Whey Processing, Functionality and Health Benefits (Institute of Food Technologists Series)

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Whey Processing, Functionality and Health Benefits

Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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The IFT Press series reflects the mission of the Institute of Food Technologists—advancing the science and technology of food through the exchange of knowledge. Developed in partnership with Wiley-Blackwell, IFT Press books serve as leading edge handbooks for industrial application and reference and as essential texts for academic programs. Crafted through rigorous peer review and meticulous research, IFT Press publications represent the latest, most significant resources available to food scientists and related agriculture professionals worldwide.

IFT Book Communications Committee Joseph H. Hotchkiss Barry G. Swanson Ruth M. Patrick Terri D. Boylston Syed S. H. Rizvi William C. Haines Mark Barrett Sajida Plauche Karen Banasiak

IFT Press Editorial Advisory Board Malcolm C. Bourne Fergus M. Clydesdale Dietrich Knorr Theodore P. Labuza Thomas J. Montville S. Suzanne Nielsen Martin R. Okos Michael W. Pariza Barbara J. Petersen David S. Reid Sam Saguy Herbert Stone Kenneth R. Swartzel

A John Wiley & Sons, Ltd., Publication

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Whey Processing, Functionality and Health Benefits

EDITORS

Charles I. Onwulata r Peter J. Huth

A John Wiley & Sons, Ltd., Publication

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Edition first published 2008 c 2008 Blackwell Publishing and the Institute of Food Technologists  Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Editorial Office 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services, and for information about how to apply for permission to reuse the copyright material in this book, please see our website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee codes for users of the Transactional Reporting Service are ISBN-13: 978-0-8138-0903-8/2008. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloguing-in-Publication Data Whey processing, functionality and health benefits / editors, Charles Onwulata, Peter Huth. – 1st ed. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-0903-8 (alk. paper) 1. Whey. 2. Whey products. 3. Whey–Health aspects. 4. Dairy processing. I. Onwulata, Charles. II. Huth, Peter (Peter J.) SF275.W5W55 2008 641.3 73–dc22

2008007432

A catalogue record for this book is available from the U.S. Library of Congress. Set in Times New Roman by Aptara Printed in Singapore by Fabulous Printers 1 2008

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Titles in the IFT Press series r Accelerating New Food Product Design and Development (Jacqueline H. Beckley, Elizabeth J. Topp, M. Michele Foley, J.C. Huang, and Witoon Prinyawiwatkul) r Advances in Dairy Ingredients (Geoffrey W. Smithers and Mary Ann Augustin) r Biofilms in the Food Environment (Hans P. Blaschek, Hua H. Wang, and Meredith E. Agle) r Calorimetry and Food Process Design (G¨on¨ul Kaletun¸c) r Food Ingredients for the Global Market (Yao-Wen Huang and Claire L. Kruger) r Food Irradiation Research and Technology (Christopher H. Sommers and Xuetong Fan) r Food Laws, Regulations and Labeling (Joseph D. Eifert) r Food Risk and Crisis Communication (Anthony O. Flood and Christine M. Bruhn) r Foodborne Pathogens in the Food Processing Environment: Sources, Detection and Control (Sadhana Ravishankar and Vijay K. Juneja) r Functional Proteins and Peptides (Yoshinori Mine, Richard K. Owusu-Apenten, and Bo Jiang) r High Pressure Processing of Foods (Christopher J. Doona and Florence E. Feeherry) r Hydrocolloids in Food Processing (Thomas R. Laaman) r Microbial Safety of Fresh Produce: Challenges, Perspectives and Strategies (Xuetong Fan, Brendan A. Niemira, Christopher J. Doona, Florence E. Feeherry, and Robert B. Gravani) r Microbiology and Technology of Fermented Foods (Robert W. Hutkins) r Multivariate and Probabilistic Analyses of Sensory Science Problems (Jean-Fran¸cois Meullenet, Rui Xiong, and Christopher J. Findlay) r Nondestructive Testing of Food Quality (Joseph Irudayaraj and Christoph Reh) r Nanoscience and Nanotechnology in Food Systems (Hongda Chen) r Nonthermal Processing Technologies for Food (Howard Q. Zhang, Gustavo V. Barbosa-C`anovas, and V.M. Balasubramaniam, Editors; C. Patrick Dunne, Daniel F. Farkas, and James T.C. Yuan, Associate Editors) r Nutraceuticals, Glycemic Health and Type 2 Diabetes (Vijai K. Pasupuleti and James W. Anderson) r Packaging for Nonthermal Processing of Food (J.H. Han) r Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions (Ross C. Beier, Suresh D. Pillai, and Timothy D. Phillips, Editors; Richard L. Ziprin, Associate Editor) r Processing and Nutrition of Fats and Oils (Ernesto M. Hernandez, Monjur Hossen, and Afaf Kamal-Eldin) r Regulation of Functional Foods and Nutraceuticals: A Global Perspective (Clare M. Hasler) r Sensory and Consumer Research in Food Product Design and Development (Howard R. Moskowitz, Jacqueline H. Beckley, and Anna V.A. Resurreccion) r Sustainability in the Food Industry (Cheryl J. Baldwin) r Thermal Processing of Foods: Control and Automation (K.P. Sandeep) r Water Activity in Foods: Fundamentals and Applications (Gustavo V. BarbosaC`anovas, Anthony J. Fontana, Jr., Shelly J. Schmidt, and Theodore P. Labuza) r Whey Processing, Functionality and Health Benefits (Charles I. Onwulata and Peter J. Huth)

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Contents

Contributors Preface Chapter 1.

ix xiii Whey Protein Production and Utilization: A Brief History Michael H. Tunick

Chapter 2.

Whey Protein Fractionation Laetitia M. Bonnaillie and Peggy M. Tomasula

Chapter 3.

Separation of β-Lactoglobulin from Whey: Its Physico-Chemical Properties and Potential Uses Raj Mehra and Brendan T. O’Kennedy

Chapter 4.

Whey Protein-Stabilized Emulsions David Julian McClements

Chapter 5.

Whey Proteins: Functionality and Foaming under Acidic Conditions Stephanie T. Sullivan, Saad A. Khan, and Ahmed S. Eissa

1

15

39

63

99

Chapter 6.

Whey Protein Films and Coatings Kirsten Dangaran and John M. Krochta

133

Chapter 7.

Whey Texturization for Snacks Lester O. Pordesimo and Charles I. Onwulata

169

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Chapter 8.

Whey Protein-Based Meat Analogs Marie K. Walsh and Charles E. Carpenter

185

Chapter 9.

Whey Inclusions K.J. Burrington

201

Chapter 10. Functional Foods Containing Whey Proteins B. Faryabi, S. Mohr, Charles I. Onwulata, and Steven J. Mulvaney Chapter 11. Whey Protein Hydrogels and Nanoparticles for Encapsulation and Controlled Delivery of Bioactive Compounds Sundaram Gunasekaran Chapter 12. Whey Proteins and Peptides in Human Health P.E. Morris and R.J. FitzGerald Chapter 13. Current and Emerging Role of Whey Protein on Muscle Accretion Peter J. Huth, Tia M. Rains, Yifan Yang, and Stuart M. Phillips

213

227

285

385

Chapter 14. Milk Whey Processes: Current and Future Trends Charles I. Onwulata

369

Appendix Index

391 393

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Contributors

Laetitia M. Bonnaillie (2) Dairy Processing and Products Research Unit, USDA-ARS-ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA K.J. Burrington (9) Center for Dairy Research, University of Wisconsin-Madison, 1605 Linden Dr, Madison, WI 53706, USA Charles E. Carpenter (8) Department of Nutrition and Food Sciences, Center for Microbial Detection and Physiology, Utah State University, 8700 Old Main Hill, NFS 318, Logan, UT 84322, USA Kirsten Dangaran (6) Dairy Processing and Products Research Unit, USDA-ARS-ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Ahmed S. Eissa (5) Department of Chemical Engineering, Cairo University, Cairo, Egypt B. Faryabi (10) Cornell University, 105 Stocking Hall, Ithaca, NY 14853, USA R.J. FitzGerald (12) Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland

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Contributors

Sundaram Gunasekaran (11) Department of Biological Systems Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA Peter J. Huth (13) Nutrition Research and Scientific Affairs, PJH Nutritional Sciences, Chicago, IL Saad A. Khan (5) Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USA John M. Krochta (6) Department of Food Science, Packaging and Biopolymer Film Laboratory, University of California-Davis, Davis, CA 95616, USA David Julian McClements (4) Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Rm 238, Amherst, MA 01003, USA Raj Mehra (3) Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland S. Mohr (10) Cornell University, 105 Stocking Hall, Ithaca, NY 14853, USA P.E. Morris (12) Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland Steven J. Mulvaney (10) Cornell University, 105 Stocking Hall, Ithaca, NY 14853, USA Brendan T. O’Kennedy (3) Moorepark Food Research Centre,Teagasc, Moorepark,Fermoy, Co. Cork, Ireland

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Contributors

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Charles I. Onwulata (7, 10, 14) Dairy Processing and Products Research Unit, USDA-ARS-ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Stuart M. Phillips (13) Department of Kinesiology—Exercise Metabolism Research Group— IWC 219B, McMaster University, Hamilton, ON L8S 4K1, Canada Lester O. Pordesimo (7) Department of Agricultural and Biological sciences, Mississippi State University, Mississippi State, MS 39762, USA Tia M. Rains (13) Provident Clinical Research and Consulting, 489 Taft Avenue, Glen Ellyn, IL 60137, USA Stephanie T. Sullivan (5) Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USA Peggy M. Tomasula (2) Dairy Processing and Products Research Unit, USDA-ARS-ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Michael H. Tunick (1) USDA-ARS-ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA Marie K. Walsh (8) Department of Nutrition and Food Sciences, Center for Microbial Detection and Physiology, Utah State University, 8700 Old Main Hill, NFS 318, Logan, UT 84322, USA Yifan Yang (13) Department of Kinesiology—Exercise Metabolism Research Group— IWC 219B, McMaster University, Hamilton, ON L8S 4K1, Canada

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Preface

Milk whey proteins have come into wider use as food ingredients only in the last 40 years, taking their proper place at an emerging frontier, where nutrition and health interface. Largely regarded in the past as a waste byproduct, advanced processing technology has propelled whey proteins to the top of the list of important nutrients, and still newer technologies will help keep it there permanently. This book provides an overview of the successes and challenges of the new whey processing industry. As food ingredients, whey proteins are used in a multitude of combinations and advanced well beyond the stage of simply delivering nutritional value by also providing essential functional and health benefits to complex food systems. The contributing authors to this book are outstanding scientists and health professionals in their fields of specialty, working diligently to enhance the utility of whey ingredients for the development of products that deliver demonstrated health benefits to consumers. The knowledge presented in this book documents the wide range of potential uses for whey proteins not only as ingredients in food formulations but also as functional components providing additional metabolic and physiological benefits beyond merely supplying essential amino acids. Health and wellness, processing and functionality, are clearly areas of continuing research and offer growth opportunity for the food industry. The benefits from this continuously growing body of knowledge will be new ingredients and innovative products that will improve the overall well-being of consumers. Topics covered in this volume will provide food scientists and manufacturers with new insight into and appreciation of the health-promoting implications of whey protein science. The topics identified below and contributed by their respective subject matter experts represent the best science knowledge base in these areas. The state of the art and science are compelling, and an

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Preface

emerging database is confirming and solidifying the human knowledge base. The compilation of knowledge on the functional and metabolic roles of whey proteins and their demonstrated biochemical efficacy in improving human health enhances the vision of the Institute of Food Technologists Book Communications Committee that supported the publication of Whey Processing: Functionality and Health Benefits. By presenting the latest information on the processing and functionality research conducted on whey proteins up to the present, this volume will accelerate new product innovation and create opportunities for the food industry. Topics covered in volume include r r r r r r r

whey utilization, its history, and progress in process technology; fractionation and separation into biological fractions with health implications; whey emulsions and stability in acidic environments; some current applications in films, coatings, and gels; new process: texturization—use of texturized whey in snacks, meat analogs, candies, and as inclusions in candies; nanoparticles in hydrogels for delivery of bioactive components; and role of whey proteins in human health.

This book serves as a valuable resource for food industry professionals in research and development, academic faculty and students in food science, human nutrition and dairy science, nutrition and health professionals, and also policy makers. Charles I. Onwulata, Ph.D.

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Chapter 1 Whey Protein Production and Utilization: A Brief History Michael H. Tunick

Introduction Whey is the liquid resulting from the coagulation of milk and is generated from cheese manufacture. Sweet whey, with a pH of at least 5.6, originates from rennet-coagulated cheese production such as Cheddar. Acid whey, with a pH no higher than 5.1, comes from the manufacture of acid-coagulated cheeses such as cottage cheese. Compositional ranges of each are shown in Table 1.1. About 9 L of whey is generated for every kilogram of cheese manufactured, and a large cheesemaking plant can generate over 1 million liters of whey daily (Jelen 2003). Cheesemaking presumably originated in the Fertile Crescent some 8,000 years ago after it was noticed that an acid-coagulated milk gel separates into curds and whey. Experimentation would have led to the first cheeses, where the original starter cultures probably consisted of some of the whey from the previous day’s cheesemaking; manufacturers of Parmigiano-Reggiano, Grana Padano, and other high-cook cheese varieties still use this method (Fox and McSweeney 2004). Reheating of the whey and recovery of the solids led to a cheese variety now known as ricotta (Italian for “recooked”). Whey not used for humans was fed to pigs or other livestock, spread as fertilizer, or simply thrown out. Whey has supplemented pig feed for centuries, and the growth of computerized systems has allowed for more precise feeding of whey and other liquid feeds to weaned pigs and lactating sows (Meat and Livestock Commission 2003). A study

1 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Whey Processing, Functionality and Health Benefits Table 1.1. Typical composition of liquid and dry whey (Jelen 2003). Product

Protein

Lactose

Minerals

Sweet whey Acid whey

g/L whey 6–10 46–52 6–8 44–46

2.5–4.7 4.3–7.2

WPC-35 WPC WPI UF permeate

g/100 g powder 35 50 65–80 4–21 88–92 <1 1 90

7.2 3–5 2–3 9

conducted in the early 1970s showed that spraying whey on the ground to a depth of 25 mm improved the yield of corn and hay without increasing groundwater pollution (Watson and Peterson 1977). Whey is a potent pollutant with a biological oxygen demand of 35–45 kg/L; 4,000 L of whey, the output of a small creamery, has the polluting strength of the sewage of 1,900 people (Marwaha and Kennedy 1988). Disposal of whey by dumping into rivers was frequently used in the United States before environmental regulations took hold (Cryan 2001). One cheese plant faced with a whey disposal problem looked at the deep abandoned well on its property, decided that “where there’s a well there’s a whey,” and discarded their surplus in it. This method ended with explosive violence in 1942 when the whey, trapped and under pressure, developed enough gas to blow off the top of the well (Anderson 1970). Nowadays whey is evolving into a sought-after product because of the lactose, minerals, and proteins it contains as well as the functional properties it imparts to food (Onwulata and Tomasula 2004). A number of products are obtained from whey processing, as shown in Figure 1.1. This chapter deals with the development of whey protein as a prized commodity.

Concentrating Whey—Early Efforts The health benefits of whey led to the development of processes to isolate the solids by concentration and drying. The initial industrial attempts

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Figure 1.1. Liquid whey processing (Marwaha and Kennedy 1988; Siso 1996).

at concentrating and drying whey were in the 1920s and involved four different methods: conventional hot roller milk driers; heating until a concentrated liquid was obtained, cooling to solidification, and then extruding in a tunnel; two-stage steam heating; and a combination of spray drying and rotary drum drying (Gillies 1974). The high cost of the process and the hygroscopic nature of the lactose in the dry product prevented much progress from being made. Roller drying, in which whey is dried on the surface of a hot drum and removed by a scraper, is still used by some processors as part of whey powder production. The first important development came in 1933 when the long-tube multiple-effect evaporator was applied to whey processing (Gillies 1974). The multiple-effect evaporator boils water in a sequence of tanks—in this case, two—with successively lower pressures. Since the boiling point of water decreases as pressure decreases, the vapor boiled off in one vessel is used to heat the next and an external heat source is needed for the first vessel only. Evaporation in the first effect takes place around 77◦ C and in the second at around 45◦ C (Kosikowski and Mistry

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1997a). Water is evaporated from the whey in long slender vertical tubes within the steam chest (Perry and Green 1997), leaving concentrated whey with 45% solids (Kosikowski 1979).

Drying Whey The second major innovation that aided whey processing was the spray drier, which was developed in the 1860s, applied to whey starting in 1937, and is still in use. The lactose, which is amorphous and hygroscopic, is cooled and crystallized to nonhygroscopic α-lactose monohydrate. The concentrated whey is then dispersed by a rotary wheel or nozzle atomizer into a drying chamber through a hot-air stream, producing a powder with 10–14% moisture. The evaporation keeps the temperature down, preventing denaturation (Deis 1997). The wet powder is dried to 3–5% moisture in a vibrating fluid bed (Kosikowski and Mistry 1997a).

Concentrating Whey—Modern Techniques Until the 1970s whey protein was available only in the heat-denatured form, a water-insoluble, gritty, yellowish-brown powder that found limited use (Wingerd 1971). Membrane filtration then arrived, which allowed for the separation and fractionation of whey proteins while retaining their solubility. Membrane filtration is a molecular sieving technique that employs a 150-μm-thick semipermeable surface supported by a more porous layer of similar material on a reinforcing base. The permeate (soluble compounds of low molecular weight) flows through while passage of the retentate (other materials) is blocked. The filter is made from cellulose acetate, ceramic, polysulfone, or zirconium oxide, and the configuration is usually spiral wound in a stainless steel housing (Henning et al. 2006; Wagner 2001). The principle of membrane filtration was developed for water desalinization in the 1950s and applied to food processing starting in 1965. Whey processors employ five types of membrane filtration, sometimes in combination: ultrafiltration (UF), microfiltration (MF), electrodialysis (ED), nanofiltration (NF), and reverse osmosis (RO). All are followed by spray drying to obtain a dry (<5% moisture) product, and

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Table 1.2. Pressure-driven membrane separation of milk components (Kelly 2003; Wagner 2001). Type

Pore size (nm)

Components retained

MF

20–4,000

UF NF RO

20–200 <2 <2

Bacteria, casein micelles, fat globules Whey proteins Lactose Ions

Molecular weight of component (kDa) 100–500 1–100 0.1–1 <0.1

combinations of these processes are utilized to create whey protein powders with different protein contents. Table 1.2 shows a comparison of the whey filtration techniques. Whey separation by UF began in 1971. The dividing line between permeate and retentate, known as the molecular weight cutoff (MWCO), is between 3 and 100 kDa for UF membranes; a MWCO of 10 kDa is usually employed for whey (Kosikowski and Mistry 1997b). Whey separation by UF is normally performed at temperatures below 55◦ C, with an inlet pressure around 300 kPa and a membrane pore size of 250 nm (Wagner 2001). Whey retentate consists of protein, fat, and insoluble salts; lactose, soluble minerals, and much of the water are in the permeate (Table 1.1). Diafiltration (DF), the addition of water to the retentate followed by a second UF, has been developed for the removal of salts and lactose (Scott 1986). Pretreatments such as pH adjustment and preconcentration may improve performance (Muller and Harper 1979). MF is similar to UF, but with a MWCO of 200 kDa. The membrane pore size may be varied from 0.05 to 10 μm, allowing for selective separation of microbial flora, the different whey proteins, and other components (Kosikowski and Mistry 1997b). This setup would have an internal volume of 800 L, a flow rate of 30,000 L/h, and a concentration ratio of 30:1, meaning that 1,000 L of concentrate and 29,000 L of permeate will be produced each hour (Wagner 2001). ED is another method for demineralizing whey. In this electrochemical process (the other membrane filtration techniques used for whey are pressure-driven processes), direct current is passed through whey inside chambers with ion-permeable walls.

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NF, sometimes called ultra-osmosis, removes particles smaller than 300–1,000 Da, and is also suitable for desalting and demineralizing whey. The method also selectively separates lactose (Kelly and Kelly 1995). An operating pressure between 1.5 and 3 MPa is sufficient to increase the total solids of whey from 5 to 40% while removing 40% of the minerals (van der Horst et al. 1995). In RO, whey is preheated to 50–55◦ C and pumped at high pressure (between 2.7 and 10 MPa) through a membrane to remove minerals (Scott 1986). The MWCO is only 150 Da, which may result in membrane-fouling problems (Muller and Harper 1979). Two-thirds of the water in whey can be removed by RO, leaving a concentrate that can be dried or shipped more efficiently (Cryan 2001).

Whey Protein Concentrate and Whey Protein Isolate UF of whey leads to a selective concentration of the protein, which when dried is whey protein concentrate (WPC). WPC may contain anywhere between 20 and 89% protein; WPC with 35% protein (WPC-35) is a common product. A combination of UF and DF removes minerals and lactose from the retentate, allowing for production of WPC with >50% protein (Kelly 2003). Almost 170 million kilograms of WPC were produced in the United States in 2005, and 86% of it was for human consumption (Gould 2006). Whey protein isolate (WPI) contains at least 90% protein, with virtually all the lactose removed. An ion-exchange tower, which separates components by ionic charge instead of molecular size, is often used in conjunction with membrane filtration (Foegeding and Luck 2003). The U.S. production of WPI in 2005 was 15.6 million kilograms (Gould 2006). Benefits of WPC and WPI in food applications include its high protein and amino acid content; low calorie, fat, and sodium content; lack of pathogens, toxic compounds, and antinutritional factors; good emulsification capacity; compatibility with other ingredients; ready availability; and the perception that it is a “natural” product (Renner and Abd El-Salam 1991). WPC and WPI are bland products with typical dairy flavors, such as sweet aromatic and cooked/milky, although heatgenerated and lipid oxidation compounds may lead to nondairy flavors (Carunchia Whetstine et al. 2005). The WPI’s high protein purity and

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solution clarity lend itself to use in beverages and nutritional supplements (Foegeding and Luck 2003).

Nutritional Aspects Whey was prescribed for therapeutic purposes, starting with Hippocrates in 460 BC and continuing through the Middle Ages (S¨usli 1956). Whey became a fashionable drink in English cities in the midseventeenth century, prompting the opening of whey houses, which were analogous to today’s coffee shops. In several of his diary entries in 1663, Samuel Pepys mentioned patronizing the whey house at the New Exchange in London (Pepys 2006). Products made with whey included whey-borse (a broth), whey-butter, whey-porridge, and wheywhig (a drink with herbs) (Trelogan 1970). Whey baths were popular in nineteenth-century spas (Trelogan 1970), and as late as World War II, Central European spas served up to 1.5 kg of whey daily to patients suffering from a variety of ailments (Holsinger et al. 1974). Serious research on the nutritive value of whey began late in the nineteenth century. Until then milk proteins were classified as casein or whey protein, and the solid obtained from heating whey was called lactalbumin. Around 1890, research showed that a precipitate, named lactoglobulin, was formed from the addition of magnesium sulfate or ammonium sulfate at low pH (Creamer and Sawyer 2003). The lactalbumin fraction was first crystallized in the 1930s (Palmer 1934) and the major whey proteins that were eventually isolated were named β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). Later discoveries included bovine serum albumin, lactoferrin, lactoperoxidase, numerous types of immunoglobulins, and other minor proteins. Table 1.3 shows the types and amounts of whey proteins and other components in bovine milk. Over half of ruminant and porcine whey protein consists of β-LG; it is not an abundant protein in the milk of other species (Walzem et al. 2002). β-LG is stable against stomach acids and proteolytic enzymes, is a rich source of the essential acid cysteine, and may be responsible for carrying the vitamin A precursor retinol from the cow to its calf (Said et al. 1989). α-LA, which binds calcium, is similar to the primary protein in human breast milk and is thus used in infant formula. The many branched-chain amino acids it contains are used by muscles for energy and protein synthesis, making it a popular

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Whey Processing, Functionality and Health Benefits Table 1.3. Typical composition of bovine milk (de Wit 1998; Fox et al. 2000). Component

Concentration (g/L)

Water Fat Lactose Ash (minerals and salts) Casein Whey proteins β-Lactoglobulin α-Lactalbumin Bovine serum albumin Immunoglobulins Lactoferrin Lactoperoxidase Enzymes

873 37 48 7 28 6.0 3.2 1.2 0.4 0.8 0.2 0.03 0.03

sports food nutrient (Tawa and Goldberg 1992). Bovine serum albumin is identical to blood serum albumin and transports insoluble free fatty acids (de Wit 1998). Immunoglobulins carry passive immunity to the newborn calf via colostrum. The iron-containing bioactive protein lactoferrin has antibacterial, antiviral, and antioxidant properties and also modulates iron metabolism and immune functions (Walzem et al. 2002). Lactoperoxidase, another bioactive protein, is part of a bactericidal system (de Wit 1998). There are also perhaps 60 enzymes in whey (Fox et al. 2000) and a few other proteins in miniscule amounts. In addition, whey protein promotes muscle synthesis, the calcium and mineral mix may mediate body composition by shifting nutrients from adipose to lean tissue, and bioactive compounds isolated from whey may improve immune function and gastrointestinal health (Ha and Zemel 2003).

Functionality Whey protein is used in many food applications because of its functionality and nutritive value. Whey protein foams well, meaning that it creates and stabilizes air bubbles in a liquid (Renner and Abd ElSalam 1991). Whey protein also remains soluble from pH 2 to 10 and

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stabilizes emulsions by forming interfacial films between hydrophobic and hydrophilic food components (Burrington 2005; Haines 2005). Ice creams, souffl´es, frothed drinks, and other food foams and emulsions are stabilized by surface-active agents for which whey protein products are frequently chosen (Foegeding et al. 2002). Processed cheeses, which are also emulsions, contain whey protein to improve melting, slicing, and spreading (Foreign Agricultural Service 2003). Acid whey powder improves the crust color and enhances flavor in bread, biscuits, crackers, and snack foods, providing a golden surface on baking (Kosikowski 1979). Whey proteins unfold and aggregate on heating, and are capable of binding large amounts of water depending on pH, ionic strength, and thermal conditions (Hudson et al. 2000). Addition of WPI to muscle protein, for instance, improves moisture retention (McCord et al. 1998). WPC has been used to increase solids and protein content in milk and cheese and to replace fat in low-fat dairy products. Protein gelation may be defined as a balance of protein–protein and protein–water interactions, enabling the formation of a threedimensional network (Renner and Abd El-Salam 1991). β-LG, the primary protein in whey, does not heat-denature until about 78◦ C, providing good thermally induced gelation properties (Paulsson and Dejmek 1990). β-LG also has a high solubility at low pH, making it an ideal active ingredient in fortified acidic beverages (Smithers et al. 1996). Lactoferrin has potential as a natural antimicrobial agent in such products as personal health items, pharmaceuticals, and specialty dietary formulations (Smithers et al. 1996). Whey proteins can also be extruded and added to other foods to improve nutritional value and texture (Onwulata and Tomasula 2004). Variations in whey functionality depend on the variety of cheeses and the processes used in manufacturing them, the severity of heat treatment required to isolate the whey protein, and other factors (Dybing and Smith 1991; Onwulata et al. 2004).

Applications WPC and WPI are used in baked goods and baking mixes; cakes and pastries; candy, chocolate, and fudge; coffee whiteners; crackers and snack foods; diet supplements; fruit beverages; gravies and sauces; infant formulas and baby food; mayonnaise; pasta; pie fillings; processed dairy

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products including ice creams; processed fruits and vegetables; salad dressings; soups, meats, and sausage; and sports drinks (Cryan 2001; Kosikowski 1979). WPC with lower protein content tends to be used more in the lower-value food products, such as dairy and bakery items, and higher concentrated WPC is generally used in higher-value products, such as meat and seafood (Foreign Agricultural Service 2003). Medicinal and nutritional products are obtained by fractionation of whey by membrane filtration into its various proteins (Zydney 1998). Outlook The cost of purchasing and installing whey-processing equipment has prevented some smaller, older cheesemaking plants from processing and selling whey. Some 30% of the whey from U.S. cheese manufacture is not sold as a result, but in modern plants in the major cheese-producing regions, whey is actively marketed and supplies approximately 11% of the revenue (Balagtas et al. 2003). Texturization of whey proteins by extrusion of WPC and WPI will soon be applied to meat extenders, snack foods, and other products (Onwulata and Tomasula 2004), and the functional properties of these texturized whey products are still being investigated (Onwulata et al. 2003; Tunick and Onwulata 2006). Whey protein has been tested in clinical trials of cancer, hepatitis B, and HIV patients with promising results (Marshall 2004). Nonfood applications such as oxygen barriers and film coatings made from denatured WPC80 and WPI show a good likelihood of adoption by industry (Balagtas et al. 2003). There is a new expression in the dairy business: “Cheese to break even, whey for profit.” This adage should hold true for the foreseeable future as new whey protein products and applications continue to be introduced. References Anderson, R.F. 1970. Whey problems of the cheese industry. In Proceedings of the First Whey Utilization Conference, pp. 24–29. Balagtas, J.V., Hutchinson, F.M., Krochta, J.M., and Sumner, D.A. 2003. Anticipating market effects of new uses for whey and evaluating returns to research and development. J. Dairy Sci. 86:1662–1672.

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Burrington, K. 2005. How to leverage the advantages of whey ingredients in beverages. Nutr. Outlook 8 (5):21–25. Carunchia Whetstine, M.E., Croissant, A.E., and Drake, M.A. 2005. Characterization of dried whey protein concentrate and isolate flavor. J. Dairy Sci. 88:3826–3839. Creamer, L.K., and Sawyer, L. 2003. Beta-lactoglobulin. In Encyclopedia of Dairy Sciences, edited by H. Roginski, J.W. Fuquay, and P.F. Fox, pp. 1932–1939. New York: Academic Press. Cryan, R. 2001. Whey: Ready for Takeoff? U.S. Dairy Mark. Outlook 7(3):1–4. Deis, R.C. 1997. Spray-drying: Innovative use of an old process. Food Product Design. Available at: www.foodproductdesign.com/articles/466/466 0597DE.html. Accessed February 12, 2008. de Wit, J.N. 1998. Nutritional and functional characteristics of whey proteins in food products. J. Dairy Sci. 81:597–608. Dybing, S.T., and Smith, D.E. 1991. Relation of chemistry and processing procedures to whey protein functionality: A review. Cult. Dairy Prod. J. 26(2):4–12. Foegeding, E.A., Davis, J.P., Doucet, D., and McGuffey, M.K. 2002. Advances in modifying and understanding whey protein functionality. Trends Food Sci. Technol. 13:151–159. Foegeding, E.A., and Luck, P.J. 2003. Whey protein products. In Encyclopedia of Dairy Sciences, edited by H. Roginski, J.W. Fuquay, and P.F. Fox, pp. 1957–1960. New York: Academic Press. Foreign Agricultural Service. 2003. U.S. Whey Exports. Available at: www.fas.usda. gov/dlp2/circular/1999/99-12Dairy/uswhey.html. Accessed February 12, 2008. Fox, P.F., Guinee, T.P., Cogan, T.M., and McSweeney, P.L.H. 2000. Chemistry of milk constituents. In Fundamentals of Cheese Science, pp. 19–44. Gaithersburg, MD: Aspen Publishers. Fox, P.F., and McSweeney, P.L.H. 2004. Cheese: Chemistry, Physics and Microbiology, Volume 1: General Aspects, 3rd ed., edited by P.F. Fox, P.L.H. McSweeney, T. Cogan, and T. Guinee, pp. 1–18. London: Elsevier Academic Press. Gillies, M.T. 1974. Whey Processing and Utilization, pp. 24–31. Park Ridge, NJ: Noyes Data Corp. Gould, B.W. 2006. Understanding Dairy Markets. Availabe at: www.aae.wisc.edu/ future. Accessed February 12, 2008. Ha, E., and Zemel, M.B. 2003. Functional properties of whey, whey components, and essential amino acids: Mechanisms underlying health benefits for active people. J. Nutr. Biochem. 14:251–258. Haines, B. 2005. The power of protein. Funct. Foods Nutraceuticals (May 2005):50–52. Henning, D.R., Baer, R.J., Hassan, A.N., and Dave, R. 2006. Major advances in concentrated and dry milk products, cheese, and milk fat-based spreads. J. Dairy Sci. 89:1179–1188. Holsinger, V.H., Posati, L.P., and DeVilbiss, E.D. 1974. Whey beverages: A review. J. Dairy Sci. 57:849–859. Hudson, H.M., Daubert, C.R., and Foegeding, E.A. 2000. Rheological and physical properties of derivitized whey protein isolate powders. J. Agric. Food Chem. 48:3112–3119.

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Jelen, P. 2003. Whey processing: Utilization and products. In Encyclopedia of Dairy Sciences, edited by H. Roginski, J.W. Fuquay, and P.F. Fox, pp. 2739–2745. New York: Academic Press. Kelly, J., and Kelly, P. 1995. Desalinisation of acid casein whey by nanofiltration. Int. Dairy J. 5:291–303. Kelly, P.M. 2003. Membrane separation. In Encyclopedia of Dairy Sciences, edited by H. Roginski, J.W. Fuquay, and P.F. Fox, pp. 1777–1786. New York: Academic Press. Kosikowski, F.V. 1979. Whey utilization and whey products. J. Dairy Sci. 62:1149– 1160. Kosikowski, F.V., and Mistry, V.V. 1997a. Whey and whey foods. In Cheese and Fermented Milk Foods, Vol. 1, pp. 422–453. Westport, CT: F.V. Kosikowski, LLC. Kosikowski, F.V., and Mistry, V.V. 1997b. Ultrafiltration, microfiltration and nanofiltration. In Cheese and Fermented Milk Foods, Vol. 1, pp. 500–519. Westport, CT: F.V. Kosikowski. Marshall, K. 2004. Therapeutic applications of whey protein. Altern. Med. Rev. 9:136– 156. Marwaha, S.S., and Kennedy, J.F. 1988. Review: Whey-pollution problem and potential utilization. Int. J. Food Sci. Technol. 23:323–336. McCord, A., Smyth, A.B., and O’Neill, E.E. 1998. Heat-induced gelation properties of salt-soluble muscle proteins as affected by non-meat proteins. J. Food Sci. 63:580– 583. Meat and Livestock Commission. 2003. General Guidelines on Liquid Feeding for Pigs. Availabe at: www.bpex.org/technical/general/pdf/ liquidfeeding.pdf. Accessed February 12, 2008. Muller, L.L., and Harper, W.J. 1979. Effects on membrane processing of pretreatments of whey. J. Agric. Food Chem. 27:662–664. Onwulata, C.I., Konstance, R.P., Cooke, P.H., and Farrell, H.M., Jr. 2003. Functionality of extrusion-texturized whey proteins. J. Dairy Sci. 86:3775–3782. Onwulata, C.I., Konstance, R.P., and Tomasula, P.M. 2004. Minimizing variations in functionality of whey protein concentrates from different sources. J. Dairy Sci. 87:749–756. Onwulata, C.I., and Tomasula, P. 2004. Whey texturization: A way forward. Food Technol. 58(7):50–54. Palmer, A.H. 1934. The preparation of a crystalline globulin from the albumin fraction of cow’s milk. J. Biol. Chem. 104:359–372. Paulsson, M., and Dejmek, P. 1990. Thermal denaturation of whey proteins in mixtures with caseins studied by differential scanning calorimetry. J. Dairy Sci. 73:590–600. Pepys, S. 2006. The Diary of Samuel Pepys. Availabe at: www.pepysdiary.com. Accessed February 12, 2008. Perry, R.H., and Green, D.W. 1997. Evaporators. In Perry’s Chemical Engineers’ Handbook, 7th ed., pp. 11-107–11-110. New York: McGraw-Hill. Renner, E., and Abd El-Salam, M.H. 1991. Ultrafiltration of whey. In Application of Ultrafiltration in the Dairy Industry, pp. 217–314. New York: Elsevier Science. Said, H.M., Ong, D.E., and Shingleton, J.L. 1989. Intestinal uptake of retinol: Enhancement by bovine milk beta-lactoglobulin. Am. J. Clin. Nutr. 49:4419–4427.

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Scott, R. 1986. Membrane filtration of milk and whey. In Cheesemaking Practice, 2nd ed., pp. 302–311. New York: Elsevier Applied Science. Siso, M.I.G. 1996. The biotechnological utilization of cheese whey: A review. Bioresour. Technol. 57:1–11. Smithers, G.W., Ballard, F.J., Copeland, A.D., De Silva, K.J., Dionysius, D.A., Francis, G.L., Goddard, C., Grieve, P.A., McIntosh, G.H., Mitchell, I.R., John, R.P., and Regester, G.O. 1996. New opportunities from the isolation and utilization of whey proteins. J. Dairy Sci. 79:1454–1459. S¨usli, H. 1956. Neue Art der Molkenverwertung: Ein Lactominerales Tafelgetr¨ank. In Proceedings of the 14th International Dairy Congress, Vol. 1, Part 2, pp. 477–486. Tawa, N.E., Jr., and Goldberg, A.L. 1992. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency. Am. J. Endocrinol. Metab. 263:E317–E325. Trelogan, H.C. 1970. Folklore, statistics and economics of whey. In Proceedings of the First Whey Utilization Conference, pp. 71–75. Tunick, M.H., and Onwulata, C.I. 2006. Rheological properties of extruded milk powders. Int. J. Food Prop. 9:835–844. Van Der Horst, H.C., Timmer, J.M.K., Robbertsen, T., and Leenders, J. 1995. Use of nanofiltration for concentration and demineralization in the dairy industry: Model for mass transport. J. Membr. Sci. 104:205–218. Wagner, J. 2001. Membrane Filtration Handbook: Practical Hints and Tips, 2nd ed., 129 pp. Minnetonka, MN: Osmonics. Walzem, R.L., Dillard, C.J., and German, J.B. 2002. Whey components: Millenia of evolution create functionalities for mammalian nutrition. What we know and what we may be overlooking. Crit. Rev. Food Sci. Nutr. 42:353–375. Watson, K.S., and Peterson, A.E. 1977. Benefits of spreading whey on agricultural land. J. Water Pollut. Control Fed. 49:24–34. Wingerd, W.H. 1971. Lactalbumin as a food ingredient. J. Dairy Sci. 54:1234–1236. Zydney, A.L. 1998. Protein separations using membrane filtration: New opportunities for whey fractionation. Int. Dairy J. 8:243–250.

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Chapter 2 Whey Protein Fractionation Laetitia M. Bonnaillie and Peggy M. Tomasula

Introduction Cheese whey is a complex watery solution containing almost 7% of solubilized solids. The solids are comprised of about 10–12% proteins, the rest being mainly lactose (74%), minerals (8%), milk fat (3%), and lactic acid (Morr 1989). Whey protein concentrates (WPCs), with protein contents ranging from 35 to 85%, and whey protein isolates (WPIs), with protein contents greater than 90%, are commercially available. The major whey proteins are α-lactalbumin (α-LA), β-lactoglobulin (β-LG), bovine serum albumin (BSA), and the heavy- and lightchain immunoglobulins (Igs). Other important proteins found in whey, but present in minor quantities, are lactoferrin (LF) and lactoperoxidase. Whey may also include the proteose-peptone components glycomacropeptides (GMPs) and low-molecular-weight products formed by the enzymatic degradation of the caseins during the cheesemaking process (De Wit 1989). The composition of whey and whey products depends on the methods of production, purification, and concentration used. Table 2.1 shows an average protein composition of sweet whey proteins. While the various commercial WPC and WPI can provide a vast array of functional, textural, and nutritional properties when added to foods, the individual whey proteins or enriched fractions of the whey proteins have the potential to enable creation of new food products with improved functional properties or enhanced nutritional aspects. In addition, the proteins may find use in personal care and cosmetic formulations. This chapter presents an up-to-date review of the wide array of techniques used to fractionate sweet whey proteins in order to enhance the 15 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Table 2.1. Sweet whey proteins: Typical composition and physical properties (Etzel 2004; Kilara and Vaghela 2004). Protein

Molecular weight Content (%) (kg/mol) Isoelectric pH

β-Lactoglobulin (β-LG) α-Lactalbumin (α-La) Glycomacropeptide (GMP) Bovine serum albumin (BSA) Immunoglobulin (Igs) Lactoferrin (LF) Lactoperoxidase

48–58 13–19 12–20 6 8–12 2 0.5

18 14 8.6 66 150 77 78

5.4 4.4 <3.8 5.1 5–8 7.9 9.6

physical, chemical, or nutritional properties offered by any one particular whey protein or enriched fraction of whey proteins. So far, the focus of research has been primarily on enriching or isolating the αLA and β-LG fractions. However, as potential benefits and applications for the minor whey proteins such as LF and GMP have become apparent (De Wit 1998; Le´on-Sicairos et al. 2006; Mangino 2005; Smithers et al. 1996; Yalcin 2006), fractionation techniques to recover purified fractions of the latter have also been developed. Utilization of Whey Protein Fractions Until recently, sweet whey has been discarded or used as animal feed. Recovering the solid components of whey is attractive for two main reasons: to reduce the organic pollution created by whey wastes when they are discarded (Ostojic et al. 2005) and, mostly, for optimal utilization of the nutritional and functional properties offered by whey proteins. To a large extent, the properties of WPCs and WPIs approximate the properties of β-LG, since it constitutes more than half of the whey proteins. To exploit the particular properties of α-LA, BSA, GMP, LF, or Ig or to emphasize the properties of β-LG, fractionation of the whey protein mixture for the isolation of one or a group of proteins is useful. The practice began in 1934, when Palmer salted out β-LG from acid whey with the addition of sodium sulfate (Na2 SO4 ) at pH 6.0 and then used the centrifugation and dialysis techniques to remove the salt and isolate β-LG crystals (Palmer 1934).

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β-LG has binding and gelling properties similar to that of egg proteins and form heat-induced gels. In a comparative study, Lorenzen and Schrader (2006) reported that WPI gels were stronger and more elastic than WPC gels because of the higher β-LG content, and because of the removal of most of the fat, lactose, and phospholipids. Reducing the amounts of GMP, nonprotein nitrogen, and proteose-peptones was also proven beneficial to gel strength. The gel strength appeared to be pH-dependent and the strongest gel was obtained with WPI at pH 6.0. Similarly, Tomasula and Yee (2001) reported that β-LG-enriched fractions showed enhanced emulsion stability, emulsion activity, viscosity, and gelling properties relative to a WPC reference. For some applications, removal of β-LG from whey is the desirable option. For example, human breast milk, like the milk of most other nonruminants, does not contain any β-LG, whereas β-LG is the main protein in the whey from ruminant’s milk. Infant formulae that approximate the amino acid composition of breast milk are desired. In addition, β-LG is reportedly an allergen (Chiancone and Gattoni 1993), and removing it would provide hypoallergenic formulae. Heine et al. (1996) proposed to incorporate extra α-LA in human infant formula in order to provide a better balance of amino acids in cow’s milk-based formulae. For example, in average formulae, there is an excess of lysine and methionine amino acids and a deficiency in cysteine and tryptophan; bovine α-LA is a tryptophan-rich protein. Addition of lactoferrin, lactoperoxidase, β-casein, and GMP has also been proposed for use in infant formulae (Jost et al. 1999). Currently the biological activity of some of the minor whey proteins and peptides that can be isolated is actively studied. For example, the microbicidal activities of lactoferrin and some of the peptides derived from lactoferrin are of interest, since they were found to kill some pathogenic microorganisms such as gram-positive bacteria (Le´onSicairos et al. 2006; van der Kraan et al. 2006). Glycomacropeptide (GMP) is a bioactive peptide whose unique composition and characteristics offer health-promoting effects with multiple possible applications. As such, GMP is a potential ingredient for various functional foods and pharmaceuticals (Mart´ın-Diana et al. 2006; Nakano et al. 2006; Thoma-Worringer et al. 2006). Immunoglobulins from milk were used for the effective treatment of various infections in newborn infants (Reiter 1978; Stephans et al. 1980).

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Some Physical and Chemical Properties of Whey Proteins Several properties that are exploited in order to fractionate the whey proteins include differences in monomer and aggregate sizes, denaturation behavior and kinetics, chemical reactivity and binding ability, surface charge distribution, polarity, and colloidal behavior. Below are a few examples of physical and behavioral differences frequently utilized during whey protein fractionation. Size The components of whey proteins show large differences in their molecular weights (Table 2.1) and variety in their conformations, which result in a wide range of monomer radii. In addition, in similar environmental conditions, some whey proteins in solution exist as monomers and others as polymers, resulting in even larger size differences. Whether a whey protein exists as a monomer or a polymer usually depends on pH, temperature and protein concentration, and this phenomenon is exploited for fractionation purposes. For example, the β-LG monomer has a molecular weight of ∼18,000 kDa. However, at low temperature (10◦ C) and low concentration (<10 mg/mL), the quaternary structure of β-LG varies highly with pH (Verheul et al. 1999). At pH below 3.4 and above 8.0, β-LG exists as a monomer. Between pH 5.2 and 8.0 (which includes the pH of milk), β-LG forms non-covalent-linked, spherical dimers that have a molecular weight of ˚ Around its isoelectric point of ∼36 kDa and a diameter of ∼18 A. pH 4.7 and more generally between pH 3.5 and 5.2, β-LG forms large octamers (Goff 1995; Verheul et al. 1999; Whitney 1977). The dependence of β-LG’s quaternary structure on pH is summarized in Figure 2.1. On the other hand, α-LA has a lower molecular weight of ∼14 kDa and α-LA monomers possess a compact, spherical tertiary structure

Figure 2.1. Quaternary structure of β-LG as a function of pH at low temperature and low concentration.

