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1914.\n\n\u2014\u2014. _Kino und Volksbildung_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1914.\n\nSerner, Walter. \"Kino und Schaulust.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 208\u2013214. Leipzig: Reclam, 1992. Originally published in _Die Schaub\u00fchne_ 9, nos. 34\/35 (1913): 807\u2013811.\n\nSiedentopf, Henry. \"\u00dcber ultramikroskopische Abbildung.\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und mikroskopische Technik_ 26 (1909): 391\u2013410.\n\n\u2014\u2014, and E. Sommerfeldt, \"\u00dcber die Anfertigung kinematographischer Mikrophotographien der Kristallisationserscheinungen.\" _Zeitschrift f\u00fcr Elektrochemie_ 13, no. 24 (14 June 1907): 325\u2013326.\n\nSimmel, Georg. \"The Metropolis and Mental Life.\" In _The Sociology of Georg Simmel_ , ed. and trans. Kurt H. Wolff, 409\u2013424. New York: Free Press, 1950.\n\nSmoluchowski, Maryan. \"Essai d'une th\u00e8orie cin\u00e8tique du movement brownien et des milieux troubles.\" _Krakau Anzeiger_ 7 (1906): 585\u2013586.\n\nSolvay, Ernest. _Notes sur le productivisme et le comptabilisme_. Brussels: Misch and Thron, 1900.\n\nSommer, Ph. \"Zur Psychologie des Kinematographen.\" _Der Kinematograph_ no. 227 (3 May 1911).\n\nSommerfeldt, Ernst. \"\u00dcber fl\u00fcssige und scheinbar lebende Kristalle; mit kinematographischen Projektionen.\" _Verhandlungen der Gesellschaft deutscher Naturforscher und \u00c4rzte_ 79, part 2 (1907): 202.\n\n\"Special Correspondence: Berlin.\" _British Medical Journal_ (5 March 1910): 598.\n\nSpencer, Herbert. _The Principles of Psychology_. 2 vols. London: Williams and Norgate, 1855.\n\nSpier, Ike. \"Die sexuelle Gefahr des Kinos.\" _Die neue Generation_ 8 (1912): 192\u2013198.\n\nStapel, Wilhelm. \"Der homo cinematicus.\" _Deutsches Volkstum_ 21 (October 1919): 319\u2013320.\n\nStein, Albert E. \"Ueber medizinisch-photographische und -kinematographische Aufnahmen.\" _Deutsche medizinische Wochenschrift_ 38 (20 June 1912): 1184\u20131186.\n\nStein, Sigmund Theodor. _Das Licht im Dienste wissenschaftlicher Forschung: Handbuch der Anwendung des Lichtes und der Photographie in der Natur- und Heilkunde_. Leipzig: Spamer, 1877.\n\n\u2014\u2014. _Die optische Projektionskunst im dienste der exakten Wissenschaften: ein Lehr- und Hilfsbuch zur unterst\u00fctzung des naturwissenschaftlichen Unterrichts_. Halle an der Saale, Germany: Knapp, 1887.\n\nStigler, R. \"\u00dcber das Flimmern der Kinematographen.\" _Archiv f\u00fcr die gesamte Physiologie des Menschen und der Tiere_ (Bonn) 123 (1908): 224\u2013232.\n\nStratz, Carl Heinrich. _Die Frauenkleidung und ihre nat\u00fcrliche Entwicklung_. Stuttgart: Enke, 1900.\n\nStrobl, Karl Hans. \"Der Kinematograph.\" In _Kein Tag ohne Kino: Schriftsteller \u00fcber den Stummfilm_ , ed. Fritz G\u00fcttinger, 50\u201354. Frankfurt: Deutsches Filmmuseum, 1984. Originally published in _Die Hilfe_ 17, no. 9 (2 March 1911).\n\nSully, James. \"Pleasure of Visual Form.\" _Mind: A Quarterly Review of Psychology and Philosophy_ 5, no. 18 (April 1880): 181\u2013201.\n\nSvedberg, Theodor. _Studien zur Lehre von der Kolloiden L\u00f6sungen_. Uppsala: Akademische Buchdruckerei Edv. Berling, 1907.\n\nTaylor, Frederick Winslow. _The Principles of Scientific Management_ [1911]. New York: Norton, 1947.\n\nThielemann, W. \"Kinematographie und biologische Forschung.\" _Bild und Film_ 3, no. 7 (1913\/1914): 171\u2013172.\n\nT\u00f6nnies, Ferdinand. _Gemeinschaft und Gesellschaft_. Leipzig: Fues, 1887. Translated by Charles Loomis as _Community and Society_ (East Lansing: Michigan State University Press, 1957).\n\nUniversum-Film A. G. _Das medizinische Filmarchiv bei der Kulturabteilung der Universum-Film A.G._ Berlin: Gahl, 1919.\n\nVan Gehuchten, Arthur. \"Coup de couteau dans la moelle lombaire. Essai de physiologie pathologique.\" _Le N\u00e9vraxe_ 9 (1907): 208\u2013232.\n\n\"Verwandte Gebiete.\" _Zentralblatt f\u00fcr R\u00f6ntgenstrahlen, Radium und verwandte Gebiete_ 1, no. 2 (1910): 78\u201380.\n\n\"Verzeichnis wissenschaftlich und technisch wervoller Films.\" _Film und Lichtbild_ 1, no. 2 (1912): 16.\n\nVirchow, Rudolph. _Post-mortem Examinations_. Translated by T. P. Smith. 3d ed. Philadelphia: Blakiston, 1895.\n\nVischer, Robert. _On the Optical Sense of Form_ (1873). In _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou, 89\u2013124. Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994.\n\nVogt, Walther. \"Hermann Braus.\" _M\u00fcnchener medizinische Wochenschrift_ 72, no. 8 (20 February 1925): 304\u2013305.\n\nVolkelt, Johannes. _System der \u00c4sthetik, Erster Band: Grundlegung der \u00c4sthetik_. 2d ed. Munich: Beck, 1927.\n\nvon Hanstein, Otfrid. _Kinematographie und Schule. Ein Vorschlag zur Reform des Anschauungs-Unterrichts_. Berlin: Lichtspiele Mozartsaal, 1911.\n\nvon Molo, Walter. \"Im Kino.\" In _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_ , ed. J\u00f6rg Schweinitz, 28\u201339. Leipzig: Reclam, 1992. Originally published in _Velhagen & Klasings Monatshefte_ 26, no. 8 (April 1912): 618\u2013627.\n\nWarstat, Willi, and Franz Bergmann. _Kino und Gemeinde_. M. Gladbach [M\u00f6nchengladbach]: Volksvereins, 1913.\n\nWassermann, A. \"Die medizinische Fakult\u00e4t.\" In _Die Universit\u00e4ten im Deutschen Reich_ , ed. W. Lexis, 146\u2013147. Berlin: Asher, 1904.\n\nWeber, Wilhelm. _Mechanik der menschlichen Gehwerkzeuge: Eine anatomisch-physiologische Untersuchung_. In _Wilhelm Weber_ ' _s Werke_ , vol. 6, ed. Der k\u00f6niglichen Gesellschaft der Wissenschaften zu G\u00f6ttingen, 1\u2013305. Berlin: Springer, 1894.\n\nWeichardt, Wilhelm. \"Erm\u00fcdungsbek\u00e4mpfung durch Antikenotoxin.\" _Deutsche milit\u00e4r\u00e4rztliche Zeitschrift_ 42, no. 1 (5 January 1913): 12\u201313.\n\nWeisenburg, T. H. \"Moving Picture Illustrations in Medicine, with Special Reference to Nervous and Mental Diseases.\" _Journal of the American Medical Association_ 59, no. 26 (28 December 1912): 2310\u20132312.\n\nWeiser, Martin. _Medizinische Kinematographie_. Dresden and Leipzig: Steinkopff, 1919.\n\n\"Wissenschaftliche Abende.\" _Film und Lichtbild_ 1, no. 3 (1912): 30\u201331.\n\n\"Wissenschaft und Lichtspiele.\" _Bild und Film_ 1, no. 2 (1912): 49\u201350.\n\nWolf-Czapek, Karl Wilhelm, ed. _Angewandte Photographie in Wissenschaft und Technik_. Berlin: Union Deutsche Verlagsgesellschaft, 1911.\n\n\u2014\u2014. \"Die Kinematographie im medizinische Unterricht.\" _Jahrbuch f\u00fcr Photographie und Reproduktionstechnik_ 22 (1908): 58\u201359.\n\n\u2014\u2014. _Die Kinematographie: Wesen, Entstehung und Ziele des lebenden Bildes_. Berlin: Union Deutsche Verlagsgesellschaft, 1908; 2d enl. ed., 1911.\n\nWolgast, Heinrich. \"Die Bedeutung der Kunst f\u00fcr die Erziehung.\" In _Die Kunsterziehungsbewegung_ , ed. Hermann Lorenzen, 17\u201320. Bad Heilbrunn, Germany: Klinkhardt, 1966.\n\nWundt, Wilhelm. _Grundz\u00fcge der physiologischen Psychologie_ , 5th ed. Leipzig: Engelmann, 1902\u20131903.\n\nWychgram, Engelhard. \"Aus optischen und mechanischen Werkst\u00e4tten IV.\" _Zeitschrift f\u00fcr wissenschaftliche Mikroskopie und f\u00fcr mikroskopishe Technik_ 28 (1911): 337\u2013361.\n\nZimmermann, Robert. _Allgemeine Aesthetik als Formwissenschaft_. Vienna: Braum\u00fcller, 1865.\n\nZuntz, Nathan, and Wilhelm Schumberg. _Studien zu einer Physiologie des Marsches_. Berlin: Hirschwald, 1901.\n\n\"Zur Er\u00f6ffnung des Ernemann-Kino in Dresden.\" _Der Kinematograph_ no. 134 (21 July 1909).\n\nSECONDARY SOURCES\n\nAbel, Richard, ed. _French Film Theory and Criticism_ , vols. 1 and 2. Princeton, N.J.: Princeton University Press, 1988.\n\n\u2014\u2014. _The Cin\u00e9 Goes to Town: French Cinema, 1896\u20131914_. Berkeley: University of California Press, 1994.\n\nAcland, Charles R., and Haidee Wasson, eds. _Useful Cinema_. Durham, N.C.: Duke University Press, 2011.\n\nAlbert, David Z. _Time and Chance_. Cambridge, Mass.: Harvard University Press, 2000.\n\nAlbisetti, James C. _Secondary School Reform in Imperial Germany_. Princeton, N.J.: Princeton University Press, 1983.\n\nAlexander, Jennifer Karns. _The Mantra of Efficiency: From Waterwheel to Social Control._ Baltimore: Johns Hopkins University Press, 2008.\n\nAllen, Ann Taylor. _Feminism and Motherhood in Germany, 1800\u20131914_. New Brunswick, N.J.: Rutgers University Press, 1991.\n\nAllesch, Christian G. _Geschichte der psychologischen \u00c4sthetik_. G\u00f6ttingen: Verlag f\u00fcr Psychologie, 1987.\n\nAlovisio, Silvio. _L'occhio sensibile. Cinema e scienze della mente nell'Italia del primo Novecento. Con una antologia di testi d'epoca_. Turin: Edizioni Kaplan, 2013.\n\nAmad, Paula. \"Archiving the Everyday: A Topos in French Film History, 1895\u20131931.\" PhD diss., University of Chicago, 2002.\n\n\u2014\u2014. _Counter-Archive: Film, The Everyday, and Albert Kahn's_ Archives de la Plan\u00e8te. New York: Columbia University Press, 2010.\n\nAndrew, Dudley. \"Film and Society: Public Rituals and Private Space.\" In _Exhibition: The Film Reader_ , ed. Ina Rae Hark, 161\u2013172. New York: Routledge, 2001. Originally published in _East-West Film Journal_ 1, no. 1 (1986): 7\u201322.\n\nAndriopoulos, Stefan. _Possessed: Hypnotic Crimes, Corporate Fiction, and the Invention of Cinema_. Chicago: University of Chicago Press, 2008.\n\n\u2014\u2014. \"Spellbound in Darkness: Hypnosis as an Allegory of Early Cinema.\" _Germanic Review_ 77, no. 2 (Spring 2002): 102\u2013116.\n\nAnz, Thomas. \"Gesundheit, Krankheit und literarische Norm: Max Nordaus 'Entartung' als Paradigma pathologisierender Kunstkritik.\" In _Gesund oder Krank?: Medizin, Moral und \u00c4sthetik in der deutschen Gegenwartsliteratur_ , 33\u201352. Stuttgart: Metzler, 1989.\n\nApfelbaum, Erika, and Gregory R. McGuire. \"Models of Suggestive Influence and the Disqualification of the Social Crowd.\" In _Changing Conceptions of Crowd Mind and Behavior_ , ed. C. F. Graumann and S. Moscovici, 27\u201350. New York and Berlin: Springer, 1986.\n\nArabatzis, Theodore. \"Experiment.\" In _The Routledge Companion to Philosophy of Science_ , ed. Stathis Psillos and Martin Curd, 159\u2013170. London and New York: Routledge, 2008.\n\nArendt, Hannah. _The Human Condition_. Chicago: University of Chicago Press, 1958.\n\nArndt, Margarete, and Barbara Bigelow. \"Toward the Creation of an Institutional Logic for the Management of Hospitals: Efficiency in the Early Nineteen Hundreds.\" _Medical Care Research and Review_ 63, no. 3 (June 2006): 369\u2013394.\n\nAsche, Susanne. \"F\u00fcrsorge, Partizipation und Gleichberechtigung\u2014die Leistungen der Karlsruherinnen f\u00fcr die Entwicklung zur Gro\u00dfstadt (1859\u20131914).\" In _Karlsruher Frauen, 1715\u20131945: Eine Stadtgeschichte_ , 171\u2013256. Karlsruhe: Badenia, 1992.\n\nAshwin, Clive. \"Pestalozzi and the Origins of Pedagogical Drawing.\" _British Journal of Educational Studies_ 29, no. 2 (June 1981): 138\u2013151.\n\nAskari, Kaveh. _Making Movies Into Art: Picture Craft from the Magic Lantern to Early Hollywood_. London: British Film Institute, 2014.\n\nAubert, Genevi\u00e8ve. \"Arthur Van Gehuchten Takes Neurology to the Movies.\" _Neurology_ 59 (November 2002): 1612\u20131618.\n\n\u2014\u2014. \"From Photography to Cinematography: Recording Movement and Gait in a Neurological Context.\" _Journal of the History of the Neurosciences_ 11, no. 3 (2002): 255\u2013264.\n\nAubin, David. \"'The Memory of Life Itself': B\u00e9nard's Cells and the Cinematography of Self-Organization.\" _Studies in History and Philosophy of Science_ 39, no. 3 (2008): 359\u2013369.\n\nBachelard, Gaston. _The New Scientific Spirit_. Translated by Arthur Goldhammer. Boston: Beacon, 1984.\n\nBaird, Davis. _Thing Knowledge: A Philosophy of Scientific Instruments_. Berkeley: University of California Press, 2004.\n\nBaptista, Tiago. \"' _Il faut voir le ma\u00eetre_ ': A Recent Restoration of Surgical Films by E.-L. Doyen (1859\u20131916).\" _Journal of Film Preservation_ 70 (November 2005): 42\u201350.\n\nBarboi, Alexandru C., Christopher G. Goetz, and Radu Musetoiu. \"The Origins of Scientific Cinematography and Early Medical Applications.\" _Neurology_ 62 (June 2004): 2082\u20132086.\n\nBarkin, Kenneth D. \"The Crisis of Modernity, 1887\u20131902.\" In _Imagining Modern German Culture, 1889\u20131910_ , ed. Fran\u00e7oise Forster-Hahn, 19\u201335. Washington, D.C.: National Gallery of Art, 1996.\n\nBarrows, Susanna. _Distorting Mirrors: Visions of the Crowd in Late Nineteenth-Century France_. New Haven: Yale University Press, 1981.\n\nBauer, Herbert. _Zur Theorie und Praxis der ersten deutschen Landerziehungsheime: Erfahrungen zur Internats- und Ganztagserziehung aus den Hermann-Lietz-Schulen_. Berlin: Volk und Wissen, 1961.\n\nBerg, Christa, ed. _Handbuch der deutschen Bildungsgeschichte_. Munich: Beck, 1991.\n\nBernard, Claude. _Introduction \u00e0 l_ ' _\u00e9tude de la m\u00e9decine exp\u00e9rimentale._ Paris: Bailli\u00e8re, 1865. Translated by Henry Copley Greene as _An Introduction to the Study of Experimental Medicine_ (New York: Macmillan, 1927).\n\nBernheimer, Charles. \"Decadent Diagnostics.\" In Charles Bernheimer, _Decadent Subjects: The Idea of Decadence in Art, Literature, Philosophy, and Culture of the_ Fin de Si\u00e8cle _in Europe_ , ed. T. Jefferson Kline and Naomi Schor, 139\u2013162. Baltimore: Johns Hopkins University Press, 2002.\n\nBerrios, G. E., and M. Gili. \"Will and Its Disorders: A Conceptual History.\" _History of Psychiatry_ 6 (1995): 87\u2013104.\n\nBettelheim, Bruno. _Freud and Man_ ' _s Soul_. New York: Knopf, 1983.\n\nBigg, Charlotte. \"Evident Atoms: Visuality in Jean Perrin's Brownian Motion Research.\" _Studies in History and Philosophy of Science_ 39 (2008): 312\u2013322.\n\n\u2014\u2014. \"A Visual History of Jean Perrin's Brownian Motion Curves.\" In _Histories of Scientific Observation_ , ed. Lorraine Daston and Elizabeth Lunbeck, 156\u2013180. Chicago: University of Chicago Press, 2011.\n\nBillings, Susan M. \"Concepts of Nerve Fiber Development, 1839\u20131930.\" _Journal of the History of Biology_ 4, no. 2 (Fall 1971): 275\u2013305.\n\nBlachut, Teodor J., and Rudolf Burkhardt. _Historical Development of Photogrammetric Methods and Instruments_. Falls Church, Va.: American Society for Photogrammetry and Remote Sensing, 1989.\n\nBlackbourn, David. _History of Germany, 1780\u20131918: The Long Nineteenth Century_ 2d ed. Malden, Mass.: Blackwell, 2003.\n\nBlackmore, John T. _Ernst Mach: His Work, Life, and Influence_. Berkeley: University of California Press, 1972.\n\nBlankenship, Janelle. \"Futurist Fantasies: Luk\u00e1cs's Early Essay 'Thoughts Toward an Aesthetic of the Cinema.'\" _Polygraph_ 13 (2001): 21\u201336.\n\nBlock, Ed Jr. \"T. H. Huxley's Rhetoric and the Popularization of Victorian Scientific Ideas, 1854\u20131874.\" In _Energy & Entropy: Science and Culture in Victorian Britain_, ed. Patrick Brantlinger, 205\u2013228. Bloomington, Ind.: Indiana University Press, 1988.\n\nBokhove, Niels W., and Karl Schuhmann. \"Bibliographie der Schriften von Theodor Lipps.\" _Zeitschrift f\u00fcr philosophische Forschung_ 45, no. 1 (January\u2013March 1991): 112\u2013130.\n\nBolz, Norbert. _Am Ende der Gutenberg-Galaxis. Die neuen Kommunikationsverh\u00e4ltnisse_. Munich: Fink, 1993.\n\nBoon, Timothy. _Films of Fact: A History of Science in Documentary Films and Television_. New York: Wallflower, 2008.\n\nBooth, Jeremy. \"A Short History of Blood Pressure Measurement.\" _Proceedings of the Royal Society of Medicine_ 70, no. 11 (November 1977): 793\u2013799.\n\nBordwell, David. _On the History of Film Style_. Cambridge, Mass.: Harvard University Press, 1997.\n\nBorell, Merriley. \"Extending the Senses: The Graphic Method.\" _Medical Heritage_ 2, no. 2 (March\/April 1986): 114\u2013121.\n\nBourdieu, Pierre. _Distinction_. Cambridge, Mass.: Harvard University Press, 1984.\n\nBowser, Eileen. _The Transformation of Cinema, 1907\u20131915_. New York: Scribner, 1990.\n\nBrain, Robert M. \"Representation on the Line: Graphic Recording Instruments and Scientific Modernism.\" In _From Energy to Information: Representation in Science and Technology, Art, and Literature_ , ed. Bruce Clarke and Linda Dalrymple Henderson, 155\u2013177. Stanford, Calif.: Stanford University Press, 2002.\n\nBrain, Robert Michael. \"The Pulse of Modernism: Experimental Physiology and Aesthetic Avant-Gardes Circa 1900.\" _Studies in History and Philosophy of Science_ 39, no. 3 (2008): 393\u2013417.\n\n\u2014\u2014. \"Self-Projection: Hugo M\u00fcnsterberg on Empathy and Oscillation in Cinema Spectatorship.\" _Science in Context_ 25, no. 3 (September 2012): 329\u2013353.\n\nBraun, Marta. _Picturing Time: The Work of \u00c9tienne-Jules Marey_. Chicago: University of Chicago Press, 1992.\n\nBreidbach, Olaf. \"Representation of the Microcosm: The Claim for Objectivity in 19th Century Scientific Microphotography.\" _Journal of the History of Biology_ 35 (2002): 221\u2013250.\n\nBrown, Elspeth H. _The Corporate Eye: Photography and the Rationalization of American Commercial Culture, 1884\u20131929_. Baltimore: Johns Hopkins University Press, 2005.\n\nBrush, Stephen G. \"A History of Random Processes: I. Brownian Movement from Brown to Perrin.\" _Archive for History of Exact Sciences_ 5, no. 1 (1968): 1\u201336.\n\n\u2014\u2014. _The Kind of Motion We Call Heat: A History of the Kinetic Theory of Gases in the 19th Century_. Amsterdam and New York: North-Holland and American Elsevier, 1976.\n\n\u2014\u2014. _Statistical Physics and the Atomic Theory of Matter from Boyle and Newton to Landau and Onsager_. Princeton, N.J.: Princeton University Press, 1983.\n\nBryson, Norman. _Vision and Painting: The Logic of the Gaze_. New Haven: Yale University Press, 1983.\n\nBucchi, Massimiano. \"Images of Science in the Classroom: Wallcharts and Science Education, 1850\u20131920.\" _British Journal for the History of Science_ 31, no. 2 (1998): 161\u2013184.\n\nBuroker, Jill Vance. \"Descartes on Sensible Qualities.\" _Journal of the History of Philosophy_ 29, no. 4 (October 1991): 585\u2013611.\n\nBurwick, Frederick, and Paul Douglass, eds. _The Crisis in Modernism: Bergson and the Vitalist Controversy_. Cambridge: Cambridge University Press, 1992.\n\nBynum, W. F. _Science and the Practice of Medicine in the Nineteenth Century_. Cambridge: Cambridge University Press, 1994.\n\nCahan, David, ed. _Hermann von Helmholtz and the Foundations of Nineteenth-Century Science_. Berkeley and Los Angeles: University of California Press, 1993.\n\n\u2014\u2014. \"The Zeiss Werke and the Ultramicroscope: The Creation of a Scientific Instrument in Context.\" In _Scientific Credibility and Technical Standards in 19th and Early 20th Century German and Britain_ , ed. Jed Z. Buchwald, 67\u2013115. Dordrecht, Netherlands: Kluwer Academic, 1996.\n\nCanales, Jimena. \"Desired Machines: Cinema and the World in Its Own Image.\" _Science in Context_ 24, no. 3 (September 2011): 329\u2013359.\n\n\u2014\u2014. \"Einstein, Bergson, and the Experiment That Failed: Intellectual Cooperation at the League of Nations.\" _Modern Language Notes_ 120, no. 5 (2006): 1168\u20131191.\n\n\u2014\u2014. \"Movement Before Cinematography: The High-Speed Qualities of Sentiment.\" _Journal of Visual Culture_ 5, no. 3 (December 2006): 275\u2013294.\n\n\u2014\u2014. \"Photogenic Venus: The 'Cinematographic Turn' in Science and Its Alternatives.\" _Isis_ 93 (2002): 585\u2013613.\n\n\u2014\u2014. \"Sensational Differences: The Case of the Transit of Venus.\" _Cahiers Fran\u00e7ois Vi\u00e8te_ 1, nos. 11\/12 (September 2007): 15\u201340.\n\n\u2014\u2014. _A Tenth of a Second: A History_. Chicago: University of Chicago Press, 2009.\n\nCanguilhem, Georges. _The Normal and the Pathological_. Translated by Carolyn R. Fawcett in collaboration with Robert S. Cohen. New York: Zone, 1991.\n\nCanning, Kathleen. _Languages of Labor and Gender: Female Factory Work in Germany, 1850\u20131914_. Ithaca, N.Y.: Cornell University Press, 1996.\n\nCarleton, H. M. \"Tissue Culture: A Critical Summary.\" _British Journal of Experimental Biology_ 1, no. 1 (October 1923): 131\u2013151.\n\nCarroll, Noel. \"Modernity and the Plasticity of Perception.\" _Journal of Aesthetics and Art Criticism_ 59, no. 1 (Winter 2001): 11\u201318.\n\nCartwright, Lisa. \"'Experiments of Destruction': Cinematic Inscriptions of Physiology.\" _Representations_ 40 (Fall 1992): 129\u2013152.\n\n\u2014\u2014. _Screening the Body: Tracing Medicine_ ' _s Visual Culture_. Minneapolis: University of Minnesota Press, 1995.\n\nChamberlin, J. Edward, and Sander L. Gilman, eds. _Degeneration: The Dark Side of Progress_. New York: Columbia University Press, 1985.\n\nChanan, Michael. _The Dream That Kicks: The Prehistory and Early Years of Cinema in Britain._ London and Boston: Routledge & Kegan Paul, 1980.\n\nCharney, Leo, and Vanessa Schwartz, eds. _Cinema and the Invention of Modern Life_. Berkeley: University of California Press, 1995.\n\nCherchi Usai, Paolo, and Lorenzo Codelli, eds. _Before Caligari: German Cinema, 1895\u20131920_. Madison: University of Wisconsin Press, 1990.\n\nChertok, L\u00e9on, and Isabelle Stengers. _A Critique of Psychoanalytic Reason: Hypnosis as a Scientific Problem from Lavoisier to Lacan_. Translated by Martha Noel Evans. Stanford, Calif.: Stanford University Press, 1992.\n\nChytry, Josef. _The Aesthetic State: A Quest in Modern German Thought_. Berkeley and Los Angeles: University of California Press, 1989.\n\nClark, Peter. \"Atomism Versus Thermodynamics.\" In _Method and Appraisal in the Physical Sciences_ , ed. Colin Howson, 41\u2013106. Cambridge: Cambridge University Press, 1976.\n\nCobley, Evelyn. _Modernism and the Culture of Efficiency: Ideology and Fiction_. Toronto and Buffalo, N.Y.: University of Toronto Press, 2009.\n\nCoopmans, Catelijne, Janet Vertesi, Michael E. Lynch, and Steve Woolgar, eds. _Representation in Scientific Practice Revisited_. Cambridge, Mass.: MIT Press, 2014.\n\nCooter, Roger, and Stephen Pumfrey. \"Separate Spheres and Public Places: Reflections on the History of Science Popularization and Science in Popular Culture.\" _History of Science_ 32, no. 97 (1994): 237\u2013267.\n\nCorrigan, Timothy. _A Cinema Without Walls: Movies and Culture After Vietnam_. New Brunswick, N.J.: Rutgers University Press, 1991.\n\nCorzo-Duchardt, Beth. \"Primal Screen: Primitivism and American Silent Film Spectatorship.\" PhD diss., Northwestern University, 2013.\n\nCowan, Michael. _Cult of the Will: Nervousness and German Modernity_. University Park: Pennsylvania State University Press, 2008.\n\nCrary, Jonathan. _Suspensions of Perception: Attention, Spectacle, and Modern Culture_. Cambridge, Mass.: MIT Press, 1999.\n\n\u2014\u2014. _Techniques of the Observer: On Vision and Modernity in the Nineteenth Century_. Cambridge, Mass.: MIT Press, 1990.\n\n\u2014\u2014. \"Unbinding Vision.\" _October_ 68 (Spring 1994): 21\u201344.\n\nCurtis, Robin. \" _Einf\u00fchlung_ and Abstraction in the Moving Image: Historical and Contemporary Reflections.\" _Science in Context_ 25, no. 3 (September 2012): 425\u2013446.\n\nCurtis, Scott. \"Between Observation and Spectatorship: Medicine, Movies, and Mass Culture in Imperial Germany.\" In _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier, 87\u201398. New Barnet, U.K.: Libbey, 2009.\n\n\u2014\u2014. \"Die kinematographische Methode. Das 'Bewegte Bild' und die Brownsche Bewegung.\" _montage\/AV: Zeitschrift f\u00fcr Theorie & Geschichte audiovisueller Kommunikation_ 14, no. 2 (2005): 23\u201343.\n\n\u2014\u2014. \"Dissecting the Medical Training Film.\" In _Beyond the Screen: Institutions, Networks and Publics of Early Cinema_ , ed. Marta Braun, Charlie Keil, Rob King, Paul Moore, and Louis Pelletier, 161\u2013167. New Barnet, U.K.: Libbey, 2012.\n\n\u2014\u2014. \"The Efficiency of Images: Educational Effectiveness and the Modernity of Motion Pictures.\" In _The Visual Culture of Modernism_ , SPELL: Swiss Papers in English Language and Literature 26, ed. Deborah L. Madsen and Mario Klarer, 41\u201359. T\u00fcbingen: Narr, 2011.\n\n\u2014\u2014. \"Einf\u00fchlung und fr\u00fche deutsche Filmtheorie.\" In _Einf\u00fchlung. Zur Geschichte und Gegenwart eines \u00e4sthetischen Konzepts_ , ed. Robin Curtis and Gertrud Koch, 61\u201384. Paderborn, Germany: Fink, 2009.\n\n\u2014\u2014. \"Images of Efficiency: The Films of Frank B. Gilbreth.\" In _Films That Work: Industrial Film and the Productivity of Media_ , ed. Vinzenz Hediger and Patrick Vonderau, 85\u201399. Amsterdam: Amsterdam University Press, 2009.\n\n\u2014\u2014. \"Photography and Medical Observation.\" In _The Educated Eye: Visual Pedagogy in the Life Sciences_ , ed. Nancy Anderson and Michael R. Dietrich, 68\u201393. Hanover, N.H.: Dartmouth College Press, 2012.\n\n\u2014\u2014. \"Science Lessons.\" _Film History_ 25, nos. 1\u20132 (2013): 45\u201354.\n\n\u2014\u2014. \"Still\/Moving: Digital Imaging and Medical Hermeneutics.\" In _Memory Bytes: History, Technology, and Digital Culture_ , ed. Lauren Rabinovitz and Abraham Geil, 218\u2013254. Durham, N.C.: Duke University Press, 2004.\n\n\u2014\u2014. \"'Tangible as Tissue': Arnold Gesell, Infant Behavior, and Film Analysis.\" _Science in Context_ 24, no. 3 (September 2011): 417\u2013442.\n\n\u2014\u2014. \"The Taste of a Nation: Training the Senses and Sensibility of Cinema Audiences in Imperial Germany.\" _Film History_ 6, no. 4 (Winter 1994): 445\u2013469.\n\n\u2014\u2014. \"Vergr\u00f6sserung und das mikroskopische Erhabene.\" _Zeitschrift f\u00fcr Medienwissenschaft_ 5 (2011): 96\u2013110.\n\nDagognet, Francois. _\u00c9tienne-Jules Marey: A Passion for the Trace_. Translated by Robert Galeta with Jeanine Herman. New York: Zone, 1992.\n\nDalton, Margaret Stieg. _Catholicism, Popular Culture, and the Arts in Germany, 1880\u20131933_. Notre Dame, Ind.: University of Notre Dame Press, 2005.\n\nDaston, Lorraine. \"On Scientific Observation.\" _Isis_ 99, no. 1 (2008): 97\u2013110.\n\n\u2014\u2014, and Elizabeth Lunbeck, eds. _Histories of Scientific Observation_. Chicago: University of Chicago Press, 2011.\n\n\u2014\u2014, and Peter Galison. \"The Image of Objectivity.\" _Representations_ 40 (Fall 1992): 81\u2013128.\n\n\u2014\u2014. _Objectivity_. New York: Zone, 2007.\n\nde Broglie, Louis. \"The Concepts of Contemporary Physics and Bergson's Ideas on Time and Motion.\" In _Bergson and the Evolution of Physics_ , ed. and trans. P. A. Y. Gunter, 45\u201362. Knoxville: University of Tennessee Press, 1969.\n\nde Chadarevian, Soraya. \"Graphical Method and Discipline: Self-Recording in Nineteenth-Century Physiology.\" _Studies in History and Philosophy of Science_ 24, no. 2 (June 1993): 267\u2013291.\n\nde Duve, Thierry. \"Time Exposure and Snapshot: The Photograph as Paradox.\" _October_ 5 (Summer 1978): 113\u2013125.\n\nde Pastre, B\u00e9atrice and Thierry Lefebvre, eds. _Filmer la science, comprendre la vie: Le cinema de Jean Comandon_. Paris: Centre national de la cin\u00e9matographie, 2012.\n\nde Rijcke, Sarah. \"Drawing Into Abstraction. Practices of Observation and Visualisation in the Work of Santiago Ram\u00f3n y Cajal.\" _Interdisciplinary Science Reviews_ 33, no. 4 (2008): 287\u2013311.\n\nDeleuze, Gilles. _Cinema 1: The Movement-Image_. Translated by Hugh Tomlinson and Barbara Habberjam. Minneapolis: University of Minnesota, 1986.\n\n\u2014\u2014. _Cinema 2: The Time-Image_. Translated by Hugh Tomlinson and Barbara Habberjam. Minneapolis: University of Minnesota, 1989.\n\nDeussing, Gottlieb Gustav. \"Der Anschauungsunterricht in der deutschen Schule von Comenius bis zur Gegenwart.\" PhD diss., Universit\u00e4t Jena, 1884.\n\nDidier, Robert. _Le Docteur Doyen: Chirurgien de la Belle \u00c9poque_. Paris: Librairie Maloine, 1962.\n\nDidi-Huberman, Georges. _Invention of Hysteria: Charcot and the Photographic Iconography of the Salp\u00eatri\u00e8re_. Translated by Alisa Hartz. Cambridge, Mass.: MIT Press, 2003.\n\nDiederichs, Helmut H. _Anf\u00e4nge deutscher Filmkritik_. Stuttgart: Fischer, 1986.\n\n\u2014\u2014. \"Fr\u00fchgeschicht deutscher Filmtheorie. Ihre Entstehung und Entwicklung bis zum Ersten Weltkrieg.\" Unpublished Habilitationsschrift, J. W. Goethe-Universit\u00e4t Frankfurt am Main, 1996.\n\n\u2014\u2014. \"Hermann H\u00e4fker.\" In _Cinegraph_ , ed. Hans-Michael Bock, Munich: edition text + kritik, 1984.\n\n\u2014\u2014. \"Kino und die Wortk\u00fcnste: Zur Diskussionen der deutschen literarischen Intelligenz 1910 bis 1915.\" _KINtop_ 13 (2004): 9\u201323.\n\n\u2014\u2014. \"Naturfilm als Gesamtkunstwerk: Hermann H\u00e4fker und sein 'Kinetographie'-Konzept.\" _Augenblick_ 8 (1990): 37\u201360.\n\n\u2014\u2014. \"The Origins of the _Autorenfilm_.\" In _Before Caligari: German Cinema, 1895\u20131920_ , ed. Paolo Cherchi Usai and Lorenzo Codelli, 380\u2013401. Madison: University of Wisconsin Press, 1990.\n\nDiederichs, Helmut H., ed. _Geschichte der Filmtheorie: Kunsttheoretische Texte von M\u00e9li\u00e8s bis Arnheim_. Frankfurt: Suhrkamp, 2004.\n\nDilthey, Wilhelm. \"The Construction of the Historical World in the Human Studies.\" In _Selected Writings_ , ed., trans., and with an introduction by H. P. Rickman, 168\u2013245. Cambridge: Cambridge University Press, 1976.\n\nDoane, Mary Ann. _The Emergence of Cinematic Time: Modernity, Contingency, the Archive_. Cambridge, Mass.: Harvard University Press, 2002.\n\nDommann, Monika, _Durchsicht, Einsicht, Vorsicht: Eine Geschichte der R\u00f6ntgenstrahlen, 1896_ \u2013 _1963_. Z\u00fcrich: Chronos, 2003.\n\ndo O'Gomes, Isabelle. \"L'oeuvre de Jean Comandon.\" In _Le cin\u00e9ma et la science_ , ed. Alexis Martinet, 78\u201385. Paris: CNRS \u00c9ditions, 1994.\n\nDouglas, Ann. _The Feminization of American Culture_. New York: Knopf, 1977.\n\nDr\u00e4ger, Horst. _Die Gesellschaft f\u00fcr Verbreitung von Volksbildung: Eine historisch-problemgeschichtliche Darstellung von 1871\u20131914_. Stuttgart: Klett, 1975.\n\nDuckwitz, Amelie, Martin Loiperdinger, and Susanne Theisen. \"'Kampf dem Schundfilm!': Kinoreform and Jugendschutz in Trier.\" _KINtop_ 9 (2000): 53\u201363.\n\nDunkel, Harold B. _Herbart and Education_. New York: Random House, 1969.\n\nDurst, David C. _Weimar Modernism: Philosophy, Politics, and Culture in Germany 1918\u20131933_. Lanham, Md.: Lexington, 2004.\n\nDuttlinger, Carolin. \"Between Contemplation and Distraction: Configurations of Attention in Walter Benjamin.\" _German Studies Review_ 30, no. 1 (February 2007): 33\u201354.\n\nEagleton, Terry. _The Ideology of the Aesthetic_. Cambridge, Mass.: Blackwell, 1990.\n\nEbel, Gerhard, and Otto L\u00fchrs. \"Urania\u2014eine Idee, eine Bewegung, eine Institution wird 100 Jahre alt.\" In _100 Jahre Urania: Wissenschaft heute f\u00fcr morgen_ , 15\u201374. Berlin: Urania Berlin, 1988.\n\nEgginton, William. \"Intimacy and Anonymity, or How the Audience Became a Crowd.\" In _Crowds_ , ed. Jeffrey T. Schnapp and Matthew Tiews, 97\u2013110. Stanford, Calif.: Stanford University Press, 2006.\n\nElias, Norbert. _The Civilizing Process_. Vol. 1, _The History of Manners_. Translated by Edmund Jephcott. New York: Pantheon, 1978.\n\nEllis, John. _Visible Fictions: Cinema, Television, Video_. London: Routledge, 1982.\n\nElsaesser, Thomas. \"Die Stadt von Morgen: Filme zum Bauen und Wohnen in der Weimarer Republik.\" In _Geschichte des dokumentarischen Film in Deutschland, Band 2: Weimarer Republik (1918\u20131933)_ , ed. Klaus Kreimeier, Antje Ehmann, and Jeanpaul Goergen, 381\u2013410. Stuttgart: Reclam, 2005.\n\n\u2014\u2014, and Michael Wedel, eds. _Kino der Kaiserzeit: Zwischen Tradition und Moderne_. Munich: edition text + kritik, 2002.\n\n\u2014\u2014, with Michael Wedel, eds. _A Second Life: German Cinema_ ' _s First Decades_. Amsterdam: Amsterdam University Press, 1996.\n\nEngstrom, Eric J. _Clinical Psychiatry in Imperial Germany: A History of Psychiatric Practice_. Ithaca, N.Y.: Cornell University Press, 2003.\n\n\u2014\u2014. \"Emil Kraepelin: Psychiatry and Public Affairs in Wilhelmine Germany.\" _History of Psychiatry_ 2, no. 6 (June 1991): 111\u2013132.\n\nErmarth, Michael. _Wilhelm Dilthey: The Critique of Historical Reason_. Chicago: University of Chicago Press, 1978.\n\nEvans, Hughes. \"Losing Touch: The Controversy Over the Introduction of Blood Pressure Instruments Into Medicine.\" _Technology and Culture_ 34, no. 4 (October 1993): 784\u2013807.\n\nFisch, J\u00f6rg. \"Zivilisation, Kultur.\" In _Geschichtliche Grundbegriffe. Historisches Lexikon zur politisch-sozialen Sprache in Deutschland_ , vol. 7, ed. Otto Brunner, Werner Conze, and Reinhardt Koselleck, 679\u2013774. Stuttgart: Klett, 1992.\n\nFitzi, Gregor. _Soziale Erfahrung und Lebensphilosophie. Georg Simmels Beziehung zu Henri Bergson_. Konstanz, Germany: UVK Verlagsgesellschaft, 2002.\n\nFlanagan, Maureen A. _America Reformed: Progressives and Progressivisms, 1890s\u20131920s._ New York: Oxford University Press, 2007.\n\nFleck, Ludwik. _Cognition and Fact: Materials on Ludwik Fleck_. Edited by Robert S. Cohen and Thomas Schnelle. Dordrecht, Netherlands, and Boston: Reidel, 1986.\n\n\u2014\u2014. _Genesis and Development of a Scientific Fact_. Edited by Thaddeus J. Trenn and Robert K. Merton. Translated by Fred Bradley and Thaddeus J. Trenn. Chicago: University of Chicago Press, 1979.\n\n\u2014\u2014. \"Scientific Observation and Perception in General [1935].\" In _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle, 59\u201378. Dordrecht, Netherlands, and Boston: Reidel, 1986.\n\n\u2014\u2014. \"Some Specific Features of the Medical Way of Thinking [1927].\" In _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle, 39\u201346. Dordrecht, Netherlands, and Boston: Reidel, 1986.\n\n\u2014\u2014. \"To Look, To See, To Know [1947].\" In _Cognition and Fact: Materials on Ludwik Fleck_ , ed. Robert S. Cohen and Thomas Schnelle, 129\u2013151. Dordrecht, Netherlands, and Boston: Reidel, 1986.\n\nFleckenstein, Karen J. \"The Mosso Plethysmograph in 19th-Century Physiology.\" _Medical Instrumentation_ 18, no. 6 (November\u2013December 1984): 330\u2013331.\n\nFoucault, Michel. _The Birth of the Clinic: An Archaeology of Medical Perception_. Translated by A. M. Sheridan Smith. New York: Vintage, 1973.\n\n\u2014\u2014. _Discipline and Punish_. Translated by Alan Sheridan. New York: Vintage, 1979.\n\n\u2014\u2014. _The History of Sexuality_. Translated by Robert Hurley. New York: Vintage, 1990.\n\nFox, Daniel M., and Christopher Lawrence. _Photographing Medicine: Images and Power in Britain and America Since 1840_. New York: Greenwood, 1988.\n\nFrank, Robert G. Jr. \"The Telltale Heart: Physiological Instruments, Graphic Methods, and Clinical Hopes, 1854\u20131914.\" In _The Investigative Enterprise: Experimental Physiology in Nineteenth-Century Medicine_ , ed. William Coleman and Frederic L. Holmes, 211\u2013290. Berkeley: University of California Press, 1988.\n\nFraser, Hilary. \"Women and the Ends of Art History: Vision and Corporeality in Nineteenth-Century Critical Discourse.\" _Victorian Studies_ 42, no. 1 (Autumn 1998): 77\u2013100.\n\nFrevert, Ute. _Krankheit als politisches Problem 1770\u20131880. Soziale Unterschichten in Preu\u00dfen zwischen medizinischer Polizei und staatlicher Sozialversicherung_. G\u00f6ttingen: Vandenhoeck & Ruprecht, 1984.\n\nFrey, Erwin, and Klaus Kroy. \"Brownian Motion: A Paradigm of Soft Matter and Biological Physics.\" _Annalen der Physik_ 14, nos. 1\u20133 (February 2005): 20\u201350.\n\nFried, Michael. _Absorption and Theatricality: Painting and Beholder in the Age of Diderot._ Chicago: University of Chicago Press, 1980.\n\nFuller, P. W. W. \"Carl Cranz, His Contemporaries, and High-Speed Photography.\" _Proceedings of SPIE_ , no. 5580, 26th International Congress on High-Speed Photography and Photonics (25 March 2005): 250\u2013260.\n\nFye, W. Bruce. \"Franz M. Groedel.\" _Clinical Cardiology_ 23, no. 2 (February 2000): 133\u2013134.\n\nGailus, Andreas. \"Of Beautiful and Dismembered Bodies: Art as Social Discipline in Schiller's _On the Aesthetic Education of Man_.\" In _Impure Reason: Dialectic of Enlightenment in Germany_ , ed. W. Daniel Wilson and Robert C. Holub, 146\u2013165. Detroit, Mich.: Wayne State University Press, 1993.\n\nGarfinkel, Harold, Michael Lynch, and Eric Livingston. \"The Work of a Discovering Science Construed with Materials from the Optically Discovered Pulsar.\" _Philosophy of the Social Sciences_ 11, no. 2 (June 1981): 131\u2013158.\n\nGaudreault, Andr\u00e9. _Film and Attraction: From Kinematography to Cinema_. Translated by Timothy Barnard. Urbana: University of Illinois Press, 2011.\n\nGay, Frederick P. \"Medical Logic.\" _Bulletin of the History of Medicine_ 7 (1939): 6\u201327.\n\nGay, Peter. _The Education of the Senses_ , vol. 1. New York: Oxford University Press, 1984.\n\nGaycken, Oliver. _Devices of Curiosity: Early Cinema and Popular Science_. Oxford and New York: Oxford University Press, 2015.\n\n\u2014\u2014. \"'A Drama Unites Them in a Fight to the Death': Some Remarks on the Flourishing of a Cinema of Scientific Vernacularization in France, 1909\u20131914.\" _Historical Journal of Film, Radio and Television_ 22, no. 3 (2002): 353\u2013374.\n\n\u2014\u2014. \"The Secret Life of Plants: Visualizing Vegetative Movement, 1880\u20131903.\" _Early Popular Visual Culture_ 10, no. 1 (2012): 51\u201369.\n\n\u2014\u2014. \"'The Swarming of Life': Moving Images, Education, and Views Through the Microscope.\" _Science in Context_ 24, no. 3 (September 2011): 361\u2013380.\n\nGebhard, Julius. _Alfred Lichtwark und die Kunsterziehungsbewegung in Hamburg._ Hamburg: Hoffmann und Campe, 1947.\n\nGeimer, Peter. \"Living and Non-living Pictures.\" In _Undead: Relations Between the Living and the Lifeless_ , ed. Peter Geimer, 39\u201351. Berlin: Max-Planck-Institut f\u00fcr Wissenschaftsgeschichte, 2003.\n\nGernsheim, Alison. \"Medical Photography in the Nineteenth Century.\" _Medical and Biological Illustration_ (London) 11, no. 2 (April 1961): 85\u201392.\n\nGeuss, Raymond. \"Kultur, Bildung, Geist.\" _History and Theory_ 35, no. 2 (May 1996): 151\u2013164.\n\nGinzburg, Carlo. \"Clues: Roots of an Evidential Paradigm.\" In _Clues, Myths, and the Historical Method_ , trans. John and Ann C. Tedeschi, 96\u2013125. Baltimore: Johns Hopkins University Press, 1989.\n\nGlasser, Otto. _Wilhelm Conrad Roentgen and the Early History of the Roentgen Rays_. Springfield, Ill.: Thomas, 1934.\n\nGordon, Rae Beth. _Why the French Love Jerry Lewis: From Cabaret to Early Cinema_. Stanford, Calif.: Stanford University Press, 2001.\n\nGreve, Ludwig, Margot Pehle, and Heidi Westhoff, eds. _H\u00e4tte ich das Kino! Die Schriftsteller und der Stummfilm_. Munich: K\u00f6sel, 1976.\n\nGrieveson, Lee. _Policing Cinema: Movies and Censorship in Early-Twentieth-Century America_. Berkeley: University of California Press, 2004.\n\nGross, David L. \" _Kultur_ and Its Discontents: The Origins of a 'Critique of Everyday Life' in Germany, 1880\u20131925.\" In _Essays on Culture and Society in Modern Germany_ , ed. Gary D. Stark and Bede Karl Lackner, 70\u201397. College Station: Texas A&M University Press, 1982.\n\nGuerlac, Suzanne. _Thinking in Time: An Introduction to Henri Bergson_. Ithaca, N.Y. : Cornell University Press, 2006.\n\nGunning, Tom. \"An Aesthetic of Astonishment: Early Film and the (In)Credulous Spectator.\" _Art and Text_ 34 (Spring 1989): 31\u201345.\n\n\u2014\u2014. \"The Cinema of Attractions: Early Film, Its Spectator and the Avant-Garde.\" In _Early Cinema: Space, Frame, Narrative_ , ed. Thomas Elsaesser, with Adam Barker, 56\u201362. London: British Film Institute, 1990.\n\n\u2014\u2014. \"Film History and Film Analysis: The Individual Film in the Course of Time.\" _Wide Angle_ 12, no. 3 (July 1990): 4\u201319.\n\n\u2014\u2014. \"Modernity and Cinema: A Culture of Shocks and Flows.\" In _Cinema and Modernity_ , ed. Murray Pomerance, 297\u2013315. New Brunswick, N.J.: Rutgers University Press, 2006.\n\n\u2014\u2014. \"Systematizing the Electric Message: Narrative Form, Gender, and Modernity in _The Lonedale Operator_.\" In _American Cinema_ ' _s Transitional Era: Audiences, Institutions, Practices_ , ed. Charlie Keil and Shelley Stamp, 15\u201350. Berkeley: University of California Press, 2004.\n\nG\u00fcttinger, Fritz, ed. _Kein Tag ohne Kino_ : _Schriftsteller \u00fcber den Stummfilm_. Frankfurt: Deutsches Filmmuseum, 1984.\n\nHacking, Ian. _Representing and Intervening: Introductory Topics in the Philosophy of Natural Science_. Cambridge: Cambridge University Press, 1983.\n\nHake, Sabine. _The Cinema_ ' _s Third Machine: Writing on Film in Germany, 1907\u20131933_. Lincoln: University of Nebraska Press, 1993.\n\nHankins, Thomas L., and Robert J. Silverman. \"The Magic Lantern and the Art of Demonstration.\" In _Instruments and the Imagination_ , 37\u201371. Princeton, N.J.: Princeton University Press, 1995.\n\nHansen, Miriam. _Babel and Babylon: Spectatorship in American Silent Film_. Cambridge, Mass.: Harvard University Press, 1991.\n\n\u2014\u2014. \"Early Silent Cinema: Whose Public Sphere?\" _New German Critique_ 29 (Spring\/Summer 1983): 147\u2013184.\n\nHansen, Miriam Bratu. _Cinema and Experience: Siegfried Kracauer, Walter Benjamin, and Theodor W. Adorno_. Berkeley: University of California Press, 2012.\n\nHarrington, Anne. _Reenchanted Science: Holism in German Culture from Wilhelm II to Hitler_. Princeton, N.J.: Princeton University Press, 1996.\n\nHarris, Stefanie. _Mediating Modernity: German Literature and the_ \" _New_ \" _Media, 1895\u20131930_. University Park: Pennsylvania State University Press, 2009.\n\nHau, Michael. \"The Holistic Gaze in German Medicine, 1890\u20131930.\" _Bulletin of the History of Medicine_ 74, no. 3 (Fall 2000): 495\u2013524.\n\nHediger, Vinzenz, and Patrick Vonderau, eds. _Films That Work: Industrial Film and the Productivity of Media_. Amsterdam: Amsterdam University Press, 2009.\n\n\u2014\u2014. \"Record, Rhetoric, Rationalization: Industrial Organization and Film.\" In _Films That Work: Industrial Film and the Productivity of Media_ , ed. Vinzenz Hediger and Patrick Vonderau, 35\u201349. Amsterdam: Amsterdam University Press, 2009.\n\nHeller, Heinz-B. _Literarische Intelligenz und Film: Zu Ver\u00e4nderungen der \u00e4sthetischen Theorie und Praxis unter dem Eindruck des Films 1910\u20131930 in Deutschland_. T\u00fcbingen: Niemeyer, 1985.\n\nHepp, Corona. _Avantgarde: Moderne Kunst, Kulturkritik und Reformbewegungen nach der Jahrhundertwende._ Munich: Deutscher Taschenbuch, 1987.\n\nHerf, Jeffrey. _Reactionary Modernism: Technology, Culture, and Politics in Weimar and the Third Reich_. Cambridge and New York: Cambridge University Press, 1984.\n\nHerr, Harry W. \"Max Nitze, the Cystoscope and Urology.\" _Journal of Urology_ 176, no. 4 (October 2006): 1313\u20131316.\n\nH\u00f6fler, G\u00fcnther A. \"La naissance de la 'nervosit\u00e9' issue de l'esprit de la modernit\u00e9 technologique. D\u00e9g\u00e9n\u00e9rescence et nomadisation chez Max Nordau et Adolph Wahrmund.\" In _Max Nordau (1849\u20131923): Critique de la D\u00e9g\u00e9n\u00e9rescence, M\u00e9diateur Franco-Allemand, P\u00e8re Fondateur du Sionisme_ , ed. Delphine Bechtel, Dominique Bourel, and Jacques Le Rider, 149\u2013160. Paris: Cerf, 1996.\n\nHoldorff, Bernd. \"Die privaten Polikliniken f\u00fcr Nervenkranke vor und nach 1900.\" In _Geschichte der Neurologie in Berlin_ , ed. Bernd Holdorff and Rolf Winau, 127\u2013137. Berlin and New York: de Gruyter, 2001.\n\n\u2014\u2014. \"Zwischen Hirnforschung, Neuropsychiatrie und Emanzipation zur klinischen Neurologie bis 1933.\" In _Geschichte der Neurologie in Berlin_ , ed. Bernd Holdorff and Rolf Winau, 157\u2013174. Berlin and New York: de Gruyter, 2001.\n\nHoof, Florian. \"'The One Best Way': Bildgebende Verfahren der \u00d6konomie und die Innovation der Managementtheorie Nach 1860.\" _montage\/AV: Zeitschrift f\u00fcr Theorie und Geschichte audiovisueller Kommunikation_ 15, no. 1 (2006): 123\u2013138.\n\nHopwood, Nick. \"Producing Development: The Anatomy of Human Embryos and the Norms of Wilhelm His.\" _Bulletin of the History of Medicine_ 74 (2000): 29\u201379.\n\nHowell, Joel D. _Technology in the Hospital: Transforming Patient Care in the Early Twentieth Century_. Baltimore: Johns Hopkins University Press, 1995.\n\nHuerkamp, Claudia. _Der Aufstieg der Arzte im 19. Jahrhundert: Vom gelehrten Stand zum professionellen Experten: Das Beispiel Preu\u00dfens_. G\u00f6ttingen: Vandenhoeck & Ruprecht, 1985.\n\n\u2014\u2014. \"The Making of the Modern Medical Profession, 1800\u20131914: Prussian Doctors in the Nineteenth Century.\" In _German Professions, 1800\u20131950_ , ed. Geoffrey Cocks and Konrad H. Jarausch, 66\u201384. New York and Oxford: Oxford University Press, 1990.\n\nHusserl, Edmund. _The Crisis of European Sciences and Transcendental Phenomenology_. Translated by David Carr. Evanston, Ill.: Northwestern University Press, 1970.\n\nIlgner, Christian, and Dietmar Linke, \"Filmtechnik: Vom Malteserkreuz zum Panzerkino.\" In _Oskar Messter: Filmpioneer der Kaiserzeit_ , ed. Martin Loiperdinger, 93\u2013134. Basel and Frankfurt: Stroemfeld\/Roter Stern, 1994.\n\nImorde, Joseph. \"Einf\u00fchlung in der Kunstgeschichte.\" In _Einf\u00fchlung. Zur Geschichte und Gegenwart eines \u00e4sthetischen Konzepts_ , ed. Robin Curtis and Gertrud Koch, 127\u2013142. Paderborn, Germany: Fink, 2009.\n\nJahoda, Gustav. \"Theodor Lipps and the Shift from 'Sympathy' to 'Empathy.'\" _Journal of the History of the Behavioral Sciences_ 4, no. 2 (2005): 151\u2013163.\n\nJarre, Hans A. \"Roentgen Cinematography.\" In _The Science of Radiology_ , ed. Otto Glasser, 198\u2013209. Springfield, Ill.: Thomas, 1933.\n\nJay, Martin. _Downcast Eyes: The Denigration of Vision in Twentieth-Century French Thought_. Berkeley and Los Angeles: University of California Press, 1993.\n\nJelavich, Peter. \"'Am I Allowed to Amuse Myself Here?': The German Bourgeoisie Confronts Early Film.\" In _Germany at the Fin de Si\u00e8cle: Culture, Politics, and Ideas_ , ed. Suzanne Marchand and David Lindenfeld, 227\u2013249. Baton Rouge: Louisiana State University Press, 2004.\n\nJenkins, Jennifer. _Provincial Modernity: Local Culture and Liberal Politics in Fin-de-Si\u00e8cle Hamburg_. Ithaca, N.Y.: Cornell University Press, 2003.\n\nJoerissen, Peter. _Kunsterziehung und Kunstwissenschaft im wilhelminischen Deutschland, 1871\u20131918_. Cologne and Vienna: B\u00f6hlau, 1979.\n\nJones, Caroline A., and Peter Galison, eds. _Picturing Science, Producing Art_. New York: Routledge, 1998.\n\nJ\u00fclich, Solveig. \"Seeing in the Dark: Early X-Ray Imaging and Cinema.\" In _Moving Images: From Edison to the Webcam_ , ed. John Fullerton and Astrid S\u00f6derberg Widding, 47\u201358. Sydney: Libbey, 2000.\n\nJung, Uli. \"Film f\u00fcr Lehre und Bildung.\" In _Geschichte des dokumentarischen Films in Deutschland_ , vol. 1, ed. Uli Jung and Martin Loiperdinger, 333\u2013340. Stuttgart: Reclam, 2005.\n\nKaes, Anton, ed. _Kino-Debatte: Texte zum Verh\u00e4ltnis von Literatur und Film 1909\u20131929_. T\u00fcbingen: Niemeyer, 1978.\n\n\u2014\u2014. \"Literary Intellectuals and the Cinema: Charting a Controversy (1909\u20131929).\" _New German Critique_ 40 (Winter 1987): 7\u201334.\n\nKaiser, C\u00e9line. _Rhetorik der Entartung: Max Nordau und die Sprache der Verletzung_. Bielefeld, Germany: Transcript, 2007.\n\nKaiser, David Aram. _Romanticism, Aesthetics, and Nationalism_. Cambridge: Cambridge University Press, 1999.\n\nKalbus, Oskar. _Pionere des Kulturfilms: Ein Beitrag zur Geschichte des Kulturfilmschaffens in Deutschland_. Karlsruhe: Neue Verlags-Gesellschaft, 1956.\n\nKater, Michael H. \"Professionalization and Socialization of Physicians in Wilhelmine and Weimar Germany.\" _Journal of Contemporary History_ 20 (1985): 677\u2013701.\n\nKaufmann, Doris. \"'Pushing the Limits of Understanding': The Discourse on Primitivism in German _Kulturwissenschaften_ , 1880\u20131930.\" _Studies in History and Philosophy of Science_ 39 (2008): 434\u2013443.\n\nKay, Carolyn. _Art and the German Bourgeoisie: Alfred Lichtwark and Modern Painting in Hamburg, 1886\u20131914_. Toronto and Buffalo, N.Y.: University of Toronto Press, 2002.\n\nKeathley, Christian. _Cinephilia and History; or, The Wind in the Trees_. Bloomington: Indiana University Press, 2006.\n\nKeele, Kenneth D. _The Evolution of Clinical Methods in Medicine_. London: Pitman, 1963.\n\nKeene, Melanie Judith. \"Object Lessons: Sensory Science Education, 1830\u20131870.\" PhD diss., University of Cambridge, 2008.\n\nKeil, Charlie. \"'To Here from Modernity': Style, Historiography, and Transitional Cinema.\" In _American Cinema_ ' _s Transitional Era: Audiences, Institutions, Practices_ , ed. Charlie Keil and Shelley Stamp, 51\u201365. Berkeley: University of California Press, 2004.\n\nKemp, Martin. \"Temples of the Body and Temples of the Cosmos: Vision and Visualization in the Vesalian and Copernican Revolutions.\" In _Picturing Knowledge: Historical and Philosophical Problems Concerning the Use of Art in Science_ , ed. Brian S. Baigrie, 40\u201384. Toronto and Buffalo, N.Y.: University of Toronto Press, 1996.\n\nKenkel, Karen J. \"The Adult Children of Early Cinema.\" _Women in German Yearbook_ (2000): 137\u2013160.\n\n\u2014\u2014. \"The Nationalisation of the Mass Spectator in Early German Film.\" In _Celebrating 1895: The Centenary of Cinema_ , ed. John Fullerton, 155\u2013162. Sydney: Libbey, 1998.\n\nKerker, Milton. \"Brownian Movement and Molecular Reality Prior to 1900.\" _Journal of Chemical Education_ 51, no. 12 (December 1974): 764\u2013768.\n\nKern, Stephen. _Anatomy and Destiny: A Cultural History of the Human Body_. Indianapolis: Bobbs-Merrill, 1975.\n\n\u2014\u2014. \"Freud and the Emergence of Child Psychology, 1880\u20131910.\" PhD diss., Columbia University, 1970.\n\nKerschensteiner, Georg. \"Begriff der Arbeitsschule.\" In _Die deutsche Reformp\u00e4dagogik_ , ed. Wilhelm Flitner and Gerhard Kudritzki, 222\u2013238. D\u00fcsseldorf and Munich: K\u00fcpper, 1961.\n\nKessler, Frank. \"The Cinema of Attractions as _Dispositif_.\" In _The Cinema of Attractions Reloaded_ , ed. Wanda Strauven, 57\u201369. Amsterdam: Amsterdam University Press, 2006.\n\n\u2014\u2014. \"Henri Bergson und die Kinematographie.\" _KINtop_ 12 (2003): 12\u201316.\n\n\u2014\u2014. \"La cin\u00e9matographie comme dispositif (du) spectaculaire.\" _CiN\u00e9MAS_ 14, no. 1 (2003): 21\u201334.\n\n\u2014\u2014. \"Viewing Change, Changing Views: The 'History of Vision' Debate.\" In _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier, 23\u201335. New Barnet, U.K.: Libbey, 2009.\n\nKessler, Frank, Sabine Lenk, and Martin Loiperdinger, eds. _Oskar Messter_ \u2014 _Erfinder und Gesch\u00e4ftsmann_. KINtop Schriften 3. Basel and Frankfurt: Stoemfeld\/Roter Stern, 1994.\n\nKillen, Andreas. _Berlin Electropolis: Shock, Nerves, and German Modernity_. Berkeley: University of California Press, 2006.\n\n\u2014\u2014. \"Psychiatry, Cinema, and Urban Youth in Early-Twentieth-Century Germany.\" _Harvard Review of Psychiatry_ 14, no. 1 (2006): 38\u201343.\n\n\u2014\u2014. \"The Scene of the Crime: Psychiatric Discourses on the Film Audience in Early Twentieth Century Germany.\" In _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier, 99\u2013111. New Barnet, U.K.: Libbey, 2009.\n\nKing, Lester S. \"Medical Logic.\" _Journal of the History of Medicine and Allied Sciences_ 33, no. 3 (July 1978): 377\u2013385.\n\n\u2014\u2014. _Medical Thinking: A Historical Preface_. Princeton, N.J.: Princeton University Press, 1982.\n\nKipp, Rudolf W. _Bilddokumente zur Geschichte des Unterrichtsfilms_. Gr\u00fcnwald, Germany: Institut f\u00fcr Film und Bild in Wissenschaft und Unterricht, 1975.\n\nKittler, Friedrich A. _Gramophone, Film, Typewriter_. Translated and with an introduction by Geoffrey Winthrop-Young and Michael Wutz. Stanford, Calif.: Stanford University Press, 1999.\n\nKlein, Martin J. \"Fluctuations and Statistical Physics in Einstein's Early Work.\" In _Albert Einstein: Historical and Cultural Perspectives_ , ed. Gerald Holton and Yehuda Elkana, 39\u201358. Princeton, N.J.: Princeton University Press, 1982.\n\nKnorr-Cetina, Karin. _The Manufacture of Knowledge: An Essay on the Constructivist and Contextual Nature of Science_. Oxford: Pergamon, 1981.\n\nKoebner, Thomas. \"Der Film als neue Kunst: Reaktionen der literarischen Intelligenz: Zur Theorie des Stummfilms (1911\u20131924).\" In _Literaturwissenschaft-Medienwissenschaft_ , ed. Volker Canaris and Helmut Kreuzer, 1\u201331. Heidelberg: Quelle und Meyer, 1977.\n\nKoenigsberger, Leo. _Hermann von Helmholtz_. Braunschweig, Germany: Viewig, 1911.\n\nKommer, Helmut. _Fr\u00fcher Film und sp\u00e4te Folgen: Zur Geschichte der Film- und Fernseherziehung_. Berlin: Basis, 1979.\n\nKoss, Juliet. \"On the Limits of Empathy.\" _Art Bulletin_ 88, no. 1 (March 2006): 139\u2013157.\n\n\u2014\u2014. _Modernism After Wagner_. Minneapolis: University of Minnesota Press, 2010.\n\nKracauer, Siegfried. _From Caligari to Hitler_. Princeton, N.J.: Princeton University Press, 1947.\n\nKubler, George. _The Shape of Time: Remarks on the History of Things_. New Haven: Yale University Press, 1962.\n\nLabisch, Alfons. _Homo Hygienicus: Gesundheit und Medizin in der Neuzeit_. Frankfurt and New York: Campus, 1992.\n\nLandecker, Hannah. \"Cellular Features: Microcinematography and Film Theory.\" _Critical Inquiry_ 31, no. 4 (2005): 903\u2013937.\n\n\u2014\u2014. \"Creeping, Drinking, Dying: The Cinematic Portal and the Microscopic World of the Twentieth-Century Cell.\" _Science in Context_ 24, no. 3 (September 2011): 381\u2013416.\n\n\u2014\u2014. _Culturing Life: How Cells Became Technologies_. Cambridge, Mass.: Harvard University Press, 2007.\n\n\u2014\u2014. \"The Life of Movement: From Microcinematography to Live-Cell Imaging.\" _Journal of Visual Culture_ 11, no. 3 (December 2012): 378\u2013399.\n\n\u2014\u2014. \"Microcinematography and the History of Science and Film.\" _Isis_ 97, no. 1 (2006): 121\u2013132.\n\n\u2014\u2014. \"New Times for Biology: Nerve Cultures and the Advent of Cellular Life in Vitro.\" _Studies in the History and Philosophy of Biological and Biomedical Sciences_ 33, no. 4 (2002): 667\u2013694.\n\n\u2014\u2014. \"Technologies of Living Substance: Tissue Culture and Cellular Life in Twentieth Century Biomedicine.\" PhD diss., Massachusetts Institute of Technology, 1999.\n\nLanzoni, Susan. \"Empathy in Translation: Movement and Image in the Psychological Laboratory.\" _Science in Context_ 25, no. 3 (September 2012): 301\u2013327.\n\nLatour, Bruno. _Science in Action_. Cambridge, Mass.: Harvard University Press, 1987.\n\n\u2014\u2014. \"Visualization and Cognition: Thinking with Eyes and Hands.\" _Knowledge and Society: Studies in the Sociology of Culture Past and Present_ 6 (1986): 1\u201340.\n\n\u2014\u2014, and Steve Woolgar. _Laboratory Life: The Social Construction of Scientific Facts_. London and Beverly Hills, Calif.: Sage, 1979.\n\nLees, Andrew. _Cities, Sin, and Social Reform in Imperial Germany._ Ann Arbor: University of Michigan Press, 2002.\n\n\u2014\u2014, and Lynn Lees, eds. _The Urbanization of European Society in the Nineteenth Century_. Lexington, Mass.: Heath, 1976.\n\nLefebvre, Thierry. \"Contribution \u00e0 l'histoire de la microcin\u00e9matographie: De Fran\u00e7ois-Franck \u00e0 Comandon.\" _1895_ 14 (June 1993): 35\u201343.\n\n\u2014\u2014. \"Die Trennung der Siamesischen Zwillinge Doodica und Radica durch Dr. Doyen.\" _KINtop_ 6 (1997): 97\u2013101.\n\n\u2014\u2014. \"Flimmerndes Licht: Zur Geschichte der Filmwahrnehmung im fr\u00fchen Kino.\" _KINtop_ 5 (1996): 71\u201380.\n\n\u2014\u2014. _La chair et le celluloid: Le cin\u00e9ma chirurgical du Docteur Doyen_. Brionne: Jean Doyen \u00e9diteur, 2004.\n\n\u2014\u2014. \"La collection des films du Dr Doyen.\" _1895_ 17 (December 1994): 100\u2013114.\n\n\u2014\u2014. \"Le cas \u00e9trange du Dr Doyen, 1859\u20131916.\" _Archives_ 29 (February 1990): 1\u201312.\n\n\u2014\u2014. \"Le Dr Doyen, un pr\u00e9curseur.\" In _Le cin\u00e9ma et la science_ , ed. Alexis Martinet, 70\u201377. Paris: CNRS \u00c9ditions, 1994.\n\n\u2014\u2014. \"The Scientia Production (1911\u20131914): Scientific Popularization Through Pictures.\" _Griffithiana_ no. 47 (May 1993): 137\u2013153.\n\nLenk, Sabine, and Frank Kessler. \"The Institutionalization of Educational Cinema: The Case of the _Kinoreformbewegung_ in Germany.\" In _The Institutionalization of Educational Cinema: Educational Cinemas in North America and Europe in the 1910s and 1920s_ , ed. Marina Dahlquist and Joel Frykholm. Bloomington: Indiana University Press, forthcoming.\n\nLevin, Tom. \"From Dialectical to Normative Specificity: Reading Luk\u00e1cs on Film.\" _New German Critique_ 40 (Winter 1987): 35\u201361.\n\nLewis, Beth Irwin. \" _Lustmord_ : Inside the Windows of the Metropolis.\" In _Berlin: Culture and Metropolis_ , ed. Charles W. Haxthausen and Heidrun Suhr, 111\u2013140. Minneapolis: University of Minnesota Press, 1990.\n\nLindstrom, J. A. \"'Getting a Hold Deeper in the Life of the City': Chicago Nickelodeons, 1905\u20131914.\" PhD diss., Northwestern University, 1998.\n\nLindstrom, Richard. \"'They All Believe They Are Undiscovered Mary Pickfords': Workers, Photography, and Scientific Management.\" _Technology and Culture_ 41, no. 4 (2000): 725\u2013751.\n\n_Lucien Bull._ Brussels: Hayez, 1967.\n\nLutz, Tom. _American Nervousness, 1903: An Anecdotal History_. Ithaca, N.Y.: Cornell University Press, 1991.\n\nLynch, Michael. \"Discipline and the Material Form of Images: An Analysis of Scientific Visuality.\" _Social Studies of Science_ 15, no. 1 (February 1985): 43\u201344.\n\n\u2014\u2014. \"The Externalized Retina: Selection and Mathematization in the Visual Documentation of Objects in the Life Sciences.\" In _Representation in Scientific Practice_ , ed. Michael Lynch and Steve Woolgar, 153\u2013186. Cambridge, Mass.: MIT Press, 1990.\n\n\u2014\u2014, and Steve Woolgar, eds. _Representation in Scientific Practice_. Cambridge, Mass.: MIT Press, 1990.\n\nMaase, Kaspar. \"Krisenbewu\u00dftsein und Reformorientierung: Zum Deutungshorizont der Gegener der modernen Popul\u00e4rk\u00fcnste 1880\u20131918.\" In _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , ed. Kaspar Maase and Wolfgang Kaschuba, 290\u2013342. Cologne: B\u00f6hlau, 2001.\n\n\u2014\u2014. \"Massenkunst und Volkserziehung: Die Regulierung von Film und Kino im deutschen Kaiserreich.\" _Archiv f\u00fcr Sozialgeschichte_ 41 (2001): 39\u201377.\n\n\u2014\u2014. \"Struggling About 'Filth and Trash': Educationalists and Children's Culture in Germany Before the First World War.\" _Paedagogica Historica_ 34, no. 1 (1998): 8\u201328.\n\nMaehle, Andreas-Holger. \"The Search for Objective Communication: Medical Photography in the Nineteenth Century.\" In _Non-verbal Communication in Science Prior to 1900_ , ed. Renato G. Mazzolini, 563\u2013586. Florence: Olschki, 1993.\n\nMaiocchi, Roberto. \"The Case of Brownian Motion.\" _British Journal for the History of Science_ 23 (September 1990): 257\u2013283.\n\nMallgrave, Harry Francis, and Eleftherios Ikonomou. \"Introduction.\" In _Empathy, Form, and Space: Problems in German Aesthetics, 1873\u20131893_ , ed. and trans. Harry Francis Mallgrave and Eleftherios Ikonomou, 1\u201385. Santa Monica, Calif.: Getty Center for the History of Art and the Humanities, 1994.\n\nMarchand, Suzanne L. _Down from Olympus: Archaeology and Philhellenism in Germany, 1750\u20131970_. Princeton, N.J.: Princeton University Press, 1996.\n\nMarcus, Laura. _The Tenth Muse: Writing About Cinema in the Modernist Period_. Oxford and New York: Oxford University Press, 2007.\n\nM\u00e1rkus, Gy\u00f6rgy. \"Life and the Soul: The Young Luk\u00e1cs and the Problem of Culture.\" In _Luk\u00e1cs Revalued_ , ed. Agnes Heller, 1\u201326. London: Blackwell, 1983.\n\nMaulitz, Russell C. \"Physician Versus Bacteriologist: The Ideology of Science in Clinical Medicine.\" In _The Therapeutic Revolution: Essays in the Social History of American Medicine,_ ed. Morris J. Vogel and Charles E. Rosenberg, 91\u2013107. Philadelphia: University of Pennsylvania Press, 1979\n\nMayer, Andreas. _Sites of the Unconscious: Hypnosis and the Emergence of the Psychoanalytical Setting._ Chicago: University of Chicago Press, 2013.\n\n\u2014\u2014. _Wissenschaft vom Gehen. Die Erforschung der Bewegung im 19. Jahrhundert_. Frankfurt: Fischer, 2013.\n\nMayer, William. \"Robert Gaupp.\" _American Journal of Psychiatry_ 108, no. 10 (April 1952): 724\u2013725.\n\nMcClelland, Charles E. \"Modern German Doctors: A Failure of Professionalization?\" In _Medicine and Modernity: Public Health and Medical Care in Nineteenth- and Twentieth-Century Germany_ , ed. Manfred Berg and Geoffrey Cocks, 81\u201397. Cambridge and New York: Cambridge University Press, 1997.\n\n\u2014\u2014. \"The Wise Man's Burden: The Role of Academicians in Imperial German Culture.\" In _Essays on Culture and Society in Modern Germany_ , ed. Gary D. Stark and Bede Karl Lackner, 45\u201369. College Station, Tex.: Texas A & M University Press, 1982.\n\nMcCormick, Richard W., and Alison Guenther-Pal, eds. _German Essays on Film_. New York: Continuum, 2004.\n\nMcCormmach, Russell. \"On Academic Scientists in Wilhelmian Germany.\" In _Science and Its Public: The Changing Relationship_ , ed. Gerald Horton and William A. Blanpied, 157\u2013171. Dordrecht, Netherlands, and Boston: Reidel, 1976.\n\nMeyer, Rudolf W. \"Bergson in Deutschland. Unter besonderer Ber\u00fccksichtigung seiner Zeitauffassung.\" In _Studien zum Zeitproblem in der Philosophie des 20. Jahrhunderts_ , ed. Rudolf W. Meyer, Ernst Wolfgang Orth, Rudolf Boehm, and Wolfgang Krewani, 10\u201364. Munich: Alber, 1982.\n\nMichaelis, Anthony R. _Research Films in Biology, Anthropology, Psychology, and Medicine_. New York: Academic, 1955.\n\nMosse, George L. _The Crisis of German Ideology: Intellectual Origins of the Third Reich_. New York: Grosset & Dunlap, 1964.\n\n_\u2014\u2014_. \"Max Nordau and His Degeneration.\" In Max Nordau, _Degeneration_ , xiii-xxxvi. New York: Fertig, 1968.\n\nM\u00fcller, Corinna. \"Der fr\u00fche Film, das fr\u00fche Kino und seine Gegner und Bef\u00fcrworter.\" In _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , ed. Kaspar Maase und Wolfgang Kaschuba, 62\u201391. Cologne, Weimar, and Vienna: B\u00f6hlau, 2001.\n\n\u2014\u2014. \"The Emergence of the Feature Film in Germany Between 1910 and 1911.\" In _Before Caligari: German Cinema, 1895\u20131920_ , ed. Paolo Cherchi Usai and Lorenzo Codelli, 94\u2013113. Madison: University of Wisconsin Press, 1990.\n\n\u2014\u2014. _Fr\u00fche deutsche Kinematographie: formale, wirtschaftliche und kulturelle Entwicklungen 1907\u20131912_. Stuttgart and Weimar: Metzler, 1994.\n\nMusser, Charles. \"A Cinema of Contemplation, A Cinema of Discernment: Spectatorship, Intertextuality and Attractions in the 1890s.\" In _The Cinema of Attractions Reloaded_ , ed. Wanda Strauven, 159\u2013179. Amsterdam: Amsterdam University Press, 2006.\n\n\u2014\u2014. _The Emergence of Cinema: The American Screen to 1907_. New York: Scribner, 1990.\n\nMyers, Greg. \"Nineteenth-Century Popularizations of Thermodynamics and the Rhetoric of Social Prophecy.