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stabilized by an internal calcium ionic bond that renders it less sensitive to pH changes above pH 4.0 (Goff 1995). Fractionation techniques that utilize the differences between protein sizes in solution include chromatography and gel and membrane filtrations and are presented later. Denaturation Behavior Whey proteins are soluble over a wide range of pH. However, various combinations of pH, temperature, and mineral composition can bring about the selective denaturation, aggregation, and precipitation of whey proteins. Heat Treatment The stability of the tertiary structure of whey proteins is determined by various noncovalent interactions and by the disulfide bonds, which are formed by two cysteine residues. β-LG has two internal disulfide bonds and one free thiol group, while α-LA has eight cysteine groups that are all involved in internal disulfide bonds (Goff 1995). In general, protein profiles for heat-treated skim milk indicate that the denaturation of the total proteins begins at 40◦ C, accelerates with increasing temperature, and becomes 95% complete at 85◦ C (Kilara and Vaghela 2004). The whey proteins, individually and in solution, denature between 64 and 85◦ C: at neutral pH, α-LA denatures at 64◦ C and β-LG denatures at 78◦ C; above 85◦ C, the whey proteins start to aggregate and gel (De Wit 1989). Changes in protein structure that occur during denaturation were shown to be irreversible (Chaplin and Lyster 1986; Hong and Creamer 2002). Whey protein denaturation generally leads to the loss of solubility of the protein. Therefore, the proportion of denatured whey protein compared to the initial population is commonly followed using solubility testing; for example, Sava et al. (2005) elucidated the correlation between heat treatment and the solubility of β-LG. At temperatures between 67 and 82◦ C and at pH 7.5, β-LG was sensitive to heat-induced sulfhydryl/disulfide (SH/SS) interchange reactions. This resulted in an increase of its surface hydrophobicity and a decrease of the slow reacting SH groups content, and therefore decreased the solubility of β-LG. Effects of Mineral Salts and pH Most proteins, including β-LG, use calcium as an intermolecular linkage that facilitates thermal aggregation. At neutral pH, calcium

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stabilizes α-LA due to internal linkages that help keep it folded even at temperatures as high as 100◦ C (Kilara and Vaghela 2004). Changing the mineral concentration in whey produces variations in the thermal stability of the various proteins. For example, the addition of calcium chloride (CaCl2 ) was shown to stabilize α-LA near its isoelectric point (pH 4.2) and make it less sensitive to heat treatment, resulting in less precipitation, while the removal of calcium ions destabilized α-LA (Bramaud et al. 1995, 1997a). The strong Ca2+ -binding site of α-LA can equally bind Mg2+ , Mn2+ , Na+ , and K+ ; α-LA also possesses several distinct Zn2+ -binding sites. The binding of zinc ions to a calcium-loaded α-LA protein was shown to decrease its thermal stability (Permyakov and Berliner 2000). After thermal aggregation of whey proteins in solution at 75◦ C and neutral pH, the aggregates can be redissolved by a careful control of the mineral composition of the solution. Cross-dialysis is one method that is utilized for the manipulation of mineral ion concentration (Havea et al. 2002). A different behavior was observed for the immunoglobulins, which were not soluble in CaCl2 solution (Bramaud et al. 1995, 1997a). Changes in pH have a considerable effect on the denaturation behaviors of α-LA and β-LG. Lowering the pH of WPC solutions results in the dissolution and removal of bound calcium ions from the center of the α-LA proteins, inducing the precipitation of α-LA. At neutral pH, α-LA contains 1 mol of calcium per mol of protein. At lower pH (3.8–4.2), calcium dissolves in the solution and α-LA unfolds (denatures) and precipitates at temperatures between 50 and 65◦ C (Bernal and Jelen 1984; Pearce 1983). After precipitation of α-LA via calcium removal using a heat/pH combination treatment, two forms of αLA can be obtained: “apo-α-LA” is the calcium-free solvated version, while “native α-LA” can be recovered by reintroducing calcium into the solution (Bramaud et al. 1997a). At higher pH (8.0 or more), divalent cations such as Ca2+ were reported to enhance the heat sensitivity of the β-LG protein, whereas monovalent salts such as NaCl increased its denaturation temperature (Varunsatian et al. 1983). The kinetics of the heat-induced and pressure-induced denaturations of the proteins of whey have been measured and the differences in the rates of denaturation are commonly exploited for fractionation purposes (Goetz and Koehler 2005; Hinrichs and Rademacher 2005; Oldfield et al. 2005; Patel et al. 2005; Sava et al. 2005; Tolkach et al. 2005). Examples of related processes are presented in the next section.

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Aggregation Whey protein aggregates formed by thermal treatment, such as spray drying, are bound together either by covalent disulfide bonds or by noncovalent bonds such as calcium bridges (Roufik et al. 2005). WPC solutions made from spray-dried sweet whey and acid whey showed different aggregation behaviors after an equal treatment at 75◦ C and neutral pH: in sweet whey, the high calcium content helped the formation of calcium bridges between protein molecules, promoting the fast formation of large aggregates of whey proteins. In acid whey, the presence of potassium limited the formation of calcium bridges, resulting in the slow formation of small, soluble, disulfide-linked aggregates during heating (Havea et al. 2002). At low pH values, acid-induced α-LA aggregates are a classic example of the molten globule state, also called “A-state,” and are promoted by zinc or lipid binding (Permyakov and Berliner 2000). Enzyme-induced aggregation may be highly selective and useful for the fractionation of whey proteins. For example, Tolkach and Kulozik (2005) have studied the selective aggregation of GMP with transglutaminase: the amino acid sequence of GMP includes two glutamine and three lysine residuals, whereby this peptide can be cross-linked by transglutaminase, whereas the native whey proteins show much less sensitivity to cross-linking with this enzyme due to their globular structure. Cross-linked GMP can then be separated from the native proteins by means of membrane technology, due to the difference in size. Membrane filtration and precipitation followed by centrifugation are just two of the many techniques commonly utilized to separate aggregates from the rest of the whey protein solution. The kinetics of the calcium-induced and heat-induced aggregation of the whey proteins have been reported and these results may also be used for fractionation purposes (Havea et al. 2002; Wu et al. 2005). A Review of Existing Techniques to Fractionate α-LA and β-LG Many separation processes have been developed for the fractionation of α-LA and β-LG as the main fraction components. These processes exploit various attributes of the whey proteins, including their denaturation properties (salt treatment processes, heat and pH treatments), the ionic nature of the proteins (electrophoresis, ion-exchange

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chromatography), the variations in protein sizes and shapes (membrane filtration, gel permeation, size exclusion chromatography), the differences in polarity (high-performance liquid chromatography), a particular chemical reactivity (complexation), or a particular physical property (coacervation, foaming). Addition of Salts Methods for isolating the proteins of whey with the addition of salts were reported as early as 1934, when Palmer (1934) and Polis et al. (1950) successfully extracted β-LG crystals from acid whey, using sodium sulfate (Na2 SO4 ) at pH 6.0 to precipitate β-LG. The salt was subsequently removed via dialysis, and β-LG crystals were isolated by centrifugation or alcohol extraction. After removal of β-LG, the first isolation of milk albumin was performed via fractionation of the remaining liquor by alcohol extraction and an infinitesimal amount of α-LA crystals was successfully obtained (Polis et al. 1950). Subsequent trials modified the method by reducing the pH with the introduction of hydrochloric acid prior to the addition of various salts. With Na2 SO4 at pH 2.0, the precipitation of all whey proteins except β-LG was obtained (Aschaffenburg and Drewry 1957). Trials using FeCl3 at low pH to precipitate β-LG showed a poor β-LG recovery and a poor purity (Kaneko et al. 1985; Kuwata et al. 1985; Zweig and Block 1954). Later, iron was found to affect the beneficial antimicrobial activity of lactoferrins, and polyphosphate salts were used instead of FeCl3 (Al-Mashikh and Nakai 1987). More recently, sodium chloride (NaCl) has been the favorite salt employed to precipitate β-LG at low pH from various whey raw materials: β-LG was separated from ultrafiltration retentate via NaCl salting-out of acid or cheese whey (Maillart and Ribadeau-Dumas 1988), while the addition of 7 wt% NaCl at pH 2.0 permitted the precipitation of β-LG from WPI; β-LG was then successfully recovered after centrifugation and diafiltration (to remove NaCl), with a β-LG recovery rate of more than 65% and a purity greater than 95% (Mat´e and Krochta 1994). Heat Treatment and pH Adjustment Most proteins are least soluble at their isoelectric point; however, whey proteins are soluble over a wide range of pH values. The following processes utilize the principle of isoelectric precipitation, combined with

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heat denaturation in order to destabilize a portion of the whey proteins for fractionation and isolation purposes. Most examples cited below precipitated α-LA along with all the minor whey proteins; Bramaud et al. (1997a) reported that BSA and immunoglobulins (Igs) always coprecipitate with α-LA. The driving principle in these fractionation methods is the difference in thermal stability of the whey proteins in acidic conditions. Pearce and coworkers (1983, 1987, 1991, 1995) has reported the precipitation of α-LA under gentle heat treatment, at temperatures ranging from 55 to 70◦ C and pH ranging from 3.8 to 5.5, with the addition of hydrochloric acid (HCl). α-LA aggregates were allowed to cool below 55◦ C for flocculation and subsequent separation from the liquid phase. A similar procedure was employed by others (Outinen et al. 1996; Rialland and Barbier 1988; Wu 2003), with a preference for pH 3.5–3.6 and a temperature of 52–55◦ C that allowed calcium to disassociate from α-LA for efficient aggregation. Precipitation of the α-LA fraction was generally followed by centrifugation or filtration to separate it from the β-LG-enriched liquor. A variation of this method consisted in using citric acid instead of HCl to acidify the whey protein solution; the double advantage of this substitution was that citric acid is generally regarded as safe (GRAS) for food applications and that citric acid provides simultaneous pH adjustment and calcium complexation, reducing the solubility of α-LA and facilitating isoelectric precipitation (Bramaud et al. 1997b). β-LG may also be precipitated under gentle heat treatment with a different pH adjustment. The aggregates of β-LG can then be recovered with ultrafiltration and electrodialysis (Amundson et al. 1982; Slack et al. 1986). After fractionation and separation of the α-LA or β-LG fraction, the pH is still highly acidic and neutralization is necessary, which introduces salts in the products. The subsequent removal of these salts necessitates extra processing steps. In recent years, Tolkach et al. (2005) extracted native α-LA with a recovery rate of 75% and a high purity (98%) at the non-acidic pH of 7.5 by selective denaturation of beta-lactoglobulin. They obtained this result by simultaneous optimization of the composition of the liquid WPC feed (protein: 5–20 g/L; lactose: 0.5 g/L; calcium: 0.55 g/L) and of the process heating time and temperature. A heat-and-pH-treatment process that does not introduce contaminants into the protein fractions is the supercritical carbon dioxide (CO2 )

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method developed by Tomasula et al. (1998). Pressurized CO2 (27– 55 bar, 54–64◦ C) was used as the acid source to lower the pH of the whey protein solution and precipitate α-LA. Solubilized CO2 was then released during extraction and depressurization of the protein fractions. This simple, economical one-step process produced individual enriched fractions of both β-LG and α-LA, with 78% recovery for β-LG and 55% recovery for α-LA, respectively (Tomasula and Parris 1999). Membrane Filtration Ultrafiltration may be used to separate the small whey proteins from the larger whey proteins and from smaller molecules such as lactose and peptides. One advantage of membrane filtration is that it causes no denaturation of the proteins (Outinen et al. 1996); however, much lower protein concentrations than in the heat-and-pH fractionation processes must be used. To modify the desired effect of ultrafiltration, one may use organic membranes or modified inorganic membranes (Lucas et al. 1998), or even membranes combined with an enzymic reactor (Sannier et al. 2000). With a cutoff size of 10 kDa, cellulose membranes reportedly retain all the whey proteins, leaving lactose and peptides in the permeate (Butylina et al. 2006). Bhattacharjee et al. (2006b) designed a two-stage ultrafiltration process with 30- and 10-kDa flat disk membranes inside a stirred rotating disk module, where the membranes’ rotation enhanced the flux and reduced the concentration polarization. The 30-kDa membrane retained BSA, LF, and Igs, while the 10-kDa membrane retained both α-LA and β-LG. Through careful optimization of the pH, the rotation speed and the transmembrane pressure, 75% pure β-LG was obtained. Nanofiltration can take the fractionation of whey one step further by separating the isolated peptides from the lactose, using a 1-kDa membrane on permeates proceeding from the 10-kDa ultrafiltration stage. The peptides/lactose membrane selectivity reportedly depends on pH (Butylina et al. 2006). Electrophoresis and Electrodialysis Electrophoresis uses the combination of protein charge and size for a precise, small-scale separation of all whey proteins, useful for the qualitative and quantitative analysis of whey proteins. Polyacrylamide

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gel electrophoresis (PAGE) is the most popular type and was first performed to quantify the various proteins of whey (Darling and Butcher 1976). Various types of gel electrophoresis are widely used nowadays for the systematic analysis of the compositions of whey protein feeds and fractions. In capillary electrophoresis, whey proteins are separated according to their migration times, in a more or less reproducible fashion. After absorbance analysis, the proteins are quantified proportionally to the area of each peak (Recio et al. 1995). Variants of capillary electrophoresis include capillary zone electrophoresis, and capillary gradient electrophoresis. Electrodialysis has been used on its own or combined with ultrafiltration (Amundson et al. 1982; Slack et al. 1986). For example, when an ultrafiltration membrane is stacked inside an electrodialysis cell, it permits the selective separation of the cationic and anionic peptides obtained from the hydrolysis of β-LG, while minimizing the fouling of the ultrafiltration membrane (Poulin et al. 2006). Ion-Exchange Chromatography This process uses the ionic properties of the whey proteins. It features the use of a resin to isolate various fractions of protein from the rest of the whey. WPIs with more than 90% proteins are easily produced from cheese whey or WPC, with less than 1% fat and lactose and little variation in properties. In addition, with a careful choice of the resin system and the eluants, α-LA and β-LG can be separated from each other even more precisely than with electrophoresis. High-performance liquid chromatography (HPLC) on various columns was used to eluate α-LA, while β-LG is adsorbed. This step was then followed by ultrafiltration for purification of the fractions (Outinen et al. 1996; Skudder 1985). Reverse-phase HPLC was also investigated for small-scale whey protein fractionation (Bobe et al. 1998). Ion-exchange chromatography can be combined with selective elution to release separately the proteins bound from cheese whey and produce several protein fractions (Etzel 2004). For example, Thuran and Etzel (2004) modified a cation-exchange chromatography method, designed to fractionate sweet whey, so that it would efficiently fractionate acid whey. This method utilized different inexpensive food-grade buffers for a two-step release of the whey proteins to obtain the α-LA fraction on one hand and a WPI containing mostly β-LG on the other hand. The rate of recovery of α-LA and the purity of the fraction (96

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and 93%, respectively) were found to be similar to that of sweet whey chromatography. De Jongh et al. (2001) applied anion-exchange affinity chromatography, with diethylaminoethyl cellulose, to the isolation of native β-LG from fresh milk. This was followed by purification with gel filtration, resulting in 80% recovery and 98% purity for β-LG in a semi-large-scale process (Heikura et al. 2005). With the ion-exchange membrane chromatography process, which was previously used by Manji et al. (1985) and Neyestani et al. (2003), the pH of the loading buffer was adjusted to facilitate the transport of βLG compared to the transport of α-LA through a strong anion-exchange membrane. With this procedure, β-LG with a high purity of 87% was obtained in the filtrate (Bhattacharjee et al. 2006a). Ayers et al. (2006) have patented a novel anion-exchange chromatography technique comprising a “water-insoluble, hydrophilic, waterswellable, hydroxyl-(C2 -C4 )-alkylated, and cross-linked regenerated cellulose, derivatized with quaternary amino (QA) groups.” This system adsorbs β-LG and is reportedly able to produce β-LG-enriched WPI directly from milk at pH 6.0–7.0. From acid whey and at pH 3.0–5.0, the extraction of acidic peptides and proteins led to the production of purified WPC. In an even more advanced, all-in-one process, crude cheese whey was passed through two serial ion exchangers, called superparamagnetic cation and anion exchangers, that allowed the sequential removal and recovery of BSA, LF, Igs, and β-LG (Heeboll-Nielsen et al. 2004). Two-Phase Partition One may exploit the differences in affinity of the various whey proteins for different material phases, either aqueous, organic, or gaseous, to selectively absorb some of the protein into the extracting phase, where it can later be easily recovered. In the context of liquid–liquid partition, systems with an aqueous phase and an organic phase are typically used (Rito-Palomares and Hernandez 1998). For example, one may exploit the selective solubility of β-LG in the aqueous phase in the presence of 3 wt% trichloroacetic acid (Fox et al. 1967; Konrad et al. 2000), or the affinity of α-LA for certain organic solvents that renders possible the solvent extraction of α-LA after heat-and-acid treatments (Freimuth and Risse 1960; Heine et al. 1992). One solvent that has been utilized for this purpose is polyethylene glycol, while the aqueous phase was

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enriched with potassium phosphate; the fractions of whey became partitioned between these two phases (Chen 1992). BSA and α-LA were absorbed into the organic phase, with partition coefficients of 10 and 27, respectively, whereas β-LG stayed in the phosphate-rich phase, with a partition coefficient of 0.07 (Capezio et al. 2005). Denatured whey proteins are good surfactants for both oil-in-water emulsions and gas-in-water foams. This amphiphilic behavior is provided by the many charges present along the peptide chain; for this reason, whey proteins form poor, unstable emulsions around their isoelectric point, when the charges are canceled (De Wit 1989). The amphiphilic properties of whey proteins were utilized in liquid–gas partition by Ekici et al. (2005) to extract whey proteins from the whey solution and concentrate them. The foam fractionation procedure consisted in creating foam from whey, which was stabilized with sodium dodecyl sulfate (SDS). At pH 2–3, the whey proteins were almost completely enriched into the foam fraction, leaving only traces in the whey solution. The resulting enrichment ratio was up to 30, and protein recovery rates ranged from 64.5 to 99.8%. Another process, developed by Fuda et al. (2005), utilized colloidal gas aphrons generated by the cationic surfactant CTAB. After optimization of the pH, ionic strength, and surfactant contents of the system, 80–90% β-LG could be precipitated in the gas phase, while most α-LA (65%), LF (75%), BSA (80%), and Igs (95%) stayed in the liquid phase. The mechanism of this process was interpreted as a succession of steps: first, β-LG was subjected to electrostatic interactions with the surfactant; denaturation ensued, followed by the precipitation of CTAB–β-LG complexes. After aggregation, the β-LG complexes were carried by flotation into the aphron phase. Finally, the colloidal gas aphrons carriers facilitated the removal of the β-LG precipitate. Chemical Reaction and Complexation Peptic Hydrolysis Native β-LG is resistant to the action of pepsin at pH 2.0 because most of its cleavages are buried in the hydrophobic core. Hence, peptic hydrolysis has been utilized to break down all the whey proteins except β-LG, which was then isolated, in its native form, by removing all the lowmolecular-weight fractions using ultrafiltration with a 30-kDa cutoff membrane (Konrad et al. 2000). Sannier et al. (2000) have performed the simultaneous purification and concentration of β-LG by combining

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the ultrafiltration membrane with an enzymic reactor, so that the process can be operated continuously. β-LG was isolated with a 74% recovery rate and a purity of 84%. Complexation Certain proteins may interact with a complexing agent, such as chitosan (made from shrimp shells), to form large complexes that can be easily removed via ultrafiltration. For example, at pH 4.6–6.5, chitosan was reported to cause the selective precipitation of β-LG from clarified cheese whey. The amount of β-LG that precipitated was increased with increasing pH and chitosan contents. At pH 6.2 and a chitosan concentration of 1.9–3.0 mg/mL, the removal of β-LG was complete, while more than 80% of the other whey proteins remained in solution (Casal et al. 2006). On the other hand, at pH 3.0, chitosan formed large soluble complexes with GMP instead of β-LG, allowing the extraction of GMP via ultrafiltration (Nakano et al. 2006). Another useful complexing agent, xanthan gum fibers, reportedly formed insoluble complexes with β-LG and some minor whey proteins, but not with α-LA. The βLG and other complexes were separated from the whey solution using ultrafiltration. Then, the xanthan gum could be recycled by redissolution at pH 10 and addition of 2-propanol, followed by ultrafiltration of the decomplexed proteins. This process was rapid and cost-effective, and yielded α-LA with a high purity (95–99% pure) (Da Fonseca and Bradley 2005).

Other Fractionation Methods Bioaffinity Separation All-trans-retinal is a bioselective ligand for β-LG. When immobilized on a porous matrix in a chromatography column (e.g., CeliteTM ), it allows the biospecific adsorption and desorption of β-LG, while α-LA is not affected. The affinity of all-trans-retinal for β-LG depends on pH and on the ionic strength: the affinity was found to be optimal at pH 5.14; at pH 7.0, the affinity decreased 44-fold, and below pH 3.5, no binding occurred (Wang and Swaisgood 1993). Vyas et al. (2002) designed a large-scale process for the separation of native β-LG from sweet whey using all-trans-retinal: all-trans-retinal was immobilized on calcium biosilicate particles inside a fluidized-bed column. The gentle

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mixing action of the fluidized bed allowed the efficient, sequential adsorption and desorption of β-LG on the biosilicate particles at pH ranging from 5.1 to 7.0, using a phosphate buffer. After ultrafiltration and freeze drying, β-LG with a purity of 95% was obtained. Coacervation With the addition of polymer to sweet whey, colloidal protein–polymer complexes can be formed, whose compositions and structures are a function of pH and polymer concentration. Capitani et al. (2005) used sodium carboxymethyl cellulose (CMC) polysaccharide polymers to produce colloids in cheese whey, adjusting the pH from 3.0 to 4.0 and the polymer concentration from 0.1 to 0.9% wt/vol. The following selective precipitates were obtained: at pH 3.0, the total proteins precipitated; at pH 3.2, α-LA–CMC complexes precipitated mostly, with a poor yield; pH 4.0 resulted in the successful precipitation of β-LG–CMC complexes mainly, with a yield of 86%. Bipolar Membrane Electroacidification At pH 4.6 and at a conductivity level of 200 micro S/cm, which was maintained with numerous additions of small volumes of potassium chloride, the precipitation of 46% of the total proteins of a WPI solution was performed, β-LG composing the main part of the precipitated protein (Bazinet et al. 2004). Separation of the Minor Whey Proteins Glycomacropeptide GMP, also called caseinomacropeptide, is a very surface-active molecule that comprises about 6.4% of dry sweet whey (Nakano et al. 2006). The methods of recovery or selective elimination of GMP from whey are numerous. The carbohydrate profile of extracted GMP varies widely and depends on the recovery method (Li and Mine 2004). These methods include ultrafiltration, ion-exchange chromatography, complexation, and solvent partition. Ultrafiltration allows the recovery of 34% of the GMP, mostly in its glycosylated form (Kawasaki et al. 1993; Li and Mine 2004).

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At pH 4.5–5.0, GMP is selectively adsorbed by anion-exchange chromatography (Ayers et al. 2006); Tek et al. (2005) reported that anionexchange chromatography performed on sweet whey permits recovery of up to 98% of GMP. They used a food-grade buffer at high-flow rate and found that GMP recovery increased with increasing pH and conductivity of the whey. GMP and β-LG bind to the same substrate; therefore, at least 0.1 M of salt was necessary to subsequently separate the bound GMP from the bound β-LG. GMP from native sweet whey can be selectively complexed with different organic molecules to form large aggregates that can easily be separated. For example, GMP was cross-linked with the transglutaminase enzyme, forming covalently-linked GMP aggregates that were removed by microfiltration or diafiltration, resulting in GMP-free native sweet whey (Tolkach and Kulozik 2005). The use of chitosan at pH 3.0 selectively produced large, soluble, chitosan–GMP complexes. Centrifugal ultrafiltration with a 100-kDa cutoff retained the chitosan–GMP complexes, but passed all other whey proteins. GMP was then recovered with the addition of NaCl, followed by dialysis (Nakano et al. 2006). Solvents utilized for the selective recovery of GMP include trichloroacetic acid and ethanol. Trichloroacetic acid fractionation recovered only glycosylated GMP, efficiently eliminating all other proteins, but had a low recovery rate (6.7%); ethanol precipitation allowed a superior (20.4%) recovery rate for GMP, mostly in its glycosylated form (Li and Mine 2004). Immunoglobulins Igs are the largest proteins in whey, with a molecular weight of 150 kDa. Bramaud et al. (1997a) recovered Igs from the α-LA fraction obtained after heat treatment at low pH of a whey protein solution acidified with citric acid. Following centrifugation, CaCl2 was added to redissolve α-LA into its native form by replenishing the calcium ions that were previously complexed by citric acid. Igs are not soluble in CaCl2 and could be separated from the α-LA fraction, resulting in 23% purer α-LA. Pessela et al. (2006) took advantage of the different adsorbing behaviors of whey proteins to isolate Igs from WPC, with a simple twostep process: first, the whey protein solution was passed through a diethylaminoethyl(DEAE)-agarose column that strongly adsorbed the BSA proteins. BSA was hereby intentionally eliminated, because of its

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ability to interact with other proteins—including Igs—and form complexes that prevents the subsequent step. The second step consisted of treating the solution containing only Igs and much smaller proteins (such as β-LG) on a low-activation aminated support where only Igs adsorbed. This procedure permitted the isolation of fairly pure Igs with an 80% recovery rate. Lactoferrin Andersson and Mattiasson (2006) extracted pure lactoferrin from WPC using a simulated moving bed chromatographic technology. This is a large-scale, continuous chromatographic process that possesses many advantages over nonmoving bed columns: a raised productivity (48%), an increased product concentration (6.5 times), a reduced buffer consumption (4.3 times), as well as a more efficient use of raw material.

Summary Advances in processing technologies have led to the industrial production of different products with varying protein contents from liquid cheese whey. These products have different biological activities and functional properties. In a recent review, Kilara and Vaghela (2004) ranked several smallscale separation methods as a function of their effectiveness in isolating each type of the whey proteins for analytical and quantification purposes: for α-LA, reverse-phase HPLC, size-exclusion HPLC, and PAGE were considered the best methods. For β-LG, the most efficient techniques were reverse-phase HPLC and SDS-PAGE. For the isolation of BSA, size-exclusion HPLC and native PAGE were preferred, while Igs were best isolated using SDS-PAGE and affinity-protein-G HPLC. When it comes to large scale separation methods, processes that use high pressure or supercritical CO2 as alternatives to mineral acids, organic solvents, or salts, are of growing interest. For example, Tomasula’s supercritical CO2 process allowed the fractionation of the whey proteins in a WPC using heat and pressurized CO2 in a pilot-scale batch process. The resulting fractions were uncontaminated and had a residual pH of 6.0, with recovery rates for α-LA and β-LG comparable to prior methods (Tomasula et al. 1998).

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Many new processes have recently been designed that permit the recovery of any of the protein fractions of cheese whey, WPC, or WPI as desired, with high recovery rates and high purities. Nowadays, one may choose a fractionation technique among the wide variety available, according to the process scale, the desired purity of the target protein fraction, and whether the desired protein must be denatured or in its native state. References Al-Mashikh, S.A., and Nakai, S. 1987. Reduction of beta-lactoglobulin content of cheese whey by polyphosphate precipitation. J. Food Sci. 52:1237–1244. Amundson, C.H., Watanawanichakorn, S., and Hill, G.G.J. 1982. Production of enriched protein fractions of β-lactoglobulin and α-lactalbumin from cheese whey. J. Food Process. Preserv. 6(2):55–71. Andersson, J., and Mattiasson, B. 2006. Simulated moving bed technology with a simplified approach for protein purification—Separation of lactoperoxidase and lactoferrin from whey protein concentrate. J. Chromatogr. A 1107(1–2):88–95. Aschaffenburg, R., and Drewry, J. 1957. Improved method for the preparation of crystalline beta-lactoglobulin and alpha-lactalbumin from cow’s milk. Biochem. J. 65(2):273–277. Ayers, J.S., Elgar, D.F., Palmano, K.P., Pritchard, M., and Bhaskar, G.B. 2006. Process for separation of whey proteins using a novel anion exchanger. U.S. Patent 7,018,665 B2: Massey University (NZ); New Zealand Dairy Board (NZ). Bazinet, L., Ippersiel, D., and Mahdavi, B. 2004. Effect of conductivity control on the separation of whey proteins by bipolar membrane electroacidification. J. Agric. Food Chem. 52(7):1980–1984. Bernal, V., and Jelen, P. 1984. Effect of calcium binding on thermal denaturation of bovine α-lactalbumin. J. Dairy Sci. 67:2452–2454. Bhattacharjee, S., Bhattacharjee, C., and Datta, S. 2006a. Studies on the fractionation of beta-lactoglobulin from casein whey using ultrafiltration and ion-exchange membrane chromatography. J. Membr. Sci. 275(1–2):141–150. Bhattacharjee, S., Ghosh, S., Datta, S., and Bhattacharjee, C. 2006b. Studies on ultrafiltration of casein whey using a rotating disk module: Effects of pH and membrane disk rotation. Desalination 195(1–3):95–108. Bobe, G., Beitz, D.C., Freeman, A.E., and Lindberg, G.L. 1998. Separation and quantification of bovine milk proteins by reversed-phase high performance liquid chromatography. J. Agric. Food Chem. 46:458–463. Bramaud, C., Aimar, P., and Daufin, G. 1995. Thermal isoelectric precipitation of α-lactalbumin from a whey protein concentrate: Influence of protein-calcium complexation. Biotechnol. Bioeng. 47(2):121–130. Bramaud, C., Aimar, P., and Daufin, G. 1997a. Optimisation of a whey protein fractionation process based on the selective precipitation of α-lactalbumin. Lait 77(3):411– 423.

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Bramaud, C., Aimar, P., and Daufin, G. 1997b. Whey protein fractionation: Isoelectric precipitation of α-lactalbumin under gentle heat treatment. Biotechnol. Bioeng. 56:391–397. Butylina, S., Luque, S., and Nystrom, M. 2006. Fractionation of whey-derived peptides using a combination of ultrafiltration and nanofiltration. J. Membr. Sci. 280(1– 2):418–426. Capezio, L., Romanini, D., Pico, G.A., and Nerli, B. 2005. Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 819(1):25–31. Capitani, C.D., Pacheco, M.T.B., Gumerato, H.F., Vitali, A., and Schmidt, F.L. 2005. Milk whey protein recuperation by coacervation with polysaccharide. Pesq. Agropec. Bras. 40(11):1123–1128. Casal, E., Montilla, A., Moreno, F.J., Olano, A., and Corzo, N. 2006. Use of chitosan for selective removal of beta-lactoglobulin from whey. J. Dairy Sci. 89(5):1384–1389. Chaplin, L.C., and Lyster, R.L.J. 1986. Irreversible heat denaturation of bovine αlactalbumin. J. Dairy Res. 53:249–258. Chen, J.-P. 1992. Partitioning and separation of α-lactalbumin and β-lactoglobulin in PEG/potassium phosphate aqueous two-phase systems. J. Ferment. Bioeng. 73:140– 147. Chiancone, E., and Gattoni, M. 1993. Selective removal of beta-lactoglobulin directly from cow’s milk and preparation of hypoallergenic formulas: a bioaffinity method. Biotechnol. Appl. Biochem. 18:1–8. Da Fonseca, L.M., and Bradley, R.L., Jr. 2005. Fractionation of whey proteins by complex formation. U.S. Patent 6,900,290 B2: Wisconsin Alumni Research Foundation, Madison, WI. Darling, D.F., and Butcher, D.W. 1976. Quantification of polyacrylamide gel electrophoresis for analysis of whey proteins. J. Dairy Sci. 59(5):863–867. de Jongh, H.H.J., Groneveld, T., and de Groot, J. 2001. Mild isolation procedure discloses new protein structural properties of β-lactoglobulin. J. Dairy Sci. Vol. 84, pp. 562–571. De Wit, J.N. 1989. Functional properties of whey proteins. In Developments in Dairy Chemistry-4, edited by P.F. Fox. New York: Elsevier Applied Science. De Wit, J.N. 1998. Marschall Rhone-Poulenc Award Lecture. Nutritional and functional characteristics of whey proteins in food products. J. Dairy Sci. 81(3):597–608. Ekici, P., Backleh-Sohrt, M., and Parlar, H. 2005. High efficiency enrichment of total and single whey proteins by pH controlled foam fractionation. Int. J. Food Sci. Nutr. 56(3):223–229. Etzel, M.R. 2004. Manufacture and use of dairy protein fractions. J. Nutr. 134(4):996S– 1002S. Fox, K.K., Holsinger, V.H., Posati, L.P., and Pallansch, M.J. 1967. Separation of βlactoglobulin from other milk serum proteins by trichloroacetic acid. J. Dairy Sci. 50:1363–1367. Freimuth, U., and Risse, I. 1960. Studies on proteins of milk serum. Part III. Experiments on the isolation of whey albumins on the basis of their solubility in organic solvents after acid treatment. Biochem. Z. 332:519–521.

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Fuda, E., Bhatia, D., Pyle, D.L., and Jauregi, P. 2005. Selective separation of betalactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB. Biotechnol. Bioeng. 90(5):532–542. Goetz, J., and Koehler, P. 2005. Study of the thermal denaturation of selected proteins of whey and egg by low resolution NMR. LWT-Food Sci. Technol. 38(5):501–512. Goff, D. 1995. Dairy science and technology. http://www.foodsci.uoguelph.ca/ dairyedu/home.html. Accessed November 2006. University of Guelph, Canada. Havea, P., Singh, H., and Creamer, L.K. 2002. Heat-induced aggregation of whey proteins: Comparison of cheese WPC with acid WPC and relevance of mineral composition. J. Agric. Food Chem. 50(16):4674–4681. Heeboll-Nielsen, A., Justesen, S.F., and Thomas, O.R. 2004. Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers. J. Biotechnol. 113(1– 3):247–262. Heikura, J., Suutari, T., Rytkonen, J., Nieminen, M., Virtanen, V., and Valkonen, K. 2005. A new procedure to isolate native beta-lactoglobulin from reindeer milk. Milchwissenschaft-Milk Sci. Int. 60(4):388–392. Heine, W., Radke, M., Wietzke, K.D., Polars, E., and Kundt, G. 1996. α-Lactalbuminenriched low-protein infant formulas: A comparison to breast milk feeding. Acta Pediatr. 85:1024–1028. Hinrichs, J., and Rademacher, B. 2005. Kinetics of combined thermal and pressureinduced whey protein denaturation in bovine skim milk. Int. Dairy J. 15(4):315–323. Hong, Y.H., and Creamer, L.K. 2002. Changed protein structures of bovine βlactoblobulin B and α-lactalbumin as a consequence of heat treatment. Int. Dairy J. 12:245–259. Jost, R., Maire, J.-C., Maynard, F., and Secretin, M.-C. 1999. Aspects of whey protein usage in infant nutrition, a brief review. Int. J. Food Sci. Tech. 34(5–6):533–542. Kaneko, T., Wu, B.T., and Nakai, S. 1985. Selective concentration of bovine immunoglobulins and α-lactalbumin from acid whey using FeCl3. J. Food Sci. 50:1531– 1536. Kawasaki, Y., Kawakami, M., Tanimoto, M., Dosako, S., Tomizawa, A., Kotake, M., and Naka-Jima, I. 1993. pH-dependent molecular weight changes of k-casein glycomacropeptide and its preparation by ultrafiltration. Milchwissenschaft 48:191–196. Kilara, A., and Vaghela, M.N. 2004. Whey proteins. In Proteins in Food Processing, edited by R.Y. Yada, pp. 72–99. Cambridge, England: Woodhead Publishing. Konrad, G., Lieske, B., and Faber, W. 2000. A large-scale isolation of native βlactoglobulin: Characterization of physicochemical properties and comparison with other methods. Int. Dairy J. 10(10):713–721. Kuwata, T., Pham, A.M., Ma, C.Y., and Nakai, S. 1985. Elimination of β-lactoglobulin from whey to simulate human milk protein. J. Food Sci. 50:605–609. Le´on-Sicairos, N., Reyes-L´opez, M., Ordaz-Pichardo, C., and de la Garza, M. 2006. Microbicidal action of lactoferrin and lactoferricin and their synergistic effect with metronidazole in Entamoeba histolytica. Biochem. Cell Biol. 84(3):327–336. Li, E.W.Y., and Mine, Y. 2004. Technical note: Comparison of chromatographic profile of glycomacropeptide from cheese whey isolated using different methods. J. Dairy Sci. 87:174–177.

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Lorenzen, P.C., and Schrader, K. 2006. A comparative study of the gelation properties of whey protein concentrate and whey protein isolate. Lait 86(4):259–271. Lucas, D., Rabiller-Baudry, M., Millesime, L., Chaufer, B., and Daufin, G. 1998. Extraction of α-lactalbumin from whey protein concentrate with modified inorganic membranes. J. Membr. Sci. 148:1–12. Maillart, P., and Ribadeau-Dumas, B. 1988. Preparation of β-lactoglobulin and βlactoglobulin-free proteins from whey retentate by NaCl salting out at low pH. J. Food Sci. 53:743–745. Mangino, M. 2005. Food Science and Nutrition: Food Proteins. Columbus, OH: Ohio State University. Manji, B., Hill, A., Kakuda, Y., and Irvine, D.M. 1985. Rapid separation of milk whey proteins by anion exchange chromatography. J. Dairy Sci. 68(12):3176–3179. Mart´ın-Diana, A.B., Gomez-Guill´en, M.C., Montero, P., and Fontecha, J. 2006. Viscoelastic properties of caseinmacropeptide isolated from cow, ewe and goat cheese whey. J. Sci. Food Agric. 86(9):1340–1349. Mat´e, J.A., and Krochta, J.M. 1994. β-Lactoglobulin separation from whey protein isolate on a large scale. J. Food Sci. 59(5):1111–1114. Morr, C.V. 1989. Whey proteins: Manufacture. In Developments in Dairy Chemistry4: Functional Milk Proteins, edited by P.F. Fox, pp. 245–284. New York: Elsevier Applied Science. Nakano, T., Ikawa, N., and Ozimek, L. 2006. Separation of glycomacropeptide from sweet whey by using chitosan and a centrifugal filter. Milchwissenschaft-Milk Sci. Int. 61(2):191–193. Neyestani, T.R., Djalali, M., and Pezeshki, M. (2003) Isolation of α-lactalbumin, βlacto-globulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Express. Purif. 29:202–208. Oldfield, D.J., Singh, H., and Taylor, M.W. 2005. Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration. J. Dairy Res. 72(3):369–378. Ostojic, S., Pavlovic, M., Zivic, M., Filipovic, Z., Gorjanovic, S., Hranisavljevic, S., and Dojcinovic, M. 2005. Processing of whey from dairy industry waste. Environ. Chem. Lett. 3(1):29–32. Outinen, M., Tossavainen, O., Tupasela, T., Koskela, P., Koskinen, H., Rantam¨aki, P., Syv¨aoja, E.-L., Antila, P., and Kankare, V. 1996. Fractionation of proteins from whey with different pilot scale processes. Lebensm.-Wiss. u.-Technol. 29:411–417. Palmer, A.H. 1934. The preparation of a crystalline globulin from the albumin fraction of cow’s milk. J. Biol. Chem. 104(2):359–372. Patel, H.A., Singh, H., Havea, P., Considine, T., and Creamer, L.K. 2005. Pressureinduced unfolding and aggregation of the proteins in whey protein concentrate solutions. J. Agric. Food Chem. 53(24):9590–9601. Pearce, R.J. 1983. Thermal separation of β-lactoglobulin and α-lactalbumin in bovine cheddar cheese whey. Aust. J. Dairy Technol. 38:144–149. Pearce, R.J. 1987. Fractionation of whey proteins. Aust. J. Dairy Technol. 212:150– 153.

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Pearce, R.J. 1991. Applications for cheese whey protein fractions. Food Res. Q. 51(1/2): 74–85. Pearce, R.J. 1995. Enriched whey protein fractions and method for the production thereof. U.S. Patent 5,455,331. Pearce, R.J., Dunkerley, J.A., and Marshall, S.C. 1991. New dairy science and technology leads to novel milk protein products. Food Res. Q. 51:145–157. Permyakov, E.A., and Berliner, L.J. 2000. α-Lactalbumin: Structure and function. FEBS Lett. 473:269–274. Pessela, B.C., Torres, R., Batalla, P., Fuentes, M., Mateo, C., Fernandez-Lafuente, R., and Guisan, J.M. 2006. Simple purification of immunoglobulins from whey proteins concentrate. Biotechnol. Prog. 22(2):590–594. Polis, B.D., Shmukler, H.W., and Custer, J.H. 1950. Isolation of a crystalline albumin from milk. J. Biol. Chem. 187(1):349–354. Poulin, J.F., Amiot, J., and Bazinet, L. 2006. Simultaneous separation of acid and basic bioactive peptides by electrodialysis with ultrafiltration membrane. J. Biotechnol. 123(3):314–328. Recio, I., Molina, E., Ramos, M., and de Frutos, M. 1995. Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries. Electrophoresis 16(4):654–658. Reiter, B. 1978. Review of the progress of dairy science: Antimicrobial systems in milk. J. Dairy Res. 45:131–147. Rialland, J.-P., and Barbier, J.-P. 1988. Process for selectively separating the αlactalbumin from the proteins of whey. U.S. Patent 4,782,138. Rito-Palomares, M., and Hernandez, M. 1998. Influence of system and process parameters on partitioning of cheese whey proteins in aqueous two-phase systems. J. Chromatogr. B Biomed. Sci. Appl. 711(1–2):81–90. Roufik, S., Paquin, P., and Britten, M. 2005. Use of high-performance size exclusion chromatography to characterize protein aggregation in commercial whey protein concentrates. Int. Dairy J. 15(3):231–241. Sannier, F., Bordenave, S., and Piot, J.-M. 2000. Purification of goat β-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor. J. Dairy Res. 67(1):43– 51. Sava, N., Van Der Plancken, I., Claeys, W., and Hendrickx, M. 2005. The kinetics of heat-induced structural changes of beta-lactoglobulin. J. Dairy Sci. 88(5):1646– 1653. Skudder, P.J. 1985. Evaluation of a porous silica-based ion-exchange medium for the production of protein fractions from rennet- and acid-whey. J. Dairy Res. 59:167– 181. Slack, A.W., Amundson, C.H., and Hill, C.G. 1986. Production of enriched βlactoglobulin and α-lactalbumin whey protein fractions. J. Food Proc. Preserv. 10:19–30. Smithers, G.W., Ballard, F.J., Copeland, A.D., De Silva, K.J., Dionysius, D.A., Francis, G.L., Goddard, C., Grieve, P.A., McIntosh, G.H., Mitchell, I.R., Pearce, R.J., and Regester, G.O. 1996. New opportunities from the isolation and utilization of whey proteins. J. Dairy Sci. 79(8):1454–1459.

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Stephans, S., Dolby, J.M., Montrenil, J., and Spike, G. 1980. Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and immunoglobulin A isolated from human milk. Immunology 41:597–603. Tek, H.N., Turhan, K.N., and Etzel, M.R. 2005. Effect of conductivity, pH, and elution buffer salinity on glycomacropeptide recovery from whey using anion exchange chromatography. J. Food Sci. 70(4):E295–E300. Thoma-Worringer, C., Sorensen, J., and Lopez-Fandino, R. 2006. Health effects and technological features of caseinomacropeptide. Int. Dairy J. 16(11):1324–1333. Thuran, K.N., and Etzel, M.R. 2004. Whey protein isolate and α-lactalbumin recovery from lactic acid whey using cation-exchange chromatography. J. Food Sci. 69(2):66– 70. Tolkach, A., and Kulozik, U. 2005. Fractionation of whey proteins and caseinomacropeptide by means of enzymatic crosslinking and membrane separation techniques. J. Food Eng. 67(1–2):13–20. Tolkach, A., Steinle, S., and Kulozik, U. 2005. Optimization of thermal pretreatment conditions for the separation of native α-lactalbumin from whey protein concentrates by means of selective denaturation of β-lactoglobulin. J. Food Sci. 70(9):E557–E566. Tomasula, P.M., and Parris, N. 1999. Whey protein fractionation using high pressure or supercritical carbon dioxide. US Patent 5,925,737. Tomasula, P.M., Parris, N., Boswell, R.T., and Moten, R.O. 1998. Preparation of enriched fractions of α-lactalbumin and β-lactoglobulin from cheese whey using carbon dioxide. J. Food Proc. Preserv. 22:463–476. Tomasula, P.M., and Yee, W.C.F. 2001. Enriched fractions of alpha-lactalbumin (α-La) and beta-lactoglobulin (β-LG) from whey protein concentrate using carbon dioxide. Functional properties in aqueous solution. J. Food Process. Preserv. 25(4):267–282. Van Der Kraan, M.I.A., Nazmi, K., van ‘t Hof, W., Amerongen, A.V.N., Veerman, E.C.I., and Bolscher, J.G.M. 2006. Distinct bactericidal activities of bovine lactoferrin peptides LFampin 268–284 and LFampin 265–284: Asp-Leu-Ile makes a difference. Biochem. Cell Biol. 84(3):358–362. Varunsatian, S., Watanabe, K., Hayakawa, S., and Nakamura, R. 1983. Effects of Ca++, Mg++, and Na+ on heat aggregation of whey protein concentrates. J. Food Sci. 48:42–48. Verheul, M., Pedersen, J.S., Roefs, S.P.F.M., and Kruif, K.G.D. 1999. Association behavior of native β-lactoglobulin. Biopolymers 49(1):11–20. Vyas, H.K., Izco, J.M., and Jimenez-Flores, R. 2002. Scale-up of native betalactoglobulin affinity separation process. J. Dairy Sci. 85(7):1639–1645. Wang, Q., and Swaisgood, H.E. 1993. Characteristics of β-lactoglobulin binding to the all-trans-retinal moiety covalently immobilized on CeliteTM . J. Dairy Sci. 76(7):1895–1901. Whitney, R. 1977. Milk proteins. In Food Colloids, edited by H.D. Graham, pp. 66–151. Westport, CT: AVI Publishing Co. Wu, C. 2003. Whey treatment process for achieving high concentration of αlactalbumin. US Patent 6,613,377.

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Wu, H., Xie, J.J., and Morbidelli, M. 2005. Kinetics of cold-set diffusion-limited aggregations of denatured whey protein isolate colloids. Biomacromolecules 6(6):3189– 3197. Yalcin, A.S. 2006. Emerging therapeutic potential of whey proteins and peptides. Curr. Pharm. Des. 12(13):1637–1643. Zweig, G., and Block, R.J. 1954. Studies on bovine whey proteins. III. The preparation of crystalline alpha-lactalbumin and beta-lactoglobulin ferrilactin. Arch. Biochem. Biophys. 51(1):200–207.

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Chapter 3 Separation of β-Lactoglobulin from Whey: Its Physico-Chemical Properties and Potential Uses Raj Mehra and Brendan T. O’Kennedy

Introduction β-Lactoglobulin (β-LG) is the major whey protein present in the milk of ruminants like cow and sheep, and is known to be absent in human and rodent milks (Sawyer 2003). It accounts for approximately 55% of the total whey protein (∼3.2 g/L). Whey processors continually show interest in fractionation of whey protein into individual whey proteinenriched fractions for nutritional, nutraceutical, and health-related end uses. The drive toward the development of the next generation of infant milk formula based on the inclusion of α-lactalbumin-enriched ingredients brings into focus the market opportunity that is presented by the availability of the “other” fraction (β-LG) produced as a coproduct. Since the latter protein fraction influences whey protein functionality for the most part, it is expected that its availability in an enriched form will lead to further enhancement of its key functional properties and stimulate further market opportunities. It is generally regarded that β-LG dictates the water-holding characteristics (viscosity and gelation) of whey protein-based ingredients. Strength, texture, water-holding capacity, and color of protein gels are influenced by type of protein, protein concentration, pH, thermal treatment, and ionic environment. There is an increasing demand for institutional researchers to investigate at a more fundamental level the 39 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Table 3.1.

Whey protein composition of bovine and human milk.

Protein

Bovine milk (g/L)

Human milk (g/L)

Total whey protein β-Lactoglobulin (β-lg) α-Lactalbumin (α-lac) Immunoglobulin (IgG) Serum albumin (BSA) Lactoferrin (LF)

5.8 3.2 (55%)a 1.2 (21%) 0.8 (14%) 0.4 (7%) 0.2 (3%)

6.6 — 2.6 (40%) 1.2 (19%) 0.6 (9%) 1.7 (26%)

a Figures

in brackets represent proportion of total whey protein.

mechanisms that influence the water-holding behavior of β-LG and the opportunities that may be presented for enhanced functional behavior arising from better scientific knowledge. This chapter attempts to review some of the more prominent β-LG separation/fractionation methodologies in the public domain, the general physico-chemical properties of β-LG, factors which may affect its behavior, functional testing of separated fractions, and some nutraceutical applications.

Separation/Fractionation of β-LG Whey contains several proteins in different amounts, each with its individual unique nutritional, nutraceutical, and health-related properties. Of these proteins, β-LG is the major whey protein present in bovine milk, while it is essentially absent in human milk. The whey protein composition of bovine and human milk is given in Table 3.1. A number of laboratory methods and pilot scale processes for fractionation of β-LG from whey have been described. The processes fall into the following categories: selective precipitation, addition of salts or precipitants, ion-exchange chromatography, enzymatic hydrolysis, and membrane filtration. Most of these processes have not been widely implemented at industrial scale due to their relatively high cost, complexity, low productivity, poor selectivity, and unsuitability for scale-up (Zydney 1998). This section will focus on pilot scale processes for fractionation of β-LG and laboratory separation methods that have potential for scale-up.

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Selective Precipitation A process for fractionating β-LG from other whey proteins by selective precipitation of β-LG due to its low solubility under conditions of reduced ionic strength and acid pH (4.65) has been reported (Amundson et al. 1982; Slack et al. 1986a). Processing steps include preconcentration of cheese whey by ultrafiltration (UF), adjustment of retentate pH to 4.65, reduction of ionic strength (demineralization) by electrodialysis or diafiltration, and recovery of precipitated β-LG by centrifugation. This leaves a supernatant enriched in α-LA. The final dried β-LG product contained high lipid levels (30.9%) and 51.6% protein. Coprecipitation of lipid material is a major disadvantage of this method for some applications such as foaming or whipping. An improved separation methodology was developed by Pearce (1983, 1987) where α-LA was selectively precipitated leaving a lipidfree β-LG stream. Cheese whey was preconcentrated by UF, adjusted to pH 4.2, heated to 64◦ C for 5 min, cooled, and aggregated α-LA separated from soluble β-LG by centrifugation. The β-LG fraction was further purified by UF and diafiltration yielding a dried product with 75.5% protein, 1.2% fat, 16.1% lactose, and 1.2% ash. The precipitation of α-LA at relatively low temperatures is related to the loss of bound calcium in the region of the isoelectric point. Several process variations, incorporating modifications and improvements on the method of Pearce (1987), have been developed. Pierre and Fauquant (1986) and Maubois et al. (1987) clarified whey by initially removing residual whey fat by the thermocalcia procedure of Fauquant et al. (1985). They adjusted the pH of the clarified whey to 3.8, concentrated by UF, and heated the retentate to 55◦ C for 30 min to aggregate and precipitate α-LA. A β-LG purity of 98% was achieved. The efficiency and selectivity of the α-LA precipitation step in this process are improved by using citric acid to adjust the pH to 3.9, and addition of citrate as a calcium sequestrant to promote destabilization of α-LA (Bramaud et al. 1997a, 1997b). This results in lowering of the free calcium concentration in the medium and displacement of the precipitation phenomenon to a lower temperature (35◦ C), thus preventing excessive denaturation of α-LA and β-LG fractions. G´esan-Guiziou et al. (1999) employed microfiltration and diafiltration to separate soluble β-LG from the precipitate resulting in a poor β-LG recovery of 51%. A β-LG purity of 85–94% was obtained, with

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the higher values achieved using acid whey, which does not contain caseinomacropeptide. Another modification of the same approach was utilized by de Wit and Bronts (1994). They pretreated whey with an ion-exchange resin to facilitate dissociation of calcium from α-LA. This promoted its destabilization on heating to 50◦ C at pH between 4.3 and 4.8 yielding a β-LG-rich fraction of 85% purity. A selective separation process was patented by Stack et al. (1995). The main differences from the process of Pearce (1987) were the initial reduction of whey calcium by electrodialysis and ion exchange, and crystallization and removal of lactose prior to heating to 35–54◦ C for 1–3 h to aggregate α-LA. This resulted in a product with a 95% β-LG purity. Use of Salts and Selected Precipitants Pure β-LG was produced from whey UF retentate by salting-out of other whey proteins at pH 2.0 with 7.0% NaCl (Mailliart and Ribadeau-Dumas 1988). The soluble β-LG left in the supernatant was subsequently saltedout by increasing the NaCl level to 30%, with a final recovery of 84%. Similarly, Mate and Krochta (1994) obtained large quantities of β-LG by selective precipitation from whey protein isolate (WPI) at pH 2.0 and 7% NaCl. A β-LG purity of greater than 95% and a recovery of 65% were reported. Addition of ferric chloride (4 mM) to cottage cheese whey and adjustment of the pH to 3.0 led to selective precipitation of α-LA and immunoglobulins, leaving β-LG in the soluble fraction (Kuwata et al. 1985). β-LG was precipitated from the supernatant by heating at pH 3.0 at 90–93◦ C for 15 min, followed by adjusting the pH to 4.5. Total nitrogen recovery in this preparation was reported to be 62%. Similarly, 90% of β-LG coprecipitated with bovine serum albumin (BSA) when acid whey was treated with 7.5 mM FeCl3 at pH 4.2 and held at 4◦ C (Kaneko 1985). Al-Mashikh and Nakai (1987) showed that 80% of β-LG precipitated with most of the BSA when sodium hexametaphosphate was added to cheese whey at pH 4.07 and 22◦ C for 1 h. Ion-Exchange Chromatography Skudder (1985) produced β-LG-enriched fractions from whey by anion exchange chromatography on Spherosil-QMA, based on the higher

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binding affinity of β-LG for this resin than other whey proteins, and the phenomenon of β-LG-induced displacement of other proteins adsorbed during passage of large quantities of whey at pH 6.5–6.63. Elution of the bound β-LG with 0.1 N HCl led to fractions with β-LG recoveries of 96% and 100%, and purities of 99% and 67.7% for acid and rennet whey, respectively. A variation of the above anion exchange method was patented by Outinen et al. (1995). Column chromatography is performed with an industrially suitable strong polystyrene-based anion exchange ˚ and a bead resin (Diaion HPA 75), with a pore size of 1,000–2,000A size diameter of 300–600 μm. β-LG binds selectively to the resin at pH 6.5 (α-LA does not adsorb under these conditions) and elution with a 2– 5% NaCl solution. A β-LG purity of >90% could be achieved with this method. Cation exchange chromatography was employed to produce a β-LG-enriched fraction from cheese whey without the use of NaCl (Etzel 1999). Enriched fractions of β-LG were obtained by application of whey, adjusted to pH 3, to a cation exchanger, and selective elution of β-LG from the resin by increasing the pH to 4.9 using a solution of 0.2 M sodium citrate. Proteolytic Hydrolysis A novel method for the preparation of highly pure and native β-LG, based on the resistance of native β-LG to peptic hydrolysis compared to other whey proteins, was developed by Kinekawa and Kitabatake (1996). WPI or whey was treated with pepsin at pH 2.0 and 37◦ C for 60 min. The unhydrolyzed native β-LG was recovered by ammonium sulfate precipitation, dialyzed, or ultrafiltered/diafiltered. The β-LG obtained from this procedure was characterized to be pure and in its native form. Based on the same principle, a simpler and more economical process was developed at pilot scale (10,000 L) level by Konrad et al. (2000). This consisted of peptic treatment of skimmed sweet whey followed by microfiltration/UF. A β-LG recovery of 67% and a purity of 94.1% were reported. However, this procedure does not allow simultaneous purification of any other individual whey proteins from the UF permeate as they are hydrolyzed. Membrane Separation Zydney (1998) suggested that whey protein could be fractionated using membrane filtration via manipulation of the electrostatic and steric

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properties of individual whey proteins in order to facilitate selective separation during membrane filtration. He had earlier observed (van Eijndhoven et al. 1995) that protein transmission through a membrane could be changed in response to changes in pH and ionic strength of the protein solution and lead to the separation of proteins with similar molecular weight (BSA from hemoglobulin). Using a cellulose membrane of 30 kDa porosity, pH 5.5, and 50 mM ionic strength, β-LG was separated from α-LA with a β-LG purification factor of 100 and a recovery of 90% from a model binary protein solution (Cheang and Zydney 2003, 2004). On the basis of this hypothesis, Mehra and Kelly (2004) were able to fractionate whey protein into two broad fractions by microfiltration: (i) immunoglobulins + BSA + LF and (ii) α-LA + βLG. Fraction (ii) was further separated into α-LA- and β-LG-enriched fractions by UF.

Physico-Chemical Properties of β-LG β-LG is the major whey protein and it generally accounts for ∼55% of the whey proteins and ∼10% of the total milk proteins. It has a molecular weight of 18,000 kDa, is a globular protein, and exists as a dimer from about pH 5 to 7. The protein exists as a dimer in solution due to the electrostatic interactions between Asp130 and Glu134 of one monomer with corresponding lysyl residues of other monomers (Creamer et al. 1983). It may also exist as an octamer at pH values between 3.5 and 5.0 or as a monomer at pH values below 3.5 (de Wit 1989). Native β-LG possesses five cysteines existing as two disulfide bonds, Cys66 –Cys160 and Cys106 –Cys119 /Cys121 , with one free thiol group varying between residues 119 and 121. The native conformation is sensitive to heat and pH and the free thiol group is of major significance on denaturation. The mechanism for heat denaturation that has evolved is that of dimer dissociation followed by monomer unfolding to permit more rapid thiol reactivity that can lead to disulfide interchange and aggregation, although noncovalent aggregation can also occur without the involvement of the thiol group (Manderson et al. 1998; Qi et al. 1995, 1997). The secondary structure of β-LG contains ∼8% α-helix, 45% β-sheet, and 47% random coil. Extremes of pH and modification of the dielectric properties of the solvent significantly alter these values.

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β-LG exists in a large number of genetically determined variants but it is generally present as either the A or the B variant. The function of β-LG is not fully understood but it has been speculated that because of its affinity for retinol the biological function of β-LG may be related to vitamin A transport (de Wit 1989). Denaturation of β-LG β-LG can be denatured by a number of different processes including heat, pressure, and chaotropic agents (e.g., urea). On denaturation, β-LG molecules, which are globular in structure, unfold to expose previously hidden reactive areas including thiol groups and disulfide bonds. Effect of Heating on Denaturation/Aggregation of β-LG β-LG as the major whey protein tends to dominate the thermal behavior of the total whey protein system. It has important functional properties in many food products, and an understanding of the details of how it unfolds and aggregates with heat treatment is important. The denaturation of β-LG is dependent on temperature, ionic strength, and pH (Li et al. 1994). Factors influencing the heat-induced denaturation/aggregation of β-LG include noncovalent interactions such as ionic, van der Waals, and hydrophobic interactions as well as thiol/disulfide exchange reactions, leading to the formation of disulfide-linked aggregates (Hoffmann and van Mil 1997; Mulvihill et al. 1990). Whether these aggregation reactions are irreversible or potentially reversible will depend on the degree of chemical or physical aggregation involved (Verheul et al. 1998) and on factors such as temperature, pH, and ionic strength (Hoffmann et al. 1996). The extent of aggregation can extend from the formation of dimers, trimers, oligomers, soluble aggregates, and so on to large aggregate formation and visible precipitation or gelation (Croguennec et al. 2004). Upon heating, β-LG undergoes intra- and intermolecular changes. Raising the temperature, shifts the β-LG monomer–dimer equilibrium at β-LG concentrations below 10 g/L toward monomers (Georges et al. 1962; Verheul et al. 1998). There is a two-step reaction scheme for the denaturation of β-LG, which consists of a first-order unfolding reaction followed by a secondorder aggregation. It is mainly physical aggregation that occurs in the

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second-order reaction at high heating temperatures. Denaturation of β-LG becomes rate limiting at high heating temperatures, pH values close to the isoelectric point of the protein, and at high NaCl concentrations (Verheul et al. 1998). Effect of pH on Heat-Induced Denaturation/Aggregation of β-LG β-LG is most stable at a pH near its isoelectric point (pH 5.2). At pH values further from the isoelectric point, the conformational stability decreases as a result of intramolecular charge repulsion (Harwalker and Ma 1989). The decreased stability at these low and high pH values leads to increasing reaction rates. pH has a strong influence on the rate of aggregation reactions and the size of aggregates of heat-treated β-LG. While denaturation/aggregation of β-LG is accelerated with increasing pH, aggregate size was found by Hoffmann and Van Mil (1999) to decrease with increasing pH. They also found that the dissociation and initiation reactions were accelerated to a large extent with increasing pH, and that due to the formation of many reactive intermediates in the initial stages of the reaction, the probability of termination reactions increased, which led to the formation of smaller aggregates. Effect of Ionic Strength and Ionic Species on Denaturation/Aggregation of β-LG The effect of calcium on the heat-induced aggregation of β-LG is probably the most studied interaction in both milk and related food systems. It has been suggested that three effects, or a combination of them, might be responsible for calcium-induced protein aggregation (Simons et al. 2002). The first phenomenon is related to intermolecular cross-linking of adjacent negatively charged or carboxylic groups by the formation of protein–Ca2+ –protein complexes (Bryant and McClements 1998; Hongsprabhas 1999; Xiong et al. 1993). The second phenomenon is the intramolecular electrostatic shielding of negative charges on the protein (Hongsprabhas and Barbut 1997). Monovalent and divalent cations both screen electrostatic interactions between charged protein molecules although the effect is greater with divalent cations (Veerman et al. 2003). The third phenomenon is ion-induced conformational changes, which lead to altered hydrophobic interactions and aggregation at elevated temperatures (Kinsella et al. 1989). However, recent observations

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demonstrated that the role of Ca2+ in the formation of intermolecular bridges was unlikely and its action was only in the screening of β-LG surface charges and that excess CaCl2 may have an inhibitory effect on the protein aggregation rate (Simons et al. 2002). Sherwin and Foegeding (1997) demonstrated that aggregation rates were affected by CaCl2 /protein stoichiometry rather than the CaCl2 and protein concentrations separately. Simons et al. (2002) and Jeyarajah and Allen (1994) suggested that calcium was bound to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges resulted in protein aggregation, driven by the partial unfolding of β-LG at elevated temperatures, which was facilitated by the absence of electrostatic repulsion. NaCl concentration also affects both steps in the denaturation/ aggregation reaction scheme. NaCl belongs to the salting-out class of salts and it stabilizes the native protein conformation and decreases the denaturation rate at high salt concentrations (Von Hippel and Schleich 1969). Chemical and physical aggregation reactions are enhanced at low salt concentrations, while at high salt concentrations physical aggregation is enhanced which leads to large aggregates. Verheul et al. (1998) observed that the maximum reaction rate with increasing NaCl concentration is caused by the combined effect of a reduced denaturation rate and increased aggregation rates. While a lot of research has focused on the denaturation/aggregation kinetics of β-LG in water or simple salt systems, relatively little research has been undertaken on the effects of milk salts on the denaturation/ aggregation behavior of β-LG. The composition of the milk serum salt solution is outlined in Table 3.2. While the calcium level is in the region of 10 mM and there is a strong ionic strength effect due mainly to the monovalent ions, the presence of citrate and phosphate brings an additional complexity to the system. The presence of these strong anions reduces the Ca2+ concentration to ∼2 mM. Due to the high ionic strength and low Ca2+ the net effect of heating β-LG in milk serum salt solutions is dominated by the ionic strength effect on the denaturation/aggregation behavior.

Role of Thiol Groups in the Aggregation of β-LG The role of the thiol group of β-LG in the heat-induced aggregation has been extensively investigated. Hoffmann and van Mil (1997) found

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Whey Processing, Functionality and Health Benefits Table 3.2. Typical composition of the milk serum salt solution. Salt

g/L

mM

Calcium Sodium Potassium Magnesium Phosphorus Chloride Citrate Sulfate Carbon dioxide

0.360 0.420 1.540 0.078 0.360 1.150 1.850 0.100 0.095

9 18 39 3.2 12 32 10 1 2

that in water at neutral and elevated pH values, the thiol group plays a crucial role in the heat-induced aggregation of β-LG by acting as an initiator of thiol–disulfide exchange reactions. The formation of intermolecular disulfide bonds prevents the reversibility of modifications in the tertiary structure of native β-LG. Noncovalent interactions including ionic bonding, van der Waals forces, and hydrophobic bonding may be involved in the heat-induced aggregation of β-LG as well as chemical aggregation by covalent intermolecular disulfide bonds. The disulfide linkage involved in the intermolecular interchange reaction is most likely the C66–C160 disulfide which is found in one of the external loops of β-LG. The other disulfide is buried and is less available for reaction (McKenzie et al. 1972; Papiz et al. 1986). Roefs and de Kruif (1994) proposed a kinetic model for the denaturation and aggregation of β-LG. There are three steps in the reaction scheme: initiation, propagation, and termination. A number of reversible reactions are involved in the initiation step where the β-LG dimer is split into two monomers, followed by an irreversible step, which is the real initiation reaction. The free thiol group reacts with the intramolecular disulfide bond by a thiol–disulfide exchange reaction in the propagation step. The termination step occurs when two reactive intermediates react with each other to form a polymer without a reactive thiol group. The dissociation and initiation reactions are accelerated to a large extent with increasing pH, and due to the formation of many

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reactive intermediates in the initial stages of the reaction, the probability of termination reactions increases, leading to the formation of smaller aggregates (Hoffmann and Van Mil 1999).

Denaturation of Genetic Variants of β-LG There are at least six genetic variants of bovine β-LG, the two most common being β-LG A and β-LG B. There are commercially significant differences between the responses of whey containing either β-LG A or β-LG B to heat treatment (Hill et al. 1997). The genetic variants A and B of β-LG exhibit a marked difference in their heat-induced denaturation/aggregation behavior (Schokker et al. 2000). It was found by Manderson et al. (1998) that the overall formation of large aggregates was greater for β-l B than for β-LG A. β-LG A was also shown to favor the formation of hydrophobically driven associations and the formation of nonnative monomers as intermediates in the aggregation pathway. At β-LG concentrations lower than 5%, the aggregation rate of the B variant is greater than that of the A variant, and at concentrations greater than 5% the opposite is found (Nielson et al. 1996). Variant A forms more intermediate-sized aggregates, whereas variant B forms a higher proportion of very large aggregates (Manderson et al. 1998). The differences in denaturation curves of β-LG A, B and C can be attributed to the structural differences within the proteins that give rise to a destabilizing cavity created by Val118 Ala (A–B) and a changed charge distribution caused by the Asp64 Gly (A–B) substitution (Qin et al. 1999).

Denaturation of β-LG in Combination with α-LA The addition of α-LA to β-LG causes a difference in the denaturation/ aggregation reactions. The addition of α-LA to β-LG A and β-LG B prior to heat treatment affects the pathway of aggregation to favor greater disulfide bonded aggregates and fewer hydrophobically associated aggregates (Schokker et al. 2000). They also found that inaccessible disulfide bonds of α-LA react with the free thiol groups of the unfolded β-LG molecules, after which they could catalyze intermolecular disulfide bond interchange.

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Heat-Induced Gelation of β-LG Mulvihill and Kinsella (1988) found that β-LG gels at pH 8.0 showed maximum gel strength in the presence of NaCl or CaCl2 . The same forces, which are responsible for denaturation/aggregation of β-LG, are believed to be responsible for maintaining the gel matrix of β-LG gels. These forces include the covalent disulfide bonds, noncovalent electrostatic forces, van der Waals forces, and hydrophobic interactions. For gel formation the critical protein concentration necessary is dependent on the pH and ionic strength of the protein solution (Paulsson et al. 1986, 1990). The addition of salt to β-LG affects its gelation properties as well as its denaturation/aggregation properties. Gels with different properties, such as transparency, shear stress and strain at fracture, and water-holding capacities are formed by varying the amount and type of salt (Bryant and McClements 2000; Hongsprabhas and Barbut 1996). At neutral pH, the β-LG gel network is supposed to be maintained by a balance between attractive bonds (mainly disulfide bonds) and repulsive interactions between negative charges on acidic amino acids (de Wit 1989). There are enhanced protein–protein interactions between denatured whey proteins near their isoelectric pH values owing to the heat-induced exposure of previously buried hydrophobic groups (Zhu and Damodaran 1994). According to Otte et al. (2000), gels formed at pH 7.0 are composed of polymers, which are internally linked by covalent bonds and associated into a network by hydrophobic interactions and electrostatic repulsions. They also proposed that at lower protein concentrations, gels formed at pH 5.0 are more dependent on noncovalent bonds than at higher protein concentrations. This was also observed for gels formed by heating at neutral pH (Gezimati et al. 1997).

Gelation of Milk: β-LG and Casein in Combination While β-LG, in isolation, can form three-dimensional structures (gels) on heating, a more significant effect is observed in food systems where both casein and β-LG are present during the heating stage. A model system, simulating the nonfat phase of yogurt milk, consists of a combination of native micellar casein and WPI in the approximate ratio that occurs in milk. The system is often used to examine the contribution of each major protein group to rheological behavior during acid gelation.

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O’Kennedy and Kelly (2000) identified two phases during acid gelation of a casein/whey protein model system. They found that the complex formation of predenatured whey proteins with casein was the dominant effect on initial gelation pH and that it is the synergistic effects on gel strength arising from casein aggregation effects that take over as acidification progresses. During the heating of milk, β-LG can covalently interact with κ-casein present at the exterior of the casein micelles (Jang and Swaisgood 1990; Singh 1993). α-Lac and the minor whey proteins can also interact with the casein micelles (Corredig and Dalgleish 1996; Oldfield et al. 2000). Heated milk is therefore a mixture of native and denatured whey proteins and casein micelles. The denatured proteins usually occur as whey protein aggregates or as whey protein aggregates associated with the casein micelles (Vasbinder et al. 2003). The degree of interaction between β-LG and κ-casein depended on the time and temperature of heating, the concentration of the proteins, pH, and the presence and concentrations of milk salts (Singh 1995). Long et al. (1963) showed that primary denaturation of β-LG precedes its interaction with κ-casein. It was also shown that only β-LG required heating for an interaction to occur. Pure κ-casein has been reported by Groves et al. (1998) to aggregate during heat treatment to form high molecular weight polymers, primarily via thiol–disulfide interactions. Cho et al. (2003) showed that the loss of the native-like character of βLG was faster in the presence of κ-casein and it increased with higher κ-casein concentrations. Alting et al. (2002) showed that the reduction of the electrostatic repulsion of the aggregates (net electric charge) is the driving force for pH-induced gelation. They also showed that the formation of additional disulfide bonds depends on the pH of gelation and that this has a clear effect on the properties of the gel. Under conditions of strong electrostatic repulsions, gels from globular proteins were transparent and had a fine-stranded structure, whereas they were opaque with a coarse, lumpy, particulate structure under conditions of weak electrostatic repulsions (Stading et al. 1993). The heat treatment of milk prior to acidification caused a shift in gelation pH to higher pH values and formed a stronger final gel (Lucey et al. 1997). It was the denatured whey protein coating the casein micelles that were thought to cause increased gel hardness and decreased syneresis as they prevented coalescence of the micelles and increased the number of contact points between the micelles (Heertje

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et al. 1985; Mottar and Bassier 1989). Vasbinder et al. (2003) showed that the formation of disulfide bonds during and after acidification of heated milk contributed strongly to the mechanical properties. They suggested that gel hardness depended on the amount of reactive thiol groups present after heating the milk.

Functional Properties and Potential Applications of β-LG-Enriched Fractions in Food Limited studies have been published on the functionality of commercial β-LG fractions or those prepared at the pilot scale using processes that have potential for commercialization. β-LG-rich fractions produced by selective precipitation at low ionic strength showed no foaming ability (Amundson et al. 1982; Slack et al. 1986b) due to the presence of the high lipid level (4.45–35.9%) in the enriched powders. Residual lipid is known to depress the foaming ability of protein powders (Rantam¨aki et al. 2000). The fraction had >85% solubility at all pH values except between pH 3 and 5 where it was insoluble (Amundson et al. 1982). However, solubility in this pH range increased to >74.5% in the presence of >0.1 M NaCl. The β-LG fraction produced by the selective isoelectric heat precipitation (Pearce 1987) exhibited total solubility and clarity over the pH range from 3.0 to 8.0. Results of a study involving ultrahigh temperature (UHT) sterilization of model acidic beverage systems containing sugar, pectin and fortified with the β-LG fraction showed that formulations containing 1–2% protein in the pH range 3.2–3.5 may be suitable for fortification of acidic beverages (Pearce 1991). Subsequent trials with fruit juice concentrates and flavors proved that β-LG fraction up to 2.5% w/w protein is ideal for protein fortification of the beverages. Pearce (1991) proposed that the separated β-LG fraction, produced essentially fat-free, was superior compared to whey powder, WPC75, and α-LA-enriched fraction in terms of foam expansion, foam stability at both low and high temperatures, and foam stability in the presence of high sugar at both ambient and high temperatures. In a comparison of the separated β-LG fraction, egg white and WPI in a meringue model system, β-LG was equal to or better than egg white in producing good meringues and thus could replace egg white in this type of food application.

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Egg white is also used as a texturing agent in processed fish and meat products to provide the desired viscosity and water-binding capacity (Pearce 1991). Stringent functional requirements on whey proteins to replace egg white replacer include formation of a uniform gel, gel breaking strength similar to or greater than egg white protein, and high water-binding capacity. Results of work on β-LG gelation indicated that it may demonstrate gel strength as much as three times greater than that of egg white protein at the same protein concentration. This suggested that β-LG fraction was capable of replacing egg white in the manufacture of processed meat and fish products. The functional properties of β-LG-enriched fractions prepared by four different pilot scale processes were compared (Outinen et al. 1996; Rantam¨aki et al. 2000; Tossavainen et al. 1998). Fractionation processes included two anion-exchange methods (QMA and HPA-75) and two based on heat aggregation of α-LA at acid pH. The four different β-LG fractions varied significantly in their functional properties. Those fractions produced by the aggregation of α-LA had higher solubilities at pH 4.6 in water (93.6 and 98.5%) than those produced by ion exchange (55.7 and 75.3%). These differences may be attributed to the denaturation of β-LG during ion exchange. All fractions were >98.9% soluble at near neutral pH (6.7). Addition of NaCl (1%) at pH 4.6 increased the solubilities of all fractions, but had little effect at pH 6.7. Emulsion studies showed small differences between the emulsion stabilities of the fractions, with QMA β-LG having the lowest stability. No differences were observed in apparent viscosities between the β-LG fractions with or without the addition of NaCl. Fractions separated by using QMA and by the method of Pearce (1987) showed high foam overruns (2,000 and 1,600, respectively) and foam stability (33 and 23 min, respectively) and were superior to egg white at the same protein concentration. The other β-LG fractions and WPC-70 failed to foam or showed poor foam properties due to the presence of high indigenous residual fat (Rantam¨aki et al. 2000). With the exception of the fraction produced by the method of Pearce (1987), all fractions displayed good gelling abilities at pH values between 6 and 8, with gel strengths ranging from 0.81 to 4.72 N (Newton) for 10% protein gels. It was concluded that the high calcium level measured in the weak gelling sample contributed to the formation of weak gels (0.12–0.70 N). Four β-LG-enriched fractions containing different mineral contents were evaluated in frankfurters for their effect on water-/fat-binding,

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textural, and sensory properties (Hayes et al. 2005). The β-LG-enriched fractions significantly improved the cook loss and tenderness of the product without having a detrimental effect on the flavor and overall acceptability of the product. WPC80 and a β-LG-enriched fraction were also assessed for their effects on the enhancement of fresh pork loins, and as possible replacement ingredients for the reduction of phosphate (Hayes et al. 2006). The study concluded that the two protein ingredients tested were comparable to salt/sodium tripolyphosphate (STPP) addition and therefore could reduce phosphate levels used in processed meat products.

Potential Health Benefits/Uses of β-LG Scientific evidence has shown that proteins high in essential amino acids (EAA), branched chain amino acids (BCAA), and particularly leucine (Leu) are associated with increased muscle protein synthesis, weight loss, body fat loss, and decreased plasma insulin and triglyceride profile (Etzel 2004). β-LG contains 17% more EAA, 33.5% more BCAA, and 74% more Leu compared to theoretical “average” values calculated for other proteins. Availability of dairy protein fractions rich in β-LG through commercial fractionation processes may allow production of β-LG-containing high protein foods that may provide nutritional benefits and tackle health issues such as obesity and diabetes. Nutritional benefits of β-LG also include reduction in energy and fat levels when used as a fat replacer in manufactured meat products. Based on the excellent heat-set gelling and water-binding properties of β-LG, a β-LG-rich whey protein fraction was modified into a hydrocolloid modified particulate gel that had textural analysis profiles and properties similar to solid fat (Pearce et al. 1998). It was successfully introduced as a fat replacer in Australian commercial meat products. The health promoting bioactivities of β-LG are more associated with hydrolyzed β-LG products than with the intact native molecule. Several peptides derived from proteolytic digestion of β-LG (using pepsin, trypsin, chymotrypsin, or other commercially available enzymes) have been reported to have antimicrobial activity (against E. coli, pathogenic E. coli, Bacillus subtilis, and Staphylococcus aureus), angiotensinconverting enzyme (ACE) activity, mitogenic activity, opioid activity, and hypocholesterolemic effects (Chatterton et al. 2006). β-LG may

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have anticancer activity as suggested from results of a study in which diets supplemented with β-LG retarded development of chemically induced colon cancer in a rat model (McIntosh et al. 1995). A chemically modified derivative of β-LG is effective in inhibiting HIV-1, HIV-2, simian immunodeficiency virus, herpes simplex virus type 1 and 2, and Chlamydia trachomatis infection in vitro, and may be effective in controlling HIV-1 infection in humans (Chatterton et al. 2006). The antiadhesion activity of β-LG has been shown to inhibit the adhesion of pathogenic strains of Klebsiella oxytoca and E. coli to immobilized ileostomy glycoproteins (Ouwehand et al. 1997). Although β-LG has been extensively researched, its physiological function is still unclear. It has been reported to bind retinol and longchain fatty acids in vitro (Sawyer 2003), and increase the intestinal uptake of retinol, triglyceride, and long-chain fatty acids in preruminant calves (Kushibiki et al. 2001). It is speculated that it may have a role in digestion, absorption, and metabolism of fatty acids in neonates, and as a carrier of retinol in food systems and in vitamin preparations.

Future Research The efficient fractionation of whey proteins requires an in-depth knowledge of the structure and inherent behavior of the native protein molecules. While the molecular structure of the β-LG molecule has been elucidated, its behavior under varying conditions of pH, ionic strength, and temperature is not fully understood. Future research on the ability of β-LG to self-associate or to associate with a secondary inclusion should provide the platform for a cost-effective methodology for large-scale fractionation of β-LG from whey. Future research in membrane and/or ion-exchange fabrication should provide a more selective choice of fractionation material to make the process more efficient. The use of biopolymers as the fractionation mechanism is a developing area of research. The formation of complex coacervates using oppositely charged biopolymers provides a viable alternative to a classical ion-exchange methodology. β-LG and a selection of charged biopolymers (chitosan, carrageenan) interact to form insoluble complexes under various conditions of pH and ionic strength. Subsequent “elution” of the native protein through alteration of the pH should provide a viable alternative methodology for fractionation. While most methodologies

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concentrate on fractionating β-LG in its native conformation, there is no reason why the protein cannot be fractionated in a nonnative form. Ongoing research into the formation of denatured but soluble aggregates of β-LG should provide the basis for additional methodologies to produce ingredients which are enriched and highly functional. The use of β-LG in food systems is limited by our understanding of how the protein denatures and subsequently aggregates. In the highly complex environment of food systems the interaction of the denatured β-LG with other components needs to be elucidated before maximum usage of the highly functional β-LG can be achieved. This would include the classical casein/β-LG interaction but also the interactions with polysaccharides, fats, and minerals. As the understanding of these interactions proceeds, the drive to fractionate β-LG in an efficient and cost-effective manner should progress accordingly.

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Long, J.E., Van Winkle, Q., and Gould, I.A. 1963. Heat-induced interaction between crude k-casein and β-lactoglobulin. J. Dairy Sci. 46:1329–1334. Lucey, J.A., Teo, C.T., Munro, P.A., and Singh, H. 1997. Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk. J. Dairy Res. 64:591–600. Mailliart, P., and Ribadeau-Dumas, B. 1988. Preparation of β-lactoglobulin and βlactoglobulin-free proteins from whey retentate by NaCl salting out at low pH. J. Food Sci. 53(3):743–752. Manderson, G.A., Hardmann, M.J., and Creamer, L.K. 1998. Effect of heat treatment on the conformation and aggregation of β-lactoglobulin A, B and C. J. Agric. Food Chem. 46:5052–5061. Mate, J.I., and Krochta, J.M. 1994. β-Lactoglobulin separation from whey protein isolate on a large scale. J. Food Sci. 59(5):1111–1113. Maubois, J.-L., Pierre, A., Fauquant, J., and Piot, M. 1987. Industrial fractionation of main whey proteins. Bull. Int. Dairy Fed. 212:154–159. McIntosh, G.H, Regester, G.O., Le Leu, R.K., Royal, P.J., and Smithers, G.W. 1995. Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats. J. Nutr. Biochem. 125(4):809–816. McKenzie, H.A., Ralston, G.B., and Shaw, D.C. 1972. Location of sulphydryl and disulphide groups in bovine β-lactoglobulins and effects of urea. Biochemistry 11:4539–4547. Mehra, R., and Kelly, P.M. 2004. Whey protein fractionation using cascade membrane filtration. Advances in fractionation and separation: Processes for novel dairy applications. Int. Dairy Fed. Bull. 389:40–44. Mottar, J., and Bassier, A. 1989. Effect of heat-induced association of whey proteins and casein micelles on yoghurt texture. J. Dairy Sci. 72:2247–2256. Mulvihill, D.M., and Kinsella, J.E. 1988. Gelation of β-lactoglobulin: Effects of sodium chloride and calcium chloride on the rheological and structural properties of gels. J. Food Sci. 53:231–236. Mulvihill, D.M., Rector, D., and Kinsella, J.E. 1990. Effects of structuring and destructuring anionic ions on the rheological properties of thermally induced βlactoglobulin gels. Food Hydrocoll. 4:267–276. Nielson, B.T., Singh, H., and Latham, J.M. 1996. Aggregation of bovine βlactoglobulins A and B on heating at 75◦ C. Int. Dairy J. 6:519–527. Oldfield, D.J., Singh, H., Taylor, M.W., and Pearce, K.N. 2000. Heat-induced interactions of β-lactoglobulin and α-lactalbumin with the casein micelle in the pHadjusted skim milk. Int. Dairy J. 10:509–518. O’Kennedy, B.T., and Kelly, P.M. 2000. Evaluation of milk protein interactions during acid gelation using a simulated yoghurt model. Milchwissenschaft 55:187–190. Otte, J., Zakora, M., and Qvist, K.B. 2000. Involvement of disulphide bonds in bovine β-lactoglobulin B gels set thermally at various pH. J. Food Sci. 65:384–389. Outinen, M., Harju, M., Tossavainen, O., and Antila, P. 1995. Process for fractionating whey proteins and the components so obtained. PCT International Patent no. WO 95/19714.

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Outinen, M., Tossavainen, O., Tupasela, T., Koskela, P., Koskinen, H., Rantam¨aki, P., Syvaoja, E.-L., Antila, P., and Kankare, V. 1996. Fractionation of proteins from whey with different pilot scale processes. Lebensm. – Wiss. U. Technol. 29:411–417. Ouwehand, A.C., Salminen, S.J., Skurnik, M., and Conway, P.L. 1997. Inhibition of pathogen adhesion by β-lactoglobulin. Int. Dairy J. 7(11):685–692. Papiz, M.Z., Sawyer, L., Eliopoulos, E.E., North, A.C.T., Findlay, J.B.C., Sivaprasadarao, R., Jones, T.A., Newcomer, M.E., and Kraulis, P.J. 1986. The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein. Nature 324:383–385. Paulsson, M., Dejmek, P., and van Vliet, T. 1990. Rheological properties of heatinduced β-lactoglobulin gels. J. Dairy Sci. 73:45–53. Paulsson, M., Hegg, P.O., and Castberg, H.B. 1986. Heat induced gelation of individual whey proteins. A dynamic rheological study. J. Food Sci. 51:87–90. Pearce, R.J. 1983. Thermal separation of β-lactoglobulin and α-lactalbumin in bovine cheddar cheese whey. Aust. J. Dairy Technol. 38:144–149. Pearce, R.J. 1987. Fractionation of whey proteins. Bull. Int. Dairy Fed. 212:150–153. Pearce, R.J., Dunkerley, J.A., Wheaton, T.W., Marshall, S.C., and Tobin, A. 1998. A solid fat replacer for manufactured meat products based on a β-lactoglobulinrich whey protein ingredient. In Proceedings of the second International Whey Conference, Chicago, USA, October 27–29, 1997, pp. 181–188. Brussels, Belgium: International Dairy Federation Publication. Pearce, R.J. 1991. Applications for cheese whey protein fractions. Food Res. Q. 51:74– 85. Pierre, A., and Fauquant, J. 1986. Principes pour un procede industriel de fractionnement des proteins du lactoserum. Le Lait 66(4):405–419. Qi, X.L., Brownlow, S., Holt, C., and Sellers, P. 1995. Thermal denaturation of βlactoglobulin—effect of protein concentration at pH 6.75 and pH 8.05. Biochim. Biophys. Acta 1248:43–49. Qi, X.L., Holt, C., McNulty, D., Clarke, D.T., Brownlow, S., and Jones, G.R. 1997. Effect of temperature on the secondary structure of β-lactoglobulin at pH 6.7 as determined by CD and IR spectroscopy: A test of the molten globule hypothesis. Biochem. J. 324:341–346. Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N., and Jameson, G.B. 1999. Structural and functional differences of variants A and B of bovine β-lactoglobulin. Protein Sci. 8:1–9. Rantam¨aki, O., Tossavainen, O., Outinen, M., Tupasela, T., Koskela, P., and Kaunismaki, M. 2000. Functional properties of the whey protein fractions produced in pilot plant processes. Foaming, water-holding capacity and gelation. Milchwissenschaft 55(10):569–572. Roefs, S.P.F.M., and De Kruif, C.G. 1994. A model for the denaturation and aggregation of β-lactoglobulin. Eur. J. Biochem. 226:883–889. Sawyer, L. 2003. β-Lactoglobulin. In Advanced Dairy Chemistry Volume 1: Proteins, 3rd ed., Part A, edited by P.F. Fox and P.L.H. McSweeney, pp. 319–386. London: Kluwer Academic/Plenum Publishers.

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Schokker, E.P., Singh, H., and Creamer, L.K. 2000. Heat induced aggregation of βlactoglobulin A and B with α-lactalbumin. Int. Dairy J. 10:843–853. Sherwin, C.P., and Foegeding, E.A. 1997. The effects of CaCl2 on aggregation of whey proteins. Milchwissenschaft 52:93–96. Simons, J.W.F.A., Kosters, H.A., Visschers, R.W., and de Jongh, H.J.J. 2002. Role of calcium as trigger in thermal β-lactoglobulin aggregation. Arch. Biochem. Biophys. 406:143–152. Singh, H. 1993. Heat induced interactions of proteins in milk. Protein & Fat Globule Modifications-IDF Seminar, pp. 191–203. Singh, H. 1995. Heat induced changes in casein, including interactions with whey proteins. In Heat-Induced Changes in Milk, 2nd ed., edited by P.F. Fox, pp. 86–104. Brussels, Belgium: IDF (International Dairy Federation Special issue no. 9501). Skudder, P.J. 1985. Evaluation of a porous silica-based ion-exchange medium for the production of protein fractions from rennet- and acid-whey. J. Dairy Res. 52:167– 181. Slack, A.W., Amundson, C.H., and Hill, C.G., Jr. 1986a. Production of enriched βlactoglobulin and α-lactalbumin whey protein fractions. J. Food Process. Preserv. 10:19–30. Slack, A.W., Amundson, C.H., and Hill, C.G., Jr. 1986b. Foaming and emulsifying characteristics of fractionated whey protein. J. Food Process. Preserv. 10:81– 88. Stack, F.M., Hennessy, M., Mulvihill, D.M., and O’Kennedy, B.T. 1995. Process for the fractionation of whey constituents. PCT International Patent Application WO 953 4216 A1. Stading, M., Langton, M., and Hermansson, A.M. 1993. Microstructure and rheological behaviour of particulate β-lactoglobulin gels. Food Hydrocoll. 7:195–212. Tossavainen, O., Rantamaki, P., Outinen, M., Tupasela, T., and Koskela, P. 1998. Functional properties of the whey protein fractions produced in pilot plant processes. Milchwissenschaft 53(8):453–457. van Eijndhoven, H.C.M., Saksena, S., and Zydney, A.L. 1995. Protein fractionation using membrane filtration: Role of electrostatic interactions. Biotechnol. Bioeng. 48:406. Vasbinder, A.J., Alting, A.C., Visschers, R.W., and De Kruif, C.G. 2003. Texture of acid milk gels: Formation of disulphide cross-links during acidification. Int. Dairy J. 13:29–38. Veerman, C., Baptist, H., Sagis, L.M.C., and Van Linden E. 2003. A new multistep Ca2+ -induced cold gelation process for β-lactoglobulin. J. Agric. Food Chem. 51:3880–3885. Verheul, M., Roefs, S.P.F.M., and de Kruif, C.G. 1998. Kinetics of heat-induced aggregation of β-lactoglobulin. J. Agric. Food Chem. 46:896–903. Von Hippel, P.H., and Schleich, T. 1969. In Structure and Stability of Biological Macromolecules, edited by S.N. Timasheff and G.D. Fasman, pp. 181–188. New York: Dekker.

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Xiong, Y.L., Dawson, K.A., and Wan, L. 1993. Thermal aggregation of b-LG: Effect of pH ionic environment and thiol reagent. J. Dairy Sci. 76:70–77. Zhu, H., and Damodaran, S. 1994. Heat induced conformational changes in whey protein isolate and its relation to foaming properties. J. Agric. Food Chem. 42:846– 855. Zydney, A.L. 1998. Protein separations using membrane filtration: New opportunities for whey fractionation. Int. Dairy J. 8:243–250.

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Chapter 4 Whey Protein-Stabilized Emulsions David Julian McClements

Introduction The major protein fractions present in bovine whey are surface-active globular proteins, i.e., β-lactoglobulin, α-lactabumin, and bovine serum albumin (Swaisgood 1996). These proteins are often used as emulsifiers because they are amphiphilic molecules that can facilitate emulsion formation, improve emulsion stability, and produce desirable physicochemical properties in emulsions (McClements 2005a; Norde 2003). Proteins adsorb to the surfaces of freshly formed oil droplets created by homogenization of oil–water–protein mixtures, where they facilitate further droplet disruption by lowering the interfacial tension and retard droplet coalescence by forming protective membranes around the droplets (Aken 2004; Dalgleish 1997; Dickinson 1992; McClements 2005a). The ability of proteins to generate repulsive interactions (e.g., steric and electrostatic) between oil droplets and to form an interfacial membrane that is resistant to rupture also plays an important role in stabilizing the droplets against flocculation and coalescence during long-term storage (Aken 2004; Dalgleish 1997; Dickinson 1992; McClements 2005a). The development of whey protein-stabilized emulsions with improved or novel physicochemical properties relies on understanding the interfacial behavior of adsorbed proteins, and on elucidating the relationship between interfacial characteristics and bulk physicochemical properties of emulsions (such as stability, rheology, and appearance). There has been a concerted research effort over many years to develop a fundamental understanding of the interfacial and functional properties of whey proteins in emulsions. Much of this work has 63 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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been carried out using simple well-defined model systems, for example, purified protein fractions, model or purified oils, fixed pH, ionic strength, and temperature. On the other hand, the whey proteins used in industrial emulsion-based products tend to be compositionally complex, and experience a variety of different solution conditions (pH, ionic strength, surfactants, biopolymers) and environmental stresses (thermal processing, chilling, freezing, drying, homogenization, and mechanical agitation) during their production, storage, and utilization. Recently, there has therefore been increasing emphasis on developing a more fundamental understanding of the influence of composition, solution conditions, ingredient interactions, and environmental stresses on whey protein functionality. This chapter focuses on recent research carried out in the area of whey protein functionality, largely drawing from work carried out in the author’s laboratory.

Emulsion Formation The process of converting two immiscible bulk phase liquids into an emulsion, or of reducing the size of the droplets in a preexisting emulsion, is known as homogenization. The mechanical device designed to carry out this process is called a homogenizer. The characteristics of some of the most commonly used homogenizers have been reviewed elsewhere, for example, high-speed mixers, high-pressure valve homogenizers, colloid mills, ultrasonic homogenizers, and membrane homogenizers (McClements 2005a; Walstra 1993, 2003). In general, homogenization can be separated into two categories depending on the nature of the starting material. The formation of an emulsion directly from two separate bulk liquids is referred to as primary homogenization, whereas the reduction in size of the droplets in an existing emulsion is referred to as secondary homogenization (Figure 4.1). The creation of a particular type of emulsion may involve the use of either of these types of homogenization, or a combination of both. In largescale food processing operations it is often more efficient to prepare an emulsion in two stages. First, the separate oil and water phases are converted to a coarse emulsion that contains fairly large droplets using one type of homogenizer (e.g., a high-speed blender). The droplets of the emulsion premix, having a low kinetic stability, are further reduced in size using a different type of homogenizer (e.g., a high-pressure

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Figure 4.1. Homogenization involves the conversion of two immiscible liquids into an emulsion (primary) or the reduction of the size of the droplets in an existing emulsion (secondary).

valve homogenizer). It should be noted that there is no clear distinction between most of the physical processes that occur during primary and secondary homogenization, for example, mixing, droplet disruption, and droplet coalescence. Finally, some homogenizers are capable of producing emulsions with small droplet sizes directly from separate oil and water phases, for example, high-intensity ultrasonicators, microfluidizers, or membrane homogenizers. Many of the important characteristics of industrial emulsions depend on the size of the droplets they contain, including their stability, texture, appearance, and functionality. Consequently, the major objective of homogenization is to create an emulsion in which the majority of droplets fall within an optimum size range that yields emulsions with properties specified by the manufacturer. The major factors that determine the size of the droplets produced after homogenization have recently been reviewed, which include emulsifier type and concentration, homogenizer type, homogenization conditions, and physicochemical properties of component phases (McClements 2005a). Whey protein emulsifiers play a number of important roles during the homogenization process. First, they decrease the interfacial tension between the oil and water phases, thereby reducing the amount of energy required to deform and disrupt the droplets. Second, they form a protective coating around the droplets that prevents them from coalescing with each other within and outside the homogenizer. The protein coating normally generates electrostatic and steric repulsive forces between the droplets that prevent them from coming into close proximity, as well as forming a viscoelastic membrane that is resistant to rupture. The size

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of the droplets produced during homogenization depends on a number of different characteristics of an emulsifier: (i) The ratio of emulsifier to dispersed phase—there must be sufficient emulsifier present to completely cover the surfaces of the droplets formed. (ii) The time required for the emulsifier to move from the bulk phase to the droplet surface—the faster the adsorption time, the smaller the droplet size. (iii) The probability that an emulsifier molecule will be adsorbed onto the surface of a droplet during an encounter between it and the droplet—the greater the adsorption efficiency, the smaller the droplet size. (iv) The amount that the emulsifier reduces the interfacial tension—the greater the amount, the smaller the droplet size. (v) The effectiveness of the emulsifier membrane in protecting the droplets against coalescence—the better the protection, the smaller the droplet size. It is usually important that whey proteins are properly dissolved in the aqueous phase prior to homogenization, and that the pH and ionic strength of the aqueous phase will not promote droplet aggregation.

Emulsion Stability Once an emulsion has been created it is important that it retains its desirable properties during storage and application. The term “emulsion stability” is used to describe the ability of an emulsion to resist changes in its properties with time. The properties of an emulsion may evolve over time due to a variety of physical, chemical, or biological processes. From a technological standpoint, it is important to identify the dominant processes occurring in the system of interest because effective strategies can then be rationally designed to overcome the problem. A number of the most important physical mechanisms responsible for the instability of emulsions are shown schematically in Figure 4.2. It is important to have knowledge of these mechanisms in order to understand how whey protein emulsifiers influence emulsion stability.

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Figure 4.2. Schematic diagram of the most common instability mechanisms occurring in food emulsions.