\" In _Energy & Entropy: Science and Culture in Victorian Britain_, ed. Patrick Brantlinger, 307\u2013338. Bloomington: Indiana University Press, 1988.\n\nNead, Lynda. _The Haunted Gallery: Painting, Photography, Film c. 1900_. New Haven and London: Yale University Press, 2007.\n\nNewman, Lex. \"Ideas, Pictures, and the Directness of Perception in Descartes and Locke.\" _Philosophy Compass_ 4, no. 1 (2009): 134\u2013154.\n\nNicholas, J. S. \"Ross Granville Harrison, 1870\u20131959.\" _Yale Journal of Biology and Medicine_ 32, no. 6 (June 1960): 407\u2013412.\n\nNichtenhauser, Adolf. \"History of Motion Pictures in Medicine.\" Unpublished manuscript. MS C 380, History of Medicine Division, National Library of Medicine, Bethesda, Md.\n\nNorth, Paul. _The Problem of Distraction_. Stanford, Calif.: Stanford University Press, 2012.\n\nNye, Mary Jo. _Molecular Reality: A Perspective on the Scientific Work of Jean Perrin_. New York: American Elsevier, 1972.\n\n\u2014\u2014. \"The Nineteenth-Century Atomic Debates and the Dilemma of an 'Indifferent Hypothesis.'\" _Studies in History and Philosophy of Science_ 7, no. 3 (1976): 245\u2013268.\n\nNye, Robert A. _Crime, Madness, and Politics in Modern France: The Medical Concept of National Decline_. Princeton, N.J.: Princeton University Press, 1984.\n\n\u2014\u2014. _The Origins of Crowd Psychology: Gustave Le Bon and the Crisis of Mass Democracy in the Third Republic._ London and Beverly Hills, Calif.: Sage, 1975.\n\nNyhart, Lynn K. \"Learning from History: Morphology's Challenges in Germany ca. 1900.\" _Journal of Morphology_ 252 (April 2002): 2\u201314.\n\n\u2014\u2014. _Modern Nature: The Rise of the Biological Perspective in Germany_. Chicago: University of Chicago Press, 2009.\n\n\u2014\u2014. \"Science, Art, and Authenticity in Natural History Displays.\" In _Models: The Third Dimension of Science_ , ed. Soraya De Chadarevian and Nick Hopwood, 307\u2013335. Stanford, Calif.: Stanford University Press, 2004.\n\nOdin, Steve. _Artistic Detachment in Japan and the West: Psychic Distance in Comparative Aesthetics_. Honolulu: University of Hawaii Press, 2001.\n\nOelkers, J\u00fcrgen. _Reformp\u00e4dagogik. Eine kritische Dogmengeschichte_. Weinheim and Munich: Juventa, 1989.\n\nOesterlen, Friedrich. _Medical Logic_. Translated by G. Whitley. London: Sydenham Society, 1855.\n\nOksiloff, Assenka. _Picturing the Primitive: Visual Culture, Ethnography, and Early German Cinema_. New York: Palgrave, 2001.\n\nOrgeron, Devin, Marsha Orgeron, and Dan Streible, eds. _Learning with the Lights Out_. New York: Oxford University Press, 2012.\n\nOstherr, Kirsten. _Cinematic Prophylaxis: Globalization and Contagion in the Discourse of World Health_. Durham, N.C.: Duke University Press, 2005.\n\n\u2014\u2014. _Medical Visions: Producing the Patient Through Film, Television, and Imaging Technologies_. New York: Oxford University Press, 2013.\n\nPasveer, Bernike. \"Knowledge of Shadows: The Introduction of X-Ray Images in Medicine.\" _Sociology of Health and Illness_ 11, no. 4 (December 1989): 361\u2013381.\n\n\u2014\u2014. \"Representing or Mediating: A History and Philosophy of X-Ray Images in Medicine.\" In _Visual Cultures of Science: Rethinking Representational Practices in Knowledge Building and Science Communication_ , ed. Luc Pauwels, 41\u201362. Lebanon, N.H.: Dartmouth College Press\/University Press of New England, 2006.\n\nPernick, Martin. _The Black Stork: Eugenics and the Death of \"Defective\" Babies in American Medicine and Motion Pictures Since 1915_. New York: Oxford University Press, 1999.\n\nPeterson, Jennifer Lynn. _Education in the School of Dreams: Travelogues and Early Nonfiction Film_. Durham, N.C.: Duke University Press, 2013.\n\nPetro, Patrice. _Joyless Streets: Women and Melodramatic Representation in Weimar Germany_. Princeton, N.J.: Princeton University Press, 1989.\n\nPflug, G\u00fcnther. \"Die Bergson-Rezeption in Deutschland.\" _Zeitschrift f\u00fcr philosophische Forschung_ 45, no. 2 (April\u2013June 1991): 257\u2013266.\n\nPodoll, K., and J. L\u00fcning. \"Geschichte des wissenschaftlichen Films in der Nervenheilkunde in Deutschland 1895\u20131929.\" _Fortschritte der Neurologie, Psychiatrie_ 66 (1998): 122\u2013132.\n\nPollmann, Inga. \"Cinematic Vitalism: Theories of Life and the Moving Image.\" PhD diss., University of Chicago, 2011.\n\nPraehauser, Ludwig. _Erfassen und Gestalten: Die Kunsterziehung als Pflege formender Kr\u00e4fte_. Salzburg: M\u00fcller, 1950.\n\nPr\u00e4ffcke, Hans. _Der Kunstbegriff Alfred Lichtwarks_. Hildesheim, Z\u00fcrich, and New York: Olms, 1986.\n\nPrigogine, Ilya. _From Being to Becoming: Time and Complexity in the Physical Sciences_. San Francisco: Freeman, 1980.\n\n\u2014\u2014, and Isabelle Stengers. _The End of Certainty: Time, Chaos, and the New Laws of Nature_. New York: Free Press, 1997.\n\n\u2014\u2014. _Order Out of Chaos: Man_ ' _s New Dialogue with Nature_. Boulder, Colo.: New Science Library, 1984.\n\nQuaresima, Leonardo. \" _Dichter, Heraus!_ The _Autorenfilm_ and German Cinema of the 1910s.\" _Griffithiana_ 38\/39 (October 1990): 101\u2013120.\n\nRabinbach, Anson. _The Human Motor: Energy, Fatigue, and the Origins of Modernity_. Berkeley and Los Angeles: University of California Press, 1990.\n\nRadder, Hans. _In and About the World: Philosophical Studies of Science and Technology_. Albany: State University of New York, 1996.\n\n\u2014\u2014. _The Material Realization of Science_. Assen and Maastricht: Van Gorcum, 1988.\n\n\u2014\u2014, ed. _The Philosophy of Scientific Experimentation_. Pittsburgh, Pa.: University of Pittsburgh Press, 2003.\n\nRadkau, Joachim. _Das Zeitalter der Nervosit\u00e4t: Deutschland zwischen Bismarck und Hitler_. Munich: Hanser, 1998.\n\nRamsey, L. J. \"Early Cineradiography and Cinefluorography.\" _History of Photography_ 7, no. 4 (October\u2013December 1983): 311\u2013322.\n\nRanci\u00e8re, Jacques. _The Emancipated Spectator_. Translated by Gregory Elliott. New York: Verso, 2009.\n\nRasmussen, Nicolas. _Picture Control: The Electron Microscope and the Transformation of Biology in America, 1940\u20131960_. Stanford, Calif.: Stanford University Press, 1997.\n\nReichert, Ram\u00f3n. \"Der Arbeitstudienfilm: Eine verborgene Geschichte des Stummfilms.\" _medien & zeit: Kommunikation in Vergangenheit und Gegenwart_ 5 (2002): 46\u201357.\n\nReill, Peter Hanns. _Vitalizing Nature in the Enlightenment_. Berkeley: University of California Press, 2005.\n\nReiser, Stanley Joel. _Medicine and the Reign of Technology_. Cambridge: Cambridge University Press, 1978.\n\nRenn, J\u00fcrgen. \"Einstein's Invention of Brownian Motion.\" _Annalen der Physik_ 14, supplement (2005): 23\u201337.\n\nRepp, Kevin. _Reformers, Critics, and the Paths of German Modernity: Anti-Politics and the Search for Alternatives, 1890\u20131914_. Cambridge, Mass.: Harvard University Press, 2000.\n\nReulecke, J\u00fcrgen. _Geschichte der Urbanisierung in Deutschland_. Frankfurt: Suhrkamp, 1985.\n\n\u2014\u2014. _Sozialer Frieden durch soziale Reform: Der Centralverein f\u00fcr das Wohl der Arbeitenden Klassen in der Fr\u00fchindustrialisierung_. Wuppertal: Hammer, 1983.\n\nRheinberger, Hans-J\u00f6rg. _Toward a History of Epistemic Things: Synthesizing Proteins in the Test Tube_. Stanford, Calif.: Stanford University Press, 1997.\n\nRich, Norman. _The Age of Nationalism and Reform, 1850\u20131890_. New York: Norton, 1970.\n\nRinger, Fritz K. _The Decline of the German Mandarins: The German Academic Community, 1890\u20131933_. Cambridge, Mass.: Harvard University Press, 1969.\n\nRitter, Christopher Owen. \"Re-presenting Science: Visual and Didactic Practice in Nineteenth-Century Chemistry.\" PhD diss., University of California, Berkeley, 2001.\n\nRitzheimer, Kara L. \"Protecting Youth from 'Trash': Anti- _Schund_ Campaigns in Baden, 1900\u20131933.\" PhD diss., State University of New York\u2013Binghamton, 2007.\n\nRosa, Hartmut, and William E. Scheuerman, eds. _High-Speed Society: Social Acceleration, Power, and Modernity_. University Park: Pennsylvania State University Press, 2009.\n\nRosen, George. \"The Efficiency Criterion in Medical Care, 1900\u20131920.\" _Bulletin of the History of Medicine_ 50, no. 1 (Spring 1976): 28\u201344.\n\nRoss, Corey. _Media and the Making of Modern Germany: Mass Communications, Society, and Politics from the Empire to the Third Reich_. New York: Oxford University Press, 2008.\n\nRossell, Deac. \"Beyond Messter: Aspects of Early Cinema in Berlin.\" _Film History_ 10 (1998): 52\u201369.\n\n\u2014\u2014. _Faszination der Bewegung: Ottomar Ansch\u00fctz zwischen Photographie und Kino_. KINtop Schriften 6. Frankfurt: Stroemfeld\/Roter Stern, 2001.\n\n\u2014\u2014. _Living Pictures: The Origins of the Movies_. Albany: State University of New York Press, 1998.\n\nRubin, Lewis Philip. \"Leo Loeb's Role in the Development of Tissue Culture.\" _Clio Medica_ 12, no. 1 (1977): 33\u201356.\n\nSach\u00dfe, Christoph. _M\u00fctterlichkeit als Beruf: Sozialarbeit, Sozialreform und Frauenbewegung, 1871\u20131929_. Frankfurt: Suhrkamp, 1986.\n\nSarasin, Philipp. \"Die Rationalisierung des K\u00f6rpers: \u00dcber 'Scientific Management' und 'biologische Rationalisierung.'\" In _Geschichtswissenschaft und Diskursanalyse_ , 61\u201399. Frankfurt: Suhrkamp, 2003.\n\nSayre, Robert, and Michael L\u00f6wy. \"Figures of Romantic Anti-capitalism.\" _New German Critique_ 32 (Spring\/Summer 1984): 42\u201392.\n\nSchaffer, Simon. \"Transport Phenomena: Space and Visibility in Victorian Physics.\" _Early Popular Visual Culture_ 10, no. 1 (February 2012): 71\u201391.\n\nScheibe, Wolfgang. _Die Reformp\u00e4dagogische Bewegung, 1900\u20131932: Eine einf\u00fchrende Darstellung_. 9th ed. Weinheim and Basel: Beltz, 1984.\n\nSchenda, Rudolf. _Die Lesestoffe der kleinen Leute: Studien zur popul\u00e4ren Literatur im 19. und 20. Jahrhundert._ Munich: Beck, 1976.\n\nSchlich, Thomas. \"'Wichtiger als der Gegenstand selbst': Die Bedeutung des fotografischen Bildes in der Begr\u00fcndung der bakteriologischen Krankheitsauffassung durch Robert Koch.\" In _Neue Wege in der Seuchengeschichte_ , ed. Martin Dinges and Thomas Schlich, 143\u2013174. Stuttgart: Steiner, 1995.\n\nSchl\u00fcpmann, Heide. _Unheimlichkeit des Blicks: Das Drama des fr\u00fchen deutschen Kinos_. Frankfurt: Stroemfeld\/Roter Stern, 1990.\n\nSchmidgen, Henning. \"1900\u2014The Spectatorium: On Biology's Audiovisual Archive.\" _Grey Room_ 43 (2011): 42\u201365.\n\n\u2014\u2014. \"Pictures, Preparations, and Living Processes: The Production of Immediate Visual Perception ( _Anschauung_ ) in Late-19th-Century Physiology.\" _Journal of the History of Biology_ 37, no. 3 (October 2004): 477\u2013513.\n\nSchmidt, Gunnar. _Anamorphotische K\u00f6rper: Medizinische Bilder vom Menschen im 19. Jahrhundert_. Cologne: B\u00f6hlau, 2001.\n\nSchmitt, Heiner. _Kirche und Film: Kirchliche Filmarbeit in Deutschland von ihren Anf\u00e4ngen bis 1945_. Boppard: Boldt, 1979.\n\nSchorr, Thomas. \"Die Film- und Kinoreformbewegung und die Deutsche Filmwirtschaft. Eine Analyse des Fachblatts _Der Kinematograph_ (1907\u20131935) unter p\u00e4dagogischen und publizistischen Aspekten.\" PhD diss., Universit\u00e4t der Bundeswehr, Munich, 1990.\n\nSchulte, Christoph. _Psychopathologie des Fin de Si\u00e8cle: Der Kulturkritiker, Arzt und Zionist Max Nordau_. Frankfurt: Fischer Taschenbuch, 1997.\n\nSchulze, Volker. \"Fr\u00fche kommunale Kinos und die Kinoreformbewegung in Deutschland bis zum Ende des ersten Weltkriegs.\" _Publizistik_ 22, no. 1 (January\u2013March 1977): 61\u201371.\n\nSchweinitz, J\u00f6rg, ed. _Prolog vor dem Film: Nachdenken \u00fcber ein neues Medium, 1909\u20131914_. Leipzig: Reclam, 1992.\n\nSecord, Anne. \"Botany on a Plate: Pleasure and the Power of Pictures in Promoting Early Nineteenth-Century Scientific Knowledge.\" _Isis_ 93, no. 1 (March 2002): 28\u201357.\n\nShapin, Steven. \"Pump and Circumstance: Robert Boyle's Literary Technology.\" _Social Studies of Science_ 14, no. 4 (November 1984): 481\u2013520.\n\n\u2014\u2014, and Simon Schaffer. _Leviathan and the Air-Pump: Hobbes, Boyle, and the Experimental Life._ Princeton, N.J.: Princeton University Press, 1985.\n\nSicard, Monique, Robert Pujade, and Daniel Wallach. _\u00c0 corps et \u00e0 raison: Photographies m\u00e9dicales, 1840\u20131920_. Paris: Marval, 1995.\n\nSinger, Ben. \"The Ambimodernity of Early Cinema: Problems and Paradoxes in the Film-and-Modernity Discourse.\" In _Film 1900: Technology, Perception, Culture_ , ed. Annemone Ligensa and Klaus Kreimeier, 37\u201351. New Barnet, U.K.: Libbey, 2009.\n\n\u2014\u2014. _Melodrama and Modernity: Early Sensational Cinema and Its Contexts_. New York: Columbia University Press, 2001.\n\nSnyder, Joel. \" _Res Ipsa Loquitur_.\" In _Things That Talk: Object Lessons from Art and Science_ , ed. Lorraine Daston, 195\u2013221. New York: Zone, 2004.\n\nSobchack, Vivian. _The Address of the Eye: A Phenomenology of Film Experience_. Princeton, N.J.: Princeton University Press, 1992.\n\nS\u00f6der, Hans-Peter. \"Disease and Health as Contexts of Modernity: Max Nordau as a Critic of Fin-de-Si\u00e8cle Modernism.\" _German Studies Review_ 14, no. 3 (October 1991): 473\u2013487.\n\nSpengler, Oswald. _The Decline of the West_. Translated by Charles Francis Atkinson. New York: Knopf, 1926\u20131928.\n\nSpringman, Luke. \"Poisoned Hearts, Diseased Minds, and American Pimps: The Language of Censorship in the _Schund und Schmutz_ Debates.\" _German Quarterly_ 68, no. 4 (Autumn 1995): 408\u2013429.\n\nStachel, John. \"Einstein on Brownian Motion.\" In _The Collected Papers of Albert Einstein_ , vol. 2, _The Swiss Years: Writings, 1900\u20131909_ , ed. John Stachel, 206\u2013222. Princeton, N.J.: Princeton University Press, 1989.\n\nStark, Gary D. \"Cinema, Society, and the State.\" In _Essays on Culture and Society in Modern Germany_ , ed. Gary D. Stark and Bede Karl Lackner, 122\u2013166. College Station: Texas A & M University Press, 1982.\n\nStarr, Paul. _The Social Transformation of American Medicine_. New York: Basic, 1982.\n\nStern, Fritz. _The Politics of Cultural Despair: A Study in the Rise of the Germanic Ideology_. Berkeley: University of California Press, 1961.\n\nStone, Judith F. _The Search for Social Peace: Reform Legislation in France, 1890\u20131914_. Albany, N.Y.: State University of New York Press, 1985.\n\nStorim, Mirjam. \"'Einer, der besser ist, als sein Ruf': Kolportageroman und Kolportagebuchhandel um 1900 und die Haltung der Buchbranche.\" In _Schund und Sch\u00f6nheit: Popul\u00e4re Kultur um 1900_ , ed. Kaspar Maase and Wolfgang Kaschuba, 252\u2013282. Cologne: B\u00f6hlau, 2001.\n\nSweeney, Dennis. \"Reconsidering the Modernity Paradigm: Reform Movements, the Social and the State in Wilhelmine Germany.\" _Social History_ 31, no. 4 (November 2006): 405\u2013434.\n\nSzczepaniak-Gillece, Jocelyn. \"Machines for Seeing: Cinema, Architecture, and Mid-century American Spectatorship.\" PhD diss., Northwestern University, 2013.\n\nSzczepanik, Petr. \"Modernism, Industry, Film: A Network of Media in the Ba\u0165a Corporation and the Town of Zl\u00edn in the 1930s.\" In _Films That Work: Industrial Film and the Productivity of Media_ , ed. Vinzenz Hediger and Patrick Vonderau, 349\u2013376. Amsterdam: Amsterdam University Press, 2009.\n\nSz\u00f6ll\u00f6si-Janze, Margit. \"Science and Social Space: Transformations in the Institutions of _Wissenschaft_ from the Wilhelmine Empire to the Weimar Republic.\" _Minerva_ 43 (2005): 339\u2013360.\n\nTakaya, Keiichi. \"The Method of _Anschauung_ : From Johann H. Pestalozzi to Herbert Spencer.\" _Journal of Educational Thought_ 37, no. 1 (2003): 77\u201399.\n\nTaurek, Renata. _Die Bedeutung der Photographie f\u00fcr die medizinische Abbildung im 19. Jahrhundert_. Cologne: Hansen, 1980.\n\nTaylor, Peter J., and Ann S. Blum. \"Pictorial Representation in Biology.\" _Biology and Philosophy_ 6, no. 2 (April 1991): 125\u2013134.\n\nTeuteberg, Hans J\u00fcrgen, ed. _Urbanisierung im 19. und 20. Jahrhundert: historische und geographische Aspekte_. Cologne: B\u00f6hlau, 1983.\n\nTosi, Virgilio. _Cinema Before Cinema: The Origins of Scientific Cinematography_. Translated by Sergio Angelini. London: British Universities Film & Video Council, 2005.\n\nTsivian, Yuri. _Early Cinema in Russia and Its Cultural Reception_. Edited by Richard Taylor. Translated by Alan Bodger. London and New York: Routledge, 1994.\n\nTucker, Jennifer. _Nature Exposed: Photography as Eyewitness in Victorian Science_. Baltimore: Johns Hopkins University Press, 2005.\n\n\u2014\u2014. \"Photography as Witness, Detective, and Imposter: Visual Representation in Victorian Science.\" In _Victorian Science in Context_ , ed. Bernard Lightman, 378\u2013408. Chicago: University of Chicago Press, 1997.\n\nUlich, Robert. \"Pestalozzi, Johann Heinrich.\" In _The Encyclopedia of Philosophy_ , vol. 6, ed. Paul Edwards, 121\u2013122. New York: Macmillan, 1967.\n\nVal\u00e9ry, Paul. _Id\u00e9e Fixe_. Translated by David Paul. New York: Pantheon, 1965.\n\nVeatch, Robert M. \"Generalization of Expertise.\" _Hastings Center Studies_ 1, no. 2 (1973): 29\u201340.\n\nVirilio, Paul. _War and Cinema: The Logistics of Perception_. Translated by Patrick Camiller. London: Verso, 1989.\n\nvom Bruch, R\u00fcdiger, ed. _Weder Kommunismus noch Kapitalismus: B\u00fcrgerliche Sozialreform in Deutschland vom Vorm\u00e4rz bis zur \u00c4ra Adenauer_. Munich: Beck, 1985.\n\n\u2014\u2014. _Wissenschaft, Politik und \u00f6ffentliche Meinung: Gelehrtenpolitik im Wilhelminischen Deutschland, 1890\u20131914_. Husum, Germany: Matthiesen, 1980.\n\nVondung, Klaus. \"Zur Lage der Gebildeten in der wilhelminischen Zeit.\" In _Das wilhelminische Bildungsb\u00fcrgertum Zur Sozialgeschichte seiner Ideen_ , ed. Klaus Vondung, 20\u201333. G\u00f6ttingen: Vandenhoeck & Ruprecht, 1976.\n\nWagner, Juliet Clare. \"Twisted Bodies, Broken Minds: Film and Neuropsychiatry in the First World War.\" PhD diss., Harvard University, 2009.\n\nWarner, John Harley. _Against the Spirit of System: The French Impulse in Nineteenth-Century American Medicine_. Princeton, N.J.: Princeton University Press, 1998.\n\n\u2014\u2014. \"Ideals of Science and Their Discontents in Late Nineteenth-Century American Medicine.\" _Isis_ 82, no. 3 (September 1991): 454\u2013478.\n\nWarnotte, Daniel. _Ernest Solvay et l_ ' _Institut de Sociologie: Contribution \u00e0 l_ ' _histoire de l_ ' _\u00e9nerg\u00e9tique sociale_. Brussels: Bruylant, 1946.\n\nWarwick, Andrew. \"X-Rays as Evidence in German Orthopedic Surgery, 1895\u20131900.\" _Isis_ 96, no. 1 (March 2005): 1\u201324.\n\nWeber, Max. \"Science as a Vocation.\" In _The Vocation Lectures_ , ed. and with an introduction by David Owen and Tracy B. Strong, trans. Rodney Livingstone, 1\u201331. Indianapolis: Hackett, 2004.\n\nWehler, Hans-Ulrich. _The German Empire, 1871\u20131918_. Leamington Spa, U.K.: Berg, 1985.\n\nWeindling, Paul. \"Bourgeois Values, Doctors and the State: The Professionalization of Medicine in Germany 1848\u20131933.\" In _The German Bourgeoisie_ , ed. David Blackbourn and Richard J. Evans, 198\u2013223. New York: Routledge, 1991.\n\n\u2014\u2014. _Health, Race, and German Politics Between National Unification and Nazism, 1870\u20131945_. Cambridge and New York: Cambridge University Press, 1989.\n\n\u2014\u2014. \"Public Health in Germany.\" In _The History of Public Health and the Modern State_ , ed. Dorothy Porter, 119\u2013131. Atlanta, Ga.: Rodopi, 1994.\n\nWesfreid, Jos\u00e9 Eduardo. \"Scientific Biography of Henri B\u00e9nard (1874\u20131939).\" In _Dynamics of Spatio-Temporal Cellular Structures: Henri B\u00e9nard Centenary Review_ , ed. Innocent Mutabazi, Jos\u00e9 Eduardo Wesfreid, and \u00c9tienne Guyon, 9\u201340. New York: Springer, 2006.\n\nWhitbeck, Caroline. \"What Is Diagnosis? Some Critical Reflections.\" _Metamedicine_ 2, no. 3 (October 1981): 319\u2013329.\n\nWilder, Kelly. _Photography and Science_. London: Reaktion, 2009.\n\nWilkending, Gisela. _Volksbildung und P\u00e4dagogik_ \" _vom Kinde aus_ \" _: Eine Untersuchung zur Geschichte der Literaturp\u00e4dagogik in den Anf\u00e4ngen der Kunsterziehungsbewegung_. Weinheim, Germany: Beltz, 1980.\n\nWinston, Brian. \"The Documentary Film as Scientific Inscription.\" In _Theorizing Documentary_ , ed. Michael Renov, 37\u201357. London and New York: Routledge, 1993.\n\nWohlring, Herman J. Review of _The Human Gait. Human Movement Science_ 8, no. 1 (February 1989): 79\u201383.\n\nYoxen, Edward. \"Seeing with Sound: A Study of the Development of Medical Images.\" In _The Social Construction of Technological Systems: New Directions in the Sociology and History of Technology_ , ed. Wiebe E. Bijker, Thomas P. Hughes, and Trevor J. Pinch, 281\u2013303. Cambridge, Mass.: MIT Press, 1987.\nINDEX\n\n**Page numbers refer to the print edition but are hyperlinked to the appropriate location in the e-book**.\n\n_ABC der Anschauung_ (Pestalozzi),\n\nabstraction: of movement, , 37\u201338, 49\u201363, 74\u201375; of spectators, 11\u201312\n\nactive and passive viewers, , . _See also_ expert\/lay distinctions\n\nAdorno, Theodor,\n\nadult education and motion pictures, 184\u201390\n\naesthetic contemplation, , , , , , 197\u2013202; agency or free will and, , , ; alertness ( _Geistesgegenwart_ ) and, ; Benjamin and, , 240\u201341; distraction and, , 233\u201335; as expert vision or observation, , , ; gender and, 231\u201332; as ground for aesthetic experience, , ; indeterminacy and, ; Kracauer and, ; Luk\u00e1cs and, 235\u201340; mass reception and, , , ; moral significance of, ; motion pictures and, , , 195\u201396, 230\u201331; movement and, , 230\u201331; as observational training, 170\u201372; passivity and, ; political implications of, ; Schiller and, ; Schopenhauer and, , . _See also_ aesthetic experience\n\naesthetic education, 143\u201347; film reform and, ; as German tradition, ; moral renewal and, , 169\u201370; museums and, ; nationalism and, 168\u201371; observational training and, , 168\u201371; Schiller and, 167\u201368, ; suggestibility and,\n\naesthetic experience, , , , __ ; aesthetic education and, ; agency within, , 202\u201314; descriptions of, 196\u2013201; detachment and, 196\u201398, , , , ; embodied vision and, 214\u201330; as emotional projection, , 220\u201321; identity and, 214\u201316; moral significance of, 197\u2013201, ; movement and, 224\u201330; as renewal of perception, ; repose and, ; Schiller and, 205\u201307; Schopenhauer and, , ; synesthesia and, 221\u201322; as system of dichotomies, 196\u2013201; taste and, ; temporality and, 204\u20137. _See also_ aesthetic contemplation\n\naesthetics, , 4\u20135; _Einf\u00fchlung_ as solution to problems in, 215\u201316; formalist and scientific approaches to, ; reception and form in, ; \"traditional,\" , , , , , 216\u201317,\n\nagency, , , 197\u201399, , 202\u201314. _See also_ free will; volition; will\n\n_Die Aktion_ ,\n\nalertness or presence of mind ( _Geistesgegenwart_ ), 212\u201314,\n\nall-at-onceness, . _See also_ observation: gaze and glance\n\nalignment of image, object, and technology, , 43\u201362\n\nAllgemeines Krankenhaus,\n\n_The Alps_ (film),\n\nAltenloh, Emilie, ,\n\nalternative public sphere, early film exhibition as,\n\nAmerican College of Surgeons,\n\nAmerican Hospital Association,\n\nanalysis and synthesis, , , 36\u201337, , , 117\u201318, ; medical observation and, 120\u201325; motion pictures and, , ; scientific cinematography's relationship to, , 117\u201318; spectatorship and,\n\nanalysis (close reading), 4\u20135, , 247\u201349\n\nanalysis of motion, , , , , , 115\u201316; Bergson's critique of, 32\u201333, ; frame-by-frame, , , ,\n\nAndreas-Salom\u00e9, Lou, 209\u201310\n\nAndrew, Dudley,\n\nAndriopolous, Stefan,\n\nanesthesia,\n\nanimal locomotion, ,\n\n_Anschaulichkeit_ (vividness), 107\u20138,\n\n_Anschauung_ (sense impression), 172\u201376\n\n_Anschauungsunterricht_ (visual means of instruction), , , , 145\u201346, 172\u201384; as apperception, 174\u201375; correlation and, 174\u201375; detail and, 173\u201374; elementary and adult education and, 176\u201377; Herbartian principles of, 182\u201383; Lichtwark and, ; motion pictures and, , 182\u201383; natural sciences and, ; as object lesson, 173\u201375; as observational training, , , , 173\u201375; Pestalozzi and, , 174\u201375; relationship to images and words, 175\u201376; self-cultivation ( _Bildung_ ) and, ; social acceleration and, 173\u201374. _See also_ object lesson\n\nAnsch\u00fctz, Ottomar, , 265n73\n\napperception, 173\u201374, 181\u201382, 226\u201327\n\n_Arbeitsschule_ (works schools),\n\n_Arbeitswissenschaft_ (the science of work),\n\narchitecture, , 224\u201327,\n\narchives, of images, , 103\u20135, 114\u201316\n\nAristotelian science,\n\nArnheim, Rudolf,\n\nart education movement ( _Kunsterziehungsbewegung_ ), , , 169\u201371,\n\nAshwin, Clive, ,\n\natomic-kinetic model of heat, , ,\n\natomic-kinetic theory of matter, 64\u201365\n\nattention ( _Aufmerksamkeit_ ), , 132\u201333, 139\u201340, , , , 198\u201399, ,\n\naura,\n\nautomatic writing,\n\nautopsies,\n\n_Autorenfilm_ (author's film),\n\nBachelard, Gaston,\n\nBal\u00e0zs, B\u00e9la, , , 279n70\n\nbearers of culture ( _Kulturtr\u00e4ger_ ), . _See also_ physicians\n\nbeautiful, theory of the, , 304n10\n\nBellevue Hospital, 104\u20135\n\nBellour, Raymond,\n\nB\u00e9nard, Henri,\n\nBenjamin, Walter, , , , 236\u201337, , ; alertness and, ; aura and, ; Bergson and, ; contemplation and, , , , 240\u201341; distraction and, , , ; Duhamel and, ; expert\/lay distinctions and, ; mode of perception and, ; optical unconscious and, ; traditional aesthetics and, ,\n\nBergson, Henri, , , , , , , , , , ; analysis and, 87\u201388; Benjamin and, ; cinematographical thinking and, , , 32\u201337; critique of science and, 32\u201337, 62\u201364; _eidos_ and, ; German counterparts to, 36\u201337; movement and, 33\u201334; synthesis and, ; time and, 74\u201376; United States and, , ,\n\nBernheim, Hippolyte,\n\n_Bildbetrachtung_ (image viewing), , ,\n\n_Bild und Film_ (trade journal), , ,\n\n_Bild und Wort_ (Image and Word society) (film exhibitions),\n\n_Bildung_ (self-cultivation), ; visual training and,\n\n_Bildungsb\u00fcrgertum_ (educated middle-class), ,\n\nBillings, Susan,\n\nBillroth, Theodor,\n\nBinet, Alfred,\n\nbiology, , , , ; cell, , , , , 76\u201388,\n\n_Der Blaue Engel_ (The Blue Angel) (film)\n\nBloch, Ernst, , 222\u201324\n\n_The Blue Angel_ (film),\n\nBorbacher Knabenmord (child murder of Borbach), 287n151\n\n_Botryllus_ (sea squirts),\n\nBourdieu, Pierre,\n\nBraun, Ludwig, 98\u2013100, 111\u201321, , ,\n\nBraune, Wilhelm, 19\u201320, 24\u201326, , 41\u201344, 62\u201363, , , , . _See alsoThe Human Gait_\n\nBraus, Hermann, , , , , __ ; nerve fibers and, , , 82\u201386,\n\nBrecht, Bertolt, ,\n\nBritish Medical Association,\n\nBrod, Max, ,\n\nBrown, Robert,\n\nBrownian motion, , 24\u201325, , 62\u201376, __ , __ , , , , 266n97; Einstein's theory of, 64\u201369, 266n94, 266n98; importance of displacement to Einstein's theory, 68\u201369\n\nBrunner, Karl, 152\u201353, 157\u201358, 292n34\n\nBrush, Stephen,\n\nBull, Lucien, ,\n\nBurrows, Montrose,\n\n_The Cabinet of Dr. Caligari_ (film),\n\ncapitalism, , , ,\n\ncardiac dynamics and mechanics, , __ ,\n\nCarlet, H. M.,\n\nCarrel, Alexis,\n\nCartwright, Lisa, ,\n\nCarvallo, Joachim-L\u00e9on, , ,\n\ncell biology, , , , , 76\u201388,\n\ncensorship, , ,\n\n_Century of the Child_ (Key),\n\nCharcot, Jean-Martin, , ,\n\nCharit\u00e9 Hospital,\n\nCharles Urban Trading Company,\n\nChase, Walter,\n\nChevroton, Lucienne, ,\n\nchild psychology, , , 162\u201364. _See also_ children and crowds: analogies between; crowd psychology\n\nchildren and crowds: analogies between, , ; aesthetic sensibilities of, ; as models for descriptions of film spectatorship, , _See also_ crowd psychology\n\nchronophotography, , , , , , 60\u201361, , ; Brownian motion and, , , ; ergonomics and, ; for _The Human Gait_ , 44\u201362, __ ; human locomotion and, 19\u201320; two-sided,\n\ncinema: theater compared to, 236\u201339\n\n_Cinema and Art_ ( _Kino und Kunst_ ) (H\u00e4fker),\n\n\"Cinema and Schoolchildren\" (Kleib\u00f6mer),\n\nCinema Reform Association, ,\n\n_The Cinematic Lesson Plan_ ( _Die kinematographische Unterrichtsstunde_ ) (Lemke), , __\n\ncinematographical thinking, , , , 32\u201337. _See also_ Bergson, Henri\n\ncinematography, 21\u201322; adaptation of for biology and physics, 76\u201377; as aid to correlation, , ; analysis and synthesis and, ; Bergson's analogy of, 32\u201337; Braun and, 111\u201314; Braus and, , 84\u201387; Brownian motion and, , 69\u201370; as confirmation of biological theory, ; high-speed, , , ; medical, 92\u201393,  , ; microcinematography, , , 92\u201393, ; science and, 32\u201337; slow-motion, , , ; as substitute for object of study, ; time-lapse, , , 84\u201385, , , , ; as optical unconscious, ; as virtual experiment, , ; as virtual witness, , ; X-rays and, , 100\u2013102, _101. See also_ chronophotography; _The Human Gait_ ; medical filmmaking; research film\n\nclass: educated middle-class, , ; ideals, ; identity, , ; warfare, 292n34; working,\n\nclass-based distinctions, , ,\n\nclassical physics, , ,\n\nClausius, Rudolf,\n\nclinical gaze, , 118\u201319, . _See also_ observation: gaze and glance\n\nclothing reform, 149\u201350\n\nCole, Lewis Gregory,\n\nColl\u00e8ge de France, ,\n\nComandon, Jean, , , , ,\n\ncommunal sense ( _sensus communis_ ),\n\ncontinuity and discontinuity, , , 32\u201334, , , , , , , 86\u201387, ,\n\ncorrelation, , , , , , , ; definition of, ; medical observation and, 115\u201317; film as aid to, , ,\n\ncountry boarding schools ( _Landerziehungsheime_ ),\n\nCranz, Carl,\n\nCrary, Jonathan, 132\u201333,\n\n_Creative Evolution_ (Bergson), , , ,\n\ncrime, hypnotism and, 135\u201336\n\ncritical method ( _kritischer Methode_ ), . _See also_ observation\n\n_Critique of Judgment_ (Kant),\n\n_Critiques_ (Kant), ,\n\nCros, Charles,\n\ncrowd psychology, , ; theories of, 164\u201365; suggestibility and, 165\u201366; will or volition and, . _See also_ children and crowds: analogies between\n\n\"The Cult of Distraction\" (Kracauer),\n\ncultural capital,\n\ncultural pessimism,\n\ncultural series,\n\n_The Culture of the Female Body as a Foundation for Women's Clothing_ (Schultze-Naumberg), 149\u201350\n\ncurricular integration,\n\nDadaists,\n\nDarwin, Charles, , , , 296n80\n\nDaston, Lorraine,\n\nde Broglie, Louis,\n\n_Degeneration_ (Nordau),\n\ndegeneration (concept),  , 131\u201332\n\nde Lagarde, Paul, ,\n\nDeleuze, Gilles, , 75\u201376\n\nDescartes, Ren\u00e9, , , 210\u201311\n\n_The Descent of Man_ (Darwin),\n\nDessoir, Max, 307n51,308n55\n\ndetachment: aesthetic,191, 196\u201398, , , , ; expert, , , . _See also_ observation: aesthetic experience\n\ndetail: as fidelity to nature, , , 107\u201308, 111\u201313, ; as ground for authenticity, 173\u201375, ; as ground for observational practice, 119\u201320, , , ; scientific management of, , , , , ; as symptom of modernity, , ,\n\nDewey, John,\n\nDiderot, Denis,\n\nDidi-Huberman, Georges,\n\nDiederichs, Helmut,\n\ndifferential reproduction, , 257n9\n\nDilthey, Wilhelm,\n\ndirect perception (immediate or sensual), , , , , , , , , , 279n70\n\ndisciplinary agendas, 2\u20133, , , , 245\u201346, ; technology and, 3\u20134, 14\u201315,\n\ndisciplinary logic, 4\u20136, 9\u201310, 14\u201315, 23\u201324, , 248\u201349; film form and, 5\u20136, 23\u201324, 111\u201325. _See also_ medical logic; patterns of use: film form and\n\ndiscontinuity. _See_ continuity and discontinuity\n\ndisease, , , , , 127\u201328; cinematography and, ; neurological, ; series photography and, 113\u201315\n\ndisinterest. _See_ detachment; aesthetic\n\ndisplacement, of particles, , 68\u201370, 72\u201373. _See also_ Brownian motion\n\n_dispositif_ , ; as experimental material, 250\u201351\n\ndissemination of media technologies, ,\n\ndistraction, , , , , ; attention and, ; Benjamin and, , , ; Kracauer and, ; proximity and,\n\nD\u00f6blin, Alfred,\n\ndocile bodies ( _aka_ working objects), 42\u201344\n\ndoctor\u2013patient relationship, ,\n\ndocumentary function, of medical filmmaking, 102\u20135\n\ndomestication of the image, 42\u201344, 51\u201362; of the body, 42\u201344. _See also_ docile bodies\n\n_Don Quixote_ (novel),\n\nDoodica (conjoined twin),\n\nDoyen, Eug\u00e8ne Louis, 106\u201310, 279n63\n\n\"Drama in the Film Theaters\" (Polgar),\n\nDriesch, Hans, ,\n\nDuenschmann, Hermann,\n\nDuhamel, Georges,\n\nduration, , , , ; cinematography and, , , . _See alsodur\u00e9e_\n\n_Duration and Simultaneity_ (Bergson),\n\n_dur\u00e9e_ (duration), 32\u201333, , ; form as interruption of, 62\u201364\n\nDuttlinger, Carolin, ,\n\nEclipse (film company), 155\u201356\n\neducated middle-class ( _Bildungsb\u00fcrgertum_ ), ,\n\neducation, , , ; art education movement, , , 169\u201371, . _See also_ adult education and motion pictures; aesthetic education, _Anschauungsunterricht_ , elementary education and motion pictures\n\neducational function, of medical filmmaking, 105\u201310\n\neffects of media technologies, 244\u201345\n\nefficiency, ; as social goal, 38\u201342; and the human body, 38\u201342; in medicine, ; of the filmic image, 109\u201310, 175\u201376, 210\u201311\n\neidetic images,\n\n_eidos_ ,\n\n_Einf\u00fchlung_ (emotional projection, feeling into), , , 215\u201322, 224\u201329, , 307n51; body as model for, ; contemplation and, , ; _Kino-Debatte_ and, , ; movement and, 224\u201328; physical response and, 228\u201330; space and, 225\u201328\n\nEinstein, Albert, 20\u201321, 24\u201325, , 66\u201369, 72\u201374, 87\u201388\n\nelementary education and motion pictures, 176\u201384\n\nElias, Norbert,\n\nembodied perception or vision, 214\u201330, . See also aesthetic experience; _Einf\u00fchlung_\n\nembourgeoisement,\n\nemotional projection. _SeeEinf\u00fchlung_\n\nempathy, . See also _Einf\u00fchlung_\n\nenemies of the cinema ( _Kinogegner_ ),\n\nenergy, conservation of, 39\u201340\n\nEnlightenment, , , ,\n\nentropy,\n\nEpstein, Jean, , , 279n70\n\nergograph,\n\nergonomics,\n\nErnemann (film company), 28\u201329,\n\n_Essay Concerning Human Understanding_ (Locke),\n\n\"The Essence of Architectural Creation\" (Schmarsow),\n\nEurope, , , , ,\n\nevidence, creation of, 24\u201325\n\nevil, children as,\n\nevolutionary theory,\n\n_Exercises in the Contemplation of Art Works_ (Lichtwark), 170\u201372,\n\nExner, Felix, , 69\u201370\n\nexperiment: in biology, 82\u201383; characteristics of, 98\u201399; differential reproduction in, 250\u201351; disciplinary logic and, 23\u201324; historiographic implications of, 250\u201351; infrastructure for, 27\u201328; in medicine, 98\u2013102; motion picture equipment manufacturers and, ; motion pictures and, 22\u201324, , ; popular audiences for, 28\u201329; time and, 139\u201340\n\nexperimental systems, 3\u20134, , 23\u201324, , 257n9\n\nexpert modes of viewing, 6\u201310, 24\u201325. _See also_ observation\n\nexpert observation. _See_ observation\n\nexpert training, , ; advantages of cinematography for, ; and professional identity, ; and research films, ,\n\nexpert viewers. _See_ observation\n\nexpert vision. _See_ observation\n\nexpert\/lay distinctions, 11\u201313, 94\u201395, ; historiographic implications of,\n\nexploratory function, of medical filmmaking, 98\u2013102\n\nEykman, P. H.,\n\nfaithful to nature ( _Naturgetreu_ ), , ,\n\nFata Morgana (film theater),\n\nfatigue, 39\u201342, ,\n\nFechner, Gustav,\n\nfeeling into. _SeeEinf\u00fchlung_\n\nfemale hysteria,\n\nfemale spectatorship, , 231\u201332\n\nfilm drama ( _Kino-drama_ ), , , ,\n\nfilm exhibition, legal restrictions on,\n\nfilm form, 4\u20135, , , , ; correspondences between form and logic, 23\u201324; disciplinary logic and, ; historiography and,\n\nfilm frame, as spatial and temporal boundary,\n\nfilmic realism, , . _See also_ _Naturgetreu_ (faithful to nature)\n\nFilm-Idea-Central,\n\n\"Film in the Service of Medicine\" (demonstration),\n\nfilm reform, 152\u201362; distribution and, 159\u201360; exhibition and, , 184\u201390; foreign films and, ; goals of, ; Kino-Kommissions and, ; Kinoreform and, ; legacy of, 160\u201362; narrative and, ; objections to motion pictures, ; positive and negative approaches to, ; production and, 156\u201359; trade journals and 152\u201353; uplift and,\n\nfilm strip as line, 74\u201376\n\nfilm studies,\n\n_Film und Lichtbild_ (periodical),\n\nfirst law of thermodynamics, ,\n\nFischer, Otto, 19\u201320, 24\u201326, , 41\u201344, . _See alsoThe Human Gait_\n\nFleck, Ludwik, 5\u20137, ,\n\nflicker effect, 130\u201331, , ,\n\nflip books,\n\nfolk medicine,\n\nForel, Auguste,\n\nform: geometry inherent in, 62\u201363, ; as temporal interruption in duration, . _See also_ abstraction: of movement\n\nform drive, ,\n\nFoucault, Michel, , 42\u201343, , , 118\u201322\n\nframe-by-frame analysis, , , ,\n\nFrance, 27\u201328, , , ,\n\nFran\u00e7ois-Franck, Charles \u00c9mile, ,\n\nFr\u00e4nkel, James,\n\n_Frankfurter Zeitung_ ,\n\nfree play of associations,\n\nfree will, , ; aesthetic contemplation and, , , ; aesthetic experience and, , 202\u201314; crowd psychology and, ; identity and, . _See also_ agency; volition; will\n\nFrench Academy of Medicine,\n\nFreud, Sigmund, 163\u201364, , , 309n72\n\n_From Caligari to Hitler_ (Kracauer), 301n145\n\n_Der Gang des Menschen_ (Braune and Fischer), . _See alsoThe Human Gait_\n\nGaudreault, Andr\u00e9, ,\n\nGaumont (film company),\n\nGaumont, Charles,\n\nGaupp, Robert, 127\u201328, 136\u201337,\n\ngaze: clinical or medical, , , 118\u201319; glance and, , 120\u201322; holistic, , , 120\u201321, . _See also_ aesthetic contemplation, all-at-onceness, observation, _Sehen_ , _Schauen_\n\n\"Gedanken zu einer Aesthetik des 'Kino' (Thoughts Toward an Aesthetic of the Cinema)\" (Luk\u00e1cs), ,\n\nGeissler tubes, , 48\u201349, 51\u201352,\n\n_Geistesgegenwart_ (alertness or presence of mind), 212\u201314,\n\ngender. _See_ women\n\ngender dynamics,\n\ngeometry, ; inherent in form, 62\u201363, . _See also_ abstraction: of movement\n\nGerman High Ministry of War,\n\nGermany, reaction to modernity, 13\u201314\n\n_Gesamtkunstwerk_ (Wagner),\n\n_Gesamtsinnlichkeit_ (sensuous totality),\n\n_Gesamtvorf\u00fchrung_ (total presentation),\n\nGesellschaft der Freunde des vaterl\u00e4ndischen Schul- und Erziehungswesens (Society of Friends of the Schools and Instruction for the Fatherland),\n\nGesellschaft deutscher Naturforscher und \u00c4rzte (Society of German Natural Scientists and Physicians), ,\n\nGesellschaft zur Verbreitung von Volksbildung (Society for the Dissemination of Popular Education) (GVV), ,\n\nGeuss, Raymond,\n\n_Gewohnheit_ (habit), ,\n\n_Giovanna d'Arco_ ( _The Maid from Orleans_ ) (film),\n\nglance, and gaze, , 120\u201322\n\nGoerke, Franz, ,\n\nGoethe, Johann Wolfgang von, ,\n\nGordon, Rae Beth,\n\nG\u00f6tze, O.,\n\nGouy, Louis-Georges,\n\nGrau, Alexander,\n\nGreat Exhibition (1851),\n\nGreat Lisbon Earthquake,\n\nGriffith, D. W.,\n\nGroedel, Franz, 101\u20132\n\nGuerlac, Suzanne,\n\nGunning, Tom, ,\n\ngutta-percha,\n\nGVV. _See_ Gesellschaft zur Verbreitung von Volksbildung\n\n_Gymnasium_ (high school), ,\n\nhabit ( _Gewohnheit_ ), ,\n\nHacking, Ian, ,\n\nH\u00e4fker, Hermann, , 152\u201353, , , 176\u201377, ; contemplation and, , ; _Kinetographie_ and, ; Lichtwark and, ; model presentations and, , ; photograph of, __ ; taste and, ; World War I and, 301n145\n\nHake, Sabine, , ,\n\nHamburg commission, , ,\n\nHansen, Miriam, , 231\u201332\n\nHarrington, Anne,\n\nHarris, Stefanie,\n\nHarrison, Ross Granville, , __ , , 82\u201383, ,\n\nHau, Michael, , ,\n\nhealth campaigns,\n\nhealth insurance,\n\nheat transference, ,\n\nHegel, Georg Wilhelm Friedrich,\n\n_Heimat_ ,\n\n_Heinrich von Ofterdingen_ (Novalis),\n\nHeld, Hans, ,\n\nHeller, Heinz-B.,\n\nHellwig, Albert, , , ,\n\nHennes, Hans, 103\u20136\n\nHenri, Victor, , 69\u201370,\n\nHerbart, Johann, , , 181\u201382, ,\n\nheterogeneity, ; of early cinema, , ,\n\nhigh school ( _Gymnasium_ ), ,\n\nhigh-speed cinematography, , ,\n\nHildebrand, Adolf, , , 224\u201325\n\nhistoriography, , ; analogy and homology in, ; disciplinary or expert use of film and, , , 246\u201347; _Kino-Debatte_ and, ; \"tactile,\" 246\u201350\n\n_History and Class Consciousness_ (Luk\u00e1cs), 235\u201336, 239\u201340\n\nholistic gaze, , , 120\u201321,\n\nHollywood, mode of production and reception,11\u201312\n\nHuelsenbeck, Richard,\n\nHuerkamp, Claudia,\n\n_The Human Gait_ (Braune and Fischer), , 44\u201362; camera placement for, __ ; coordinates graph for, __ , __ ; coordinates table for, __ ; determination of coordinates for, __ ; instrument for coordinate measurements, __ ; measurement of coordinate, __ ; military recruit for, __ ; resulting chronophotograph for, __ ; subject at rest, __ ; tridimensional model for, __ , __\n\nhuman locomotion, 19\u201320, , 38\u201339, 41\u201344. _See alsoThe Human Gait_\n\nhuman sciences,\n\nHusserl, Edmund, 58\u201359\n\nhypnotism, __ ; cinema and, 136\u201337; as model for observation, 138\u201340; as model for spectatorship, 135\u201337\n\nhysteria, ,\n\nIbsen, Henrik,\n\nidentity: aesthetic experience and, , 214\u201330; class, , ; free will and, ; professional, , , , , ; self-identity, , , , ,\n\n_Image and Word_ ( _Bild und Wort_ society) (film exhibitions),\n\nimage archives, , 103\u20135, 114\u201316\n\nimage viewing ( _Bildbetrachtung_ ), , ,\n\nimagination, , , ; architecture and, ; repose and,\n\nimaginative faculty, 209\u201310\n\nimmutability,\n\nimproper viewing, , 94\u201395. _See also_ spectatorship\n\nincremental exposures, . _See also_ series photography\n\nIndustrial Revolution, , 147\u201348\n\n\"Infantile Sexuality\" (Freud),\n\ninfantilization, of audience, . _See also_ children and crowds: analogies between\n\ninner child,\n\n_Innerlichkeit_ (inwardness), 239\u201340\n\ninner movement, . See also movement and: aesthetic contemplation; movement and: aesthetic experience\n\ninscription devices, , 30\u201331\n\nInstitut de Sociologie,\n\nInstitut Marey, ,\n\ninterdisciplinary research,\n\ninwardness ( _Innerlichkeit_ ), 239\u201340\n\n_Die Irrfahrten des Odysseus_ ( _The Wanderings of Odysseus_ ) (film), , __\n\nJacobj, Carl,\n\nJanssen, Jules,\n\nJelavich, Peter,\n\njournals, trade 152\u201353\n\nKade, August,\n\nKaes, Anton, , ,\n\nKaiserin-Friedrich-Haus,\n\nKammer-Lichtspiele Theater, __\n\nKant, Immanuel, , , 214\u201315; aesthetic contemplation and, ; aesthetic experience and, , 195\u201399; citizenship and, 140\u201341; taste and,\n\nK\u00e4stle, C.,\n\nKeene, Melanie,\n\nKelvin, Lord. _See_ Thomson, William\n\nKey, Ellen,\n\nKienzl, Hermann, 209\u201310\n\nKiesler, Frederick,\n\nKillen, Andreas, 284n122\n\n_Der Kinematograph_ (trade journal), 152\u201353, , ,\n\n_Kinematographie und Schule_ (Mendel),\n\n_Die kinematographische Unterrichtsstunde_ ( _The Cinematic Lesson Plan_ ) (Lemke), , __\n\n_Kinetographie_ ,\n\n_Kinobuch_ (Pinthus),\n\n_Kino-Debatte_ , 16\u201317, 193\u201395, 201\u20132, , ; aesthetic experience and, , ; alertness and, ; _Einf\u00fchlung_ and, , ; historiography and, ; literary emphasis and, ; somnambulism and, ; traditional aesthetics and,\n\n_Kino-drama_ (film drama), , , ,\n\n_Kinogegner_ (enemies of the cinema),\n\n_Kino-Kommissions_ ,\n\n_Kinoreform_ , 153\u201355, , , . _See also_ film reform\n\n_Kino und Kunst_ ( _Cinema and Art_ ) (H\u00e4fker),\n\n\"Kino und Schaulust\" (Serner),\n\n_Kitsch_ ,\n\nKleib\u00f6mer, Georg, 202\u20133\n\nKoch, Robert,\n\nKosmographia,\n\nKracauer, Siegfried, , , 233\u201334, , , 301n145\n\nKraepelin, Emil, ,\n\nKretz, Richard,\n\nKrieger, Ernst,\n\n_kritischer Methode_ (critical method), . _See also_ observation\n\n_Kultur_ and _Zivilisation_ , 144\u201345\n\n_Kulturtr\u00e4ger_ (bearers of culture), . _See also_ physicians\n\n_Kunsterziehungsbewegung_ (art education movement), , , 169\u201371,\n\n_Der Kunstwart_ ,\n\nKutner, Robert, ,\n\nkymograph,\n\nlaboratories, , , ; motion pictures in, , , , 27\u201330, ,\n\n_Ladenkinos_ (storefront cinemas), , __\n\nLandecker, Hannah,\n\n_Landerziehungsheime_ (country boarding schools),\n\nLangbehn, August Julius, 168\u201369\n\nLange, Konrad, , , , 169\u201370, ; _Kino-drama_ and, ; taste and,\n\nLatour, Bruno, 30\u201331,\n\nLaycock, Thomas,\n\nlay viewers, , . _See also_ spectatorship\n\n_Lebensphilosophie_ ,\n\nLe Bon, Gustave, ,\n\nlegibility, , , ,\n\nlegitimacy, , , , , ; of motion pictures within a discipline, 1\u20132, 4\u20135, , , , , 122\u201323, ,\n\nLemke, Hermann, , , , , , , , ; Cinema Reform Association and, , ; Film-Idea-Central and, ; Herbart and, 181\u201382; Society for the Dissemination of Adult Education and, ; Society for the Dissemination of Popular Education and,\n\nLevin, Tom,\n\n_Lichtbild-B\u00fchne_ (trade periodical), , __\n\nLichtbilderei (film institute), 159\u201360\n\nLichtwark, Alfred, , 169\u201372,\n\nLipps, Theodor, 227\u201328,\n\nlocal exhibition rights, 295n61\n\nLocke, John, 210\u201311\n\nlocomotion: animal, , ; human, 19\u201320, , 38\u201339, 41\u201344. _See alsoThe Human Gait_\n\nlogic: disciplinary, 4\u20136, 9\u201310, 14\u201315, 23\u201324, , 248\u201349; film form and, 5\u20136, 23\u201324, 111\u201325; medical, , , 90\u201391, 111\u201325. _See also_ patterns of use: film form and\n\nLombroso, Cesare,\n\nLomon, Andr\u00e9,\n\nLonde, Albert,\n\n_The Lonedale Operator_ (Griffith),\n\nLuisen-Kino, __\n\nLuk\u00e1cs, Georg, , , , , 288n9, 311n98; child psychology and, ; contemplation and, 235\u201337, ; Romanticism and, ; time and,\n\nLumi\u00e8re (film company),\n\nLynch, Michael, , ,\n\nMach, Ernst,\n\nMacintyre, John,\n\nmagazines, film 152\u201353\n\n_The Maid from Orleans_ ( _Giovanna d'Arco_ ) (film),\n\nMaiocchi, Roberto, ,\n\nMantell, Gideon Algernon,\n\nMarey, \u00c9tienne-Jules, , 40\u201342, , , ; graphic method of, , , ; movement and, ; single-camera system of,\n\nMarinescu, Gheorghe,\n\n_Marital Hygiene_ (film),\n\nMarxism,\n\nmasculinity, , 150\u201351,\n\nmathematical immanent in matter. _See_ geometry\n\n_Matter and Memory_ (Bergson),\n\nmean square displacement, . _See also_ Brownian motion\n\n\"Measurement of the Temperature Dependency of Brownian Motion\" (Seddig),\n\nmedia ensemble ( _Medienverbund_ ), ,\n\nmedia technology, 1\u20134, , ,\n\nmedical demonstrations, 103\u20134\n\nmedical filmmaking, 92\u201393, 90\u201396, , , ; documentary function of, 102\u20135; educational function of, 105\u201310; exploratory function of, 98\u2013102; multiple functions of, 96\u2013110; observation and, 110\u201325. See also cinematography, research film\n\nmedical gaze, , , 118\u201319. _See also_ gaze; medical perception; observation\n\nmedical hermeneutics,\n\nmedicalization, of society,\n\nmedical logic, , , 90\u201391, 111\u201325. _See also_ disciplinary logic, patterns of use: film form and\n\nmedical perception, 119\u201320. _See also_ gaze; medical gaze; observation\n\nmedical way of thinking, . _See also_ medical logic; disciplinary logic; patterns of use: film form and\n\nmedicine, , , 15\u201316, , , , ; Doyen and, 106\u201307; folk, ; photography and, , ; social, ; spectacle and, 279n63\n\n_Medienverbund_ (media ensemble), ,\n\n\"Melody in the Cinema, or Immanent and Transcendental Music\" (Bloch),\n\nMendel, Georg Victor,\n\nmental development,\n\nMesster, Oskar, 28\u201329\n\nmetaphysics,\n\n\"The Metropolis and Mental Life\" (Simmel),\n\nMichaelis, Anthony,\n\nmicrocinematography, , , 92\u201393; medical filmmaking and,\n\nmicrophotographs,\n\nmilitary,\n\nmind, comparisons with motion pictures and modernity, 208\u201312\n\nmodel presentations ( _Mustervorstellungen_ ), , ,\n\nmode of perception (Benjamin), 12\u201313\n\n_Modern Hospital_ ,\n\nmodernity, , , , , , , ; cultural and social, ; excesses of, , , , , , , ; German reaction to, 13\u201314, 149\u201351; motion pictures as emblem of, 125\u201326, , , , , 208\u20139, ; motion pictures as potential haven from, ; pace of change within, 131\u201334, 203\u20134, ; as series of shocks, , . _See also_ social acceleration\n\nmodernity thesis,\n\nMoll, Albert,\n\nmonopoly system, 295n61\n\nmoral weakness,\n\nMosso, Angelo, ,\n\n_Le mouvement_ (Marey),\n\nmovement, , ; aesthetic education and, 178\u201379; analysis of, , , , , , 115\u201316; animal locomotion, , ; architecture and, ; Bergson and, 33\u201334, , ; contemplation and, , 230\u201331; education and, ; _Einf\u00fchlung_ and, 224\u201328; human locomotion, 19\u201320, , 38\u201339, 41\u201344; immobility and, 37\u201338; inner, ; Marey and, ; pathological, . _See also_ Brownian motion; _The Human Gait_\n\n_Moving Picture World_ ,\n\nmulticellular theory of nerve development,\n\nM\u00fcnsterberg, Hugo, ,\n\nmusic, 222\u201324\n\nMusser, Charles,\n\n_Mustervorstellungen_ (model presentations), , ,\n\nMuybridge, Eadweard, 26\u201327,\n\nNature, 190\u201391\n\nnature films, ,\n\n_Naturgetreu_ (faithful to nature), , ,\n\nnegative space, spectatorship as, ,\n\nneo-Kantian tradition,\n\nnerve fibers, 76\u201388, __\n\nnervousness,\n\nneurasthenia,\n\nneurological diseases,\n\nneurology, ; cinematography and, 103\u20135\n\nNewton, Isaac, ,\n\nNielsen, Asta, ,\n\nNietzsche, Friedrich, ,\n\nNogu\u00e8s, Pierre,\n\nnontheatrical uses of film, ; disciplinary agendas and,\n\nNordau, Max, , __ , 131\u201334,\n\nNovalis,\n\nnovels, ; serialized,\n\nNye, Mary Jo, 68\u201369\n\nobjectivity, , ,\n\nobject lesson, , , , 173\u201375, 177\u201379, , 183\u201384. _See alsoAnschauungsunterricht_\n\nobservation: the accommodation of motion pictures to, 5\u20136, 9\u201311, 111\u201312, ; aesthetic contemplation and, , , 170\u201372, ; aesthetic education and, , 168\u201371; _Anschauungsunterricht_ and, , , , 172\u201376; analysis and synthesis and, , ; attention and, ; in biology, 80\u201382; as correlation, , 111\u201317, ; detail and, 119\u201320, , , ; disciplinary logic and, , 9\u201310, 23\u201324, , , 111\u201325; as engine of progress, ; experiment and, , ; gaze and glance as, , 119\u201322; gender and, , , 231\u201332; hypnotism and, , 138\u201340; as ideology, 8\u20139, ; in medicine, , , , 110\u201325; as method of ordering thought, ; modernity and, 132\u201334, , , 191\u201392; photography and, ; as practice, , , 118\u201320; spectatorship and, , , , , , , , , 139\u201340, , , 191\u201392, , ; temporality of, 118\u201326; theory and, , 66\u201367, , 80\u201381; training and, 6\u20137, 10\u201311, , , , , 145\u201346. _See also_ all-at-onceness, analysis and synthesis; \"critical method\"; expert modes of viewing\n\n_On the Optical Sense of Form_ (Vischer),\n\nopera,\n\noptical unconscious,\n\n\"ordinary perception\" (Bergson), , 33\u201336, , , ,\n\norgan function,\n\n\"The Origin of Geometry\" (Husserl),\n\nOstwald, Wilhelm,\n\noutgrowth theory of nerve development, , ,\n\npace: human liberty or potential and, ; of modern life. , , , , , , , , ; of motion pictures, , , , , , , , , , , , ; of observation, , , ; superficiality and, , 203\u20134; tastelessness and, . _See also_ modernity; observation; social acceleration\n\nPaget, Sir James, , ,\n\nPAGU (film company),\n\nParnaland, Ambroise-Fran\u00e7ois,\n\nparticle displacement, , 68\u201370, 72\u201373\n\npassive and active viewers, , . _See also_ expert\/lay distinctions\n\npassivity, ,\n\nPasveer, Bernike,\n\nPath\u00e9 Fr\u00e9r\u00e8s, ,\n\npathological movement,\n\npathology,\n\npatterns of use: film form and, 20\u201322, 248\u201351; historiography and, 20\u201322, , 248\u201349; medicine and, , 121\u201323; professional identity and,\n\nperception: apperception, 173\u201374, 181\u201382, 226\u201327; embodied, 215\u201317, , ; direct (immediate or sensual), , , , , , , , ; mode of (Benjamin), 12\u201313; \"ordinary\" (Bergson), , 33\u201336, , , , . _See also_ aesthetic experience; medical gaze; medical perception; observation\n\nPerrin, Jean, ,\n\nPestalozzi, Johann Heinrich, , , 172\u201376,\n\nPfeffer, Wilhelm,\n\nPfemfert, Franz,\n\nphotogrammetry,\n\nphotography, , 29\u201330, , , , ; Brownian motion and, 66\u201367, 69\u201370, __ , 73\u201375; medicine and, , , 104\u201305, 112\u201314, ; _Naturgetreu_ and, ; series, 112\u201315, , . _See also_ chronophotography\n\nphysical sciences,\n\nphysicians: as bearers of culture ( _Kulturtr\u00e4ger_ ), ; doctor\u2013patient relationship, , ; as experts, 126\u201327\n\nphysics, , , 76\u201377; classical, , , ; metaphysics,\n\nphysiology, , , ,\n\nPinthus, Kurt, ,\n\nPizon, Antoine, ,\n\nPlato,\n\nplay drive, 206\u20137\n\npleasure, visual, 228\u201329\n\nPolgar, Alfred, , ,\n\nPolimanti, Osvaldo,\n\npolitical consciousness,\n\npolitics, of contemplation, 230\u201342\n\nPopulist movement,\n\nPorten, Henny,\n\npositivism,\n\npresence, , , , 237\u201338\n\nprimitivism, 296n80\n\n_The Problem of Form in the Fine Arts_ (Hildebrand),\n\n_Proceedings of the Royal Saxon Society for Sciences_ ,\n\nprofessional identity, , , , ,\n\nProgressive movement,\n\nprojection, , , , , ; emotional. _See_ Einf\u00fchlung; slow-motion, ; speeds of, , ,\n\nproper viewing, , 94\u201395. _See also_ observation\n\nprotoplasmic bridge theory of nerve development, , ,\n\npsychoanalysis, ,\n\npsychology: of children, , , 162\u201364; of crowds, , , 164\u201366; social, ,\n\npublic health, ,\n\npublic sphere, , ; women in,\n\npulp fiction ( _Schundliteratur_ ), ,\n\nquantitative frame analysis, , , ,\n\n_Quo Vadis?_ (film),\n\nRabinbach, Anson, , , ,\n\nRadica (conjoined twin),\n\nradiology, ,\n\nRam\u00f3n y Cajal, Santiago,\n\nRanci\u00e8re, Jacques, 199\u2013200\n\n_Raumgestaltung_ (spatial creation),\n\nRauscher, Ulrich, 211\u201312\n\nreadability. _See_ legibility\n\nrealism. _See_ filmic realism\n\nRefining the Cinema ( _Veredelung des Kinos_ ),\n\nreform: of clothing, 149\u201350; of education, 148\u201349, ; of film, , , 152\u201362, , ; in Germany, 147\u201352; _Kinoreform_ , , 154\u201355, , , ; _Kulturkritik_ and, ; modernity and, ; spirit of, 147\u201362\n\n_Reformkinos_ ,\n\nregulation, of cinemas, 158\u201359\n\nReicher, Karl,\n\nReich Health Office, 284n122\n\n_Rembrandt as Educator_ (Langbehn), 168\u201369\n\nrepeatability, ,\n\nrepose, , ,\n\nresearch film: , 19\u201326, , , , ; Bergson and, 32\u201337; Brownian motion and, 62\u201376, __ , __ ; disciplinary contexts of, ; distribution of, ; documentary function of, 102\u20135; educational function of, 105\u201310; exploratory function of, 98\u2013102; medical, 92\u201393, 96\u2013125; nerve fibers and, 76\u201388, __ ; overview of, 26\u201331; public screening of, ; science of work and, 37\u201344; tissue cultures and, 76\u201388. _See also_ chronophotography; cinematography; _The Human Gait_ ; medical filmmaking\n\nRheinberger, Hans-J\u00f6rg, ,\n\nRicher, Paul,\n\nRieder, H.,\n\nRies, Julius, ,\n\nRitter, Christopher,\n\nRitzheimer, Kara,\n\nRockefeller Institute,\n\nRomanticism,\n\nRosenthal, J.,\n\nRoss, Corey,\n\nRousseau, Jean-Jacques,\n\nRunge, Philipp Otto,\n\nRussia,\n\nR\u00fcswald, K. (teacher),\n\nSaint-Louis Hospital,\n\nSamuleit, Paul,\n\nscanning ( _Schauen_ ), ,\n\nSchaar, Eckard,\n\n_Schauen_ (scanning), ,\n\n_Schaulust_ (scopophilia), , 228\u201330, 309n72\n\nSchelling, Friedrich Wilhelm Joseph,\n\nSchenk, Paul,\n\nSchiller, Friedrich, , , , ; aesthetic contemplation and, , ; aesthetic education and, 167\u201368, ; aesthetic experience and, , , 195\u201398, 204\u20137; form drive and, , ; play drive and, 206\u20137; sense drive and, , ; temporality and, 204\u20137; vision and,\n\nSchlanger, Benjamin,\n\nSchl\u00fcpmann, Heide, 231\u201332\n\nSchmarsow, August, 225\u201327,\n\nSchopenhauer, Arthur, , , , , ; aesthetic experience and, , , , ; self and, ; temporality and,\n\nSchultze-Naumberg, Paul,\n\n_Sch\u00fclervorstellungen_ (commercial screenings for children),\n\n_Schulvorstellungen_ (screenings for classes),\n\nSchumberg, Wilhelm,\n\n_Schund_ campaigns, 151\u201352,\n\n_Schundfilme_ (trashy films), , ,\n\n_Schundliteratur_ (pulp fiction), ,\n\nSchuster, Paul, ,\n\nscientific filmmaking. _See_ research film. _See also_ chronophotography; cinematography; _The Human Gait_ ; medical filmmaking\n\nscientific method, , , , , , , , , , , . _See also_ experiment; observation\n\nscientific observation. _See_ observation\n\nscientific theater,\n\nscopophilia ( _Schaulust_ ), , 228\u201330, 309n72\n\nsea squirts ( _Botryllus_ ),\n\nsecond law of thermodynamics, ,\n\nSeddig, Max, 20\u201321, 25\u201326, , , 69\u201375, 87\u201388\n\n_Sehen_ (seeing), ,\n\nself, loss of, ,\n\nself-awareness, ,\n\nself-cultivation ( _Bildung_ ), ,\n\nself-identity, , ,\n\nself-mastery,\n\nSellmann, Adolf, , , ,\n\nsense drive, ,\n\nsense impression ( _Anschauung_ ), 172\u201373,\n\nsensuality, 142\u201343\n\nsensuous totality ( _Gesamtsinnlichkeit_ ),\n\n_sensus communis_ (communal sense),\n\nsequential images,\n\nserialized novels,\n\nseries photography, 112\u201315, , . _See also_ chronophotography; medicine; photography\n\nSerner, Walter, , 228\u201329\n\nsexual arousal, 228\u201329\n\nsexual images, ,\n\nsexuality,\n\nShapin, Steven,\n\nshocks, modernity as series of, ,\n\nSiedentopf, Henry,\n\nSimmel, Georg,\n\nslow-motion cinematography, , ,\n\nslow-motion projection,\n\nSmoluchowski, Maryan,\n\nSobchack, Vivian,\n\nsocial acceleration, , , , , , ; _Anschauungsunterricht_ and, 173\u201374. _See also_ modernity; pace: human liberty or potential and\n\nSocial Democratic Party of Germany (SPD), 292n34\n\nsocial psychology, ,\n\nSociety for the Dissemination of Popular Education (Gesellschaft zur Verbreitung von Volksbildung) (GVV), ,\n\nSociety of Friends of the Schools and Instruction for the Fatherland (Gesellschaft der Freunde des vaterl\u00e4ndischen Schul- und Erziehungswesens),\n\nSociety of German Natural Scientists and Physicians (Gesellschaft deutscher Naturforscher und \u00c4rzte), ,\n\nSolvay, Ernest,\n\n\"Some Specific Features of the Medical Way of Thinking\" (Fleck),\n\nsomnambulism, 212\u201313\n\nsoullessness, 239\u201340\n\nspatial creation ( _Raumgestaltung_ ),\n\nSPD. _See_ Social Democratic Party of Germany\n\nspectacle, in medicine, 279n63\n\nspectatorship, , , 200\u2013202, ; analysis, synthesis and, ; children, crowds and, 162\u201365; embodied, , 220\u201325; gender and, , 231\u201332; hypnotism and, 135\u201337; observation and, , , , , , , , , 139\u201340, , , 191\u201392, , ; theories of film, 11\u201312. _See also_ improper viewing; lay viewers; negative space\n\n\"Spectacles of the Earth\" (H\u00e4fker),\n\nSpencer, Herbert,\n\n\"The Stage Considered as a Moral Institution\" (Schiller),\n\nstaining techniques, 84\u201385\n\nStapel, Wilhelm, ,\n\nstatistical aspect, of medical logic,\n\nstillness and movement, , , ,\n\nstimulus shield,\n\nstorefront cinemas ( _Ladenkinos_ ), , __\n\nStratz, Carl Heinrich,\n\nstrobe effect, 51\u201352\n\nStrobl, Karl Hans, , ,\n\n_Studien zu einer Physiologie des Marsches_ (Zuntz and Schumberg),\n\nsubjectivity, , , , , ; objectivity and, ,\n\nsublime, theory of the,\n\nsuggestibility, , , 164\u201365,\n\nsurgery of the dead,\n\n\"Surrealism\" (Benjamin),\n\nsynesthesia, 221\u201322,\n\nsynthesis. _See_ analysis and synthesis\n\nSzczepaniak-Gillece, Jocelyn,\n\ntaste, , , , , , , , ; aesthetics; morality and, 167\u201368; geometry of, __ ; H\u00e4fker and, 186\u201389; ideology and, ; nation and, 170\u201371; as _sensus communis_ , ; and vision, 145\u201346,\n\nTausk, Viktor,\n\ntechnological reproducibility,\n\ntechnology, ; formal relationships in, ; media, 1\u20134,\n\ntemperance movement,\n\ntemporal discontinuity. _See_ continuity and discontinuity\n\ntemporal interruption, ,\n\ntemporality: of aesthetic experience, 202\u201314; of observation, 118\u201326\n\ntemporal phenomena,\n\nTews, Johannes,\n\ntextual analysis. _See_ analysis (close reading)\n\ntheater: cinema compared to, 236\u201339; scientific,\n\ntheory: disciplinary logic and, ; observation and, , 66\u201367, , 80\u201381\n\n_Theory of the Novel_ (Luk\u00e1cs), 235\u201336,\n\nthermodynamics, , ,\n\nThomson, William (Lord Kelvin),\n\n\"Thoughts Toward an Aesthetic of the Cinema (Gedanken zu einer Aesthetik des 'Kino')\" (Luk\u00e1cs), ,\n\nthought styles, , . _See also_ disciplinary logic\n\n_Three Essays on Sexuality_ (Freud),\n\ntime, , ; Luk\u00e1cs and, ; reversibility of, 74\u201375; spatialization of, 62\u201364, ; spectatorship and will and, 125\u201341. _See also_ duration; _dur\u00e9e_ ; temporality\n\ntime-lapse cinematography, , , 84\u201385, , , , , ; Braus and, ; medical filmmaking and,\n\n_Tirol in Waffen_ ( _Tirol in Arms_ ) (film),\n\ntissue cultures, 76\u201388\n\nTitchener, Edward B.,\n\nTolstoy, Leo,\n\nT\u00f6nnies, Ferdinand, 288n9\n\ntotal presentation ( _Gesamtvorf\u00fchrung_ ),\n\ntrade journals and magazines, 152\u201353\n\ntraditional aesthetics. _See_ aesthetics\n\ntraining film,\n\ntrashy films ( _Schundfilme_ ), , ,\n\ntriangulation,\n\nTucker, Jennifer,\n\ntutelage,\n\ntwo-sided chronophotography, . _See also_ chronophotography; _The Human Gait_\n\nUFA, , ; cultural division of ( _Kulturabteilung_ ),\n\nultramicroscope, ,\n\nUnion-Theater, __\n\nUnited Kingdom, , , , 147\u201348\n\nUnited States, , , , , , 147\u201348; Bergson and, , , ; object lesson and,\n\nUniversity of T\u00fcbingen, , , ,\n\nuplift and educational film,\n\nuseful cinema,\n\nVal\u00e9ry, Paul,\n\nVan Gehuchten, Arthur,\n\n_Veredelung des Kinos_ (Refining the Cinema),\n\nVierordt, Hermann,\n\nVirchow, Rudolph,\n\nvirtual experiments, ,\n\nvirtual witnessing, ,\n\nVischer, Robert, , , , , 224\u201325\n\nvisual means of instruction. _SeeAnschauungsunterrich_\n\nvisual pleasure, 228\u201329\n\nvitalism and mechanism, 39\u201340\n\nvividness ( _Anschaulichkeit_ ), 107\u20138,\n\nVl\u00e8s, Fred,\n\nvolition, , 286n143. _See also_ agency; free will\n\nvon Bergmann, Ernst,\n\nvon Helmholtz, Hermann, ,\n\nWagner, Richard, ,\n\n_The Wanderings of Odysseus_ ( _Die Irrfahrten des Odysseus_ ) (film), , __\n\n_Wanderkino_ ,\n\nWarstat, Willi,\n\nWeber, Max,\n\nWeber, Wilhelm, 263n58\n\nWeber brothers,\n\nWeichardt, Wilhelm,\n\nWeimar Republic,\n\nWeisenburg, Theodore,\n\n\"What Is Enlightenment?\" (Kant),\n\nwholeness and fragmentation, 36\u201337\n\nWilhelm II (kaiser), ,\n\n_Wilhelm Meister_ (Goethe),\n\nwill 139. _See also_ agency; free will\n\nWolf-Czapek, K. W.,\n\nWolgast, Heinrich,\n\nwomen, ; clothing reform and, 149\u201350; elite and, ; hysteria and, ; in public sphere, 150\u201351, ; spectatorship by, , 231\u201332\n\n_Women's Clothing and Its Natural Development_ (Stratz),\n\nworking class,\n\n\"Work of Art\" (Benjamin), , , , ,\n\nworking objects. _See_ docile bodies\n\n_The World as Will and Representation_ (Schopenhauer),\n\nWorld War I, , , 161\u201362, , , , ; class and, ; gender dynamics and, ; H\u00e4fker and, 301n145; health campaigns and, ; _Kulturabteilung_ and, ; X-ray cinematography and,\n\nWundt, Wilhelm,\n\nX-rays, , 113\u201314; cinematography, , 100\u2013102, __\n\nZeiss, Carl, ,\n\n_Zivilisation_ and _Kultur_ , 144\u201345\n\nZoological Gardens,\n\nZsigmondy, Richard,\n\nZuntz, Nathan, \nFILM AND CULTURE\n\nA series of Columbia University Press\n\nEdited by John Belton\n\n_What Made Pistachio Nuts? Early Sound Comedy and the Vaudeville Aesthetic_ , Henry Jenkins\n\n_Showstoppers: Busby Berkeley and the Tradition of Spectacle_ , Martin Rubin\n\n_Projections of War: Hollywood, American Culture, and World War II_ , Thomas Doherty\n\n_Laughing Screaming: Modern Hollywood Horror and Comedy_ , William Paul\n\n_Laughing Hysterically: American Screen Comedy of the 1950s_ , Ed Sikov\n\n_Primitive Passions: Visuality, Sexuality, Ethnography, and Contemporary Chinese Cinema_ , Rey Chow\n\n_The Cinema of Max Ophuls: Magisterial Vision and the Figure of Woman_ , Susan M. White\n\n_Black Women as Cultural Readers_ , Jacqueline Bobo\n\n_Picturing Japaneseness: Monumental Style, National Identity, Japanese Film_ , Darrell William Davis\n\n_Attack of the Leading Ladies: Gender, Sexuality, and Spectatorship in Classic Horror Cinema_ , Rhona J. Berenstein\n\n_This Mad Masquerade: Stardom and Masculinity in the Jazz Age_ , Gaylyn Studlar\n\n_Sexual Politics and Narrative Film: Hollywood and Beyond_ , Robin Wood\n\n_The Sounds of Commerce: Marketing Popular Film Music_ , Jeff Smith\n\n_Orson Welles, Shakespeare, and Popular Culture_ , Michael Anderegg\n\n_Pre-Code Hollywood: Sex, Immorality, and Insurrection in American Cinema, 1930\u20131934_ , Thomas Doherty\n\n_Sound Technology and the American Cinema: Perception, Representation, Modernity_ , James Lastra\n\n_Melodrama and Modernity: Early Sensational Cinema and Its Contexts_ , Ben Singer\n\n_Wondrous Difference: Cinema, Anthropology, and Turn-of-the-Century Visual Culture_ , Alison Griffiths\n\n_Hearst Over Hollywood: Power, Passion, and Propaganda in the Movies_ , Louis Pizzitola\n\n_Masculine Interests: Homoerotics in Hollywood Film_ , Robert Lang\n\n_Special Effects: Still in Search of Wonder_ , Michele Pierson\n\n_Designing Women: Cinema, Art Deco, and the Female Form_ , Lucy Fischer\n\n_Cold War, Cool Medium: Television, McCarthyism, and American Culture_ , Thomas Doherty\n\n_Katharine Hepburn: Star as Feminist_ , Andrew Britton\n\n_Silent Film Sound_ , Rick Altman\n\n_Home in Hollywood: The Imaginary Geography of Hollywood_ , Elisabeth Bronfen\n\n_Hollywood and the Culture Elite: How the Movies Became American_ , Peter Decherney\n\n_Taiwan Film Directors: A Treasure Island_ , Emilie Yueh-yu Yeh and Darrell William Davis\n\n_Shocking Representation: Historical Trauma, National Cinema, and the Modern Horror Film_ , Adam Lowenstein\n\n_China on Screen: Cinema and Nation_ , Chris Berry and Mary Farquhar\n\n_The New European Cinema: Redrawing the Map_ , Rosalind Galt\n\n_George Gallup in Hollywood_ , Susan Ohmer\n\n_Electric Sounds: Technological Change and the Rise of Corporate Mass Media_ , Steve J. Wurtzler\n\n_The Impossible David Lynch_ , Todd McGowan\n\n_Sentimental Fabulations, Contemporary Chinese Films: Attachment in the Age of Global Visibility_ , Rey Chow\n\n_Hitchcock's Romantic Irony_ , Richard Allen\n\n_Intelligence Work: The Politics of American Documentary_ , Jonathan Kahana\n\n_Eye of the Century: Film, Experience, Modernity_ , Francesco Casetti\n\n_Shivers Down Your Spine: Cinema, Museums, and the Immersive View_ , Alison Griffiths\n\n_Weimar Cinema: An Essential Guide to Classic Films of the Era_ , Edited by Noah Isenberg\n\n_African Film and Literature: Adapting Violence to the Screen_ , Lindiwe Dovey\n\n_Film, A Sound Art_ , Michel Chion\n\n_Film Studies: An Introduction_ , Ed Sikov\n\n_Hollywood Lighting from the Silent Era to Film Noir_ , Patrick Keating\n\n_Levinas and the Cinema of Redemption: Time, Ethics, and the Feminine_ , Sam B. Girgus\n\n_Counter-Archive: Film, the Everyday, and Albert Kahn's Archives de la Plan\u00e8te_ , Paula Amad\n\n_Indie: An American Film Culture_ , Michael Z. Newman\n\n_Pretty: Film and the Decorative Image_ , Rosalind Galt\n\n_Film and Stereotype: A Challenge for Cinema and Theory_ , J\u00f6rg Schweinitz\n\n_Chinese Women's Cinema: Transnational Contexts_ , Edited by Lingzhen Wang\n\n_Hideous Progeny: Disability, Eugenics, and Classic Horror Cinema_ , Angela M. Smith\n\n_Hollywood's Copyright Wars: From Edison to the Internet_ , Peter Decherney\n\n_Electric Dreamland: Amusement Parks, Movies, and American Modernity_ , Lauren Rabinovitz\n\n_Where Film Meets Philosophy: Godard, Resnais, and Experiments in Cinematic Thinking_ , Hunter Vaughan\n\n_The Utopia of Film: Cinema and Its Futures in Godard, Kluge, and Tahimik_ , Christopher Pavsek\n\n_Hollywood and Hitler, 1933\u20131939_ , Thomas Doherty\n\n_Cinematic Appeals: The Experience of New Movie Technologies_ , Ariel Rogers\n\n_Continental Strangers: German Exile Cinema, 1933\u20131951_ , Gerd Gem\u00fcnden\n\n_Deathwatch: American Film, Technology, and the End of Life_ , C. Scott Combs\n\n_After the Silents: Hollywood Film Music in the Early Sound Era, 1926\u20131934_ , Michael Slowik\n\n_\"It's the Pictures That Got Small\": Charles Brackett on Billy Wilder and Hollywood's Golden Age_ , Edited by Anthony Slide\n\n_Plastic Reality: Special Effects, Technology, and the Emergence of 1970s Blockbuster Aesthetics_ , Julie A. Turnock\n\n_Maya Deren: Incomplete Control_ , Sarah Keller\n\n_Dreaming of Cinema: Spectatorship, Surrealism, and the Age of Digital Media_ , Adam Lowenstein\n\n_Motion(less) Pictures: The Cinema of Stasis_ , Justin Remes\n\n_The Lumi\u00e8re Galaxy: Seven Key Words for the Cinema to Come_ , Francesco Casetti\n\n_The End of Cinema? A Medium in Crisis in the Digital Age_ , Andr\u00e9 Gaudreault and Philippe Marion\n\n_Studios Before the System: Architecture, Technology, and the Emergence of Cinematic Space_ , Brian R. Jacobson\n","meta":{"redpajama_set_name":"RedPajamaBook"}}
+{"text":" \nPraise for\n\nA UNIVERSE FROM NOTHING\n\n\"Krauss possesses a rare talent for making the hardest ideas in astrophysics accessible to the layman, due in part to his sly humor . . . one has to hope that this book won't appeal only to the partisans of the culture wars\u2014it's just too good and interesting for that. Krauss is genuinely in awe of the 'wondrously strange' nature of our physical world, and his enthusiasm is infectious.\"\n\n\u2014Associated Press\n\n\"An eloquent guide to our expanding universe . . . There have been a number of fine cosmology books published recently but few have gone so far, and none so eloquently, in exploring why it is unnecessary to invoke God to light the blue touchpaper and set the universe in motion.\"\n\n\u2014Financial Times\n\n\"How physicists came up with the current model of the cosmos is quite a story, and to tell it in his elegant A Universe from Nothing, physicist Lawrence Krauss walks a carefully laid path . . . It would be easy for this remarkable story to revel in self-congratulation, but Krauss steers it soberly and with grace . . . His asides on how he views each piece of science and its chances of being right are refreshingly honest . . . unstable nothingness, as described by Krauss . . . is also invigorating for the rest of us, because in this nothingness there are many wonderful things to see and understand.\"\n\n\u2014Nature\n\n\"[An] excellent guide to cutting-edge physics . . . As Krauss elegantly argues in A Universe from Nothing, the accelerating expansion, indeed the whole existence of the cosmos, is most likely powered by 'nothing.' Krauss is an exemplary interpreter of tough science, and the central part of the book, where he discusses what we know about the history of the universe\u2014and how we know it\u2014is perfectly judged. It is detailed but lucid, thorough but not stodgy . . . Space and time can indeed come from nothing; nothing, as Krauss explains beautifully, being an extremely unstable state from which the production of \"'something\"' is pretty much inevitable . . . A Universe from Nothing is a great book: readable, informative and topical.\"\n\n\u2014New Scientist\n\n\"With its mind-bending mechanics, Krauss argues, our universe may indeed have appeared from nowhere, rather than at the hands of a divine creator. There's some intellectual heavy lifting here\u2014Einstein is the main character, after all\u2014but the concepts are articulated clearly, and the thrill of discovery is contagious. 'We are like the early terrestrial mapmakers,' Krauss writes, puzzling out what was once solely the province of our imaginations.\"\n\n\u2014Mother Jones\n\n\"His arguments for the birth of the universe out of nothingness from a physical, rather than theological, beginning not only are logical but celebrate the wonder of our natural universe. Recommended.\"\n\n\u2014Library Journal\n\n\"Lively and humorous as well as informative . . . Readers will find the result of Krauss's '[celebration of our] absolutely surprising and fascinating universe' as compelling as it is intriguing.\"\n\n\u2014Publishers Weekly\n\n\"The author delivers plenty of jolts in this enthusiastic and lucid but demanding overview of the universe, which includes plenty of mysteries\u2014but its origin isn't among them. A thoughtful, challenging book\u2014but not for the faint of heart or those not willing to read carefully.\"\n\n\u2014Kirkus Reviews\n\n\"Krauss is a lucid . . . writer, as well as a sparkling speaker and wit, an all-purpose science communicator . . . [I]t is an account of how to untie a paradox, scientifically. And it's also a scientist's hymn\u2014a song of secular appreciation\u2014to the unseen.\"\n\n\u2014cbcnews.ca\n\n\"In A Universe from Nothing, Lawrence Krauss has written a thrilling introduction to the current state of cosmology\u2014the branch of science that tells us about the deep past and deeper future of everything. As it turns out, everything has a lot to do with nothing\u2014and nothing to do with God. This is a brilliant and disarming book.\"\n\n\u2014Sam Harris, author of The Moral Landscape\n\n\"People always say you can't get something from nothing. Thankfully, Lawrence Krauss didn't listen. In fact, something big happens to you during this book about cosmic nothing, and before you can help it, your mind will be expanding as rapidly as the early universe.\"\n\n\u2014Sam Kean, author of The Disappearing Spoon\n\n\"Nothing is not nothing. Nothing is something. That's how a cosmos can be spawned from the void\u2014a profound idea conveyed in A Universe From Nothing that unsettles some yet enlightens others. Meanwhile, it's just another day on the job for physicist Lawrence Krauss.\"\n\n\u2014Neil deGrasse Tyson, astrophysicist, American Museum of Natural History\n\n\"With characteristic wit, eloquence, and clarity Lawrence Krauss gives a wonderfully illuminating account of how science deals with one of the biggest questions of all: how the universe's existence could arise from nothing. It is a question that philosophy and theology get themselves into muddle over, but that science can offer real answers to, as Krauss's lucid explanation shows. Here is the triumph of physics over metaphysics, reason and enquiry over obfuscation and myth, made plain for all to see: Krauss gives us a treat as well as an education in fascinating style.\"\n\n\u2014A. C. Grayling, author of The Good Book\n\n\"Astronomers at the beginning of the twentieth century were wondering whether there was anything beyond our Milky Way Galaxy. As Lawrence Krauss lucidly explains, astronomers living two trillion years from now, will perhaps be pondering precisely the same question! Beautifully navigating through deep intellectual waters, Krauss presents the most recent ideas on the nature of our cosmos, and of our place within it. A fascinating read.\"\n\n\u2014Mario Livio, author of Is God A Mathematician? and The Golden Ratio\n\n\"In this clear and crisply written book, Lawrence Krauss outlines the compelling evidence that our complex cosmos has evolved from a hot, dense state and how this progress has emboldened theorists to develop fascinating speculations about how things really began.\"\n\n\u2014Sir Martin Rees, author of Our Final Hour\n\n\"A series of brilliant insights and astonishing discoveries have rocked the Universe in recent years, and Lawrence Krauss has been in the thick of it. With his characteristic verve, and using many clever devices, he's made that remarkable story remarkably accessible. The climax is a bold scientific answer to the great question of existence: Why is there something rather than nothing?\"\n\n\u2014Frank Wilczek, Nobel Laureate and Herman Feshbach professor of Physics at MIT, and author of The Lightness of Being\nThank you for purchasing this Free Press eBook.\n\n* * *\n\nSign up for our newsletter and receive special offers, access to bonus content, and info on the latest new releases and other great eBooks from Free Press and Simon & Schuster.\n\nCLICK HERE TO SIGN UP\n\nor visit us online to sign up at  \neBookNews.SimonandSchuster.com\n\n## CONTENTS\n\nPreface to the Paperback Edition\n\nPreface\n\nChapter 1: A Cosmic Mystery Story: Beginnings\n\nChapter 2: A Cosmic Mystery Story: Weighing the Universe\n\nChapter 3: Light from the Beginning of Time\n\nChapter 4: Much Ado About Nothing\n\nChapter 5: The Runaway Universe\n\nChapter 6: The Free Lunch at the End of the Universe\n\nChapter 7: Our Miserable Future\n\nChapter 8: A Grand Accident?\n\nChapter 9: Nothing Is Something\n\nChapter 10: Nothing Is Unstable\n\nChapter 11: Brave New Worlds\n\nEpilogue\n\nAfterword by Richard Dawkins\n\nAbout the Author\n\nQ & A with the Author\n\nIndex\n_To Thomas, Patty, Nancy, and Robin, for helping inspire me to create something from nothing . . ._\n_On this site in 1897,_  \n _Nothing happened._  \n\u2014Plaque on wall of Woody Creek Tavern,  \nWoody Creek, Colorado\n\n## PREFACE TO THE PAPERBACK EDITION\n\nSince the hardcover version of this book first appeared, a visceral negative reaction among some commentators to the very idea of a universe arising from nothing has been balanced by a major scientific discovery that supports this possibility. The confirmation of the Higgs boson refines our understanding of the relationship between seemingly empty space and our existence. I want to elaborate on both the Higgs boson and the negative reactions to A Universe from Nothing in this new preface.\n\nWhen I chose to subtitle this book Why There Is Something Rather Than Nothing, I wanted to connect the remarkable discoveries of modern science to a question that has fascinated theologians, philosophers, natural philosophers, and the general public for more than two millennia. But I wasn't fully aware of how my choice of words might lead to the same kind of confusion that occurs whenever one says in public that Evolution is a theory.\n\nIn popular parlance, theory means something very different from its scientific sense. So too nothing is a hot-button issue for some people, a line in the sand that some people are not willing to cross, so that even using the word, just as using the word God, can be so polarizing that it obfuscates more important issues. A similar remark can be made about the question \"Why?\": using why and nothing together can be as explosive as mixing diesel fuel and fertilizer.\n\nIn chapter 9 of this book I mention a fact that I now want to introduce first here. Whenever one asks \"Why?\" in science, one actually means \"How?\". \"Why?\" is not really a sensible question in science because it usually implies purpose and, as anyone who has been the parent of a small child knows, one can keep on asking \"Why?\" forever, no matter what the answer to the previous question. Ultimately, the only way to end the conversation seems to be to say \"Because!\"\n\nScience changes the meaning of questions, especially why-like questions, as it progresses. Here is an early example of this fact, which illustrates a number of features in common with the more recent revelations I treat in this book.\n\nThe renowned astronomer Johannes Kepler claimed in 1595 to have had an epiphany when he suddenly thought he had answered a profoundly important why question: \"Why are there six planets?\" The answer, he believed, lay in the view of the five Platonic solids, those sacred objects from geometry whose faces can be composed of regular polygons\u2014triangles, squares, etc.\u2014and that could be circumscribed by spheres whose size would increase as the number of faces of the solid increased. If these spheres then separated the orbits of the six known planets, he conjectured, perhaps their relative distances from the sun and the fact that there were just six of them could be understood as revealing, in a profound and deep sense, the mind of God, the mathematician. (The idea that geometry was sacred goes back as far as Pythagoras.) \"Why are there six planets?\"\u2014then, in 1595\u2014was considered a meaningful question, one that revealed purpose to the universe.\n\nNow, however, we understand the question is meaningless. In the first place, we know there are not six planets, there are nine planets. (Pluto will always be a planet for me. Not only do I like to annoy my friend Neil deGrasse Tyson by so insisting, but my daughter did her fourth-grade science project on Pluto, and I don't want that to have been in vain!) More important, however, we know our solar system is not unique, which Kepler and his era did not know. More than two thousand planets orbiting other stars have been discovered (by a satellite named Kepler, coincidentally!).\n\nThe important question then becomes not \"Why?\" but \"How does our solar system have nine planets?\" (or, eight planets, depending upon your count). Since clearly lots of different solar systems exist, with very different features, what we really want to know is how typical we are, what specific conditions might have existed allowing our solar system to have four rocky planets closest to the sun, surrounded by a number of far larger gas giants. The answer to this question might shed light on the likelihood of finding life elsewhere in the universe, for example.\n\nMost important, however, we realize that there is nothing profound about six (or eight or nine), nothing that points to purpose or design . . . no evidence of \"purpose\" in the distribution of planets in the universe. Not only has \"why\" become \"how\" but \"why\" no longer has any verifiable meaning.\n\nSo too, when we ask \"Why is there something rather than nothing?\" we really mean \"How is there something rather than nothing?\" This brings me to the second confusion engendered by my choice of words. There are many seeming \"miracles\" of nature that appear so daunting that many have given up trying to find an explanation of how we came to be and, instead, blame it all on God. But the question I really care about, and the one that science can actually address, is the question of how all the \"stuff\" in the universe could have come from no \"stuff,\" and how, if you wish, formlessness led to form. That is what seems so astounding and nonintuitive. It seems to violate everything we know about the world\u2014in particular the fact that energy in its various forms, including mass, is conserved. Common sense suggests that \"nothing,\" in this sense the absence of \"something,\" should have zero total energy. Therefore, where did the 400 billion or so galaxies that make up the observable universe come from?\n\nThe fact that we need to refine what we mean by \"common sense\" in order to accommodate our understanding of nature is, to me, one of the most remarkable and liberating aspects of science. Reality liberates us from the biases and misconceptions that have arisen because our intellects evolved through our animal ancestors, whose survival was based on whether predators might lurk behind trees or in caves and not on understanding the wave function of electrons in atoms.\n\nOur modern conception of the universe is so foreign to what even scientists generally believed a mere century ago that it is a tribute to the power of the scientific method and the creativity and persistence of humans who want to understand it. That is worth celebrating. As I describe in this book, the question and the possible answers to how something might come from nothing are even more interesting than merely the possibility of galaxies manifesting from empty space. Science provides a possible road map for the creation of space (and time) itself\u2014and perhaps also an understanding of how the laws of physics that govern the dynamics of space and time can arise haphazardly.\n\nFor many people, however, the fascinating possible resolutions of these age-old mysteries are not sufficient. The deeper question of nonexistence overwhelms them. Can we understand how absolute nothingness, without even the potential for anything at all to exist, does not still reign supreme? Can one ever say anything other than the fact that the nothing that became our something was a part of \"something\" else, in which the potential for our existence, or any existence, was always implicit?\n\nIn the book I take a rather flippant attitude toward this concern, because I don't think it adds anything to the productive discussion, which is \"What questions are actually answerable by probing the universe?\" I have discounted this philosophical issue, but not because I think those people who occupy themselves with certain aspects of it are not trying hard to define logical questions. Rather, I discount this aspect of philosophy here because I think it bypasses the really interesting and answerable physical questions associated with the origin and evolution of our universe. No doubt some will view this as my own limitation, and maybe it is. But it is within that context that people should read this book. I don't make any claims to answer any questions that science cannot answer, and I have tried very carefully within the text to define what I mean by \"nothing\" and \"something.\" If those definitions differ from those you would like to adopt, so be it. Write your own book. But don't discount the remarkable human adventure that is modern science because it doesn't console you.\n\nNow, the good news! This past summer, physicists around the world, including me, were glued to computers at very odd hours to watch live as scientists at the Large Hadron Collider, outside Geneva, announced that they had found one of the most important missing pieces of the jigsaw puzzle that is nature\u2014the Higgs particle (or Higgs boson).\n\nProposed almost fifty years ago to allow for consistency between theoretical predictions and experimental observations in elementary particle physics, the Higgs particle's discovery caps one of the most remarkable intellectual adventures in human history\u2014one that anyone interested in the progress of knowledge should at least be aware of\u2014and makes even more remarkable the precarious accident that allowed our existence to form from nothing, the subject of this book. The discovery is further proof that the universe of our senses is just the tip of a vast, largely hidden cosmic iceberg and that seemingly empty space can provide the seeds for our existence.\n\nThe prediction of the Higgs particle accompanied a remarkable revolution that completely changed our understanding of particle physics in the latter part of the twentieth century. Just fifty years ago, in spite of the great advances of physics in the previous half century, we understood only one of the four fundamental forces of nature\u2014electromagnetism\u2014as a fully consistent quantum theory. In just one subsequent decade, however, not only had three of the four known forces surrendered to our investigations, but a new elegant unity of nature had been uncovered. It was found that all of the known forces could be described using a single mathematical framework\u2014and that two of the forces, electromagnetism and the weak force (which governs the nuclear reactions that power the sun), were actually different manifestations of a single underlying force.\n\nHow could two such different forces be related? After all, the photon, the particle that conveys electromagnetism, has no mass, while the particles that convey the weak force are very massive\u2014almost one hundred times as heavy as the particles that make up atomic nuclei, a fact that explains why the weak force is weak.\n\nBritish physicist Peter Higgs and several others showed that, if there exists an otherwise invisible background field (Higgs field) permeating all of space, then the particles that convey some force like electromagnetism can interact with this field and effectively encounter resistance to their motion and slow down, like a swimmer moving through molasses. As a result, these particles can behave as if they are heavy, as if they have a mass. The physicist Steven Weinberg (and somewhat later Abdus Salam) applied this idea to a model of the weak and electromagnetic forces previously proposed by Sheldon L. Glashow, and everything fit together.\n\nThis idea can be extended to the rest of particles in nature, including particles like those that make up the protons and neutrons, as well as fundamental particles like electrons, all of which combine to make up the atoms in our bodies. If some particle interacts more strongly with this background field, it ends up acting heavier. If it interacts more weakly, it acts lighter. If it doesn't interact at all, like the photon, it remains massless.\n\nIf anything sounds too good to be true, this is it. The miracle of mass\u2014indeed, of our very existence (because if not for the Higgs, there would be no stars, no planets, and no people)\u2014is apparently possible because of some otherwise hidden background field whose only effect seems to be to allow the world to look the way it does.\n\nBut relying on invisible miracles is the stuff of religion, not science. To ascertain whether this remarkable accident was real, physicists relied on another facet of the quantum world. Associated with every background field is a particle, and if you pick a point in space and hit it hard enough, you may whack out real particles. The trick is hitting it hard enough over a small enough volume. And that's the rub. After fifty years of trying, including a failed attempt in the United States to build an accelerator to test these ideas, no sign of the Higgs had appeared. In fact, I was betting against it, since a career in theoretical physics has taught me that nature usually has a far richer imagination than we do.\n\nUntil July.\n\nThe apparent discovery of the Higgs boson may not result in a better toaster or a faster car. But it provides a remarkable celebration of the human mind's capacity to uncover nature's secrets, and of the technology we have built to control them. Hidden in what seems like empty space\u2014indeed, like nothing\u2014appear to be the very elements that allow for our existence.\n\nThe discovery of a Higgs field further validates many of the ideas I discuss in this book. The idea that the very early universe went through a period of superluminal expansion, called inflation, that basically produced almost all the space and matter in the observable universe from almost nothing relies heavily on the possibility that another field, much like the Higgs field we seem to have discovered this past year, momentarily held sway in early times.\n\nThe existence of a Higgs field permeating all of space today also begs several important questions, most notably \"What conditions in the early universe led to such a cosmic accident?\" \"Why does the field have the value it is measured to have?\" \"Could it have been different?\" \"Could the laws of physics, had initial conditions been slightly different, have resulted in a universe without matter as we observe it today?\" These are precisely the kind of questions I discuss near the end of this book.\n\nWhatever the ultimate resolution of these puzzles, and others that I shall discuss in this book, our discoveries in fundamental physics and astronomy over the past forty years have changed our understanding of our place in the universe in profound ways, by changing not only the questions we ask, but the very meaning of the questions we have asked. That, as I want to stress once again, is perhaps the greatest legacy of modern science, a legacy it shares with great music, great literature, and great art, and one that needs to be shared more widely.\n\n## PREFACE\n\n_Dream or nightmare, we have to live our experience as it is, and we have to live it awake. We live in a world which is penetrated through and through by science and which is both whole and real. We cannot turn it into a game simply by taking sides._\n\n\u2014JACOB BRONOWSKI\n\nIn the interests of full disclosure right at the outset I must admit that I am not sympathetic to the conviction that creation requires a creator, which is at the basis of all of the world's religions. Every day beautiful and miraculous objects suddenly appear, from snowflakes on a cold winter morning to vibrant rainbows after a late-afternoon summer shower. Yet no one but the most ardent fundamentalists would suggest that each and every such object is lovingly and painstakingly and, most important, purposefully created by a divine intelligence. In fact, many laypeople as well as scientists revel in our ability to explain how snowflakes and rainbows can spontaneously appear, based on simple, elegant laws of physics.\n\nOf course, one can ask, and many do, \"Where do the laws of physics come from?\" as well as more suggestively, \"Who created these laws?\" Even if one can answer this first query, the petitioner will then often ask, \"But where did that come from?\" or \"Who created that?\" and so on.\n\nUltimately, many thoughtful people are driven to the apparent need for First Cause, as Plato, Aquinas, or the modern Roman Catholic Church might put it, and thereby to suppose some divine being: a creator of all that there is, and all that there ever will be, someone or something eternal and everywhere.\n\nNevertheless, the declaration of a First Cause still leaves open the question, \"Who created the creator?\" After all, what is the difference between arguing in favor of an eternally existing creator versus an eternally existing universe without one?\n\nThese arguments always remind me of the famous story of an expert giving a lecture on the origins of the universe (sometimes identified as Bertrand Russell and sometimes William James), who is challenged by a woman who believes that the world is held up by a gigantic turtle, who is then held up by another turtle, and then another . . . with further turtles \"all the way down!\" An infinite regress of some creative force that begets itself, even some imagined force that is greater than turtles, doesn't get us any closer to what it is that gives rise to the universe. Nonetheless, this metaphor of an infinite regression may actually be closer to the real process by which the universe came to be than a single creator would explain.\n\nDefining away the question by arguing that the buck stops with God may seem to obviate the issue of infinite regression, but here I invoke my mantra: The universe is the way it is, whether we like it or not. The existence or nonexistence of a creator is independent of our desires. A world without God or purpose may seem harsh or pointless, but that alone doesn't require God to actually exist.\n\nSimilarly, our minds may not be able to easily comprehend infinities (although mathematics, a product of our minds, deals with them rather nicely), but that doesn't tell us that infinities don't exist. Our universe could be infinite in spatial or temporal extent. Or, as Richard Feynman once put it, the laws of physics could be like an infinitely layered onion, with new laws becoming operational as we probe new scales. _We simply don't know!_\n\nFor more than two thousand years, the question, \"Why is there something rather than nothing?\" has been presented as a challenge to the proposition that our universe\u2014which contains the vast complex of stars, galaxies, humans, and who knows what else\u2014might have arisen without design, intent, or purpose. While this is usually framed as a philosophical or religious question, it is first and foremost a question about the natural world, and so the appropriate place to try and resolve it, first and foremost, is with science.\n\nThe purpose of this book is simple. I want to show how modern science, in various guises, can address and _is_ addressing the question of why there is something rather than nothing: The answers that have been obtained\u2014from staggeringly beautiful experimental observations, as well as from the theories that underlie much of modern physics\u2014all suggest that getting something from nothing is not a problem. Indeed, something from nothing may have been _required_ for the universe to come into being. Moreover, all signs suggest that this is how our universe _could_ have arisen.\n\nI stress the word _could_ here, because we may never have enough empirical information to resolve this question unambiguously. But the fact that a universe from nothing is even plausible is certainly significant, at least to me.\n\nBefore going further, I want to devote a few words to the notion of \"nothing\"\u2014a topic that I will return to at some length later. For I have learned that, when discussing this question in public forums, nothing upsets the philosophers and theologians who disagree with me more than the notion that I, as a scientist, do not truly understand \"nothing.\" (I am tempted to retort here that theologians are experts at nothing.)\n\n\"Nothing,\" they insist, is not any of the things I discuss. Nothing is \"nonbeing,\" in some vague and ill-defined sense. This reminds me of my own efforts to define \"intelligent design\" when I first began debating with creationists, of which, it became clear, there is no clear definition, except to say what it isn't. \"Intelligent design\" is simply a unifying umbrella for opposing evolution. Similarly, some philosophers and many theologians define and redefine \"nothing\" as not being any of the versions of nothing that scientists currently describe.\n\nBut therein, in my opinion, lies the intellectual bankruptcy of much of theology and some of modern philosophy. For surely \"nothing\" is every bit as physical as \"something,\" especially if it is to be defined as the \"absence of something.\" It then behooves us to understand precisely the physical nature of both these quantities. And without science, any definition is just words.\n\nA century ago, had one described \"nothing\" as referring to purely empty space, possessing no real material entity, this might have received little argument. But the results of the past century have taught us that empty space is in fact far from the inviolate nothingness that we presupposed before we learned more about how nature works. Now, I am told by religious critics that I cannot refer to empty space as \"nothing,\" but rather as a \"quantum vacuum,\" to distinguish it from the philosopher's or theologian's idealized \"nothing.\"\n\nSo be it. But what if we are then willing to describe \"nothing\" as the absence of space and time itself? Is this sufficient? Again, I suspect it would have been . . . at one time. But, as I shall describe, we have learned that space and time can themselves spontaneously appear, so now we are told that even this \"nothing\" is not really the nothing that matters. And we're told that the escape from the \"real\" nothing requires divinity, with \"nothing\" thus defined by fiat to be \"that from which only God can create something.\"\n\nIt has also been suggested by various individuals with whom I have debated the issue that, if there is the \"potential\" to create something, then that is not a state of true nothingness. And surely having laws of nature that give such potential takes us away from the true realm of nonbeing. But then, if I argue that perhaps the laws themselves also arose spontaneously, as I shall describe might be the case, then that too is not good enough, because whatever system in which the laws may have arisen is not true nothingness.\n\nTurtles all the way down? I don't believe so. But the turtles are appealing because science is changing the playing field in ways that make people uncomfortable. Of course, that is one of the purposes of science (one might have said \"natural philosophy\" in Socratic times). Lack of comfort means we are on the threshold of new insights. Surely, invoking \"God\" to avoid difficult questions of \"how\" is merely intellectually lazy. After all, if there were no potential for creation, then God couldn't have created anything. It would be semantic hocus-pocus to assert that the potentially infinite regression is avoided because God exists outside nature and, therefore, the \"potential\" for existence itself is not a part of the nothingness from which existence arose.\n\nMy real purpose here is to demonstrate that in fact science _has_ changed the playing field, so that these abstract and useless debates about the nature of nothingness have been replaced by useful, operational efforts to describe how our universe might actually have originated. I will also explain the possible implications of this for our present and future.\n\nThis reflects a very important fact. When it comes to understanding how our universe evolves, religion and theology have been at best irrelevant. They often muddy the waters, for example, by focusing on questions of nothingness without providing any definition of the term based on empirical evidence. While we do not yet fully understand the origin of our universe, there is no reason to expect things to change in this regard. Moreover, I expect that ultimately the same will be true for our understanding of areas that religion now considers its own territory, such as human morality.\n\nScience has been effective at furthering our understanding of nature because the scientific ethos is based on three key principles: (1) follow the evidence wherever it leads; (2) if one has a theory, one needs to be willing to try to prove it wrong as much as one tries to prove that it is right; (3) the ultimate arbiter of truth is experiment, not the comfort one derives from one's a priori beliefs, nor the beauty or elegance one ascribes to one's theoretical models.\n\nThe results of experiments that I will describe here are not only timely, they are also unexpected. The tapestry that science weaves in describing the evolution of our universe is far richer and far more fascinating than any revelatory images or imaginative stories that humans have concocted. Nature comes up with surprises that far exceed those that the human imagination can generate.\n\nOver the past two decades, an exciting series of developments in cosmology, particle theory, and gravitation have completely changed the way we view the universe, with startling and profound implications for our understanding of its origins as well as its future. Nothing could therefore not be more interesting to write about, if you can forgive the pun.\n\nThe true inspiration for this book comes not so much from a desire to dispel myths or attack beliefs, as from my desire to celebrate knowledge and, along with it, the absolutely surprising and fascinating universe that ours has turned out to be.\n\nOur search will take us on a whirlwind tour to the farthest reaches of our expanding universe, from the earliest moments of the Big Bang to the far future, and will include perhaps the most surprising discovery in physics in the past century.\n\nIndeed, the immediate motivation for writing this book now is a profound discovery about the universe that has driven my own scientific research for most of the past three decades and that has resulted in the startling conclusion that most of the energy in the universe resides in some mysterious, now inexplicable form permeating all of empty space. It is not an understatement to say that this discovery has changed the playing field of modern cosmology.\n\nFor one thing, this discovery has produced remarkable new support for the idea that our universe arose from precisely nothing. It has also provoked us to rethink both a host of assumptions about the processes that might govern its evolution and, ultimately, the question of whether the very laws of nature are truly fundamental. Each of these, in its own turn, now tends to make the question of why there is something rather than nothing appear less imposing, if not completely facile, as I hope to describe.\n\nThe direct genesis of this book hearkens back to October of 2009, when I delivered a lecture in Los Angeles with the same title. Much to my surprise, the YouTube video of the lecture, made available by the Richard Dawkins Foundation, has since become something of a sensation, with nearly a million viewings as of this writing, and numerous copies of parts of it being used by both the atheist and theist communities in their debates.\n\nBecause of the clear interest in this subject, and also as a result of some of the confusing commentary on the web and in various media following my lecture, I thought it worth producing a more complete rendition of the ideas that I had expressed there in this book. Here I can also take the opportunity to add to the arguments I presented at the time, which focused almost completely on the recent revolutions in cosmology that have changed our picture of the universe, associated with the discovery of the energy and geometry of space, and which I discuss in the first two-thirds of this book.\n\nIn the intervening period, I have thought a lot more about the many antecedents and ideas constituting my argument; I've discussed it with others who reacted with a kind of enthusiasm that was infectious; and I've explored in more depth the impact of developments in particle physics, in particular, on the issue of the origin and nature of our universe. And finally, I have exposed some of my arguments to those who vehemently oppose them, and in so doing have gained some insights that have helped me develop my arguments further.\n\nWhile fleshing out the ideas I have ultimately tried to describe here, I benefitted tremendously from discussions with some of my most thoughtful physics colleagues. In particular I wanted to thank Alan Guth and Frank Wilczek for taking the time to have extended discussions and correspondence with me, resolving some confusions in my own mind and in certain cases helping reinforce my own interpretations.\n\nEmboldened by the interest of Leslie Meredith and Dominick Anfuso at Free Press, Simon & Schuster, in the possibility of a book on this subject, I then contacted my friend Christopher Hitchens, who, besides being one of the most literate and brilliant individuals I know, had himself been able to use some of the arguments from my lecture in his remarkable series of debates on science and religion. Christopher, in spite of his ill health, kindly, generously, and bravely agreed to write a foreword. For that act of friendship and trust, I will be eternally grateful. Unfortunately, Christopher's illness eventually overwhelmed him to the extent that completing the foreword became impossible, in spite of his best efforts. Nevertheless, in an embarrassment of riches, my eloquent, brilliant friend, the renowned scientist and writer Richard Dawkins, had earlier agreed to write an afterword. After my first draft was completed, he then proceeded to produce something in short order whose beauty and clarity was astounding, and at the same time humbling. I remain in awe. To Christopher, Richard, then, and all of those above, I issue my thanks for their support and encouragement, and for motivating me to once again return to my computer and write.\n\n## CHAPTER 1\n\nA COSMIC MYSTERY STORY: BEGINNINGS\n\n_The Initial Mystery that attends any journey is: how did the traveler reach his starting point in the first place?_\n\n\u2014LOUISE BOGAN, _Journey Around My Room_\n\n_It was a dark and stormy night._\n\nEarly in 1916, Albert Einstein had just completed his greatest life's work, a decade-long, intense intellectual struggle to derive a new theory of gravity, which he called the general theory of relativity. This was not just a new theory of gravity, however; it was a new theory of space and time as well. And it was the first scientific theory that could explain not merely how objects move through the universe, but also how the universe itself might evolve.\n\nThere was just one hitch, however. When Einstein began to apply his theory to describing the universe as a whole, it became clear that the theory didn't describe the universe in which we apparently lived.\n\nNow, almost one hundred years later, it is difficult to fully appreciate how much our picture of the universe has changed in the span of a single human lifetime. As far as the scientific community in 1917 was concerned, the universe was static and eternal, and consisted of a single galaxy, our Milky Way, surrounded by a vast, infinite, dark, and empty space. This is, after all, what you would guess by looking up at the night sky with your eyes, or with a small telescope, and at the time there was little reason to suspect otherwise.\n\nIn Einstein's theory, as in Newton's theory of gravity before it, gravity is a purely attractive force between all objects. This means that it is impossible to have a set of masses located in space at rest forever. Their mutual gravitational attraction will ultimately cause them to collapse inward, in manifest disagreement with an apparently static universe.\n\nThe fact that Einstein's general relativity didn't appear consistent with the then picture of the universe was a bigger blow to him than you might imagine, for reasons that allow me to dispense with a myth about Einstein and general relativity that has always bothered me. It is commonly assumed that Einstein worked in isolation in a closed room for years, using pure thought and reason, and came up with his beautiful theory, independent of reality (perhaps like some string theorists nowadays!). However, nothing could be further from the truth.\n\nEinstein was always guided deeply by experiments and observations. While he performed many \"thought experiments\" in his mind and did toil for over a decade, he learned new mathematics and followed many false theoretical leads in the process before he ultimately produced a theory that was indeed mathematically beautiful. The single most important moment in establishing his love affair with general relativity, however, had to do with observation. During the final hectic weeks that he was completing his theory, competing with the German mathematician David Hilbert, he used his equations to calculate the prediction for what otherwise might seem an obscure astrophysical result: a slight precession in the \"perihelion\" (the point of closest approach) of Mercury's orbit around the Sun.\n\nAstronomers had long noted that the orbit of Mercury departed slightly from that predicted by Newton. Instead of being a perfect ellipse that returned to itself, the orbit of Mercury precessed (which means that the planet does not return precisely to the same point after one orbit, but the orientation of the ellipse shifts slightly each orbit, ultimately tracing out a kind of spiral-like pattern) by an incredibly small amount: 43 arc seconds (about 1\/ **100** of a degree) per century.\n\nWhen Einstein performed his calculation of the orbit using his theory of general relativity, the number came out just right. As described by an Einstein biographer, Abraham Pais: \"This discovery was, I believe, by far the strongest emotional experience in Einstein's scientific life, perhaps in all his life.\" He claimed to have heart palpitations, as if \"something had snapped\" inside. A month later, when he described his theory to a friend as one of \"incomparable beauty,\" his pleasure over the mathematical form was indeed manifest, but no palpitations were reported.\n\nThe apparent disagreement between general relativity and observation regarding the possibility of a static universe did not last long, however. (Even though it did cause Einstein to introduce a modification to his theory that he later called his biggest blunder. But more about that later.) Everyone (with the exception of certain school boards in the United States) now knows that the universe is not static but is expanding and that the expansion began in an incredibly hot, dense Big Bang approximately 13.72 billion years ago. Equally important, we know that our galaxy is merely one of perhaps 400 billion galaxies in the observable universe. We are like the early terrestrial mapmakers, just beginning to fully map the universe on its largest scales. Little wonder that recent decades have witnessed revolutionary changes in our picture of the universe.\n\nThe discovery that the universe is not static, but rather expanding, has profound philosophical and religious significance, because it suggested that our universe had a beginning. A beginning implies creation, and creation stirs emotions. While it took several decades following the discovery in 1929 of our expanding universe for the notion of a Big Bang to achieve independent empirical confirmation, Pope Pius XII heralded it in 1951 as evidence for Genesis. As he put it:\n\nIt would seem that present-day science, with one sweep back across the centuries, has succeeded in bearing witness to the august instant of the primordial Fiat Lux [Let there be Light], when along with matter, there burst forth from nothing a sea of light and radiation, and the elements split and churned and formed into millions of galaxies. Thus, with that concreteness which is characteristic of physical proofs, [science] has confirmed the contingency of the universe and also the well-founded deduction as to the epoch when the world came forth from the hands of the Creator. Hence, creation took place. We say: \"Therefore, there is a Creator. Therefore, God exists!\"\n\nThe full story is actually a little more interesting. In fact, the first person to propose a Big Bang was a Belgian priest and physicist named Georges Lema\u00eetre. Lema\u00eetre was a remarkable combination of proficiencies. He started his studies as an engineer, was a decorated artilleryman in World War I, and then switched to mathematics while studying for the priesthood in the early 1920s. He then moved on to cosmology, studying first with the famous British astrophysicist Sir Arthur Stanley Eddington before moving on to Harvard and eventually receiving a second doctorate, in physics from MIT.\n\nIn 1927, before receiving his second doctorate, Lema\u00eetre had actually solved Einstein's equations for general relativity and demonstrated that the theory predicts a nonstatic universe and in fact suggests that the universe we live in is expanding. The notion seemed so outrageous that Einstein himself colorfully objected with the statement \"Your math is correct, but your physics is abominable.\"\n\nNevertheless, Lema\u00eetre powered onward, and in 1930 he further proposed that our expanding universe actually began as an infinitesimal point, which he called the \"Primeval Atom\" and that this beginning represented, in an allusion to Genesis perhaps, a \"Day with No Yesterday.\"\n\nThus, the Big Bang, which Pope Pius so heralded, had first been proposed by a priest. One might have thought that Lema\u00eetre would have been thrilled with this papal validation, but he had already dispensed in his own mind with the notion that this scientific theory had theological consequences and had ultimately removed a paragraph in the draft of his 1931 paper on the Big Bang remarking on this issue.\n\nLema\u00eetre in fact later voiced his objection to the pope's 1951 claimed proof of Genesis via the Big Bang (not least because he realized that if his theory was later proved incorrect, then the Roman Catholic claims for Genesis might be contested). By this time, he had been elected to the Vatican's Pontifical Academy, later becoming its president. As he put it, \"As far as I can see, such a theory remains entirely outside of any metaphysical or religious question.\" The pope never again brought up the topic in public.\n\nThere is a valuable lesson here. As Lema\u00eetre recognized, whether or not the Big Bang really happened is a scientific question, not a theological one. Moreover, even if the Big Bang had happened (which all evidence now overwhelmingly supports), one could choose to interpret it in different ways depending upon one's religious or metaphysical predilections. You can choose to view the Big Bang as suggestive of a creator if you feel the need or instead argue that the mathematics of general relativity explain the evolution of the universe right back to its beginning without the intervention of any deity. But such a metaphysical speculation is independent of the physical validity of the Big Bang itself and is irrelevant to our understanding of it. Of course, as we go beyond the mere existence of an expanding universe to understand the physical principles that may address its origin, science can shed further light on this speculation and, as I shall argue, it does.\n\nIn any case, neither Lema\u00eetre nor Pope Pius convinced the scientific world that the universe was expanding. Rather, as in all good science, the evidence came from careful observations, in this case done by Edwin Hubble, who continues to give me great faith in humanity, because he started out as a lawyer and then became an astronomer.\n\nHubble had earlier made a significant breakthrough in 1925 with the new Mount Wilson 100-inch Hooker telescope, then the world's largest. (For comparison, we are now building telescopes more than ten times bigger than this in diameter and one hundred times bigger in area!) Up until that time, with the telescopes then available, astronomers were able to discern fuzzy images of objects that were not simple stars in our galaxy. They called these nebulae, which is basically Latin for \"fuzzy thing\" (actually \"cloud\"). They also debated whether these objects were in our galaxy or outside of it.\n\nSince the prevailing view of the universe at the time was that our galaxy was all that there was, most astronomers fell in the \"in our galaxy\" camp, led by the famous astronomer Harlow Shapley at Harvard. Shapley had dropped out of school in fifth grade and studied on his own, eventually going to Princeton. He decided to study astronomy by picking the first subject he found in the syllabus to study. In seminal work he demonstrated that the Milky Way was much larger than previously thought and that the Sun was not at its center but simply in a remote, uninteresting corner. He was a formidable force in astronomy and therefore his views on the nature of nebulae held considerable sway.\n\nOn New Year's Day 1925, Hubble published the results of his two-year study of so-called spiral nebulae, where he was able to identify a certain type of variable star, called a Cepheid variable star, in these nebulae, including the nebula now known as Andromeda.\n\nFirst observed in 1784, Cepheid variable stars are stars whose brightness varies over some regular period. In 1908, an unheralded and at the time unappreciated would-be astronomer, Henrietta Swan Leavitt, was employed as a \"computer\" at the Harvard College Observatory. (\"Computers\" were women brought in to catalogue the brightness of stars recorded on the observatory's photographic plates; women were not allowed to use the observatory telescopes at the time.) Daughter of a Congregational minister and a descendant of the Pilgrims, Leavitt made an astounding discovery, which she further illuminated in 1912: she noticed that there was a regular relationship between the brightness of Cepheid stars and the period of their variation. Therefore, if one could determine the distance to a single Cepheid of a known period (subsequently determined in 1913), then measuring the brightness of other Cepheids of the same period would allow one to determine the distance to these other stars!\n\nSince the observed brightness of stars goes down inversely with the square of the distance to the star (the light spreads out uniformly over a sphere whose area increases as the square of the distance, and thus since the light is spread out over a bigger sphere, the intensity of the light observed at any point decreases inversely with the area of the sphere), determining the distance to faraway stars has always been the major challenge in astronomy. Leavitt's discovery revolutionized the field. (Hubble himself, who was snubbed for the Nobel Prize, often said Leavitt's work deserved the prize, although he was sufficiently self-serving that he might have suggested it only because he would have been a natural contender to share the prize with her for his later work.) Paperwork had actually begun in the Royal Swedish Academy to nominate Leavitt for the Nobel in 1924 when it was learned that she had died of cancer three years earlier. By dint of his force of personality, knack for self-promotion, and skill as an observer, Hubble would become a household name, while Leavitt, alas, is known only to aficionados of the field.\n\nHubble was able to use his measurement of Cepheids and Leavitt's period-luminosity relation to prove definitively that the Cepheids in Andromeda and several other nebulae were much too distant to be inside the Milky Way. Andromeda was discovered to be another island universe, another spiral galaxy almost identical to our own, and one of the more than 100 billion other galaxies that, we now know, exist in our observable universe. Hubble's result was sufficiently unambiguous that the astronomical community\u2014including Shapley, who, incidentally, by this time had become director of the Harvard College Observatory, where Leavitt had done her groundbreaking work\u2014quickly accepted the fact that the Milky Way is not all there is around us. Suddenly the size of the known universe had expanded in a single leap by a greater amount than it had in centuries! Its character had changed, too, as had almost everything else.\n\nAfter this dramatic discovery, Hubble could have rested on his laurels, but he was after bigger fish or, in this case, bigger galaxies. By measuring ever fainter Cepheids in ever more distant galaxies, he was able to map the universe out to ever-larger scales. When he did, however, he discovered something else that was even more remarkable: the universe is expanding!\n\nHubble achieved his result by comparing the distances for the galaxies he measured with a different set of measurements from another American astronomer, Vesto Slipher, who had measured the spectra of light coming from these galaxies. Understanding the existence and nature of such spectra requires me to take you back to the very beginning of modern astronomy.\n\nOne of the most important discoveries in astronomy was that star stuff and Earth stuff are largely the same. It all began, as did many things in modern science, with Isaac Newton. In 1665, Newton, then a young scientist, allowed a thin beam of sunlight, obtained by darkening his room except for a small hole he made in his window shutter, through a prism and saw the sunlight disperse into the familiar colors of the rainbow. He reasoned that white light from the sun contained all of these colors, and he was correct.\n\nA hundred fifty years later, another scientist examined the dispersed light more carefully, discovered dark bands amidst the colors, and reasoned that these were due to the existence of materials in the outer atmosphere of the sun that were absorbing light of certain specific colors or wavelengths. These \"absorption lines,\" as they became known, could be identified with wavelengths of light that were measured to be absorbed by known materials on Earth, including hydrogen, oxygen, iron, sodium, and calcium.\n\nIn 1868, another scientist observed two new absorption lines in the yellow part of the solar spectrum that didn't correspond to any known element on Earth. He decided this must be due to some new element, which he called helium. A generation later, helium was discovered on Earth.\n\nLooking at the spectrum of radiation coming from other stars is an important scientific tool for understanding their composition, temperature, and evolution. Starting in 1912, Slipher observed the spectra of light coming from various spiral nebulae and found that the spectra were similar to those of nearby stars\u2014except that all of the absorption lines were shifted by the same amount in wavelength.\n\nThis phenomenon was by then understood as being due to the familiar \"Doppler effect,\" named after the Austrian physicist Christian Doppler, who explained in 1842 that waves coming at you from a moving source will be stretched if the source is moving away from you and compressed if it is moving toward you. This is a manifestation of a phenomenon we are all familiar with, and by which I am usually reminded of a Sidney Harris cartoon where two cowboys sitting on their horses out in the plains are looking at a distant train, and one says to the other, \"I love hearing that lonesome wail of the train whistle as the magnitude of the frequency changes due to the Doppler effect!\" Indeed, a train whistle or an ambulance siren sounds higher if the train or ambulance is moving toward you and lower if it is moving away from you.\n\nIt turns out that the same phenomenon occurs for light waves as sound waves, although for somewhat different reasons. Light waves from a source moving away from you, either due to its local motion in space or due to the intervening expansion of space, will be stretched, and therefore appear redder than they would otherwise be, since red is the long-wavelength end of the visible spectrum, while waves from a source moving toward you will be compressed and appear bluer.\n\nSlipher observed in 1912 that the absorption lines from the light coming from all the spiral nebulae were almost all shifted systematically toward longer wavelengths (although some, like Andromeda, were shifted toward shorter wavelengths). He correctly inferred that most of these objects therefore were moving away from us with considerable velocities.\n\nHubble was able to compare his observations of the distance of these spiral galaxies (as they were by now known to be) with Slipher's measurements of the velocities by which they were moving away. In 1929, with the help of a Mount Wilson staff member, Milton Humason (whose technical talent was such that he had secured a job at Mount Wilson without even having a high school diploma), he announced the discovery of a remarkable empirical relationship, now called Hubble's law: There is a linear relationship between recessional velocity and galaxy distance. Namely, galaxies that are ever more distant are moving away from us with faster velocities!\n\nWhen first presented with this remarkable fact\u2014that almost all galaxies are moving away from us, and those that are twice as far away are moving twice as fast, those that are three times away three times as fast, etc.\u2014it seems obvious what this implies: _We are the center of the universe!_\n\nAs some friends suggest, I need to be reminded on a daily basis that _this is not the case_. Rather, it was consistent with precisely the relationship that Lema\u00eetre had predicted. Our universe is indeed expanding.\n\nI have tried various ways to explain this, and I frankly don't think there is a good way to do it unless you think outside the box\u2014in this case, outside the universal box. To see what Hubble's law implies, you need to remove yourself from the myopic vantage point of our galaxy and look at our universe from the outside. While it is hard to stand outside a three-dimensional universe, it is easy to stand outside a two-dimensional one. On the next page I have drawn one such expanding universe at two different times. As you can see, the galaxies are farther apart at the second time.