Gravitational Separation Gravitational separation is one of the most common forms of instability in emulsions and may result in either creaming or sedimentation depending on the relative densities of the dispersed and continuous phases. Creaming is the upward movement of droplets due to the fact that their density is lower than that of the surrounding liquid, whereas sedimentation is the downward movement of droplets due to the fact that they have a higher density than the surrounding liquid (Figure 4.2). The creaming velocity of an isolated rigid spherical particle suspended in a Newtonian liquid obeys Stokes’ law (Equation (4.1)): vStokes = −

2gr 2 (ρ2 − ρ1 ) 9η1

(4.1)

where r is the radius of the particle, g is the acceleration due to gravity, ρ is the density, η is the shear viscosity, and the subscripts 1 and 2 refer to the continuous and dispersed phases, respectively. The sign of vStokes determines whether the droplet moves upward (+) or downward (−), i.e., creams or sediments. Stokes’ law highlights a number of strategies that industrial manufacturers of emulsions can use to retard gravitational

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separation, i.e., decreasing the density contrast between the two phases, decreasing the droplet radius, or increasing the viscosity of the continuous phase. Each of these strategies is used in industry, with the most appropriate one or a combination of them depending on the nature of the emulsion. It should be stressed that Stokes’ law is inappropriate for accurately predicting gravitational separation in many industrial emulsions because they do not exist as dilute suspensions of noninteracting rigid spheres suspended in a Newtonian fluid. For this reason, the theory has been extended to take into account various other factors, such as droplet fluidity, droplet concentration, particle–particle interactions, and nonNewtonian continuous phases (McClements 2005a). Whey protein emulsifiers can influence the stability of oil-in-water (O/W) emulsions to creaming in a variety of ways. First, they may facilitate the formation of small droplets during homogenization, thereby reducing the creaming velocity. Second, they may influence the tendency for droplet flocculation to occur, which increases the effective size of the particles in the emulsion. In dilute systems, flocculation usually decreases the stability of the emulsion to creaming due to the increase in creaming velocity, but in concentrated systems flocculation may actually increase the creaming stability due to the formation of a network of aggregated particles that prevents their movement. Droplet Aggregation The droplets in emulsions frequently encounter their neighbors because they are in continual motion because of the effects of thermal energy, gravity, or applied mechanical forces. After an encounter, emulsion droplets may either move apart or remain aggregated, depending on the relative magnitude of the attractive and repulsive interactions between them (Dalgleish 1997; Dickinson 1992; McClements 2005a). Droplets tend to aggregate when the attractive forces dominate the repulsive forces. The three major types of aggregation in emulsions are flocculation, coalescence, and partial coalescence. Flocculation Droplet flocculation is the process whereby two or more droplets come together to form an aggregate in which the droplets retain their individual integrity (Figure 4.2). Flocculation may be either beneficial or

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detrimental to emulsion quality depending on the nature of the product. As mentioned above, flocculation accelerates gravitational separation in dilute emulsions, which is undesirable because it reduces their shelf life. It may also cause an appreciable increase in emulsion viscosity (“thickening”), and may even lead to the formation of a gel. Some emulsified industrial products are expected to have a low viscosity and therefore flocculation is detrimental. In other products, a controlled amount of flocculation may be advantageous because it leads to the creation of a desirable texture. Flocculation may occur in emulsions through a variety of different processes that either increase the attractive forces or decrease the repulsive forces between the droplets, for example, reduction in electrostatic repulsion by changing pH or ionic strength; increase in depletion attraction due to the presence of nonadsorbed polymer; increase in hydrophobic attraction due to an increase in the surface hydrophobicity of the droplets, for instance, due to thermal denaturation of adsorbed proteins; and adsorption of polymers onto more than one droplet leading to bridge formation (Dalgleish 1997; Dickinson 1992; McClements 2005a). Examples of some of these mechanisms are given below for whey protein-stabilized emulsions. Coalescence Coalescence is the process whereby two or more liquid droplets merge together to form a single larger droplet (Figure 4.2). Coalescence causes emulsion droplets to cream or sediment more rapidly because of the droplet size increase. In oil-in-water emulsions, coalescence eventually leads to the formation of a separate oil layer, a process that is referred to as oiling off. Coalescence requires that the liquid contained within two or more emulsion droplets come into direct contact. Coalescence may occur immediately after two droplets come into contact with each other, or after the droplets have been in contact for prolonged periods. The latter case may occur in highly concentrated emulsions, flocculated emulsions, or creamed layers where the droplets remain in close proximity for some time. In the subsequent step, the thin layer of continuous phase separating the droplets, as well as the interfacial protein layers coating the droplets, must be disrupted to allow the lipid molecules to come into direct contact. The rate at which coalescence proceeds and the physical mechanism by which it occurs is thus highly dependent on the nature of the emulsifier used to stabilize the system. Improving the stability of an emulsion to coalescence may be achieved by preventing

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droplet flocculation, preventing formation of a creamed layer, reducing the droplet concentration, and altering the rheological properties of the interfacial membrane to improve rupture resistance. Recent experiments have shown that the susceptibility of protein-coated droplets to coalescence is often increased when they are sheared or adsorbed onto air–water interfaces (Aken 2004). Partial Coalescence Partial coalescence occurs when two or more partially crystalline oil droplets come into contact and form an irregularly shaped aggregate (Walstra 2003). It is initiated when a fat crystal from one partially crystalline droplet penetrates into the liquid portion of another partially crystalline droplet. Over time, the droplets may partly fuse together to reduce the surface area of lipid that is exposed to water. Nevertheless, the aggregates partly retain the shape of droplets from which they were formed due to the low mobility of molecules in fat crystal networks. Partial coalescence only occurs in emulsions that contain partially crystalline regions. This is because one of the key requirements for partial coalescence is penetration into the liquid phase. If all droplets were completely liquid they would undergo normal coalescence. If all droplets were completely solid they would undergo flocculation rather than partial coalescence because of the lack of liquid lipid regions that had sufficient molecular mobility required for merging. Thus, one can expect an “optimum” crystal content at which partial coalescence would be highest. Indeed it has been found that increasing the solid content of the droplets causes an initial increase in the partial coalescence rate until a maximum value is reached, after which the partial coalescence rate decreases (Walstra 2003). The solid content at which this maximum rate occurs depends on the morphology and location of the crystals within the droplets, as well as the magnitude of the applied shear stresses. Effect of Whey Protein on Aggregation Whey protein emulsifiers can influence the stability of emulsions to droplet aggregation (flocculation, coalescence, and partial coalescence) in a number of ways. First, adsorbed whey proteins that are more effective in producing highly charged and thick interfaces are more likely to increase the repulsive interactions between droplets, thereby preventing flocculation. Second, adsorbed whey proteins that are capable of producing highly visco–elastic interfaces that are resistant to deformation are more likely to prevent coalescence and partial coalescence. Third,

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nonadsorbed whey proteins that are capable of thickening or gelling the continuous phase may prevent droplets from coming into close contact for extended periods by slowing down their movement, hence reducing the likelihood of flocculation or coalescence. Fourth, nonadsorbed whey proteins may increase the osmotic attraction between droplets, thereby promoting depletion flocculation. Ostwald Ripening Ostwald ripening is the process whereby large droplets grow at the expense of smaller ones because of diffusion of dispersed phase molecules from one droplet to another through the intervening continuous phase (Kabalnov 2001; Kabalnov and Shchukin 1992). The origin of this effect is the fact that the solubility of solute molecules within a spherical particle in the surrounding solvent increases as the size of the particle decreases. This leads to a thermodynamic driving force that favors movement of solute molecules from small droplets to large droplets, leading to a net increase in the mean droplet diameter in the emulsion over time. The rate of droplet growth is mainly determined by the solubility of the solute molecules in the continuous phase, and is usually only important in systems where the solute has an appreciable solubility. In principle, whey proteins may retard droplet Ostwald ripening by forming interfaces that are resistant to compression or expansion and that therefore provide a mechanical resistance to droplet shrinkage or growth. In practice, the mechanical strength of whey protein films does not appear to be high enough to prevent Ostwald ripening (Dickinson et al. 1999). Phase Inversion Phase inversion is the process whereby a system changes from an oilin-water emulsion to a water-in-oil emulsion, or vice versa (Figure 4.2). Phase inversion is an essential step in the manufacture of a number of important industrial products, including butter, and margarine. On the other hand, phase inversion is undesirable in other products because it has an adverse effect on the product’s appearance, texture, or stability, for example, coagulation of cream. Phase inversion can be triggered by some alteration under the composition or environmental conditions of an emulsion, for example, disperse phase volume fraction, emulsifier type, emulsifier concentration,

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solvent conditions, temperature, or mechanical agitation (Aoki et al. 2005; Walstra 2003). Only certain types of emulsion are capable of undergoing phase inversion, rather than being completely broken down into their component phases. These emulsions are capable of existing in a kinetically stable state both before and after the phase inversion has taken place. It is usually necessary to agitate an emulsion during the phase inversion process; otherwise it will separate into its component phases. The physicochemical basis of phase inversion is believed to be extremely complex, involving aspects of flocculation, coalescence, partial coalescence, and emulsion formation. Whey proteins may influence the likelihood of phase inversion by reducing the tendency for flocculation, coalescence, or partial coalescence to occur (see discussion of these mechanisms in previous sections).

Bulk Physicochemical Properties of Emulsions Emulsion Rheology Rheology is the science concerned with the relationship between applied stresses and the deformation and/or flow of matter (Rao 1999). It is important in the food industry because it influences the texture, mouthfeel, shelf life, and processing of many food materials. Rheological testing usually involves measuring the flow and/or deformation of a material when a defined stress is applied. The extent of the deformation and/or flow depends on the physicochemical properties of the material, and can provide valuable information about product properties and functionality. A manufacturer must therefore be able to design and produce a product that has the rheological properties expected by the consumer. Sophisticated and sensitive analytical techniques based on these principles are available for characterizing the rheological behavior of emulsions, and are widely used in industrial, government, and academic laboratories (Rao 1999). Industrially, emulsions are compositionally and structurally complex soft materials that can exhibit a wide range of different rheological behavior, ranging from relatively low viscosity fluids (such as milk or beverages) to solid-like materials with relatively high elastic modulus (such as refrigerated butter or margarine). A variety of mathematical models have been developed to relate the rheological properties of

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Figure 4.3. Schematic representation of the change in relative viscosity (η/η1 ) of an oil-in-washer emulsion with droplet concentration for a nonflocculated and flocculated system.

emulsions to their composition and microstructure. In general, the apparent viscosity of an emulsion can be described by the following equation (Aoki et al. 2005; Dickinson 1992): η = f (η1 , η2 , φ, r, w(h), τ )

(4.2)

where η1 is the viscosity of the continuous phase, η2 is the viscosity of the dispersed phase, φ is the dispersed phase volume fraction, r is the droplet radius, w(h) is the interaction potential between the droplets, and τ is the applied shear stress. The precise form of Equation (4.3) used to describe the rheological properties of an emulsion depends on the characteristics of the system, for example, droplet concentration, droplet interactions, and continuous phase rheology. Exact expressions of the relationship between the rheology of colloidal suspensions and their composition/structure are only available in certain limiting cases, such as Einstein’s equation for a dilute suspension of rigid spherical particles: η = η1 (1 + 2.5φ)

(4.3)

This equation illustrates that the rheology of a dilute emulsion is proportional to the rheology of the continuous phase and increases with increasing droplet concentration. The Einstein equation can be extended to account for the effects of droplet fluidity, particle–particle interactions, droplet flocculation and non-Newtonian continuous phase rheology (Aoki et al. 2005; Dickinson 1992). A typical plot of the dependence of emulsion viscosity on droplet concentration is shown in Figure 4.3 for

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nonflocculated and flocculated emulsions. In nonflocculated emulsions, the viscosity increases gradually as the droplet concentration increases until a critical droplet concentration (φc ) is reached where the droplets become close packed and then the viscosity increases steeply. Emulsions that have droplet concentrations appreciably higher than φc tend to exhibit viscoplastic type rheological behavior, for example, mayonnaise. In flocculated emulsions, the droplet concentration where a sudden increase in viscosity occurs is much lower because network formation occurs between the droplets. In addition, the viscosity of flocculated emulsions is much higher than that of nonflocculated emulsions because the effective volume fraction of the particles is larger. It should also be noted that many industrial emulsions show highly shear-thinning rheological behavior, particularly if they contain thickening agents in the continuous phase and/or if the droplets are flocculated (Aoki et al. 2005; Dickinson 1992). Whey proteins may influence the rheological properties of oil-inwater emulsions in a variety of ways. First, they will affect the sign, magnitude, and range of the various colloidal interactions between the oil droplets, which may alter the amount of droplet flocculation in the system (Aoki et al. 2005). As mentioned earlier, a flocculated emulsion tends to have a higher viscosity and a greater propensity for shear thinning to occur than a nonflocculated emulsion with the same droplet concentration. Second, whey proteins may appreciably increase the rheological properties of the continuous aqueous phase (η1 ), particularly if they are aggregated due to heating. Indeed, heat denatured whey proteins may form highly viscous solutions or gels, which would greatly affect the rheology of the overall emulsion. Consequently, any factor that influences the aggregation of adsorbed or nonadsorbed whey proteins will tend to alter emulsion rheology (e.g., pH, ionic strength, and temperature). Emulsion Appearance The optical properties of many industrial emulsions play an important role in determining their quality, as it is usually the first sensory impression that a consumer makes of a product (McClements 2002; McClements et al. 1998). It is therefore often important to understand the factors that determine emulsion appearance as this would aid in the design of emulsion-based products with improved quality. When a

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Figure 4.4. Dependence of the lightness of an oil-in-water emulsion on droplet concentration. The emulsion lightness increases steeply from 0 to 10 wt% oil, and then does not change appreciably at higher oil contents.

light beam is incident upon the surface of an emulsion a portion of the incident light beam is transmitted through the emulsion while another portion is reflected. The relative proportions of light transmitted and reflected at different wavelengths depend on the scattering and absorption of the light wave by the emulsion. Light scattering and absorption depend on the size, concentration, refractive index, and spatial distribution of droplets, as well as the presence of any chromophoric materials (e.g., dyes). Hence, the overall appearance of an emulsion is influenced by its structure and composition. Scattering is largely responsible for the “turbidity,” “opacity,” or “lightness” of an emulsion, whereas absorption is largely responsible for “chromaticity” (blueness, greenness, redness, etc.). It should be stressed that the overall appearance of an emulsion also depends on the nature of the light source and the detector used. Mathematical models have recently been developed to predict the color of emulsions from knowledge of their droplet characteristics (McClements 2002). For example, the tristimulus coordinates (L , a, b values) of emulsions can be predicted from knowledge of their droplet radius (r ), disperse phase volume fraction (φ), and dye visible absorption spectrum (α(λ)): L , a, b = f (r, φ, α(λ)). Theoretical predictions and experimental measurements indicate that the “lightness” of an emulsion increases and the “color intensity” decreases with increasing droplet concentration (particularly between 0 and 5% droplets), which is due to increased light scattering (Figure 4.4). This has important implications for the development of reduced fat products, since reducing the concentration of oil droplets in an O/W emulsion below a certain level causes an appreciable change in product appearance.

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Whey proteins may influence the optical properties of oil-in-water emulsions in a variety of ways. First, the use of whey proteins as emulsifiers will determine the size of the oil droplets produced during homogenization, which has previously been shown to influence the lightness and color of oil-in-water emulsions (Chantrapornchai et al. 1999; McClements 2002). In general, the lightness of an emulsion first increases with increasing droplet diameter, reaches a maximum value (around 0.1–1 μm), and then decreases. Second, the use of whey proteins as emulsifiers may influence the degree of droplet flocculation in an emulsion, and therefore the mean effective particle size. Nevertheless, studies have shown that droplet flocculation does not have a large impact on the appearance of moderately concentrated oil-in-water emulsions (Chantrapornchai et al. 2001).

Influence of Environmental Stresses on Emulsion Stability In this section we examine some of the important environmental stresses that may influence the stability of oil-in-water emulsions stabilized by whey proteins. Aging After adsorption onto an oil–water interface a globular protein may undergo substantial changes in its conformation and interactions due to the change in its molecular environment (Norde 2003). In an aqueous phase a protein is surrounded primarily by water molecules, but at an oil– water interface it is surrounded by water molecules on one side and oil molecules on the other side. Proteins undergo conformational changes after adsorption in order to maximize the number of favorable interactions and minimize the number of unfavorable interactions in their new environment. The time taken for these conformational changes to occur depends on the molecular flexibility and packing of the adsorbed molecules (Norde 2003). Relatively flexible proteins (such as casein) undergo relatively rapid conformational changes, whereas more rigid globular whey proteins (such as β-lactoglobulin or BSA) take much longer (e.g., hours or days). Protein conformational changes resulting from adsorption of the protein molecules onto an interface are usually referred to as “surface denaturation.” The extent of the conformational

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changes that occur in whey proteins after adsorption onto oil droplet surfaces in oil-in-water emulsions depends on the nature of the oil phase. The extent of conformational changes has been shown to be larger for more nonpolar oils, which may be because the hydrophobic driving force for protein unfolding is greater. For many globular proteins, surface denaturation leads to an increased exposure of nonpolar and sulfhydryl-containing amino acids to the aqueous phase (Kim et al. 2002b). When the droplets in globular proteinstabilized oil-in-water emulsions are in close proximity (e.g., pH close to isoelectric point or high ionic strength) surface denaturation can promote droplet flocculation through increased hydrophobic attraction and disulfide bond formation between proteins adsorbed onto different droplets (Kim et al. 2002b). On the other hand, when the droplets are prevented from coming into close proximity (e.g., pH far from isoelectric point or low ionic strength) extensive protein–protein interactions at an interface may lead to the formation of an interfacial membrane that is more difficult to disrupt, which may therefore provide better protection against droplet coalescence under quiescent conditions. Thermal Processing In many practical applications it is important to subject proteinstabilized emulsions to thermal processing, for example, cooking, pasteurization, or sterilization (Kim et al. 2005). Emulsions stabilized by globular proteins are particularly sensitive to thermal treatments, because these proteins unfold when the temperature exceeds a critical value exposing reactive groups originally located in their interiors, for example, nonpolar or sulfhydryl groups (Monahan et al. 1995, 1996). These reactive groups increase the attractive interactions between proteins that are adsorbed either onto the same or onto different droplets, thereby altering the susceptibility of emulsions to droplet flocculation and coalescence. At room temperature, whey protein-stabilized emulsions (pH 7) are stable to flocculation in the absence of added salt because of the relatively strong electrostatic repulsion between the droplets (Keowmaneechai and McClements 2002a; Kim et al. 2002a). On the other hand, they become unstable to flocculation when a sufficiently high level of salt (150 mM) is added to the continuous phase because this screens the electrostatic repulsion between the droplets (Figure 4.5). At

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Figure 4.5. Influence of thermal processing and NaCl concentration on βlactoglobulin protein-stabilized oil-in-water emulsions (pH 7). An increase in the mean particle diameter (d32 ) indicates increased flocculation.

relatively low temperatures (<65◦ C), droplet flocculation is due to surface denaturation of the globular whey proteins after adsorption (see above). When β-lactoglobulin-stabilized emulsions are heated above the thermal-denaturation temperature of the adsorbed globular proteins (Tm ∼ 70◦ C) in the presence of salt (150 mM NaCl), protein unfolding becomes much more extensive, which leads to an increase in the extent of droplet flocculation (Figure 4.5). Nevertheless, it is interesting to note that very little droplet flocculation is observed when a β-lactoglobulinstabilized emulsion is heated above the thermal-denaturation temperature in the absence of salt, and salt is then added after the emulsion has been cooled to room temperature. These results suggest that interactions between proteins adsorbed onto different droplets are favored when the droplets are in close proximity during heating (i.e., high salt), but that interactions between proteins adsorbed onto the same droplets are favored when droplets are not in close proximity during heating (i.e., low salt). Presumably in the latter case, the extensive molecular rearrangements and intradroplet protein–protein interactions that occur above Tm reduce the surface hydrophobicity and/or the number of exposed sulfhydryl groups at the droplet surfaces. This knowledge may provide a useful practical method of reducing the susceptibility of whey protein-stabilized emulsions to droplet flocculation during heat processing. The extent of droplet flocculation and the structure of the flocs formed when whey protein-stabilized emulsions (pH 7) were subjected

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to thermal processing (55–95◦ C; 0–2 h) has been found to depend on the holding temperature and time, as well as on the nonadsorbed protein concentration (Sliwinski et al. 2003a, b). The flocs formed at relatively short-holding times were progressively disrupted when the emulsions were held at elevated temperatures. The stability and rheology of whey protein-stabilized emulsions may therefore be manipulated by controlling the protein concentration and the heating conditions. Emulsions stabilized by proteins that do not undergo extensive heat-induced conformational changes (such as caseins), are usually less susceptible to droplet aggregation during heating (Dalgleish 1997). Mechanical Stresses Droplet coalescence occurs in oil-in-water emulsions when the thin film of material (the continuous phase separating the droplets and/or the interfacial layers coating the droplets) separating the lipid phase is ruptured and the fluids within the droplets (the dispersed phase) merge together. Under quiescent conditions, protein-stabilized emulsions are highly stable to droplet coalescence because the interfacial membrane formed by the proteins generates strong short-range repulsive forces and is resistant to rupture (Aken 2004). Nevertheless, there are a number of situations where droplet coalescence may be promoted due to the application of mechanical stresses, such as shearing, centrifugation, or homogenization (Aken 2004): Insufficient Emulsifier If there is insufficient emulsifier present in a system to completely cover all the oil–water interfaces present, then there will be gaps in the interfacial membranes surrounding the droplets. Coalescence could then occur if two gaps on different droplets came into close proximity. This type of coalescence is likely to be most important during homogenization where new surfaces are continually being created by the intense forces generated within a homogenizer. Film Stretching If a sufficiently large stress is applied parallel to an interface that is covered with an emulsifier, then some of the emulsifier molecules may be dragged along the interface, leaving some regions where there is an excess of emulsifier and other regions where there is a depletion

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of emulsifier. Coalescence could then occur if two emulsifier-depleted regions on different droplets came into close proximity during a droplet– droplet encounter. This process is likely to be important only if the adsorption of the emulsifier is relatively slow compared to the duration of the applied stresses and droplet encounter frequency, otherwise the emulsifier would have sufficient time to adsorb onto the droplet surfaces and cover the gaps. Film stretching is likely to be important in emulsions that are subjected to intense mechanical stresses, especially if the droplets are in close proximity, for example, in flocculated or concentrated emulsions. Film Tearing If a sufficiently large stress is applied parallel to an interface that comprises a highly cohesive layer of emulsifier molecules, then it may cause the interfacial membrane to buckle and tear, leaving exposed emulsifierdepleted patches that promote coalescence. This mechanism is likely to be important in systems where the interfacial membranes are highly cohesive (e.g., protein membranes with extensive cross-linking), particularly under high applied mechanical stresses, for example, shearing, homogenization, centrifugation. It is likely to be less important in dilute, nonflocculated, and quiescent emulsions because the forces generated in these systems are not strong enough to tear the interfacial membranes. Experiments have shown that emulsions stabilized by whey proteins that have undergone extensive covalent cross-linking at the oil–water interface are more susceptible to coalescence under shearing conditions than under quiescent conditions, which has been attributed to the film tearing mechanism mentioned above (Aken 2004). Homogenization One of the most important roles of emulsifiers is to facilitate the formation of small emulsion droplets during homogenization of the oil and aqueous phases. An effective emulsifier should rapidly adsorb onto the freshly formed droplet surfaces, reduce the interfacial tension by an appreciable amount to facilitate droplet disruption, and provide a protective coating that prevents the droplets from aggregating with their neighbors (Aoki et al. 2005; Walstra 1993, 2003). Protein emulsifiers differ in the rate at which they adsorb onto droplet surfaces during

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homogenization, in the minimum amount that is required to saturate the droplet surfaces, and in their ability to protect droplets against coalescence under different environmental conditions. For example, casein micelles adsorb more rapidly onto droplet surfaces than individual casein molecules during high-pressure valve homogenization, but more protein is required to saturate the droplet surfaces for casein micelles than individual casein molecules. The amount of total protein present also plays a major role in determining the stability of the droplets to aggregation during homogenization. It is convenient to divide the influence of emulsifier concentration on the droplet size into two regions: insufficient emulsifier and excess emulsifier (Tcholakova et al. 2002, 2003, 2005, 2006). Insufficient Emulsifier When the emulsifier concentration is limiting (i.e., there is insufficient emulsifier present to cover all the droplet surface area created by the homogenizer), then the droplet size is governed primarily by the emulsifier concentration, rather than by the energy input of the homogenizer. The surface load of the emulsifier remains relatively constant in this regime and is close to the value of the excess surface concentration of the emulsifier at saturation (sat ). Under these conditions the mean droplet size produced by homogenization is determined by the maximum amount of surface area that can be covered by the available emulsifier (Equation (4.4)): rmin =

3sat φ 3sat φ =  cS c S (1 − φ)

(4.4)

where sat is the excess surface concentration of the emulsifier at saturation (in kg m−2 ), φ is the disperse phase volume fraction, c S is the concentration of the emulsifier in the emulsion (in kg m−3 ), and cS is the concentration of the emulsifier in the continuous phase (in kg m−3 ). Different proteins have different sat values, which means that the size of the droplets produced using a given amount of protein depends on the protein type in this regime. Typically, β-lactoglobulin has a sat value of around 1–2 mg m−2 . Excess Emulsifier When the emulsifier concentration is in excess (i.e., there is more emulsifier present than is required to completely cover all the droplet surface

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area created by the homogenizer), then the droplet size is relatively independent of emulsifier concentration and depends primarily on the energy input of the homogenizer. Under these circumstances the mean droplet diameter that can be produced depends on the flow conditions prevalent in the homogenizer (e.g., laminar, turbulent, and/or cavitational) and the physicochemical properties of the component phases (e.g., interfacial tension and viscosities). If the emulsifier does not form multiple layers at the interface, then the surface load of the emulsifier will remain relatively constant in this regime ( ∼ sat ). Alternatively, if the emulsifier is capable of forming multiple layers at the interface, then the surface load may increase as the overall concentration of the emulsifier in the system is increased ( > sat ). This latter effect has been observed for globular proteins, such as whey proteins, where the surface load increases up to a certain level as the overall emulsifier concentration is increased. Whey proteins may undergo appreciable denaturation during the homogenization process, which has been attributed to surface denaturation of the proteins after adsorption onto the droplet surfaces rather than due to the high-pressure gradients generated within the homogenizer (Rampon et al. 2003). This kind of protein denaturation may have an appreciable influence on the subsequent stability and properties of emulsions, although little work has been done in this area. Chilling and Freezing There are many potential applications for oil-in-water emulsions that can be chilled or frozen during storage and then warmed or thawed prior to use, for example, refrigerated and frozen foods (Hartel 2001). Storage at reduced temperatures can be used to protect product quality against microbial growth, enzymatic reactions, and chemical degradation. Nevertheless, many oil-in-water emulsions are highly unstable when they are chilled and frozen, and rapidly breakdown after reheating. The design and manufacture of whey protein-stabilized emulsions with improved stability to cold storage therefore depends on understanding the basic physicochemical mechanisms that occur during chilling and freezing. A variety of different physicochemical processes may occur when a food emulsion is cooled, including fat crystallization, ice formation, freeze concentration, interfacial phase transitions, and biopolymer conformational changes (Hartel 2001; Walstra 2003).

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When oil-in-water emulsions are cooled to temperatures where the fat phase becomes partially crystallized (but the aqueous phase remains liquid) they become susceptible to partial coalescence, which promotes droplet aggregation and emulsion instability (see above). When oil-inwater emulsions are cooled to temperatures where the water crystallizes, there are a number of additional physicochemical processes that occur that can also promote emulsion instability (Aoki et al. 2005; Hartel 2001; Thanasukarn et al. 2004a, b; Vanapalli and Coupland, 2001; Vanapalli et al. 2002; Walstra 2003). First, when ice crystals form in the aqueous phase the oil droplets are forced into closer proximity. Second, there may be insufficient free water present to fully hydrate the protein molecules adsorbed onto the droplet surfaces, which can promote increased droplet–droplet interactions. Third, ice crystallization leads to an increase in the ionic strength of any freeze-concentrated nonfrozen aqueous phase surrounding the emulsion droplets, which may screen electrostatic repulsion between the protein-coated droplets thereby promoting flocculation. Fourth, it is possible that ice crystals formed during freezing may penetrate into the oil droplets and disrupt their interfacial protein layers, thus making them more prone to coalescence once they are thawed. Fifth, protein molecules may adsorb onto the surface of ice crystals, which would reduce the amount left to cover the emulsion droplets. Finally, globular proteins may lose their functionality when the temperature is decreased below a certain level, for example, cold denaturation. At present there is still a relatively poor understanding of the relative importance of these various mechanisms of emulsion instability in protein-stabilized emulsions. The influence of chilling and freezing on the stability of whey protein isolate (WPI) stabilized emulsions has been compared with that of casein and Tween 20 stabilized emulsions (Thanasukarn et al. 2004a, b). These studies have shown that dairy proteins (WPI and casein) provide better protection against droplet coalescence than small molecule surfactants when the oil phase is partially crystalline, and that they can only provide a limited degree of protection against droplet coalescence when both water and oil crystallization occur. This has been attributed to the ability of the proteins to form relatively thick interfacial layers around the droplets that are difficult to penetrate by fat crystals. The stability of emulsions to water crystallization can be improved by incorporating cryoprotectants (e.g., sucrose) or by creating multiple layers of proteins and polysaccharides around the oil droplets (Thanasukarn et al. 2006).

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Drying Protein-stabilized oil-in-water emulsions are often dried to form powders that can be used as food ingredients in other products (e.g., flavorings) or that can be hydrated by consumers prior to use (e.g., dairy creamers, soups) (Christensen et al. 2001; Faldt and Bergenstahl 1996a, b; Hogan et al. 2001a, b; Madene et al. 2006; Millqvist-Fureby 2003; Millqvist-Fureby et al. 2001; Park et al. 2005). The emulsion should maintain its desirable physicochemical and stability characteristics after the drying and hydration processes. A variety of different unit operations are utilized to dry food emulsion products, such as spray-drying and freeze-drying. Spray-drying may cause denaturation of globular whey proteins because of the relatively high temperatures involved or because of the generation of air–liquid interfaces in the system where whey proteins can adsorb and undergo surface denaturation. Recently, it has been shown that oil-in-water emulsions stabilized predominantly by caseins are more stable to spray-drying than those stabilized predominantly by whey proteins, which was attributed to denaturation of the globular whey proteins (Sliwinski et al. 2003a, b). Freeze-drying is often used in the preparation of specialized protein ingredients. During the freezing step the aqueous protein solution separates into ice crystals dispersed in a freezeconcentrated solution that contains free proteins and emulsion droplets (McClements 2002). During the drying step the water is removed in two stages: (i) the water in the ice crystals is removed by sublimation and (ii) the water in the freeze-concentrated solution is removed by evaporation. In the absence of cosolvents (such as sugars or polyols), proteins usually lose their functionality during drying processes, but in the presence of certain cosolvents protein functionality can be retained. At least three different physicochemical mechanisms have been proposed to account for the ability of cosolvents to enhance protein stability during dehydration processes (McClements 2002). First, cosolvents that are preferentially excluded from protein surfaces tend to favor folded over unfolded states of protein molecules, thereby retarding cold-, heat-, surface-, and pressure-denaturation processes. Second, some cosolvents are capable of forming hydrogen bonds with the surface of dried proteins, thereby inhibiting protein unfolding and aggregation by taking the place of water molecules. Third, some cosolvents are capable of forming a highly viscous “glass” phase around the protein molecules that

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retards protein degradation by decreasing the molecular mobility of the system. It is likely that all these mechanisms play some role in enhancing whey protein stability to dehydration, but the relative importance of each mechanism still needs to be established for particular systems. Influence of Aqueous Phase Composition The composition and properties of the aqueous phase surrounding whey protein-coated droplets in oil-in-water emulsions may vary widely depending on the nature of the food. For example, foods have different pH values (typically between 3 and 7), different ionic strengths (typically from 1 to 1000 mM), and may contain various other functional ingredients (typically, sugars, polyols, biopolymers, etc.). In this section, we examine some of the effects of aqueous phase composition on the properties of whey protein-stabilized emulsions. pH and Ionic Strength In practical applications proteins may have to exhibit their desirable functional properties in products that have a wide range of different pH values and ionic strengths (Aoki et al. 2005). The interfacial membranes formed by whey proteins are usually relatively thin (1 to 10 nm) and electrically charged (−80 to +80 mV); hence the major mechanism preventing droplet flocculation in protein-stabilized emulsions is electrostatic repulsion, rather than steric repulsion (McClements 2004, 2005a). Protein-stabilized emulsions are therefore particularly sensitive to pH and ionic strength effects. They tend to flocculate at pH values close to the isoelectric point of the adsorbed proteins (low surface charge density) and when the ionic strength exceeds a particular level (high electrostatic screening), because the electrostatic repulsion between the droplets is then no longer sufficiently strong to overcome the various attractive interactions, for example, van der Waals, hydrophobic, or depletion. Multivalent counter-ions are particularly efficient at promoting emulsion instability because they are more effective in screening electrostatic interactions (reducing the Debye screening length) and because they can bind to droplet surfaces thereby reducing the ζ-potential (Aoki et al. 2005; Keowmaneechai and McClements 2002a; Kulmyrzaev et al. 2000). The difference in the effectiveness of

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Figure 4.6. Influence of pH and salt concentration on the flocculation stability of whey protein-stabilized oil-in-water emulsions. An increase in the mean particle diameter (d32 ) indicates increased flocculation.

monovalent (K+ ) and divalent (Ca2+ ) counter-ions in promoting droplet flocculation in a whey protein-stabilized emulsion (pH 7) is highlighted in Figure 4.6. A variety of strategies have been developed to improve the stability of protein-stabilized emulsions to droplet flocculation induced by pH or ionic strength effects: r

Multivalent counter-ions, such as Ca2+ , Fe2+ , or Fe3+ , can be sequestered by binding to chelating agents (such as EDTA, citrates, phosphates) or by binding to biopolymers (such as casein macropeptide, phosvitin, feritin) (Diaz et al. 2003; Keowmaneechai and McClements 2002a, b, 2006; Klinkesorn et al. 2005). r Ionic surfactants that adsorb onto the surface of the oil droplets can be added to a protein-stabilized emulsion to change the pH dependence of the ζ -potential of the droplets, thereby changing the range of pH values that the emulsion is stable to flocculation (Kelley and McClements 2003b).

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r

Electrically charged biopolymers that adsorb onto the surface of oppositely charged droplets can be added to a protein-stabilized emulsion to change the pH dependence of the ζ-potential of the droplets, thereby changing the range of pH values that the emulsion is stable to flocculation (Dickinson 2003; Guzey 2004; McClements 2005b). r Insoluble colloidal forms of minerals can also be used that do not release the free multivalent cations into the aqueous phase, such as calcium carbonate. Sugars and Polyols The aqueous phase surrounding the oil droplets in many commercial emulsion-based products often contains a variety of different kinds of small polar molecules, such as sugars and polyols (McClements 2002). These relatively small molecules may influence the molecular and functional properties of the adsorbed whey proteins in oil-in-water emulsions in a variety of different ways, and therefore it is important to understand their influence on whey protein functionality in emulsions. The influence of a neutral cosolvent (sucrose) on the kinetics of droplet aggregation promoted by surface denaturation in βlactoglobulin-stabilized oil-in-water emulsions has recently been examined (Kim et al. 2003). When sucrose was added to the emulsions immediately after homogenization, the rate of particle growth decreased as the sucrose concentration was increased (0–40 wt%). On the other hand, when sucrose was added to the emulsions after a few hours of aging it actually promoted a greater extent of droplet flocculation. A number of physicochemical mechanisms were postulated to account for the observed effects of sucrose on the stability of the emulsions to droplet flocculation. First, sucrose increases the viscosity of the continuous phase, which should slow down the rate of droplet–droplet collisions. Second, sucrose alters the physicochemical properties of the aqueous solution surrounding the droplets (e.g., density, dielectric constant, refractive index, osmotic pressure), which may change the height of the repulsive energy barrier in the droplet–droplet interaction potential thereby altering the fraction of droplet collisions leading to aggregation. Third, sucrose puts the proteins under osmotic stress, which may slow down the kinetics of protein surface denaturation. Fourth, sucrose increases the attractive interactions between emulsion droplets through a short-range “molecular depletion interaction,” which may strengthen the bonds between flocculated droplets. More research is required to

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determine the relative importance of these and other mechanisms in specific emulsion systems. The influence of sucrose on the extent of droplet flocculation occurring in oil-in-water emulsions stabilized by a globular protein after thermal processing has also been examined (Kim et al. 2003). Sucrose (0–40 wt%) and NaCl (150 mM) were added to n-hexadecane oil-inwater emulsions stabilized by β-lactoglobulin (β-LG, pH 7.0) either before or after isothermal heat treatment (30–95◦ C for 20 min). When salt was added to emulsions before heat treatment, appreciable droplet flocculation was observed below the thermal-denaturation temperature of the adsorbed β-LG (Tm ∼ 70◦ C), and more extensive flocculation was observed above Tm . On the other hand, when salt was added to emulsions after heat treatment, appreciable droplet flocculation still occurred below Tm , but little flocculation was observed above Tm (see above). Addition of sucrose to the emulsions increased Tm , and either promoted or suppressed droplet flocculation depending on whether it was added before or after heat treatment. These results were interpreted in terms of the influence of sucrose on protein conformational stability, protein–protein interactions, and the physicochemical properties of aqueous solutions. The presence of high levels of sucrose increased the attraction between emulsion droplets once the protein molecules had unfolded, but it meant that the emulsions had to be heated to a higher temperature to promote protein unfolding. Surfactants Emulsions often contain small molecule surfactants, which may be either nonionic or ionic, for example, lecithin, Tweens, Spans, DATEM, and SLS (Aoki et al. 2005). These surfactants can alter the stability of protein-stabilized emulsions either directly or indirectly. Surfactants may directly influence protein functionality by binding to them, because this can lead to substantial changes in protein conformation and interactions (Kelley and McClements 2003a). Ionic surfactants may bind to protein molecules through a combination of electrostatic and hydrophobic interactions, whereas nonionic surfactants may bind to proteins through hydrophobic interactions. Once a surfactant has bound to a protein, it may either stabilize or destabilize the protein structure and/or it may either promote or oppose protein aggregation depending on

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surfactant type, surfactant concentration, and environmental conditions. These alterations in the molecular characteristics of globular proteins may lead to changes in their ability to absorb onto interfaces and to stabilize emulsions. It is therefore important to improve our understanding of the origin and nature of protein–surfactant interactions and of their influence on protein functionality. Small molecule surfactants may also influence protein functionality in a more indirect manner. Surfactants are surface-active molecules that normally compete with proteins for the available oil–water interface, and thus can displace some or all the proteins from the interface (Wilde 2000; Wilde et al. 2002; Woodward et al. 2004). The composition of an interface depends on the relative surface activity and concentration of the protein and surfactant molecules, as well as the time that each component is introduced into the system. Interfacial composition alters the magnitude, range, and sign of the interactions between droplets, as well as the rheology of the interface, and therefore it may alter the overall physicochemical properties and stability of emulsions. The competition between protein and surfactant molecules at interfaces may therefore play an important role in determining protein functionality in food emulsions. The application of new imaging techniques and computer simulations has provided a good understanding of the microstructure of interfaces containing a mixture of proteins and small molecule surfactants (Wilde et al. 2002). When a protein-coated interface is brought into contact with an aqueous solution containing surfactant a sequence of events occurs that depends on the surfactant concentration. At relatively low surfactant concentrations, surfactant molecules adsorb onto the interface and form small islands of surfactant located within the protein network. As the surfactant concentration is increased the size of the surfactant-rich regions expands, restricting the protein network to a smaller surface area. At relatively high surfactant concentrations, the protein region increases appreciably in thickness and eventually the protein molecules are completely displaced from the interface. The two-dimensional phase separation of the interface into protein-rich and surfactant-rich domains can clearly be observed using microscopy techniques and computer simulations (Wilde 2000; Wilde et al. 2002; Woodward et al. 2004). The structure of the domains formed depends on the strength of the attractive interactions between the adsorbed proteins. Proteins such as β-lactoglobulin, which have strong interfacial interactions, tend to form irregular-shaped domains, whereas those such as

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casein, which have weak interfacial interactions, tend to form circularshaped domains (Pugnaloni et al. 2004). Biopolymers Protein-stabilized oil-in-water emulsions may contain one or more types of biopolymers in the continuous phase. These biopolymers could be a fraction of the protein used as an emulsifier that did not adsorb onto the droplet surfaces because they were already saturated with protein, or they could be other functional ingredients, such as thickening agents or gelling agents. These biopolymers may interact with the adsorbed proteins, either directly or indirectly, and influence the stability of the emulsion through a variety of mechanisms (Dickinson 2003; McClements 2005b): Depletion Flocculation The presence of nonadsorbing biopolymers in the aqueous phase of a protein-stabilized emulsion causes an increase in the attractive force between the droplets due to an osmotic effect associated with the exclusion of the biopolymers from a narrow region surrounding each droplet. This attractive force increases as the concentration of biopolymers increases, until eventually it may become large enough to overcome the repulsive interactions between the droplets and cause them to flocculate. This type of droplet aggregation is usually referred to as depletion flocculation. A wide variety of different biopolymers have previously been shown to be capable of inducing depletion flocculation when added in sufficiently high concentrations, including polysaccharides (xanthan gum, gum arabic, modified starch, maltodextrin, pectin, and carrageenan) and proteins (whey and caseinate). The lowest concentration required to cause depletion flocculation is referred to as the critical flocculation concentration (CFC). The CFC decreases as the size of the emulsion droplets increases and the effective volume fraction of the biopolymers increases. The flocculation rate initially increases as the concentration of nonadsorbing biopolymers is increased because of the enhanced attraction between the droplets, i.e., a higher collision efficiency. However, once the concentration of biopolymers exceeds a certain concentration the flocculation rate often decreases because the viscosity of the continuous phase increases so much that the movement of the droplets is severely retarded, i.e., a lower collision frequency.

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Figure 4.7. Influence of pectin concentration on droplet flocculation in whey proteinstabilized oil-in-water emulsions (pH 7). An increase in creaming index indicates increased depletion flocculation.

In general, any change under solution conditions that alters the effective volume of the nonadsorbed biopolymers will influence the CFC, for example, temperature, ionic strength, solvent quality. An example of depletion flocculation due to nonadsorbed polysaccharide (pectin) in a whey protein-stabilized oil-in-water emulsion (pH 7.0) is shown in Figure 4.7. At this pH, the β-lactoglobulin-coated droplets and the pectin molecules are both negatively charged so that the pectin does not adsorb onto the droplet surfaces. Once a critical pectin concentration is exceeded the attractive depletion interaction between the droplets is sufficient to overcome the various repulsion interactions and droplet flocculation occurs. Bridging Flocculation Electrically charged biopolymers are capable of adsorbing onto the surfaces of oppositely charged emulsion droplets through electrostatic interactions (Dickinson 2003). At certain biopolymer and droplet concentrations, the biopolymers promote droplet flocculation due to charge neutralization and bridging effects (McClements 2005b; Mun et al. 2005). Since the driving force for this type of bridging flocculation is electrostatic in origin, the stability of the emulsions is strongly dependent on pH and ionic strength, as well as the electrical characteristics of the adsorbed proteins and the biopolymers. A large number of studies have examined the factors that influence bridging flocculation in protein-stabilized emulsions. Emulsions stabilized by β-lactoglobulin undergo bridging flocculation by charged polysaccharides (e.g., pectin,

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Figure 4.8. Influence of pectin concentration on droplet flocculation in whey proteinstabilized oil-in-water emulsions (pH 4). An increase in the mean particle diameter (d) indicates increased bridging flocculation.

carrageenan, dextran sulfate) at pH values where the polysaccharide and protein have opposite charges (Galazka et al. 1999; Gu et al. 2004a, b, 2005a, b; Guzey et al. 2004). Bridging flocculation is most pronounced at polysaccharide concentrations where the droplet surfaces are only partially covered. If there is a high concentration of polysaccharide present in the continuous phase, then the propensity for bridging flocculation to occur is reduced because there is sufficient biopolymer to completely cover the droplet surfaces (McClements 2005b). Nevertheless, if the free biopolymer concentration gets sufficiently high then depletion flocculation may occur. An example of bridging flocculation due to adsorbed polysaccharide (pectin) in a whey protein-stabilized oil-in-water emulsion (pH 4.0) is shown in Figure 4.8. At this pH, the β-lactoglobulin-coated droplets are positively charged and the pectin molecules are negatively charged so that the pectin adsorbs onto the droplet surfaces. At intermediate pectin concentrations there is insufficient polysaccharide to completely coat the droplet surfaces and so bridging flocculation occurs. At higher pectin concentrations, the droplets are completely coated by the pectin and so multilayer formation occurs and the emulsion is stable to flocculation (see below). Multiple-Layer Formation Electrically charged biopolymers are capable of adsorbing onto the surfaces of oppositely charged emulsion droplets (McClements 2005b). At sufficiently high biopolymer concentrations and low droplet concentrations, biopolymers tend to adsorb onto the surface of protein-coated

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droplets to form an interfacial layer consisting of protein + biopolymer. Recent experiments in our laboratory have shown that droplets coated by multilayered interfacial membranes often have improved stability to environmental stresses (such as thermal treatments, drying, freezing, and mechanical agitation) than those stabilized by single-layered membranes because of the increase in interfacial thickness and rheology (Galazka and Dickinson 1995; Gu et al. 2004a, b, 2005a, b, c; Guzey et al. 2004; Moreau et al. 2003). Conclusions There have been significant advances in our understanding of the factors that influence the functionality of whey proteins in oil-in-water emulsions. This chapter has mainly focused on advances in the understanding of the influence of solution composition and environmental stresses on whey protein functionality in emulsions. Improved knowledge of these effects should facilitate the rational and systematic design, and production of whey protein-stabilized emulsion-based products. References Aken, G.A.V. 2004. Coalescence mechanisms in protein stabilized emulsions. In Food Emulsions, edited by Friberg, Larsson and Sjoblom, pp. 299–326. New York: Marcel Dekker. Aoki, T., Decker, E.A., and McClements, D.J. 2005. Influence of environmental stresses on stability of O/W emulsions containing droplets stabilized by multilayered membranes produced by a layer-by-layer electrostatic deposition technique. Food Hydrocoll. 19:209–220. Chantrapornchai, W., Clydesdale, F., and McClements, D.J. 1999. Theoretical and experimental study of spectral reflectance and color of concentrated oil-in-water emulsions. J. Colloid Interface Sci. 218:324–330. Chantrapornchai, W., Clydesdale, F.M., and McClements, D.J. 2001. Influence of flocculation on optical properties of emulsions. J. Food Sci. 66:464–469. Christensen, K.L., Pedersen, G.P., and Kristensen, H.G. 2001. Preparation of redispersible dry emulsions by spray drying. Int. J. Pharm. 212:187–194. Dalgleish, D.G. 1997. Adsorption of protein and the stability of emulsions. Trends Food Sci Technol. 8:1–6. Diaz, M., Dunn, C.M., McClements, D.J., and Decker, E.A. 2003. Use of caseinophosphopeptides as natural antioxidants in oil-in-water emulsions. J. Agric. Food Chem. 51:2365–2370.