\n\nNow imagine that you are living in one of the galaxies at the second time, t2 which I shall mark in white, at time t2.\n\nTo see what the evolution of the universe would look like from this galaxy's vantage point, I simply superimpose the right image on the left, placing the galaxy in white on top of itself.\n\nVoil\u00e0! From this galaxy's vantage point every other galaxy is moving away, and those that are twice as far have moved twice the distance in the same time, those that are three times as far away have moved three times the distance, etc. As long as there is no edge, those on the galaxy feel as if they are at the center of the expansion.\n\nIt doesn't matter what galaxy one chooses. Pick another galaxy, and repeat:\n\nDepending upon your perspective, then, either _every place_ is the center of the universe, or _no place_ is. It doesn't matter; Hubble's law is consistent with a universe that is expanding.\n\nNow, when Hubble and Humason first reported their analysis in 1929, they not only reported a linear relationship between distance and recession velocity, but also gave a quantitative estimate of the expansion rate itself. Here are the actual data presented at the time:\n\nAs you can see, Hubble's guess of fitting a straight line to this data set seems a relatively lucky one. (There is clearly some relationship, but whether a straight line is the best fit is far from clear on the basis of this data alone.) The number for the expansion rate they obtained, derived for the plot, suggested that a galaxy a million parsecs away (3 million light-years)\u2014the average separation between galaxies\u2014is moving away from us with a speed of 500 kilometers\/second. This estimate was not so lucky, however.\n\nThe reason for this is relatively simple to see. If everything is moving apart today, then at earlier times they were closer together. Now, if gravity is an attractive force, then it should be slowing the expansion of the universe. This means the galaxy we see moving away from us at 500 kilometers\/second today would have been moving faster earlier.\n\nIf for the moment, though, we just assume that the galaxy had always been carried away with that velocity, we can work backward and figure out how long ago it would have been at the same position as our galaxy. Since galaxies twice as far away are moving twice as fast, if we work backward we find out that they were superimposed on our position at exactly the same time. Indeed, the entire observable universe would have been superimposed at a single point, the Big Bang, at a time that we can estimate in this way.\n\nSuch an estimate is clearly an upper limit on the age of the universe, because, if the galaxies were once moving faster, they would have gotten where they are today in less time than this estimate would suggest.\n\nFrom this estimate based on Hubble's analysis, the Big Bang happened approximately 1.5 billion years ago. Even in 1929, however, the evidence was already clear (except to some scriptural literalists in Tennessee, Ohio, and a few other states) that the Earth was older than 3 billion years old.\n\nNow, it is embarrassing for scientists to find that the Earth is older than the universe. More important, it suggests something is wrong with the analysis.\n\nThe source of this confusion was simply the fact that Hubble's distance estimates, derived using the Cepheid relations in our galaxy, were systematically incorrect. The distance ladder based on using nearby Cepheids to estimate the distance of farther away Cepheids, and then to estimate the distance to galaxies in which yet more distant Cepheids were observed, was flawed.\n\nThe history of how these systematic effects have been overcome is too long and convoluted to describe here and, in any case, no longer matters because we now have a much better distance estimator.\n\nOne of my favorite Hubble Space Telescope photographs is shown below:\n\nIt shows a beautiful spiral galaxy far far away, long long ago (long long ago because the light from the galaxy takes some time\u2014more than 50 million years\u2014to reach us). A spiral galaxy such as this, which resembles our own, has about 100 billion stars within it. The bright core at its center contains perhaps 10 billion stars. Notice the star on the lower left corner that is shining with a brightness almost equal to these 10 billion stars. On first sighting it, you might reasonably assume that this is a much closer star in our own galaxy that got in the way of the picture. But in fact, it is a star in that same distant galaxy, more than 50 million light-years away.\n\nClearly, this is no ordinary star. It is a star that has just exploded, a supernova, one of the brightest fireworks displays in the universe. When a star explodes, it briefly (over the course of about a month or so) shines in visible light with a brightness of 10 billion stars.\n\nHappily for us, stars don't explode that often, about once per hundred years per galaxy. But we are lucky that they do, because if they didn't, we wouldn't be here. One of the most poetic facts I know about the universe is that essentially every atom in your body was once inside a star that exploded. Moreover, the atoms in your left hand probably came from a different star than did those in your right. We are all, literally, star children, and our bodies made of stardust.\n\nHow do we know this? Well, we can extrapolate our picture of the Big Bang back to a time when the universe was about 1 second old, and we calculate that all observed matter was compressed in a dense plasma whose temperature should have been about 10 billion degrees (Kelvin scale). At this temperature nuclear reactions can readily take place between protons and neutrons as they bind together and then break apart from further collisions. Following this process as the universe cools, we can predict how frequently these primeval nuclear constituents will bind to form the nuclei of atoms heavier than hydrogen (i.e., helium, lithium, and so on).\n\nWhen we do so, we find that essentially no nuclei\u2014beyond lithium, the third lightest nucleus in nature\u2014formed during the primeval fireball that was the Big Bang. We are confident that our calculations are correct because our predictions for the cosmic abundances of the lightest elements agree bang-on with these observations. The abundances of these lightest elements\u2014hydrogen, deuterium (the nucleus of heavy hydrogen), helium, and lithium\u2014vary by 10 orders of magnitude (roughly 25 percent of the protons and neutrons, by mass, end up in helium, while 1 in every 10 billion neutrons and protons ends up within a lithium nucleus). Over this incredible range, observations and theoretical predictions agree.\n\nThis is one of the most famous, significant, and successful predictions telling us the Big Bang really happened. _Only a hot Big Bang can produce the observed abundance of light elements and maintain consistency with the current observed expansion of the universe._ I carry a wallet card in my back pocket showing the comparison of the predictions of the abundance of light elements and the observed abundance so that, each time I meet someone who doesn't believe that the Big Bang happened, I can show it to them. I usually never get that far in my discussion, of course, because data rarely impress people who have decided in advance that something is wrong with the picture. I carry the card anyway and reproduce it for you later in the book.\n\nWhile lithium is important for some people, far more important to the rest of us are all the heavier nuclei like carbon, nitrogen, oxygen, iron, and so on. These were _not_ made in the Big Bang. The only place they can be made is in the fiery cores of stars. And the only way they could get into your body today is if these stars were kind enough to have exploded, spewing their products into the cosmos so they could one day coalesce in and around a small blue planet located near the star we call the Sun. Over the course of the history of our galaxy, about 200 million stars have exploded. These myriad stars sacrificed themselves, if you wish, so that one day you could be born. I suppose that qualifies them as much as anything else for the role of saviors.\n\nIt turns out a certain type of exploding star, called a Type Ia supernova, has been shown by careful studies performed over the 1990s to have a remarkable property: with high accuracy, those Type Ia supernovae that are intrinsically brighter also shine longer. The correlation, while not fully understood theoretically, is empirically very tight. This means that these supernovae are very good \"standard candles.\" By this we mean that these supernovae can be used to calibrate distances because their intrinsic brightness can be directly ascertained by a measurement that is independent of their distance. If we observe a supernova in a distant galaxy\u2014and we can because they are very bright\u2014then by observing how long it shines, we can infer its intrinsic brightness. Then, by measuring its apparent brightness with our telescopes, we can accurately infer just how far away the supernova and its host galaxy are. Then, by measuring the \"redshift\" of the light from the stars in the galaxy, we can determine its velocity, and thus can compare velocity with distance and infer the expansion rate of the universe.\n\nSo far so good, but if supernovae explode only once every hundred years or so per galaxy, how likely are we ever to be able to see one? After all, the last supernova in our own galaxy witnessed on Earth was seen by Johannes Kepler in 1604! Indeed, it is said that supernovae in our galaxy are observed only during the lifetimes of the greatest astronomers, and Kepler certainly fits the bill.\n\nStarting out as a humble mathematics teacher in Austria, Kepler became assistant to the astronomer Tycho Brahe (who himself had observed an earlier supernova in our galaxy and was given an entire island by the king of Denmark in return), and using Brahe's data on planetary positions in the sky taken over more than a decade, Kepler derived his famous three laws of planetary motion early in the seventeenth century:\n\n1. Planets move around the Sun in ellipses.\n\n2. A _line_ connecting a planet and the Sun sweeps out equal _areas_ during equal intervals of time.\n\n3. The _square_ of the _orbital period_ of a planet is directly _proportional_ to the _cube_ (3rd power) of the _semi-major axis_ of its orbit (or, in other words, of the \"semi-major axis\" of the ellipse, half of the distance across the widest part of the ellipse).\n\nThese laws in turn lay the basis for Newton's derivation of the universal law of gravity almost a century later. Besides this remarkable contribution, Kepler successfully defended his mother in a witchcraft trial and wrote what was perhaps the first science fiction story, about a journey to the moon.\n\nNowadays, one way to see a supernova is simply to assign a different graduate student to each galaxy in the sky. After all, one hundred years is not too different, in a cosmic sense at least, from the average time to do a PhD, and graduate students are cheap and abundant. Happily, however, we don't have to resort to such extreme measures, for a very simple reason: the universe is big and old and, as a result, rare events happen all the time.\n\nGo out some night into the woods or desert where you can see stars and hold up your hand to the sky, making a tiny circle between your thumb and forefinger about the size of a dime. Hold it up to a dark patch of the sky where there are no visible stars. In that dark patch, with a large enough telescope of the type we now have in service today, you could discern perhaps 100,000 galaxies, each containing billions of stars. Since supernovae explode once per hundred years per, with 100,000 galaxies in view, you should expect to see, on average, about three stars explode on a given night.\n\nAstronomers do just this. They apply for telescope time, and some nights they might see one star explode, some nights two, and some nights it might be cloudy and they might not see any. In this way several groups have been able to determine Hubble's constant with an uncertainty of less than 10 percent. The new number, about 70 kilometers per second for galaxies on average of 3 million light-years apart, is almost a factor of 10 smaller than that derived by Hubble and Humason. As a result, we infer an age of the universe of closer to 13 billion years, rather than 1.5 billion years.\n\nAs I shall describe later, this too is in complete agreement with independent estimates of the age of the oldest stars in our galaxy. From Brahe to Kepler, from Lema\u00eetre to Einstein and Hubble, and from the spectra of stars to the abundance of light elements, four hundred years of modern science have produced a remarkable and consistent picture of the expanding universe. Everything holds together. The Big Bang picture is in good shape.\n\n## CHAPTER 2\n\nA COSMIC MYSTERY STORY: WEIGHING THE UNIVERSE\n\n_There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we know we don't know. But there are also unknown unknowns. There are things we don't know we don't know._\n\n\u2014DONALD RUMSFELD\n\nHaving established that the universe had a beginning, and that that beginning was a finite and measurable time in the past, a natural next question to ask is, \"How will it end?\"\n\nIn fact, this was the very question that led me to move from my home territory, particle physics, into cosmology. During the 1970s and 1980s, it became increasingly clear from detailed measurements of the motion of stars and gas in our galaxy, as well as from the motion of galaxies in large groups of galaxies called clusters, that there was much more to the universe than meets either the eye or the telescope.\n\nGravity is the chief force operating on the enormous scale of galaxies, so measuring the motion of objects on these scales allows us to probe the gravitational attraction that drives this motion. Such measurements took off with the pioneering work of the American astronomer Vera Rubin and her colleagues in the early 1970s. Rubin had graduated with her doctorate from Georgetown after taking night classes while her husband waited in the car because she didn't know how to drive. She had applied to Princeton, but that university didn't accept women into their graduate astronomy program until 1975. Rubin rose to become only the second woman ever to be awarded the Gold Medal of the Royal Astronomical Society. That prize and her many other well-deserved honors stemmed from her groundbreaking measurements of the rotation rate of our galaxy. By observing stars and hot gas that were ever-farther from the center of our galaxy, Rubin determined that these regions were moving much faster than they should have been if the gravitational force driving their movement was due to the mass of all the observed objects within the galaxy. Due to her work, it eventually became clear to cosmologists that the only way to explain this motion was to posit the existence of significantly more mass in our galaxy than one could account for by adding up the mass of _all_ of this hot gas and stars.\n\nThere was a problem, however, with this view. The very same calculations that so beautifully explain the observed abundance of the light elements (hydrogen, helium, and lithium) in the universe also tell us more or less how many protons and neutrons, the stuff of normal matter, must exist in the universe. This is because, like any cooking recipe\u2014in this case nuclear cooking\u2014the amount of your final product depends upon how much of each ingredient you start out with. If you double the recipe\u2014four eggs instead of two, for example\u2014you get more of the end product, in this case an omelet. Yet the initial density of protons and neutrons in the universe arising out of the Big Bang, as determined by fitting to the observed abundance of hydrogen, helium, and lithium, accounts for about twice the amount of material we can see in stars and hot gas. Where are those particles?\n\nIt is easy to imagine ways to hide protons and neutrons (snowballs, planets, cosmologists . . . none of them shines), so many physicists predicted that as many protons and neutrons lie in dark objects as visible objects. However, when we add up how much \"dark matter\" has to exist to explain the motion of material in our galaxy, we find that the ratio of total matter to visible matter is not 2 to 1, but closer to 10 to 1. If this is not a mistake, then the dark matter cannot be made of protons and neutrons. There are just not enough of them.\n\nAs a young elementary particle physicist in the early 1980s, learning of this possibility of the existence of exotic dark matter was extremely exciting to me. It implied, literally, that the dominant particles in the universe were not good old-fashioned garden-variety neutrons and protons, but possibly some new kind of elementary particle, something that didn't exist on Earth today, but something mysterious that flowed between and amidst the stars and silently ran the whole gravitational show we call a galaxy.\n\nEven more exciting, at least for me, this implied three new lines of research that could fundamentally reilluminate the nature of reality.\n\n1. If these particles were created in the Big Bang, like the light elements I have described, then we should be able to use ideas about the forces that govern the interactions of elementary particles (instead of the interactions of nuclei relevant to determine elemental abundance) to estimate the abundance of possible exotic new particles in the universe today.\n\n2. It might be possible to derive the total abundance of dark matter in the universe on the basis of theoretical ideas in particle physics, or it might be possible to propose new experiments to detect dark matter\u2014either of which could tell us how much total matter there is and hence what the geometry of our universe is. The job of physics is not to invent things we cannot see to explain things we can see, but to figure out how to see what we cannot see\u2014to see what was previously invisible, the known unknowns. Each new elementary particle candidate for dark matter suggests new possibilities for experiments to detect directly the dark matter particles parading throughout the galaxy by building devices on Earth to detect them as the Earth intercepts their motion through space. Instead of using telescopes to search for faraway objects, if the dark matter particles are in diffuse bunches permeating the entire galaxy, they are here with us now, and terrestrial detectors might reveal their presence.\n\n3. If we could determine the nature of the dark matter, and its abundance, we might be able to determine how the universe will end.\n\nThis last possibility seemed the most exciting of all, so I will begin with it. Indeed, I got involved in cosmology because I wanted to be the first person to know how the universe would end.\n\nIt seemed like a good idea at the time.\n\nWhen Einstein developed his theory of general relativity, at its heart was the possibility that space could curve in the presence of matter or energy. This theoretical idea became more than mere speculation in 1919 when two expeditions observed starlight curving around the Sun during a solar eclipse in precisely the degree to which Einstein had predicted should happen if the presence of the Sun curved the space around it. Einstein almost instantly became famous and a household name. (Most people today think it was the equation E = mc2, which came fifteen years earlier, that did it, but it wasn't.)\n\nNow, if space is potentially curved, then the geometry of our whole universe suddenly becomes a lot more interesting. Depending upon the total amount of matter in our universe, it could exist in one of three different types of geometries, so-called _open, closed,_ or _flat_.\n\nIt is hard to envisage what a curved three-dimensional space might actually look like. Since we are three-dimensional beings, we can no more easily intuitively picture a curved three-dimensional space than the two-dimensional beings in the famous book _Flatland_ could imagine what their world would look like to a three-dimensional observer if it were curved like the surface of a sphere. Moreover, if the curvature is very small, then it is hard to imagine how one might actually detect it in everyday life, just as, during the Middle Ages at least, many people felt the Earth must be flat because from their perspective it looked flat.\n\nCurved three-dimensional universes are difficult to picture\u2014a closed universe is like a three-dimensional sphere, which sounds pretty intimidating\u2014but some aspects are easy to describe. If you looked far enough in one direction in a closed universe, you would see the back of your head.\n\nWhile these exotic geometries may seem amusing or impressive to talk about, operationally there is a much more important consequence of their existence. General relativity tells us unambiguously that a closed universe whose energy density is dominated by matter like stars and galaxies, and even more exotic dark matter, _must_ one day recollapse in a process like the reverse of a Big Bang\u2014a Big _Crunch,_ if you will. An open universe will continue to expand forever at a finite rate, and a flat universe is just at the boundary, slowing down, but never quite stopping.\n\nDetermining the amount of dark matter, and thus the total density of mass in the universe, therefore promised to reveal the answer to the age-old question (at least as old as T. S. Eliot anyway): Will the universe end with a bang or a whimper? The saga of determining the total abundance of dark matter goes back at least a half century, and one could write a whole book about it, which in fact I have already done, in my book _Quintessence_. However, in this case, as I shall now demonstrate (with both words _and_ then a picture), it is true that a single picture is worth at least a thousand (or perhaps a hundred thousand) words.\n\nThe largest gravitationally bound objects in the universe are called _superclusters_ of galaxies. Such objects can contain thousands of individual galaxies or more and can stretch across tens of millions of light-years. Most galaxies exist in such superclusters, and indeed our own galaxy is located within the Virgo supercluster of galaxies, whose center is almost 60 million light-years away from us.\n\nSince superclusters are so large and so massive, basically anything that falls into anything will fall into clusters. So if we could weigh superclusters of galaxies and then estimate the total density of such superclusters in the universe, we could then \"weigh the universe,\" including all the dark matter. Then, using the equations of general relativity, we could determine whether there is enough matter to close the universe or not.\n\nSo far so good, but how can we weigh objects that are tens of millions of light-years across? Simple. Use gravity.\n\nIn 1936, Albert Einstein, following the urgings of an amateur astronomer, Rudi Mandl, published a short paper in the magazine _Science_ titled \"Lens-Like Action of a Star by the Deviation of Light in the Gravitational Field.\" In this brief note Einstein demonstrated the remarkable fact that space itself could act like a lens, bending light and magnifying it, just like the lenses in my own reading glasses.\n\nIt was a kindlier, gentler time in 1936, and it is interesting to read the informal beginning of Einstein's paper, which after all was published in a distinguished scientific journal: \"Some time ago, R. W. Mandl paid me a visit and asked me to publish the results of a little calculation, which I had made at his request. This note complies with his wish.\" Perhaps this informality was accorded to him because he was Einstein, but I prefer to suppose that it was a product of the era, when scientific results were not yet always couched in language removed from common parlance.\n\nIn any case, the fact that light followed curved trajectories if space itself curved in the presence of matter was the first significant new prediction of general relativity and the discovery that led to Einstein's international fame, as I have mentioned. So it is perhaps not that surprising (as was recently discovered) that in 1912, well before Einstein had in fact even completed his general relativity theory, he had performed calculations\u2014as he tried to find some observable phenomenon that would convince astronomers to test his ideas\u2014that were essentially identical to those he published in 1936 at the request of Mr. Mandl. Perhaps because he reached the same conclusion in 1912 that he stated in his 1936 paper, namely \"there is no great chance of observing this phenomenon,\" he never bothered to publish his earlier work. In fact, after examining his notebooks for both periods, we can't say for sure that he later even remembered having done the original calculations twenty-four years before.\n\nWhat Einstein did recognize on both occasions is that the bending of light in a gravitational field could mean that, if a bright object was located well behind an intervening distribution of mass, light rays going out in various directions could bend around the intervening distribution and converge again, just as they do when they traverse a normal lens, producing either a magnification of the original object or the production of numerous image copies of the original object, some of which might be distorted (see figure below).\n\nWhen he calculated the predicted effects for lensing of a distant star by an intervening star in the foreground, the effect was so small that it appeared absolutely unmeasurable, which led him to make the remark mentioned above\u2014that it was unlikely that such a phenomenon could ever be observed. As a result, Einstein figured that his paper had little practical value. As he put it in his covering letter to the editor of _Science_ at the time: \"Let me also thank you for your cooperation with the little publication, which Mister Mandl squeezed out of me. It is of little value, but it makes the poor guy happy.\"\n\nEinstein was not an astronomer, however, and it would take one to realize that the effect Einstein had predicted might be not only measurable, but also useful. Its usefulness came from applying it to the lensing of distant objects by much larger systems such as galaxies or even clusters of galaxies, not to the lensing of stars by stars. Within months of Einstein's publication, the brilliant Caltech astronomer Fritz Zwicky submitted a paper to the _Physical Review_ in which he demonstrated the practicality of precisely this possibility (and also indirectly put down Einstein for his ignorance regarding the possible effect of lensing by galaxies rather than stars).\n\nZwicky was an irascible character and way ahead of his time. As early as 1933 he had analyzed the relative motion of galaxies in the Coma cluster and determined, using Newton's laws of motion, that the galaxies were moving so fast that they should have flown apart, destroying the cluster, unless there was far more mass in the cluster, by a factor more than 100, than could be accounted for by the stars alone. He thus should properly be considered as having discovered dark matter, though at the time his inference was so remarkable that most astronomers probably felt there might be some other less exotic explanation for the result he got.\n\nZwicky's one-page paper in 1937 was equally remarkable. He proposed three different uses for gravitational lensing: (1) testing general relativity, (2) using intervening galaxies as a kind of telescope to magnify more distant objects that would otherwise be invisible to telescopes on earth, and, most important, (3) resolving the mystery of why clusters appear to weigh more than can be accounted for by visible matter: _\"Observations on the deflection of light around nebulae may provide the most direct determination of nebular masses and clear up the above-mentioned discrepancy.\"_\n\nZwicky's paper is now seventy-four years old but reads instead like a modern proposal for using gravitational lensing to probe the universe. Indeed, each and every suggestion he made has come to pass, and the final one is the most significant of all. Gravitational lensing of distant quasars by intervening galaxies was first observed in 1987, and in 1998, sixty-one years after Zwicky proposed weighing nebulae using gravitational lensing, the mass of a large cluster was determined by using gravitational lensing.\n\nIn that year, physicist Tony Tyson and colleagues at the now defunct Bell Laboratories (which had such a noble and Nobel tradition of great science, from the invention of the transistor to the discovery of the cosmic microwave background radiation) observed a distant large cluster, colorfully labeled CL 0024 + 1654, located about 5 billion light-years away. In this beautiful image from the Hubble Space Telescope, a spectacular example of the multiple image of a distant galaxy located another 5 billion light-years behind the cluster can be seen as highly distorted and elongated images amidst the otherwise generally rounder galaxies.\n\nLooking at this image provides fuel for the imagination. First, every spot in the photo is a galaxy, not a star. Each galaxy contains perhaps 100 billion stars, along with them probably hundreds of billions of planets, and perhaps long-lost civilizations. I say long-lost because the image is 5 billion years old. The light was emitted 500 million years before our own Sun and Earth formed. Many of the stars in the photo no longer exist, having exhausted their nuclear fuel billions of years ago. Beyond that, the distorted images show precisely what Zwicky argued would be possible. The large distorted images to the left of the center of the image are highly magnified (and elongated) versions of this distant galaxy, which otherwise would probably not be visible at all.\n\nWorking backward from this image to determine the underlying mass distribution in the cluster is a complicated and complex mathematical challenge. To do so, Tyson had to build a computer model of the cluster and trace the rays from the source through the cluster in all possible different ways, using the laws of general relativity to determine the appropriate paths, until the fit they produced best matched the researchers' observations. When the dust settled, Tyson and collaborators obtained a graphical image that displayed precisely where the mass was located in this system pictured in the original photograph:\n\nSomething strange is going on in this image. The spikes in the graph represent the location of the visible galaxies in the original image, but most of the mass of the system is located _between_ the galaxies, in a smooth, dark distribution. In fact, more than 40 times as much mass is between the galaxies as is contained in the visible matter in the system (300 times as much mass as contained in the stars alone with the rest of visible matter in hot gas around them). Dark matter is clearly not confined to galaxies, but also dominates the density of clusters of galaxies.\n\nParticle physicists like myself were not surprised to find that dark matter also dominates clusters. Even though we didn't have a shred of direct evidence, we all hoped that the amount of dark matter was sufficient to result in a flat universe, which meant that there had to be more than 100 times as much dark matter as visible matter in the universe.\n\nThe reason was simple: a flat universe is the only mathematically beautiful universe. Why? Stay tuned.\n\nWhether or not the total amount of dark matter was sufficient to produce a flat universe, observations such as these obtained by gravitational lensing (I remind you that gravitational lensing results from the local curvature of space around massive objects; the flatness of the universe relates to the global average curvature of space, ignoring the local ripples around massive objects) and more recent observations from other areas of astronomy have confirmed that the total amount of dark matter in galaxies and clusters is far in excess of that allowed by the calculations of Big Bang nucleosynthesis. We are now virtually certain that the dark matter\u2014which, I reiterate, has been independently corroborated in a host of different astrophysical contexts, from galaxies to clusters of galaxies\u2014must be made of something entirely new, something that doesn't exist normally on Earth. This kind of stuff, which isn't star stuff, isn't Earth stuff either. But it _is_ something!\n\nThese earliest inferences of dark matter in our galaxy have spawned a whole new field of experimental physics, and I am happy to say that I have played a role in its development. As I have mentioned above, dark matter particles are all around us\u2014in the room in which I am typing, as well as \"out there\" in space. Hence we can perform experiments to look for dark matter and for the new type of elementary particle or particles of which it is comprised.\n\nThe experiments are being performed in mines and tunnels deep underground. Why underground? Because on the surface of the Earth we are regularly bombarded by all manner of cosmic rays, from the Sun and from objects much farther away. Since dark matter, by its very nature, doesn't interact electromagnetically to produce light, we assume that its interactions with normal material are extremely weak, so it will be extremely difficult to detect. Even if we are bombarded every day by millions of dark matter particles, most will go through us and the Earth, without even \"knowing\" we are here\u2014and without our noticing. Thus, if you want to detect the effects of the very rare exceptions to this rule, dark matter particles that actually bounce off atoms of matter, you had better be prepared to detect very rare and infrequent events. Only underground are you sufficiently shielded from cosmic rays for this to be possible even in principle.\n\nAs I write this, however, an equally exciting possibility is arising. The Large Hadron Collider, outside of Geneva, Switzerland, the world's largest and most powerful particle accelerator, has just begun running. But we have many reasons to believe that, at the very high energies at which protons are smashed together in the device, conditions similar to those in the very early universe will be re-created, albeit over only microscopically small regions. In such regions the same interactions that may have first produced what are now dark matter particles during the very early universe may now produce similar particles in the laboratory! There is thus a great race going on. Who will detect dark matter particles first: the experimenters deep underground or the experimentalists at the Large Hadron Collider? The good news is that, if either group wins the race, no one loses. We all win, by learning what the ultimate stuff of matter really is.\n\nEven though the astrophysical measurements I described don't reveal the identity of dark matter, they do tell us how much of it exists. A final, direct determination of the total amount of matter in the universe came from the beautiful inferences of gravitational lensing measurements like the one I have described combined with other observations of X-ray emissions from clusters. Independent estimates of the clusters' total mass is possible because the temperature of the gas in clusters that are producing the X-rays is related to the total mass of the system in which they are emitted. The results were surprising, and as I have alluded, disappointing to many of us scientists. For when the dust had settled, literally and metaphorically, the total mass in and around galaxies and clusters was determined to be only about 30 percent of the total amount of mass needed to result in a flat universe today. (Note that this is more than 40 times as much mass as can be accounted for by visible matter, which therefore makes up less than 1 percent of the mass needed to make up a flat universe.)\n\nEinstein would have been amazed that his \"little publication\" ultimately was far from useless. Supplemented by remarkable new experimental and observational tools that opened new windows on the cosmos, new theoretical developments that would have amazed and delighted him, and the discovery of dark matter that probably would have raised his blood pressure, Einstein's small step into the world of curved space had ultimately turned into to a giant leap. By the early 1990s, the holy grail of cosmology had apparently been achieved. Observations had determined that we live in an open universe, one that would therefore expand forever. Or had they?\n\n## CHAPTER 3\n\nLIGHT FROM THE BEGINNING OF TIME\n\n_As it was in the beginning, is now, and shall ever be._\n\n\u2014GLORIA PATRI\n\nIf you think about it, trying to determine the net curvature of the universe by measuring the total mass contained within it and then using the equations of general relativity to work backward has huge potential problems. Inevitably, you have to wonder whether matter is hidden in ways that we cannot uncover. For instance, we can only probe for the existence of matter within these systems using the gravitational dynamics of visible systems like galaxies and clusters. If significant mass somehow resided elsewhere, we'd miss it. It would be much better to measure the geometry of the whole visible universe directly.\n\nBut how can you measure the three-dimensional geometry of the whole visible universe? It's easier to start with a simpler question: How would you determine if a two-dimensional object like the Earth's surface was curved if you couldn't go around the Earth or couldn't go above it in a satellite and look down?\n\nFirst, you could ask a high school student, What is the sum of the angles in a triangle? (Choose the high school carefully, however . . . a European one is a good bet.) You would be told 180 degrees, because the student no doubt learned Euclidean geometry\u2014the geometry associated with flat pieces of paper. On a curved two-dimensional surface like a globe, you can draw a triangle, the sum of whose angles is far greater than 180 degrees. For example, consider drawing a line along the equator, then making a right angle, going up to the North Pole, then another right angle back down to the equator, as shown below. Three times 90 is 270, far greater than 180 degrees. Voil\u00e0!\n\nIt turns out that this simple, two-dimensional thinking extends directly and identically to three dimensions, because the mathematicians who first proposed non-flat, or so-called non-Euclidean, geometries realized that the same possibilities could exist in three dimensions. In fact, the most famous mathematician of the nineteenth century, Carl Friedrich Gauss, was so fascinated by the possibility that our own universe might be curved that he took data in the 1820s and '30s from geodetic survey maps to measure large triangles between the German mountain peaks of Hoher Hagen, Inselberg, and Brocken to determine if he could detect any curvature of space itself. Of course, the fact that the mountains are on the curved surface of the Earth means that the two-dimensional curvature of the surface of the Earth would have interfered with any measurement he was performing to probe for curvature in the background three-dimensional space in which the Earth is situated, which he must have known. I assume he was planning to subtract any such contribution from his final results to see if any possible leftover curvature might be attributable to a curvature of the background space.\n\nThe first person to try to measure the curvature of space definitively was an obscure mathematician, Nikolai Ivanovich Lobachevsky, who lived in remote Kazan in Russia _._ Unlike Gauss, Lobachevsky was actually one of two mathematicians who had the temerity to propose in print the possibility of so-called hyberbolic curved geometries, where parallel lines could diverge. Remarkably, Lobachevsky published his work on hyperbolic geometry (which we now call \"negatively curved\" or \"open\" universes) in 1830.\n\nShortly thereafter, when considering whether our own three-dimensional universe might be hyperbolic, Lobachevsky suggested that it might be possible to \"investigate a stellar triangle for an experimental resolution of the question.\" He suggested that observations of the bright star Sirius could be taken when the Earth was on either side of its orbit around the Sun, six months apart. From observations, he concluded that any curvature of our universe must be _at least_ 166,000 times the radius of the Earth's orbit.\n\nThis is a big number, but it is trivially small on cosmic scales. Unfortunately, while Lobachevsky had the right idea, he was limited by the technology of his day. One hundred and fifty years later, however, things have improved, thanks to the most important set of observations in all of cosmology: measurements of the cosmic microwave background radiation, or CMBR.\n\nThe CMBR is nothing less than the afterglow of the Big Bang. It provides another piece of direct evidence, in case any is needed, that the Big Bang really happened, because it allows us to look back directly and detect the nature of the very young, hot universe from which all the structures we see today later emerged.\n\nOne of the many remarkable things about the cosmic microwave background radiation is that it was discovered in New Jersey, of all places, by two scientists who really didn't have the slightest idea what they were looking for. The other thing is that it existed virtually under all our noses for decades, potentially observable, but was missed entirely. In fact, you may be old enough have seen its effects without realizing it, if you remember the days before cable television, when channels used to end their broadcast days in the wee morning hours and not run infomercials all night. When they went off the air, after showing a test pattern, the screen would revert to static. About 1 percent of that static you saw on the television screen was radiation left over from the Big Bang.\n\nThe origin of the cosmic microwave background radiation is relatively straightforward. Since the universe has a finite age (recall it is 13.72 billion years old), and as we look out at ever more distant objects, we are looking further back in time (since the light takes longer to get to us from these objects), you might imagine that if we looked out far enough, we would see the Big Bang itself. In principle this is not impossible, but in practice, between us and that early time lies a wall. Not a physical wall like the walls of the room in which I am writing this, but one that, to a great extent, has the same effect.\n\nI cannot see past the walls in my room because they are opaque. They absorb light. Now, as I look out in the sky back further and further in time, I am looking at the universe as it was younger and younger, and also hotter and hotter, because it has been cooling ever since the Big Bang. If I look back far enough, to a time when the universe was about 300,000 years old, the temperature of the universe was about 3,000 degrees (Kelvin scale) above absolute zero. At this temperature the ambient radiation was so energetic that it was able to break apart the dominant atoms in the universe, hydrogen atoms, into their separate constituents, protons and electrons. Before this time, neutral matter did not exist. Normal matter in the universe, made of atomic nuclei and electrons, consisted of a dense \"plasma\" of charged particles interacting with radiation.\n\nA plasma, however, can be opaque to radiation. The charged particles within the plasma absorb photons and reemit them so that radiation cannot easily pass through such a material uninterrupted. As a result, if I try to look back in time, I cannot see past the time when matter in the universe was last largely comprised of such a plasma.\n\nOnce again, it is like the walls in my room. I can see them only because electrons in atoms on the surface of the wall absorb light from the light in my study and then reemit it, and the air between me and the walls is transparent, so I can see all the way to the surface of the wall that emitted the light. So too with the universe. When I look out, I can see all the way back to that \"last scattering surface,\" which is the point at which the universe became neutral, where protons combined with electrons to form neutral hydrogen atoms. After that point, the universe became largely transparent to radiation, and I can now see the radiation that was absorbed and reemitted by the electrons as matter in the universe became neutral.\n\nIt is therefore a _prediction_ of the Big Bang picture of the universe that there should be radiation coming at me from all directions from that \"last scattering surface.\" Since the universe has expanded by a factor of about 1,000 since that time, the radiation has cooled on its way to us and is now approximately 3 degrees above absolute zero. And that is precisely the signal that the two hapless scientists found in New Jersey in 1965, and for whose discovery they were later awarded the Nobel Prize.\n\nActually a second Nobel Prize was given more recently for observations of the cosmic microwave background radiation, and for good reason. If we could take a photo of the surface of the last scattering surface, we would get a picture of the neonatal universe a mere 300,000 years into its existence. We could see all the structures that would one day collapse to form galaxies, stars, planets, aliens, and all the rest. Most important, these structures would have been unaffected by all the subsequent dynamical evolution that can obscure the underlying nature and origin of the first tiny primordial perturbations in matter and energy which were presumably created by exotic processes in the earliest moments of the Big Bang.\n\nMost important for our purpose, however, on this surface there would be a characteristic scale, which is imprinted by nothing other than time itself. One can understand this as follows: If one considers a distance spanning about 1 degree on the last scattering surface as seen by an observer on Earth, this would correspond to a distance on that surface of about 300,000 light-years. Now, since the last scattering surface reflects a time when the universe itself was about 300,000 years old, and since Einstein tells us that no information can travel through space faster than the speed of light, this means that no signal from one location could travel across this surface at that time by more than about 300,000 light-years.\n\nNow consider a lump of matter smaller than 300,000 light-years across. Such a lump will have begun to collapse due to its own gravity. But a lump larger than 300,000 light-years across won't even begin to collapse, because it doesn't yet even \"know\" it is a lump. Gravity, which itself propagates at the speed of light, cannot have traveled across the full length of the lump. So just as Wile E. Coyote runs straight off a cliff and hangs suspended in midair in the Road Runner cartoons, the lump will just sit there, waiting to collapse when the universe becomes old enough for it to know what it is supposed to do!\n\nThis singles out a special triangle, with one side 300,000 light-years across, a known distance away from us, determined by the distance between us and the last scattering surface, as shown below:\n\nThe largest lumps of matter, which will have already begun to collapse and in so doing will produce irregularities on the image of the microwave background surface, will span this angular scale. If we are able to obtain an image of this surface as it looked at that time, we would expect such hot spots to be, on average, the largest significant lumps we see in the image.\n\nHowever, whether the angle spanned by this distance is precisely 1 degree will in fact be determined by the geometry of the universe. In a flat universe, light rays travel in straight lines. In an open universe, however, light rays bend outward as one follows them back in time. In a closed universe, light rays converge as one follows them backward. Thus, the actual angle spanned on our eyes by a ruler that is 300,000 light-years across, located at a distance associated with the last scattering surface, depends upon the geometry of the universe, as shown below:\n\nThis provides a direct, clean test of the geometry of the universe. Since the size of the largest hot spots or cold spots in the microwave background radiation image depends just upon causality\u2014the fact that gravity can propagate only at the speed of light, and thus the largest region that can have collapsed at that time is simply determined by the farthest distance a light ray can have propagated at that time\u2014and because the angle that we see spanned by a fixed ruler at a fixed distance from us is just determined by the curvature of the universe, a simple picture of the last scattering surface can reveal to us the large-scale geometry of space-time.\n\nThe first experiment to attempt such an observation was a ground-launched balloon experiment in Antarctica in 1997 called BOOMERANG. While the acronym stands for _B_ alloon _O_ bservations _o_ f _M_ illimetric _E_ xtragalactic _R_ adiation _an_ d _G_ eophysics, the real reason it was called this name is simpler. A microwave radiometer was attached to a high-altitude balloon as shown below:\n\nThe balloon then went around the world, which is easy to do in Antarctica. Actually, at the South Pole it is really easy to do, since you can just turn around in a circle. However, from McMurdo Station the round trip around the continent, aided by the polar winds, took two weeks, after which the device returned to its starting point, hence the name BOOMERANG.\n\nBoomerang path around Antarctica.\n\nThe purpose of the balloon trip was simple. To get a view of the microwave background radiation, reflecting a temperature of 3 degrees above absolute zero (Kelvin scale), which is not contaminated by the far hotter material on Earth (even in Antarctica temperatures are more than two hundred degrees hotter than the temperature of the cosmic microwave background radiation), we want to go as far as possible above the ground, and even above most of the atmosphere of the Earth. Ideally we use satellites for this purpose, but high-altitude balloons can do much of the job for far less money.\n\nIn any case, after two weeks, BOOMERANG returned an image of a small part of the microwave sky displaying hot and cold spots in the radiation pattern coming from the last scattering surface. Shown below is one image of the region the BOOMERANG experiment observed (with \"hot spots\" and \"cold spots\" being shaded dark and light respectively), superimposed upon the original photo of the experiment:\n\nThis image serves two purposes as far as I am concerned. First, it displays the actual physical scale of the hot and cold spots as seen in the sky by BOOMERANG, with the foreground images for comparison. But it also illustrates another important aspect of what can only be called our cosmic myopia. When we look up on a sunny day, we see a blue sky, as shown in the previous image of the balloon. But this is because we have evolved to see visible light. We have done so, no doubt, both because the light from the surface of our Sun peaks in the visible region, and also because many other wavelengths of light get absorbed in our atmosphere, so they cannot reach us on the Earth's surface. (This is fortunate for us, since much of this radiation could be harmful.) In any case, if we had instead evolved to \"see\" microwave radiation, the image of the sky we would see, day or night, as long as we weren't looking directly at the Sun, would take us directly back to an image of the last scattering surface, more than 13 billion light-years away. This is the \"image\" returned by the BOOMERANG detector.\n\nThe first flight of BOOMERANG, which produced this image, was remarkably fortunate. Antarctica is a hostile, unpredictable environment. On a later flight, in 2003, the entire experiment was nearly lost due to a balloon malfunction and subsequent storm. A last-minute decision to cut free from the balloon before it was blown to some inaccessible location saved the day and a search-and-rescue mission located the payload on the Antarctic plain and recovered the pressurized vessel containing the scientific data.\n\nBefore interpreting the BOOMERANG image, I want to emphasize one more time that the actual physical size of the hot spots and cold spots recorded on the BOOMERANG image are fixed by simple physics associated with the last scattering surface, while the _measured_ sizes of the hot spots and cold spots in the image derive from the geometry of the universe. A simple two-dimensional analogy may help further explain the result: In two dimensions, a **closed geometry** resembles the surface of a sphere, while an **open geometry** resembles the surface of a saddle. If we draw a triangle on these surfaces, we observe the effect I described, as straight lines converge on a sphere, and diverge on a saddle, and, of course, remain straight on a flat plane:\n\nSo the million-dollar question now is, How big _are_ the hot spots and cold spots in the BOOMERANG image? To answer this, the BOOMERANG collaboration prepared several simulated images on their computer of hot spots and cold spots as would be seen in closed, flat, and open universes, and compared this with (another false color) image of the actual microwave sky.\n\nIf you examine the image on the lower left, from a simulated closed universe, you will see that the average spots are larger than in the actual universe. On the right, the average spot size is smaller. But, just like Baby Bear's bed in Goldilocks, the image in the middle, corresponding to a simulated flat universe, is \"just right.\" The mathematically beautiful universe hoped for by theorists seemed to be vindicated by this observation, even though it appears to conflict strongly with the estimate made by weighing clusters of galaxies.\n\nIn fact, the agreement between the predictions for a flat universe and the image obtained by BOOMERANG is almost embarrassing. Examining the spots and searching for the largest ones that had time to collapse significantly inward at the time reflected in the last scattering surface, the BOOMERANG team produced the following graph:\n\nThe data are the points. The solid line gives the prediction for a flat universe, with the largest bump occurring close to 1 degree!\n\nSince the BOOMERANG experiment published its results, a far more sensitive satellite probe of the microwave background radiation was launched by NASA, the Wilkinson Microwave Anisotropy Probe (WMAP). Named after the late Princeton physicist David Wilkinson, who was one of the original Princeton physicists who should have discovered the CMBR had they not been scooped by the Bell Labs scientists, WMAP was launched in June 2001. It was sent out to a distance of one million miles from the Earth, where, on the far side of the Earth from the Sun, it could view the microwave sky without contamination from sunlight. Over a period of seven years it imaged the whole microwave sky (not just a portion of the sky as BOOMERANG did, since BOOMERANG had to contend with the presence of the Earth below it) with unprecedented accuracy.\n\nHere the entire sky is projected on a plane, just as the surface of a globe can be projected on a flat map. The plane of our galaxy would lie along the equator, and 90 degrees above the plane of our galaxy is the North Pole on this map and 90 degrees below the plane of our galaxy is the South Pole. The image of the galaxy, however, has been removed from the map in order to reflect purely the radiation coming from the last scattering surface.\n\nWith this kind of exquisite data a much more precise estimate can be made of the geometry of the universe. A WMAP plot that is analogous to the one shown for the BOOMERANG image confirms to an accuracy of 1 percent that we live in a flat universe! The expectations of theorists were correct. Yet once again, we cannot ignore the apparent obvious inconsistency of this result with the result I described in the last chapter. Weighing the universe by measuring the mass of galaxies and clusters yields a value a factor of 3 smaller than the amount needed to result in a flat universe. Something has to give.\n\nWhile theorists may have been patting themselves on the back for guessing that the universe is flat, almost no one was prepared for the surprise that nature had in store to resolve the contradictory estimates of the geometry of the universe coming from measuring mass versus measuring curvature directly. The missing energy needed to result in a flat universe turned out to be hiding right under our noses, literally.\n\n## CHAPTER 4\n\nMUCH ADO ABOUT NOTHING\n\n_Less is more._\n\n\u2014LUDWIG MIES VAN DER ROHE,  \nAFTER ROBERT BROWNING\n\nOne step forward, two steps back, or so it seemed in our search for understanding our universe and accurately giving it a face. Even though observations had finally definitively determined the curvature of our universe\u2014and in the process validated long-held theoretical suspicions\u2014suddenly, even though it was known that ten times as much matter exists in the universe as could be accounted for by protons and neutrons, even that massive amount of dark matter, comprising 30 percent of what was required to produce a flat universe, was nowhere near sufficient to account for all the energy in the universe. The direct measurement of the geometry of the universe and the consequent discovery that the universe is indeed flat meant that 70 percent of the energy of the universe was still missing, neither in nor around galaxies or even clusters of galaxies!\n\nThings were not quite as shocking as I have made them out to be. Even before these measurements of the curvature of the universe, and the determination of the total clustered mass within it (as described in chapter 2), there were signs that the by-then conventional theoretical picture of our universe\u2014with sufficient dark matter (three times as much as we now know exists, in fact) to be spatially flat\u2014was just not consistent with observations. Indeed, as early as 1995, I wrote a heretical paper with a colleague of mine, Michael Turner, from the University of Chicago, suggesting that this conventional picture couldn't be correct, and in fact the only possibility that appeared consistent with both a flat universe (our theoretical preference at the time) and observations of the clustering of galaxies and their internal dynamics was a universe that was far more bizarre and that hearkened back to a crazy theoretical idea Albert Einstein had in 1917 to solve the apparent contradiction between the predictions of his theory and the static universe he thought we lived in and which he later abandoned.\n\nAs I recall, our motivation at the time was more to show that something was wrong with the prevailing wisdom than it was to suggest a definitive solution to the problem. The proposal seemed too crazy to really believe, so I don't think anyone was more surprised than we were when it turned out, three years later, that our heretical suggestion was precisely on the money after all!\n\nLet's return to 1917. Recall that Einstein had developed general relativity and had heart palpitations of joy over discovering that he could explain the precession of the perihelion of Mercury, even as he had to confront that fact that his theory couldn't explain the static universe in which he thought he was living.\n\nHad he had greater courage of his convictions, he might have _predicted_ that the universe couldn't be static. But he didn't. Instead, he realized that he could make a small change in his theory, one that was completely consistent with the mathematical arguments that had led him to develop general relativity in the first place, and one that looked like it might allow a static universe.\n\nWhile the details are complex, the general structure of Einstein's equations in general relativity is relatively straightforward. The left-hand side of the equations describes the curvature of the universe, and with it, the strength of the gravitational forces acting on matter and radiation. These are determined by the quantity on the right-hand side of the equation, which reflects the total density of all kinds of energy and matter within the universe.\n\nEinstein realized that adding a small extra constant term to the left-hand side of the equation would represent a small extra constant _repulsive_ force throughout all of space in addition to the standard gravitational attraction between distant objects that falls off as the distance between them increases. If it were small enough, this extra force could be undetectable on human scales or even on the scale of our solar system, where Newton's law of gravity is observed to hold so beautifully. But he reasoned that, because it was constant throughout all of space, it could build up over the scale of our galaxy and be large enough to counteract the attractive forces between very distant objects. He thus reasoned that this could result in a static universe on the largest scales.\n\nEinstein called this extra term the _cosmological term._ Because it is simply a constant addition to the equations, it is now, however, conventional to call this term the _cosmological constant._\n\nOnce he recognized that the universe is actually expanding, Einstein dispensed with this term and is said to have called the decision to add it to his equations his biggest blunder.\n\nBut getting rid of it is not so easy. It is like trying to put the toothpaste back in the tube after you have squeezed it out. This is because we now have a completely different picture of the cosmological constant today, so that, if Einstein had not added the term, someone else would have in the intervening years.\n\nMoving Einstein's term from the left-hand side of his equations to the right-hand side is a small step for a mathematician but a giant leap for a physicist. While it is trivial mathematically to do so, once this term is on the right-hand side, where all the terms contributing to the energy of the universe reside, it represents something completely different from a physical perspective\u2014namely a new contribution to the total energy. But what kind of stuff could contribute such a term?\n\nThe answer is, _nothing._\n\nBy _nothing,_ I do not mean nothing, but rather _nothing_ \u2014in this case, the nothingness we normally call empty space. That is to say, if I take a region of space and get rid of everything within it\u2014dust, gas, people, and even the radiation passing through, namely absolutely _everything_ within that region\u2014if the remaining empty space _weighs something,_ then that would correspond to the existence of a cosmological term such as Einstein invented.\n\nNow, this makes Einstein's cosmological constant seem even crazier! For any fourth grader will tell you how much energy is contained in nothing, even if they don't know what energy is. The answer must be nothing.\n\nAlas, most fourth graders have not taken quantum mechanics, nor have they studied relativity. For when one incorporates the results of Einstein's special theory of relativity into the quantum universe, empty space becomes much stranger than it was before. So strange in fact that even the physicists who first discovered and analyzed this new behavior were hard-pressed to believe that it actually existed in the real world.\n\nThe first person to successfully incorporate relativity into quantum mechanics was the brilliant, laconic British theoretical physicist Paul Dirac, who himself had already played a leading role in developing quantum mechanics as a theory.\n\nQuantum mechanics was developed from 1912 to 1927, primarily through the work of the brilliant and iconic Danish physicist Niels Bohr and the brilliant young hot-shots Austrian physicist Erwin Schr\u00f6dinger and German physicist Werner Heisenberg. The quantum world first proposed by Bohr, and refined mathematically by Schr\u00f6dinger and Heisenberg, defies all commonsense notions based on our experience with objects on a human scale. Bohr first proposed that electrons in atoms orbit around the central nucleus, as planets do around the Sun, but demonstrated that the observed rules of atomic spectra (the frequencies of light emitted by different elements) could only be understood if somehow the electrons were restricted to have stable orbits in a fixed set of \"quantum levels\" and could not spiral freely toward the nucleus. They could move between levels by absorbing or emitting only discrete frequencies, or quanta, of light\u2014the very quanta that Max Planck had first proposed in 1905 as a way of understanding the forms of radiation emitted by hot objects.\n\nBohr's \"quantization rules\" were rather ad hoc, however. In the 1920s, Schr\u00f6dinger and Heisenberg independently demonstrated that it was possible to derive these rules from first principles if electrons obeyed rules of dynamics that were different from those applied to macroscopic objects like baseballs. Electrons could behave like waves as well as particles, appearing to spread out over space (hence, Schr\u00f6dinger's \"wave function\" for electrons), and the results of measurements of the properties of electrons were shown to yield only probabilistic determinations, with various combinations of different properties not being exactly measurable at the same time (hence, Heisenberg's \"Uncertainty Principle\").\n\nDirac had shown that the mathematics proposed by Heisenberg to describe quantum systems (for which Heisenberg won the 1932 Nobel Prize) could be derived by careful analogy with the well-known laws governing the dynamics of classical macroscopic objects. In addition, he was also later able to show that the mathematical \"wave mechanics\" of Schr\u00f6dinger could also be so derived and was formally equivalent to Heisenberg's formulation. But Dirac also knew that the quantum mechanics of Bohr, Heisenberg, and Schr\u00f6dinger, as remarkable as it was, applied only to systems where Newton's laws, and not Einstein's relativity, would have been the appropriate laws governing the classical systems that the quantum systems were built with by analogy.\n\nDirac liked to think in terms of mathematics rather than pictures, and as he turned his attention to trying to make quantum mechanics consistent with Einstein's laws of relativity, he started playing with many different sorts of equations. These included complicated multicomponent mathematical systems that were necessary to incorporate the fact that electrons have \"spin\"\u2014that is to say they spin around like small tops and have angular momentum, and they also can spin both clockwise and anticlockwise around any axis.\n\nIn 1929, he hit pay dirt. The Schr\u00f6dinger equation had beautifully and accurately described the behavior of electrons moving at speeds much slower than light. Dirac found that if he modified the Schr\u00f6dinger equation into a more complex equation using objects called matrices\u2014which actually meant that his equation really described a set of four different coupled equations\u2014he could consistently unify quantum mechanics with relativity, and thus in principle describe the behavior of systems where the electrons were moving at much faster speeds.\n\nThere was a problem, however. Dirac had written down an equation meant to describe the behavior of electrons as they interacted with electric and magnetic fields. But his equation appeared also to require the existence of new particles just like electrons but with opposite electric charge.\n\nAt the time, there was only one elementary particle in nature known with a charge opposite that of the electron\u2014the proton. But protons are not at all like electrons. To begin with, they are 2,000 times heavier!\n\nDirac was flummoxed. In an act of desperation he argued that the new particles were in fact protons, but that somehow when moving through space the interactions of protons would cause them to act as if they were heavier. It didn't take long for others, including Heisenberg, to show that this suggestion made no sense.\n\nNature quickly came to the rescue. Within two years of the time Dirac proposed his equation, and a year after he had capitulated and accepted that, if his work was correct, then a new particle must exist, experimenters looking at cosmic rays bombarding the Earth discovered evidence for new particles identical to electrons but with an opposite electric charge, which were dubbed positrons.\n\nDirac was vindicated, but he also recognized his earlier lack of confidence in his own theory by later saying that his equation was smarter than he was!\n\nWe now call the positron the \"antiparticle\" of the electron, because it turns out that Dirac's discovery was ubiquitous. The same physics that required an antiparticle for the electron to exist requires one such particle to exist for almost every elementary particle in nature. Protons have antiprotons, for example. Even some neutral particles, like neutrons, have antiparticles. When particles and antiparticles meet, they annihilate into pure radiation.\n\nWhile all this may sound like science fiction (and indeed antimatter plays an important role in _Star Trek_ ), we create antiparticles all the time at our large particle accelerators around the world. Because antiparticles otherwise have the same properties as particles, a world made of antimatter would behave the same way as a world of matter, with antilovers sitting in anticars making love under an anti-Moon. It merely is an accident of our circumstances, due, we think, to rather more profound factors we will get to later, that we live in a universe that is made up of matter and not antimatter or one with equal amounts of both. I like to say that while antimatter may seem strange, it is strange in the sense that Belgians are strange. They are not really strange; it is just that one rarely meets them.\n\nThe existence of antiparticles makes the observable world a much more interesting place, but it also turns out to make empty space much more complicated.\n\nLegendary physicist Richard Feynman was the first person to provide an intuitive understanding of why relativity requires the existence of antiparticles, which also yielded a graphic demonstration that empty space is not quite so empty.\n\nFeynman recognized that relativity tells us that observers moving at different speeds will make different measurements of quantities such as distance and time. For example, time will appear to slow down for objects moving very fast. If somehow objects could travel faster than light, they would appear to go backward in time, which is one of the reasons that the speed of light is normally considered a cosmic speed limit.\n\nA key tenet of quantum mechanics, however, is the Heisenberg Uncertainty Principle, which, as I have mentioned, states that it is impossible to determine, for certain pairs of quantities, such as position and velocity, exact values for a given system at the same time. Alternatively, if you measure a given system for only a fixed, finite time interval, you cannot determine its total energy exactly.\n\nWhat all this implies is that, for very short times, so short that you cannot measure their speed with high precision, quantum mechanics allows for the possibility that these particles act as if they are moving faster than light! But, if they are moving faster than light, Einstein tells us they must be behaving as if they are moving backward in time!\n\nFeynman was brave enough to take this apparently crazy possibility seriously and explore its implications. He drew the following diagram for an electron moving about, periodically speeding up in the middle of its voyage to faster-than-light speed.\n\nHe recognized that relativity would tell us that another observer might alternatively measure something that would appear as shown below, with an electron moving forward in time, then backward in time, and then forward again.\n\nHowever, a negative charge moving backward in time is mathematically equivalent to a positive charge moving forward in time! Thus, relativity would require the existence of positively charged particles with the same mass and other properties as electrons.\n\nIn this case one can reinterpret Feynman's second drawing as follows: a single electron is moving along, and then at another point in space a positron-electron pair is created out of nothing, and then the positron meets the first electron and the two annihilate. Afterward, one is left with a single electron moving along.\n\nIf this doesn't bother you, then consider the following: for a little while, even if you start out with just a single particle, and end with a single particle, for a short time there are three particles moving about:\n\nIn the brief middle period, for at least a little while, something has spawned out of nothing! Feynman beautifully describes this apparent paradox in his 1949 paper, \"A Theory of Positrons,\" with a delightful wartime analogy:\n\nIt is as though a bombardier watching a single road through the bomb-sight of a low-flying plane suddenly sees three roads and it is only when two of them come together and disappear again that he realizes that he has simply passed over a long switchback in a single road.\n\nAs long as this time period during this \"switchback\" is so short that we cannot measure all the particles directly, quantum mechanics and relativity imply that not only is this weird situation allowed, it is required. The particles that appear and disappear in timescales too short to measure are called _virtual_ particles.\n\nNow inventing a whole new set of particles in empty space that you cannot measure sounds a lot like proposing a large number of angels sitting on the head of a pin. And it would be about as impotent an idea if these particles had no other measurable effects. However, while they are not directly observable, it turns out their _indirect_ effects produce most of the characteristics of the universe we experience today. Not only this, but one can calculate the impact of these particles more precisely than any other calculation in science.\n\nConsider, for example, a hydrogen atom\u2014the system Bohr tried to explain by developing his quantum theory and Schr\u00f6dinger later tried to describe by deriving his famous equation. The beauty of quantum mechanics was that it could explain the specific colors of light emitted by hydrogen when it was heated up by arguing that electrons orbiting around the proton could exist only in discrete energy levels, and when they jumped between levels they absorbed or emitted only a fixed set of frequencies of light. The Schr\u00f6dinger equation allows one to calculate the predicted frequencies, and it gets the answer almost exactly right.\n\nBut not exactly.\n\nWhen the spectrum of hydrogen was observed more carefully, it was seen to be more complicated than had previously been estimated, with some additional small splittings between levels observed, called the \"fine structure\" of the spectrum. While these splittings had been known since Bohr's time, and it was suspected that perhaps relativistic effects had something do to with them, until a fully relativistic theory was available, no one could confirm this suspicion. Happily, Dirac's equation managed to improve the predictions compared to Schr\u00f6dinger's equation and reproduced the general structure of the observations, including fine structure.\n\nSo far so good, but in April of 1947, United States experimentalist Willis Lamb and his student Robert C. Retherford performed an experiment that might otherwise seem incredibly ill motivated. They realized that they had the technological ability to measure the energy-level structure of the level of hydrogen atoms with an accuracy of 1 part in 100 million.\n\nWhy would they bother? Well, whenever experimentalists find a new method to measure something with vastly greater precision than was possible before, that is often sufficient motivation for them to go ahead. Whole new worlds are often revealed in the process, as when the Dutch scientist Antonie Philips van Leeuwenhoek first stared at a drop of seemingly empty water with a microscope in 1676 and discovered it was teeming with life. In this case, however, the experimenters had more immediate motivation. Up until the time of Lamb's experiment, the available experimental precision could not test Dirac's prediction in detail.\n\nThe Dirac equation did predict the general structure of the new observations, but the key question that Lamb wanted to answer was whether it predicted it in detail. This was the only way to actually test the theory. And when Lamb tested the theory, it seemed to give the wrong answer, at a level of about 100 parts per billion, well above the sensitivity of his apparatus.\n\nSuch a small disagreement with experiment may not seem like a lot, but the predictions of the simplest interpretation of the Dirac theory were unambiguous, as was the experiment, and they differed.\n\nOver the next few years, the best theoretical minds in physics jumped into the fray and tried to resolve the discrepancy. The answer came after a great deal of work, and when the dust had settled, it was realized that the Dirac equation actually gives precisely the correct answer, but only if you include the effect of virtual particles. Pictorially, this can be understood as follows. Hydrogen atoms are usually pictured in chemistry books something like this, with a proton at the center and an electron orbiting around it, jumping between different levels:\n\nHowever, once we allow for the possibility that electron-positron pairs can spontaneously appear from nothing for a bit before annihilating each other again, over any short time the hydrogen atom really looks like this:\n\nAt the right of the figure I have drawn such a pair, which then annihilate at the top. The virtual electron, being negatively charged, likes to hang around closer to the proton, while the positron likes to stay farther away. In any case, what is clear from this picture is that the actual charge distribution in a hydrogen atom is _not,_ at any instant, described by simply a single electron and proton.\n\nRemarkably, we physicists have learned (after all the hard work by Feynman and others) that we can use Dirac's equation to calculate to an arbitrarily high precision, the impact on the spectrum of hydrogen of all the possible virtual particles that may exist intermittently in its vicinity. And when we do, we come up with _the best, most accurate prediction in all of science_. All other scientific predictions pale in comparison. In astronomy, the most recent observations of the cosmic microwave background radiation allow us to compare with theoretical predictions at the level of perhaps 1 part in 100,000, which is remarkable. However, using Dirac's equation, and the predicted existence of virtual particles, we can calculate the value of atomic parameters and compare them with observations and have remarkable agreement at the level of about 1 part in a billion or better!\n\nVirtual particles therefore exist.\n\nWhile the spectacular precision available in atomic physics is hard to match, there is nevertheless another place where virtual particles play a key role that may actually be more relevant to the central issue of this book. It turns out that they are responsible for most of your mass, and that of everything that is visible in the universe.\n\nOne of the great successes in the 1970s in our fundamental understanding of matter came with the discovery of a theory that accurately describes the interactions of quarks, the particles that make up the protons and neutrons that form the bulk of material from which you and everything you can see are made. The mathematics associated with the theory is complex, and it took several decades before techniques were developed that could handle it, particularly in the regime where the strong interaction between the quarks became appreciable. A herculean effort was launched, including building some of the most complicated parallel processing computers, which simultaneously utilize tens of thousands of individual processors, in order to try to calculate the fundamental properties of protons and neutrons, the particles we actually measure.\n\nAfter all of this work, we now have a good picture of what the inside of a proton actually looks like. There may be three quarks contained therein, but there is also a lot of other stuff. In particular, virtual particles reflecting the particles and fields that convey the strong force between quarks are popping in and out of existence all the time. Here is a snapshot of how things actually look. It is not a real photograph of course, but rather an artistic rendering of the mathematics governing the dynamics of quarks and the fields that bind them. The odd shapes and different shadings reflect the strength of the fields interacting with one another and with the quarks inside the proton as virtual particles spontaneously pop in and out of existence.\n\nThe proton is intermittently full of these virtual particles and, in fact, when we try to estimate how much they might contribute to the mass of the proton, we find that the quarks themselves provide very little of the total mass and that the fields created by these particles contribute most of the energy that goes into the proton's rest energy and, hence, its rest mass. The same is true for the neutron, and since you are made of protons and neutrons, the same is true for you!\n\nNow, if we can calculate the effects of virtual particles on the otherwise empty space in and around atoms, and we can calculate the effects of virtual particles on the otherwise empty space inside of protons, then shouldn't we be able to calculate the effects of virtual particles on truly empty space?\n\nWell, this calculation is actually harder to do. This is because, when we calculate the effect of virtual particles on atoms or on the proton mass, we are actually calculating the total energy of the atom or proton _including_ virtual particles; then, we calculate the total energy that the virtual particles would contribute without the atom or proton present (i.e., in empty space); and _then_ we subtract the two numbers in order to find the net impact upon the atom or proton. We do this because it turns out that each of these two energies is formally infinite when we attempt to solve the appropriate equations, but when we subtract the two quantities, we end up with a finite difference, and moreover one that agrees precisely with the measured value!\n\nHowever, if we want to calculate the effect of virtual particles on empty space alone, we have nothing to subtract, and the answer we get is therefore infinite.\n\nInfinity is not a pleasant quantity, however, at least as far as physicists are concerned, and we try to avoid it whenever possible. Clearly, the energy of empty space (or anything else, for that matter) cannot be physically infinite, so we have to figure out a way to do the calculation and get a finite answer.\n\nThe source of the infinity is easy to describe. When we consider all possible virtual particles that can appear, the Heisenberg Uncertainty Principle (which I remind you says that the uncertainty in the measured energy of a system is inversely proportional to the length of time over which you observe it) implies that particles carrying ever more energy can appear spontaneously out of nothing as long as they then disappear in ever-shorter times. In principle, particles can therefore carry almost infinite energy as long as they disappear in almost infinitesimally short times.\n\nHowever, the laws of physics as we understand them apply only for distances and times larger than a certain value, corresponding to the scale where the effects of quantum mechanics must be considered when trying to understand gravity (and its associated effects on space-time). Until we have a theory of \"quantum gravity,\" as it is called, we can't trust extrapolations that go beyond these limits.\n\nThus, we might hope that the new physics associated with quantum gravity will somehow cut off the effects of virtual particles that live for less time than the \"Planck-time,\" as it is called. If we then consider the cumulative effects of only virtual particles of energies equal to or lower than that allowed by this temporal cutoff, we arrive at a finite estimate for the energy that virtual particles contribute to nothing.\n\nBut there is a problem. This estimate turns out to be about 1,000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000,\u00ad000 times larger than the energy associated with all the known matter in the universe, including dark matter!\n\nIf the calculation of the atomic energy level spacings including virtual particles is the best computation in all of physics, this estimate of the energy space\u2014120 orders of magnitude larger than the energy of everything else in the universe\u2014is undoubtedly the worst! If the energy of empty space were anywhere near this large, the repulsive force induced (remember the energy of empty space corresponds to a cosmological constant) would be large enough to blow up the Earth today, but more important, it would have been so great at early times that everything we now see in our universe would have pushed apart so quickly in the first fraction of a second of the Big Bang that no structure, no stars, no planets, and no people would ever have formed.\n\nThis problem, appropriately called the Cosmological Constant Problem, has been around since well before I was a graduate student, first made explicit by the Russian cosmologist Yakov Zel'dovich around 1967. It remains unsolved and is perhaps the most profound unsolved fundamental problem in physics today.\n\nIn spite of the fact that we have had no idea how to solve the problem for more than forty years, we theoretical physicists knew what the answer had to be. Like the fourth grader who I suggested would have guessed that the energy of empty space had to be zero, we too felt that when an ultimate theory was derived, it would explain how the effects of virtual particles would cancel, leaving empty space with precisely zero energy. Or nothing. Or rather, Nothing.\n\nOur reasoning was better than the fourth grader's, or so we thought. We needed to reduce the magnitude of the energy of empty space from the truly gargantuan value that the na\u00efve estimate suggested to a value consistent with the upper limits allowed by observation. This would require some way to subtract from a very large positive number another very large positive number so the two would cancel to 120 decimal places, leaving something non-zero in the 121st decimal place! But there is no precedent in science for canceling two large numbers to such accuracy, with only something minuscule left over.\n\nHowever, zero is a number that is easy to produce. Symmetries of nature often allow us to demonstrate that there are precisely equal and opposite contributions coming from different parts of a calculation, canceling out exactly, with precisely nothing left over. Or, again, Nothing.\n\nThus, we theorists were able to rest easy and sleep at night. We didn't know how to get there, but we were sure what the final answer had to be.\n\nNature, however, had something different in mind.\n\n## CHAPTER 5\n\nTHE RUNAWAY UNIVERSE\n\n_It is mere rubbish, thinking at present of the origin of life; one might as well think of the origin of matter._\n\n\u2014CHARLES DARWIN\n\nWhat Michael Turner and I argued in 1995 was heretical in the extreme. Based on little more than theoretical prejudice, we presumed the universe was flat. (I should stress once again here that a \"flat\" three-dimensional universe is not flat like a two-dimensional pancake is flat, but is rather the three-dimensional space that all of us intuitively picture, in which light rays travel in straight lines. This is to be contrasted with the much harder to picture curved three-dimensional spaces in which light rays, which trace the underlying curvature of space, do not travel in straight lines.) Then we inferred that all available cosmological data at the time were consistent with a flat universe only if about 30 percent of the total energy resided in some form of \"dark matter,\" as observations seemed to indicate existed around galaxies and clusters, but much more strangely than even this, that the remaining 70 percent of the total energy in the universe resided not in any form of matter, but rather in empty space itself.\n\nOur idea was crazy by any standards. In order to result in a value for the cosmological constant consistent with our claim, the estimated value for this quantity described in the last chapter would have to be reduced somehow by 120 orders of magnitude and still not be precisely zero. This would involve the most severe fine-tuning of any physical quantity known in nature, without the slightest idea how to adjust it.\n\nThis was one of the reasons that, as I lectured at various universities about the quandary of a flat universe, I evoked mostly smiles and no more. I don't think many people took our proposal seriously, and I am not even sure Turner and I did. Our point in raising eyebrows with our paper was to illustrate graphically a fact that was beginning to dawn not just on us, but also on several of our theorist colleagues around the world: something looked wrong with the by-then \"standard\" picture of our universe, in which almost all the energy required by general relativity to produce a flat universe today was assumed to reside in exotic dark matter (with a pinch of baryons\u2014i.e., us Earthlings, stars, visible galaxies\u2014to salt the mix).\n\nA colleague recently reminded me that for the two years following our modest proposal, it was referenced only a handful of times in subsequent papers, and apparently all but one or two of these were in papers written by Turner or me! As perplexing as our universe is, the bulk of the scientific community believed it couldn't be as crazy as Turner and I suggested it was.\n\nThe simplest alternative way out of the contradictions was the possibility that the universe wasn't flat but open (one in which parallel light rays today would curve apart if we traced their trajectory backward. This was of course before the cosmic microwave background measurements made it clear that this option was not viable.) However, even this possibility had its own problems, though the situation there remained far from clear as well.\n\nAny high school physics student will happily tell you that gravity sucks\u2014that is, it is universally attractive. Of course, like so many things in science, we now recognize that we have to expand our horizons because nature is more imaginative than we are. If for the moment we assume that the attractive nature of gravity implies that the expansion of the universe has been slowing down, recall that we get an upper limit on the age of the universe by assuming that the velocity of a galaxy located at a certain distance from us has been constant since the Big Bang. This is because, if the universe has been decelerating, then the galaxy was once moving away faster from us than it is now, and therefore it would have taken less time to get to its current position than if it had always been moving at its current speed. In an open universe dominated by matter, the deceleration of the universe would be slower than in a flat universe, and therefore the inferred age of the universe would be greater than it would be for a flat universe dominated by matter, for the same current measured expansion rate. It would in fact be much closer to the value we estimate by assuming a constant rate of expansion over cosmic time.\n\nRemember that a non-zero energy of empty space would produce a cosmological constant\u2014like gravitational repulsion\u2014implying that the expansion of the universe would instead speed up over cosmic time, and therefore galaxies would previously have been moving apart more slowly than they are today. This would imply that it would have taken even longer to get to their present distance than it would for a constant expansion. Indeed, for a given measurement of the Hubble constant today, the longest possible lifetime of our universe (about 20 billion years) is obtained by including the possibility of a cosmological constant along with the measured amount of visible and dark matter, if we are free to adjust its value along with the density of matter in the universe today.\n\nIn 1996, I worked with Brian Chaboyer and our collaborators Pierre Demarque at Yale and postdoc Peter Kernan at Case Western Reserve to put a lower limit on the age of these stars to be about 12 billion years. We did this by modeling the evolution of millions of different stars on high-speed computers and comparing their colors and brightness with actual stars observed in globular clusters in our galaxy, which were long thought to be among the oldest objects in the galaxy. Assuming about a billion years for our galaxy to form, this lower limit effectively ruled out a flat universe dominated by matter and favored one with a cosmological constant (one of the factors that had weighed on the conclusions in my earlier paper with Turner), while an open universe teetered on the hairy edge of viability.\n\nHowever, the ages of the oldest stars involve inferences based on observations at the edge of the then current sensitivity and, in 1997, new observational data forced us to revise our estimates downward by about 2 billion years, leading to a somewhat younger universe. So the situation became much murkier, and all three cosmologies once again appeared viable, sending many of us back to the drawing board.\n\nAll of this changed in 1998, coincidentally the same year that the BOOMERANG experiment demonstrated that the universe is flat.\n\nIn the intervening seventy years since Edwin Hubble measured the expansion rate of the universe, astronomers had worked harder and harder to pin down its value. Recall that in the 1990s they had finally found a \"standard candle\"\u2014that is, an object whose intrinsic luminosity observers felt they could independently ascertain, so that, when they measured its apparent luminosity, they could then infer its distance. The standard candle seemed to be reliable and was one that could be observed across the depths of space and time.\n\nA certain type of exploding star called Type Ia supernova had recently been demonstrated to exhibit a relationship between brightness and longevity. Measuring how long a given Type Ia supernova remained bright required, for the first time, taking into account time dilation effects due to the expansion of the universe, which imply that the measured lifetime of such a supernova is actually longer than its actual lifetime in its rest frame. Nonetheless, we could infer the absolute brightness and measure its apparent brightness with telescopes and ultimately determine the distance to the host galaxy in which the supernova had exploded. Measuring the redshift of the galaxy at the same time allowed us to determine velocity. Combining the two allows us to measure, with increasing accuracy, the expansion rate of the universe.\n\nBecause supernovae are so bright, they provide not only a great tool to measure the Hubble constant, they also allow observers to look back to distances that are a significant fraction of the total age of the universe.\n\nThis offered a new and exciting possibility, which observers viewed as a much more exciting quarry: measuring how Hubble's constant changes over cosmic time.\n\nMeasuring how a constant is changing sounds like an oxymoron, and it would be except for the fact that we humans live such brief lives, at least on a cosmic scale. On a human timescale the expansion rate of the universe is indeed constant. However, as I have just described, the expansion rate of the universe will change over cosmic time due to the effects of gravity.\n\nThe astronomers reasoned that if they could measure the velocity and distance of supernovae located far away\u2014across the far reaches of the visible universe\u2014then they could measure the rate at which the expansion of the universe was slowing down (since everyone assumed the universe was acting sensibly, and the dominant gravitational force in the universe was attractive). This in turn they hoped would reveal whether the universe was open, closed, or flat, because the rate of slowing as a function of time is different for each geometry.\n\nIn 1996, I was spending six weeks visiting Lawrence Berkeley Laboratory, lecturing on cosmology and discussing various science projects with my colleagues there. I gave a talk about our claim that empty space might have energy, and afterward, Saul Perlmutter, a young physicist who was working on detecting distant supernovae, came up to me and said, \"We will prove you wrong!\"\n\nSaul was referring to the following aspect of our suggestion of a flat universe, 70 percent of the energy of which should be contained in empty space. Recall that such energy would produce a cosmological constant, leading to a repulsive force that would then exist throughout all of space and that would dominate the expansion of the universe, causing its expansion to _speed up,_ not slow down.\n\nAs I have described, if the expansion of the universe was speeding up over cosmic time, then the universe would be older today than we would otherwise infer had the expansion been slowing down. This would then imply that the look-back in time to galaxies with a given redshift would be longer than it would otherwise be. In turn, if they have been receding from us for a longer time, this would imply that the light from them originated from farther away. The supernovae in galaxies at some given measured redshift would then appear fainter to us than if the light originated closer. Schematically, if one was measuring velocity versus distance, the slope of the curve for relatively nearby galaxies would allow us to determine the expansion rate today, and then whether the curve bent upward or downward for distant supernovae would tell us whether the universe was speeding up or slowing down over cosmic time.\n\nTwo years after our meeting, Saul and his collaborators, part of an international team called the Supernova Cosmology Project, published a paper based on early preliminary data that indeed suggested we were wrong. (Actually, they did not argue that Turner and I were wrong, since they, along with most of the other observers, really didn't give much credence to our proposal.) Their data suggested that the distance-versus-redshift plot curved downward, and thus that an upper limit on the energy of empty space had to be well below what would have been required to make a significant contribution to the total energy today.\n\nHowever, as often happens, the first data that come in might not be representative of all the data\u2014either you are simply statistically unlucky, or unexpected systematic errors might affect the data, which are not manifest until you have a much bigger sample. This was the case with data that the Supernova Cosmology Project published, and so the conclusions were incorrect.\n\nAnother international supernova search project, called the High-Z Supernova Search Team, led by Brian Schmidt at Mount Stromlo Observatory in Australia, was carrying out a program with the same goal, and they began to obtain different results. Brian recently told me that when their first significant High-Z Supernova determination came in, suggesting an accelerating universe with significant vacuum energy, they were turned down for telescope time and informed by a journal that they must be wrong because the Supernova Cosmology Project had already determined that the universe was indeed flat, and dominated by matter.\n\nThe detailed history of the competition between these two groups will undoubtedly be replayed many times, especially after they share the Nobel Prize, which they undoubtedly will.* This is not the place to worry about priority. Suffice it to say that by early 1998, Schmidt's group published a paper demonstrating that the universe appeared to be accelerating. About six months later, Perlmutter's group announced similar results and published a paper confirming the High-Z Supernova result, in effect acknowledging their earlier error\u2014and lending more credence to a universe dominated by the energy of empty space or, as it is now more commonly called, dark energy.\n\nThe speed with which these results were adopted by the scientific community\u2014even though they required a wholesale revision of the entire accepted picture of the universe\u2014provides an interesting study in scientific sociology. Almost overnight, there appeared to be universal acceptance of the results, even though, as Carl Sagan has emphasized, \"Extraordinary claims require extraordinary evidence.\" This was certainly an extraordinary claim if ever one was.\n\nI was shocked when, in December 1998, _Science_ magazine called the discovery of an accelerating universe the Scientific Breakthrough of the Year, producing a remarkable cover with a drawing of a shocked Einstein.\n\nI wasn't shocked because I thought that the result wasn't worthy of a cover. Quite the contrary. If true, it was one of the most important astronomical discoveries of our time, but the data at the time were merely strongly suggestive. They required such a change in our picture of the universe that I felt that we should all be more certain that other possible causes of the effects observed by the teams could be ruled out definitively before everyone jumped on the cosmological constant bandwagon. As I told at least one journalist at the time, \"The first time I didn't believe in a cosmological constant was when observers claimed to discover it.\"\n\nMy somewhat facetious reaction may seem strange, given that I had been promoting the possibility in one form or another for perhaps a decade. As a theorist, I feel that speculation is fine, especially if it promotes new avenues for experiment. But I believe in being as conservative as possible when examining real data, perhaps because I reached scientific maturity during a period when so many new and exciting but tentative claims in my own field of particle physics turned out to be spurious. Discoveries ranging from a claimed new fifth force in nature to the discovery of new elementary particles to the supposed observation that our universe is rotating as a whole have come and gone with much hoopla.\n\nThe biggest concern at the time regarding the claimed discovery of an accelerating universe was that distant supernovae may appear dimmer than they would otherwise be expected to be, not because of an accelerated expansion, but merely because either (a) they _are_ dimmer, or (b) perhaps some intergalactic or galactic dust present at early times partially obscures them.\n\nIn the intervening decade, it has nevertheless turned out that the evidence for acceleration has become overwhelming, almost unimpeachable. First, many more supernovae at high redshift have been measured. From these, a combined analysis of the supernovae from the two groups done within a year of the original publication yielded the following plot:\n\nAs a guide to the eye, to help you see whether the distance-versus-redshift curve bends upward or downward, the observers have drawn a dotted straight line in the upper half of the plot from the bottom left to the top right corner that goes through the data that represent nearby supernovae. The slope of this line tells us the expansion rate today. Then, in the lower half of the figure they have made that same straight line horizontal, to guide the eye. If the universe were decelerating, as had been expected in 1998, the distant supernovae at a redshift (z) close to 1 would fall below the straight line. But as you can see, most of them fall above the straight line. This is due to either one of two reasons:\n\n1. the data are wrong, or\n\n2. the expansion of the universe _is_ accelerating.\n\nIf we take, for the moment, the second alternative and ask, \"How much energy would we have to put in empty space in order to produce the observed acceleration?\" the answer we come up with is remarkable. The solid curve, which fits the data best, corresponds to a flat universe, with 30 percent of the energy in matter and 70 percent in empty space. This is, remarkably, precisely what is needed in order to make a flat universe consistent with the fact that only 30 percent of the required mass exists in and around galaxies and clusters. An apparent concordance has been achieved.\n\nNevertheless, because the claim that 99 percent of the universe is invisible (1 percent visible matter embedded in a sea of dark matter surrounded by energy in empty space) fits into the category of an extraordinary claim, we should seriously consider the first of the two possibilities I mention above: namely, that the data are wrong. In the intervening decade, all the rest of the data from cosmology has continued to solidify the general concordance picture of a cockamamie, flat universe in which the dominant energy resides in empty space and in which everything we can see accounts for less than 1 percent of the total energy, with the matter we can't see being composed mostly of some yet unknown, new type of elementary particles.\n\nFirst, new data on stellar evolution have improved as new satellites have provided us with information on the elemental abundances in old stars. Using these, my colleague Chaboyer and I were able, in 2005, to demonstrate definitively that the uncertainties in the estimates of the age of the universe using these data were now small enough to rule out lifetimes younger than about 11 billion years. This was inconsistent with any universe in which empty space itself contained a significant amount of energy. Again, since we are not certain that this energy is due to a cosmological constant, it now goes by the simpler name \"dark energy,\" in analogue to the moniker of \"dark matter\" that dominates galaxies.\n\nThis estimate for the age of our universe was vastly improved in about 2006 when new precision measurements of the cosmic microwave background using the WMAP satellite allowed observers to precisely measure the time since the Big Bang. We now know the age of the universe to four significant figures. It is 13.72 billion years old!\n\nI would never have figured that, in my lifetime, we would obtain such accuracy. But now that we have it, we can confirm that there is no way that a universe with the measured expansion rate today could be this old without dark energy, and in particular, dark energy that behaves essentially like the energy represented by a cosmological constant would behave. In other words, it is energy that appears to remain constant over time.\n\nIn the next scientific breakthrough, observers were able to measure accurately how matter, in the form of galaxies, has clustered together over cosmic time. The result depends upon the expansion rate of the universe, as the attractive force pulling galaxies together has to compete with the cosmic expansion driving matter apart. The larger the value of the energy of empty space, the sooner it will come to dominate the energy of the universe, and the sooner the increasing expansion rate will eventually stop the gravitational collapse of matter on ever larger scales.\n\nBy measuring gravitational clustering, therefore, observers have been able to confirm, once again, that the only flat universe that is consistent with observed large-scale structure in the universe is one with approximately 70 percent dark energy and, once again, that dark energy behaves more or less like the energy represented by a cosmological constant.\n\nIndependent of these indirect probes of the expansion history of the universe, the supernova observers have done extensive tests of possibilities that could induce systematic errors in their analysis, including the possibility of increased dust at large distances that make supernovae look dimmer, and ruled them out one by one.\n\nOne of their most important tests involved searching back in time.\n\nEarlier in the history of the universe, when what is now our currently observable region was much smaller in size, the density of matter was much greater. However, the energy density of empty space remains the same over time if it reflects a cosmological constant\u2014or something like it. Thus, when the universe was less than about half its present size, the energy density of matter would have exceeded the energy density of empty space. For all times before this time matter, and not empty space, would have produced the dominant gravitational force acting on the expansion. As a result, the universe would have been decelerating.\n\nIn classical mechanics there is a name for the point at which a system changes its acceleration and, in particular, goes from decelerating to accelerating. It is called a \"jerk.\" In 2003, I organized a conference at my university to examine the future of cosmology and invited one of the High-Z Supernova survey members, Adam Riess, who had told me he would have something exciting to report at the meeting. He did. The next day, the _New York Times,_ which was reporting on the meeting, ran a photo of Adam accompanied by the headline \"Cosmic Jerk Discovered.\" I have kept that photo and turn to it for amusement from time to time.\n\nThe detailed mapping of the expansion history of the universe, demonstrating that it shifted from a period of deceleration to acceleration, added substantial weight to the claim that the original observations, which implied the existence of dark energy, were in fact correct. With all of the other evidence now available, it is very difficult to imagine that, by adhering to this picture, somehow we are being led on a cosmic wild-goose chase. Like it or not, dark energy seems here to stay, or at least to stay until it changes in some way.\n\nThe origin and nature of dark energy is without a doubt the biggest mystery in fundamental physics today. We have no deep understanding of how it originates and why it takes the value it has. We therefore have no idea of why it has begun to dominate the expansion of the universe and only relatively recently, in the past 5 billion years or so, or whether that is a complete accident. It is natural to suspect that its nature is tied in some basic way to the origin of the universe. And all signs suggest that it will determine the future of the universe as well.\n\n* * *\n\n* Indeed, as this book goes to print I just learned that Saul and Brian, along with Adam Reiss, who was part of the High-Z Supernova project, were awarded the Nobel Prize in Physics for 2011 for their discovery.\n\n## CHAPTER 6\n\nTHE FREE LUNCH AT THE END OF THE UNIVERSE\n\n_Space is big. Really big. You just won't believe how vastly, hugely, mind-bogglingly big it is. I mean, you may think it's a long way down the road to the chemist's, but that's just peanuts to space._\n\n\u2014DOUGLAS ADAMS, _The Hitchhiker's Guide to the Galaxy_\n\nOne out of two isn't bad, I suppose. We cosmologists had guessed, correctly it turned out, that the universe is flat, so we weren't that embarrassed by the shocking revelation that empty space indeed has energy\u2014and enough energy in fact to dominate the expansion of the universe. The existence of this energy was implausible, but even more implausible is that the energy is not enough to make the universe uninhabitable. For if the energy of empty space were as large as the a priori estimates I described earlier suggested it should be, the expansion rate would have been so great that everything that we now see in the universe would quickly have been driven beyond the horizon. The universe would become cold, dark, and empty well before stars, our Sun, and our Earth could have formed.\n\nOf all the reasons to suppose that the universe was flat, perhaps the simplest to understand arose from the fact that the universe had been well-known to be almost flat. Even in the early days, before dark matter was discovered, the known amount of visible material in and around galaxies accounted for perhaps 1 percent of the total amount of matter needed to result in a flat universe.\n\nNow, 1 percent may not seem like much, but our universe is very old, billions of years old. Assuming that the gravitational effects of matter or radiation dominate the evolving expansion, which is what we physicists always thought was the case, then if the universe is not precisely flat, as it expands, it moves further and further away from being flat.\n\nIf it is open, the expansion rate continues at a faster rate than it would for a flat universe, driving matter farther and farther apart compared to what it would be otherwise, reducing its net density and very quickly yielding an infinitesimally small fraction of the density required to result in a flat universe.\n\nIf it is closed, then it slows the expansion down faster and eventually causes it to recollapse. All the while, the density first decreases at a slower rate than for a flat universe, and then as the universe recollapses, it starts to increase. Once again, the departure from the density expected for a flat universe increases with time.\n\nThe universe has increased in size by a factor of almost a trillion since it was 1 second old. If, at that earlier moment, the density of the universe was not almost exactly that expected of a flat universe but was, say, only 10 percent of that appropriate for a flat universe at the time, then today the density of our universe would differ from that of a flat universe by at least a factor of a trillion. This is far greater than the mere factor of 100 that was known to separate the density of the visible matter in the universe from the density of what would produce a flat universe today.\n\nThis problem was well-known, even in the 1970s, and it became known as the Flatness Problem. Considering the geometry of the universe is like imagining a pencil balancing vertically on its point on a table. The slightest imbalance one way or the other and it will quickly topple. So it is for a flat universe. The slightest departure from flatness quickly grows. Thus, how could the universe be so close to being flat today if it were not _exactly_ flat?\n\nThe answer is simple: it must be essentially flat today!\n\nThat answer's actually not so simple, because it begs the question, How did initial conditions conspire to produce a flat universe?\n\nThere are two answers to this second, more difficult question. The first goes back to 1981, when a young theoretical physicist and postdoctoral researcher at Stanford University, Alan Guth, was thinking about the Flatness Problem and two other related problems with the standard Big Bang picture of the universe: the so-called Horizon Problem and the Monopole Problem. Only the first need concern us here, since the Monopole Problem merely exacerbates both the Flatness and Horizon problems.\n\nThe Horizon Problem relates to the fact that the cosmic microwave background radiation is extremely uniform. The small temperature deviations, which I described earlier, represented density variations in matter and radiation back when the universe was a few hundred thousand years old, of less than 1 part in 10,000 compared to the otherwise uniform background density and temperature. So while I was focused on the small deviations, a deeper, more urgent question was, How did the universe get to be so uniform in the first place?\n\nAfter all, if instead of the earlier image of the CMBR (where temperature variations of a few parts in 100,000 are reflected in different colors), I showed a temperature map of the microwave sky on a linear scale (with variations in shades representing variations in temperature, of say, \u00b10.03 degree [Kelvin] about the mean background temperature of about 2.72 degrees above absolute zero, or a variation of 1 part in 100 about the mean), the map would look like this:\n\nCompare this image, which contains nothing discernible in the way of structure, to a similar projection of the surface of the Earth, with only slightly greater sensitivity, with color variations representing variations about the mean radius by about 1 part in 500 or so:\n\nThe universe is, therefore, on large scales, _incredibly uniform_!\n\nHow could this be? Well, one might simply assume that, at early times, the early universe was hot, dense, and in thermal equilibrium. This means that any hot spots would have cooled, and cold spots would have heated up until the primordial soup reached the same temperature throughout.\n\nHowever, as I pointed out earlier, when the universe was a few hundred thousand years old, light could have traveled only a few hundred thousand light-years, representing a small percentage of what is now the total observable universe (this former distance would represent merely an angle of about 1 degree on a map of the complete cosmic microwave background last scattering surface as it is observed today). Since Einstein tells us that no information can propagate faster than light, in the standard Big Bang picture, there is simply no way that one part of what is now the observable universe at that time would have been affected by the existence and temperature of other parts on angular scales of greater than about 1 degree. Thus, there is no way that the gas on these scales could have thermalized in time to produce such a uniform temperature throughout!\n\nGuth, a particle physicist, was thinking about processes that could have occurred in the early universe that might have been relevant for understanding this problem when he came up with an absolutely brilliant realization. If, as the universe cooled, it underwent some kind of phase transition\u2014as occurs, for example, when water freezes to ice or a bar of iron becomes magnetized as it cools\u2014then not only could the Horizon Problem be solved, but also the Flatness Problem (and, for that matter, the Monopole Problem).\n\nIf you like to drink really cold beer, you may have had the following experience: you take a cold beer bottle out of the refrigerator, and when you open it and release the pressure inside the container, suddenly the beer freezes completely, during which it might even crack part of the bottle. This happens because, at high pressure, the preferred lowest energy state of the beer is in liquid form, whereas once the pressure has been released, the preferred lowest energy state of the beer is the solid state. During the phase transition, energy can be released because the lowest energy state in one phase can have lower energy than the lowest energy state in the other phase. When such energy is released, it is referred to as \"latent heat.\"\n\nGuth realized that, as the universe itself cooled with the Big Bang expansion, the configuration of matter and radiation in the expanding universe might have gotten \"stuck\" in some meta-stable state for a while until ultimately, as the universe cooled further, this configuration then suddenly underwent a phase transition to the energetically preferred ground state of matter and radiation. The energy stored in the \"false vacuum\" configuration of the universe before the phase transition completed\u2014the \"latent heat\" of the universe, if you will\u2014could dramatically affect the expansion of the universe during the period before the transition.\n\nThe false vacuum energy would behave just like that represented by a cosmological constant because it would act like an energy permeating empty space. This would cause the expansion of the universe at the time to speed up ever faster and faster. Eventually, what would become our observable universe would start to grow faster than the speed of light. This is allowed in general relativity, even though it seems to violate Einstein's special relativity, which says nothing can travel faster than the speed of light. But one has to be like a lawyer and parse this a little more carefully. Special relativity says nothing can travel _through space_ faster than the speed of light. But _space itself_ can do whatever the heck it wants, at least in general relativity. And as space expands, it can carry distant objects, which are at rest in the space where they are sitting, apart from one another at superluminal speeds.\n\nIt turns out that the universe could have expanded during this inflationary period by a factor of more than 1028. While this is an incredible amount, it amazingly could have happened in a fraction of a second in the very early universe. In this case, everything within our entire observable universe was once, before inflation happened, contained in a region much smaller than we would have traced it back to if inflation had not happened, and most important, so small that there would have then been enough time for the entire region to thermalize and reach exactly the same temperature.\n\nInflation made another relatively generic prediction possible. When a balloon gets blown up larger and larger, the curvature at its surface gets smaller and smaller. Something similar happens for a universe whose size is expanding exponentially, as can occur during inflation\u2014driven by a constant and large false vacuum energy. Indeed, by the time inflation ends (solving the Horizon Problem), the curvature of the universe (if it is non-zero to begin with) gets driven to an absurdly small value so that, even today, the universe appears essentially flat when measured accurately.\n\nInflation is the only currently viable explanation of both the homogeneity and flatness of the universe, based on what could be fundamental and calculable microscopic theories of particles and their interactions. But more than this, inflation makes another, perhaps even more remarkable prediction possible. As I have described already, the laws of quantum mechanics imply that, on very small scales, for very short times, empty space can appear to be a boiling, bubbling brew of virtual particles and fields wildly fluctuating in magnitude. These \"quantum fluctuations\" may be important for determining the character of protons and atoms, but generally they are invisible on larger scales, which is one of the reasons why they appear so unnatural to us.\n\nHowever, during inflation, these quantum fluctuations can determine when what would otherwise be different small regions of space end their period of exponential expansion. As different regions stop inflating at slightly (microscopically) different times, the density of matter and radiation that results when the false vacuum energy gets released as heat energy in these different regions is slightly different in each one.\n\nThe pattern of density fluctuations that result after inflation\u2014arising, I should stress, from the quantum fluctuations in otherwise empty space\u2014turns out to be precisely in agreement with the observed pattern of cold spots and hot spots on large scales in the cosmic microwave background radiation. While consistency is not proof, of course, there is an increasing view among cosmologists that, once again, if it walks like a duck and looks like a duck and quacks like a duck, it is probably a duck. And if inflation indeed is responsible for all the small fluctuations in the density of matter and radiation that would later result in the gravitational collapse of matter into galaxies and stars and planets and people, then it can be truly said that we all are here today because of quantum fluctuations in what is essentially _nothing_.\n\nThis is so remarkable I want to stress it again. Quantum fluctuations, which otherwise would have been completely invisible, get frozen by inflation and emerge afterward as density fluctuations that produce everything we can see! If we are all stardust, as I have written, it is also true, if inflation happened, that we all, literally, emerged from quantum nothingness.\n\nThis is so strikingly nonintuitive that it can seem almost magical. But there is at least one aspect of all of this inflationary prestidigitation that might seem particularly worrisome. Where does all the energy come from in the first place? How can a microscopically small region end up as a universe-sized region today with enough matter and radiation within it to account for everything we can see?\n\nMore generally, we might ask the question, How is it that the density of energy can remain constant in an expanding universe with a cosmological constant, or false vacuum energy? After all, in such a universe, space expands exponentially, so that if the density of energy remains the same, the total energy within any region will grow as the volume of the region grows. What happened to the conservation of energy?\n\nThis is an example of something that Guth coined as the ultimate \"free lunch.\" Including the effects of gravity in thinking about the universe allows objects to have\u2014amazingly\u2014\"negative\" as well as \"positive\" energy. This facet of gravity allows for the possibility that positive energy stuff, like matter and radiation, can be complemented by negative energy configurations that just balance the energy of the created positive energy stuff. In so doing, gravity can start out with an empty universe\u2014and end up with a filled one.\n\nThis may also sound kind of fishy, but in fact it is a central part of the real fascination that many of us have with a flat universe. It is also something that you might be familiar with from high school physics.\n\nConsider throwing a ball up in the air. Generally, it will come back down. Now throw it harder (assuming you are not indoors). It will travel higher and stay aloft longer before returning. Finally, if you throw it hard enough, it will not come down at all. It will escape the Earth's gravitational field and keep heading out into the cosmos.