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Dickinson, E. 1992. An Introduction to Food Colloids. Oxford: Oxford Science. Dickinson, E. 2003. Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocoll. 17:25–39. Dickinson, E., Ritzoulis, C., Yamamoto, Y., and Logan, H. 1999. Ostwald ripening of protein-stabilized emulsions: Effect of transglutaminase crosslinking. Colloids Surf. B, Biointerfaces 12:139–146. Faldt, P., and Bergenstahl, B. 1996a. Spray-dried whey protein/lactose/soybean oil emulsions: 2. Redispersability, wettability and particle structure. Food Hydrocoll. 10:431–439. Faldt, P., and Bergenstahl, B. 1996b. Spray-dried whey protein/lactose/soybean oil emulsions: 1. Surface composition and particle structure. Food Hydrocoll. 10:421– 429. Galazka, V.B., and Dickinson, E. 1995. Surface-properties of protein layers adsorbed from mixtures of gelatin with various caseins. J. Texture Stud. 26:401–409. Galazka, V.B., Smith, D., Ledward, D.A., and Dickinson, E. 1999. Interactions of ovalbumin with sulphated polysaccharides: Effects of pH, ionic strength, heat and high pressure treatment. Food Hydrocoll. 13:81–88. Gu, Y.S., Decker, E.A., and McClements, D.J. 2004a. Influence of iota-carrageenan on droplet flocculation of beta-lactoglobulin-stabilized oil-in-water emulsions during thermal processing. Langmuir 20:9565–9570. Gu, Y.S., Decker, E.A., and McClements, D.J. 2004b. Influence of pH and iotacarrageenan concentration on physicochemical properties and stability of betalactoglobulin-stabilized oil-in-water emulsions. J. Agric. Food Chem. 52:3626– 3632. Gu, Y.S., Decker, A.E., and McClements, D.J. 2005a. Production and characterization of oil-in-water emulsions containing droplets stabilized by multilayer membranes consisting of beta-lactoglobulin, iota-carrageenan and gelatin. Langmuir 21:5752– 5760. Gu, Y.S., Decker, E.A., and McClements, D.J. 2005b. Influence of pH and carrageenan type on properties of beta-lactoglobulin stabilized oil-in-water emulsions. Food Hydrocoll. 19:83–91. Gu, Y.S., Regnier, L., and McClements, D.J. 2005c. Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by beta-lactoglobulin-iota-carrageenan membranes. J. Colloid Interface Sci. 286:551– 558. Guzey, D., Kim, H.J., and McClements, D.J. 2004. Factors influencing the production of o/w emulsions stabilized by beta-lactoglobulin-pectin membranes. Food Hydrocoll. 18:967–975. Hartel, R.W. 2001. Crystallization in Foods. Gaithersburg, MD: Aspen Publishers. Hogan, S.A., McNamee, B.F., O’Riordan, E.D., and O’Sullivan, M. 2001a. Microencapsulating properties of whey protein concentrate 75. J. Food Sci. 66:675–680. Hogan, S.A., McNamee, B.F., O’Riordan, E.D., and O’Sullivan, M. 2001b. Microencapsulating properties of sodium caseinate. J. Agric. Food Chem. 49:1934–1938. Kabalnov, A. 2001. Ostwald ripening and related phenomena. J. Dispersion Sci. Technol. 22:1–12.

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Kabalnov, A.S., and Shchukin, E.D. 1992. Ostwald ripening theory—applications to fluorocarbon emulsion stability. Adv. Colloid Interface Sci. 38:69–97. Kelley, D., and McClements, D.J. 2003a. Interactions of bovine serum albumin with ionic surfactants in aqueous solutions. Food Hydrocoll. 17:73–85. Kelley, D., and McClements, D.J. 2003b. Influence of sodium dodecyl sulfate on the thermal stability of bovine serum albumin stabilized oil-in-water emulsions. Food Hydrocoll. 17:87–93. Keowmaneechai, E., and McClements, D.J. 2002a. Effect of CaCl2 and KCl on physiochemical properties of model nutritional beverages based on whey protein stabilized oil-in-water emulsions. J. Food Sci. 67:665–671. Keowmaneechai, E., and McClements, D.J. 2002b. Influence of EDTA and citrate on physicochemical properties of whey protein-stabilized oil-in-water emulsions containing CaCl2. J. Agric. Food Chem. 50:7145–7153. Keowmaneechai, E., and McClements, D.J. 2006. Influence of EDTA and citrate on thermal stability of whey protein stabilized oil-in-water emulsions containing calcium chloride. Food Res. Int. 39:230–239. Kim, H.J., Decker, E.A., and McClements, D.J. 2002a. Impact of protein surface denaturation on droplet flocculation in hexadecane oil-in-water emulsions stabilized by beta-lactoglobulin. J. Agric. Food Chem. 50:7131–7137. Kim, H.J., Decker, E.A., and McClements, D.J. 2002b. Role of postadsorption conformation changes of beta-lactoglobulin on its ability to stabilize oil droplets against flocculation during heating at neutral pH. Langmuir 18:7577–7583. Kim, H.J., Decker, E.A., and McClements, D.J. 2003. Influence of sucrose on droplet flocculation in hexadecane oil-in-water emulsions stabilized by beta-lactoglobulin. J. Agric. Food Chem. 51:766–772. Kim, H.J., Decker, E.A., and McClements, D.J. 2005. Influence of protein concentration and order of addition on thermal stability of beta-lactoglobulin stabilized n-hexadecane oil-in-water emulsions at neutral pH. Langmuir 21:134–139. Klinkesorn, U., Sophanodora, P., Chinachoti, P., Decker, E.A., and McClements, D.J. 2005. Encapsulation of emulsified tuna oil in two-layered interfacial membranes prepared using electrostatic layer-by-layer deposition. Food Hydrocoll. 19:1044– 1053. Kulmyrzaev, A., Sivestre, M.P.C., and McClements, D.J. 2000. Rheology and stability of whey protein stabilized emulsions with high CaCl2 concentrations. Food Res. Int. 33:21–25. Madene, A., Jacquot, M., Scher, J., and Desobry, S. 2006. Flavour encapsulation and controlled release—a review. Int. J. Food Sci. Technol. 41:1–21. McClements, D.J. 2002. Modulation of globular protein functionality by weakly interacting cosolvents. Crit. Rev. Food Sci. Nutr. 42:417–471. McClements, D.J. 2004. Protein-stabilized emulsions. Curr. Opin. Colloid Interface Sci. 9:305–313. McClements, D.J. 2005a. Food Emulsions: Principles, Practice, and Techniques. Boca Raton, FL: CRC Press. McClements, D.J. 2005b. Theoretical analysis of factors affecting the formation and stability of multilayered colloidal dispersions. Langmuir 21:9777–9785.

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McClements, D.J., Chantrapornchai, W., and Clydesdale, F. 1998. Prediction of food emulsion color using light scattering theory. J. Food Sci. 63:935–939. Millqvist-Fureby, A. 2003. Characterisation of spray-dried emulsions with mixed fat phases. Colloids Surf. B 31:65–79. Millqvist-Fureby, A., Elofsson, U., and Bergenstahl, B. 2001. Surface composition of spray-dried milk protein-stabilised emulsions in relation to pre-heat treatment of proteins. Colloids Surf. B 21:47–58. Monahan, F.J., McClements, D.J., and German, J.B. 1995. Disulfide-mediated polymerization of whey proteins in whey-protein isolate-stabilized emulsions. Abstr. Pap. Am. Chem. Soc. 210:190-AGFD. Monahan, F.J., McClements, D.J., and German, J.B. 1996. Disulfide-mediated polymerization reactions and physical properties of heated WPI-stabilized emulsions. J. Food Sci. 61:504–509. Moreau, L., Kim, H.J., Decker, E.A., and McClements, D.J. 2003. Production and characterization of oil-in-water emulsions containing droplets stabilized by betalactoglobulin-pectin membranes. J. Agric. Food Chem. 51:6612–6617. Mun, S., Decker, E.A., and McClements, D.J. 2005. Influence of droplet characteristics on the formation of oil-in-water emulsions stabilized by surfactant-chitosan layers. Langmuir 21:6228–6234. Norde, W. 2003. Colloids and Interfaces in Life Sciences. New York: Marcel Dekker. Park, E.Y., Murakami, H., Mori, T., and Matsumura, Y. 2005. Effects of protein and peptide addition on lipid oxidation in powder model system. J. Agric. Food Chem. 53:137–144. Pugnaloni, L.A., Dickinson, E., Ettelaie, R., Mackie, A.R., and Wilde, P.J. 2004. Competitive adsorption of proteins and low-molecular-weight surfactants: Computer simulation and microscopic imaging. Adv. Colloid Interface Sci. 107:27–49. Rampon, V., Riaublanc, A., Anton, M., Genot, C., and McClements, D.J. 2003. Evidence that homogenization of BSA-stabilized hexadecane-in-water emulsions induces structure modification of the nonadsorbed protein. J. Agric. Food Chem. 51:5900– 5905. Rao, M.A. 1999. Rheology of Fluids and Semisolid Foods: Principles and Applications. New York: Springer. Sliwinski, E.L., Lavrijsen, B.W.M., Vollenbroek, J.M., Van Der Stege, H.J., van Boekel, M.A.J.S., and Wouters, J.T.M. 2003a. Effects of spray drying on physicochemical properties of milk protein-stabilised emulsions. Colloids Surf. B, Biointerfaces 31:219–229. Sliwinski, E.L., Roubos, P.J., Zoet, F.D., van Boekel, M.A.J.S., and Wouters, J.T.M. 2003b. Effects of heat on physicochemical properties of whey protein-stabilised emulsions. Colloids Surf. B, Biointerfaces 31:231–242. Swaisgood, H.E. 1996. Characteristics of milk, in Food Chemistry, 3rd edition, edited by O.R. Fennema, New York: Marcel Dekker, chapter 14. Tcholakova, S., Denkov, N.D., Ivanov, I.B., and Campbell, B. 2002. Coalescence in beta-lactoglobulin-stabilized emulsions: Effects of protein adsorption and drop size. Langmuir 18:8960–8971.

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Tcholakova, S., Denkov, N.D., Sidzhakova, D. and Campbell, B. 2006. Effect of thermal treatment, ionic strength, and pH on the short-term and long-term coalescence stability of beta-lactoglobulin emulsions. Langmuir 22:6042–6052. Tcholakova, S., Denkov, N.D., Sidzhakova, D., Ivanov, I.B., and Campbell, B. 2003. Interrelation between drop size and protein adsorption at various emulsification conditions. Langmuir 19:5640–5649. Tcholakova, S., Denkov, N.D., Sidzhakova, D., Ivanov, I.B., and Campbell, B. 2005. Effects of electrolyte concentration and pH on the coalescence stability of betalactoglobulin emulsions: Experiment and interpretation. Langmuir 21:4842–4855. Thanasukarn, P., Pongsawatmanit, R., and McClements, D.J. 2004a. Influence of emulsifier type on freeze-thaw stability of hydrogenated palm oil-in-water emulsions. Food Hydrocoll. 18:1033–1043. Thanasukarn, P., Pongsawatmanit, R., and McClements, D.J. 2004b. Impact of fat and water crystallization on the stability of hydrogenated palm oil-in-water emulsions stabilized by whey protein isolate. Colloids Surf. A 246:49–59. Thanasukarn, P., Pongsawatmanit, R., and McClements, D.J. 2006. Utilization of layerby-layer interfacial deposition technique to improve freeze-thaw stability of oil-inwater emulsions. Food Res. Int. 39:721–729. Vanapalli, S.A., and Coupland, J.N. 2001. Emulsions under shear–the formation and properties of partially coalesced lipid structures. Food Hydrocoll. 15:507–512. Vanapalli, S.A., Palanuwech, J., and Coupland, J.N. 2002. Stability of emulsions to dispersed phase crystallization: Effect of oil type, dispersed phase volume fraction, and cooling rate. Colloids Surf. 204:227–237. Walstra, P. 1993. Principles of emulsion formation. Chem. Eng. Sci. 48:333. Walstra, P. 2003. Physical Chemistry of Foods. New York: Marcel Dekker. Wilde, P.J. 2000. Interfaces: Their role in foam and emulsion behaviour. Curr. Opin. Colloid Interface Sci. 5:176–181. Wilde, P.J., Husband, F.A., Mackie, A.R., Ridout, M.J., and Morris, V.J. 2002. Proteinsurfactant interactions at interfaces, their influence on interfacial structure, and the stability of foams and emulsions. Abstr. Pap. Am. Chem. Soc. 223:U453. Woodward, N.C., Wilde, P.J., Mackie, A.R., Gunning, A.P., Gunning, P.A., and Morris, V.J. 2004. Effect of processing on the displacement of whey proteins: Applying the orogenic model to a real system. J. Agric. Food Chem. 52:1287–1292.

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Chapter 5 Whey Proteins: Functionality and Foaming under Acidic Conditions Stephanie T. Sullivan, Saad A. Khan, and Ahmed S. Eissa

Introduction The acidic environment is of particular significance for the behavior of whey proteins. At pH values around 5, that is, close to the isoelectric point, whey proteins tend to aggregate extensively and form large aggregates on the order of several microns. The extent of aggregation depends on the thermal treatment and other physical conditions. At lower pH values (3 or lower) on the other hand, whey protein molecules possess fine-stranded structure. The molecules have an overall positive charge and repel each other. Typically for acidic conditions (pH < 4), disulfide interchange reaction does not proceed upon thermal denaturation of protein molecules, causing aggregation to be weak and merely physical in nature. The absence of chemical bonds under these acidic conditions motivated several researchers to induce chemical cross-links enzymatically. Other researchers induced disulfide interchange reaction at neutral or alkaline conditions prior to acidification. The structural properties under acidic conditions affect the properties of whey proteins such as gelation, emulsification, and foaming. Methods for protein structure determination and prediction have improved significantly in recent years making it convenient to examine whey proteins under different conditions (Schmid 2001). In this regard, understanding the characteristics of whey proteins at acidic pH is highly desirable as such a condition increases product shelf-life and stability including those for pharmaceutical and food applications. Whey 99 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Figure 5.1. Typical percentages of whey protein in bovine milk.

proteins consist primarily of β-lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine serum albumin (BSA), immunoglobulin (Ig), and lactoferrin (LF). The composition of bovine whey protein is shown in Figure 5.1. From a functional standpoint, the behavior of β-LG is known to dominate the ingredients of whey proteins, especially those with higher protein content such as whey protein isolate (WPI) and some whey protein concentrate (WPC) (Table 5.1) (Chandan 2006; Eissa 2005; Kinsella and Whitehead 1989). Whey proteins are considered to have many beneficial health attributes including reducing the risk of heart disease and cancer as well as lowering blood pressure (Chandan and Shah 2006). Because of this, whey protein is considered to have significant nutritional value and is used in both human and animal food manufacture, including formulations for nutritional supplementation. For example, formulators of nutritious drinks such as yogurt use low pH acid-heat denatured whey Table 5.1.

Some characteristics of whey proteins.

Whey protein

No. of amino acid residues

Approx. mol. wt. (Da)

Isoelectric point

β-Lactoglobulin α-Lactalbumin Bovine serum albumin Immunoglobulins Proteose peptone

162 123 582 — —

18,277 14,175 69,000 150,000–1,000,000 4,000–40,000

5.2 5.1 4.8 4.6–6.0 3.7

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Functional properties of whey proteins.

Functionality

Properties of whey proteins

Water binding Solubility Viscosity Gelation Emulsification Foaming Flavor binding

Water binding capacity increase with denaturation of protein Soluble at all pH levels. If denatured, insoluble at pH 5 Low for native protein; higher if denatured Heat gelation at 70◦ C or higher and influenced by pH and salts Good except at pH 4–5, if heat denatured Good foam/overrun, β-lactoglobulin better than α-lactalbumin Retention varies with degree of denaturation

proteins due to their high solubility (Pearce 1992). This chapter aims to review the mechanisms of acidic whey protein on some functional properties of whey proteins such as gelation and foaming (Table 5.2).

Effect of Acidic Conditions on Protein Structure and Stability The structures of whey proteins including those at acidic pH are published in the Protein Data Bank (Berman et al. 2000), including that of the β-LG variant A at pH 2.6. It has been established that at acidic pH, β-LG exists as a monomer (Tromelin et al. 2006; Uhrinova et al. 2000). Figure 5.2 presents the amino acid sequence of the β-LG variant A, which comprises 162 amino acid residues, a relative molecular mass of 18,300 Da, and a diameter of 3.0 nm (Chandan 2006; Eissa 2005). Whey proteins undergo conformational changes upon heating, changing with conditions such as protein content, pH, salt concentration, and salt composition. Changing conformation results in the formation of a variety of gels “from fine-stranded particles to large particulate gel structures” (Roefs and Peppelman 2001), thus making whey proteins a

Figure 5.2. Amino acid sequence of β-LG variant A.

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β-Lactoglobulin characteristics with pH.

β-LG characteristics with pH

pH

Solubility limits Monomer structure with two disulfide bonds

2–10 <3.5 >8.0 7.5 >7.0 >7.0

Conformational changes and molecule expansion Increase in reactivity of thiol group Dissociation of the dimer

highly versatile food ingredient due to the ability to control its structure and texture by modulating different conditions. Whey proteins are considered globular proteins with a compact and ordered molecular structure that can (a) be organized in tight conformations (α-helices and β-sheets), (b) be stabilized by physical (hydrogen and hydrophobic) and covalent (primarily disulfide bridges) bonds, and upon adsorption, (c) retain secondary structure while undergoing changes in tertiary structure (van Aken 2004). These characteristics influence the ability of whey proteins to form gels, emulsions, and foams. Alizadeh-Pasdar et al. (2002) used three data-processing techniques—analysis of variance (ANOVA), principal component analysis (PCA), and principal component similarity (PCS) of Raman spectra to look at the effects of pH on whey protein and β-LG. The characteristic conformational structure of β-LG at different pH ranging from 5 to 9 is shown in Table 5.3. Alizadeh-Pasdar et al. (2002) showed that pH has a significant influence on the secondary structure of whey protein, due to “C–H” stretching vibrations of hydrocarbon side chains. Pearce (1992) summarized properties of whey proteins influenced by acidic pH as follows: (1) heat denatured WPC was more than 90% soluble at pH 2–4; (2) whole or partially concentrated whey formed little precipitate at pH 2.5–3.5 when heated for 15 min at 88–90◦ C; (3) when pH was changed to 4.5, centrifugation did collect precipitate; and (4) concentrated whey 2.7:1 had higher recoveries at pH 3.5, but lower solubility. Whey proteins stability is affected by physical conditions. The concentration of hydrogen ions (pH) is an important factor affecting protein function and stability (Alberts et al. 1994). The effect of pH on protein structure is primarily due to the change of the ionization form of the side groups of the different amino acids. It is understood that as pH

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decreases, the positive charge on whey protein molecules increases. Under native conditions, the overall charge of whey protein molecules is negative. Negativity tends to decrease with decreasing pH till the isoelectric point is reached (∼5.2 for β-LG and 5.1 for α-LA). A further decrease in pH makes whey protein molecules have an overall positive charge. Studies on protein stability found no correlation between overall charge of the protein and protein stability (Alexov 2004). However, for whey proteins, calorimetric studies show that whey proteins under acidic conditions are more difficult to denature and hence more stable compared to neutral conditions. Thermal denaturation of β-LG at pH 3.5 was found to occur at 78◦ C, which is about 16◦ higher than the denaturation temperature at pH 7 (Relkin 1994). The higher stability of β-LG at pH 3.5 as compared to neutral conditions is visible in Figure 5.3. In mixtures of whey proteins with other macromolecules, protein stability is affected by the interaction and conjugation with the other molecules (Kazmierski et al. 2003).

Gelation Properties of Whey Proteins The scientific term “gel” was first introduced by Thomas Graham (1869). Since then, many definitions have been introduced to describe the gel state. Gelation involves the construction of a continuous network of macroscopic dimensions (Ziegler and Foegeding 1990). Characterization of a sample to define whether it is a gel or not is yet a debatable matter. However, the widely accepted criterion is that a sample is a gel when its elastic modulus is higher than its viscous modulus and independent of frequency. Network formation requires denaturation of the protein molecules followed by intermolecular interactions (Wong 1989). Denaturation is required to expose the reactive functional groups of the protein. Interactions between the exposed functional groups involve chemical bonding and physical linkages. A schematic representation of the gelation mechanism is shown in Figure 5.4. Chemical bonding typically involves disulfide bond formation, which is crucial for protein aggregation (Roefs and de Kruif 1994; Sawyer 1968; Shimada and Cheftel 1989). Physical interactions include van der Waals interactions, hydrogen bonding, electrostatic, and hydrophobic interactions (Alting et al. 2003). A minimum protein concentration is required for gelation. This concentration is a function of temperature, pH, and ionic strength.

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Heat flow (mW)

Heating curve Sigmoidal baseline

(a)

Temperature ( C)

Heat flow (mW)

Heating curve Sigmoidal baseline

(b)

Temperature ( C)

Figure 5.3. Examples of β-lactoglobulin heating curves (upward curves) (4.95% concentration, ∼40 mg, 10◦ C/min from 20 to 120◦ C) and sigmoidal baseline (downward curves): a, pH 3.5; b, pH 7.

104

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Figure 5.4. Schematic representation of gelation of globular proteins. Solid short connections refer to chemical bonding (disulfide), while dotted connections refer to physical interactions (hydrogen bonding, hydrophobic interactions, and electrostatic interactions).

Gelation is usually the result of heat treatment at a temperature higher than the denaturation temperature. This is known as thermally induced—or heat induced—gelation. Several publications have extensively examined thermally induced gelation of whey proteins (Gezimati et al. 1996a, b; Kavanagh et al. 2000a, b, 2002; Tobitani and RossMurphy 1997). The gel types are usually categorized as fine (filamentous) or particulate according to the nature of the aggregates. When the primary aggregates are linear, fine gels are obtained: whereas particulate gels are obtained when the primary aggregates are close to the isoelectric point or when charges are screened via salt addition. A schematic representation of fine and particulate gel formation is shown in Figure 5.5. Particulate gel formation typically occurs from the aggregation of the primary molecules due to a lack of molecular repulsion, typically at high salt concentrations and pH values around 5. However, if the protein concentration is less than the minimum concentration needed for gelation, we obtain a soluble protein polymer aggregate (Britten and Giroux 2001). Cold gelation is another mechanism for gel formation. Cold gelation—also known as cold-set gelation—involves gelation of soluble proteins by changing their physical environment such as pH or ionic strength. This type of gelation is what typically occurs when milk is acidified into cheese curd (Bryant and McClements 1998). Whey protein gels are usually characterized by their rheological properties. Rheological characterization includes small strain and large

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Whey Processing, Functionality and Health Benefits Viscous solution

Particulate gel

Increasing protein concentration Unaggregated native protein

Particulate formation Viscous solution

Aggregated particulates Filamentous gel

No salt

Unaggregated native protein

Filament formation

Aggregated filaments

Figure 5.5. Development of particulate and fine (filamentous) gel structures. (Reproduced with kind permission of Bryant and McClements.)

strain properties. Rheological properties of whey protein gels have been extensively discussed in various publications (Britten and Giroux 2001; Chantrapornchai and McClements 2002a, b; Ikeda 2003; Lowe et al. 2003). Figures 5.6–5.7 show gel development in terms of rheological properties—upon heating or upon acidification by the addition of salt to polymerized whey proteins (Ikeda 2003; Resch et al. 2005). Large strain properties of gels are represented in terms of fracture stress and strain (Figure 5.8). Values of stress and strain at the fracture point reflect the strength and deformability of the gels, respectively. Mouthfeel and sensory properties are believed to be directly linked to the rheological properties measured by small and large strain instruments (Akhtar et al. 2006; Barrangou et al. 2006; Prinz et al. 2007; Truong et al. 2002). Examination of the whey protein gel microstructure is frequently done via electron microscopy (Boye et al. 2000; Hongsprabhas

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tan d

Complex modulus, G* (Pa)

1,000

−1

2,000

4,000

6,000

8,000

Figure 5.6. G* (solid square) and tan δ developments in 7% w/w β-LG aqueous solution containing 0.1 mol/dm3 NaCl at pH 7 during isothermal heating at 70◦ C. Values of tan δ were determined at 1 rad/s (open circle).31 10,000

Complex modulus (Pa)

1,000

0

2,000

4,000

6,000

8,000

10,000

12,000

Figure 5.7. Complex modulus development over time for 12% (w/v protein) βlactoglobulin solutions at pH 3.35 prepared with different acidulants and heated for 3 h at 80◦ C.

107

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108 3,000

Apparent stress (Pa)

2,500

2,000

1,500

1,000

500

Apparent strain

Figure 5.8. Apparent fracture stress and strain determined by the vane method for 12% (w/v protein) β-lactoglobulin gels prepared with different acidulants.

et al. 1999; Langton et al. 1996; Ngarize et al. 2005; Verheul and Roefs 1998). Fine gels appear under the electron microscope to be made of small and homogeneous building flocs, while particulate gels appear to be made of large lumps of protein flocs neighboring large interstitial voids. Figures 5.9 (Eissa et al. 2004) and 5.10 (Stading et al. 1995) reveal typical micrographs of whey protein gels under various conditions. Whey protein gels have high capability for imbibing water in their matrix. Gels can typically contain more than 95% of its content as water (Hinrichs et al. 2003). Water-holding capacity is closely related to the microstructure. Fine-stranded gels are characterized by higher waterholding capacity, while water is easily drained from the large pores of the particulate gels (Chantrapornchai and McClements 2002b). Various methodologies have been followed to assess the water-holding capacity. The formation of large aggregates is usually related to a significant decrease in the water-holding capacity (Barbut 1995a, b). Figure 5.11 shows the effect of salt content on the water-holding capacity of whey protein gels.

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5 KV

109

X10,000

(a)

5 KV

X10,000

(b)

Figure 5.9. SEM micrographs of acidic whey protein gels (pH 4) obtained under different conditions: (a) thermal gelation, (b) heated at pH 7 and then cold set by acidification to pH 4 using glucono-δ-lactone acid. All samples are shown at a magnification of 10,000×. Bars correspond to 1μm.

Gelation Properties of Whey Proteins under Acidic Conditions Gelation of whey proteins at low pH may be thermally induced (Lupano 1994; Lupano et al. 1992, 1996) or cold set (Alting 2003; Britten and Giroux 2001). Thermally induced gelation at pH close to isoelectric point creates opaque gels with particulate microstructures, compared to transparent gels far from the isoelectric point. Several studies on gelling properties of whey proteins under acidic conditions have been undertaken including that of Shimada and Cheftel (1988) and Stading et al. (1995). Water-holding capacity and protein solubility were found to decrease noticeably as pH approached the isoelectric point. Lupano et al. (1996) also reported that noncovalent bonds were responsible for the maintenance of the gel structure at pH 4.0, but in case of the gels prepared at pH 4.25, disulfide bonds were also involved in maintaining the structure of the gel.

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Figure 5.10. Transmission electron micrographs show the fine-stranded network structure of 12% β-lactoglobulin gels formed at (a) pH 3.5 and (b) 7.5. Scanning electron micrographs show the particulate network structure of 10% β-lactoglobulin gels at pH 5.3 formed at (c) 12 and (d) 0.1◦ C/min.

Noncovalent interactions are thought to dominate the gel structure at pH ≤4 with no disulfide bonds (Lupano et al. 1996). Fracture stress and strain are also affected profoundly by the absence of disulfide bonds (Errington and Foegeding 1998). Stading and Hermanson (1991)

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Figure 5.11. Transmittance at 660 nm (T660) and water-holding capacity (WHC) of heated (87◦ C, 45 min) protein dispersions (0.1% protein, w/v) or gels (10% protein, w/w) from WPC, as a function of pH. The bars show standard deviation.

studied β-LG gels at different pH values and found that gels formed at low pH (<4) were brittle and exhibited a low strain and stress at fracture, as opposed to those formed at high pH, which were rubber-like with high strain and stress at fracture. Errington and Foegeding (1998) studied whey protein gels and found that WPI gels (14%) formed at pH 3.0 and 2.5 were weak (fracture stress 17–19 kPa) and brittle (fracture strain 0.33–0.39) compared to fracture stress of 59–92 kPa, and fracture strain of 1.7–1.2 at pH 7 and 6.5. The fracture stress and strain for 10% whey protein gels at various pH values (Stading and Heranson 1991) are presented in Figure 5.12. The following summarizes various gelation studies under acidic conditions (Table 5.4). The textural properties of whey protein gels can be enhanced at low pH through acid induced, cold-set gelation, in which disulfide bonds are created in a neutral pH and then the pH value is dropped to the required acidic pH (Errington and Foegeding 1998). An alternative route is to induce enzymatic crosslinks to strengthen the protein network and

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Figure 5.12. Fracture properties of whey protein isolate gels. Gels formed by heating 10% (w/v) protein solutions, containing 100 mM NaCl, at 80◦ C for 30 min. Gel pH is indicated.

enhance the texture (Eissa 2005; Eissa et al. 2004, 2006; Eissa and Khan 2005, 2006.

Enhancing Textural Properties of Gels by Enzymatic Crosslinking Enzymatic crosslinking has been studied with emphasis on transglutaminase as the crosslinking enzyme (Eissa et al. 2004, 2006; Eissa and Khan 2005, 2006). Eissa et al. (2004, 2006) crosslinked whey proteins at pH 8, creating ε-(γ-glutamyl)lysine bonds. Upon acidification of the polymerized whey proteins using GDL under cold-set conditions, gels (pH ∼ 4) exhibited higher fracture/yield stress and strain compared to coldset gels with no enzyme and conventional heat-set gels at 80◦ C (Eissa et al. 2004). An alternative protocol for crosslinking has proved to be even more effective, in which a preheating step (80◦ C for 1 h) at neutral pH is conducted, followed by subsequent enzyme treatment and final

β-lactoglobulin

WPC

WPI

Yamul and Lupano (2005)

113

Eissa et al. (2004)

4

3.75 and 7

3.35

3.5–6

β-Lactoglobulin

Stading et al. (1995)

Thermal gelation (80◦ C for 1 h) and acid induced cold gelation

Thermal gelation (87◦ C for 45 min)

Thermal gelation (80◦ C for 3 h)

Thermal gelation at 80◦ C

Thermal gelation (80◦ C for 30 min)

Gelation methodology

Thermal gelation led to weak and brittle gels while acid induced gelation increased strength and deformability noticeably

The presence of wheat flour produced an increase in the cohesiveness of gels prepared at pH 3.75, whereas at neutral pH a decrease in cohesiveness was observed

Using different acidulants resulted in wide variation in gels’ rheological properties (fracture stress and strain)

A particulate network at pH (4–6) and a fine-stranded network below and above. Gels are weak and brittle at pH 3. Gels are strong and brittle at pH 5.5

Gels formed at pH 3.0 and 2.5 were weak and brittle due to the absence of disulfide bonds

Conclusions

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Resch et al. (2005)

2.5 and 3

WPI

Errington and Foegeding (1998)

pH

Form of whey

Selected findings of studies of whey protein gels under acidic conditions.

Author(s)

Table 5.4.

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cold-set gelation through acidulation with GDL (Eissa and Khan, 2005). The disulfide and ε-(γ-glutamyl)lysine bonds formed through heat and enzyme treatment, respectively, produced polymerized whey protein solutions. The molecular weight of these samples increased with protein concentration with the role of additional enzyme crosslinking manifesting as higher molecular weights only at higher protein concentration. This seemed to suggest that enzyme crosslinking was predominantly intramolecular at lower protein concentrations. During acidification in the absence of the enzyme, the gel modulus (G  ) showed a maximum as the pH approached the isolectric point and then decreased considerably afterward. However, the presence of ε-(γ-glutamyl)lysine bonds retarded the modulus decrease and made the network more stable against electrostatic repulsion. Moreover, the fracture strain and stress revealed a significant increase with enzyme treatment, but without affecting the microstructure in the length scales probed using confocal microscopy protein solutions.

Emulsifying Properties of Whey Proteins Whey proteins play an important role in fluid emulsion stability (Dalgeish 1996; McClements 1999). The surface activity of the proteins is due to their amphiphilic nature. This characteristic leads to their adsorption at the oil–water interface during emulsification. Similar to conventional surfactants, proteins help the breakage of oil drops during emulsification by lowering the interfacial tension between oil and water. Whey proteins also facilitate stabilization of the drops against coalescence. The adsorption of the proteins produces an emulsifying viscoelastic layer or film that prevents coalescence of the dispersed phase (Damodaran 1997). It is worth mentioning that emulsions are unstable thermodynamic mixtures that tend to reach minimum energy by phase separation after a certain period of time, known as the shelflife (Harper 1992). The shelf-life of a mixture emulsified by protein molecules is dependent on the ability of the protein molecules to resist the local mechanical stresses between the two immiscible phases. The ability of protein molecules to stabilize certain emulsions depends to a large extent on the structure, conformation, and flexibility of protein molecules (Das and Kinsella 1990). In the native state of whey proteins, hydrophobic residues are hidden inside the globular structure of the

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protein. Upon denaturation—either thermally or by changing solvent quality—hydrophobic residues are exposed to the surface leading to an improvement in protein functionality. A proper balance in hydrophilic– lipohphilic properties of the protein is required for proper emulsification properties. Controlling the extent of denaturation is a key factor in tailoring such properties (Figure 5.13; Tcholakova et al. 2006). Several methods are used to enhance whey protein’s emulsifying properties. These include hydrolysis, enzymatic treatment, or conjunction with polysaccharides (Einhorn-Stoll et al. 2005; Herceg et al. 2007; Mishra et al. 2001; Neirynck et al. 2004) or with casein (Parkinson and Dickinson 2007). Controlled hydrolysis (10–30% hydrolysis) of whey proteins enhanced emulsification properties. Hydrolysis of the residual fats in whey proteins is another means of enhancing the emulsification properties (Blecker et al. 1997). Crosslinking of whey proteins using oxireductase enzymes increases film elasticity and enhances emulsion stability. However, excessive crosslinking is sometimes detrimental to emulsion stability.

Emulsifying Properties of Whey Proteins under Acidic Conditions Protein-stabilized oil-in-water emulsions at pH values below the isoelectric point of the protein produce cationic emulsion droplets that decrease the oxidation of lipids by decreasing iron–lipid interactions. WPI-stabilized algal oil emulsions at pH 3.0 had good physical and oxidative stability after pasteurization (Djordjevic et al. 2004). The factors presumably associated with the superior emulsifying properties of whey proteins are large molecular weight and intact hydrophobic areas (Gauthier et al. 1993). Tcholakova et al. (2006) studied emulsification properties of whey proteins at pH range of 4–7. The effect of pH on the density of the adsorbed protein per unit area of oil droplets is shown in Figure 5.13 for emulsions stabilized by 0.02 wt% β-LG. For different electrolyte concentrations studied (10 and 150 mM) the adsorption passes through a maximum,  ≈ 2.5 mg/m2 —where  is the surface density of adsorbed protein—at pH around the isoelectric point. This behavior is attributed to the suppressed electrostatic repulsion between the protein molecules. The intact protein molecules were adsorbed as monolayer onto the surface. The effect of pH on  for emulsions stabilized by 0.1 wt% β-LG is shown in Figure 5.14 for

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Specific surface area (m2/g)

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d3,2 (μm)

(a)

(b)

Protein content (wt %)

Figure 5.13. (a) Dependence of emulsion droplet surface area on protein concentration for sodium caseinate (•); whey protein concentrate (); milk protein concentrate () and skim milk powder (); (b) dependence of mean emulsion droplet size (d3,2) on protein concentration for sodium caseinate (•); whey protein concentrate (); milk protein concentrate (), and skim milk powder (∇).

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0.1 wt% β-LG CEL = 150 mM 0.02 wt% β-LG CEL = 150 mM

0.02 wt% β-LG CEL = 10 mM

Figure 5.14. Protein adsorption surface density, , as a function of pH, for emulsions stabilized by 0.02 wt% β-lactoglobulin at CEL (concentration of electrolyte) = 10 mM or 150 mM, and by 0.1 wt% β-LG at CEL = 150 mM.

electrolyte concentration of 150 mM showing similar dependences of  on pH. A deep minimum in emulsion stability was observed at pH 5.0 (Figure 5.15) showing low coalescence stability at pH 5.0. The low stability at pH 5.0 was attributed to the different protein conformation and structure formed from charged protein molecules (away from the isoelectric point). The conformation under such a condition is relatively rigid, behaving as a fragile, easy-to-break film, allowing drop–drop coalescence upon small mechanical disturbances. Polymerization of whey protein via thermal treatment may enhance emulsion stability due to steric hindrance of the resulting polymer. However, Tcholakova et al. (2006) found no change in emulsion stability after the thermal treatment of β-LG at pH 5. This was due to the conformational properties of β-LG. Close to the isoelectric point the reactive sulfhydryl and disulfide groups remain hidden in the interior of the molecules, impeding formation of the protein polymer. Data shown in Figure 5.16 demonstrate this.

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Critical osmotic pressure (kPa)

13:59

CEL = 10 mM

CEL = 150 mM

Figure 5.15. Critical osmotic pressure for coalescence as a function of pH, in emulsions stabilized by 0.02 wt% β-lactoglobulin, at two different electrolyte concentrations, CEL = 10 mM and 150 mM.

Whey Proteins: Making the Connection between Emulsion and Foam Formation Theoretical understanding and analysis of emulsion and foam properties are very similar. However, as Walstra notes, the magnitudes of emulsion and foam properties are different (Walstra 1987). Table 5.5 lists the magnitudes of some of the commonly used properties. Some researchers have evaluated both emulsion and foaming properties simultaneously. Kresic et al. (2006) studied the impact of highpressure treatments on a variety of properties for both WPC and WPI. The treatments improved the foaming ability of WPI, but decreased that of WPC; still foams of both had improved stability. This group later expanded their analysis of whey protein emulsion and foaming properties with the addition of carbohydrates (Herceg et al. 2007). The addition of glucose and sucrose enhanced foam stability and expansion as well as the maximum foam stability of WPC, WPI, or β-LG

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Critical osmotic pressure (kPa)

0.1 wt% β-LG CEL = 150 mM

Nonheated

Figure 5.16. Critical osmotic pressure as a function of pH for non-heated and heated at 85◦ C emulsions, stabilized by 0.1 wt% β-LG at CEL = 150 mM.

Table 5.5.

Emulsion and foam property magnitudes.

Property

Emulsion value

Foam value

Particle diameter (m)

2 × 10−7 –10−5

10−4 –3 × 10−3

0.01–0.8

0.5–0.97

Density difference (kg/m )

10–100

1000

Compressibility of dispersed phase (N−1 m2 )

5 × 10−10

10−5

Interfacial tension (N m−1 )

10−3 –10−2

0.03–0.05

Laplace pressure (N m−2 )

104

102

Solubility of dispersed phase in continuous phase (vol%)

0 (oil-in-water; 0.15 (water-in-oil)

2.2

Particle volume fraction 3

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solutions; however, addition of starch or inulin showed different trends. Emulsion activity and stability index depended on the purity of the whey protein, with highest values for the most pure β-LG followed by WPI, and then WPC. When carbohydrates were added, whether or not the emulsion characteristics improved depended on which carbohydrate was added. When glucose or sucrose was added, emulsion behavior improved. When starch or inulin was added, emulsion activity and stability decreased (Herceg et al. 2007). Aqueous solution pH ranged from 5.99 to 7.10. So the opportunity to measure and compare these studied foam and emulsion characteristics at acidic pH exists. Chevalier et al. (2001) evaluated the solubility, heat stability, emulsifying, and foaming of β-LG variant A in the native and heated state, as well as with the addition by the Maillard reaction of various sugars (glycated β-LG). For solubility and heat stability studies, the pH was varied from acidic to neutral, for emulsifying analysis pH was maintained at either 5 or 7, and foaming property determination pH was kept at 7 only. Emulsifying properties were determined by measuring oil droplet size distribution mean diameter, and for all systems evaluated, this mean diameter was higher at pH 5. As noted earlier, the ability of whey protein to form an emulsion is related to its solubility; Kinsella and Whitehead (1989) and Chevalier et al. (2001) demonstrated the changes in solubility with pH. Therefore, “such effects close to pH value near the pI of β-LG could be at the origin of the decrease of emulsion observed in the (mean diameter) values.” The foaming studies measured a variety of foaming properties based on liquid drainage, including the foam density and a foam stability index. Chevalier et al. (2001) concluded that systems with moderate glycation achieved best foam properties at the pH studied.

Foaming Properties of Whey Proteins and Effect of pH In the development of milk products, foaming can be desirable (i.e., whipping of cream, achieving desirable texture of yogurt and frozen dessert products) or undesirable (i.e., handling skim milk) (Chandan 2006). Foaming characteristics of whey proteins have been widely studied, but few have focused on whey protein at acidic pH (Bals and Kulozik 2003a, b; Davis et al. 2004, 2005; Davis and Foegeding 2004; Dickinson 2006; Ekici et al. 2005; Firebaugh and Daubert 2005; Foegeding et al. 2006; Havn et al. 2006; Herceg et al. 2007; Ibanoglu and Karatas 2001;

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Kresic et al. 2006; Luck et al. 2002; Martin-Diana et al. 2006; Pernell et al. 2000; Relkin et al. 2006; Richert 1979; Tosi et al. 2007; Van Der Ven et al. 2002; Zhang et al. 2004). Roth et al. (2005) studied the effect of pH on β-LG films, measuring the viscosity and the time-independent interfacial shear elasticity, G, as a function of the age of the film. For both, the viscosity and G were lowest at a pH of 2.0, while the highest values were at a pH of 5.6—the isoelectric point. This agrees with the findings that β-LG moves from a dimeric to monomeric state as pH moves in either direction from the isoelectric point. Bals and Kulozik (2003b) devised a new membrane foaming method and studied WPI foaming properties at pH 4.6, demonstrating that with heating and thus increasing β-LG denaturation, solution viscosity, foam texture coarseness, and bubble size increase. However, as expected, bubble development was more difficult in solutions with higher viscosity. The methods used included image analysis of the foams using a reversed phase light microscope. Foam capacity, rigidity, stability, and bubble size were also measured. Overall, they concluded that heat treatment enabled production of “good foaming properties and high foam stability.” They described the details of this new foaming method elsewhere (Bals and Kulozik 2003a). Interaction of whey proteins with other compounds is of interest for product development, such as binding with flavor compounds. β-LG has been studied more than other whey proteins for its binding properties and stable nature, although it exists in many different oligomeric states that vary with pH as well as temperature and concentration. Tromelin et al. (2006) discuss these structural changes as well as the effect of pH on whey proteins foaming properties. They cite one study that concluded that pH 5 maximized foaming stability that increased with heat treatment. At other pH of 4 and 7, heat treatment had no effect. Adding certain flavor compounds to β-LG seemed to enhance its foaming properties. On the processing side, Ekici et al. (2005) studied foam fractionation by varying solution pH. The foam fractionation process at acidic pH between 2 and 3 enriched almost all whey proteins from solution into the foam. However, this was positively assisted by the addition of the surfactant sodium dodecyl Sulfate (SDS). This result agrees with that of Townsend and Nakai (1983) that the foaming capacity increases with an increase in protein hydrophobicity, which in turn is influenced

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by solution pH. Using the method of foam fractionation that was first used approximately 100 years ago, whey proteins were effectively separated from dilute whey solutions to evaluate optimal process conditions. Eissa and Khan et al. (2005) have evaluated hydrophobic interactions in whey proteins influenced by the addition of dithiothreitol, enzyme as well as SDS at pH 6.9. SDS addition to samples containing no enzyme dropped the viscosity drastically, revealing the presence of dominant hydrophobic interactions in the absence of enzyme (Eissa and Khan 2006). This agrees with the improved foaming capacity with increased protein hydrophobicity obtained via addition of SDS. In addition, one would expect that foaming capacity would also increase with a decrease in the solution viscosity. Foegeding et al. (2006) have evaluated whey proteins foaming at various pH values. It is noted that at pH 3.0, reduction in foam yield stress and stability is caused by the adsorbed WPI layer’s weaker dilatational elasticity. One study of foaming properties of hydrolyzed β-LG did not control pH (Davis et al. 2005). However, another varied WPI and β-LG pH from 3.0 to 7.0 (Davis et al. 2004). Surface tensions of WPI and β-LG solutions were higher at pH 3 and 7 than at 5, which is near the isoelectric point. Interfacial rheological measurements indicate that WPI at pH 7 can form a much stronger viscoelastic network than at pH 3.0. Davis et al. (2004) found a positive correlation between foam yield stress and dilatational elasticity. In spite of initial indications that foam stability may be reduced at pH 3 compared to 7, studies comparing the WPI and β-LG to that of glycated β-LG at acidic pH still show variability in the dilatational interfacial values of products utilizing whey protein at acidic pH. Foaming and Whey According to Dickinson (1992), food foaming is primarily done in three ways: whipping, bubble formation at an orifice, and bubble generation in situ. Whipping entraps air in a viscous liquid, but control over how much air is entrapped is poor. Laboratories use the latter ways to better control the foaming process. Dickinson explains the relationship of the forces acting on a bubble emerging from an orifice as a balance of buoyancy and surface tension so that the bubble radius can be predicted. The third bubble formation method is by heterogeneous nucleation. Properties that influence bubble size in the latter two cases include the surface

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tension and density of the solution. Dickinson (1992) identifies the key performance factor in foaming of whey and other proteins as the solubility, and pH influences this solubility. Dickinson (1992) states that the relationship between foaming properties and the soluble protein concentration is stronger than their relationship to pH, and that at a molecular level, those proteins that foam best have both sufficient hydrophobicity and backbone flexibility. Foam Stability and Whey Protein According to Damodaran (2005), factors impacting foam stability include disjoining pressure, surfactant film viscoelasticity, and interfacial tension. Proteins like β-LG behave like surfactants due to their amphiphilic nature which are also enhanced by mixing with other surfactants. Damodaran also notes that macroscopic processes such as liquid drainage and gas disproportionation control foam stability. The following are noted by Damodaran as aspects to be considered in analyzing foam stability: r r r r r

Gas bubble volume fraction Bubble shape Disjoining pressure Interfacial tension Liquid drainage—rate of film drainage V = 2h 3 (P)/3μR 2 where h is the lamella film thickness, μ is the dynamic viscosity, P is the difference between the capillary hydrostatic pressure and the disjoining pressure between lamella film interfaces, and R is the bubble radius. r Surfactant film physicochemical properties Viscoelasticity—controls small bubble rate of shrinkage and collapse Ostwald ripening (gas disproportionation)–gas diffusion from small to large bubbles Rate of surfactant (protein) desorption: slow for proteins → as bubble shrinks increases surface dilatational modulus of adsorbed layer which retards disproportionation rate. Marangoni effect−influences drainage Recently, an expression for foam drainage has been derived mathematically by applying Darcy’s law, resulting in a foam drainage equation as

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follows (Stone et al. 2003):   1/2   ρg γ δε k (ε) ∂ε ∇ε = 0 +∇ • εk (ε) − ∇ • ∂t μ 2μL (ε)1/2

(5.1)

where ε is the liquid volume fraction, ρ is the liquid density, k is the foam permeability, L is the channel length, δ is a system-determined geometrical constant, and γ is the interfacial tension. Stone et al. (2003) challenge that quantitative predictions using the foam drainage equation require an understanding of an individual bubble behavior and plateau borders (drainage channels), and thus permeability. Schmitt et al. (2005) calculated a film gas permeability, K , in diminishing bubble experiments of the whey protein β-LG as well as mixtures of β-LG, and acacia gum at pH of 4.2. Schmitt et al. (2005) also measured foam properties of dispersions of these two systems by sparging gas through a glass frit. By this method, they controlled the porosity and thus gas flow, forming air bubbles 10–16 μm in diameter. Studies on proteins and complexes of proteins with surfactants, lipids, or polysaccharides and their surface activity are ongoing, but much remains to be understood in terms of the ability of proteins to form emulsions and foams. Dickinson et al. (2002) studied four proteins including WPI and pure β-LG at pH 7.0 using a bubble apparatus and did not find evidence of foam stabilization for any of the proteins evaluated. But utilizing the experimental data gathered, they performed a theoretical analysis based on gas disproportionation, yielding a predictive model for the variation of bubble radius versus time as follows (Equation (5.2)): t = τ

ri

r2 dr α(r/L)

(5.2)

r (t)

where α is a correction function of r/L, with r the bubble radius and L the distance of the edge of the bubble away from the air–liquid interface, and the time constant τ = P0 /2Dγ SRT with P0 the pressure in gas above liquid, S the solubility constant of the gas in the liquid, γ the surface tension, D the diffusion coefficient of the gas in the liquid phase, R the ideal gas constant, and T the temperature. The experimental data agreed very well with this model for the WPI. Some correction was required for β-LG and another protein, but they concluded that assuming constant

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surface tension was not ideal, as one would expect surface tension to change with time and decreasing bubble size (Dickinson et al. 2002). This group later developed a new apparatus to study bubble stability with additional results for these proteins and found good agreement with this model. They also examined the effects of adding sugars and gelatin on bubble stability (Murray et al. 2005). Since variation of whey protein solubility with pH has been evaluated (Pelegrine and Gasparetto 2005), performing similar experiments with whey protein at acidic pH and comparing the data to these models is possible. Ibanoglu and Karatas (2001) investigated the effects of high pressure processing on foam stability of WPI at pH ranging from 5 to 7. Similar to other studies, the pH closer to the isoelectric point had diminished foaming properties, and this was attributed to increased aggregation at this pH. Foam Capacity Proteins exhibit a direct correlation between foaming capacity and the average hydrophobicity of proteins. Two measures of protein hydrophobicity have been presented in the literature. First, Bigelow calculated an average protein hydrophobicity from amino acid residue hydrophobicities measured thermodynamically and weighted by protein amino acid composition. Dickinson discusses data correlating foaming capacity and Bigelow’s average hydrophobicity and notes that due to the data scatter, other molecular factors must be at play. Dickinson (1992) also mentions Nakai’s surface hydrophobicity and that although data correlates well for emulsion activity, foaming capacities do not, due to slower formation process and opportunity for the protein’s hydrophobic regions to be more readily exposed at the air–water interface (Dickinson 2006).