\n\nHow do we know when the ball will escape? We use a simple matter of energy accounting. A moving object in the gravitational field of the Earth has two kinds of energy. One, the energy of motion, is called _kinetic energy,_ from the Greek word for motion. This energy, which depends upon the speed of the object, is always positive. The other component of the energy, called _potential energy_ (related to the potential to do work), is generally negative.\n\nThis is the case because we define the total gravitational energy of an object located at rest infinitely far away from any other object as being zero, which seems reasonable. The kinetic energy is clearly zero, and we define the potential energy as zero at this point, so the total gravitational energy is zero.\n\nNow, if the object is not infinitely far away from all other objects but is close to an object, like the Earth, it will begin to fall toward it because of the gravitational attraction. As it falls, it speeds up, and if it smacks into something on the way (say, your head), it can do work by, say, splitting it open. The closer it is to the Earth's surface when it is let go, the less work it can do by the time it hits the Earth. Thus, potential energy _decreases_ as you get closer to the Earth. But if the potential energy is zero when it is infinitely far away from the Earth, it must get more and more negative the closer it gets to the Earth because its potential to do work decreases the closer it gets.\n\nIn classical mechanics, as I defined it here, the definition of potential energy is arbitrary. I could have set the potential energy of an object as zero at the Earth's surface, and then it would be some large number when the object is infinitely far away. Setting the total energy to zero at infinity does make physical sense, but it is, at least at this point in our discussion, merely a convention.\n\nRegardless of where one sets the zero point of potential energy, the wonderful thing about objects that are subject to only the force of gravity is that the _sum_ of their potential and kinetic energies remains a constant. As objects fall, potential energy is converted to the kinetic energy of motion, and as they bounce back up off the ground, kinetic energy is converted back to potential, and so on.\n\nThis allows us a marvelous bookkeeping tool to determine how fast one needs to throw something up in the air in order to escape the Earth, since if it eventually is to reach infinitely far away from the Earth, its total energy must be greater than or equal to zero. I then simply have to ensure that its total gravitational energy at the time it leaves my hand is greater than or equal to zero. Since I can control only one aspect of its total energy\u2014namely the speed with which it leaves my hand\u2014all I have to do is find the magic speed where the positive kinetic energy of the ball equals the negative potential energy it has due to the attraction at the Earth's surface. Both the kinetic energy and the potential energy of the ball depend precisely the same way on the mass of the ball, which therefore cancels out when these two quantities are equated, and one finds a single \"escape velocity\" for all objects from the Earth's surface, namely about 7 miles per second, when the total gravitational energy of the object is precisely zero.\n\nWhat has all this got to do with the universe in general, and inflation in particular, you may ask? Well, the exact same calculation I just described for a ball that I throw up from my hand at the Earth's surface applies to every object in our expanding universe.\n\nConsider a spherical region of our universe centered on our location (in the Milky Way galaxy) and large enough to encompass a lot of galaxies but small enough so that it is well within the largest distances we can observe today:\n\nIf the region is large enough but not too large, then the galaxies at the edge of the region will be receding from us uniformly due to the Hubble expansion, but their speeds will be far less than the speed of light. In this case, the laws of Newton apply, and we can ignore the effects of special and general relativity. In other words, every object is governed by physics that is identical to that which describes the balls that I have just imagined trying to eject from the Earth.\n\nConsider the galaxy shown above, moving away from the center of the distribution as shown. Now, just as for the ball from the Earth, we can ask whether the galaxy will be able to escape from the gravitational pull of all the other galaxies within the sphere. And the calculation we would perform to determine the answer is precisely the same as the calculation we performed for the ball. We simply calculate the total gravitational energy of the galaxy, based on its motion outward (giving it positive energy), and the gravitational pull of its neighbors (providing a negative energy piece). If its total energy is greater than zero, it will escape to infinity, and if less than zero, it will stop and fall inward.\n\nNow, remarkably, it is possible to show that we can rewrite the simple Newtonian equation for the total gravitational energy of this galaxy in a way that reproduces _exactly_ Einstein's equation from general relativity for an expanding universe. And the term that corresponds to the total gravitational energy of the galaxy becomes, in general relativity, the term that describes the curvature of the universe.\n\nSo what do we then find? In a flat universe, and _only_ in a flat universe, the total average Newtonian gravitational energy of each object moving with the expansion is _precisely zero_!\n\nThis is what makes a flat universe so special. In such a universe the positive energy of motion is exactly canceled by the negative energy of gravitational attraction.\n\nWhen we begin to complicate things by allowing for empty space to have energy, the simple Newtonian analogy to a ball being thrown up in the air becomes incorrect, but the conclusion remains essentially the same. In a flat universe, even one with a small cosmological constant, as long as the scale is small enough that velocities are much less than the speed of light, the Newtonian gravitational energy associated with every object in the universe is zero.\n\nIn fact, with a vacuum energy, Guth's \"free lunch\" becomes even more dramatic. As each region of the universe expands to ever larger size, it becomes closer and closer to being flat, so that the total Newtonian gravitational energy of everything that results after the vacuum energy during inflation gets converted to matter and radiation becomes precisely zero.\n\nBut you can still ask, Where does all the energy come from to keep the density of energy constant during inflation, when the universe is expanding exponentially? Here, another remarkable aspect of general relativity does the trick. Not only can the gravitational energy of objects be negative, but their relativistic \"pressure\" can be negative.\n\nNegative pressure is even harder to picture than negative energy. Gas, say in a balloon, exerts pressure on the walls of the balloon. In so doing, if it expands the walls of the balloon, it does work on the balloon. The work it does causes the gas to lose energy and cool. However, it turns out that the energy of empty space is gravitationally repulsive precisely because it causes empty space to have a \"negative\" pressure. As a result of this negative pressure, the universe actually does work _on_ empty space as it expands. This work goes into maintaining the constant energy density of space even as the universe expands.\n\nThus, if the quantum properties of matter and radiation end up endowing even an infinitesimally small region of empty space with energy at very early times, this region can grow to be arbitrarily large and arbitrarily flat. When the inflation is over, one can end up with a universe full of stuff (matter and radiation), and the total Newtonian gravitational energy of that stuff will be as close as one can ever imagine to zero.\n\nSo when all the dust is settled, and after a century of trying, we have measured the curvature of the universe and found it to be zero. You can understand why so many theorists like me have found this not only very satisfying, but also highly suggestive.\n\nA universe from Nothing . . . indeed.\n\n## CHAPTER 7\n\nOUR MISERABLE FUTURE\n\n_The future ain't what it used to be._\n\n\u2014YOGI BERRA\n\nIn one sense it is both remarkable and exciting to find ourselves in a universe dominated by nothing. The structures we can see, like stars and galaxies, were all created by quantum fluctuations from nothing. And the average total Newtonian gravitational energy of each object in our universe is equal to nothing. Enjoy the thought while you can, if you are so inclined, because, if all this is true, we live in perhaps the worst of all universes one can live in, at least as far as the future of life is concerned.\n\nRemember that barely a century ago, Einstein was first developing his general theory of relativity. Conventional wisdom then held that our universe was static and eternal. In fact, Einstein not only ridiculed Lema\u00eetre for suggesting a Big Bang, but also invented the cosmological constant for the purpose of allowing a static universe.\n\nNow, a century later we scientists can feel smug for having discovered the underlying expansion of the universe, the cosmic microwave background, dark matter, and dark energy.\n\nBut what will the future bring?\n\nPoetry . . . of a sort.\n\nRecall that the domination of the expansion of our universe by the energy of seemingly empty space was inferred from the fact that this expansion is speeding up. And, just as with inflation, as described in the last chapter, our observable universe is at the threshold of expanding faster than the speed of light. And with time, because of the accelerated expansion, things will only get worse.\n\nThis means that, the longer we wait, the less we will be able to see. Galaxies that we can now see will one day in the future be receding away from us at faster-than-light speed, which means that they will become invisible to us. The light they emit will not be able to make progress against the expansion of space, and it will never again reach us. These galaxies will have disappeared from our horizon.\n\nThe way this works is a little different than you might imagine. The galaxies do not suddenly disappear or twinkle out of existence in the night sky. Rather, as their recession speed approaches the speed of light, the light from these objects gets ever more redshifted. Eventually, all their visible light moves to infrared, microwave, radio wave, and so on, until the wavelength of light they emit ends up becoming larger than the size of the visible universe, at which point they become officially invisible.\n\nWe can calculate about how long this will take. Since the galaxies in our local cluster of galaxies are all bound together by their mutual gravitational attraction, they will not recede with the background expansion of the universe discovered by Hubble. Galaxies just outside our group are about 1\/5000th the distance out to the point where the recession velocity of objects approaches the speed of light. It will take them about 150 billion years, about 10 times the current age of the universe, to get there, at which point all the light from the stars within the galaxies will have redshifted by a factor of about 5,000. By about 2 trillion years, their light will have redshifted by an amount that will make their wavelength equal to the size of the visible universe, and the rest of the universe will literally have disappeared.\n\nTwo trillion years may seem like a long time, and it is. In a cosmic sense, however, it is nowhere near an eternity. The longest-lived \"main sequence\" stars (which have the same evolutionary history as our Sun) have lifetimes far longer than our Sun and will still be shining in 2 trillion years (even as our own Sun dies out in about only 5 billion years). And so in the far future there may be civilizations on planets around those stars, powered by solar power, with water and organic materials. And there may be astronomers with telescopes on those planets. But when they look out at the cosmos, essentially everything we can now see, all 400 billion galaxies currently inhabiting our visible universe, will have disappeared!\n\nI have tried to use this argument with Congress to urge the funding of cosmology now, while we still have time to observe all that we can! For a congressperson, however, two years is a long time. Two trillion is unthinkable.\n\nIn any case, those astronomers in the far future would be in for a big surprise, if they had any idea what they were missing, which they won't. Because not only will the rest of the universe have disappeared, as my colleague Robert Scherrer of Vanderbilt and I recognized a few years ago, but essentially all of the evidence that now tells us we live in an expanding universe that began in a Big Bang will also have disappeared, along with all evidence of the existence of the dark energy in empty space that will be responsible for this disappearance.\n\nWhile less than a century ago conventional wisdom still held that the universe was static and eternal, with stars and planets coming and going, but on its largest scales the universe itself perduring, in the far future, long after any remnants of our planet and civilization have likely receded into the dustbin of history, the illusion that sustained our civilization until 1930 will be an illusion that will once again return, with a vengeance.\n\nThere are three main observational pillars that have led to the empirical validation of the Big Bang, so that, even if Einstein and Lema\u00eetre had never lived, the recognition that the universe began in a hot, dense state would have been forced upon us: the observed Hubble expansion; the observation of the cosmic microwave background; and the observed agreement between the abundance of light elements\u2014hydrogen, helium, and lithium\u2014we have measured in the universe with the amounts predicted to have been produced during the first few minutes in the history of the universe.\n\nLet's begin with the Hubble expansion. How do we know the universe is expanding? We measure the recession velocity of distant objects as a function of their distance. However, once all visible objects outside of our local cluster (in which we are gravitationally bound) have disappeared from our horizon, there will no longer be any tracers of the expansion\u2014no stars, galaxies, quasars, or even large gas clouds\u2014that observers could track. The expansion will be so efficient that it will have removed all objects from our sight that are actually receding from us.\n\nMoreover, on a timescale of less than a trillion years or so, all the galaxies in our local group will have coalesced into some large meta-galaxy. Observers in the far future will see more or less precisely what observers in 1915 thought they saw: a single galaxy housing their star and their planet, surrounded by an otherwise vast, empty, static space.\n\nRecall also that all evidence that empty space has energy comes from observing the rate of speed-up of our expanding universe. But, once again, without tracers of the expansion, the acceleration of our expanding universe will be unobservable. Indeed, in a strange coincidence, we are living in the only era in the history of the universe when the presence of the dark energy permeating empty space is likely to be detectable. It is true that this era is several hundred billion years long, but in an eternally expanding universe it represents the mere blink of a cosmic eye.\n\nIf we assume that the energy of empty space is roughly constant, as would be the case for a cosmological constant, then in much earlier times the energy density of matter and radiation would have far exceeded that in empty space. This is simply because, as the universe expands, the density of matter and radiation decreases along with the expansion because the distance between particles grows, so there are fewer objects in each volume. At earlier times, say earlier than about 5 billion to 10 billion years ago, the density of matter and radiation would have been far greater than it is today. The universe at this time and earlier was therefore dominated by matter and radiation, with their consequent gravitational attraction. In this case, the expansion of the universe would have been slowing down at these early times, and the gravitational impact of the energy of empty space would have been unobservable.\n\nBy the same token, far in the future, when the universe is several hundred billion years old, the density of matter and radiation will have decreased even further, and one can calculate that dark energy will have a mean energy density far in excess of a thousand billion times greater than the density of all remaining matter and radiation in the universe. It will, by then, completely govern the gravitational dynamics of the universe on large scales. However, at that late age, the accelerating expansion will have become essentially unobservable. In this sense, the energy of empty space ensures, by its very nature, that there is a finite time during which it is observable, and, remarkably, we live during this cosmological instant.\n\nWhat about the other major pillar of the Big Bang, the cosmic microwave background radiation, which provides a direct baby picture of the universe? First, as the universe expands ever faster in the future, the temperature of the CMBR will fall. When the presently observable universe is about 100 times larger than it is now, the temperature of the CMBR will have fallen by a factor of 100, and its intensity, or the energy density stored within it, will have fallen by a factor of 100 million, making it about 100 million times harder to detect than it currently is.\n\nBut, after all, we have been able to detect the cosmic microwave background amidst all the other electronic noise on Earth, and we can imagine that observers in the far future will be 100 million times smarter than those we are blessed with today, so that all hope is not lost. Alas, it turns out that even the brightest observer one could imagine, with the most sensitive instrument one could build, will still be essentially out of luck in the distant future. This is because in our galaxy (or the meta-galaxy that will form when our galaxy merges with its neighbors, beginning with Andromeda in about 5 billion years) there is hot gas between stars, and this gas is ionized, so that it contains free electrons, and thus behaves like a plasma. As I described earlier, such a plasma is opaque to many types of radiation.\n\nThere is something called a \"plasma frequency,\" below which radiation cannot permeate a plasma without absorption. Based on the currently observed density of free electrons in our galaxy, we can estimate the plasma frequency in our galaxy, and if we do this, we find that the bulk of the CMB radiation from the Big Bang will be stretched, by the time the universe gets to be about 50 times its present age, to long enough wavelengths, and hence low enough frequencies, that it will be below our future (meta-) galaxy's plasma frequency at that time. After that, the radiation will essentially not be able to make it into our (meta-)galaxy to be observed, no matter how tenacious the observer. The CMBR, too, will have disappeared.\n\nSo no observed expansion, no leftover afterglow of the Big Bang. But what about the abundance of the light elements\u2014hydrogen, helium, and lithium\u2014which also provides a direct signature of the Big Bang?\n\nIndeed, as I described in chapter 1, whenever I meet someone who doesn't believe in the Big Bang, I like to show them the following figure that I keep as a card in my wallet. I then say: \"See! There was a Big Bang!\"\n\nThis figure looks very complicated, I know, but it actually shows the relative predicted abundance of helium, deuterium, helium-3, and lithium, compared to hydrogen, based on our current understanding of the Big Bang. The green curve, going up and to the right, displays the predicted abundance of helium, the second most abundant element in the universe, by weight, compared with hydrogen (the most abundant element). The red and dark blue curves, going down and to the right represent the predicted abundances of deuterium and helium-3, respectively, not by weight but by number of atoms compared to hydrogen. Finally, the purple lower curve represents the predicted abundance of the next lightest element, lithium, again by number.\n\nThe predicted abundances are plotted as functions of the assumed total density of normal matter (made of atoms) in the universe today. If varying this quantity produced no combination of all the predicted elemental abundances that fit with our observations, it would be strong evidence against their production in a hot Big Bang. Note that the predicted abundances of these elements vary by almost 10 orders of magnitude.\n\nThe unshaded boxes associated with each curve represent the allowed range of the actual estimated primordial abundance of these elements based on observations of old stars and hot gas in and outside of our galaxy\n\nThe vertical blue band then represents that region where all the predictions and observations _do_ agree. It is hard to imagine more concrete support than this agreement between predictions and observations, again for elements whose predicted abundances vary by 10 orders of magnitude, for an early, hot Big Bang where all the light elements were first produced.\n\nIt is worth repeating the implications of this remarkable agreement more forcefully: Only in the first seconds of a hot Big Bang, with an initial abundance of protons and neutrons that would result in something very close to the observed density of matter in visible galaxies today, and a density of radiation that would leave a remnant that would correspond precisely to the observed intensity of the cosmic microwave background radiation today, would nuclear reactions occur that could produce precisely the abundance of light elements, hydrogen and deuterium, helium and lithium, that we infer to have comprised the basic building blocks of the stars that now fill the night sky.\n\nAs Einstein might have put it, only a very malicious (and, therefore, in his mind unimaginable) God would have conspired to have created a universe that so unambiguously points to a Big Bang origin without its having occurred.\n\nIndeed, when the rough agreement between the inferred helium abundance in the universe with the predicted helium abundance arising from a Big Bang was first demonstrated in the 1960s, this was one of the key bits of data that helped the Big Bang picture win out over the then very popular steady-state model of the universe championed by Fred Hoyle and his colleagues.\n\nIn the far future, however, things will be quite different. Stars burn hydrogen, producing helium, for example. At the present time only about 15 percent or so of all the observed helium in the universe could have been produced by stars in the time since the Big Bang\u2014once again, a compelling bit of evidence that a Big Bang was required to produce what we see. But in the far future this will not be the case, because many more generations of stars will have lived and died.\n\nWhen the universe is a trillion years old, for example, far more helium will have been produced in stars than will have been produced in the Big Bang itself. This situation is displayed in the following chart:\n\nWhen 60 percent of the visible matter in the universe is comprised of helium, there will be no necessity for production of primordial helium in a hot Big Bang in order to produce agreement with observations.\n\nObservers and theorists in some civilization in the far future will, however, be able to use this data to infer that the universe must have had a finite age. Because stars burn hydrogen to helium, there will be an upper limit on how long stars could have existed in order not to further deplete the ratio between hydrogen and helium. Thus, future scientists will estimate that the universe in which they live is less than about a trillion years old. But any direct signature that the beginning involved a Big Bang, rather than some other kind of spontaneous creation of our future single (meta-)galaxy, will be lacking.\n\nRemember that Lema\u00eetre derived his claim of a Big Bang purely on the basis of thinking about Einstein's general relativity. We can assume that any advanced civilization in the far future will discover the laws of physics, electromagnetism, quantum mechanics, and general relativity. Will some Lema\u00eetre of the far future therefore be able to derive a similar claim?\n\nLema\u00eetre's conclusion that our universe had to begin in a Big Bang was unavoidable, but it was based on an assumption that will not be true for the observable universe of the far future. A universe with matter stretching out uniformly in all directions, one that is isotropic and homogenous, cannot be static, for the reasons Lema\u00eetre and eventually Einstein recognized. However, there is a perfectly good solution of Einstein's equations for a single massive system surrounded by an otherwise empty static space. After all, if such a solution did not exist, then general relativity would not be able to describe isolated objects like neutron stars or, ultimately, black holes.\n\nLarge mass distributions like our galaxy are unstable, so eventually our (meta-)galaxy will itself collapse to form a massive black hole. This is described by a static solution of Einstein's equation called the Schwarzschild solution. But the time frame for our galaxy to collapse to form a massive black hole is much longer than the time frame for the rest of the universe to disappear. Thus, it will seem natural for scientists of the future to imagine that our galaxy could have existed for a trillion years in empty space without significant collapse and without requiring an expanding universe surrounding it.\n\nOf course, speculations about the future are notoriously difficult. I am writing this, in fact, while at the World Economic Forum in Davos, Switzerland, which is full of economists who invariably predict the behavior of future markets and revise their predictions when they turn out to be horribly wrong. More generally, I find any predictions of the far future, and even the not-so-far future, of science and technology to be even sketchier than those of \"the dismal science.\" Indeed, whenever I'm asked about the near future of science or what the next big breakthrough will be, I always respond that if I knew, I would be working on it right now!\n\nThus, I like to think of the picture I have presented in this chapter as something like the picture of the future presented by the third ghost in Dickens's _A Christmas Carol_. This is the future as it _might be_. After all, since we have no idea what the dark energy permeating empty space is, we also therefore cannot be certain that it will behave like Einstein's cosmological constant and remain constant. If it doesn't, the future of the universe could be far different. The expansion may not continue to accelerate, but instead may once again slow down over time so that distant galaxies will not disappear. Alternatively, perhaps there will be some new observable quantities we cannot yet detect that may provide astronomers in the future with evidence that there was once a Big Bang.\n\nNevertheless, based on everything we know about the universe today, the future I have sketched out is the most plausible one, and it is fascinating to consider whether logic, reason, and empirical data might still somehow induce future scientists to infer the correct underlying nature of our universe, or whether it will forever remain obscured behind the horizon. Some brilliant future scientist exploring the fundamental nature of forces and particles might derive a theoretical picture that will suggest that inflation must have happened, or that there must be an energy in empty space, which would further explain why there are no galaxies within the visible horizon. But I am not so sanguine about this.\n\nPhysics is, after all, an empirical science, driven by experiment and observation. Had we not observationally inferred the existence of dark energy, I doubt any theorist would have been bold enough to suggest its existence today. And while it is also possible to imagine tentative signatures that might suggest something is wrong with the picture of a single galaxy in a static universe without a Big Bang\u2014perhaps some observation of elemental abundances that appears anomalous\u2014I suspect that Occam's razor will suggest that the simplest picture is the correct one, and that the anomalous observations might be explained by some local effects.\n\nEver since Bob Scherrer and I laid out the challenge that future scientists will use falsifiable data and models\u2014the very paragon of good science\u2014but in the process that they will come up with a false picture of the universe, many of our colleagues have tried to suggest ways to probe that the universe is actually expanding in the far future. I too can imagine possible experiments. But I cannot see that they would be well motivated.\n\nFor example, you would need to eject bright stars from our galaxy and send them off into space, wait a billion years or so for them to explode, and try to observe their recession velocities as a function of the distance they reach before they explode in order to probe to see if they are getting any extra kick from a possible expansion of space. A tall order, but even if you could imagine somehow pulling this off, I cannot see the National Science Foundation of the future actually funding the experiment without at least some other motivation for arguing on behalf of an expanding universe. And if somehow stars from our galaxy are naturally ejected and detectable as they move out toward the horizon, it is not clear to me that observing an anomalous acceleration of some of these objects would be interpreted in terms of such a bold and strange proposal as an expanding universe dominated by dark energy.\n\nWe can consider ourselves lucky that we live at the present time. Or as Bob and I put it in one of the articles we wrote: \"We live at a very special time . . . the only time when we can observationally verify that we live at a very special time!\"\n\nWe were being somewhat facetious, but it is sobering to suggest that one can use the best observational tools and theoretical tools at one's disposal and nevertheless come up with a completely false picture of the large-scale universe.\n\nI should point out, nevertheless, that even though incomplete data _can_ lead to a false picture, this is far different from the (false) picture obtained by those who choose to ignore empirical data to invent a picture of creation that would otherwise contradict the evidence of reality (young earthers, for example), or those who instead require the existence of something for which there is no observable evidence whatsoever (like divine intelligence) to reconcile their view of creation with their a priori prejudices, or worse still, those who cling to fairy tales about nature that presume the answers before questions can even be asked. At least the scientists of the future will be basing their estimates on the best evidence available to them, recognizing as we all do, or at least as scientists do, that new evidence may cause us to change our underlying picture of reality.\n\nIn this regard, it is worth adding that perhaps we are missing something even today that might have been observable had only we lived 10 billion years ago or perhaps could see if we lived 100 billion years into the future. Nevertheless, I should stress that the Big Bang picture is too firmly grounded in data from every area to be proved invalid in its general features. But some new, nuanced understanding of the fine details of the distant past or distant future, or of the origin of the Big Bang and its possible uniqueness in space, might easily emerge with new data. In fact, I hope it will. One lesson that we can draw from the possible future end of life and intelligence in the universe is that we need to have some cosmic humility in our claims, even if such a thing is difficult for cosmologists.\n\nEither way, the scenario I have just described has a certain poetic symmetry, even if it is equally tragic. Long into the future, scientists will derive a picture of the universe that will hearken back to the very picture we had at the beginning of the last century, which itself ultimately served as the catalyst for investigations that led to the modern revolutions in cosmology. Cosmology will have come full circle. I for one find that remarkable, even if it underscores what some may view as the ultimate futility of our brief moment in the sun.\n\nRegardless, the fundamental problem illustrated by the possible future end of cosmology is that we have only one universe to test\u2014the one we live in. While test it we must if we want to have any hope of understanding how what we now observe arose, we nevertheless are limited in both what we can measure and in our interpretations of the data.\n\nIf many universes exist, and if we could somehow probe more than one, we might have a better chance of knowing which observations are truly significant and fundamental and which arise only as an accident of our circumstances.\n\nAs we shall see next, while the latter possibility is unlikely, the former is not, and scientists are pressing forward with new tests and new proposals to further our understanding of the unexpected and strange features of our universe.\n\nBefore proceeding, however, it is perhaps worth ending with another, more literary picture of the likely future I have presented here and one that is particularly relevant to the subject of this book. It comes from Christopher Hitchens's response to the scenario I have just described. As he put it, \"For those who find it remarkable that we live in a universe of Something, just wait. Nothingness is heading on a collision course right toward us!\"\n\n## CHAPTER 8\n\nA GRAND ACCIDENT?\n\n_Once you assume a creator and a plan, it makes humans objects in a cruel experiment whereby we are created to be sick and commanded to be well._\n\n\u2014CHRISTOPHER HITCHENS\n\nWe are hardwired to think that everything that happens to us is significant and meaningful. We have a dream that a friend is going to break her arm, and the next day we find out that she sprained her ankle. Wow! Cosmic! Clairvoyant?\n\nThe physicist Richard Feynman used to like to go up to people and say: \"You won't believe what happened to me today! You just won't believe it!\" And when they would inquire what happened, he would say, \"Absolutely nothing!\" By this he was suggesting that when something like the dream I described above happens, people ascribe significance to it. But they forget the myriad nonsense dreams they had that predicted absolutely nothing. By forgetting that most of the time nothing of note occurs during the day, we then misread the nature of probability when something unusual does occur: among any sufficiently large number of events, something unusual is bound to happen just by accident.\n\nHow does this apply to our universe?\n\nUntil the discovery that, inexplicably, the energy of empty space is not only not zero, but takes a value that is 120 orders of magnitude smaller than the estimate I described based on ideas from particle physics suggests, the conventional wisdom among physicists was that every fundamental parameter we measured in nature _is_ significant. By this I mean that, somehow, on the basis of fundamental principles, we would eventually be able to understand things such as why gravity is so much weaker than the other forces of nature, why the proton is 2,000 times heavier than the electron, and why there are three families of elementary particles. Put another way, once we understood the fundamental laws that govern the forces of nature at its smallest scales, all of these current mysteries would be revealed as natural consequences of these laws.\n\n(A purely religious argument, on the other hand, could take significance to an extreme by suggesting that each fundamental constant is significant because God presumably chose each one to have the value it does as part of a divine plan for our universe. In this case, nothing is an accident, but by the same token, nothing is predicted or actually explained. It is an argument by fiat that goes nowhere and yields nothing useful about the physical laws governing the universe, other than perhaps providing consolation for the believer.)\n\nBut the discovery that empty space has energy started a revision in thinking among many physicists about what is required in nature and what may be accidental.\n\nThe catalyst for this new gestalt originates from the argument I gave in the last chapter: dark energy is measurable today because \"now\" is the only time in the history of the universe when the energy in empty space is comparable to the energy density in matter.\n\nWhy should we be living at such a \"special\" time in the history of the universe? Indeed, this flies in the face of everything that has characterized science since Copernicus. We have learned that the Earth is not the center of the solar system and that the Sun is a star on the lonely outer edges of a galaxy that is merely one out of 400 billion galaxies in the observable universe. We have come to accept the \"Copernican principle\" that there is nothing special about our place and time in the universe.\n\nBut with the energy of empty space being what it is, we _do_ appear to live at a special time. This is shown best by the following illustration of a \"brief history of time.\"\n\nThe two curves represent the energy density of all matter in the universe, and the energy density of empty space (presuming it is a cosmological constant) as a function of time. As you can see, the density of matter falls, as the universe expands (as the distance between galaxies becomes ever greater and matter therefore gets \"diluted\"), just as you would expect. However, the energy density in empty space remains constant, because, one might argue, with empty space there is nothing to dilute! (Or, as I have somewhat less facetiously described, the universe does work on empty space as it expands.) The two curves cross relatively close to the present time, which is the source of the strange coincidence I have described.\n\nNow consider what would happen if the energy in empty space were, say, 50 times greater than the value we estimate today. Then the two curves would cross at a different, earlier time, as shown in the figure below.\n\nThe time that the two curves cross for the upper, enlarged value of the energy of empty space is the time when galaxies first formed, about a billion years after the Big Bang. But remember that the energy of empty space is gravitationally repulsive. If it had come to dominate the energy of the universe before the time of galaxy formation, the repulsive force due to this energy would have outweighed (literally) the normal attractive gravitational force that caused matter to clump together. And galaxies would never have formed!\n\nBut if galaxies hadn't formed, then stars wouldn't have formed. And if stars hadn't formed, planets wouldn't have formed. And if planets hadn't formed, then astronomers wouldn't have formed!\n\nSo, in a universe with an energy of empty space merely 50 times bigger than that we observe, apparently no one would have been around today to try to measure the energy.\n\nCould this be telling us something? Shortly after the discovery of our accelerating universe, physicist Steven Weinberg proposed, based on an argument he had developed more than a decade earlier\u2014before the discovery of dark energy\u2014that the \"Coincidence Problem\" could therefore be solved if perhaps the value of the cosmological constant that we measure today were somehow \"anthropically\" selected. That is, if somehow there were many universes, and in each universe the value of the energy of empty space took a randomly chosen value based on some probability distribution among all possible energies, then only in those universes in which the value is not that different from what we measure would life as we know it be able to evolve. So maybe we find ourselves in a universe with a tiny energy in empty space because we couldn't find ourselves in one with a much larger value. Put another way, it is not too surprising to find that we live in a universe in which we can live!\n\nThis argument, however, makes mathematical sense only if there is a possibility that many different universes have arisen. Talking about many different universes can sound like an oxymoron. After all, traditionally the notion of universe has become synonymous with \"everything that exists.\"\n\nMore recently, however, _universe_ has come to have a simpler, arguably more sensible meaning. It is now traditional to think of \"our\" universe as comprising simply the totality of all that we can now see and all that we could ever see. Physically, therefore, our universe comprises everything that either once could have had an impact upon us or that ever will.\n\nThe minute one chooses this definition for a universe, the possibility of other \"universes\"\u2014regions that have always been and always will be causally disconnected from ours, like islands separated from any communication with one another by an ocean of space\u2014becomes possible, at least in principle.\n\nOur universe is so vast that, as I have emphasized, something that is not impossible is virtually guaranteed to occur somewhere within it. Rare events happen all the time. You might wonder whether the same principle applies to the possibility of many universes, or a _multiverse,_ as the idea is now known. It turns out that the theoretical situation is actually stronger than simply a possibility. A number of central ideas that drive much of the current activity in particle theory today appear to require a multiverse.\n\nI want to stress this because, in discussions with those who feel the need for a creator, the existence of a multiverse is viewed as a cop-out conceived by physicists who have run out of answers\u2014or perhaps questions. This may eventually be the case, but it is not so now. Almost every logical possibility we can imagine regarding extending laws of physics as we know them, on small scales, into a more complete theory, suggests that, on large scales, our universe is not unique.\n\nThe phenomenon of inflation provides perhaps the first, and perhaps best, rationale. In the inflationary picture, during the phase when a huge energy temporarily dominates some region of the universe, this region begins to expand exponentially. At some point, a small region within this \"false vacuum\" may exit inflation as a phase transition occurs within the region and the field within it relaxes to its true, lower energy value; the expansion within this region will then cease to be exponential. But the space _between_ such regions will continue to expand exponentially. At any one time, unless the phase transition completes through all of space, then almost all of space lies within an inflating region. And the inflating region will separate those regions that first exit inflation by almost unfathomable distances. It is like lava pouring out of a volcano. Some of the rock will cool and solidify, but those rocks will be carried far apart from one another as they float on a sea of liquid magma.\n\nThe situation can be even more dramatic. In 1986, Andrei Linde, who along with Alan Guth has been one of the chief architects of modern inflationary theory, promoted and explored a possibly even more general scenario. This was also anticipated in some sense by another inventive Russian cosmologist in the United States, Alex Vilenkin. Both Linde and Vilenkin have the inner confidence that one finds in great Russian physicists, but their history is quite different. Linde thrived in the old Soviet physics establishment before immigrating to the United States after the fall of the Soviet Union. Brash, brilliant, and funny, he has continued to dominate much of theoretical particle cosmology in the interim. Vilenkin emigrated far earlier, before he was a physicist, and worked in the United States in various jobs, including as a night watchman, while he studied. And while he was always interested in cosmology, he accidentally applied to the wrong school for graduate work and ended up doing a thesis in condensed matter physics\u2014the physics of materials. He then got a job as a postdoctoral researcher at Case Western Reserve University, where I later became Chair. During this period, he asked his supervisor, Philip Taylor, if he could spend several days a week working on cosmology in addition to his assigned projects. Philip later told me that, even with this part-time labor, Alex was the most productive postdoc he had ever had.\n\nIn any case, what Linde recognized is that, while quantum fluctuations during inflation may often push the field that drives inflation toward its lowest energy state, and thus provide a graceful exit, there is always the possibility that, in some regions, quantum fluctuations will drive the field toward yet higher energies, and hence away from values where inflation will end, so that inflation will continue unabated. Because such regions will expand for longer periods of time, there will be far more space that is inflating than that which is not. Within these regions, quantum fluctuations again will drive some subregions to exit inflation and thus stop expanding exponentially, but again there will be regions where quantum fluctuations will cause inflation to persist even longer. And so on.\n\nThis picture, which Linde dubbed \"chaotic inflation,\" indeed resembles more familiar chaotic systems on Earth. Take boiling oatmeal, for example. At any point a bubble of gas may burst from the surface, reflecting regions where liquid at high temperature completes a phase transition to form a vapor. But between the bubbles the oatmeal is roiling and flowing. On large scales there is regularity\u2014there are always bubbles popping somewhere. But locally, things are quite different depending upon where one looks. So it would be in a chaotically inflating universe. If one happened to be located in a \"bubble\" of true ground state that had stopped inflating, one's universe would appear very different from the vast bulk of space around it, which would still be inflating.\n\nIn this picture, inflation is eternal. Some regions, indeed most of space, will go on inflating forever. Those regions that exit inflation will become separate, causally disconnected universes. I want to stress that a multiverse is _inevitable_ if inflation is eternal, and eternal inflation is by far the most likely possibility in most, if not all, inflationary scenarios. As Linde put it in his 1986 paper:\n\nThe old question why our universe is the only possible one is now replaced by the question in which theories [of] the existence of mini-universes of our type [are] possible. This question is still very difficult, but it is much easier than the previous one. In our opinion, the modification of the point of view on the global structure of the universe and on our place in the world is one of the most important consequences of the development of the inflationary universe scenario.\n\nAs Linde emphasized, and has since become clear, this picture also provides another new possibility for physics. It could easily be that there are many possible low-energy quantum states of the universe present in nature that an inflating universe might ultimately decay into. Because the configuration of the quantum states of these fields will be different in each such region, the character of the fundamental laws of physics in each region\/universe can then appear different.\n\nHere arose the first \"landscape\" in which the anthropic argument, provided earlier, could play itself out. If there are many different states in which our universe could end up in after inflation, perhaps the one we live in, one in which there is non-zero vacuum energy that is small enough so galaxies could form, is just one of a potentially infinite family and the one that is selected for inquisitive scientists because it supports galaxies, stars, planets, and life.\n\nThe term \"landscape\" did not, however, first arise in this context. It was promoted by a much more effective marketing machine associated with the juggernaut that has been driving particle theory for much of the past quarter century\u2014string theory. String theory posits that elementary particles are made up of more fundamental constituents, not particles, but objects that behave like vibrating strings. Just as string vibrations on a violin can create different notes, so too in this theory different sorts of vibrations produce objects that might, in principle, behave like all the different elementary particles we find in nature. The catch, however, is that the theory is not mathematically consistent when defined in merely four dimensions, but appears to require many more to make sense. What happens to the other dimensions is not immediately obvious, nor is the issue of what other objects besides strings may be important to define the theory\u2014just some of the many unsolved challenges that have presented themselves and dulled some of the early enthusiasm for this idea.\n\nHere is not the place to thoroughly review string theory, and in fact a thorough review is probably not possible, because if one thing has become clear in the past twenty-five years, it is that what was formerly called string theory is clearly something much more elaborate and complicated, and something whose fundamental nature and makeup is still a mystery.\n\nWe still have no idea if this remarkable theoretical edifice actually has anything to do with the real world. Nevertheless, perhaps no theoretical picture has ever so successfully permeated the consciousness of the physics community without having yet demonstrated its ability to successfully resolve a single experimental mystery about nature.\n\nMany people will take the last sentence as a criticism of string theory, but although I have been branded in the past as a detractor, that is not really my intent here, nor has it been my intent in the numerous lectures and well-intentioned public debates I have had with my friend Brian Greene, one of string theory's main proponents, on the subject. Rather, I think it is simply important to cut through the popular hype for a reality check. String theory involves fascinating ideas and mathematics that might shed light on one of the most fundamental inconsistencies in theoretical physics\u2014our inability to cast Einstein's general relativity in a form that can be combined with the laws of quantum mechanics to result in sensible predictions about how the universe behaves on its very smallest scales.\n\nI have written a whole book about how string theory has attempted to circumvent this problem, but for our purposes here, only a very brief summary is necessary. The central proposal is simple to state, if difficult to implement. On very small scales, appropriate to the scale where the problems between gravity and quantum mechanics might first be encountered, elementary strings may curl up into closed loops. Amidst the set of excitations of such closed loops there always exists one such excitation that has the properties of the particle that, in quantum theory, conveys the force of gravity\u2014the graviton. Thus, the quantum theory of such strings provides, in principle, the playing field on which a true quantum theory of gravity might be built.\n\nSure enough, it was discovered that such a theory might avoid the embarrassing infinite predictions of the standard quantum approaches to gravity. There was one hitch, however. In the simplest version of the theory, such infinite predictions can be obviated only if the strings that make up elementary particles are vibrating, not merely in the three dimensions of space and one of time that we are all familiar with, but rather in twenty-six dimensions!\n\nYou might expect that such a leap of complexity (and, perhaps, faith) would be enough to turn off most physicists about the theory, but in the mid-1980s some beautiful mathematical work by a host of individuals, most notably Edward Witten at the Institute for Advanced Study, demonstrated that the theory could in principle do far more than just provide a quantum theory of gravity. By introducing new mathematical symmetries, most notably a remarkably powerful mathematical framework called \"supersymmetry,\" it became possible to reduce the number of dimensions required for consistency of the theory from twenty-six to merely ten.\n\nMore important, however, it looked like it might be possible, within the context of string theory, to unify gravity with the other forces in nature in a single theory, and moreover possible to explain the existence of every single elementary particle known in nature! Finally, it appeared as if there might be a single unique theory in ten dimensions that would reproduce everything we see in our four-dimensional world.\n\nClaims of a \"Theory of Everything\" began to propagate, not just in the scientific literature, but in popular literature as well. As a result, perhaps more people are familiar with \"superstrings\" than are familiar with \"superconductivity\"\u2014the latter being the remarkable fact that when some materials are cooled to extremely low temperatures, they can conduct electricity without any resistance whatsoever. This is not only one of the most remarkable properties of matter ever observed, but it has already transformed our understanding of the quantum makeup of materials.\n\nAlas, the intervening twenty-five years or so have not been kind to string theory. Even as the best theoretical minds in the world began to focus their attention on it, producing volumes of new results and a great deal of new mathematics in the process (Witten went on to win the highest prize in mathematics, for example), it became clear that the \"strings\" in string theory are probably not the fundamental objects at all. Other, more complicated structures, called \"branes,\" named after membranes in cells, which exist in higher dimensions, probably control the behavior of the theory.\n\nWhat is worse, the uniqueness of the theory began to disappear. After all, the world of our experience is not ten-dimensional, but rather four-dimensional. Something has to happen to the remaining six spatial dimensions, and the canonical explanation of their invisibility is that they are somehow \"compactified\"\u2014that is, they are curled up on such small scales that we cannot resolve them on our scales or even on the tiny scales that are probed by our highest energy particle accelerators today.\n\nThere is a difference between these proposed hidden domains and the domains of spirituality and religion, even though they may not appear so different on the surface. In the first place, they are accessible in principle if one could build a sufficiently energetic accelerator\u2014beyond the bounds of practicality perhaps, but not beyond the bounds of possibility. Second, one might hope, as one does for virtual particles, to find some indirect evidence of their existence via the objects we can measure in our four-dimensional universe. In short, because these dimensions were proposed as part of a theory developed to actually attempt to explain the universe, rather than justify it, they might ultimately be accessible to empirical testing, even if the likelihood is small.\n\nBut beyond this, the possible existence of these extra dimensions provides a huge challenge to the hope that our universe is unique. Even if one starts with a unique theory in ten dimensions (which, I repeat, we do not yet know exist), then every different way of compactifying the invisible six dimensions can result in a different type of four-dimensional universe, with different laws of physics, different forces, different particles, and governed by differing symmetries. Some theorists have estimated that there are perhaps 10500 different possible consistent four-dimensional universes that could result from a single ten-dimensional string theory. A \"Theory of Everything\" had suddenly become a \"Theory of Anything\"!\n\nThis situation was exemplified sarcastically in a cartoon from one of my favorite scientific comic strips, called _xkcd_. In this strip one person says to another: \"I just had an awesome idea. What if all matter and energy is made of tiny vibrating strings.\" The second person then says, \"Okay. What would that imply?\" To which the first person responds: \"I dunno.\"\n\nOn a slightly less facetious note, the Nobel Prize\u2013winning physicist Frank Wilczek has suggested that string theorists have invented a new way of doing physics, reminiscent of a novel way of playing darts. First, one throws the dart against a blank wall, and then one goes to the wall and draws a bull's-eye around where the dart landed.\n\nWhile Frank's comment is an accurate reflection of much of the hype that has been generated, it should be stressed that at the same time those working on the theory are honestly trying to uncover principles that might govern the world in which we live. Nevertheless, the plethora of possible four-dimensional universes, which used to be such an embarrassment for string theorists, has now become a virtue of the theory. One can imagine that, in a ten-dimensional \"multiverse\" one can embed a host of different four-dimensional universes (or five-dimensional ones, or six-dimensional ones, or so on . . .), and each one can have different laws of physics, and moreover, in each one the energy of empty space can be different.\n\nWhile it sounds like a convenient fabrication, it appears to be an automatic consequence of the theory, and it does create a true multiverse \"landscape\" that might provide a natural framework for developing an anthropic understanding of the energy of empty space. In this case, we do not need an infinite number of possible universes separated in three-dimensional space. Rather, we can imagine an infinite number of universes stacked up above a single point in our space, invisible to us, but each of which could exhibit remarkably different properties.\n\nI want to emphasize that this theory is not as trivial as the theological musing of Saint Thomas Aquinas about whether several angels could occupy the same place, an idea that was derided by later theologians as fruitless speculations on how many angels could fit on the point of a needle\u2014or most popularly, on the head of a pin. Aquinas actually answered this question himself by saying that more than one angel could not occupy the same space\u2014of course, without any theoretical or experimental justification! (And if they were bosonic quantum angels, he would have been wrong in any case.)\n\nPresented with such a picture, and adequate mathematics, one might hope, in principle, to actually make physical predictions. For example, one might derive a probability distribution describing the likelihood of finding different types of four-dimensional universes embedded in a larger dimensional multiverse. One might find, for example, that the bulk of such universes that have small vacuum energy also have three families of elementary particles and four different forces. Or one might find that only in universes with small vacuum energy could there exist a long-range force of electromagnetism. Any such result might provide reasonably compelling evidence that a probabilistic anthropic explanation of the energy of empty space\u2014in other words, finding that a universe that looks like ours with small vacuum energy is not improbable\u2014makes solid physical sense.\n\nYet the mathematics has not yet brought us this far, and it may never do so. But in spite of our current theoretical impotence, this does not mean that this possibility is not actually realized by nature.\n\nNevertheless, in the meantime, particle physics has taken anthropic reasoning a step further.\n\nParticle physicists are way ahead of cosmologists. Cosmology has produced one totally mysterious quantity: the energy of empty space, about which we understand virtually nothing. However, particle physics has not understood many more quantities for far longer!\n\nFor example: Why are there three generations of elementary particles\u2014the electron, and its heavier cousins the muon and tauon, for example, or the three different sets of quarks, of which the lowest energy set makes up the bulk of matter we find on Earth? Why is gravity so much weaker than the other forces in nature, such as electromagnetism? Why is the proton 2,000 times heavier than the electron?\n\nSome particle physicists have now jumped on the anthropic bandwagon in the extreme, perhaps because their efforts to explain these mysteries according to physical causes have not yet been successful. After all, if one fundamental quantity in nature is actually an environmental accident, why aren't most or all of the other fundamental parameters? Maybe all of the mysteries of particle theory can be solved by invoking the same mantra: if the universe were any other way, we could not live in it.\n\nOne might wonder if such a solution of the mysteries of nature is any solution at all or, more important, whether it describes science as we understand it. After all, the goal of science, and in particular physics, over the past 450 years has been to explain why the universe must be the way we measure it to be, rather than why in general the laws of nature would produce universes that are quite different.\n\nI have tried to explain why this is not quite the case, namely why many respectable scientists have turned to the anthropic principle and why a number have worked quite hard to see if we might learn something new about our universe based on it.\n\nLet me now go further and try to explain how the existence of forever undetectable universes\u2014either removed from us by virtually infinite distances in space or, right beyond the tip of our noses, removed from us by microscopic distances in possible extra dimensions\u2014might nevertheless be subject to some kind of empirical testing.\n\nImagine, for example, that we devised a theory based on unifying at least three of the four forces of nature in some Grand Unified Theory, a subject of continued intense interest in particle physics (among those who have not given up looking for fundamental theories in four dimensions). Such a theory would make predictions about the forces of nature that we measure and about the spectrum of elementary particles that we probe at our accelerators. Should such a theory make a host of predictions that are subsequently verified in our experiments, we would have very good reason to suspect that it contains a germ of truth.