Conclusion The utilities of whey protein are numerous, including formation of gels, emulsions, and foams. These and other functionalities of whey proteins at acidic pH warrant further investigation. For example, earlier discussions of whey protein foaming and the impact of pH indicate that being able to manipulate pH may improve foaming. However, the effect of pH appears extremely complex and dependent upon several other variables.

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Many studies to date have examined whey protein functionalities as well as the influence of pH. Additional understanding of the influence of pH on whey protein characteristics is required for better control and enhancement of whey protein product development and manufacturing processes.

References Akhtar, M., Murray, B.S., and Dickinson, E. 2006. Perception of creaminess of model oil-in-water dairy emulsions: Influence of the shear-thinning nature of a viscositycontrolling hydrocolloid. Food Hydrocoll. 20:839–847. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. 1994. Molecular Biology of the Cell. New York: Garland Publishing. Alexov, E. 2004. Numerical calculations of the pH of maximal protein stability. The effect of the sequence composition and three-dimensional structure. Eur. J. Biochem. 271:173–185. Alizadeh-Pasdar, N., Nakai, S., and Li-Chan, E.C.Y. 2002. Principal component similarity analysis of Raman spectra to study the effects of pH, heating, and kappacarrageenan on whey protein structure. J. Agric. Food Chem. 50:6042–6052. Alting, A.C., Hamer, R.J., de Kruif, C.G., de Jongh, H.H.J., Simons, J.W.F.A., and Visschers, R.W. 2003. Physical and chemical interactions in pH-induced aggregation and gelation of whey proteins. In Food Colloids, Biopolymers and Materials. Cambridge, UK: Royal Society of Chemistry. Alting, A.C., de Jongh, H.J.J., Visschers, R.W., and Simons, J.F.A. 2003. Physical and chemical interactions in pH-induced aggregation and gelation of whey proteins. Spec. Publ. R. Soc. Chem. 284:49–57. Bals, A., and Kulozik, U. 2003a. The influence of the pore size, the foaming temperature and the viscosity of the continuous phase on the properties of foams produced by membrane foaming. J. Membr. Sci. 220:5–11. Bals, A., and Kulozik, U. 2003b. Effect of pre-heating on the foaming properties of whey protein isolate using a membrane foaming apparatus. Int. Dairy J. 13:903–908. Barbut, S. 1995a. Effect of sodium level on the microstructure and texture of whey protein isolate gels. Food Res. Int. 28:437–443. Barbut, S. 1995b. Effects of calcium level on the structure of pre-heated whey protein isolate gels. Lebensmittel-Wissenschaft und-Technologie 28:598–603. Barrangou, L.M., Drake, M., Daubert, C.R. and Foegeding, E.A. 2006. Textural properties of agarose gels: II. Relationships between rheological properties and sensory texture. Food Hydrocoll. 20:196–203. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., and Bourne, P.E. 2000. The Protein Data Bank. Nucleic Acids Res. 28:235–242.

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Blecker, C., Paquot, M., Lamberti, I., Sensidoni, A., Lognay, G., and Deroanne, C. 1997. Improved emulsifying and foaming of whey proteins after enzymic fat hydrolysis. J. Food Sci. 62:48–52. Boye, J.I., Kalab, M., Alli, I., and Ma, C.Y. 2000. Microstructural properties of heatset whey protein gels: Effect of pH. Lebensmittel-Wissenschaft und-Technologie 33:165–172. Britten, M., and Giroux, H.J. 2001. Acid-induced gelation of whey protein polymers: Effects of pH and calcium concentration during polymerization. Food Hydrocoll. 15:609–617. Bryant, C.M., and McClements, D.J. 1998. Molecular basis of protein functionality with special consideration of cold-set gels derived from heat-denatured whey. Trends Food Sci. Technol. 9:143–151. Chandan, R.C., and Shah, N.P. 2006. Manufacturing Yogurt and Fermented Milks, edited by R.C. Chandan, pp. 311–325. Ames, IA: Blackwell Publishing. Chandan, R.C. 2006. Manufacturing Yogurt and Fermented Milks, edited by R.C. Chandan, pp. 17–39. Ames, IA: Blackwell Publishing. Chantrapornchai, W., and McClements, D.J. 2002a. Influence of glycerol on optical properties and large-strain rheology of heat-induced whey protein isolate gels. Food Hydrocoll. 16:461–466. Chantrapornchai, W., and McClements, D.J. 2002b. Influence of NaCl on optical properties, large-strain rheology and water holding capacity of heat-induced whey protein isolate gels. Food Hydrocoll. 16:467–476. Chevalier, F., Chobert, J., Popineau, Y., Nicolas, M.G., and Haertle, T. 2001. Improvement of functional properties of [beta]-lactoglobulin glycated through the Maillard reaction is related to the nature of the sugar. Int. Dairy J. 11:145– 152. Dalgeish, D.G. 1996. Conformations and structures of milk proteins adsorbed to oil– water interfaces. Food Res. Int. 29:541–547. Damodaran, S. 1997. Food Proteins and Their Applications, edited by S. Damodaran and A. Paraf, pp. 57–110. New York: Marcel Dekker. Damodaran, S. 2005. Protein stabilization of emulsions and foams. J. Food Sci. 70:R54–R66. Das, K.P., and Kinsella, J.E. 1990. Stability of food emulsions: Physicochemical role of protein and non-protein emulsfiers. Adv. Food Nutr. Res. 34:201. Davis, J.P., and Foegeding, E.A. 2004. Foaming and interfacial properties of polymerized whey protein isolate. J. Food Sci. 69:C404–C410. Davis, J.P., Doucet, D., and Foegeding, E.A. 2005. Foaming and interfacial properties of hydrolyzed [beta]-lactoglobulin. J. Colloid Interface Sci. 288:412–422. Davis, J.P., Foegeding, E.A., and Hansen, F.K. 2004. Electrostatic effects on the yield stress of whey protein isolate foams. Colloids Surf. B 34:13–23. Dickinson, E. 1992. An Introduction to Food Colloids, p. 207. Oxford: Oxford University Press. Dickinson, E. 2006. Structure formation in casein-based gels, foams, and emulsions. Colloids Surf. A 288:3–11.

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Dickinson, E., Ettelaie, R., Murray, B.S., and Du, Z. 2002. Kinetics of disproportionation of air bubbles beneath a planar air–water interface stabilized by food proteins. J. Colloid Interface Sci. 252:202–213. Djordjevic, D., McClements, D.J., and Decker, E.A. 2004. Oxidative stability of whey protein-stabilized oil-in-water emulsions at pH 3: Part B. Potential omega-3 fatty acid delivery systems . J. Food Sci. 69:C356–C362. Einhorn-Stoll, U., Ulbrich, M., Sever, S., and Kunzek, H. 2005. Formation of milk protein–pectin conjugates with improved emulsifying properties by controlled dry heating. Food Hydrocoll. 19:329–340. Eissa, A.S. 2005. Enzymatic modification of whey protein gels at low pH. Ph.D. Thesis, North Carolina State University, Raleigh, NC. Eissa, A.S., and Khan, S.A. 2005. Acid induced gelation of preheated, enzymatically modified whey proteins. J. Agric. Food Chem. 53:5010–5017. Eissa, A.S., and Khan, S.A. 2006. Modulation of hydrophobic interactions in denatured whey proteins by transglutaminase enzyme. Food Hydrocoll. (Special issue: WCFS Food Summit) 20:543–547. Eissa, A.S., Bisram, S., and Khan, S.A. 2004. Polymerization and gelation of whey protein isolates at low pH using transglutaminase enzyme. J. Agric. Food Chem. 52:4456–4464. Eissa, A.S., Puhl, C.F., Kadla, J., and Khan, S.A. 2006. Enzymatic crosslinking of β-lactoglobulin: Conformation properties using FTIR spectroscopy. Biomacromolecules 7:1707–1713. Ekici, P., Backleh-Sohrt, M., and Parlar, H. 2005. High efficiency enrichment of total and single whey proteins by pH controlled foam fractionation. Int. J. Food Sci. Nutr. 56:223–229. Errington, A.D., and Foegeding, E.A. 1998. Factors determining fracture stress and strain of fine-stranded whey protein gels. J. Agric. Food Chem. 46:2963–2967. Firebaugh, J.D., and Daubert, C.R. 2005. Emulsifying and foaming properties of a derivatized whey protein ingredient. Int. J. Food Prop. 8:243–253. Foegeding, E.A., Luck, P.J., and Davis, J.P. 2006. Factors determining the physical properties of protein foams. Food Hydrocoll. 20:284–292. Gauthier, S.F., Paquin, P., Pouliot, Y., and Turgeon, S. 1993. Surface activity and related functional properties of peptides obtained from whey proteins. J. Dairy Sci. 76:321– 328. Gezimati, J., Singh, H., and Creamer, L.K. 1996a. Aggregation and gelation of bovine β-lactoglobulin, α-lactalbumin, and serum albumin. ACS Symp. Ser. 650:113–123. Gezimati, J., Singh, H., and Creamer, L.K. 1996b. Heat-induced interactions and gelation of mixtures of bovine β-lactoglobulin and serum albumin. J. Agric. Food Chem. 44:804–810. Harper, W.J. 1992. Functional properties of whey protein concentrate and their relationship to ultrafiltration. New Applications of Membrane Process. International Dairy Federation Issue. Havn, S.S., Ipsen, R., Nielsen, P.M., and Lilbaek, H. M. 2006. Improving the foaming properties and heat stability of whey protein concentrates by phospholipase treatment. Milchwissenschaft-Milk Sci. Int. 61:188–191.

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Herceg, Z., Reˇzek, A., Lelas, V., Kreˇsi´c, G., and Franetovi´c, M. 2007. Effect of carbohydrates on the emulsifying, foaming and freezing properties of whey protein suspensions. J. Food Eng. 79:279–286. Hinrichs, R., G¨otz, J., and Weisser, H. 2003. Water-holding capacity and structure of hydrocolloid-gels, WPC-gels and yogurts characterised by means of NMR. Food Chem. 82:155–160. Hongsprabhas, P., Barbut, S., and Marangoni, A.G. 1999. The structure of cold-set Whey Protein Isolate gels prepared with Ca++ . Lebensmittel-Wissenschaft undTechnologie 32:196–202. Ibanoglu, E., and Karatas, S. 2001. High pressure effect on foaming behaviour of whey protein isolate. J. Food Eng. 47:31–361. Ikeda, S. 2003. Heat-induced gelation of whey proteins observed by rheology, atomic force microscopy, and Raman scattering spectroscopy. Food Hydrocoll. 17:399–406. Kavanagh, G.M., Clark, A.H., and Ross-Murphy, S.B. 2000a. Heat-induced gelation of globular proteins: Part 3. Molecular studies on low pH β-lactoglobulin gels. Int. J. Biol. Macromol. 28:41–50. Kavanagh, G.M., Clark, A.H., and Ross-Murphy, S.B. 2000b. Heat-induced gelation of globular proteins: Part 4. Gelation kinetics of low pH β-lactoglobulin gels. Langmuir 16:9584–9594. Kavanagh, G.M., Clark, A.H., and Ross-Murphy, S.B. 2002. Heat-induced gelation of globular proteins: Part 5. Creep behavior of β-lactoglobulin gels. Rheol. Acta 41:276–284. Kazmierski, M., Agboola, S., and Corredig, M. 2003. Optimizing stability of orange juice fortified with whey protein at low pH values. J. Food Qual. 26:227–352. Kinsella, J.E., and Whitehead, D.M. 1989. Proteins in whey: Chemical, physical and functional proteins. Adv. Food Nutr. Res. 33:343–438. Kresic, G., Lelas, V., Herceg, Z., and Rezek, A. 2006. Effects of high pressure on functionality of whey protein concentrate and whey protein isolate. Lait 86:303– 315. Langton, M., Astr¨om, A., and Hermansson, A. 1996. Texture as a reflection of microstructure. Food Qual. Prefer. 7:185–191. Lowe, L.L., Foegeding, E.A., and Daubert, C.R. 2003. Rheological properties of finestranded whey protein isolate gels. Food Hydrocoll. 17:515–522. Luck, P.J., Bray, N., and Foegeding, E.A. 2002. Factors determining yield stress and overrun of whey protein foams. J. Food Sci. 67:1677–1681. Lupano, C.E. 1994. Effect of heat treatments in very acidic conditions on whey protein isolate properties. J. Dairy Sci. 77:2191–2198. Lupano, C.E., Dumay, E., and Cheftel, J.C. 1992. Gelling properties of whey protein isolate: Influence of calcium removal by dialysis or diafiltration at acid or neutral pH. Int. J. Food Sci. Technol. 27A:615–628. Lupano, C. E., Renzi, L.A., and V.R. 1996. Gelation of whey protein concentrate in acidic conditions: Effect of pH. J. Agric. Food Chem. 44:3010–3014. Martin-Diana, A.B., Frias, J., and Fontecha, J. 2006. Comparison of foaming capacity of caseinmacropeptide from different species with whey protein concentrate. Milchwissenschaft-Milk Sci. Int. 61:134–138.

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McClements, D.J. 1999. Food Emulsions: Principles, Practice, and Techniques. Boca Raton, FL: CRC Press. Mishra, S., Mann, B., and Joshi, V.K. 2001. Functional improvement of whey protein concentrate on interaction with pectin. Food Hydrocoll. 15:9–15. Murray, B.S., Dickinson, E., Lau, C.K., Nelson, P.V., and Schmidt, E. 2005. Coalescence of protein-stabilized bubbles undergoing expansion at a simultaneously expanding planar air–water interface. Langmuir 21:4622–4630. Neirynck, N., Meeren, P.V.d., Gorbe, S.B., Dierckx, S., and Dewettinck, K. 2004. Improved emulsion stabilizing properties of whey protein isolate by conjugation with pectins. Food Hydrocoll. 18:949–957. Ngarize, S., Adams, A., and Howell, N. 2005. A comparative study of heat and high pressure induced gels of whey and egg albumen proteins and their binary mixtures. Food Hydrocoll. 19:984–996. Parkinson, E.L., and Dickinson, E. 2007. Synergistic stabilization of heat-treated emulsions containing mixtures of milk proteins. Int. Dairy J. 17:95–103. Pearce, R.J. 1992. In Whey and Lactose Processing, edited by J.G. Zadow, p. 271. London: Elsevier Applied Science. Pelegrine, D.H.G., and Gasparetto, C.A. 2005. Whey proteins solubility as function of temperature and pH. Lebensmittel-Wissenschaft und-Technologie 38:77– 802005/2. Pernell, C.W., Foegeding, E.A., and Daubert, C.R. 2000. Measurement of the yield stress of protein foams by vane rheometry. J. Food Sci. 65:110–114. Prinz, J.F., Janssen, A.M., and Wijk, R.A.d. 2007. In vitro simulation of the oral processing of semi-solid foods. Food Hydrocoll. 21:39–401. Relkin, P. 1994. Differential scanning calorimetry: A useful tool for studying protein denaturation. Thermochimica Acta 246:371–386. Relkin, P., Sourdet, S., Smith, A.K., Goff, H.D., and Cuvelier, G. 2006. Effects of whey protein aggregation on fat globule microstructure in whipped-frozen emulsions. Food Hydrocoll. 20:1050–1056. Resch, J.J., Daubert, C.R., and Foegeding, E.A. 2005. The effects of acidulant type on the rheological properties of β-lactoglobulin gels and powders derived from these gels. Food Hydrocoll. 19:851–860. Richert, S.H. 1979. Physical-chemical properties of whey protein foams. J. Agric. Food Chem. 27:665–664. Roefs, P.F.M., and de Kruif, C.G. 1994. Heat-induced denaturation and aggregation of β-lactoglobulin. Prog. Colloid Polym. Sci. 97:262–266. Roefs, S.P.F.M., and Peppelman, H.A. 2001. Food Colloids: Fundamentals of Formulation, edited by E. Dickinson and R. Miller, pp. 358–368. London: The Royal Society of Chemistry. Roth, S., Murray, B.S., and Dickinson, E. 2005. Interfacial shear rheology of aged and heat-treated beta-lactoglobulin films. Displacement by nonionic surfactant. J. Agric. Food Chem. 48:1491–1497. Sawyer, W.H. 1968. Heat denaturation of bovine β-lactoglobulins and relevance of disulfide aggregation. J. Dairy Sci. 51:323–329.

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Chapter 6 Whey Protein Films and Coatings Kirsten Dangaran and John M. Krochta

Introduction Edible films and coatings have been involved in food preservation for centuries. Since the twelfth century, civilizations have been using wax coatings as a method to lengthen shelf life of foods (Debeaufort et al. 1998). The main purpose was to prevent the loss of moisture and to maintain quality and texture during storage. Today, waxes are still commonly used to preserve fruits, vegetables, and meats for extended shipping and shelf life, but there are now more materials available for edible films and coatings. These alternative sources, along with improved processing techniques, have extended the use of edible films and coatings beyond simple moisture barriers. Since the early 1900s, food-grade shellac resins have been used for improving the appearance of foods (Beckett 2000; Minifie 1982). While these particular edible coatings have provided nominal protection or improvement to the physical appearance of foods, their uses are limited. Research in the area of edible films has improved the technology and created more options. Carbohydrates and proteins are biopolymers that can form films like waxes, lipids, or resins. Corn zein has been employed commercially as a food coating since World War II to enhance food appearance (Lawton 2002). The diverse structures and chemistries of carbohydrates and proteins offer a wide array of films and coating properties. Protein- and carbohydrate-based edible films are generally more cohesive and flexible than wax films and possess better gas-barrier properties at certain conditions. Biopolymers also

133 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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have the advantage of having more possibilities for adjusting film properties for specific application development through chemistry and processing. Many publications have been written reviewing the properties of edible films and coatings formed from biopolymers such as wheat gluten, soy protein, starch, cellulose, and casein (Brandenberg et al. 1993; Gennadios 2002; Gennadios and Weller 1990; Krochta 1997, 2002; Krochta and De Mulder-Johnston 1997; Krochta et al. 1994; Psomiadou et al. 1996; Tomasula et al. 1998). While many biopolymers have been studied as edible films and coatings, this chapter focuses on whey protein. The specific chemistry of whey protein that allows it to form films has been discussed, as well as the properties and applications. Throughout this chapter, the terms edible film and edible coating have been used interchangeably, depending on the research or application. Specifically, an edible film is a thin continuous sheet formed from a biopolymer matrix that is cohesive enough and has the physical integrity to stand alone. The thickness of an edible film is typically 2–10 mils (0.050–0.250 mm). Depending on their thermal properties and surface chemistry, edible films can be formed into pouches or laminated onto other packaging substrates. The main purpose for edible films from biopolymers is to control mass transfer of multiple compounds including gas, aroma, oil, and water vapor into or out of a food, preserving food quality. Edible films must also be both strong and flexible to withstand forces experienced during handling and processing. Edible coatings are edible films formed directly on the surface of a food or material. They are typically thinner than stand-alone edible films. While edible coatings themselves can improve the physical integrity of a coated product, they do not necessarily need to be as tough and resilient as a stand-alone film because of the underlying support of food. Additionally, edible coatings can improve appearance of a product by adding color or gloss, making it more appealing to consumers. Edible films are the form used to study mechanical, barrier, and surface properties. Coatings are studied as one type of application of an edible film. Based on their properties, edible films and coatings have the potential to reduce the need for some petroleum-based layers in packaging systems. Because they are made from renewable resources, edible films and coatings are an environmentally friendly alternative to synthetic packaging from oil.

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Edible Film and Coating Formation Solvent Casting of Edible Films Films can be formed via several processes depending on the starting material. Lipid and wax films can be formed through solidification of the melted material (Fennema and Kester 1991). Biopolymers in solution can form films by changing the conditions of the solution. Heating, adding salt, or changing the pH may alter conditions in the solution such that the biopolymers aggregate in a separate film phase. This process is called coascervation (Debeaufort et al. 1998; Krochta et al. 1994). Most edible films are formed by removing the solvent, called solvent casting. For years, solvent casting has been the main method of forming whey protein films for research. Food-grade water and ethanol are the two possible solvent choices to keep the films safe for consumption. Solvent casting starts with a dilute solution of biopolymer. The solution is spread in a thin layer on a level surface, and the solvent evaporates to form a film. Various levels of equipment are available for solvent casting of whey protein film from simple casting-plate setups to more advanced batch and continuous lab coaters. Because it is effective and cost-efficient, the most commonly used method of forming whey protein film samples for research is manually spreading film solutions into level petri dishes or plates and then drying in ambient conditions. More advanced equipment can make large whey protein films by mechanically spreading the solution at a fixed thickness. Edible film drying can occur under ambient conditions, with hot air, infrared energy, or microwave energy. Method of drying can significantly affect the physical properties of the final film. There are two periods during drying of the films: the constant rate period (CRP) and the falling rate period (FRP) (Kozempel et al. 2003). During the CRP, mass transfer between the surface of the film and air is the major phenomenon. Once the surface comes into equilibrium with air conditions, the FRP begins. During this period of drying, the mass transfer of water from the film to the air is limited by diffusion of water from the inside of the film to the surface. Air drying rate is dependent on the (1) exposed surface area, (2) drying air temperature, (3) drying air relative humidity (RH), (4) drying air velocity, and (5) drying period (Alcantara et al. 1998). Infrared

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drying has an energy source and an energy acceptor. Similar to air drying, infrared drying rate is dependent on film heat transfer, which can be affected by (1) surface temperature of the heating and receiving materials, (2) surface properties of the emitting body (emissivity) and receiving body (absorption coefficient), and (3) shapes of the emitting and receiving bodies (Brennan and Cowell 1990; Fellows 2000; Singh and Heldman 1993). Microwave drying will affect the FRP of drying differently than infrared or hot air drying. Microwave energy can penetrate more quickly into films, leading to faster diffusion of water from the center of the film to the film surface. Different drying conditions can change edible film morphology, which affects appearance, barrier, and mechanical properties (Perez-Gago and Krochta 2000). Extrusion of Edible Films As an alternative to solvent casting, some edible biopolymer films can be formed through extrusion of the biopolymer material with little or no addition of solvent. Elevated temperature and shear in the extruder soften and melt the polymer to allow for a cohesive film matrix to form. Extrusion of biopolymers into films has certain advantages over solvent casting. Evaporation of solvent in solvent casting is energy expensive and time-consuming. Equipment cost for continuous solvent-casting equipment is weighted heavily by the cost of the drying oven. Aqueousbased film-forming solutions involve slower drying with longer ovens than do organic solvent solutions, adding to the final cost of commercialized edible film products. Extrusion of biopolymer into films is faster and requires less energy. The savings in processing may bring the cost of biopolymer film formation to a competitive range with synthetic film production. Edible Coating Formation Edible coatings are formed in processes with the same formation mechanism as in solvent casting of films. Typical methods for forming a film as a coating on a product are panning, fluidized-bed coating, spray coating, and dipping. Panning, a method used by both the pharmaceutical and confectionery industry, entails putting the product to be coated into a large rotating bowl referred to as the pan (Minifie 1982). The coating

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solution is ladled or sprayed into the rotating pan, and the product is tumbled to evenly distribute the solution over the surface of the food or pharmaceutical product. Air that is forced into the pan is used to dry the coating. Fluidized-bed coating is a method used commonly by the pharmaceutical industry to coat tablets. However, its application to coating nuts has also been explored (Lin and Krochta 2006). Coating solution is sprayed into a fluidized bed of product. Spraying of coating solution is interrupted with periods of drying to allow gradual formation of the coating. The action of the fluidized bed during drying of the coating appears to reduce formation of clusters of coated product stuck to each other that can be a problem in panning. Spray coating applies a uniform coating over a food surface. Spray coating is a potentially more controllable method of coating application than pan coating or fluidized-bed coating. However, the bottom surface of the product must be coated in a separate operation after drying and then turning the product to expose the bottom. Dipping, the other possible method of forming edible coatings on the surfaces of food, is best for irregularly shaped food objects. Final formed coatings may be less uniform than coatings applied using other methods, thus multiple dippings with draining and drying in between may be necessary to ensure full coverage (Krochta et al. 1994).

Whey Protein Film and Coating Formation Whey Proteins Whey is a by-product of the cheese-making process. The technical definition of whey proteins is “those that remain in the milk serum after coagulation of the caseins at pH 4.6 and temperature 20◦ C” (Morr and Ha 1993). Whey proteins are not aggregated into the cheese curd to a significant degree (Walzem et al. 2002). The protein interactions that occur between chains determine film network formation and film properties. There are several individual proteins within the mixture of whey protein, with β-lactoglobulin, α-lactalbumin, bovine serum albumin (BSA), and immunoglobins being the main proteins (deWit and Klarenbeek 1983; Kinsella 1984). The most abundant and important of these proteins for film formation is β-lactoglobulin.

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β-Lactoglobulin is an 18.3-kDa protein consisting of around 160 amino acids, depending on the genetic variant. It is insensitive to changes in pH but is thermolabile. The secondary structure is dominated by an eight-stranded β-barrel. There is also a three-turn α-helix. Under neutral conditions, β-lactoglobulin exists as a dimer. As pH is lowered to less than 3, β-lactoglobulin dissociates into the monomeric form. When pH is raised to greater than 8, the protein forms an oligomer of eight associated protein molecules. For film formation, there is an important free sulfhydryl group, cysteine 121 (CYS121), in the amino acid sequence and two disulfide bonds between cysteine 66 and cysteine 160 and between cysteine 106 and cysteine 119. In the native form, CYS121 is hidden by the α-helix (Sawyer et al. 1999). When analyzed by differential scanning calorimetry (DSC), a denaturation temperature of 78◦ C was measured for β-lactoglobulin in 0.7 M phosphate buffer and pH 6 (deWit and Klarenbeek 1983) α-Lactalbumin is the second most abundant whey protein, with a molecular weight of 14.2 kDa. It contains four internal disulfide bridges that are thought to be responsible for α-lactalbumin’s characteristic resistance to heat denaturation compared to the other whey proteins. BSA is the longest single-chain whey protein, with a molecular weight of 66 kDa. It is prone to precipitation around 40–45◦ C due to increased hydrophobic binding between the chains (deWit and Klarenbeek 1983). The immunoglobulins are a mixture of proteins that are very thermolabile. Solvent Casting of Whey Protein Films Solvent-cast whey protein films, for which the solvent is water, can form from native proteins through the electrostatic interactions, hydrogen bonding, and van der Waals forces that occur between the protein chains as the water evaporates. Native films are cohesive, but the protein film network can be improved, and the resulting solvent-cast film tensile and barrier properties improved through heat denaturation and crosslinking of the whey protein chains (Perez-Gago and Krochta 1999). Thus, most research on whey protein films has involved heat denaturing of the whey protein in aqueous solvent and then casting the solution to form a film with cross-linked whey protein upon evaporation of the water. Heat denaturation and subsequent polymerization of whey protein chains has been studied, and the changes to β-lactoglobulin by heat

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have been documented. As temperature increases, there are three main events leading to polymerization of the β-lactoglobulin. First, the dimer dissociates when heated to above 40◦ C under neutral pH (deWit and Klarenbeek 1983). Around 60–65◦ C, the α-helix changes conformation and the free thiol group at CYS121 is exposed. Finally, polymerization occurs through intermolecular disulfide exchange if the temperature is held at >60–65◦ C (Galani and Apenten 1999). Free cysteine can reduce disulfide bridges, allowing for the formation of new disulfide bonds, which can be formed both intra- or intermolecularly, with the latter enhancing polymerization. The reactivity of CYS121 is both pHand temperature-dependent. When pH increases above 6.8, the disulfide bond formation rate increases. Polymerization is not the only chemical reaction involved in whey protein film network formation. Noncovalent aggregation also occurs through new hydrophobic, ionic, and van der Waals interactions that occur between newly exposed groups of the heatdenatured whey protein. These interactions increase as pH decreases toward the isoelectric point of whey protein (Kinsella 1984; Kinsella and Whitehead 1989). There are other methods for inducing protein chain cross-linking besides heat denaturation. Irradiation has been successfully used to cross-link casein proteins, as well as soy proteins (Brault et al. 1997; Lacroix et al. 2002). A hypothesized mechanism is radical polymerization through tyrosine and the formation of bityrosine linkages between protein chains. However, whey proteins are low in tyrosine residues, and irradiation alone does not produce a significant increase in molecular weight of whey protein (Vachon et al. 2000). However, whey protein can be cross-linked with other proteins like casein using irradiation (Lacroix et al. 2002) to increase molecular weight and change protein film properties. Cross-linking of whey proteins has also been induced both chemically and enzymatically. Formaldehyde, glutaraldehyde, tannic and lactic acids have been used to cross-link whey proteins through lysine residues. However, the cross-linked products are no longer edible due to the toxicity of the cross-linking agents (Galietta et al. 1998). Transglutaminase is a food-grade enzyme that uses the acyl transferase mechanism to link the γ -carboxyamide (acyl donor) of a glutamine residue to the γ -amine (acyl acceptor) of lysine residues along protein chains (Mahmoud and Savello 1992). The molecular weight of α-lactalbumin, β-lactoglobulin, and α-lactalbumin/β-lactoglobulin mixtures was shown to increase after

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transglutaminase treatment. Moreover, the treated proteins were more heat stable (Truong et al. 2004). The reaction is pH dependent and the extent of whey protein cross-linking decreases as the pH increases to greater than 7.5 (Aboumahmoud and Savello 1990). The cross-linked networks are less soluble, which would affect permeability properties of films formed from the cross-linked proteins. The whey protein film network can also be affected by changing the free volume of the matrix. This has been accomplished by the addition of a plasticizer or by hydrolyzing the whey protein prior to film formation. Plasticizers are small-molecular-weight chemicals added to polymers and biopolymers to solvate and lubricate the chains, thus allowing more movement (Kern Sear and Darby 1982; Marcilla and Beltran 2002). There are two types of plasticizers: internal and external. Internal plasticizers are molecules that chemically modify a chain, for example, through acylation or carboxylation. This increases the free volume of the chains by creating steric hindrances between amino acids. External plasticizers act as a lubricant in whey protein films by interrupting protein–protein interactions and hydrogen bonding, thus allowing more movement of the chains. Plasticizers have to be compatible with a polymer in terms of size, shape, and chemistry to be effective. Hydrolyzed whey proteins have been enzymatically treated to cleave some of the peptide bonds in the protein chains. The shorter chains have more free volume and movement. Extrusion of Whey Protein Films Much less work has been done on extruded whey protein films. In studies by Hernandez et al. (2005, 2006) the feed composition and extruder operation parameters to successfully extrude transparent, flexible whey protein sheets were determined. Sheets with different glycerol contents ranging from 46 to 52% (dry basis) were formed in a corotating twin screw extruder with six heating zones, ranging from 20 to 130◦ C, and a product temperature at the slit die of 143–150◦ C (Hernandez 2007). The extruder had a length-to-diameter ratio of 30:1, and the screw speed used was 250 rpm. These extruder dimensions and operating conditions allowed for sufficient heat denaturing and cross-linking of the whey protein to produce sheets that had improved tensile properties compared to solvent-cast heat-denatured whey protein films. Hernandez (2007) also found that it was possible to obtain continuous, transparent flexible whey protein sheets containing 49% glycerol, using

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extruder screw speeds ranging from 200 to 275 rpm. Screw speeds of 300 rpm and higher resulted in shorter residence times and, therefore, higher throughputs in the extruder. However, the shorter residence times at these higher screw speeds did not allow time for the whey protein powder particles and glycerol plasticizer being extruded to heat sufficiently and interact long enough to form a cohesive sheet. Given the advantages of extrusion for stand-alone films, more research on extrusion of whey protein films is needed.

Properties of Solvent-Cast Whey Protein Films The mechanical, barrier, and appearance properties are the most important characteristics of edible films, because they determine under what conditions they can be applied and used. As with traditional plastic film packaging, the most significant mechanical properties of interest are tensile strength, elastic modulus, and percent elongation. The most important barrier properties are determined as film oxygen permeability and water vapor permeability. Carbon dioxide, oil, and aroma permeability properties are also of interest, but the information is of value for more specific applications. The most important appearance properties are transparency, color, and gloss. Tensile Properties Tensile properties—tensile strength, elastic modulus, percent elongation, and resiliency—are indicators of protein–protein interactions in whey protein film matrices. They can reflect the type and extent of chain bonding, presence of crystalline domains, and the free volume in the whey protein film. Tensile properties are determined using an instrument that applies uniaxial force at a constant rate. Values for the tensile properties of whey protein films are determined from the stress–strain plots of films (measured resistive force versus distance stretched). Tensile strength is the maximum amount of force applied to a film per unit original cross-sectional area before film breakage. Young’s modulus, or elastic modulus, is the initial strain response of a film to applied stress. It is the slope of the stress–strain plot, and the higher the value, the stiffer the film (Sperling 2001). Elongation is the distance the film will stretch before breaking divided by the original film length. Resiliency is the film’s overall toughness. It can be estimated by multiplying tensile

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strength by percent elongation. Whey protein films have strong protein– protein chain interactions, which result in strong, stiff, brittle films with very small elongation. The tensile properties can be adjusted to make more flexible, stretchable, resilient films by changing the state of the protein or by the addition of plasticizers. The state of the protein, which is affected by processing temperature and/or shear, affects the tensile properties. Table 6.1 shows some measured tensile properties of whey protein films of different composition. Increased cross-linking that occurs during denaturation leads to stronger and stiffer films with greater elongation (Perez-Gago and Krochta 1999) compared to films made with whey protein in the native form. The crosslinking of whey protein chains produces stronger films, but also allows for greater deformation of the films. The amount and type of plasticizer in a whey protein film also affects tensile properties. Plasticizers are small molecules that solvate and/or lubricate protein chains. They interrupt protein–protein interactions and increase free volume in the film, making them more flexible. Plasticizer efficiency, or how well a plasticizer adjusts tensile properties, is dependent on the size, shape, and compatibility of the plasticizer with the protein. McHugh and Krochta (1994b) found increasing glycerol content caused a decrease in tensile strength and an increase in elongation. Comparing glycerol-plasticized films to sorbitol-plasticized films, percent elongation of the glycerolplasticized films was much greater but the films were weaker. Sorbitol is crystalline at room temperature. Microscopic domains of crystallized plasticizer can act as cross-linkers, thus increasing the film strength. However, since crystals have no flexibility or stretchability, they increase film elastic modulus and lower film elongation. In a study by Sothornvit and Krochta (2001), five plasticizers differing in size, shape, and composition were studied in film made from β-lactoglobulin, the major protein fraction in whey protein. Plasticizer efficiency ratings were determined for glycerol, sucrose, sorbitol, PEG200, and PEG400. Shape and waterbinding capacity of plasticizers were determined to be the major issues affecting plasticizer efficiency. The bulky-ringed plasticizer sucrose was less efficient at plasticizing β-lactoglobulin than the linear plasticizers. Glycerol was the most efficient plasticizer overall. Besides its small size, glycerol is more hygroscopic, thus attracting more water to help plasticize the whey protein film. Compared to synthetics like polyethylene, polypropylene, and polystyrene, whey protein films have lower resilience (toughness). Thus, either making pouches from whey protein

3 7 29 14 18 1 2 19–44 22–31 45–83 31–38

WPIa :gly (30%b ) WPIc :gly (30%) WPIc :gly (15%) WPIc :sor (30%) WPIc :sor (40%) WPIc :gly (30%), 5.5% DHd WPIc :gly (30%), 10% DHd LDPEe HDPEf Polystyrene Polypropylene

143

Native whey protein isolate. Glycerol content, dry basis. c Heat-denatured whey protein isolate. d Degree of hydrolysis of whey protein. e Low-density polyethylene. f High-density polyethylene.

b

100 199 1,100 1,040 625 6 100 280–410 1,000–1,600 2,620–3,380 1,170–1,730

Elastic modulus (MPa) 7 41 4 3 5 40 4 600 10–1,200 1–4 100–600

Elongation (%)

Perez-Gago and Krochta (1999) Perez-Gago and Krochta (1999) McHugh and Krochta (1994b) McHugh and Krochta (1994b) McHugh and Krochta (1994b) Sothornvit and Krochta (2000a) Sothornvit and Krochta (2000a) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000)

References

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a

Tensile strength (MPa)

Tensile properties of solvent-cast whey protein films (25◦ C, 50% RH).

Film

Table 6.1.

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films for relatively small amounts of foods or forming films as coatings on foods are more viable application routes until tensile properties can be improved. Permeability Properties Mass transfer of a molecule through a film takes place in three steps: (1) absorption into one side of the film, (2) diffusion through the film, and (3) desorption from the other side of the film. There are standard methods developed by the American Society for Testing and Materials (ASTM) for measuring oxygen, water, and carbon dioxide permeability through a thin sheet of plastic based on Fick’s Law of steady-state diffusion. Permeability through a film is dependent on two things: the diffusion coefficient and solubility of the permeate in the film matrix. P = DS where P is permeability, D is the diffusion coefficient, and S is solubility. The diffusion coefficient is specific for a given permeate-protein system and relates the flux to concentration gradients. Diffusion coefficients and solubility are influenced by (1) chemistry of the protein and penetrating molecule, (2) amount of amorphous and crystalline areas in the polymer since diffusion occurs through amorphous regions, (3) temperature, (4) glass transition temperature, and (5) plasticizer content (Hernandez et al. 2000). Oxygen Permeability Oxygen permeability is one of the most important properties in terms of application potential of whey protein films. Oxygen is a hydrophobic molecule and, thus, has low solubility in hydrophilic whey protein films; consequently, whey protein films have been determined to be excellent barriers to oxygen permeability (Table 6.2). Common equipment for determining oxygen permeability consists of a permeability cell that seals a film sample between two chambers, an oxygen detector and a way to establish different concentrations of oxygen on either side of the film. Adjustments to relative humidity and temperature are also included since both can significantly affect the permeability of oxygen in whey protein films by adjusting the free volume of the film and mobility of the permeant within the film. McHugh and Krochta (1994b) found an almost exponential increase in oxygen

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Oxygen-barrier properties of whey proteina films (25◦ C, 50% RH). O2 permeability (cm3 μm/kPa d m2 ) References

WPI:gly (30%) 76 WPI:gly (15%) 19 WPI:sor (30%) 4 WPI:sor (50%) 8 WPI:gly (30%), 10% DHb 90 β-Lacc :gly (30%) 38 β-Lac:sor (30%) 5 LDPE 1,900 HDPE 260 Nylon 6 25 Polypropylene 620 EVOHd 0.2

McHugh and Krochta (1994b) McHugh and Krochta (1994b) McHugh and Krochta (1994b) McHugh and Krochta (1994b) Sothornvit and Krochta (2000a) Sothornvit and Krochta (2000b) Sothornvit and Krochta (2000b) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000)

a

All whey protein films in this table are heat denatured. Degree of hydrolysis. c beta-lactoglobulin. d Ethylene vinyl alcohol copolymer. b

permeability as relative humidity of the test conditions increased for whey protein films. Other protein-based films have been studied, and an exponential relationship between the oxygen permeability and temperature was also found (Gennadios et al. 1993). Mat´e and Krochta (1996) found an Arrhenius relationship between whey protein films and temperature. As with tensile properties, the oxygen-barrier properties of whey protein films can be affected by plasticizer content, as well as by relative humidity and temperature. Increasing glycerol content from 15 to 30% increased oxygen permeability from 18.5 to 76.1 (cm3 μm)/(kPa d m2 ), respectively (McHugh and Krochta 1994b). Using a plasticizer that is solid at room temperature significantly lowers oxygen permeability by an order of magnitude when a comparison to glycerol is made at plasticizer levels, giving equivalent tensile properties. The oxygen permeability of a whey protein film plasticized with 30% sorbitol is 4.3 (cm3 μm)/(kPa d m2 ) comparatively (McHugh and Krochta 1994b). The improved permeability of whey protein films plasticized with sorbitol and other solid-at-room-temperature molecules may be due to their larger size and/or bulkier shape, making them less efficient. Such

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Film

Water vapor permeability (g mm/kPa h m2 ) References

WPIa :gly (30%b ) WPIc :gly (30%) WPIc :gly (15%) WPIc :beeswax (20%, 1 μm) WPIc :beeswax (60%, 1 μm) WPIc :beeswax (60%, 2.5 μm) WPIc :gly (30%), 5.5% DHd WPIc :gly (30%), 10%d DH LDPEe HDPEf Polypropylene EVOH

5 5 2 2 1 2 4 4 0.002 0.0003 0.001 0.001

Perez-Gago and Krochta (1999) Perez-Gago and Krochta (1999) Mat´e and Krochta (1996) Perez-Gago and Krochta (2001) Perez-Gago and Krochta (2001) Perez-Gago and Krochta (2001) Sothornvit and Krochta (2000c) Sothornvit and Krochta (2000c) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000) Hernandez et al. (2000)

a

Native whey protein isolate. Glycerol content, dry basis. c Heat-denatured whey protein isolate. d Degree of hydrolysis of whey protein. e Low-density polyethylene. f High-density polyethylene. b

plasticizers may also create impenetrable crystalline domains within the film, lowering permeability (Rogers 1985), although the crystalline domains would also negatively affect tensile properties. With the appropriate plasticizer choice, the oxygen permeability of whey protein films is competitive with traditionally used packaging barriers like nylon and ethylene vinyl alcohol (Table 6.2). Moreover, the oxygen permeability of whey protein films is 1–2 orders of magnitude lower than polypropylene, low-density polyethylene and high-density polyethylene permeabilities. Water Vapor Permeability The water vapor permeability of whey protein films has been measured for various compositions (Table 6.3). Because it is hydrophilic protein, whey protein films are only moderate barriers to moisture at best. The method for determining water vapor permeability is a simple technique to perform, but the mathematics used to describe the phenomenon is complex. The method uses an established relative humidity gradient

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and circulating air to prevent stagnation. The gradient is made with distilled water for 100% relative humidity and various saturated salt solutions to lower the relative humidity. Two different relative humidity environments are established on either side of the whey protein film. The technique is based on the ASTM method for measuring the water vapor permeability of hydrophobic packaging materials (ASTM 1995). However, when the method is applied to hydrophilic films a correction factor is needed. McHugh et al. (1993) found that there is a partial pressure gradient on the inside layer (side without air circulation) during testing of the low-barrier protein films due to the low resistance to diffusion of water in the films. The extent of the gradient is dependent on both the film thickness and the height of the gap between the surface of the humidity source and the film. Using a method for calculating diffusion of water vapor through air, mass transfer through the stagnant layer was accounted for and a corrected partial pressure at the underside of the film can be estimated and used to more accurately determine the water vapor permeability of whey protein films. Alhough the water vapor permeability of whey protein films is high, the barrier properties of the films have been improved through addition of hydrophobic materials like waxes and lipids. Whey protein–lipid films can be formed in two ways: by forming a bilayer of protein and lipid or through forming emulsions (Morillon et al. 2002). Although bilayers have solid layers of lipid or wax, which are excellent barriers to water, this film-forming technique has had inconsistent results, perhaps due to separation of the layers. In addition, the two-step process generally required for a bilayer film is difficult and unlikely to be practical for applications. For emulsion-based whey protein–lipid films, the size of the lipid particles in the film matrix significantly affects the permeability. In a study by McHugh and Krochta (1994a), the effect of lipid particle size in whey protein films was studied. As particle size decreased, water vapor permeability also decreased. A possible explanation is increased path tortuosity of the permeate. As particle size decreases for a given lipid content, the number of particles in the film increases. Water does not diffuse through lipid but must travel around it. As the number of particles increases, the path of the water in essence gets longer within the film. Perez-Gago and Krochta (2001) however found that there is a connection to particle size and amount of lipid in the film. In a low-level (20%) beeswax whey protein film, decreasing particle size did not affect water vapor permeability. At high levels

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(60% beeswax) decreasing particle size did have an effect. In addition to increasing tortuosity, decreasing lipid particle size increases the size of the protein–lipid interface. Protein chains are immobilized at the interface, thereby lowering free volume and consequently water vapor permeability. As with lipid particle size, lipid type can affect the water vapor permeability of whey protein films. Shellhammer and Krochta (1997) looked at four lipids and waxes: carnauba wax, candelia wax, milkfat fraction, and beeswax. They determined that beeswax and milkfat fraction are more viscoelastic than the carnauba and candelia waxes. When incorporated in whey protein films, the viscoelastic milkfat and beeswax improve the water vapor permeability more than candelia or carnauba wax (Shellhammer et al. 1997). They hypothesized that the more plastic nature of the milkfat and beeswax particles allowed them to deform during drying to form an intact lipid network inside the film, resulting in a better barrier. The moisture barrier of whey protein films with milkfat fraction or beeswax at 40% was 2–4 times better than whey protein films without lipids, respectively. Also, likely due to their viscoelastic behavior, milkfat and beeswax could be incorporated to higher amounts in whey protein films without film cracking, resulting in additional improvement in barrier properties. While the addition of lipids and waxes can greatly improve the water vapor permeability of whey protein films, their effect on tensile properties must be considered. At high levels, especially for brittle waxes, tensile strength and elongation decrease and films become brittle and hard to handle without breaking (Shellhammer and Krochta 1997). When candelia or carnauba wax is incorporated at level of greater than 40%, whey protein films crack upon drying. However, there is a positive effect on tensile properties of decreasing particle size of insoluble additives in protein films (Dangaran et al. 2006). Perez-Gago and Krochta (2001) found tensile strength and elongation significantly increased when particle size of beeswax in whey protein films decreased. Compare to synthetics, whey protein films are only moderate moisture barriers. Even with the inclusion of lipids, whey protein films still have higher water vapor permeabilities than low-density polyethylene (LDPE), high-density polyethylene (HDPE), and nylon. In terms of applications, whey protein films may be best for food products needing a low to moderate moisture barrier to avoid condensation from forming on the surface. Moreover, the appearance needs to be considered because

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including lipids and waxes confers some opaqueness. They may have some short-term use inside food as protective layers between high and low water activity layers like cookies and cream fillings or piecrusts and fruit fillings. Aroma and Oil Permeability Whey protein films have been found to be excellent barriers to aroma compounds and oil (Han and Krochta 2001; Miller et al. 1998). This is consistent with the findings that whey protein limits the flavor perception of benzaldehyde, citral, and D-limonene (Hansen and Heinis 1992), and that β-lactoglobulin has been found to be a binder of aromatic compounds (Farrell et al. 1987). Miller and Krochta (1998) developed a method for determining permeability of aroma compounds through films. They used the method to determine that whey protein films had better barrier to D-limonene than vinylidene chloride copolymer (coVDC) by 250–15,000 times, depending on relative humidity, but not as good a barrier as ethylene vinyl alcohol copolymer (EVOH). The oil-barrier properties of whey protein films were examined as coatings on paper. Chan and Krochta (2001b) found that denatured whey protein coatings were better barriers to oil than native whey protein, due to the cross-linked protein network produced in denaturation. Moreover, the performance of the denatured whey protein coating was similar to polyvinyl alcohol (PVOH) or fluorocarbon coating commercially used to make paperboard grease resistant. By monitoring the penetration of dyed vegetable oil on whey protein-coated paper over time, Lin and Krochta (2003) compared whey protein coatings with various plasticizers as barriers to oil. They found whey protein plasticized with glycerol (1.3 M) prevented the penetration of oil into the paper for at least 16 h. PEG200 was also found to be good choice for a plasticizer in whey protein coatings as barriers to grease. Appearance Properties Whey protein forms films are transparent and highly glossy—two characteristics very important to coating applications. Trezza and Krochta (2000) found whey protein–glycerol films had gloss values (90.8) similar to shellac (92.9) and higher than hydroxylpropyl methylcellulose (64.7), a carbohydrate biopolymer used as a food and pharmaceutical coating. The gloss of whey protein films can be affected by plasticizer

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choice. Lee et al. (2002a) found sucrose-plasticized whey protein coatings had the highest gloss compared to glycerol-, propylene glycol-, or PEG200-plasticized coatings. They hypothesized that the refractive index (RI) of the plasticizer affected the gloss of the final film. Amorphous sucrose has RI higher than other commonly used plasticizers. The amount of light a surface reflects is related to the RI—the higher the RI, the more the light is reflected. Dangaran and Krochta (2003) found that as sucrose content increased, the gloss of whey protein films and coatings significantly increased. However, crystallization of the sucrose that occurred over time gave the whey protein films a hazy appearance and lowered the gloss. To be acceptable coatings, crystallization of the plasticizer needed to be controlled. Dangaran and Krochta (2006a) found that sucrose crystallization in whey protein films could be hindered by the addition of inhibitors. Raffinose and modified starch prevented crystal growth in whey protein–sucrose films for at least 1,800 h of storage at 53% relative humidity. Whey protein films without inhibitors had noticeable crystallization after 50 h of storage. When applied as coatings to chocolates for a glossy finish, the whey protein– sucrose coatings with raffinose inhibitors maintained gloss longer than whey protein coatings containing only sucrose (Dangaran and Krochta 2006b).

Properties of Extruded Whey Protein Films Hernandez (2007) used a corotating twin-screw extruder with dimensions and operating conditions that allowed formation of plasticized whey protein sheets that were homogeneous, transparent, and flexible. The sheets produced by extrusion had greater strength and elongation properties compared to solvent-cast heat-denatured whey protein films, indicating greater extent of heat denaturing and protein cross-linking with the extrusion process. Similar to solvent-cast whey protein films, increasing plasticizer content of extruded sheets significantly decreased their strength and stiffness. However, contrary to the usual increase in elongation for solventcast films, elongation of the extruded whey protein films was unaffected (Hernandez 2007; Hernandez et al. 2006). Extruded sheets with a thickness of 1.31 ± 0.02 mm displayed thermoplastic behavior that allowed them to be compression-molded into

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thinner films with a thickness of 0.18 ± 0.02 mm. Thermal transitions of these films were determined by DSC and then used as a guide to selection of film heat-sealing temperatures. The films could be heatsealed with an impulse heat-sealer over a range of temperatures, pressures, and sealing times. Degradation of the seal area occurred at around 204◦ C, corresponding to degradation temperatures observed by DSC. Seal strengths of extruded/compression-molded films with 49% (db) glycerol and thickness of 0.18 ± 0.02 mm were measured. Solvent-cast films with 40% (db) glycerol content and thickness of 0.13 ± 0.01 mm were found to have significantly higher seal strength, likely mainly due to their lower glycerol content. Better comparisons between extruded and solvent-cast films should be made using films with similar plasticizer content and thickness. More research on extruded films is highly desirable. Extruded whey protein films are more likely to be practical than solvent-cast films for heat-sealing into pouches.

Whey Protein Coating Applications Based on their inherent properties, some specific applications of whey protein films formed as coatings have been researched and developed. Table 6.4 shows some examples of designed applications and the film properties involved. By taking advantage of the passive gas barrier, glossy appearance properties or active film capabilities, whey protein films and coatings have been designed to be coatings that lengthen shelf life, improve consumer acceptability, or raise the level of food safety for a product. Nuts and Peanuts By taking advantage of the excellent oxygen-barrier properties, whey protein films formed as coatings have been investigated for use in protecting foods that are high in polyunsaturated fats, specifically nuts and peanuts, which are susceptible to lipid oxidation. Nutmeat is quickly oxidized when exposed to oxygen and forms rancid off-notes, which make the product unacceptable to consumers and shortens shelf life. Commonly, nuts and peanuts are packaged in metal, glass, or multilayer metallized plastic packaging with nitrogen flushing or vacuum to

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Product

Function

Film property

Nuts/peanuts

Protect from lipid oxidation Carry protective antioxidants Extend shelf life

Oxygen barrier Antioxidant carrier (i.e., active film)

Confectioneries

Add smooth finish Add gloss Add color

Gloss/transparency

Eggs

Prevent weight loss Extend shelf life

Moisture barrier Gas barrier

Fresh cut products

Carry antibrowning agents Carry texture enhancers Extend shelf life

Antibrowning agent carrier (i.e., active film) Oxygen barrier

Meat products

Carry antimicrobials Extend shelf life

Antimicrobial carrier (i.e., active film)

Plastics and paper

Reduce oxygen permeability Prevent oil migration

Oxygen barrier Oil barrier

protect them from oxygen. However, once the packaging is opened, the nuts are exposed to oxygen once again. A whey protein coating applied directly to the nut surface allows the protective layer to remain with the food, also reducing the high-performance barrier requirement for the outside product packaging. In a study by Mate et al. (1996), peanuts coated with whey protein isolate (WPI) had lower peroxide and hexanal formation during storage as compared to uncoated peanuts. Peroxide and hexanal are by-products and chemical indicators of lipid oxidation. Lee and Krochta (2002) found that the whey protein coatings could extend the shelf life of peanuts to 273 days at 25◦ C compared to 136 days for uncoated nuts. By including vitamin E, an antioxidant, shelf life was estimated to be 330 days. A major issue for whey protein coating effectiveness as an oxygen-barrier coatings is complete surface coverage. Lin and Krochta (2005) found that using a surfactant in the whey protein coating significantly increased coating efficiency, improving the application potential.

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Eggs Another application explored for whey protein films is improving shelf life of eggs. During storage, eggs lose albumen, yolk quality, and weight. In addition, the internal pH changes. Loss of water and carbon dioxide through the shell are the major causes for loss of egg quality. In a study by Caner (2005b), the shelf life of whey protein-coated grade A eggs was 1 week longer than that of uncoated eggs, when stored under ambient laboratory conditions. The color, yolk index (yolk height and yolk width), and pH changed slower and remained at higher quality levels significantly longer than uncoated eggs. Moreover, a consumer study performed by Caner (2005a) found that the surface tactile and appearance properties of whey protein-coated eggs were significantly more preferred than uncoated eggs using a hedonic scale. A longer shelf life may help defer part of the estimated $10 million per year loss for the egg industry.

Confectionery Products Taking advantage of the glossy, transparent properties of edible films, whey protein coatings can be used to impart a smooth, glossy finish to dried food or confectionery products. Currently, shellac is used to finish chocolates, jelly beans, and other panned candies with a smooth, glossy surface. The edible shellac is a resin that must be first dissolved in ethanol before application. Large amounts of volatile organic compounds (VOCs) are released into the atmosphere as the shellac glaze dries, adding to air pollution. EPA policies are requiring the confectionery industry to reduce their VOC emissions. A way to accomplish this is to use a water-based coating instead of shellac. Whey protein coatings are a viable alternative. Studies by Trezza and Krochta (2001) found that glycerol-plasticized WPI films were highly glossy, comparable in gloss value to shellac films. Lee et al. (2002a) applied the WPI films as coatings to panned chocolate candies and found sucrose-plasticized WPI coatings to be the glossiest. Due to nonoptimized pan-coating conditions, the gloss of WPI coatings on chocolate was significantly lower than shellac coatings. However, in a consumer study, the lower level of WPI gloss coatings was preferred overall (Lee et al. 2002b). In an effort to improve the WPI coatings, Dangaran and Krochta (2003) adjusted the level of sucrose in the WPI coatings and the coating conditions to

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create a durable, smooth, glossy water-based coating for chocolates that can potentially replace shellac. Meat Products Whey protein film coatings have been studied as an added layer of protection for roasted turkey, smoked salmon, and sausage products (Cagri et al. 2002; Min et al. 2006a, b). All three ready-to-eat meat products are susceptible to contamination during slicing and packaging, and there have been Listeria monocytogenes, Escherichia coli, or Salmonella outbreaks associated with these meat foods (Cagri et al. 2002; Min et al. 2005b). Whey protein coatings with active antimicrobials have been shown to be effective at inhibiting growth of these pathogenic bacteria, thus increasing food safety and extending product shelf life. Smoked salmon samples were stored for 35 days in 4 and 10◦ C conditions (Min et al. 2005c). Samples were either uncoated, coated with lactoperoxidase-containing WPI film prior to inoculation, or coated after inoculation with L . monocytogenes (4 log CFU/g). Uncoated salmon stored in 4 and 10◦ C had at least 4 log CFU/g Listeria after 21 or 3 days, respectively. When coating was applied prior to inoculation, no Listeria was detected after 35 days of storage in all samples stored in 4◦ C or samples stored in 10◦ C. When simulated bacterial contamination occurred before coating, salmon samples initially decreased in Listeria levels from >1 log CFU/g to undetectable levels during the first 3 days of storage and remained there (Min et al. 2006a). Sorbic acid and p-aminobenzoic acid were incorporated into whey protein coatings for application to bologna and sliced summer sausage. The active whey protein coatings had antimicrobial activity for 21 days of storage against L. monocytogenes, E. coli, and Salmonella typhimirium DT104 (Cagri et al. 2002). Fruits and Vegetables A growing trend in food products is convenience foods. Fresh cut fruits and vegetables are growing in popularity; however, once cut, the produce becomes highly perishable. The respiration rate of fresh cut fruits can be 1.2–7 times higher than unprocessed fruit, according to Lee et al. (2003). Polyphenol oxidase activity is elevated with the increased exposure to oxygen, causing the cut produce to brown. Ethylene production

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increases inducing ripening and texture begins to deteriorate. Sulfites can be used to reduce browning, but there have been questions raised concerning sulfite use in foods and health. High levels of ascorbic acid or citric acid can be used to prevent browning; however, flavor may be affected (Perez-Gago et al. 2003). Modified atmosphere packaging could slow oxidation, but if oxygen levels are reduced too low, anaerobic conditions could be created and this creates the risk of anaerobic bacterial growth. Edible coatings that have moderate oxygen, carbon dioxide, and water vapor permeability can be applied to the surface of fresh cut product to extend shelf life by delaying ripening, delaying browning, reducing water loss, reducing aroma loss, carrying antioxidants, or carrying texture enhancer (Olivas and Barbosa-Canovas 2005). In a study by Le Tien et al. (2001), whey protein coatings significantly delayed browning in fresh cut Macintosh apples and russet potatoes. Similarly, Perez-Gago et al. (2003) found WPI-beeswax coatings reduced the rate of enzymatic browning in Golden Delicious apples. Fuji apples coated with various formulations of whey protein concentrate (WPC) coatings were tested by Lee et al. (2003). They determined that WPC coatings that contained ascorbic acid for color and calcium chloride for texture enhancement produced cut apples with the highest consumer acceptance after 2 weeks of storage compared to uncoated cut apples or cut apples coated with carrageenan. Packaging Whey protein coatings have potential applications beyond food products. They can be applied to traditional packaging materials like paper and plastic films to impart a new functional property. Paper is the most widely used packaging material because of its versatility, printability, and easy recyclability. However, since it is made from cellulose, which is hydrophilic, paper is a poor water vapor barrier. Moreover, paper loses its strength and integrity when wet. It is often coated with wax or polyethylene to improve the moisture-barrier properties. Han and Krochta (1999) found that paper coated with WPI had increased wettability and water absorption, which allow for printing, but decreased water vapor permeability. Paper can be coated with a plastic laminate or aluminum foil to infer grease-barrier properties. In another study, Han and Krochta (2001) found WPI-coated paper had significantly reduced oil absorption. Chan and Krochta (2001a, b) found that paperboard

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coated with glycerol-plasticized WPI had excellent grease barrier, oxygen barrier, color, and gloss compared to commercial PVOH and fluorocarbon coating. Lin and Krochta (2003) found that grease-barrier function was maintained when 80% WPC coatings plasticized with sucrose were applied to paperboard. The results of these studies are important for application of paper and paperboards for packaging greasy foods such as chips, hamburgers, and pizza. As well as coating paper, WPI has been used to coat polyethylene, polypropylene, and polyvinyl chloride (Hong and Krochta 2003; Hong and Krochta 2004; Hong et al. 2004). Multilayer packaging is used, as each layer can impart a separate barrier function. When an oxygen barrier is needed, ethylene vinyl alcohol or metal is often incorporated into the packaging system. However, multilayer packaging cannot be recycled. An edible coating applied to the surface of plastic films can impart improved barrier properties as well as allow for easier recycling of the plastic. Depending on the edible coating, an enzymatic treatment or simple chemical wash would remove the coating, and the plastic can then be recycled. In studies by Hong and Krochta (2003, 2004, 2006) low-density polyethylene and polypropylene were coated with WPI. Oxygen permeability was significantly reduced by the WPI coating until the films were exposed to an environment with a relative humidity ≥80%. Active Whey Protein Films Because of their inherent characteristics, whey protein films are excellent oxygen, aroma, and oil barriers without adjustment. They can be passive barriers and add a layer of protection to foods by being incorporated into the product as a film layer or a coating. They serve parallel functions to traditional packaging materials. A next step in both edible film and traditional packaging technology is the incorporation of functional compounds that confer another protective action to the system creating what is known as active packaging. By definition, active packaging interacts directly with the food or headspace of the product (Han 2000; Ozdemir and Floros 2004). Some typical purposes of active packaging are given in Table 6.5. In traditional packaging systems, the active compounds may be toxic and therefore cannot touch the food directly. To prevent contamination, the active compounds may be incorporated into complex multilayer

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Active packaging examples.

Oxygen scavenger Carbon dioxide absorber/emitter Antimicrobial

Time–temperature indicator pH indicator

packaging. As stated previously, layered packaging is difficult to recycle and most often ends up as waste in landfills. Edible films and coatings can also be active layers, but have the benefit of being nontoxic concerning contact with food. Whey protein films can carry such bioactive compounds as flavors, natural oxygen scavengers, and antimicrobials without the concern of toxicity. Flavor Carriers As a carrier of flavor compounds, edible films have been commercialized into oral flavor strips, with the most successful product being Listerine Pocket PacksR . The success of these oral strips may be connected to an increased interest in commercialization of edible films. Industry is pursing edible films as carriers of vitamins, nutrients, and over-thecounter medications. The market size of edible films is predicted to grow to over $350 million industry by 2008 (Anonymous 2006) and was just $1 million in 1999. Antioxidant Carriers Edible films from whey protein that incorporate natural, nontoxic antioxidants can be safer alternatives to oxygen scavenger sachets or butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) antioxidants that are currently often used in packaging. Sachets that contain iron complexes or other oxygen scavengers could be potentially toxic if mistakenly swallowed; BHA and BHT, which vaporize into the package headspace and then absorb on the food surface, are considered to be a possible safety risk by some groups. It should be noted that, thus far, BHA and BHT are considered safe by Food and Drug Administration at these low levels. But, the Center for Science in the Public Interest advises consumers to avoid them (Center for Science in the Public Interest, 2007). Ascorbic acid, tocopherols, β-carotene, albumin, and

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bilirubin all possess oxygen-scavenging activity while being safe for consumption (Niki 1991). In a study by Janjarasskul and Krochta (2006), whey protein films containing ascorbic acid were found to have antioxidant function that enhanced the oxygen-barrier function of the film. In an application, these ascorbic acid-containing whey protein films were coated onto roasted peanuts, which oxidize quickly due to their high polyunsaturated fat content. Min and Krochta (2006) found a significantly lower level of peroxide compounds in ascorbic acid–WPI-coated nuts compared to uncoated and WPI-only coated nuts. After 14 days of storage at elevated temperature, uncoated peanuts had 21 meq peroxide/kg (determined by thiobarbituric acid reactive substances assay), while WPI-coated and ascorbic acid–WPI-coated peanuts has 14 and 9 meq peroxide/kg, respectively. Antimicrobial Carriers Incorporation of natural antimicrobial compounds in WPI films has been introduced in the subsection on “Meat Products.” Perhaps the greatest potential for active edible films concerns food safety. In the United States, most class I product recalls are caused by postprocess contamination (Cagri et al. 2004). In the produce sector, fungal or bacterial attack can cause a significant postharvest loss in product. Chemical antimicrobial compounds have been and continue to be used. However, there is a growing concern about the use of synthetic pesticides and chemicals with foods (Sloan 2001; Wilcock et al. 2004). Natural antimicrobials have been researched as effective and socially acceptable alternatives. Whey protein films incorporating organic acids and various bioactive peptides have been tested against both spoilage and pathogenic organisms. Postprocessing contamination can cause loss of product quality or food safety hazards. Whey protein films containing antimicrobials provide another layer of protection while potentially reducing the amount of antimicrobials needed for efficacy. Protective compounds like organic acids have been sprayed onto food surfaces; however, they can quickly diffuse into the food interior, leaving the surface susceptible to bacterial contamination. Incorporation of the antimicrobials in edible films can slow the rate of diffusion of the compound, thus maintaining a minimum inhibitory concentration for a longer time on the surface where it can be most effective. The two issues for active edible films containing antimicrobials are the minimum inhibitory concentrations against

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different levels of contamination from specific microorganisms and diffusion constants of the antimicrobials in the film and in the food. Both have been investigated for whey protein films. The bioactive proteins lysozyme, lactoferrin, and lactoperoxidase have been extensively investigated as antimicrobials in whey protein films. It was determined that at least one of the proteins was effective against studied spoilage mold and pathogenic gram-positive or gramnegative bacteria. Lysozyme, lactoferrin, and lactoperoxidase are all naturally occurring active compounds that can be found in various foods or animals, including humans. Since they are already present in daily life, there is no concern of introducing new antibiotics to humans or new antibiotic resistance. Lysozyme hydrolyses linkages in peptidoglycan cell walls causing cell lysis. Lactoferrin chelates iron, an essential nutrient for bacterial growth, making it unavailable. Lactoperoxidase systems (LPOSs) oxidize thiocyanate to hypothiocyanate, which then oxidized sulfhydryl groups in microbial enzymes. In all studies, postprocessing contamination was simulated for situations when a protective edible film was applied either before or after contamination. This was done either by first inoculating an appropriate agar with a target mold or bacteria then placing an active whey protein film on top to determine inhibition or by placing the active whey protein film onto the agar then inoculating. LPOS was found to be effective against Salmonella enterica and E. coli O157:H7 when incorporated into whey protein films (Min et al. 2005b). A concentration of 0.15 g LPOS/g film was needed to inhibit 4 log CFU/cm2 of both pathogenic bacteria. LPOS in WPI films was also found to have inhibitory effect against 4.2 log CFU/cm2 L. monocytogenes when the films had 29 mg LPOS/g film.In a storage study, LPOS-WPI films inhibited growth for 35 days at 4◦ C. At 10◦ C, the simulation of an active film over a contaminated food surface inhibited growth for 21 days, while the simulation of active film applied prior to contamination inhibited growth for 35 days. There is possibly more efficient contact of the coating with cells in the coating-inoculation simulation. Rough surfaces of foods in the inoculation-then-coating simulation may protect pathogenic cells from LPOS. Min et al. (2005c) found that lysozyme-WPI films were also active against L. monocytogenes. A minimum of 204 mg/g of lysozyme in film inhibited growth of inoculum (4.4 CFU/cm2 ). LPOS was also determined to be effective against Penicillium commune, a spoilage mold found on bread, nuts,

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meats, and dairy. On DRBC agar, 59 mg/g LPOS inhibited growth of up to 104 mold spores. Another active peptide, the bacteriocin nisin, was investigated in WPI films in a study by Ko et al. (2001). Nisin is a natural antimicrobial with GRAS (generally regarded as safe) status for certain food products in the United States. When nisin was incorporated into the WPI films at 6,000 IU/g at pH 3, a 2.42-log reduction of L. monocytogenes was seen by the WPI-nisin film. Cagri et al. (2001) looked at the effectiveness of organic acids in WPI films at inhibiting Listeria, E. coli O157:H7, and S. typhimirium. They tested p-aminobenzoic acid and sorbic acid. At a minimum concentration of 0.5% acid in the films, both active WPI films showed zones of inhibition on TSAYE agar that was inoculated with Listeria, E. coli O157:H7, or S. typhimirium DT104. To get the acids in their more effective form, undissociated, pH 5.2 was used for testing. This indicated these GRAS organic acids would be good active film components for lower pH foods like cheeses of fermented meats. Concerning rate of diffusion of active compound in whey protein films, the effect of film composition has been investigated, including plasticizer and lipid content. It is necessary to have the appropriate film matrix for a targeted rate of controlled release of the active antimicrobial. Depending on the structure and chemistry of the antimicrobial, the film matrix may need to be hydrophilic or hydrophobic. Potassium sorbate, a commonly used antimycotic agent, was incorporated into whey protein films, and the effect of glycerol content and beeswax content on the diffusion coefficient was determined by Franssen et al. (2004). They found an increase in diffusion coefficient (D) as glycerol content went up and free volume increased, but lipid content did not affect D values. In a similar study, Ozdemir and Floros (2003) investigated potassium sorbate diffusion in whey protein films made with sorbitol and beeswax. Both studies found D values around 10−11 m2 /s. Franssen et al. (2004) also explored use of natamycin, another antimycotic often used in the cheese industry. A much larger molecule than potassium sorbate, the D values were determined to be around 10−14 m2 /s. Min et al. (2006b, c) measured the diffusion of antimicrobial peptides, lysozyme, and lactoperoxidase, and/or their active by-products in whey protein films. Thiocyanate and hypothiocyanate are the active antimicrobial products that are produced by lactoperoxidase. Lysozyme D values in whey protein films were between 3.1 × 10−16 and 2.9 × 10−13 m2 /s,

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depending on temperature (4–22◦ C) and glycerol content (25–50% dry basis). Thiocyanite and hypothiocyanate had D values between 1.9 × 10−13 and 5.2 × 10−12 m2 /s and between 1.3 × 10−14 and 6.5 × 10−13 m2 /s, respectively, depending on glycerol content. By altering whey protein film formation, the desired diffusion rate of active compounds can be attained.

Future Trends The future of whey protein films and coatings goes hand in hand with the interests of consumers. They have been the subject of much research and will likely have increasing use by the food industry. Research into edible films in general will continue, especially with the support of such Presidential Initiatives as 13101 and 13134, which call for increase use and study of bio-based products. There is an increasing need for packaging materials that are alternatives to petroleum-based sources. As oil prices continue to go up, so do packaging costs. Renewable sources of materials for packaging will create a steady, reliable supply. Edible films from whey protein are “green” alternatives to traditional plastics. Based on its excellent oxygen-barrier properties, whey protein films can be competitive biodegradable materials replacing EVOH, nylon, or polyesters, which are typically used as oxygen barriers. Biodegradable packaging is estimated to grow 20% over the next few years, taking up a larger share of the packaging market. Protein-based films and coatings offer alternative properties to the carbohydrate-based packaging materials that have already been successfully accepted into the market. Moreover, proteins provide more opportunities for change in chemical structure and, thus, future property improvement than carbohydrates. More and more companies are seeking out biodegradable agriculturally based packaging and seeing where it can fit into their packaging needs. An economic study on some of the proposed applications of whey protein coatings covered in this chapter has been assessed (Balagtas et al. 2003). Based on interest, the gloss and nut-coating applications are most likely to be accepted and commercialized, creating a potential increase of $5–22.4 million/year for the dairy industry and creating new outlets for whey. Hurdle technology to improve food safety can also take advantage of the active film function demonstrated with whey protein films.

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Hernandez, V.M., McHugh, T.H., Berrios, J., Olson, D., Pan, J., and Krochta, J.M. 2006. Glycerol content effect on the tensile properties of whey protein sheets formed by twin-screw extrusion. IFT Annual Meeting and Food Expo, Orlando. FL. Hernandez, V.M. 2007. Thermal properties, extrusion and heat-sealing of whey protein edible films. PhD Dissertation, University of California, Davis, Davis, CA Hong, S.I., and Krochta, J.M. 2003. Oxygen barrier properties of whey protein isolate coatings on polypropylene films. J. Food Sci. 68:224–228. Hong, S.I., and Krochta, J.M. 2004. Whey protein isolate coating on LDPE film as a novel oxygen barrier in the composite structure. Packag. Technol. Sci. 17:13–21. Hong, S.I., and Krochta, J.M. 2006. Oxygen barrier performance of whey protein coated plastic films as affected by temperature, relative humidity, base film and protein type. J. Food Eng. 77:736–745. Hong, S.I., Han, J.H., and Krochta, J.M. 2004. Optical and surface properties of whey protein isolate coatings on plastic films as influenced by substrate, protein concentration, and plasticizer type. J. Appl. Polym. Sci. 92:335–343. Janjarasskul, T., and Krochta, J.M. 2006. WPI film/coating with oxygen scavenging function by the incorporation of ascorbic acid. IFT Annual Meeting and Food Expo, Orlando, FL. Kern Sear, J., and Darby, J.R. 1982. Technology of Plasticizers, 1180 pp. Hoboken, NJ: John Wiley and Sons. Kinsella, J.E., and Whitehead, D.M. 1989. Proteins in whey: Chemical, physical, and functional properties. Adv. Food Nutr. Res. 33:343–427. Kinsella, J.E. 1984. Milk proteins: Physicochemical and functional properties. Crit. Rev. Food Sci. Nutr. 21:197–262. Ko, S., Janes, N.S., Hettiarachchy, N.S., and Johnson, M.G. 2001. Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes. J. Food Sci. 66:1006–1011. Kozempel, M., McAloon, A.J., and Tomasula, P.M. 2003. Drying kinetics of calcium caseinate. J. Agric. Food Chem. 51:773–776. Krochta, J.M. 1997. Edible protein films and coatings. In Food Proteins and Their Applications in Foods, edited by S. Damodaran, and A. Paaraf, p. 694. New York: Marcel Dekker. Krochta, J.M. 2002. Proteins as raw materials for films and coatings: Definitions, current status and opportunities. In Protein-Based Films and Coatings, edited by A. Gennadios, p. 672. Boca Raton, FL: CRC Press. Krochta, J.M., Baldwin, E.A., and Nisperos-Carriedo, M.O. 1994. Edible Coatings and Films to Improve Food Quality, p. 392. Boca Raton, FL: CRC Press. Krochta, J.M., and De Mulder-Johnston, C. 1997. Edible and biodegradable polymer films: Challenges and opportunities. Food Technol. 51:61–74. Lacroix, M., Le, T.C., Ouattara, B., Yu, H., Letendre, M., Sabato, S.F., Mateescu, M.A., and Patterson, G. 2002. Use of gamma-irradiation to produce films from whey, casein, and soya proteins: Structure and functionals characteristics. Radiat. Phys. Chem. 63:827–832. Lawton, J.W. 2002. Zein: A history of processing and use. Cereal Chem. 79:1–18. Le Tien, C., Vachon, C., Mateescu, M.A., and Lacroix, M. 2001. Milk protein coatings prevent oxidative browning of apples and potatoes. J. Food Sci. 66:512–516.

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Lee, J.Y., Park, H.J., Lee, C.Y., and Choi, W.Y. 2003. Extending shelf-life of minimally processed apples with edible coatings and antibrowning agents. Lebensm. Wiss. Technol. 36:323–329. Lee, S.-Y., Dangaran, K.L., and Krochta, J.M. 2002a. Gloss stability of wheyprotein/plasticizer coating formulations on chocolate surface. J. Food Sci. 67:1121– 1125. Lee, S.Y., and Krochta, J.M. 2002. Accelerated shelf-life testing of whey-proteincoated peanuts analyzed by static headspace gas chromatography. J. Agric. Food Chem. 50:2022–2028. Lee, S.Y., Dangaran, K.L., Guinard, J.X., and Krochta, J.M. 2002b. Consumer acceptance of whey-protein-coated as compared with shellac-coated chocolate. J. Food Sci. 67:2764–2769. Lin, S.Y., and Krochta, J.M. 2003. Plasticizer effect on grease barrier and color properties of whey-protein coatings on paperboard. J. Food Sci. 68:229–333. Lin, S.Y., and Krochta, J.M. 2006. Fluidized-bed system for whey protein film-coating on peanuts. J. Food Process Eng. 29:532–546. Lin, S.-Y.D., and Krochta, J.M. 2005. Whey protein coating efficiency of surfactantmodified hydrophobic surfaces. J. Agric. Food Chem. 53:5018–5023. Mahmoud, R., and Savello, P. 1992. Mechanical properties of and water vapor transferability through whey protein films. J. Dairy Sci. 75:942–946. Marcilla, A., and Beltran, M. 2002. Mechanisms of plasticizers action. In Handbook of Plasticizers, edited by G. Wypych, pp. 107–119. Norwich, NJ: Noyes Publications. Mat´e, J.I., and Krochta, J.M. 1996. Comparison of oxygen and water vapor permeabilities of whey protein isolate and beta-lactoglobulin edible films. J. Agric. Food Chem. 44:3001–3004. Mate, J.I., Frankel, E.N., and Krochta, J.M. 1996. Whey protein isolate edible coatings: Effect on the rancidity process of dry roasted peanuts. J. Food Sci. 44:1736–1740. McHugh, T.H., Avena-Bustillos, R.J., and Krochta, J.M. 1993. Hydrophilic edible films: Modified procedure for water vapor permeability and explanation of thickness effects. J. Food Sci. 58:899–903. McHugh, T.H., and Krochta, J.M. 1994a. Dispersed phase particle size effects on water vapor permeability of whey protein-beeswax edible emulsion films. J. Food Process. Preserv. 18:173–188. McHugh, T.H., and Krochta, J.M. 1994b. Sorbitol- vs. glycerol- plasticized whey protein edible films: Integrated oxygen permeability and tensile property evaluation. J. Agric. Food Chem. 42:841–845. Miller, K.S., and Krochta, J.M. 1998. Measuring aroma transport in polymer films. Trans. Am. Soc. Agric. Eng. 41:427–433. Miller, K.S., Upadhyaya, S.K., and Krochta, J.M. 1998. Permeability of d-limonene in whey protein films. J. Food Sci. 63:244–247. Min, S., and Krochta, J.M. 2006. Ascorbic acid-containing whey protein film-coatings for control of peanut oxidation. IFT Annual Meeting and Food Expo, Orlando, FL. Min, S., Harris, L.J., and Krochta, J.M. 2006a. Inhibition of Salmonella enterica and Escherichia coli O157:H7 on roasted turkey by edible whey protein coatings incorporating lactoperoxidase system. J. Food Prot. 69:784–793.

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Min, S., Rumsey, T.R., and Krochta, J.M. 2006b. Lysozyme diffusion in smoked salmon coated with whey protein films incorporating lysozyme. IFT Annual Meeting and Food Expo, Orlando, FL. Min, S., Rumsey, T.R., and Krochta, J.M. 2006c. Diffusion of thiocyanate and hypothiocyanate in whey protein films incorporating the lactoperoxidase system. IFT Annual Meeting and Food Expo, Orlando, FL. Min, S., Harris, L.J., Han, J.H., and Krochta, J.M. 2005a. Listeria monocytogenes inhibition by whey protein films and coatings incorporating lysozyme. J. Food Prot. 68:2317–2325. Min, S., Harris, L.J., and Krochta, J.M. 2005b. Antimicrobial effects of lactoferrin, lysozyme, and the lactoperoxidase system and edible whey protein films incorporating the lactoperoxidase system against Salmonella enterica and Escherichia coli O157:H7. J. Food Sci. 70:M332–M338. Min, S., Harris, L.J., Han, J.H., and Krochta, J.M. 2005c. Listeria monocytogenes inhibition by whey protein films and coatings incorporating the lysozyme. J. Food Prot. 68:2317–2325. Minifie, B.W. 1982. Chocolate, Cocoa and Confectionery: Science and Technology, 2nd ed., p. 735. Westport, CT: AVI Publishing. Morillon, V., Debeaufort, F., Blond, G., Capelle, M., and Voilley, A. 2002. Factors affecting the moisture permeability of lipid-based edible films: A review. Crit Rev. Food Sci. Nutr. 42:67–89. Morr, C.V., and Ha, E.Y.W. 1993. Whey protein concentrates and isolates: Processing and functional properties. Crit. Rev. Food Sci. Nutr. 33:431–476. Niki, E. 1991. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54:1119S–1124S. Olivas, G.I., and Barbosa-Canovas, G.V. 2005. Edible coatings for fresh-cut fruits. Crit Rev. Food Sci. Nutr. 45:657–670. Ozdemir, M., and Floros, J.D. 2003. Film composition effects on diffusion of potassium sorbate through whey protein films. J. Food Sci. 68:511–515. Ozdemir, M., and Floros, J.D. 2004. Active food packaging technologies. Crit. Rev. Food Sci. Nutr. 44:185–193. Perez-Gago, M.B., and Krochta, J.M. 1999. Water vapor permeability, solubility, and tensile properties of heat-denatured versus native whey protein films. J. Food Sci. 64:1034–1037. Perez-Gago, M.B., Serra, M., Alonso, M., Mateos, M., and Del Rio, M.A. 2003. Effect of solid content and lipid content of whey protein isolate-beeswax edible coatings on color change of fresh-cup apples. J. Food Sci. 68:2186–2191. Perez-Gago, M., and Krochta, J.M. 2000. Drying temperature effect on water vapor permeability and mechanical properties of whey protein-lipid emulsion films. J. Agric. Food Chem. 48:2687–2692. Perez-Gago, M., and Krochta, J.M. 2001. Lipid particle size effect on water vapor permeability and mechanical properties of whey protein/beeswax emulsion films. J. Agric. Food Chem. 49:996–1002. Psomiadou, E., Arvanitoyannis, I., and Yamamoto, N. 1996. Edible films made from natural resources; microcrystalline cellulose (MCC), methylcellulose (MC) and corn starch and polyols—part 2. Carbohydr. Polym. 31:193–204.

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Rogers, C.E. 1985. Permeation of gases and vapours in polymers. In Polymer Permeability, edited by J. Comyn, pp. 11–74. London, England: Kluwer Academic Publishers. Sawyer, L., Kontopidis, G., and Wu, S.-Y. 1999. Beta-lactoglobulin—a threedimensional perspective. Int. J. Food Sci. Technol. 34:409–418. Shellhammer, T.H., and Krochta, J.M. 1997. Whey protein emulsion film performance as affected by lipid type and amount. J. Food Sci. 62:390–394. Shellhammer, T.H., Rumsey, T.R., and Krochta, J.M. 1997. Viscoelastic properties of edible lipids. J. Food Eng. 33:305–350. Singh, R.P., and Heldman, D.R. 1993. Introduction to Food Engineering, 2nd ed., p. 499. San Diego, CA: Academic Press. Sloan, A.E. 2001. Top 10 trends to watch and work on. Food Technol. 55:38–58. Sothornvit, R., and Krochta, J.M. 2000a. Oxygen permeability and mechanical properties of films from hydrolyzed whey protein. J. Agric. Food Chem. 48:3913–3916. Sothornvit, R., and Krochta, J.M. 2000b. Plasticizer effect on oxygen permeability of beta-lactoglobulin films. J. Agric. Food Chem. 48:6298–6302. Sothornvit, R., and Krochta, J.M. 2000c. Water vapor permeability and solubility of films from hydrolyzed whey protein. J. Food Sci. 65:700–703. Sothornvit, R., and Krochta, J.M. 2001. Plasticizer effect on mechanical properties of beta-lactoglobulin films. J. Food Eng. 50:149–155. Sperling, L.H. 2001. Introduction to Physical Polymer Science, 3rd ed. New York: John Wiley and Sons. Tomasula, P.M., Parris, N., Yee, W., and Coffin, D. 1998. Properties of films made from CO2 -precipitated casein. J. Agric. Food Chem. 11:4470–4474. Trezza, T.A., and Krochta, J.M. 2000. The gloss of edible coatings as affected by surfactants, lipids, relative humidity, and time. J. Food Sci. 65:658–662. Trezza, T.A., and Krochta, J.M. 2001. Specular reflection, gloss, roughness and surface heterogeneity of biopolymer coatings. J. Appl. Polym. Sci. 79:2221–2229. Truong, V.-D., Clare, D.A., Catignani, G.L., and Swaisgood, H.E. 2004. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase. J. Agric. Food Chem. 52:1170–1176. Vachon, C., Yu, H.-L., Yefsah, R., Alain, R., St-Gelais, D., and Lacroix, M. 2000. Mechanical and structural properties of milk protein edible films cross-linked by heating and gamma-irradiation. J. Agric. Food Chem. 48:3202–3209. Walzem, R.L., Dillard, C.J., and German, J.B. 2002. Whey components: Milennia of evolution create functionalities for mammalian nutrition: What we know and what we may be overlooking. Crit. Rev. Food Sci. Nutr. 42:353–375. Wilcock, A., Pun, M., Khanona, J., and Aung, M. 2004. Consumer attitudes, knowledge and behaviour: A review of food safety issues. Trends Food Sci. Technol. 15:56–66.

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Chapter 7 Whey Texturization for Snacks Lester O. Pordesimo and Charles I. Onwulata

Introduction To improve the nutritional profile of crunchy snacks, incorporation of whey proteins in their processing is an idea that has been widely broached and continues to be researched. The general perception is that snack foods, many of which are starch-based, high-energy, low-nutrientdense foods, are principal contributors to the higher incidence of obesity and diabetes worldwide. With surging health consciousness among consumers (O’Donnell and O’Donnell 2006), whey proteins have become the protein of choice for the nutritional enhancement of food products because of the accumulating body of evidence supporting impressive health benefits of whey proteins coupled with the suitability of whey proteins for a wide range of food applications (Berry 2006). The ascension of whey proteins to this level of application was highlighted by the 2005 International Whey Conference objective of showcasing whey protein as a value-added ingredient with tremendous health and nutritional benefits. Moreover, fortifying crunchy snacks with whey proteins presents an avenue for both increased and higher value utilization of whey proteins. This potentially increases the utilization of whey products in foods, which is still below 50% of total production (American Dairy Products Institute 2005). Many crunchy snacks are produced through extrusion; some would be simply extruded snacks while others would be categorized as fabricated snacks. Fabricated snacks are textured snacks resulting from the processing of mixtures with potato and other starchbased ingredients. In a broad sense, this would include first-generation (direct-expanded) snacks, such as corn curls, second-generation snacks (half products or pellets, coextruded products, masa-based snacks, and 169 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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crispbreads) (Lusas and Rooney 2001). Since blending (mixing) is accomplished within the extrusion process, there already exists an avenue for protein fortification of extruded snacks that can be accomplished directly at formulation and actual product production as opposed to doing this postproduction like with coatings. The focus of this chapter is on the fortification of crunchy snacks with whey proteins.

Benefits of Whey Proteins Whey, the coproduct of the cheese-making process, is a source of high-quality protein that provides all the essential amino acids necessary for good health. Whey protein has the highest bioavailability, or protein efficiency ratio, of any protein, which means the human body can more efficiently metabolize this protein. Being one of the richest sources of bioactive materials, whey protein has many benefits beyond basic nutrition. Accumulating evidence suggests that whey proteins may have beneficial effects for other health concerns, including cancer, kidney disease, osteoporosis, cognitive function, obesity, and possibly lowering the potential for insulin resistance in diabetics. Bioactive components derived from whey fractions, such as immunoglobulins, glycomacropeptides, and whey-derived minerals are reported to have specific health benefits, such as enhanced immune function and antioxidant activity, relief of metabolic stress, positive stress responses, improved muscle functionality, greater strength, and improved general health. The major whey protein components αlactalbumin and β-lactoglobulin contain many bioactive sequences which show angiotensin-converting enzyme (ACE) inhibitory activity. Also, regulatory peptides released by enzymatic proteolysis of whey proteins are potential modulators of intestinal digestion of foods, and may provide healthful benefits such as boosting the immune systems and antihypertensive activities (Philanto-Leppala 2001). Over the immediate past few years, obesity and weight management have become major issues for health professionals in the United States. Nutrition is certainly involved in this issue and a contributing dietary habit to this trend may be the increasing consumption of low-nutrientdense, high glycemic snacks, such as corn puffs or potato chips, which are mostly carbohydrates. Glycemic index measures the rapid increases

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in blood sugar following starch consumption. In 2001, the U.S. Surgeon General called for the prevention and decrease of obesity by changing the dietary habits of U.S. consumers and combining this with regular physical activity. The former can also be achieved by taking advantage of dietary habits and improving the nutritional profile of the crunchy snack foods compulsively consumed by consumers because of their convenience, widespread availability, and eating satisfaction. Blending corn or other edible starches with whey proteins creates crunchy snack foods that are lower in carbohydrates and have a better nutritional balance. These nutritionally enhanced snacks may even become a component of weight management diets. In a way, this strategy is already supported by the change in U.S. foodservice regulations taking effect in March 2000 allowing the use of whey proteins and certain other dairy ingredients as alternate components for meat products in the National School Lunch Program in the United States (Federal Register, March 9, 2000). Research has shown that diets with a reduced ratio of carbohydrates to protein are beneficial for weight loss; also, the role of leucine, a branched chain amino acid (BCAA), in weight loss diets and glucose management has also been reported (Wester et al. 2000). Whey proteins contain higher levels of both BCAAs and leucine (26 g and 14 g, respectively, per 100 g of protein) than are found in any other food protein (muscle: 18 g and 8g, soy: 18 g and 8 g, or wheat: 15 g and 7 g) (Layman 2003). Studies in whey protein show that in isocaloric diets, after 16 weeks with the combined effect of diet plus exercise, the subjects in the protein group lost significantly more weight than the subjects in the carbohydrate group (Layman 2003). Superior metabolism of whey proteins, which are rich in the sulfurcontaining amino acids cysteine and methionine, has made them the protein of choice by athletes seeking to maintain and/or bulk body mass in order to achieve enhanced sports performance. Taking off from this, various whey protein ingredients have been added to commercially available nutritional products for the everyday athlete who wants to be in a better physical shape to achieve enhanced physical performance. The fact that whey proteins are abundant, cost-effective ingredients, and have a neutral taste has also contributed to their wide use. Recent studies have confirmed the ability of whey proteins or amino acid mixtures with a composition similar to whey protein to promote whole body and muscle protein synthesis (Ha and Zemel 2003).

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Processing Whey proteins can be directly processed into crunchy snacks from the point of formulation through two pathways involving twin-screw extrusion. The first approach, resulting in an essentially finished product, is to blend whey proteins and cereal carbohydrates, such a corn or wheat starch, through twin-screw extrusion under high shear at moderate temperatures. The product is finished by a gentle drying to produce the snack. This has been a subject of many research efforts (Aguilera and Kosikowski 1978; Harper 1986; Kim and Maga 1987; Singh et al. 1991; Smietana et al. 1998). The second approach involves first texturizing the whey proteins in a preliminary process to produce ingredients that would have better functionality in a fabricated snack product produced in a subsequent extrusion step. Texturized proteins are defined as food products made from edible protein sources characterized by having structural integrity and identifiable texture that enables them to withstand hydration in cooking and other preparations (Liu 1997). Texturized whey proteins (TWP) have been shown to work nicely in directly expanded snacks. Direct Extrusion Twin-screw extrusion to combine whey proteins and carbohydrate food polymers such as corn or wheat starches in crunchy snacks has been demonstrated (Aguilera and Kosikowski 1978; Harper 1986; Holay 1982; Kim and Maga 1987; Matthey and Hanna 1997; Singh et al. 1991; Smietana et al. 1998). Twin-screw extrusion can enhance mechanical energy transfer which could minimize the negative textural effects of whey protein inclusion in snack products produced through single-screw extrusion (Barres et al. 1990; Edemir et al. 1992). However, supplementation with native whey proteins (term used by Kester and Richardson (1984) and Martinez-Serna and Villota (1992) to refer to widely available ingredients that are the direct result of concentrating protein from whey) has been limited to no more than 10% of the main starch ingredient due to adverse effects on the texture of the final product. Interactions of lipids, protein, and starch, which occur during extrusion at temperatures ranging from 80 to 150◦ C, resulted in the loss of protein quality through Maillard reactions, discoloration of product, and loss of texture from the production of dextrinized starch (Camire 1990).

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A negative deviation in the mechanical properties and structures of whey protein-fortified snack products from existing commercial products has also been reported (Alavi et al. 1999; Gogoi et al. 2000). The interaction of protein and starch matrices fundamentally changes the character of products by decreasing brittleness, making the snack products unacceptable (Gi-Hung 1997; Sokhey et al. 1994). Reduction in product quality is caused by the collapse of whey proteins within the starch matrix, resulting in reduced expansion and increased hardness (Kim and Maga 1987; Smietana et al. 1998). The result is that such nonexpanded products are not acceptable to a sensory or consumer panel (Onwulata and Heymann 1994). Research efforts by Onwulata and his coworkers focused on using twin-screw extrusion to incorporate whey proteins into expanded snack products to increase their protein content, for example, increasing the protein content of corn puffs from 2 to 20%. The aim of whey protein supplementation is to improve the nutritive content of expanded snacks while maintaining the quality of expanded snacks that has become expected in the marketplace by the careful adjustment of extrusion process variables that affect expanded snacks (Booth 1990; Lusas and Rooney 2001). Whey proteins in the form of whey protein concentrate (WPC34) and sweet whey solids were extruded with corn meal, wheat starch, and rice and barley flours substituting 15–35% of the carbohydrate (Onwulata et al. 2001) (Figure 7.1). By controlling the extrusion processes—operating at high shear, high temperatures (100–140◦ C), and low moisture (10–15%)—up to 25% of the carbohydrate was substituted with WPC to obtain a fair product (Onwulata et al. 1998). The products were brittle and crunchy but not expanded. The whey protein supplemented products were not as puffed as the straight corn product because the whey proteins held water and collapsed within the starch matrices. Some of the formulated products were also dense and discolored suffering in quality due to the Maillard

Figure 7.1. Shows some of these extruded snacks.