\n\nNow, suppose this theory also predicts a period of inflation in the early universe, and in fact predicts that our inflationary epoch is merely one of a host of such episodes in an eternally inflating multiverse. Even if we could not explore the existences of such regions beyond our horizon directly, then, as I have said earlier, if it walks like a duck and quacks like a duck . . . Well, you know.\n\nFinding possible empirical support for the ideas surrounding extra dimensions is more far-fetched but not impossible. Many bright young theorists are devoting their professional careers to the hope of developing the theory to the point where there might be some evidence, even indirect, that it is correct. Their hopes might be misplaced, but they have voted with their feet. Perhaps some evidence from the new Large Hadron Collider near Geneva will reveal some otherwise hidden window into this new physics.\n\nSo, after a century of remarkable, truly unprecedented progress in our understanding of nature, we have found ourselves able to probe the universe on scales that were previously unimaginable. We have understood the nature of the Big Bang expansion back to its earliest microseconds and have discovered the existence of hundreds of billions of new galaxies, with hundreds of billions of new stars. We have discovered that 99 percent of the universe is actually invisible to us, comprising dark matter that is most likely some new form of elementary particle, and even more dark energy, whose origin remains a complete mystery at the present time.\n\nAnd after all of this, it may be that physics will become an \"environmental science.\" The fundamental constants of nature, so long assumed to take on special importance, may just be environmental accidents. If we scientists tend to take ourselves and our science too seriously, maybe we also have taken our universe too seriously. Maybe literally, as well as metaphorically, we are making much ado about nothing. At least we may be making too much of the nothing that dominates our universe! Maybe our universe is rather like a tear buried in a vast multiversal ocean of possibilities. Maybe we will never find a theory that describes why the universe has to be the way it is.\n\nOr maybe we will.\n\nThat, finally, is the most accurate picture I can paint of reality as we now understand it. It is based on the work of tens of thousands of dedicated minds over the past century, building some of the most complex machines ever devised and developing some of the most beautiful and also the most complex ideas with which humanity has ever had to grapple. It is a picture whose creation emphasizes the best about what it is to be human\u2014our ability to imagine the vast possibilities of existence and the adventurousness to bravely explore them\u2014without passing the buck to a vague creative force or to a creator who is, by definition, forever unfathomable. We owe it to ourselves to draw wisdom from this experience. To do otherwise would do a disservice to all the brilliant and brave individuals who helped us reach our current state of knowledge.\n\nIf we wish to draw philosophical conclusions about our own existence, our significance, and the significance of the universe itself, our conclusions should be based on empirical knowledge. A truly open mind means forcing our imaginations to conform to the evidence of reality, and not vice versa, whether or not we like the implications.\n\n## CHAPTER 9\n\nNOTHING IS SOMETHING\n\n_I don't mind not knowing. It doesn't scare me._\n\n\u2014RICHARD FEYNMAN\n\nIsaac Newton, perhaps the greatest physicist of all time, profoundly changed the way we think about the universe in many ways. But perhaps the most important contribution he made was to demonstrate the possibility that the entire universe is explicable. With his universal law of gravity, he demonstrated for the first time that even the heavens might bend to the power of natural laws. A strange, hostile, menacing, and seemingly capricious universe might be nothing of the sort.\n\nIf immutable laws governed the universe, the mythical gods of ancient Greece and Rome would have been impotent. There would have been no freedom to arbitrarily bend the world to create thorny problems for mankind. What held for Zeus would also apply to the God of Israel. How could the Sun stand still at midday if the Sun did not orbit the Earth but its motion in the sky was actually caused by the revolution of the Earth, which, if suddenly stopped, would produce forces on its surface that would destroy all human structures and humans along with them?\n\nOf course, supernatural acts are what miracles are all about. They are, after all, precisely those things that circumvent the laws of nature. A god who can create the laws of nature can presumably also circumvent them at will. Although why they would have been circumvented so liberally thousands of years ago, before the invention of modern communication instruments that could have recorded them, and not today, is still something to wonder about.\n\nIn any case, even in a universe with no miracles, when you are faced with a profoundly simple underlying order, you can draw two different conclusions. One, drawn by Newton himself, and earlier espoused by Galileo and a host of other scientists over the years, was that such order was created by a divine intelligence responsible not only for the universe, but also for our own existence, and that we human beings were created in her image (and apparently other complex and beautiful beings were not!). The other conclusion is that the laws themselves are all that exist. These laws themselves require our universe to come into existence, to develop and evolve, and we are an irrevocable by-product of these laws. The laws may be eternal, or they too may have come into existence, again by some yet unknown but possibly purely physical process.\n\nPhilosophers, theologians, and sometimes scientists continue to debate these possibilities. We do not know for certain which of them actually describes our universe, and perhaps we shall never know. But the point is, as I emphasized at the very beginning of this book, the final arbiter of this question will not come from hope, desire, revelation, or pure thought. It will come, if it ever does, from an exploration of nature. Dream or nightmare, as Jacob Bronowski said in the opening quote in the book\u2014and one person's dream in this case can easily be another's nightmare\u2014we need to live our experience as it is and with our eyes open. The universe is the way it is, whether we like it or not.\n\nAnd here, I think it is _extremely significant_ that a universe from nothing\u2014in a sense I will take pains to describe\u2014that arises naturally, and even inevitably, is increasingly consistent with everything we have learned about the world. This learning has _not_ come from philosophical or theological musings about morality or other speculations about the human condition. It is instead based on the remarkable and exciting developments in empirical cosmology and particle physics that I have described.\n\nI want thus to return to the question I described at the beginning of this book: Why is there something rather than nothing? We are now presumably in a better position to address this, having reviewed the modern scientific picture of the universe, its history, and its possible future, as well as operational descriptions of what \"nothing\" might actually comprise. As I also alluded to at the beginning of this book, this question too has been informed by science, like essentially all such philosophical questions. Far from providing a framework that forces upon us the requirement of a creator, the very meaning of the words involved have so changed that the sentence has lost much of its original meaning\u2014something that again is not uncommon, as empirical knowledge shines a new light on otherwise dark corners of our imagination.\n\nAt the same time, in science we have to be particularly cautious about \"why\" questions. When we ask, \"Why?\" we usually mean \"How?\" If we can answer the latter, that generally suffices for our purposes. For example, we might ask: \"Why is the Earth 93 million miles from the Sun?\" but what we really probably mean is, \"How is the Earth 93 million miles from the Sun?\" That is, we are interested in what physical processes led to the Earth ending up in its present position. \"Why\" implicitly suggests purpose, and when we try to understand the solar system in scientific terms, we do not generally ascribe purpose to it.\n\nSo I am going to assume what this question really means to ask is, \"How is there something rather than nothing?\" \"How\" questions are really the only ones we can provide definitive answers to by studying nature, but because this sentence sounds much stranger to the ear, I hope you will forgive me if I sometimes fall into the trap of appearing to discuss the more standard formulation when I am really trying to respond to the more specific \"how\" question.\n\nEven here, from the perspective of actual _understanding,_ this particular \"how\" question has been supplanted by a host of operationally more fruitful questions, such as, \"What might have produced the properties of the universe that most strikingly characterize it at the present time?\" or, perhaps more important, \"How can we find out?\"\n\nHere I want to once again beat what I wish were a dead horse. Framing questions in this way allows the production of new knowledge and understanding. This is what differentiates them from purely theological questions, which generally presume the answers up front. Indeed, I have challenged several theologians to provide evidence contradicting the premise that theology has made no contribution to knowledge in the past five hundred years at least, since the dawn of science. So far no one has provided a counterexample. The most I have ever gotten back was the query, \"What do you mean by knowledge?\" From an epistemological perspective this may be a thorny issue, but I maintain that, if there were a better alternative, someone would have presented it. Had I presented the same challenge to biologists, or psychologists, or historians, or astronomers, none of them would have been so flummoxed.\n\nThe answers to these sorts of fruitful questions involve theoretical predictions that can be tested via experiments to drive our operational knowledge of the universe forward more directly. Partly for this reason, I have focused on such fruitful questions up to this point in this book. Nevertheless, the \"something from nothing\" question continues to have great currency, and therefore probably needs to be confronted.\n\nNewton's work dramatically reduced the possible domain of God's actions, whether or not you attribute any inherent rationality to the universe. Not only did Newton's laws severely constrain the freedom of action of a deity, they dispensed with various requirements for supernatural intervention. Newton discovered that the motion of planets around the Sun does not require them to be continually pushed along their paths, but rather, and highly nonintuitively, requires them to be pulled by a force acting toward the Sun, thus dispensing of the need for the angels who were often previously invoked as guiding the planets on their way. While dispensing with this particular use of angels has had little impact on people's willingness to believe in them (polls suggest far more people believe in angels in the United States than believe in evolution), it is fair to say that progress in science since Newton has even more severely constrained the available opportunities for the hand of God to be manifest in his implied handiwork.\n\nWe can describe the evolution of the universe back to the earliest moments of the Big Bang without specific need for anything beyond known physical laws, and we have also described the universe's likely future history. There are certainly still puzzles about the universe that we don't understand, but I am going to assume that readers of this book are not wedded to a \"God of the Gaps\" picture, whereby God is invoked whenever there is something specific about our observations that seems puzzling or not fully understood. Even theologians recognize that such recourse not only diminishes the grandeur of their supreme being, but it also opens that being up to being removed or further marginalized whenever new work explains or removes the puzzle.\n\nIn this sense, the \"something from nothing\" argument really tries to focus on the original act of creation and asks whether a scientific explanation can ever be logically complete and fully satisfying in addressing this specific issue.\n\nIt turns out that, given our current understanding of nature, there are three different, separate meanings for the \"something from nothing\" question. The short answer to each is \"quite plausibly yes,\" and I shall discuss each in turn in the rest of this book as I attempt to explain why or, as I have argued just now, better yet how.\n\nOccam's razor suggests that, if some event is physically plausible, we don't need recourse to more extraordinary claims for its being. Surely the requirement of an all-powerful deity who somehow exists outside of our universe, or multiverse, while at the same time governing what goes on inside it, is one such claim. It should thus be a claim of last, rather than first, resort.\n\nI have already argued in the preface to this book that merely defining \"nothingness\" as \"nonbeing\" is not sufficient to suggest that physics, and more generally science, is not adequate to address the question. Let me give an additional, more specific argument here. Consider an electron-positron pair that spontaneously pops out of empty space near the nucleus of an atom and affects the property of that atom for the short time the pair exists. In what sense did the electron or positron exist before? Surely by any sensible definition they didn't. There was potential for their existence, certainly, but that doesn't define _being_ any more than a potential human being exists because I carry sperm in my testicles near a woman who is ovulating, and she and I might mate. Indeed, the best answer I have ever heard to the question of what it would be like to be dead (i.e., be nonbeing) is to imagine how it felt to be before you were conceived. In any case, if potential to exist were the same as existence, then I am certain that by now masturbation would be as hot button a legal issue as abortion now is.\n\nThe Origins Project at Arizona State University, which I direct, recently ran a workshop on the Origin of Life, and I cannot help but view the present cosmological debate in this context. We do not yet fully understand how life originated on Earth. However, we have not only plausible chemical mechanisms by which this might be conceivable, but we are also homing in closer and closer every day to specific pathways that might have allowed biomolecules, including RNA, to arise naturally. Moreover, Darwinian evolution, based on natural selection, provides a compellingly accurate picture of how complex life emerged on this planet following whatever specific chemistry produced the first faithfully self-replicating cells with a metabolism that captured energy from their environment. (As good a definition of life as I can come up with for the moment.)\n\nJust as Darwin, albeit reluctantly, removed the need for divine intervention in the evolution of the modern world, teeming with diverse life throughout the planet (though he left the door open to the possibility that God helped breathe life into the first forms), our current understanding of the universe, its past, and its future make it more plausible that \"something\" can arise out of nothing without the need for any divine guidance. Because of the observational and related theoretical difficulties associated with working out the details, I expect we may never achieve more than plausibility in this regard. But plausibility itself, in my view, is a tremendous step forward as we continue to marshal the courage to live meaningful lives in a universe that likely came into existence, and may fade out of existence, without purpose, and certainly without us at its center.\n\nLet's now return to one of the most remarkable features of our universe: it is as close to being flat as we can measure. I remind you of the unique facet of a flat universe, at least on scales where it is dominated by matter in the form of galaxies, and where a Newtonian approximation remains valid: in a flat universe, and only in a flat universe, the average Newtonian gravitational energy of every object participating in the expansion is precisely zero.\n\nI emphasize that this was a falsifiable postulate. It didn't have to be this way. Nothing required this except theoretical speculations based on considerations of a universe that could have arisen naturally from nothing, or at the very least, from _almost nothing_.\n\nI cannot overstress the importance of the fact that, once gravity is included in our considerations of nature, one is no longer free to define the total energy of a system arbitrarily, nor the fact that there are both positive and negative contributions to this energy. Determining the total gravitational energy of objects being carried along by the expansion of the universe is _not_ subject to arbitrary definition any more than the geometric curvature of the universe is a matter of definition. It is a property of space itself, according to general relativity, and this property of space is determined by the energy contained within it.\n\nI say this because it has been argued that the statement that the average total Newtonian gravitational energy of every galaxy in a flat, expanding universe is zero is arbitrary, and that any other value would be just as good, but that scientists \"define\" the zero point to argue against God. So claimed Dinesh D'Souza, anyway, in his debates with Christopher Hitchens on the existence of God.\n\nNothing could be further from the truth. The effort to determine the curvature of the universe was an undertaking carried out over half a century by scientists who devoted their lives to determining the actual nature of the universe, not to imposing their own desires upon it. Even well after the theoretical arguments about why the universe should be flat were first proposed, my observational colleagues, during the 1980s and even early 1990s, remained bent on proving otherwise. For, after all, in science one achieves the greatest impact (and often the greatest headlines) not by going along with the herd, but by bucking against it.\n\nNevertheless, the data have had the last word, and the last word is in. Our observable universe is as close to being flat as we can measure. The Newtonian gravitational energy of galaxies moving along with the Hubble expansion _is_ zero\u2014like it or not.\n\nI would now like to describe how, if our universe arose from nothing, a flat universe, one with zero total Newtonian gravitational energy of every object, is precisely what we should expect. The argument is a little subtle\u2014subtler than I have been able to describe in my popular lectures on the subject\u2014so I am happy to have the space here to carefully try to lay it out.\n\nFirst, I want to be clear about what kind of \"nothing\" I am discussing at the moment. This is the simplest version of nothing, namely empty space. For the moment, I will assume space exists, with nothing at all in it, and that the laws of physics also exist. Once again, I realize that in the revised versions of nothingness that those who wish to continually redefine the word so that no scientific definition is practical, this version of nothing doesn't cut the mustard. However, I suspect that, at the times of Plato and Aquinas, when they pondered why there was something rather than nothing, empty space with nothing in it was probably a good approximation of what they were thinking about.\n\nAs we saw in chapter 6, Alan Guth has explained precisely how we can get something from this kind of nothing\u2014the ultimate free lunch. Empty space can have a non-zero energy associated with it, even in the absence of any matter or radiation. General relativity tells us that space will expand exponentially, so that even the tiniest region at early times could quickly encompass a size more than large enough to contain our whole visible universe today.\n\nAs I also described in that chapter, during such a rapid expansion, the region that will eventually encompass our universe will get flatter and flatter even as the energy contained within empty space grows as the universe grows. This phenomenon happens without the need for any hocus pocus or miraculous intervention. This is possible because the gravitational \"pressure\" associated with such energy in empty space is actually negative. This \"negative pressure\" implies that, as the universe expands, the expansion dumps energy _into_ space rather than vice versa.\n\nAccording to this picture, when inflation ends, the energy stored in empty space gets turned into an energy of real particles and radiation, creating effectively the traceable beginning of our present Big Bang expansion. I say the traceable beginning because inflation effectively erases any memory of the state of the universe before it began. All complexities and irregularities on initially large scales (if the initial preexisting universe or metaverse were large, even infinitely large) get smoothed out and\/or driven so far outside our horizon today that we will always observe an almost uniform universe after enough inflationary expansion has taken place.\n\nI say almost uniform because I also described in chapter 6 how quantum mechanics will always leave some residual, small-density fluctuations that get frozen during inflation. This results in the second amazing implication of inflation, that small-density fluctuations in empty space due to the rules of quantum mechanics will later be responsible for all the structure we observe in the universe today. So we, and everything we see, result out of quantum fluctuations in what is essentially nothingness near the beginning of time, namely during the inflationary expansion.\n\nAfter all the dust is settled, the generic configuration of the matter and radiation will be that of an essentially flat universe, one in which the average Newtonian gravitational energy of all objects will appear to be zero. This will almost always be the case, unless one could very carefully fine-tune the amount of inflation.\n\nTherefore, our observable universe can start out as a microscopically small region of space, which can be essentially empty, and still grow to enormous scales containing eventually lots of matter and radiation, all without costing a drop of energy, with enough matter and radiation to account for everything we see today!\n\nThe important point worth stressing in this brief summary of the inflationary dynamics discussed in chapter 6 is that something can arise from empty space _precisely_ because the energetics of empty space, in the presence of gravity, are _not_ what common sense would have guided us to suspect before we discovered the underlying laws of nature.\n\nBut no one ever said that the universe is guided by what we, in our petty myopic corners of space and time, might have originally thought was sensible. It certainly seems sensible to imagine that a priori, matter cannot spontaneously arise from empty space, so that _something,_ in this sense, cannot arise from _nothing_. But when we allow for the dynamics of gravity and quantum mechanics, we find that this commonsense notion is no longer true. This is the _beauty_ of science, and it should not be threatening. Science simply forces us to revise what is sensible to accommodate the universe, rather than vice versa.\n\nTo summarize then: the observation that the universe is flat and that the local Newtonian gravitational energy is essentially zero today is strongly suggestive that our universe arose though a process like that of inflation, a process whereby the energy of empty space (nothing) gets converted into the energy of something, during a time when the universe is driven closer and closer to being essentially exactly flat on all observable scales.\n\nWhile inflation demonstrates how empty space endowed with energy can effectively create everything we see, along with an unbelievably large and flat universe, it would be disingenuous to suggest that empty space endowed with energy, which drives inflation, is really _nothing_. In this picture one must assume that space exists and can store energy, and one uses the laws of physics like general relativity to calculate the consequences. So if we stopped here, one might be justified in claiming that modern science is a long way from really addressing how to get something from nothing. This is just the first step, however. As we expand our understanding, we will next see that inflation can represent simply the tip of a cosmic iceberg of nothingness.\n\n## CHAPTER 10\n\nNOTHING IS UNSTABLE\n\nFiat justitia\u2014ruat caelum. _(Do justice, and let the skies fall.)_\n\n\u2014ANCIENT ROMAN PROVERB\n\nThe existence of energy in empty space\u2014the discovery that rocked our cosmological universe and the idea that forms the bedrock of inflation\u2014only reinforces something about the quantum world that was already well established in the context of the kinds of laboratory experiments I have already described. Empty space is complicated. It is a boiling brew of virtual particles that pop in and out of existence in a time so short we cannot see them directly.\n\nVirtual particles are manifestations of a basic property of quantum systems. At the heart of quantum mechanics is a rule that sometimes governs politicians or CEOs\u2014as long as no one is watching, anything goes. Systems continue to move, if just momentarily, between all possible states, including states that would not be allowed if the system were actually being measured. These \"quantum fluctuations\" imply something essential about the quantum world: nothing always produces something, if only for an instant.\n\nBut here's the rub. The conservation of energy tells us that quantum systems can misbehave for only so long. Like embezzling stockbrokers, if the state that a system fluctuates into requires sneaking some energy from empty space, then the system has to return that energy in a time short enough so that no one measuring the system can detect it.\n\nAs a result, you might presume to safely argue that this \"something\" that is produced by quantum fluctuations is ephemeral\u2014not measurable, unlike, say, you or I or the Earth on which we live. But this ephemeral creation, too, is subject to the circumstances associated with our measurements. For example, consider the electric field emanating from a charged object. It is definitely real. You can feel the static electric force on your hair or watch a balloon stick to a wall. However, the quantum theory of electromagnetism suggests that the static field is due to the emission, by the charged particles involved in producing the field, of virtual photons that have essentially zero total energy. These virtual particles, because they have zero energy, can propagate across the universe without disappearing, and the field due to the superposition of many of them is so real it can be felt.\n\nSometimes conditions are such that real, massive particles can actually pop out of empty space with impunity. In one example, two charged plates are brought close together and, once the electric field gets strong enough between them, it becomes energetically favorable for a real particle-antiparticle pair to \"pop\" out of the vacuum, with the negative charge heading toward the positive plate and the positive charge toward the negative one. In so doing, it is possible that the reduction in energy arising from reducing the net charge on each of the plates and hence the electric field between them can be greater than the energy associated with the rest mass energy required to produce two real particles. Of course, the strength of the field has to be huge for such a condition to be possible.\n\nThere is actually a place where strong fields of a different kind might allow a phenomenon similar to that described above to occur\u2014but in this case due to gravity. This realization actually made Stephen Hawking famous among physicists in 1974, when he showed that it might be possible for black holes\u2014out of which, in the absence of quantum mechanical considerations at least, nothing can ever escape\u2014to radiate physical particles.\n\nThere are many different ways to try to understand this phenomenon, but one of these is strikingly familiar to the situation I described above with electric fields. Outside of the core of black holes is a radius called the \"event horizon.\" Inside an event horizon, no object can classically escape because the escape velocity exceeds the speed of light. Thus, even light emitted inside this region will not make it outside the event horizon.\n\nNow imagine a particle-antiparticle pair nucleates out of empty space just outside of the event horizon due to quantum fluctuations in that region. It is possible, if one of the particles actually falls within the event horizon, for it to lose enough gravitational energy by falling into the black hole that this energy exceeds twice the rest mass of either particle. This means that the partner particle can fly off to infinity and be observable without any violation of energy conservation. The total positive energy associated with the radiated particle is more than compensated by the loss of energy experienced by its partner particle falling into the black hole. The black hole can therefore radiate particles.\n\nThe situation is even more interesting, however, precisely because the energy lost by the infalling particle is greater than the positive energy associated with its rest mass. As a result, when it falls into the black hole, the net system of the black hole plus the particle actually has less energy than it did before the particle fell in! The black hole therefore actually gets _lighter_ after the particle falls in by an amount that is equivalent to the energy carried away by the radiated particle that escapes. Eventually the black hole may radiate away entirely. At this point we do not know because the final stages of black hole evaporation involve physics on such small distance scales that general relativity alone cannot tell us the final answer. On these scales, gravity must be treated as a fully quantum mechanical theory, and our current understanding of general relativity is not sufficient to allow us to determine precisely what will happen.\n\nNevertheless, all of these phenomena imply that, under the right conditions, not only can nothing become something, it is required to.\n\nAn early example in cosmology of the fact that \"nothing\" can be unstable and form something comes from efforts to understand why we live in a universe of matter.\n\nYou probably don't wake up each morning wondering about this, but the fact that our universe contains matter is remarkable. What is particularly remarkable about this is that, as far as we can tell, our universe does not contain substantial amounts of antimatter, which you will recall is required to exist by quantum mechanics and relativity, so that for every particle that we know of in nature, there can exist an equivalent antiparticle with opposite charge and the same mass. Any sensible universe at its inception, one might think, would contain equal amounts of both. After all, the antiparticles of normal particles have the same mass and similar other properties, so if particles were created at early times, it would have been equally easy to create antiparticles.\n\nAlternatively, we could even imagine an antimatter universe in which all of the particles that make up the stars and galaxies were replaced with their antiparticles. Such a universe would appear to be almost identical to the one we live in. Observers in such a universe (themselves made of antimatter) would no doubt call what we call antimatter as matter. The name is arbitrary.\n\nHowever, if our universe began sensibly, with equal amounts of matter and antimatter, and stayed that way, we wouldn't be around to ask \"Why?\" or \"How?\" This is because all particles of matter would have annihilated with all particles of antimatter in the early universe, leaving nothing but pure radiation. No matter or antimatter would be left over to make up stars, or galaxies, or to make up lovers or antilovers who might otherwise one day gaze out and be aroused by the spectacle of the night sky in each other's arms. No drama. History would consist of emptiness, a radiation bath that would slowly cool, leading ultimately to a cold, dark, bleak universe. Nothingness would reign supreme.\n\nScientists began to understand in the 1970s, however, that it is possible to begin with equal amounts of matter and antimatter in an early hot, dense Big Bang, and for plausible quantum processes to \"create something from nothing\" by establishing a small asymmetry, with a slight excess of matter over antimatter in the early universe. Then, instead of complete annihilation of matter and antimatter, leading to nothing but pure radiation today, all of the available antimatter in the early universe could have annihilated with matter, but the small excess of matter would have had no comparable amount of antimatter to annihilate with, and would then be left over. This would then lead to all the matter making up stars and galaxies we see in the universe today.\n\nAs a result, what might otherwise seem a small accomplishment (establishing a small asymmetry at early times) might instead be considered almost as the moment of creation. Because once an asymmetry between matter and antimatter was created, nothing could later put it asunder. The future history of a universe full of stars and galaxies was essentially written. Antimatter particles would annihilate with the matter particles in the early universe, and the remaining excess of matter particles would survive through the present day, establishing the character of the visible universe we know and love and inhabit.\n\nEven if the asymmetry were 1 part in a billion there would be enough matter left over to account for everything we see in the universe today. In fact, an asymmetry of 1 part in a billion or so is precisely what was called for, because today there are roughly 1 billion photons in the cosmic microwave background for every proton in the universe. The CMBR photons are the remnants, in this picture, of the early matter-antimatter annihilations near the beginning of time.\n\nA definitive description of how this process could have happened in the early universe is currently lacking because we have not yet fully and empirically established the detailed nature of the microphysical world at the scales where this asymmetry was likely to have been generated. Nevertheless, a host of different plausible scenarios has been explored based on the current best ideas we have about physics at these scales. While they differ in the details, they all have the same general characteristics. Quantum processes associated with elementary particles in the primordial heat bath can inexorably drive an empty universe (or equivalently an initially matter-antimatter symmetric universe) almost imperceptibly toward a universe that will be dominated by matter or antimatter.\n\nIf it could have gone either way, was it then just a circumstantial accident that our universe became dominated by matter? Imagine standing on top of a tall mountain and tripping. The direction you fall was not preordained, but rather is an accident, depending upon which direction you were looking in or at what point in your stride you tripped. Perhaps similarly our universe is like that, and even if the laws of physics are fixed, the ultimate direction of the asymmetry between matter and antimatter was driven by some random initial condition (just as in the case of tripping down the mountain, the law of gravity is fixed and determines that you will fall, but your direction may be an accident). Once again, our very existence in that case would be an environmental accident.\n\nIndependent of this uncertainty, however, is the remarkable fact that a feature of the underlying laws of physics can allow quantum processes to drive the universe away from a featureless state. Physicist Frank Wilczek, who was one of the first theorists to explore these possibilities, has reminded me that he utilized precisely the same language I have used previously in this chapter, in the 1980 _Scientific American_ article he wrote on the matter-antimatter asymmetry of the universe. After describing how a matter-antimatter asymmetry might plausibly be generated in the early universe based on our new understanding of particle physics, he added a note that this provided one way of thinking about the answer to the question of why there is something rather than nothing: _nothing_ is unstable.\n\nThe point Frank was emphasizing is that the measured excess of matter over antimatter in the universe appears on first glance to be an obstacle to imagining a universe that could arise from an instability in empty space, with nothingness producing a Big Bang. But if that asymmetry could arise dynamically after the Big Bang, that barrier is removed. As he put it:\n\nOne can speculate that the universe began in the most symmetrical state possible and that in such a state no matter existed; the universe was a vacuum. A second state existed, and in it matter existed. The second state had slightly less symmetry, but was also lower in energy. Eventually a patch of less symmetrical phase appeared and grew rapidly. The energy released by the transition found form in the creation of particles. This event might be identified with the big bang . . . The answer to the ancient question \"Why is there something rather than nothing?\" would be that \"nothing\" is unstable.\n\nBefore I proceed, however, I am again reminded of the similarities between the discussion I have just given of a matter-antimatter asymmetry and the discussions we had at our recent Origins workshop to explore our current understanding of the nature of life in the universe and its origin. My words were different, but the fundamental issues are remarkably similar: What specific physical process in the early moments of the Earth's history could have led to the creation of the first replicating biomolecules and metabolism? As in the 1970s in physics, the recent decade has seen incredible progress in molecular biology. We learned of natural organic pathways, for example, that could produce, under plausible conditions, ribonucleic acids, long thought to be the precursors to our modern DNA-based world. Until recently it was felt that no such direct pathway was possible and that some other intermediate forms must have played a key role.\n\nNow few biochemists and molecular biologists doubt that life can arise naturally from nonlife, even though the specifics are yet to be discovered. But, as we discussed all of this, a common subtext permeated our proceedings: Did the life that first formed on Earth _have_ to have the chemistry that it did, or are there many different, equally viable possibilities?\n\nEinstein once asked a question that, he said, was the one thing he really wanted to know about nature. I admit it is the most profound and fundamental question that many of us would like answered. He put it as follows: \"What I want to know is whether _God_ [ _sic_ ] had any choice in the creation of the universe.\"\n\nI have annotated this because Einstein's God was not the God of the Bible. For Einstein, the existence of order in the universe provided a sense of such profound wonder that he felt a spiritual attachment to it, which he labeled, motivated by Spinoza, with the moniker \"God.\" In any case, what Einstein really meant in this question was the issue I have just described in the context of several different examples: Are the laws of nature unique? And is the universe we inhabit, which has resulted from these laws, unique? If you change one facet, one constant, one force, however slight, would the whole edifice crumble? In a biological sense, is the biology of life unique? Are we unique in the universe? We will return to discuss this most important question later in this book.\n\nWhile such a discussion will cause us to further refine and generalize notions of \"nothing\" and \"something,\" I want to return to taking an intermediate step in making the case for the inevitable creation of something.\n\nAs I have defined it thus far, the relevant \"nothing\" from which our observed \"something\" arises is \"empty space.\" However, once we allow for the merging of quantum mechanics and general relativity, we can extend this argument to the case where space itself is forced into existence.\n\nGeneral relativity as a theory of gravity is, at its heart, a theory of space and time. As I described in the very beginning of this book, this means that it was the first theory that could address the dynamics not merely of objects moving through space, but also how space itself evolves.\n\nHaving a quantum theory of gravity would therefore mean that the rules of quantum mechanics would apply to the properties of space and not just to the properties of objects existing in space, as in conventional quantum mechanics.\n\nExtending quantum mechanics to include such a possibility is tricky, but the formalism Richard Feynman developed, which led to a modern understanding of the origin of antiparticles, is well suited to the task. Feynman's methods focus on the key fact to which I alluded at the beginning of this chapter: quantum mechanical systems explore all possible trajectories, even those that are classically forbidden, as they evolve in time.\n\nIn order to explore this, Feynman developed a \"sum over paths formalism\" to make predictions. In this method, we consider all possible trajectories between two points that a particle might take. We then assign a probability weighting for each trajectory, based on well-defined principles of quantum mechanics, and then perform a sum over all paths in order to determine final (probabilistic) predictions for the motion of particles.\n\nStephen Hawking was one of the first scientists to fully exploit this idea to the possible quantum mechanics of space-time (the union of our three-dimensional space along with one dimension of time to form a four-dimensional unified space-time system, as required by Einstein's special theory of relativity). The virtue of Feynman's methods was that focusing on all possible paths ends up meaning that the results can be shown to be independent of the specific space and time labels one applies to each point on each path. Because relativity tells us that different observers in relative motion will measure distance and time differently and therefore assign different values to each point in space and time, having a formalism that is independent of the different labels that different observers might assign to each point in space and time is particularly useful.\n\nAnd it is most useful perhaps in considerations of general relativity, where the specific labeling of space and time points becomes completely arbitrary, so that different observers at different points in a gravitational field measure distances and times differently, and all that ultimately determines the behavior of systems are geometric quantities like curvature, which turn out to be independent of all such labeling schemes.\n\nAs I have alluded to several times, general relativity is not fully consistent with quantum mechanics, at least as far as we can tell, and therefore there is no completely unambiguous method to define Feynman's sum-over-paths technique in general relativity. So we have to make some guesses in advance based on plausibility and check to see if the results make sense.\n\nIf we are to consider the quantum dynamics of space and time then, one must imagine that in the Feynman \"sums,\" one must consider every different possible configuration that can describe the different geometries that space can adopt during the intermediate stages of any process, when quantum indeterminacy reigns supreme. This means we must consider spaces that are arbitrarily highly curved over short distances and small times (so short and so small that we cannot measure them so that quantum weirdness can reign free). These weird configurations would then not be observed by large classical observers such as us when we attempt to measure the properties of space over large distances and times.\n\nBut let's consider even stranger possibilities. Remember that, in the quantum theory of electromagnetism, particles can pop out of empty space at will as long as they disappear again on a time frame determined by the Uncertainty Principle. By analogy, then, in the Feynman quantum sum over possible space-time configurations, should one consider the possibility of small, possibly compact spaces that themselves pop in and out of existence? More generally, what about spaces that may have \"holes\" in them, or \"handles\" like donuts dunking into space-time?\n\nThese are open questions. However, unless one can come up with a good reason for excluding such configurations from the quantum mechanical sum that determines the properties of the evolving universe, and to date no such good reason exists that I know of, then under the general principle that holds everywhere else I know of in nature\u2014namely that anything that is not proscribed by the laws of physics must actually happen\u2014it seems most reasonable to consider these possibilities.\n\nAs Stephen Hawking has emphasized, a quantum theory of gravity allows for the creation, albeit perhaps momentarily, of space itself where none existed before. While in his scientific work he was not attempting to address the \"something from nothing\" conundrum, effectively this is what quantum gravity may ultimately address.\n\n\"Virtual\" universes\u2014namely the possible small compact spaces that may pop into and out of existence on a timescale so short we cannot measure them directly\u2014are fascinating theoretical constructs, but they don't seem to explain how something can arise from nothing over the long term any more than do the virtual particles that populate otherwise empty space.\n\nHowever, recall that a nonzero real electric field, observable at large distances away from a charged particle, can result from the coherent emission of many virtual zero energy photons by the charge. This is because virtual photons that carry zero energy do not violate energy conservation when they are emitted. The Heisenberg Uncertainty Principle, therefore, does not constrain them to exist for only very brief times before they must be reabsorbed and disappear back into nothingness. (Again recall that the Heisenberg Uncertainty Principle states that the uncertainty with which we measure the energy of a particle, and hence the possibility that its energy may change slightly by the emission and absorption of virtual particles, is inversely proportional to the length of time over which we observe it. Hence, virtual particles that carry away zero energy can do so essentially with impunity\u2014namely they can exist for arbitrarily long times and travel arbitrarily far away before being absorbed . . . leading to the possible existence of long-range interactions between charged particles. If the photon was not massless, so that photons always carried away non-zero energy due to a rest mass, the Heisenberg Uncertainty Principle would imply that the electric field would be short range because photons could propagate only for short times without being reabsorbed again.)\n\nA similar argument suggests that one can imagine one specific type of universe that might spontaneously appear and need not disappear almost immediately thereafter because of the constraints of the Uncertainty Principle and energy conservation. Namely, a compact universe with zero total energy.\n\nNow, I would like nothing better than to suggest that this is precisely the universe we live in. This would be the easy way out, but I am more interested here in being true to our current understanding of the universe than in making an apparently easy and convincing case for creating it from nothing.\n\nI have argued, I hope compellingly, that the average Newtonian gravitational energy of every object in our flat universe is zero. And it is. But that is not the whole story. Gravitational energy is not the total energy of any object. To this energy we must add its rest energy, associated with its rest mass. Put another way, as I have described earlier, the gravitational energy of an object at rest isolated from all other objects by an infinite distance is zero, because if it is at rest, it has no kinetic energy of motion, and if it is infinitely far away from all other particles, the gravitational force on it due to other particles, which could provide potential energy to do work, is also essentially zero. However, as Einstein told us, its total energy is not merely due to gravity, but also includes the energy associated with its mass, so that, as is famously known, E = mc2.\n\nIn order to take this rest energy into account, we have to move from Newtonian gravity to general relativity, which, by definition, incorporates the effects of special relativity (and E = mc2) into a theory of gravity. And here things get both subtler and more confusing. On small scales compared to the possible curvature of a universe, and as long as all objects within these scales are moving slowly compared to the speed of light, the general relativistic version of energy reverts to the definition we are familiar with from Newton. However, once these conditions no longer hold, all bets are off, almost.\n\nPart of the problem is that it turns out that energy as we normally think of it elsewhere in physics is not a particularly well-defined concept on large scales in a curved universe. Different ways of defining coordinate systems to describe the different labels that different observers may assign to points in space and time (called different \"frames of reference\") can lead, on large scales, to different determinations of the total energy of the system. In order to accommodate this effect, we have to generalize the concept of energy, and, moreover, if we are to define the total energy contained in any universe, we must consider how to add up the energy in universes that may be infinite in spatial extent.\n\nThere is a lot of debate over precisely how to do this. The scientific literature is replete with claims and counterclaims in this regard.\n\nOne thing is certain, however: There is one universe in which the total energy is definitely and precisely zero. It is not, however, a flat universe, which is in principle infinite in spatial extent, and therefore the calculation of total energy becomes problematic. It is a closed universe, one in which the density of matter and energy is sufficient to cause space to close back upon itself. As I have described, in a closed universe, if you look far enough in one direction, you will eventually see the back of your head!\n\nThe reason the energy of a closed universe is zero is really relatively simple. It is easiest to consider the result by analogy with the fact that in a closed universe the total electric charge must also be zero.\n\nSince the time of Michael Faraday we think of electric charge as being the source of an electric field (due in modern quantum parlance to the emission of the virtual photons I described above). Pictorially, we imagine \"field lines\" emanating out radially from the charge, with the number of field lines being proportional to the charge, and the direction of field lines being outward for positive charges and inward for negative charges, as shown below.\n\nWe imagine these field lines going out to infinity, and as they spread out, getting farther apart. This implies that the strength of the electric field gets weaker and weaker. However, in a closed universe, the field lines associated with a positive charge, for example, may start out spreading apart but eventually, just as the lines of longitude on a map of the Earth come together at the North and South Poles, the field lines from the positive charge will come together again on the far side of the universe. When they converge, the field will get stronger and stronger again until there is enough energy to create a negative charge that can \"eat\" the field lines at this antipodal point of the universe.\n\nIt turns out a very similar argument, in this case associated not with the \"flux\" of field lines but with the \"flux\" of energy in a closed universe, tells us that the total positive energy, including that associated with the rest masses of particles, must be exactly compensated for by a negative gravitational energy, so that the total energy is precisely zero.\n\nSo if the total energy of a closed universe is zero, and if the sum-over-paths formalism of quantum gravity is appropriate, then quantum mechanically such universes could appear spontaneously with impunity, carrying no net energy. I want to emphasize that these universes would be completely self-contained space-times, disconnected from our own.\n\nThere is a hitch, however. A closed expanding universe filled with matter will in general expand to a maximum size and then recollapse just as quickly, ending up in a space-time singularity where the no-man's land of quantum gravity at present cannot tell us what its ultimate fate will be. The characteristic lifetime of tiny closed universes will therefore be microscopic, perhaps on the order of the \"Planck time,\" the characteristic scale over which quantum gravitational processes should operate, about 10\u221244 seconds or so.\n\nThere is a way out of this dilemma, however. If, before such a universe can collapse, the configuration of fields within it produces a period of inflation, then even an initially tiny closed universe can rapidly, exponentially expand, becoming closer and closer to an infinitely large flat universe during this period. After one hundred or so doubling times of such inflation, the universe will be so close to flat that it could easily last much longer than our universe has been around without collapsing.\n\nAnother possibility actually exists, one that always gives me a slight twinge of nostalgia (and envy), because it represented an important learning experience for me. When I was first a postdoc at Harvard, I was playing with the possible quantum mechanics of gravitational fields, and I learned of a result by a good friend from graduate school, Ian Affleck. A Canadian who had been a graduate student at Harvard when I was at MIT, Affleck joined the Society of Fellows a few years before I did and had used the mathematical theory of Feynman that we now use for dealing with elementary particles and fields, called quantum field theory, to calculate how particles and antiparticles could be produced in a strong magnetic field.\n\nI realized that the form of the solution that Ian had described, something called an \"instanton,\" resembled very much an inflating universe, if one took over his formalism to the case of gravity. But it looked like an inflating universe that began from nothing! Before writing up this result, I wanted to address my own confusion about how to interpret what physics such a mathematical solution might correspond to. I soon learned, however, that while I was cogitating, just down the road the very creative cosmologist I mentioned earlier, Alex Vilenkin, who has since become a friend, had actually just written a paper that described in exactly this fashion how quantum gravity indeed might create an inflating universe directly from nothing. I was scooped, but I couldn't be that upset because (a) I frankly didn't understand in detail at that point what I was doing, and (b) Alex had the boldness to propose something that at the time I didn't. I have since learned that one doesn't have to understand all the implications of one's work in order to publish. Indeed, there are several of my own most important papers that I only fully understood well after the fact.\n\nIn any case, while Stephen Hawking and his collaborator Jim Hartle have proposed a very different scheme for trying to determine the \"boundary conditions\" on universes that may begin from nothing at all, the important facts are these:\n\n1. In quantum gravity, universes can, and indeed always will, spontaneously appear from nothing. Such universes need not be empty, but can have matter and radiation in them, as long as the total energy, including the negative energy associated with gravity, is zero.\n\n2. In order for the closed universes that might be created through such mechanisms to last for longer than infinitesimal times, something like inflation is necessary. As a result, the only long-lived universe one might expect to live in as a result of such a scenario is one that today appears flat, just as the universe in which we live appears.\n\nThe lesson is clear: quantum gravity not only appears to allow universes to be created from nothing\u2014meaning, in this case, I emphasize, the absence of space and time\u2014it may require them. \"Nothing\"\u2014in this case no space, no time, no anything!\u2014 _is_ unstable.\n\nMoreover, the general characteristics of such a universe, if it lasts a long time, would be expected to be those we observe in our universe today.\n\nDoes this prove that our universe arose from nothing? Of course not. But it does take us one rather large step closer to the plausibility of such a scenario. And it removes one more of the objections that might have been leveled against the argument of creation from nothing as described in the previous chapter.\n\nThere, \"nothing\" meant empty but preexisting space combined with fixed and well-known laws of physics. Now the requirement of space has been removed.\n\nBut, remarkably, as we shall next discuss, even the laws of physics may not be necessary or required.\n\n## CHAPTER 11\n\nBRAVE NEW WORLDS\n\n_It was the best of times. It was the worst of times._\n\n\u2014CHARLES DICKENS\n\nThe central problem with the notion of creation is that it appears to require some externality, something outside of the system itself, to _preexist,_ in order to create the conditions necessary for the system to come into being. This is usually where the notion of God\u2014some external agency existing separate from space, time, and indeed from physical reality itself\u2014comes in, because the buck seems to be required to stop somewhere. But in this sense _God_ seems to me to be a rather facile semantic solution to the deep question of creation. I think this is best explained within the context of a slightly different example: the origin of morality, which I first learned from my friend Steven Pinker.\n\nIs morality external and absolute, or is it derived solely within the context of our biology and our environment, and thus can it be determined by science? During a debate on this subject organized at Arizona State University, Pinker pointed out the following conundrum.\n\nIf one argues, as many deeply religious individuals do, that without God there can be no ultimate right and wrong\u2014namely that God determines for us what is right and wrong\u2014one can then ask the questions: What if God decreed that rape and murder were morally acceptable? Would that make them so?\n\nWhile some might answer yes, I think most believers would say no, God would not make such a decree. But why not? Presumably because God would have some reason for not making such a decree. Again, presumably this is because reason suggests that rape and murder are not morally acceptable. But if God would have to appeal to reason, then why not eliminate the middleman entirely?\n\nWe may wish to apply similar reasoning to the creation of our universe. All of the examples I have provided thus far indeed involve creation of something from what one should be tempted to consider as nothing, but the _rules_ for that creation, i.e., the laws of physics, were preordained. Where do the rules come from?\n\nThere are two possibilities. Either God, or some divine being who is not bound by the rules, who lives outside of them, determines them\u2014either by whim or with malice aforethought\u2014or they arise by some less supernatural mechanism.\n\nThe problem with God determining the rules is that you can at least ask what, or who, determined God's rules. Traditionally the response to this is to say that God is, among the Creator's many other spectacular attributes, the _cause of all causes,_ in the language of the Roman Catholic Church, or the _First Cause_ (as per Aquinas), or in the language of Aristotle, moving the _prime mover_.\n\nInterestingly, Aristotle recognized the problem of a first cause, and decided that for this reason the universe must be eternal. Moreover, God himself, whom he identified as pure self-absorbed thought, the love of which motivated the prime mover to move, had to be eternal, not causing motion by creating it, but rather by establishing the end purpose of motion, which itself Aristotle deemed had to be eternal.\n\nAristotle felt that equating First Cause with God was less than satisfying, in fact that the Platonic notion of First Cause was flawed, specifically because Aristotle felt every cause must have a precursor\u2014hence, the requirement that the universe be eternal. Alternatively, if one takes the view of God as the cause of all causes, and therefore eternal even if our universe is not, the _reductio ad absurdum_ sequence of \"why\" questions does indeed terminate, but as I have stressed, only at the expense of introducing a remarkable all-powerful entity for which there is simply no other evidence.\n\nIn this regard, there is another important point to stress here. The apparent logical necessity of First Cause is a real issue for any universe that has a beginning. Therefore, on the basis of logic alone one cannot rule out such a deistic view of nature. But even in this case it is vital to realize that this deity bears no logical connection to the personal deities of the world's great religions, in spite of the fact that it is often used to justify them. A deist who is compelled to search for some overarching intelligence to establish order in nature will not, in general, be driven to the personal God of the scriptures by the same logic.\n\nThese issues have been debated and discussed for millennia, by brilliant and not-so-brilliant minds, many of the latter making their current living by debating them. We can return to these issues now because we are simply better informed by our knowledge of the nature of physical reality. Neither Aristotle nor Aquinas knew about the existence of our galaxy, much less the Big Bang or quantum mechanics. Hence the issues they and later medieval philosophers grappled with must be interpreted and understood in the light of new knowledge.\n\nConsider, in the light of our modern picture of cosmology, for example, Aristotle's suggestion that there are no First Causes, or rather that causes indeed go backward (and forward) infinitely far in all directions. There is no beginning, no creation, no end.\n\nWhen I have thus far described how something almost always can come from \"nothing,\" I have focused on either the creation of something from preexisting empty space or the creation of empty space from no space at all. Both initial conditions work for me when I think of the \"absence of being\" and therefore are possible candidates for nothingness. I have not addressed directly, however, the issues of what might have existed, if anything, before such creation, what laws governed the creation, or, put more generally, I have not discussed what some may view as the question of First Cause. A simple answer is of course that either empty space or the more fundamental nothingness from which empty space may have arisen, preexisted, and is eternal. However, to be fair, this does beg the possible question, which might of course not be answerable, of what, if anything, fixed the rules that governed such creation.\n\nOne thing is certain, however. The metaphysical \"rule,\" which is held as an ironclad conviction by those with whom I have debated the issue of creation, namely that \" _out of nothing nothing comes,_ \" has no foundation in science. Arguing that it is self-evident, unwavering, and unassailable is like arguing, as Darwin falsely did, when he made the suggestion that the origin of life was beyond the domain of science by building an analogy with the incorrect claim that matter cannot be created or destroyed. All it represents is an unwillingness to recognize the simple fact that nature may be cleverer than philosophers or theologians.\n\nMoreover, those who argue that out of nothing nothing comes seem perfectly content with the quixotic notion that somehow God can get around this. But once again, if one requires that the notion of true nothingness requires not even the _potential_ for existence, then surely God cannot work his wonders, because if he does cause existence from nonexistence, there must have been the potential for existence. To simply argue that God can do what nature cannot is to argue that _supernatural_ potential for existence is somehow different from regular natural potential for existence. But this seems an arbitrary semantic distinction designed by those who have decided in advance (as theologians are wont to do) that the supernatural (i.e., God) must exist so they define their philosophical ideas (once again completely divorced from any empirical basis) to exclude anything but the possibility of a god.\n\nIn any case, to posit a god who could resolve this conundrum, as I have emphasized numerous times thus far, often is claimed to require that God exists outside the universe and is either timeless or eternal.\n\nOur modern understanding of the universe provides another plausible and, I would argue, far more physical solution to this problem, however, which has some of the same features of an external creator\u2014and moreover is logically more consistent.\n\nI refer here to the multiverse. The possibility that our universe is one of a large, even possibly infinite set of distinct and causally separated universes, in each of which any number of fundamental aspects of physical reality may be different, opens up a vast new possibility for understanding our existence.\n\nAs I have mentioned, one of the more distasteful but potentially true implications of these pictures is that physics, at some fundamental level, is merely an environmental science. (I find this distasteful because I was brought up on the idea that the goal of science was to explain why the universe had to be the way it is and how that came to be. If instead the laws of physics as we know them are merely accidents correlated to our existence, then that fundamental goal was misplaced. However, I will get over my prejudice if the idea turns out to be true.) In this case, the fundamental forces and constants of nature in this picture are no more fundamental than the Earth-Sun distance. We find ourselves living on Earth rather than Mars not because there is something profound and fundamental about the Earth-Sun distance, but rather simply if Earth were located at a different distance, then life as we know it could not have evolved on our planet.\n\nThese anthropic arguments are notoriously slippery, and it is almost impossible to make specific predictions based on them without knowing explicitly both the probability distribution among all possible universes of the various fundamental constants and forces\u2014namely, which may vary and which don't, and what possible values and forms they may take\u2014and also exactly how \"typical\" we are in our universe. If we are not \"typical\" life forms, then anthropic selection, if it occurs at all, may be based on different factors from those we would otherwise attribute it to.\n\nNevertheless, a multiverse, either in the form of a landscape of universes existing in a host of extra dimensions, or in the form of a possibly infinitely replicating set of universes in a three-dimensional space as in the case of eternal inflation, changes the playing field when we think about the creation of our own universe and the conditions that may be required for that to happen.\n\nIn the first place, the question of what determined the laws of nature that allowed our universe to form and evolve now becomes less significant. If the laws of nature are themselves stochastic and random, then there is no prescribed \"cause\" for our universe. Under the general principle that anything that is not forbidden is allowed, then we would be guaranteed, in such a picture, that some universe would arise with the laws that we have discovered. No mechanism and no entity is required to fix the laws of nature to be what they are. They could be almost anything. Since we don't currently have a fundamental theory that explains the detailed character of the landscape of a multiverse, we cannot say. (Although to be fair, to make any scientific progress in calculating possibilities, we generally assume that certain properties, like quantum mechanics, permeate all possibilities. I have no idea if this notion can be usefully dispensed with, or at least I don't know of any productive work in this regard.)\n\nIn fact, there may be no fundamental theory at all. Although I became a physicist because I hoped that there was such a theory, and because I hoped that I might one day help contribute to discovering it, this hope may be misplaced, as I have already lamented. I take solace in the statement by Richard Feynman, which I summarized briefly before, but want to present in its entirety here:\n\nPeople say to me, \"Are you looking for the ultimate laws of physics?\" No, I'm not. I'm just looking to find out more about the world, and if it turns out there is a simple ultimate law that explains everything, so be it. That would be very nice to discover. If it turns out it's like an onion with millions of layers, and we're sick and tired of looking at layers, then that's the way it is . . . My interest in science is to simply find out more about the world, and the more I find out, the better it is. I like to find out.\n\nOne can carry the argument further and in a different direction, which also has implications for the arguments at the core of this book. In a multiverse of any of the types that have been discussed, there could be an infinite number of regions, potentially infinitely big or infinitesimally small, in which there is simply \"nothing,\" and there could be regions where there is \"something.\" In this case, the response to why there is something rather than nothing becomes almost trite: there is something simply because if there were nothing, we wouldn't find ourselves living there!\n\nI recognize the frustration inherent in such a trivial response to what has seemed such a profound question throughout the ages. But science has told us that anything profound or trivial can be dramatically different from what we might suppose at first glance.\n\nThe universe is far stranger and far richer\u2014more wondrously strange\u2014than our meager human imaginations can anticipate. Modern cosmology has driven us to consider ideas that could not even have been formulated a century ago. The great discoveries of the twentieth and twenty-first centuries have not only changed the world in which we operate, they have revolutionized our understanding of the world\u2014or worlds\u2014that exist, or may exist, just under our noses: the reality that lies hidden until we are brave enough to search for it.\n\nThis is why philosophy and theology are ultimately incapable of addressing by themselves the truly fundamental questions that perplex us about our existence. Until we open our eyes and let nature call the shots, we are bound to wallow in myopia.\n\nWhy is there something rather than nothing? Ultimately, this question may be no more significant or profound than asking why some flowers are red and some are blue. \"Something\" may always come from nothing. It may be required, independent of the underlying nature of reality. Or perhaps \"something\" may not be very special or even very common in the multiverse. Either way, what is really useful is not pondering this question, but rather participating in the exciting voyage of discovery that may reveal specifically how the universe in which we live evolved and is evolving and the processes that ultimately operationally govern our existence. That is why we have science. We may supplement this understanding with reflection and call that philosophy. But only via continuing to probe every nook and cranny of the universe that is accessible to us will we truly build a useful appreciation of our own place in the cosmos.\n\nBefore concluding, I want to raise one more aspect of this question that I haven't touched upon, but which strikes me as worth ending with. Implicit in the question of why there is something rather than nothing is the solipsistic expectation that \"something\" will persist\u2014that somehow the universe has \"progressed\" to the point of our existence, as if we were the pinnacle of creation. Far more likely, based on everything we know about the universe, is the possibility that the future, perhaps the infinite future, is one in which nothingness will once again reign.\n\nIf we live in a universe whose energy is dominated by the energy of nothing, as I have described, the future is indeed bleak. The heavens will become cold and dark and empty. But the situation is actually worse. A universe dominated by the energy of empty space is the worst of all universes for the future of life. Any civilization is guaranteed to ultimately disappear in such a universe, starved of energy to survive. After an unfathomably long time, some quantum fluctuation or some thermal agitation may produce a local region where once again life can evolve and thrive. But that too will be ephemeral. The future will be dominated by a universe with nothing in it to appreciate its vast mystery.\n\nAlternatively, if the matter that makes us up was created at the beginning of time by some quantum processes, as I have described, we are virtually guaranteed that it, too, will disappear once again. Physics is a two-way street, and beginnings and endings are linked. Far, far into the future, protons and neutrons will decay, matter will disappear, and the universe will approach a state of maximum simplicity and symmetry.\n\nMathematically beautiful perhaps, but devoid of substance. As Heraclitus of Ephesus wrote in a slightly different context, \"Homer was wrong in saying: 'Would that strife might perish from among gods and men!' He did not see that he was praying for the destruction of the universe; for if his prayers were heard, all things would pass away.\" Or, as Christopher Hitchens has restated it, \"Nirvana _is_ nothingness.\"\n\nA more extreme version of this eventual retreat into nothingness may be inevitable. Some string theorists have argued, on the basis of complex mathematics, that a universe like ours, with a positive energy in empty space, _cannot_ be stable. Eventually, it must decay to a state in which the energy associated with space will be negative. Our universe will then recollapse inward to a point, returning to the quantum haze from which our own existence may have begun. If these arguments are correct, our universe will then disappear as abruptly as it probably began.\n\nIn this case, the answer to the question, \"Why is there something rather than nothing?\" will then simply be: \"There won't be for long.\"\n\n## EPILOGUE\n\n_The sanction of experienced fact as a face of truth is a profound subject, and the mainspring which has moved our civilization since the Renaissance._\n\n\u2014JACOB BRONOWSKI\n\nI began this book with another quote from Jacob Bronowski:\n\nDream or nightmare, we have to live our experience as it is, and we have to live it awake. We live in a world which is penetrated through and through by science and which is both whole and real. We cannot turn it into a game simply by taking sides.\n\nAs I have also argued, one person's dream is another person's nightmare. A universe without purpose or guidance may seem, for some, to make life itself meaningless. For others, including me, such a universe is invigorating. It makes the fact of our existence even more amazing, and it motivates us to draw meaning from our own actions and to make the most of our brief existence in the sun, simply because we are here, blessed with consciousness and with the opportunity to do so. Bronowski's point, however, is that it doesn't really matter either way, and what we would like for the universe is irrelevant. Whatever happened, happened, and it happened on a cosmic scale. And whatever is about to happen on that scale will happen independent of our likes and dislikes. We cannot affect the former, and we are unlikely to affect the latter.\n\nWhat we can do, however, is try to understand the circumstances of our existence. I have described in this book one of the most remarkable journeys of exploration humanity has ever taken in its evolutionary history. It is an epic quest to explore and understand the cosmos on scales that simply were unknown a century ago. The journey has pushed the limits of the human spirit, combining the willingness to follow evidence wherever it might lead with the courage to devote a lifetime to exploring the unknown with the full knowledge that the effort might go nowhere, and finally requiring a mixture of creativity and persistence to address the often tedious tasks of sorting through endless equations or endless experimental challenges.\n\nI have always been attracted to the myth of Sisyphus and have likened the scientific effort at times to his eternal task of pushing a boulder up a mountain, only to have it fall back each time before he reaches the top. As Camus imagined, Sisyphus was smiling, and so should we. Our journey, whatever the outcome, provides its own reward.\n\nThe phenomenal progress we have made in the past century has brought us to the cusp, as scientists, of operationally addressing the deepest questions that have existed since we humans took our first tentative steps to understand who we are and where we came from.\n\nAs I have described here, in the process the very meaning of these questions has evolved along with our understanding of the universe. \"Why is there something rather than nothing?\" must be understood in the context of a cosmos where the meaning of these words is not what it once was, and the very distinction between something and nothing has begun to disappear, where transitions between the two in different contexts are not only common, but required.\n\nAs such, the question itself has been sidelined as we strive in our quest for knowledge. Instead, we are driven to understand the processes that govern nature in a way that allows us to make predictions and, whenever possible, to affect our own future. In so doing, we have discovered that we live in a universe in which empty space\u2014what formerly could have passed for nothing\u2014has a new dynamic that dominates the current evolution of the cosmos. We have discovered that all signs suggest a universe that could and plausibly did arise from a deeper nothing\u2014involving the absence of space itself\u2014and which may one day return to nothing via processes that may not only be comprehensible but also processes that do not require any external control or direction. In this sense, science, as physicist Steven Weinberg has emphasized, does not make it impossible to believe in God, but rather makes it possible to not believe in God. Without science, everything is a miracle. With science, there remains the possibility that nothing is. Religious belief in this case becomes less and less necessary, and also less and less relevant.\n\nThe choice to turn to the notion of divine creation falls to each of us, of course, and I don't expect the ongoing debate to die down anytime soon. But as I have stressed, I believe that if we are to be intellectually honest, we must make an informed choice, informed by fact, not by revelation.\n\nThat has been the purpose of this book, to provide an informed picture of the universe as we understand it and to describe the theoretical speculations that currently are driving physics forward as we scientists attempt to separate the wheat from the chaff in our observations and theories.\n\nI have made clear my own predilection: the case that _our_ universe arose from nothing seems by far the most compelling intellectual alternative to me at the present time. You will draw your own conclusion.\n\nI want to end my discussion by returning to a question that I personally find even more intellectually fascinating than the question of something from nothing. It is the question Einstein asked about whether God had any choice in the creation of the universe. This question provides the basic motivation for almost all research into the fundamental structure of matter, space, and time\u2014the research that has occupied me for much of my professional life.\n\nI used to think there was a stark choice in the answer to this question, but in the process of writing this book, my views have altered. Clearly, if there is a single theory involving a unique set of laws that describes and, indeed, prescribes how our universe came into being and the rules that have governed its evolution ever since\u2014the goal of physics since Newton or Galileo\u2014then the answer would appear to be, \"No, things had to be the way they were, and are.\"\n\nBut if our universe is not unique, and it is a part of a vast and possibly infinite multiverse of universes, would the answer to Einstein's question be a resounding \"Yes, there is a host of choices for existence\"?\n\nI am not so sure. It could be that there is an infinite set of different combinations of laws and varieties of particles and substances and forces and even distinct universes that may arise in such a multiverse. It may be that only a certain very restricted combination, one that results in the universe of the type in which we live or one very much like it, can support the evolution of beings who can ask such a question. Then the answer to Einstein will still remain negative. A God or a Nature that could encompass a multiverse would be as constrained in the creation of a universe in which Einstein could ask the question as either would be if there is only one choice of a consistent physical reality.\n\nI find oddly satisfying the possibility that, in either scenario, even a seemingly omnipotent God would have no freedom in the creation of our universe. No doubt because it further suggests that God is unnecessary\u2014or at best redundant.\n\n## AFTERWORD\n\nby Richard Dawkins\n\nNothing expands the mind like the expanding universe. The music of the spheres is a nursery rhyme, a jingle to set against the majestic chords of the Symphonie Galactica. Changing the metaphor and the dimension, the dusts of centuries, the mists of what we presume to call \"ancient\" history, are soon blown off by the steady, eroding winds of geological ages. Even the age of the universe, accurate\u2014so Lawrence Krauss assures us\u2014to the fourth significant figure at 13.72 billion years, is dwarfed by the trillennia that are to come.\n\nBut Krauss's vision of the cosmology of the remote future is paradoxical and frightening. Scientific progress is likely to go into reverse. We naturally think that, if there are cosmologists in the year 2 trillion AD, their vision of the universe will be expanded over ours. Not so\u2014and this is one of the many shattering conclusions I take away on closing this book. Give or take a few billion years, ours is a very propitious time to be a cosmologist. Two trillion years hence, the universe will have expanded so far that all galaxies but the cosmologist's own (whichever one it happens to be) will have receded behind an Einsteinian horizon so absolute, so inviolable, that they are not only invisible but beyond all possibility of leaving a trace, however indirect. They might as well never have existed. Every trace of the Big Bang will most likely have gone, forever and beyond recovery. The cosmologists of the future will be cut off from their past, and from their situation, in a way that we are not.\n\nWe know we are situated in the midst of 100 billion galaxies, and we know about the Big Bang because the evidence is all around us: the redshifted radiation from distant galaxies tells us of the Hubble expansion and we extrapolate it backward. We are privileged to see the evidence because we look out on an infant universe, basking in that dawn age when light can still travel from galaxy to galaxy. As Krauss and a colleague wittily put it, \"We live at a very special time . . . the only time when we can observationally verify that we live at a very special time!\" The cosmologists of the third trillennium will be forced back to the stunted vision of our early twentieth century, locked as we were in a single galaxy which, for all that we knew or could imagine, was synonymous with the universe.\n\nFinally, and inevitably, the flat universe will further flatten into a nothingness that mirrors its beginning. Not only will there be no cosmologists to look out on the universe, there will be nothing for them to see even if they could. Nothing at all. Not even atoms. Nothing.\n\nIf you think that's bleak and cheerless, too bad. Reality doesn't owe us comfort. When Margaret Fuller remarked, with what I imagine to have been a sigh of satisfaction, \"I accept the universe,\" Thomas Carlyle's reply was withering: \"Gad, she'd better!\" Personally, I think the eternal quietus of an infinitely flat nothingness has a grandeur that is, to say the least, worth facing off with courage.\n\nBut if something can flatten into nothing, can nothing spring into action and give birth to something? Or why, to quote a theological chestnut, is there something rather than nothing? Here we come to perhaps the most remarkable lesson that we are left with on closing Lawrence Krauss's book. Not only does physics tell us how something could have come from nothing, it goes further, by Krauss's account, and shows us that nothingness is unstable: something was almost bound to spring into existence from it. If I understand Krauss aright, it happens all the time: The principle sounds like a sort of physicist's version of two wrongs making a right. Particles and antiparticles wink in and out of existence like subatomic fireflies, annihilating each other, and then re-creating themselves by the reverse process, out of nothingness.\n\nThe spontaneous genesis of something out of nothing happened in a big way at the beginning of space and time, in the singularity known as the Big Bang followed by the inflationary period, when the universe, and everything in it, took a fraction of a second to grow through twenty-eight orders of magnitude (that's a 1 with twenty-eight zeroes after it\u2014think about it).\n\nWhat a bizarre, ridiculous notion! Really, these scientists! They're as bad as medieval Schoolmen counting angels on pinheads or debating the \"mystery\" of the transubstantiation.\n\nNo, not so, not so with a vengeance and in spades. There is much that science still doesn't know (and it is working on it with rolled-up sleeves). But some of what we do know, we know not just approximately (the universe is not mere thousands but billions of years old): we know it with confidence and with stupefying accuracy. I've already mentioned that the age of the universe is measured to four significant figures. That's impressive enough, but it is nothing compared to the accuracy of some of the predictions with which Lawrence Krauss and his colleagues can amaze us. Krauss's hero Richard Feynman pointed out that some of the predictions of quantum theory\u2014again based on assumptions that seem more bizarre than anything dreamed up by even the most obscurantist of theologians\u2014have been verified with such accuracy that they are equivalent to predicting the distance between New York and Los Angeles to within one hairsbreadth.\n\nTheologians may speculate about angels on pinheads or whatever is the current equivalent. Physicists might seem to have their own angels and their own pinheads: quanta and quarks, \"charm,\" \"strangeness,\" and \"spin.\" But physicists can count their angels and can get it right to the nearest angel in a total of 10 billion: not an angel more, not an angel less. Science may be weird and incomprehensible\u2014more weird and less comprehensible than any theology\u2014but science works. It gets results. It can fly you to Saturn, slingshotting you around Venus and Jupiter on the way. We may not understand quantum theory (heaven knows, I don't), but a theory that predicts the real world to ten decimal places cannot in any straightforward sense be wrong. Theology not only lacks decimal places: it lacks even the smallest hint of a connection with the real world. As Thomas Jefferson said, when founding his University of Virginia, \"A professorship of Theology should have no place in our institution.\"\n\nIf you ask religious believers why they believe, you may find a few \"sophisticated\" theologians who will talk about God as the \"Ground of all Isness,\" or as \"a metaphor for interpersonal fellowship\" or some such evasion. But the majority of believers leap, more honestly and vulnerably, to a version of the argument from design or the argument from first cause. Philosophers of the caliber of David Hume didn't need to rise from their armchairs to demonstrate the fatal weakness of all such arguments: they beg the question of the Creator's origin. But it took Charles Darwin, out in the real world on HMS _Beagle,_ to discover the brilliantly simple\u2014and non-question-begging\u2014alternative to design. In the field of biology, that is. Biology was always the favorite hunting ground for natural theologians until Darwin\u2014not deliberately, for he was the kindest and gentlest of men\u2014chased them off. They fled to the rarefied pastures of physics and the origins of the universe, only to find Lawrence Krauss and his predecessors waiting for them.\n\nDo the laws and constants of physics look like a finely tuned put-up job, designed to bring us into existence? Do you think some agent must have caused everything to start? Read Victor Stenger if you can't see what's wrong with arguments like that. Read Steven Weinberg, Peter Atkins, Martin Rees, Stephen Hawking. And now we can read Lawrence Krauss for what looks to me like the knockout blow. Even the last remaining trump card of the theologian, \"Why is there something rather than nothing?\" shrivels up before your eyes as you read these pages. If _On the Origin of Species_ was biology's deadliest blow to supernaturalism, we may come to see _A Universe from Nothing_ as the equivalent from cosmology. The title means exactly what it says. And what it says is devastating.\n\n## ABOUT THE AUTHOR\n\nLawrence M. Krauss is Foundation Professor in the School of Earth and Space Exploration and the Physics Department at Arizona State University, as well as Co-Director of the Cosmology Initiative and Inaugural Director of the Origins Project. The Origins program involves new and wide-ranging interdisciplinary research, teaching, and outreach focusing on all aspects of origins: from the origins of the cosmos to human origins, to the origins of consciousness and culture. Krauss is an internationally known theoretical physicist with broad research interests, including the interface between elementary particle physics and cosmology. He received his PhD in physics from the Massachusetts Institute of Technology in 1982, and joined the Harvard Society of Fellows. In 1985, he joined the faculty of physics at Yale University, and then moved to Case Western Reserve University as Ambrose Swasey Professor in 1993. From 1993 to 2005, he served as chairman of the physics department at Case. He is the recipient of numerous international awards for his research and writing, and is the only physicist to receive awards from all three major US physics societies, the American Physical Society, the American Association of Physics Teachers, and the American Institute of Physics.\n\nKrauss is also one of the few prominent scientists today to have actively crossed the chasm between science and popular culture, causing him to be heralded as a unique \"public intellectual\" by _Scientific American_ magazine. For example, besides his books and radio and television work, and his newspaper and magazine commentaries, Krauss has performed solo with the Cleveland Orchestra, narrating Gustav Holst's _The Planets_ at the Blossom Music Center in the most highly attended concert at that venue, and he was nominated for a Grammy Award for his liner notes for a Telarc CD of music from _Star Trek_. In 2005, he also served as a jury member at the Sundance Film Festival.\nAlso by Lawrence M. Krauss\n\n_The Fifth Essence_\n\n _Fear of Physics_\n\n _The Physics of Star Trek_\n\n_Beyond Star Trek:_  \n _From Alien Invasions to the End of Time_\n\n_Quintessence:_  \n _The Mystery of the Missing Mass_\n\n_Atom:_  \n _A Single Oxygen Atom's Journey from the Big Bang to Life on Earth . . . and Beyond_\n\n_Hiding in the Mirror:_  \n _The Quest for Alternate Realities, from Plato to String Theory_\n\n_Quantum Man:_  \n _Richard Feynman's Life in Science_\n\n## Q & A WITH THE AUTHOR\n\n1. What do you really mean by \"nothing\"?\n\nAs I describe in the book, I believe it is most useful to base our definitions on empirically discovered realities rather than abstract philosophical precepts. For me, independent of the question of \"non-existence,\" which takes one off on lots of potentially deep philosophical issues but rather impotent physics ideas, the really seemingly miraculous aspect of our universe, which I also believe has inspired much of the debate about this topic over the centuries, is how all of the stuff we see could have arisen from a universe in which that stuff did not already exist. It seems at the very least to violate the conservation of energy, and more significantly, common sense. But one of the great things about science that I want to convey is that common sense is not necessarily a good guide to understanding nature at the forefront. Our common sense should derive from the universe, rather than vice versa. And the remarkable non-miraculous miracle is that combining quantum mechanics with gravity allows stuff to arise from no-stuff.\n\nNow, that state of no-stuff may not be \"nothing\" in a classical sense, but it is a remarkable transformation nevertheless. So, the first form of \"nothing\" is just empty space. But one is perfectly reasonable in questioning whether this is really \"nothing\" because space is there, as is time. I then describe how it is possible that space and time themselves could have arisen from no space and time, which is certainly closer to absolute nothing. Needless to say, one can nevertheless question whether that is nothing, because the transition is mediated by some physical laws. Where did they come from? That is a good question, and one of the more modern answers is that even the laws themselves may be random, coming into existence along with universes that may arise. This may still beg the question of what allows any of this to be possible, but at some level it is, as I describe at the beginning of the book, \"turtles all the way down.\" There are questions we can address effectively via empirical methods and questions we can ask that don't lead to physical insights and predictions. The trick is to tell the difference between the two.\n\n2. Why \"How?\" and not \"Why?\"\n\n\"Why\" questions are laced with intellectual baggage that is usually unintended. We can ask \"Why are there nine planets around our sun?\" (since for me Pluto will always be a planet!) but by that we don't ascribe significance or purpose to the number nine, as if the universe was designed so there would be nine planets around the sun. If our sun was the only star, then one might ascribe some significance to that particular number (as Kepler did when he tried to explain six planets in terms of Platonic solids). But we are much more interested in the different ways solar systems can arise and how. Asking \"Why?\" presumes some purpose, which need not exist. Ultimately one can keep asking why forever, and the ultimate answer may simply be \"Because,\" but that doesn't illuminate much.\n\n3. If you get rid of God, then does life lose all purpose?\n\nFor me it certainly doesn't\u2014quite the opposite. I would find little purpose living in a world ruled by some divine Saddam Hussein\u2014like character, as my late friend Christopher Hitchens put it, who not only makes all the rules, but punishes those who disobey them with eternal damnation. I find living in a universe without purpose to be amazing, because it makes the accident of our existence and our consciousness even more precious\u2014something to be valued during our brief moment in the sun.\n\n4. What do you mean by \"flat\"? Is the universe flat as a pancake?\n\nI wish I had described this a little more carefully in the hardcover edition, and I have expanded the discussion in this edition. A flat three-dimensional space is just the kind of space you already thought you lived in, where light rays travel in straight lines, and perpendicular axes (x,y, and z) remain perpendicular. In curved three-dimensional spaces neither statement is true. Since mass and energy can curve space locally (i.e., around the sun and earth for example), the big question is what about the global structure of space on the largest scales: Is it curved or not? And it turns out on the largest observable scales, it isn't. And that fact is very telling, as I describe in the book, because it is what one would expect from a universe that arose from nothing.\n\n5. Isn't science just another kind of faith?\n\nAbsolutely not. Scientists change their minds, admit they are wrong, and are happy and eager to throw out ideas that turn out not to work. We don't presume to know for certain the answers to questions before we ask them. So yes, we have faith that the universe is comprehensible, but the greatest thing about science is that our faith is shakable. At any moment we can give up believing in anything we once believed in, if nature suggests otherwise.\n\n6. Does the search for the Higgs boson at the Large Hadron Collider have cosmological significance? What if we discover it? What if we don't?\n\nI discuss this issue in the new preface to this paperback edition. The search for the Higgs boson reflects the capstone of a remarkable intellectual journey that began over fifty years ago, and if it is discovered at the LHC as initial results reported in 2011 suggest, it would validate a theoretical edifice that otherwise would be on shaky ground. In that sense it would be remarkable if our ideas about the Higgs are correct, because usually nature surprises us. Most theories are in fact wrong. If that weren't the case, anyone could do physics. But in any case if the Higgs exists, it means that another aspect of our existence is a cosmic accident. Particles would get their masses by interactions with a background, otherwise invisible, field, like trying to swim through molasses. That means if such a field had not become established in the early universe, we wouldn't be here . . . yet more something from nothing! At the same time, a Higgs discovery at the LHC will likely raise more questions than answers: Why does it have the mass it does? How can we understand its existence in the context of all four known forces in nature? And so on.\n\n7. I have read it claimed that the fundamental laws of nature have nothing to say on the subject of where observed forces came from, or of why the world should have consisted of the particular kinds of particles and fields it does, or why there should have been a world in the first place. Can you comment?\n\nIn fact, it is one of the great developments in particle physics in the past forty years to realize that the properties of the universe we see, which forces are manifest, which particular kinds of fields can exist on observable scales, and which particles have mass and which don't, can arise spontaneously as an accident of our circumstances. This phenomenon is called \"spontaneous symmetry breaking,\" and it basically says that as the universe evolves and cools, some background field can develop throughout space, just like an ice crystal spontaneously forms on your window sill and just as the Higgs field is predicted to have done. (The nature of the specific patterns on your window sill on a frosty day is not predetermined at the beginning of time but arises dynamically.)\n\nWhen this background field develops, it causes some particles to become massive (and therefore become unstable to decay to other particles and disappear) and others to remain massless. It also determines which forces operate at long distances, like electromagnetism, and which don't, like the weak interaction. As for why a world can exist in the first place, once again, spontaneous symmetry breaking\u2014in this case including the possibility of gravity acting\u2014can cause some universes to expand indefinitely and be long lived, while others will disappear in an instant. Thus it can also explain why some worlds exist long enough to ask the question: \"Why is there something rather than nothing?\"\n\n8. Isn't it presumptuous to claim that we know the universe came from nothing, and that science has answered all the outstanding questions of cosmology?\n\nIt is amusing to read this criticism, usually launched by people who haven't read the book. One of the central points of my book is that we don't know all the answers, but what we have learned is remarkably tantalizing, while at the same time butting us up against some profound fundamental questions that may never be truly amenable to empirical falsification.\n\n9. Isn't science compatible with religion? After all, they both explore the same questions, don't they?\n\nScience is compatible with some basic form of deism\u2014namely, we cannot say that a universe, even one that comes from nothing by natural physical processes, was not created with some underlying purpose that may not be evident. (The fact that there is no evidence of purpose makes it of course harder to argue for one, but never mind.) But having said that, science is not compatible with all the strict doctrines of all the world's major religions, and that includes Christianity, Judaism, Islam, as well as some of the minor ones, like Mormonism and Buddhism. And there is good reason for this: The doctrines were written down by people who didn't know how the world worked. Except for Mormonism, which is recent, they were written down when we didn't know that the Earth orbited the Sun!\n\n10. Are you an atheist?\n\nNot in the sense that I can claim definitively that there is no God or purpose to the universe. I cannot claim definitively that there isn't a teapot orbiting Jupiter, as Bertrand Russell once said. It is highly unlikely, of course. But what I can claim definitively is that I wouldn't want to live in a universe with a God\u2014that makes me an anti-theist, as my friend Christopher Hitchens was.\nWe hope you enjoyed reading this Free Press eBook.\n\n* * *\n\nSign up for our newsletter and receive special offers, access to bonus content, and info on the latest new releases and other great eBooks from Free Press and Simon & Schuster.\n\nCLICK HERE TO SIGN UP\n\nor visit us online to sign up at  \neBookNews.SimonandSchuster.com\n\n## INDEX\n\nabsorption lines, , \u201311\n\nAdams, Douglas,\n\nAffleck, Ian, \u201369\n\nAndromeda, , , ,\n\nAntarctic, , ,\n\nanthropic arguments, \u201326, , , \u201337\n\non fundamental forces and constants, \u201376\n\non something-rather-than-nothing question, \u201378\n\nantiparticles:\n\nas appearing to move backward in time, \u201365\n\nasymmetry between particles and, \u201362, \u201360\n\ncreation in electric field of, ,\n\nin Dirac's electron equation, \u201361\n\nas required by quantum mechanics, \u201365, , \u201362\n\nas required by relativity theory, \u201365,\n\n_see also_ positrons; virtual particles\n\nAristotle, \u201373\n\nAtkins, Peter,\n\nbaryons,\n\nBig Bang, xvii, , , , ,\n\nCMBR left from, _see_ cosmic microwave background radiation\n\ndating of, , \u201316, ,\n\ndensity of protons and neutrons in, \u201325\n\nelements created in, , \u201325, , \u201314\n\nevidence for, , , , , \u201315, ,\n\nfuture disappearance of evidence for, \u201319,\n\nGod and, , \u20136\n\nknown physical laws as explanation of,\n\nnucleosynthesis in,\n\nas predicted by relativity theory, , , \u201315\n\nwall between us and, \u201344\n\nBig Crunch,\n\nbiology, , \u201391\n\nblack holes, , \u201356\n\nBogan, Louise,\n\nBohr, Niels, \u201359,\n\nBOOMERANG (Balloon Observations Of Millimetric Extragalactic Radiation and Geophysics), \u201354,\n\nbosons,\n\nBrahe, Tycho, ,\n\nbranes,\n\nBronowski, Jacob, xi, ,\n\nBrowning, Robert,\n\nCamus, Albert,\n\nCepheid variable stars, \u20139,\n\nChaboyer, Brian, , \u201387\n\nchaotic inflation,\n\n_Christmas Carol, A_ (Dickens),\n\nCL 0024 + 1654,\n\nclosed universe, \u201328\n\nas appearing flat, ,\n\nhot and cold spots in, \u201352\n\ninflation in, , \u201370\n\nlight rays as converging in, ,\n\nrate of expansion in, ,\n\ntotal energy in, \u201368\n\nclusters, , , , , , ,\n\ngravitational lensing by, , \u201333\n\nmeasurement of, \u201388\n\nComa cluster,\n\nCopernican principle,\n\ncosmic microwave background radiation (CMBR):\n\nBOOMERANG's attempt to photograph, \u201354\n\ndiscovery of, , , , , , ,\n\nas evidence for Big Bang, , , ,\n\nflat universe implied by, \u201354,\n\nhot spots and cold spots in, , \u201349, \u201352, , ,\n\nmeasurements of, \u201343, \u201354,\n\nobservation vs. prediction of, \u201369\n\nphotons in,\n\nuniformity of, \u201395\n\nas unobservable in far future, \u201311\n\ncosmic myopia, \u201350\n\ncosmic rays, ,\n\ncosmological constant,\n\nas \"anthropically selected,\" \u201326\n\ndark energy as represented by, , \u201373, \u201376, , , , \u201384, , , , , \u201324,\n\nEinstein's introduction and regret of, , \u201358,\n\nCosmological Constant Problem, \u201373\n\ncosmology, xii, xvi, xviii, , ,\n\npossible future end of, \u201319, \u201388\n\ncreator, creationism, xi, xii, xiv, , ,\n\n_see also_ God; theology\n\ndark energy, xii,\n\nage of universe and,\n\namount of, ,\n\nin anthropic argument,\n\nbleak future implied by, \u201319, \u201380\n\ncosmic jerk and, \u201389\n\ncosmological constant as representative of, , \u201373, \u201376, , , , \u201384, , , , , \u201324,\n\ndensity of, , \u201324\n\ndiscovery of, xviii, , \u201317, , ,\n\nevidence for, \u201389, \u20139\n\nexpansion of universe as dominated by, , \u201389, , , , \u20139\n\nfalse vacuum energy as,\n\nin flat universe, , , \u201388,\n\nKrauss and Turner's proposal of, , \u201376,\n\nmeasurement of, \u201373,\n\norigin of, , ,\n\nreal particles and radiation created from, ,\n\nin string theory,\n\nzero gravitational energy allowed by,\n\ndark matter, ,\n\namount of, \u201326, \u201328, , , \u201356\n\nin closed universe,\n\ncomposition of, , \u201335\n\ndensity of galaxy clusters as dominated by,\n\ndetection of, , , , \u201337, , ,\n\nexperiments on, \u201336\n\nflat universe as caused by, , , , , ,\n\nratio of visible matter to, , , , ,\n\nZwicky's early prediction of,\n\nDarwin, Charles, , , , \u201391\n\nDawkins, Richard, xix, \u201391\n\nDemarque, Pierre,\n\ndeuterium, abundance of, ,\n\nDickens, Charles, ,\n\ndimensions, extra:\n\npossible evidence for, \u201338\n\nin string theory, , , , , \u201335\n\nundetectable universes in, ,\n\nDirac, Paul, , \u201360, \u201369,\n\nDoppler effect,\n\nD'Souza, Dinesh,\n\nE = mc2, ,\n\nEddington, Arthur Stanley, \u20135\n\nEinstein, Albert, , ,\n\nexperiment as important to, \u20133\n\ngravitational lensing paper of, \u201331, \u201337\n\nidea of expanding universe rejected by, , \u201357,\n\nand question of God's choice, \u201361, \u201385\n\nuniversal speed limit discovered by,\n\n_see also_ relativity, general theory of; relativity, special theory of\n\nelectric fields, , ,\n\nelectromagnetism, , ,\n\nquantum theory of, ,\n\nelectrons, , , ,\n\nantimatter form of, \u201360\n\nin early universe,\n\nmass of, , ,\n\nas moving faster than light, \u201365\n\npotential for existence of,\n\nin quantum levels,\n\nspin of,\n\nwave function of, , , \u201367\n\nelementary particles, ,\n\n_see also specific particles_\n\nempty space, xvi\n\nenergy of, _see_ dark energy\n\nreal particles created in,\n\nvirtual particles and fields in, \u201365, \u201371, \u201398, ,\n\n_see also_ nothing\n\nenergy:\n\nconservation of, \u2013104, , , ,\n\ndark, _see_ dark energy\n\ndefinition of,\n\nkinetic, \u2013101\n\nnegative, \u2013104,\n\npotential, \u2013101\n\nrest,\n\nenergy density, , , \u20134\n\nevent horizon,\n\nevolution, biological, xiii, ,\n\nfalse vacuum energy, , , , ,\n\nFaraday, Michael,\n\nFeynman, Richard, ,\n\non accuracy of quantum theory, \u201390\n\non fundamental laws, xiii,\n\nnecessity of antiparticles demonstrated by, \u201365, \u201362\n\nsum over paths formalism developed by, \u201363, \u201369\n\nfield lines, \u201367\n\n_Flatland_ (Abbott),\n\nFlatness Problem, ,\n\nflat universe, , ,\n\nclosed universe as giving appearance of, ,\n\ndark energy in, , , \u201388,\n\ndark matter as cause of, , , , , ,\n\nevidence against, \u201354, \u201356,\n\nevidence for, , , \u201356, \u201376, , \u201393, ,\n\nexpansion rate of, , \u201388,\n\nas expected of universe arising from nothing, \u201352\n\nas explained by inflation, , , \u201351, ,\n\nhot and cold spots in,\n\nKrauss and Turner's prediction of, , \u201376,\n\nlight rays as straight in, ,\n\nslowed expansion of,\n\ntotal mass needed for, , ,\n\nzero gravitational energy in, ,\n\nzero total energy in,\n\nfundamental constants of nature, , , \u201376\n\nfundamental forces, , , \u201376\n\n_see also specific forces_\n\ngalaxies:\n\ngravitational lensing by,\n\ninstability of,\n\nredshift of, \u201311, , \u20137\n\nrotation of, \u201324\n\nspiral, _see_ spiral galaxies (spiral nebulae)\n\nsuperclusters of,\n\n_see also_ clusters; Hubble's Law\n\nGalileo Galilei, ,\n\nGauss, Carl Friedrich, \u201341\n\nGenesis, ,\n\ngeodetic survey maps, \u201341\n\ngeometry, \u201341\n\nof universe, _see_ closed universe; flat universe; open universe\n\nGod, ,\n\nBig Bang and, , \u20136\n\nDarwin's removal of need for, , \u201391\n\nEinstein's \"choice\" question to, \u201361, \u201385\n\nfundamental constants and,\n\ninfinite regress and, xii, xv\n\nin Newton's work, ,\n\nOccam's Razor and,\n\nand origin of morality,\n\nPius XII's attempt to prove existence of, ,\n\nredundancy of,\n\nsomething from nothing as created by, xv, \u201375\n\nand total gravitational energy,\n\n_see also_ theology\n\nGod of the Gaps, \u201346\n\nGrand Unified Theory,\n\ngravitational lensing, \u201335\n\ndark matter detected by, ,\n\nEinstein's paper on, \u201331, \u201337\n\nZwicky's proposed use for, \u201332,\n\ngraviton,\n\ngravity, xvi\n\nas attractive, , ,\n\nof dark matter,\n\nand expansion of universe, , \u201380,\n\nmotion of galaxies affected by, \u201324\n\nnegative energy allowed by, \u2013104,\n\nnegative pressure of,\n\nNewton's theory of, , , , , , \u201366\n\nspeed of, ,\n\nas unified with other forces,\n\nweakness of, ,\n\nweighing superclusters of galaxies with,\n\n_see also_ relativity, general theory of\n\nGreene, Brian,\n\nGuth, Alan, xviii, , , , , ,\n\nHarris, Sydney,\n\nHartle, Jim,\n\nHawking, Stephen, , \u201364, ,\n\nHeisenberg, Werner, \u201359\n\n_see also_ Uncertainty Principle\n\nhelium, ,\n\nabundance of, , \u201325, , \u201312,\n\nHeraclitus of Ephesus,\n\nHigh-Z Supernova Search Team, ,\n\nHilbert, David, \u20133\n\nHitchens, Christopher, xviii\u2013xix, , , ,\n\n_Hitchhiker's Guide to the Galaxy, The_ (Adams),\n\nHomer,\n\nHorizon Problem, , ,\n\nHoyle, Fred,\n\nHubble, Edwin, ,\n\nage of universe estimated by, \u201316\n\nexpansion of universe discovered by, , , , , , , ,\n\nnew galaxies discovered by, \u20139\n\nHubble's constant, \u201316, , \u201380\n\nHubble's law, \u201316, ,\n\nHubble Space Telescope, ,\n\nHumason, Milton, , ,\n\nHume, David,\n\nhydrogen, , , \u201368\n\nabundance of, , \u201325, , \u201312,\n\nhyperbolic geometry,\n\ninfinite regress, xii, xv\n\ninfinities, xii\u2013xiii,\n\ninflation, , , ,\n\nas allowed in general relativity, \u201397\n\nas beginning from nothing,\n\nin closed universe, , \u201370\n\nconservation of energy and, \u2013104\n\nenergy density and, , \u20134\n\nas eternal, \u201329, ,\n\nflat universe explained by, , , \u201351, ,\n\nhomogeneous universe explained by, , \u201351\n\nHorizon Problem solved by,\n\nmultiverses created by, \u201329\n\nquantum fluctuations and, \u2013104,\n\nsmall-density fluctuations in, \u201351\n\ntotal gravitational energy and, \u20133\n\nzero gravitational energy and, , ,\n\ninstanton,\n\niron, ,\n\nJames, William, xii\n\nJefferson, Thomas,\n\njerk, \u201389\n\n_Journey Around My Room_ (Bogan),\n\nKepler, Johannes, \u201320,\n\nthree laws of planetary motion discovered by,\n\nKernan, Peter,\n\nkinetic energy, \u2013101\n\nKrauss, Lawrence, xvi\u2013xviii, , \u201376, , , , \u201387, \u201318\n\nLamb, Willis, \u201367\n\nlandscape, \u201330, \u201335, \u201377\n\nLarge Hadron Collider, \u201336,\n\nlast scattering surface, \u201347, , \u201354,\n\nBOOMERANG's attempt to picture, \u201354\n\ncharacteristic scale of, \u201345\n\nas earliest visible point of universe, \u201344\n\ngeometry of universe revealed by, \u201347,\n\nLeavitt, Henrietta Swan, \u20138\n\nLeeuwenhook, Antonie Philips van,\n\nLema\u00eetre, Georges, \u20136, , , , , \u201315\n\n\"Lens-Like Action of a Star by the Deviation of Light in the Gravitational Field\" (Einstein), \u201331, \u201337\n\nlife, origin of, , , ,\n\nlight:\n\nbending of, \u201327, \u201330, , ,\n\ngeometry of universe and,\n\nhuman ability to see, \u201350\n\nquanta of,\n\nwavelengths of, , ,\n\nlight speed, as cosmic speed limit, , , , , ,\n\nLinde, Andrei, \u201328,\n\nlithium:\n\nabundance of, , \u201325, , \u201312,\n\nLobachevsky, Nikolai Ivanovich, \u201342\n\nMandl, Rudi, , ,\n\nmass:\n\ndensity in universe of,\n\nof electrons, , ,\n\nof photons,\n\nof protons, \u201361, ,\n\nrest, ,\n\ntotal amount of, , , , , ,\n\nmatter:\n\nconstant creation of,\n\nas created by quantum fluctuation, \u201351,\n\nas created in electric field, ,\n\ndensity of, , , , , ,\n\ndisappearance of,\n\nenergy density of, \u201324\n\norigin of, ,\n\nratio of dark matter to, , , , ,\n\nMercury, orbit of, ,\n\nmetabolism, ,\n\nmeta-galaxy, , \u201311, ,\n\nMies van der Rohe, Ludwig,\n\nMilky Way, , , , , ,\n\nmiracles, \u201342\n\nMonopole Problem, ,\n\nmorality, xvi, \u201372\n\nmotion, Newton's laws of,\n\nMount Stromlo Observatory,\n\nMount Wilson Observatory, ,\n\nmultiverse, , \u201329, \u201335, \u201377,\n\nGod's choice and, \u201385\n\nmuons,\n\nnebulae, \u20137, \u201332\n\n_see also_ spiral galaxies (spiral nebulae)\n\nnegative pressure, ,\n\nneutrons, ,\n\namount of, ,\n\nantiparticle of,\n\ndecay of,\n\ninitial density of,\n\nnuclear reactions of,\n\nneutron stars,\n\nNewton, Isaac:\n\nGod as viewed by, ,\n\ngravitational theory of, , , , , , \u201366\n\nlaws of motion formulated by,\n\nprism experiment of,\n\nsingle theory sought by,\n\nNobel Prize, , ,\n\nnothing:\n\ndefinitions of, xiii\u2013xv,\n\nas effected by virtual particles, \u201371,\n\nenergy in,\n\nas possible end of universe, \u201380,\n\nuniverse as originating from, xvii, \u201343, , \u201370\n\nas unstable, \u201370, , ,\n\nweight of,\n\n_see also_ empty space; something-rather-than-nothing question\n\n_On the Origin of Species_ (Darwin),\n\nopen universe,\n\nin computer simulation,\n\ndeceleration of,\n\neternal expansion of, ,\n\nevidence for, \u201337,\n\nexpansion rate of, ,\n\nhot and cold spots in,\n\nlight rays as bent in, , ,\n\nLobachevsky's investigation of, \u201342\n\nOrigin of Life workshop, ,\n\nOrigins Project, ,\n\noxygen, ,\n\nPais, Abraham,\n\nparticle accelerators, \u201336,\n\nparticle physicists, , ,\n\nparticle theory, xvi\n\nperihelion, ,\n\nperiod-luminosity relationship, \u20138\n\nPerlmutter, Saul, , ,\n\nphase transitions, \u201396, ,\n\nphilosophy, xiii\u2013xiv,\n\nphotons, , , \u201367\n\nphysical laws, xiii, , ,\n\nas arising from nothing, xv, , \u201380\n\nand asymmetry of matter and antimatter, \u201359\n\nas complete and necessary for describing universe's evolution,\n\nmiracles vs., \u201342\n\norigins of, xi\u2013xii\n\npossible randomness of, xvii, \u201376\n\n_Physical Review,_\n\nPinker, Steven, \u201372\n\nPius XII, Pope, ,\n\nPlanck, Max,\n\nPlanck-time, ,\n\nplanets, motion of, ,\n\nplasma, , , \u201311\n\nPlato, xii, ,\n\npositrons, \u201360, \u201365, \u201368,\n\npotential energy, \u2013101\n\npressure, negative, ,\n\nprotons,\n\namount of, ,\n\nantimatter of,\n\ndecay of,\n\nin early universe, ,\n\nmass of, \u201361, ,\n\nnuclear reactions of,\n\npositrons mistaken for, \u201361\n\nsmashing of, \u201336\n\nstructure of, \u201370\n\nquantum fluctuations, \u201398\n\nconservation of energy as limitation to, \u201354\n\nas explanation of everything, , , \u201351, , , ,\n\nas important in inflation, \u2013104,\n\n_see also_ inflation\n\nquantum gravity, ,\n\nblack hole radiation and,\n\nand fate of closed expanding universes,\n\ninflation created by,\n\nspace created by, \u201364\n\nvirtual particles and, \u201372\n\nquantum levels,\n\nquantum mechanics, ,\n\naccuracy of predictions of, \u201369, \u201390\n\ndevelopment of, \u201359\n\nrelativity theory and, \u201359, ,\n\nof space-time, ,\n\n_see also_ Uncertainty Principle\n\nquantum vacuum, xiv\n\nquarks, \u201370,\n\nquasars, \u201332\n\n_Quintessence_ (Krauss),\n\nradiation:\n\nas caused by meeting of particle and antiparticle,\n\nas created by quantum fluctuation, \u201351\n\ndensity of, ,\n\nin early universe, \u201344\n\nquanta of,\n\nredshift, \u201311, , \u201382, \u201386,\n\ndistance-versus-, , \u201382, \u201386,\n\nin far future, \u20137\n\nRees, Martin,\n\nrelativity, general theory of:\n\napparent problem with, , ,\n\nBig Bang predicted by, , , \u201315\n\ncollapse of closed universe predicted in,\n\ncurvature of universe in, , ,\n\ndark matter required for flat universe in,\n\ndensity of universe in,\n\nequations of, \u201358\n\nevolution of universe explained by, , ,\n\nexperimental evidence for, \u20133, \u201327\n\nfaster-than-light growth of space allowed by, \u201397\n\nlabeling of space and time as arbitrary in,\n\nlight bent in, \u201327, \u201334\n\nnegative energy in, , ,\n\nnegative pressure in,\n\nnonstatic universe predicted by, \u20132, , , \u201357\n\nquantum mechanics and, \u201360, ,\n\nrest energy in,\n\nspace as curved in, \u201327, \u201334, , \u201342,\n\ntotal energy in, \u201366\n\nweighing the universe with,\n\n_see also_ cosmological constant\n\nrelativity, special theory of, ,\n\nantiparticles required by, \u201365,\n\nand observer-dependent measurements, ,\n\nreligion, _see_ God; theology\n\nRetherford, Robert C.,\n\nRichard Dawkins Foundation, xvii\n\nRiess, Adam,\n\nRNA, ,\n\nRoman Catholic Church, xii, , ,\n\nRubin, Vera,\n\nRumsfeld, Donald,\n\nRussell, Bertrand, xii\n\nSagan, Carl, \u201383\n\nScherrer, Robert, , \u201318\n\nSchmidt, Brian,\n\nSchr\u00f6dinger, Erwin, \u201359\n\n_see also_ electrons, wave function of\n\nSchwarzchild solution,\n\nscience, xiii\u2013xiv\n\nGod and,\n\npurposes of, xv\n\nsomething-rather-than-nothing question probed by, xiii\n\nthree key principles of, xvi\n\n_Science,_ , ,\n\n_Scientific American,_\n\nself-replicating cells,\n\nShapley, Harlow, \u20137,\n\nsingularity,\n\nSlipher, Vesto, ,\n\nsnowflakes, xi\n\nsolar eclipse, \u201327\n\nsomething-rather-than-nothing question, \u201345, \u201389,\n\nanthropic answer to, \u201378\n\nboundary conditions for, \u201370\n\nas \"how\" question,\n\nand instability of nothing,\n\nmetaphysical answer to, \u201375\n\norigin of universe and, xiii\n\nphysical laws as complete for, \u201352\n\nphysical laws unnecessary for, , \u201380\n\nas possibly insignificant, \u201379\n\nquantum fluctuations as answer to, \u201351, , ,\n\nquantum gravity's answer to,\n\nas scientific, xiii\n\nshifting meaning of, \u201383\n\nspace unnecessary for, \u201370\n\nspace:\n\nas arising from nothing, xiv\u2013xv, \u201370\n\nas created by quantum gravity, \u201364\n\ncurvature of, \u201327, \u201335, , \u201342, ; _see also_ closed universe; flat universe; open universe\n\nfaster-than-light growth of, \u201397\n\nin general theory of relativity, , \u201327, \u201329, ,\n\ninflation of, _see_ inflation\n\nas possibly infinite, xiii\n\nquantum theory as applying to,\n\nspace-time, quantum mechanics, ,\n\nspectrum, \u201310, ,\n\nSpinoza, Baruch,\n\nspiral galaxies (spiral nebulae), ,\n\nabsorption lines of, \u201311\n\n\"standard candle,\" , \u201379\n\nstars:\n\nbrightness of, \u20138,\n\ncomposition of, ,\n\nelements made in, \u201319, \u201314\n\nmain sequence,\n\nvariable,\n\n_Star Trek,_\n\nstatic electricity,\n\nStenger, Victor,\n\nstring theory, , \u201335,\n\nsum over paths formalism, \u201363, \u201369\n\nSun, spectrum of,\n\nsuperclusters,\n\nsuperconductivity,\n\nsupernaturalism, \u201342\n\nsupernova:\n\nBrahe's observation of,\n\nelements created in, \u201318\n\nnumber of, , \u201321\n\nand rate of expansion of universe, \u201382, \u201386,\n\nType a, , , \u201382, \u201386,\n\nSupernova Cosmology Project, \u201382\n\nsuperstrings,\n\nsupersymmetry,\n\nsymmetry, , ,\n\nSysiphus,\n\ntauons,\n\ntheology, xi, xiii\u2013xiv, xvi, , \u201346, ,\n\n_see also_ God\n\nTheory of Everything, ,\n\n\"Theory of Positrons, A\" (Feynman), \u201365\n\nThomas Aquinas, xii, , , ,\n\nthought experiments,\n\ntime:\n\nantiparticles as appearing to move backward in, \u201365\n\nas arising from nothing, xiv\u2013xv\n\ncharacteristic scale imprinted on last scattering surface by, \u201345\n\nin general theory of relativity, ,\n\nas possibly infinite, xiii\n\n_see also_ space-time\n\ntotal energy:\n\nin closed universe, \u201368\n\nin flat universe,\n\nproblems in measurement of,\n\nas total gravitational energy plus energy associated with mass,\n\ntotal gravitational energy, \u20133,\n\ndefinition of,\n\nin inflation, \u20133\n\nNewtonian equation for,\n\nas nonaribtrary, \u201349\n\nin total energy,\n\nin universe arising from nothing, \u201352,\n\ntriangles, sum of angles in, \u201340,\n\nTurner, Michael, , \u201376, ,\n\nType Ia supernova, , , \u201382, \u201386,\n\nTyson, Tony, , \u201334\n\nUncertainty Principle, , \u201365, , , ,\n\nuniverse:\n\nage of, , \u201316, , , , \u201387, , ,\n\nalleged rotation of,\n\naverage density of,\n\nboundary conditions of creation from nothing of, \u201370\n\ncooling of, ,\n\nend of, , , ,\n\nas eternal, \u201374\n\nhomogeneity of, ,\n\nmeasurement of curvature of, \u201354, , ,\n\nmultiverse and, , \u201329\n\norigins of, xv, xvi, xvii, xviii, , , , ; _see also_ Big Bang\n\nphase transition of, \u201396,\n\nas possibly infinite, xiii\n\nrecollapse and disappearance of,\n\nsize of, \u20139\n\nstatic view of, , , , \u201357, , , ,\n\ntotal mass in, , , , , ,\n\nweight of, \u201337,\n\n_see also_ closed universe; flat universe; inflation; open universe\n\nuniverse, expansion of, xii, xvii, \u20134, , , , , , , ,\n\ndark energy and, , \u201389, , , , \u20139,\n\ngravity and, , \u201380,\n\nrate of, \u201315, , \u201389, , , ,\n\nas unobservable in far future, \u201319\n\n_see also_ Hubble's law\n\nVilenkin, Alex, ,\n\nVirgo supercluster,\n\nvirtual particles:\n\nDirac equation and, \u201369\n\nempty space effected by, \u201369, \u201371, \u201398, , , ,\n\nas explanation of static electricity,\n\nFeynman's proof of, \u201365\n\nindirect evidence for, \u201367, \u201371,\n\nin quantum gravity,\n\nshort lifetime of, \u201368,\n\nzero total energy of,\n\nvirtual photons, , \u201367\n\nvirtual universes,\n\nWeinberg, Steven, ,\n\nWilczek, Frank, xviii, \u201335,\n\nWilkinson, David,\n\nWilkinson Microwave Anisotropy Probe (WMAP), \u201354,\n\nWitten, Edward, \u201333\n\nX-ray emissions,\n\nZel'dovich, Yakov, \u201373\n\nZwicky, Fritz, \u201332,\n\nFREE PRESS  \nA Division of Simon & Schuster, Inc.  \n1230 Avenue of the Americas  \nNew York, NY 10020  \nwww.SimonandSchuster.com\n\nCopyright \u00a9 2012 by Lawrence M. Krauss\n\nAll rights reserved, including the right to reproduce this book or portions thereof in any form whatsoever. For information address Free Press Subsidiary Rights Department, 1230 Avenue of the Americas, New York, NY 10020.\n\nFirst Free Press hardcover edition January 2012\n\nFREE PRESS and colophon are trademarks of Simon & Schuster, Inc.\n\nJacket design by James Perales  \nCover photograp (stars) \u00a9 Getty\/Luke Peterson Photography\/Flickr  \nAuthor photo \u00a9 Nancy Dahl-Tacconi\n\nThe Simon & Schuster Speakers Bureau can bring authors to your live event. For more information or to book an event contact the Simon & Schuster Speakers Bureau at 1-866-248-3049 or visit our website at www.simonspeakers.com.\n\nLibrary of Congress Cataloging-in-Publication Data  \nKrauss, Lawrence Maxwell.  \nA universe from nothing : why there is something rather than nothing\/ Lawrence M. Krauss ; with an afterword by Richard Dawkins.  \np. cm.  \nIncludes index.  \n1. Cosmology. 2. Beginning. 3. End of the universe. I. Title.  \nQB981.K773 2012 2011032519  \n523.1'8\u2014dc23\n\nISBN 978-1-4516-2445-8  \nISBN 978-1-4516-2447-2 (ebook)\n","meta":{"redpajama_set_name":"RedPajamaBook"}}