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reaction. Matthey and Hanna (1997) working also with WPC and starch blends similarly encountered reduced expansion and increased hardness in their test products. Working with whey protein isolate (WPI), Martinez-Serna and Villota reported a 30% reduction in expansion ratio due to the addition of 20% WPI. Most food extrusion cooking is thermal, high-temperature, short-time processing, with most of the energy coming from friction and the heated barrels (Harper 1986). The heat is needed to convert water into superheated steam at high pressure that then produces the puffed products. The high temperature needed to puff the products (120–170◦ C) is counterintuitive to the inclusion of whey and other dairy proteins which are heat sensitive. Beneficial nutrients such as vitamins and carotenoids containing input ingredients in the extruder are also degraded because of the high process temperatures (Ilo and Berghofer 1998; Lee et al. 1978). Additionally, high-temperature, short-time extrusion of proteins causes greater wear on the extruder screws and barrel, thereby shortening the maintenance and replacement service cycle. A solution to the problem of including heat labile ingredients into directly expanded extrusion products is to use supercritical CO2 as a puffing agent. Supercritical fluid extrusion processing (SCFX) developed by Rizvi and Mulvaney (1993) at Cornell University allows for the production of puffed snacks with extruder at temperatures less than 100◦ C. Through this modified extrusion process they were able to produce a product containing 40–60% WPC34 that still had an expanded and crispy texture (Alavi et al. 1999; Gogoi et al. 2000; Sokhey et al. 1996). Gi-Hung (1997) was also able to achieve the same results.

Texturization Protein texturization is the process of imparting a structure to proteinaceous food ingredients so that visible forms such as fibers or crumbles are created. Texturization involves the restructuring of the protein molecules into a layered crosslinked mass which is resistant to disruption upon further heating and/or processing (Harper 1986). Whey proteins could be texturized or denatured before their inclusion in food products, especially those produced through cooking, such as by extrusion, to minimize the effects of further heat processing and/or to have some (hypothetically better) control on the extent of heat denaturation. Kester and Richardson (1984) had proposed that subjecting native whey

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proteins to thermal conditions that promote only partial denaturation may be a practical way to produce a unique and desirable blend of functional properties. Also, partial denaturation, or combining partially denatured whey protein with native protein, was suggested by Ryan (1977) as a technique for intentional modification of functionality. Texturization causes the whey proteins to interact together to form macroscopic three-dimensional structures and alters their chemical and physical reactivity with other ingredients in a food product, particularly food polymers. Texturized proteins are defined as food products made from edible protein sources characterized by having structural integrity and identifiable texture that enables them to withstand hydration in cooking and other preparations (Lockmiller 1972). Textured or texturized products are a combination of proteins and starches such as soy flour modified by different structure-inducing means, mostly by extrusion processing, to create chewy or stringy texture (Shen and Morr 1979). Texturization of proteins can be accomplished through several different processes (Harper 1986), but extrusion has the advantages of process simplicity and the involvement of less equipment. Several food processing unit operations are accomplished simultaneously in the extruder, by just a single piece of equipment. For these reasons, extrusion has seemingly been the favored process starting with the texturization of soy proteins.

Cooking Extrusion Extrusion at elevated temperatures and high moistures serves to unfold, denature, and crosslink the proteins into a new molten state (Harper 1979), imparting fibrous structure and improving such textural characteristics as elasticity, chewiness, and toughness. These dense fibrous structures are created from such globular proteins as whey protein or soy protein under high moisture conditions (Shen and Morr 1979). Commercially, it is mainly through this extrusion process that soy concentrates are transformed into a texturized product resembling meat in texture and form. Walsh and coworkers have employed similar techniques to create whey protein-fortified meat analogs (see Chapter 8). It is the extensive body of knowledge in texturizing soy proteins since the late 1960s that is the foundation for research in the texturization of whey proteins. The use of extrusion to texturize soy protein for their

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use as meat extenders (Atkinson 1970) has long been recognized as one of the most significant developments in food processing. The process, products, and applications of texturized soy proteins have been discussed in many reviews and articles (Harper 1986; Kearns et al. 1989; Kinsella 1978; Lusas and Riaz 1996; Noguchi and Isobe 1989; Rhee et al. 1981). The result of shearing by extrusion at elevated temperatures is the formation of fibrous networks. Harper (1979) explained that these networks are formed through disulfide bonds, and crosslinking of protein chains, through amide bonds between free-carboxyl and amino side groups on the protein chains. Early work on the extrusion of whey proteins such as that by Queguiner et al. (1992) was limited to the coagulation of whey proteins as the endpoint. These led to investigations in the use of extrusion to create whey protein gels (Martinez-Serna and Villota 1992). Extruding WPI at an extruder screw speed of 150 rpm and barrel temperatures ranging from 20 to 110◦ C resulted in a firm, spread-like thermocoagulated gel (Queguiner et al. 1992; Szpendowski et al. 1994; Wolkenstein 1988). The extruded whey gels are used in low-calorie substitute foods such as cheese spreads and ice cream (Cheftel et al. 1992; Fichtali et al. 1995; Queguiner et al. 1992). There is a substantial body of knowledge on twin-screw extrusion of casein that also provides insight into and serves as a reference basis for the extrusion of whey proteins (Barraquio and Van De Voort 1991; Cavalier et al. 1990; Fichtali et al. 1995; Suchkov et al. 1988; Van De Voort et al. 1984). Some studies, such as that of Mulvaney et al. (1997) and Visser (1988), were limited to the development of continuous extrusion processes to convert casein to caseinates. Cavalier et al. (1990) developed a process for the manufacture of cheese analogs using twinscrew texturization processing. Casein is generally extruded at temperatures as low as 80◦ C to create base products for the dry spinning process. Caseinate extrusion and process conditions have been adapted to develop the parameters for extrusion cooking of cheddar cheeses (Mulvaney et al. 1997). Referencing information on the extrusion of both soy and dairy proteins, Onwulata and his coworkers at USDAARS and Walsh and her coworkers at Utah State University separately developed twin-screw extrusion processes for producing TWP (Onwulata and Tomasula 2004a, b; Walsh and Carpenter 2003). Their research constitutes the more recent efforts in whey protein texturization reported in literature. Kester and Richardson (1984) discussed that modification

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Extrusion melt temperatures of whey proteins.

Product

Melt temperature (◦ C)

Pre-extrusion (%)

Post-extrusion (%)

WPC80 WLAC WPI

70b 75a 74a

40.9b 68.7a 28.0a

59.9b 94.4a 94.8a

WPC80: whey protein concentrate, 80% protein; WLAC: whey lactalbumin; WPI: whey protein isolate: number reported is mean of three samples. Means with different letters within a column are significantly ( p < 0.05) different.

of whey proteins to improve functionality can be accomplished by chemical, enzymatic, or physical means. They noted further that the physical means of changing the whey protein’s functional performance could be achieved through thermal treatment, biopolymer complexing, or texturization. By employing a combination of thermomechanical treatment, by means of extrusion, and biopolymer complexing to achieve a physical modification of whey proteins and a consequential change in their functionality, the efforts of these two research groups have involved a combination of all methods of physically modifying proteins noted by Kester and Richardson (1984). Whey texturization work was the offshoot of the efforts to directly include whey proteins in finished crunchy extruded products (Onwulata et al. 1998). Initial efforts involved biopolymer complexing WPC34 and sweet whey solids (Onwulata et al. 2001). Extruded products containing up to 65% starch and 35% whey proteins were created by extruding at low moisture (Onwulata and Tomasula 2004a). More recent research efforts involved extruding whey protein concentrate (WPC80), whey lactalbumin, and WPI unblended with any cereal carbohydrate (Onwulata et al. 2006a, b; Onwulata and Tomasula 2004b). Extruding the whey proteins at a cook temperature below 100◦ C, as a case in point, resulted in varying degrees of melt temperatures and denaturation of the different whey protein products (Table 7.1). WPC80 was the least denatured after extrusion while lactalbumin and WPI were more significantly denatured. Varying extrusion cook temperature allowed a new controlled rate of denaturation, indicating that a texturized ingredient with a predetermined functionality based on the degree of denaturation could be created. Thermally denatured WPI is a unique ingredient that has the potential to be used in large amounts.

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The process they developed to create textured WPIs is a modification of that used for manufacturing texturized vegetable proteins described by Snyder and Kwon (1987). Walsh and Carpenter (2003) developed a process for directly texturing WPC80 with an edible biopolymer such as cornstarch in the extruder. Their major adjustments to the extrusion process to produce the patented product called Texturized Whey Protein (TWP) included varying extruded section temperatures, changing screw and paddle configuration, and fitting a cooling die to a twin-screw extruder to cause the alignment of proteins in the melted dough. This extruded product blend containing 20–40% starch and 60–80% WPC can be added in as ingredients for puffed snacks or serve as meat extenders. Walsh and coworkers have however focused their research and development efforts on utilizing this product as a meat extender (see Chapter 8). Based on a consumer study, it was found that beef patties extended with up to 40% TWP and cornstarch were as acceptable to consumers as 100% beef patties (Hale et al. 2002; Taylor and Walsh 2002). In addition, physical and instrumental analysis showed that the patties had less cook loss, diameter reduction, and change in thickness than all beef patties, and were not easily distinguishable from 100% beef.

Cold Extrusion With the elevated temperatures in cooking extrusion, the Maillard reaction causes discoloration of whey proteins, especially WPI. Furthermore, severe food processing conditions such as high temperatures and extreme pH changes induce transformations and racemization of proteins during crosslinking destroying protein nutritive qualities (Friedman 1999) in the process of intentionally altering the protein functionality. These are drawbacks with texturization through high temperature cooking extrusion. Walkenstrom and Hermansson (1997) showed that shear alone was adequate to induce structuring of particulate whey to create gels. Most foods that are extruded actually undergo gelling before being shaped upon their exiting through the die. This is the basis for cold extrusion to effect whey protein texturization. Cold or nonthermal extrusion should minimize the loss of protein quality caused by high heat reactions (Camire 1990). In cold extrusion, the molten gel temperatures are not achieved and only shear-induced gels, which are similar to

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cold-set gels, are encountered (Cho et al. 1995). This makes mechanical energy input a critical factor in nonthermal extrusion. In their review of cold denaturation of proteins under high pressure, Kunugi and Tanaka (2002) pointed out that cold-denatured proteins are in a new state, similar to the molten globular state of heat-denatured proteins. Cold denaturation also follows a two-step process: primary disassociation and unfolding and secondary refolding and realignment of the native protein. The most stable results were at 10◦ C with results at temperatures greater than 54◦ C proving to be unstable. Bryant and McClements (1998) reviewed the molecular basis of protein functionality with a special consideration of cold-set gels derived from denatured whey and showed unique functionalities such as increased gelation, thickening, and water-binding. To create cold-set gels, whey proteins are first denatured by heating to achieve unfolding and aggregation, before quickly cooling to form gels (Resch and Daubert 2002). The particular application for these cold-set gels is in comminuted meats, fish products, desserts, sauces, and dips. The technology of cold extrusion cooking is relatively new and offers the food industry the opportunity to modernize, shorten their processing time, and ultimately achieve significant cost savings (Reifsteck and Jeon 2000). Extrusion is termed nonthermal or cold extrusion cooking when process temperature is kept below 50◦ C. Nonthermal extrusion creates favorable conditions such as diminished thermal effect, increased viscosity, and shear (Cho et al. 1995). The literature on cold extrusion is extremely limited (Beckett et al. 1994; Cho et al. 1995; Osburn et al. 1995). Beckett et al. (1994) reported the use of extrusion shear to plasticize milk chocolate isothermally below its normal melting point (27– 32◦ C), showing that a continuous cold extrusion process can be used to produce a textured milk product. Cho et al. (1995) used cold extrusion to develop natural flavors in a starch and methionine or cysteine matrix. Methionine and cysteine would have been destroyed by hightemperature extrusion. Osburn et al. (1995) showed that cold extruded restructured porcine protein products had desirable sensory and textural properties resulting from partial realignment of muscle fibers. In preliminary experiments, Onwulata and coworkers found that WPIs are denatured within 45–90 s when extruded in a twin-screw extruder at 50◦ C. Also, they found that the degree of denaturation (texturization), ranging from 40 to 90% of WPI, might be adjusted through the proper selection of such extrusion conditions as moisture, temperature, and shear rates. They anticipated that because of the low extrusion

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temperatures the denatured or texturized WPI would maintain its nutritive quality, and may then be subsequently extruded together with corn or wheat flour to make puffed snack products without collapsing them. Subsequent work has shown that this is so. Summary and Conclusions The direct nutritional and health benefits of adding whey proteins to extruded crunchy snacks that are characteristically low in protein is to improve their overall protein profile and boost their nutritional value. Successful incorporation of whey proteins into extruded snack products will also enhance their consumer appeal in a consumer market environment that has become and will continue to be very health conscious. Although progress has been made in directly extruding native whey proteins with carbohydrates to form crunchy snacks, generally speaking, the quality of formulations with the desired higher levels of whey protein inclusion has not compared favorably with the snacks with no added protein. Texturization, which involves a restructuring of the whey proteins into a layered crosslinked mass which is resistant to disruption upon further heating and/or processing, apparently is the means to including whey proteins in crunchy snacks at higher inclusion levels. Research efforts have demonstrated that extrusion processing is an effective method for denaturing whey proteins to create texturized products. Extrusion processing denatured WPC, whey lactalbumin, and WPI, with the greatest amount of denaturing occurring with WPI. Denatured WPI retained its native protein value, functionality, and digestibility when extruded at 50◦ C or below; changes in functionality occurred at 75 and 100◦ C. Through the selection of extrusion conditions, denatured whey proteins with unique functionalities were produced. It is highly possible that a better understanding of texturization and the process variables affecting this could lead to the development of textured whey protein tailor made for specific food applications. References Aguilera, J.M., and Kosikowski, F.V. 1978. Soybean extruded products: A response surface analysis. J. Food Sci. 41:1200–1212.

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Alavi, S.H., Gogoi, B.K., Khan, M., Bowman, B.J., and Rizvi, S.S.H. 1999. Structural properties of protein-stabilized starch-based supercritical fluid extrudates. Food Res. Int. 32:107–118. American Dairy Products Institute. 2005. Dairy Products: Utilization and Production Trends 2004. Elmhurst, IL: American Dairy Products Institute. Atkinson, W.T. 1970. Meat-like protein food products. U.S. Patent 3,488,770. Barraquio, V.L., and Van De Voort, F.R. 1991. Sodium caseinate from skim milk powder by extrusion processing: Physicochemical and functional properties. J. Food Sci. 56:1552–1556, 1561. Barres, C., Vergnes, B., Tayeb, J., and Della Valle, G. 1990. Transformation of wheat flour by extrusion cooking: Influence of screw configuration and operating conditions. Cereal Chem. 67:427–433. Beckett, S.T., Craig, M.A., Gurney, R.J., Ingleby, B.S., Mackley, M.R., and Parsons, T.C.L. 1994. The cold extrusion of chocolate. Food Bioprod. Process. 72:47–54. Berry, D. 2006. The future for dairy proteins. Dairy Foods 107:34–36, 38, 40, 42. Booth, R.G. 1990. Snack Food. New York: Van Nostrand Reinhold Company. Bryant, C.M., and McClements, D.J. 1998. Molecular basis of protein functionality with special consideration of cold-set gels derived from heat-denatured whey. Trends Food Technol. 9:143–151. Camire, M.E. 1990. Chemical and nutritional changes in foods during extrusion. Crit. Rev. Food Sci. Nutr. 29:35–57. Cavalier, C., Queguiner, C., Dumay, E., and Cheftel, J.C. 1990. Preparation of cheese analogs by extrusion cooking. In Processing and Quality of Foods, Vol. 1, edited by P. Zeuthen et al., pp. 373–383. London: Elsevier Applied Science. Cheftel, J.C., Kitagawa, M., and Queguiner, C. 1992. New protein texturization processes by extrusion cooking at high moisture levels. Food Rev. Int. 8:235–275. Cho, M.H., Zheng, X., Wang, S.S., Kim, Y., and Ho, C.T. 1995. Production of natural flavors using a cold extrusion process. In Flavor Technology: Physical Chemistry, Modification, and Process. ACS Symposium Series 610, pp. 120–128. Washington, DC: American Chemical Society Edemir, M.M., Edwards, R.H., and McCarthy, K.L. 1992. Effect of screw configuration on mechanical energy transfer during twin-screw extrusion of rice flour. Lebensm.Wiss.u-Technol. 25:502–508. Fichtali, J., Van-De-Voort, F.R., and Diosady, L.L. 1995. Performance evaluation of acid casein neutralization process by twin-screw extrusion. J. Food Eng. 26:301–318. Field, A.L., Austin, S.B., Gillman, M.W., Rosner, B., Rockett, H.R., and Colditz, G.A. 2004. Snack food intake does not predict weight change among children and adolescents. Int. J. Obes. Relat. Metab. Disord. 28:1210–1216. Friedman, M. 1999. Chemistry, nutrition, microbiology of D-amino acids. J. Agric. Food 47:3457–3479. Gi-Hung, R., and Mulvaney, S.J. 1997. Analysis of physical properties and mechanical energy input of cornmeal extrudates fortified with dairy products by carbon dioxide injection. Korean J. Food Sci. Technol. 29:947–954. Gogoi, B.K., Alavi, S.H., and Rizvi, S.S.H. 2000. Mechanical properties of proteinstabilized starch-based supercritical fluid extrudates. Int. J. Food Prop. 3:37–58.

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Ha, E., and Zemel, M.B. 2003. Functional properties of whey, whey components, essential amino acids: Mechanisms underlying health benefits for active people (review). J. Nutr. Biochem. 14:251–258. Hale, A.B., Carpenter, C.E., and Walsh, M.K. 2002. Instrumental and consumer evaluation of beef patties extended with extrusion-textured whey proteins. J. Food Sci. 67:1267–1270. Harper, J.M. 1979. Extruder not prerequisite for texture formation. J. Food Sci. 44:97– 100. Harper, J.M. 1986. Extrusion texturization of foods. Food Technol. 40:70–76. Holay, S.H.A.H. 1982. Influence of the extrusion shear environment on plant protein texturization. J. Food Sci. 47:1869–1875. Ilo, S., and Berghofer, E. 1998. Kinetics of thermochemical destruction of thiamin during extrusion cooking. J. Food Sci. 63:312–316. Kearns, J.P., Rokey, G.J., and Huber, G.R. 1989. Extrusion of texturized proteins. In Proceedings of the World Congress: Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, edited by T.H. Applewhite, p. 353. Champaign, IL: American Oil Chemists’ Society. Kester, J.J., and Richardson, T. 1984. Modification of whey proteins to improve functionality. J. Dairy Sci. 67:2757–2774. Kim, C.H., and Maga, J.A. 1987. Properties of extruded whey protein concentrate and cereal flour blends. Lebensm. Wiss. U -Technol. 20:311–318. Kinsella, J.E. 1978. Texturized proteins: Fabrication, flavoring and nutrition. Crit. Rev. Food Sci. Nutr. 10:147–207. Kunugi, S., and Tanaka, N. 2002. Cold denaturation of proteins under high pressure. Biochim. Biophys. Acta—Protein Struct. Mol. Enzymol. 1595:329–344. Layman, D.K. 2003. The role of leucine in weight loss diets and glucose homeostatsis. J. Nutr. 133:261S–267S. Lee, T.C., Chen, T., Alid, G., and Chichester, C.O. 1978. Stability of vitamin A and provitamin A (carotenoids) in extrusion cooking process. AIChE J. 74:192–195. Liu, K. 1997. Soybeans: Chemistry, Technology and Utilization. London: Chapman and Hall. Lockmiller, N.R. 1972. Texture protein products. Food Technol. 26:56. Lusas, E.W., and Riaz, M.N. 1996. Texturized food proteins from fullfat soybeans at low cost. Extrusion Commun. 9:15–18. Lusas, E.W., and Rooney, L.W. 2001. Snack Foods Processing. Lancaster, PA: Technomic Publishing. Martinez-Serna, M.D., and Villota, R. 1992. Reactivity, functionality, extrusion performance of native and chemically modified whey. In Food Extrusion Science and Technology, edited by J.L. Kokini et al., pp. 387–414. New York: Marcel Dekker. Matthey, F.P., and Hanna, M.A. 1997. Physical and functional properties of twin-screw extruded whey protein concentrate-corn starch blends. Lebens.-Wiss. U.-Technol. 30:359–366. Mulvaney, S., Rong, S., Barbano, D.M., and Yun, J.J. 1997. Systems analysis of the plastication and extrusion processing of Mozzarella cheese. J. Dairy Sci. 80:3030– 3039.

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Noguchi, A., and Isobe, S. 1989. New food proteins, extrusion process and products in Japan. In Proceedings of the World Congress: Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, edited by T.H. Applewhite, p. 375. Champaign, IL: American Oil Chemists’ Society. O’Donnell, J.A., and O’Donnell, C.D. 2006. Building better foods and supplements. Prepared Foods 175:NS3–NS4, NS6, NS8, NS10–NS11. Onwulata, C.I., and Heymann, H. 1994. Sensory properties of extruded corn meal related to the spatial distribution of process conditions. J. Sens. Stud. 9:101–112. Onwulata, C.I., and Tomasula, P.M. 2004a. Whey texturization: A way forward. Food Technol. 58:50–54. Onwulata, C.I., and Tomasula, P.M. 2004b. Processes for creating textured whey protein products. In Proceedings of the Fourth International Whey Conference, pp. 221– 234. Onwulata, C.I., Konstance, R.P., Smith, P.W., and Holsinger, V.H. 1998. Physical properties of extruded products as affected by cheese whey. J. Food Sci. 63: 814–818. Onwulata, C.I., Konstance, R.P., Smith, P.W., and Holsinger, V.H. 2001. Incorporation of whey products in extruded corn, potato or rice snacks. Food Res. Int. 34:679–687. Onwulata, C.I., Konstance, R.P., Cooke, P.H., and Farrell, H.M., Jr. 2006a. Functionality of extrusion—texturized whey proteins. J. Dairy Sci. 86:3775–3782. Onwulata, C.I., Isobe, S., Tomasula, P.M., and Cooke, P.H. 2006b. Properties of whey protein isolates extruded under acidic and alkaline conditions. J. Dairy Sci. 89:71– 81. Osburn, W.N., Mandigo, R.W., and Kuber, P.S. 1995. Utilization of twin screw cold extrusion to manufacture restructured chops from lower-valued pork. Bulletin #94219-A. University of Nebraska, College of Agriculture Extension and Home Economics, Lincoln, NE. Philanto-Leppala, A. 2001. Bioactive peptides derived from bovine whey proteins: opiod and aceinhibitory peptides. Trends Food Sci. Technol. 11:347–356. Queguiner, C., Dumay, E., Salou-Cavalier, C., and Cheftel, J.C. 1992. Microcoagulation of a whey protein isolate by extrusion cooking at acid pH. J. Food Sci. 57: 610–616. Reifsteck, B.M., and Jeon, I.J. 2000. Retension of volatile flavors in confections by extrusion processing. Food Rev. Int. 16:435–452. Resch, J.J., and Daubert, C.R. 2002. Rheological and physicochemical properties of derivatized whey protein concentrate powders. Int. J. Food Prop. 5:419–434. Rhee, K.C., Kuo, C.K., and Lusas, E.W. 1981. Texturization. ACS Symp. Ser. 147:52. Rizvi, S.S.H., and Mulvaney, S.J. 1993. Extrusion processing with supercritical fluids. Food Technol. 43:74, 76–82. Ryan, D.S. 1977. Determinants of functional properties of proteins and protein derivatives in foods. In Food Proteins: Improvement Through Chemical and Enzymatic Modification, edited by R.E. Feeney, and J.R. Whitaker. Advances in Chemistry Series No. 160. Washington, DC: American Chemical Society. Shen, J.L., and Morr, C.V. 1979. Physicochemical aspects of texturization: Fiber formation from globular proteins. J. Am. Oil Chem. Soc. 56:638–708.

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Singh, R.K., Nielsen, S.S., Chambers, J.V., Martinez-Serna, M., and Villota, R. 1991. Selected characteristic of extruded blends of milk protein raffinate of nonfat dry milk with corn flour. J. Food Proc. Preserv. 15:285–302. Smietana, Z., Formal, L., Szpendowski, J., and Soral-Smietana, M. 1998. Utilization of milk protein and cereal starches to obtain coextrudates. Nahrung 32:545–552. Snyder, H.E., and Kwon, T.W. 1987. Protein products, In Soybean Utilization, edited by H.E. Snyder, and T.W. Kwon. New York: Van Nostrand Rheinhold Company. Sokhey, A.S., Kollengode, A.N., and Hanna, M.A. 1994. Screw configuration effects on cornstarch expansion during extrusion. J. Food Sci. 59:895–898, 908. Sokhey, A.S., Rizvi, S.S.H., and Mulvaney, S.J. 1996. Application of supercritical fluid extrusion to cereal processing. Cereal Foods World 41:29–34. Suchkov, V.V., Grinberg, V., Muschiolik, G., Schmandke, H., and Tulstoguzov, V.B. 1988. Mechanical and functional properties of anisotropic gel fibers obtained from two-phase system of water–casein–sodium alginate. Nahrung 32:661–668. Szpendowski, J., Smietana, Z., Chojnowski, W., and Swigon, J. 1994. Modification of the structure of casein preparations in the course of extrusion. Nahrung 37:1–4. Taylor, B.J., and Walsh, M.K. 2002. Development and sensory analysis of a textured whey protein meatless patty. J. Food Sci. 67:1555–1558. Van De Voort, F.R., Stanley, D.W., and Edamura, R. 1984. Improved utilization of dairy proteins: Coextrusion of casein and wheat flour. J. Dairy Sci. 67:749–758. Visser, J. 1988. Dry spinning of milk protein. In Food Structures—Its Creation and Evaluation, edited by J.M.V. Blanshard, and J.R. Mitchell, pp. 197–218. London: Butterworth-Heinemann. Walkenstrom, P., and Hermansson, A.M. 1997. Mixed gels of gelatine and whey proteins formed by combining temperature and high pressure. Food Hydrocoll. 11:457– 470. Walsh, M.K., and Carpenter, C.E. 2003. Textured whey protein product and method. U.S. Patent 6,607,777. Wester, T.J., Lobley, G.E., Birnie, L.M., and Lomax, A.X. 2000. Insulin stimulates phenylalanine uptake across the hind limb in feed lambs. J. Nutr. Biochem. 130:608– 611. Wolkenstein, M. 1988. CALO fats, cholesterol and calories. In Low-Calories Products, edited by G.G. Birch, and M.G. Lindley, pp. 43–61. London: Elsevier Applied Science.

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Chapter 8 Whey Protein-Based Meat Analogs Marie K. Walsh and Charles E. Carpenter

Overview of Meat Analogs and Extenders Extrusion cooking has been used for processing many kinds of foods including cereals, snacks, pet foods, and texturized products from proteins. The most popular raw material for the production of texturized vegetable proteins in an extrusion system has been defatted soy flour. Soy flour (50% protein, 30% carbohydrate) is the largest source for the manufacture of textured protein products worldwide. The U.S. soyfoods market is valued at over $4 billion with the meat alternative category accounting for 14% of sales (Golbitz 2006). Other vegetable protein sources that have also been extrusion-textured, individually or in blends, for meat extenders/analogs include defatted wheat gluten, corn, rice, sesame flour, conola, rapeseed concentrates, and peanut flour. Consumers can find texturized vegetable protein in forms such as bacon bits, pepperoni, Canadian bacon, sliced lunch meats, sausages, patties, and nuggets. Texturized vegetable protein analogs are sought after by those looking for a healthier alternative (no cholesterol, low fat) to meat, who for religious reasons may be vegetarians or vegans, or are concerned about the microbial safety of meat products. Once only sold in health food stores, alternative meat products can now be found in all supermarkets and club stores. A large market for texturized vegetable protein exists in its use as an extender in U.S. schools and military. Prior to 2000, the type of protein used in the U.S. School Lunch Program was termed “textured vegetable protein” and generally consisted of textured soy protein (TSP) and the amount used was limited to 30% as a meat extender. In the new Code of Federal Regulations (CFR) the name was changed from “vegetable protein products” to 185 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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“alternate protein products” to remove the requirement that the protein source only be of plant origin. The biological quality of the protein in the alternate protein product must be at least 80% that of casein and must contain at least 18% protein when fully hydrated or formulated. Schools, institutions, and service institutions may use a single type of meat alternative product or a blend of meat alternate products to meet 100% of the meat/meat alternative requirements. The restriction that the alternate protein product could only constitute 30% of the meat component was removed in the revised CFR. Extrusion Twin-screw (TS) extruders can be used at higher moisture conditions (>40%) as compared to single-screw extruders (<35%). TS extruders have advantages over the single-screw extruders due to the interlocking screws. They can effectively mix a wider range of ingredients into a dough which is melted by the thermomechanical action of the extruder barrel and screws (Akdogan 1999; Lee et al. 2005). Meat extenders can be produced via thermoplastic extrusion on a single-screw extruder under low moisture conditions. The products from low moisture extrusion are usually expanded, have a sponge-like structure, absorb water rapidly, and are often used as meat extenders (Lin et al. 2002). TS extrusion under high moisture conditions (>40% water), also known as wet extrusion, can produce meat extenders or analogs with a fibrous texture (Lin et al. 2000, 2002; Noguchi 1989). The TS extruder is fitted with a cooling die that is essential for proper texture formation in meat replacers and analogs. The cooling die is attached to the end of the extruder and helps the proteins align in the melted dough. The three steps for protein texturization in TS extruders include melting the protein dough at shear and high temperature, steady pumping of the melt from the extruder barrel to the cooling die, and the development of laminar flow in the cooling die which results in fiber formation (Cheftel et al. 1992; Lee et al. 2005). Since the use of TS extruders for fibrous-texture formation is nowadays more common, the focus of this chapter will be on the use of TS extruders to produce whey-type meat extenders. During thermoplastic protein extrusion, proteins are heat- denatured under conditions of shear and high temperature (Kitabatake and Doi 1992; Tolstoguzov 1993). The high temperatures keep the denatured proteins in a molten state, allowing them to align in the direction of the

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material flow. Whether a protein exists in a molten state depends on the temperature and moisture content. If the extrusion mix is heterogeneous, usually accomplished by adding a polysaccharide, incompatibilities of the components lead to a separation of phases. Laminar flow within the extruder and die creates a layering of phases, the structure of which is fixed upon cooling. The texture of the extrudate is stabilized by the protein crosslinks formed during extrusion. Extrusion is a complicated process and there are many factors that influence the texture of an extruded product (Akdogan 1999; Cheftel et al. 1992; Harper 1986; Kitabatake and Doi 1992; Tolstoguzov 1993). These factors can be grouped into the two main categories of physiochemical parameters and configuration parameters. Physiochemical parameters include the formulation of the extrusion mix and the temperatures and pressures used during extrusion. Configuration parameters include items such as the extruder screw and barrel configuration and the dimensions of a cooling die. The physiochemical and configuration parameters are obviously not mutually exclusive. For example, screw configuration will influence the pressure profile along the extruder barrel, which in turn affects the physical state of the proteins and their interactions. Unfortunately, extruder science has not yet been developed to the point where the main effects of these parameters are fully understood, let alone the interactions. There is a good empirical understanding of the configuration parameters necessary for developing a layered and meat-like texture in extruded products and the specifics seem to remain fairly constant over a wide range of protein sources including soy and other vegetable proteins (Cheftel et al. 1992). The basic extruder configuration is defined as follows (Akdogan 1999; Harper 1986; Tolstoguzov 1993). The first section of the extruder is configured to provide for the mixing of the formulation components. The second section of the extruder is configured to produce the temperatures and pressures necessary to melt the proteins and polysaccharides. Optimum temperatures and moisture levels for thermoplastic extrusion of textured vegetable proteins range between approximately 100 and 180◦ C and 35 and 70% water, respectively (Akdogan 1999; Cheftel et al. 1992; Tolstoguzov 1993). At the end of the barrel, there must be a section that allows laminar flow to develop a layered separation of the protein and polysaccharide phases (Figure 8.1). The third section of the extruder needs to continue the laminar flow while cooling the dough to allow the layered structure to solidify with

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Figure 8.1. Simplified drawing of a twin-screw extruder.

minimal expansion upon the product exit. This is accomplished either in the extruder barrel if there is sufficient length or in a specially designed cooling die. Lastly, the die exit provides the final shaping of the dough. The cooling die is a major component of the extruder when the production of fibrous products is sought. The die is attached directly to the end of the extruder barrel and can consist of a cooling die jacket. Along with the alignment of the protein molecules, there is a layering of the protein and starch in the melt which is reduced to ambient pressure before being expelled from the die resulting in very minimal steam flash-off and expansion of the product. The die dissipates the thermal and mechanical energy accumulated in the food mix. The steam in the product is able to condense in the cooling die and the formation of longitudinally orientated bubbles favors the resulting product that has the typical layered characteristics of meat (Harper 1981; Lee et al. 2005). In contrast to the configuration parameters of extrusion, which are similar from protein to protein source, the physiochemical parameters vary significantly depending on the proteins to be texturized. Catalogued within the physiochemical parameters are such things as protein type and content, polysaccharide type and content, water content, pH, ionic strength, and temperature. The primary effect of most of these factors is on the extent and the type of protein–protein bonds that form. Many different polysaccharides have been employed for the extrusion of textured proteins, including maltodextrins, carboxymethyl cellulose, and cornstarch (Cheftel et al. 1992; Tolstoguzov 1993). Even at low concentrations of less than 5%, these polysaccharides are incompatible with proteins in an aqueous solution and the proteins and polysaccharides

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separate into distinct phases. When a section of laminar flow is encountered in the extruder, the protein and polysaccharide phases layer themselves, thereby giving texture to the extruded product. Because of the separation of phases, the polysaccharide may not directly interfere with protein–protein interactions. However, the polysaccharide phase tends to have a greater affinity for water than the protein phase, thereby causing the protein concentration in the protein phase to be slightly greater than otherwise expected. This may have a small indirect effect on protein– protein interactions. The moisture content during extrusion also influences the fiber formation in high moisture extrusion using a cooling die. Lin et al. (2000, 2002) characterized soy protein meat analogs extruded under various moisture conditions (60, 65, and 70% moisture content) and showed that the lower moisture products contained more nondisulfide covalent crosslinks. A descriptive sensory analysis showed that the 60% moisture products were more layered, cohesive, springy, and chewy compared to the 70% moisture products. The fiber formation in products produced at high moisture extrusion was digitally imaged by Ranasinghesagara et al. (2005) and Yao et al. (2004). Textured soy samples produced at lower moisture (60% ) showed higher fiber formation than the products produced at higher moisture (70%). A textured product made from a blend of soy and whey protein concentrate (3:2) showed fiber formation at moisture contents of 60 and 65% (Ranasinghesagara et al. 2005). The resistance of the fabricated texture to degradation during cooking and consumption depends on the nature of the protein crosslinks that were formed during extrusion. The types of protein interactions that can occur at extrusion temperatures and moisture levels include hydrophobic interactions, ionic bonds, and covalent bonds. Covalent crosslinking can occur via nonenzymatic browning, formation of isopeptides, and formation of disulfide bonds (Stanley 1989). Weak hydrophobic and hydrogen bonds can be easily disrupted with water or buffer, while stronger covalent and ionic bonds resist disruption to retain product texture. The extent of covalent crosslinking is estimated by measuring the protein solubility of the extrudate after treating with water, buffer, sodium dodecyl sulfate (SDS), and/or 2-mercaptoethanol (Harper 1986). We have shown that in extruded–expanded whey protein products the type of interactions formed varied depending on the type of starch used (Allen et al. 2006). In extruded–expanded samples containing whey protein and normal cornstarch, covalent complexes between amylose and protein were

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likely formed with approximately 50% of the protein solubilized in a solution containing SDS and 2-mercaptoethanol. A different trend was observed in extruded–expanded samples containing whey protein and pregelatinized waxy starch; covalent protein–protein interactions were favored, not protein:starch with these ingredients.

Fibrous-Textured Whey Protein The functional properties (gelling, foaming) of whey proteins are comparable to those of soy proteins. Because of the chemical similarities between whey and soy proteins, it is likely that thermoplastic extrusion can produce a textured whey protein (TWP) similar to the TSPs that are widely used as extenders of coarse-ground meat products. Investigations on texturizing milk proteins for use in meat products began in the early 1970s. Initial attempts at texturizing milk proteins were targeted at developing a meat analog of whole muscle, rather than developing a replacer of ground product. Previous research on TWPs for use as a meat extender or analog included microwave expansion and extrusion. Burgess et al. (1978) and Tuohy (1980a, b) textured whey proteins with microwave expansion and created a product similar in texture to cooked, minced beef. The microwave-expanded product exhibited minimum texture values at pH 5 and maximum values at pH 7–9. Cuddy and Zall (1982) extruded acid whey containing 30% soy oil, but found the product had little cohesiveness with a crumbly texture. Trial runs using acid whey alone were not successful because of clogging and difficulty in getting a moistened mixture to flow through the extruder (Cuddy and Zall 1982). Martinez-Serna and Villota (1992) investigated the reactivity, functionality, and extrusion performance of products produced from cornstarch and native and chemically modified whey proteins. The addition of whey proteins led to a 30% decrease in the expansion ratio of the extruded products. The highest degree of fiber formation and alignment occurred in the acetylated WPI–cornstarch extrudates, although the product was hard and brittle. The alkaline whey protein–cornstarch extrudates showed textural properties similar to those of soy protein extrudates. The types of protein bonds formed in the extruded products were determined to be disulfide, hydrophobic, and ionic. When disulfide bonds predominated, as in alkaline whey proteins, the extrudate

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was tough and inelastic. When hydrophobic interactions were stronger, as in the acetylated whey proteins, the extrudate was more brittle and less cohesive. Other research on extruded whey proteins focuses on microcoagulation for the production of fat substitutes, increasing the gelling properties of whey protein after controlled shearing, and altering the functionality of whey proteins. In the work by Queguiner et al. (1992), whey protein concentrate (WPC) (20% protein) was coagulated by extrusion to produce nonaggregated semisolid spreads for use as fat replacers. Nonaggregated semisolid spreads were obtained only in the pH range of 3.5–3.9 at 90–100◦ C. At higher pH values (4.5–6.8) more intermolecular disulfide exchange reactions took place, resulting in a product with a grainy texture. Ker and Toledo (1992) used controlled shearing by extruding whey protein isolate (WPI) at 25◦ C to pretreat proteins prior to heat-induced gelation. The sheared WPI resulted in a gel of increased strength due to increased protein–protein interactions. Onwulata et al. (2003, 2006) have investigated the use of low-temperature extrusion (<100◦ C) at various pH values to alter the functionality of whey protein. We have developed a TWP product that we have used as an extender of beef patties and to form meatless patties (Hale et al. 2002; Taylor and Walsh 2002; Walsh and Carpenter 2003). Our laboratory research was conducted on an APV Baker MPF19 TS extruder (L/D 25) fitted with a cooling die (22 cm × 1 cm × 2 mm) at the end of the barrel. Temperature zones along the barrel were controlled and monitored with CAL3200 Autotune temperature controllers (Cal Controllers, Inc., Libertyville, IL). The dry mix was a blend of whey protein concentrate containing 80% protein (WPC 80) and normal cornstarch containing 25% amylose and 75% amylopectin. The liquid source was fed into the extruder through the fluid line set 5 cm from dry input, and hydrated the dry mix immediately after it passed through the funnel. The set of five barrel temperature zones was 25, 25, 130, 140, and 140◦ C, which resulted in temperatures of 55, 90, 130, 140, and 140◦ C during product collection. The product melt temperature was approximately 162◦ C with pressures ranging from 250 to 350 psi. Our original process was at a 32% moisture content (Hale et al. 2002) which was modified to a 50% moisture level in the later studies (Taylor and Walsh 2002) which increased the fibrous nature of the product. Our preliminary extrusion studies focused on screw and paddle configurations that would allow the whey protein/starch sample to pass

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through the extruder without surging or backing-up, varying the liquid source, the addition of calcium to the dry feed, and varying the amount of whey protein. The optimal screw and paddle configurations were found to be low shear to allow product conveyance through the extruder. The liquid sources investigated included water, acid (0.1, 0.2, and 1 M HCl), base (0.1, 0.2, and 1 M NaOH), and the addition of calcium chloride per protein basis to the dry mix (0.5, 0.9, and 1.7%) with water as the liquid source. The protein content ranged from 40 to 72%. Liquid Source Extruded samples were initially analyzed by measuring pH and observing physical characteristics. The pH measurements showed that pH of the samples ranged from 4.0 to 7.5 (Table 8.1). In general, pH decreased ( p < 0.05) in samples extruded with acid, and pH increased in samples extruded with base. There was also a decrease in pH in samples extruded with calcium, which may be due to the acidity of dissociated calcium chloride salt. There were no significant differences between the total protein of the samples as determined by the Kjeldahl nitrogen method. The average percentage of the total protein content of TWP samples was 50.55% ± 0.56. Table 8.1.

The pH and physical properties of TWP samples.

Variable

PH

Color

Opacity

TWP (0.1 M HCl) TWP (0.2 M HCl) TWP (1 M HCl) TWP (0.1 M NaOH) TWP (0.2 M NaOH) TWP (1.0 M NaOH) TWP (0.5% Ca) TWP (0.9% Ca) TWP (1.7% Ca) TWP (H2 0)

6.25 ± 0.02B,C,D 5.93 ± 0.09D 4.02 ± 0.51E 6.58 ± 0.02B,C 6.73 ± 0.11B 7.48 ± 1.27A 6.37 ± 0.09B,C,D 6.18 ± 0.10B,C,D 6.03 ± 0.02C,D 6.45 ± 0.04B,C,D

Caramel brown Light brown Tan/orange Light yellow Dark brown Nearly black Brown Light brown Grayish-brown Caramel brown

Opaque Opaque Opaque Opaque Translucent Translucent Opaque Opaque Opaque Opaque

The pH values are the average of two replications with two samples in each replication. Statistics were calculated using analysis of variance in SAS (Cary, NC). Differences were calculated using the least-squared difference test (LSD, p < 0.05). Means sharing superscript letters are not different at p > 0.05. Other values are observations made on extruded and dried products.

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The acid, base, and added calcium level affected the physical properties of TWP. Observations of physical characteristics imply that TWP differed in color, opacity, and structure. Color was darker at higher pH, most likely due to increased Malliard browning. TWP extruded at high pH contrasted with other samples in opacity, structure, and texture, which may indicate a different protein structure in those samples. In gelation, whey proteins exhibit either linear or globular aggregation mechanisms, depending on the physiochemical parameters. These aggregation mechanisms lead to different gelling properties. During extrusion, the physiochemical parameters may influence the aggregation mechanism of whey proteins in the initial stages of extrusion. The bonds formed in initial aggregation, though later replaced by more stable bonds, influenced the properties and functionality of the final product. Protein Solubility The amount of protein solubilized by different solvents is shown in Figure 8.2. For this analysis, TWP samples were crushed to <60 mesh and slurries of sample (3.85% w/v in solution) were shaken for 1.5 h at 150 rpm. The solutions used were water, 2% SDS, 0.5 N NaCl, or 0.02% 2-mercaptoethanol (BME). Samples were then centrifuged for

Figure 8.2. Percent soluble protein in water, sodium dodecyl sulfate (SDS), sodium chloride (NaCl), and beta-mercaptoethanol (BME) of textured whey samples extruded with water (control), acid, or base as the liquid source or with calcium chloride added to the dry mix extruded with water. The columns are the pooled means of 10 extruded samples inn each treatment (three levels of acid, base, and added calcium). Means sharing superscript letter are not different at p > 0.05.

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15 min at 5,000 × g and the protein content of the supernatant was determined spectrophotometrically using the BCA assay (Pierce Chem Co., Rockford, IL). The columns in Figure 8.2 are the pooled means of 10 extruded samples in each treatment (three levels of acid, base, and added calcium). The amount of protein solubilized by water was generally the lowest among the four treatments while the amount of protein solubilized in SDS was generally higher. Samples produced with acid or calcium as the liquid source result in lower soluble protein. This may be due to a difference in the initial aggregation mechanism of the proteins. Globular aggregation is favored in the presence of salt or at a pH near the isoeletric point, exhibited by samples extruded with acid. In globular aggregation, proteins aggregate, unhindered, into large clumps, which results in low WHC and dense structures that may hinder solubilization of proteins. Additional information on the types of bonds stabilizing insoluble aggregates was obtained by comparing protein solubility in 2% SDS (disrupts noncovalent interactions), 0.5 M NaCl (disrupts electrostatic interactions), and 0.02% BME (cleaves disulfide bonds). Water solubilizes protein held by only the weakest noncovalent interactions. The additional protein solubilized in each buffer (SDS, NaCl, and BME), as compared to the protein solubilized in water, reflects the relative extent of noncovalent, ionic, and disulfide bonds, respectively. In this research, no more than 12% of the total protein was solubilized by extraction in any one solvent. These results indicate that multiple types of bonds stabilized the TWP including nonspecific covalent bonds. Other researchers have suggested that high temperatures (100–150◦ C) are known to lead to covalent bond formation. These irreversible chemical changes include Malliard reactions, cysteine breakdown, and the possible breakdown of disulfide bonds (DeWit and Klarenbeek 1984; Li-Chan 1983). Also, at high pH, cysteine breakdown increases, dehydro-alanine forms, and if lactose is present, lysine is destroyed (DeWit and Klarenbeek 1984). Initial bond formation mediates protein aggregation, and the bonds are later replaced by stronger covalent bonds that stabilize the networks. This is not surprising since the TWP melt temperature was over 160◦ C. Protein Concentration Table 8.2 lists the samples extruded at different protein concentrations and descriptions of the products formed. We were able to produce a

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195

Protein concentrations in TWP production.

WPC/cornstarch

Protein (%)

Characteristics of TWP products

1:1 3:2 2:1 3:1 4:1 5:1 6:1 9:1

40 48 52 60 64 66 69 72

No fibrous texture formed Fibrous texture formed Original product, fibrous texture formed Fibrous texture formed Fibrous texture formed Very difficult to extrude, can get fibrous texture Too difficult to extrude Too difficult to extrude

textured product with protein levels ranging from 48 to 64%. Assuming that a carbohydrate is needed to form a heterogenous mixture with the protein and that the phases separate during cooling to form a textured product, there was not enough cornstarch in the blends with protein concentrations of 66% and higher to allow fiber formation. The opposite is shown at 40% protein, which was below the protein content needed for fiber formation. Textured Whey Protein as Meat Extenders/Analogs Based on the results presented here, we typically use a protein level of 50% in order to produce a TWP for additional research. The base liquid source we currently use is 0.2 M NaOH based on the fiber formation in these samples and the results of consumer evaluation of beef patties extended with extrudates produced with base (Hale et al. 2002). Hale et al. (2002) found that consumers who evaluated beef patties extended with 30% of TWP extruded with base were not significantly different from the all beef control with respect to tenderness, juiciness, texture, flavor, and overall acceptability (Table 8.3). The lowest scores were given to TWP extruded with base and TSP. In addition, beef patties extended with up to 40% TWP were not significantly different than the all beef control with respect to tenderness, juiciness, texture, beef flavor, and overall acceptability with a consumer panel (Figure 8.3). The TWP was also formulated into patties using egg white, wheat gluten, and xanthan gum as binders (Taylor and Walsh 2002). The TWP patties contained approximately 30% protein, 4% fat, and from 8 to

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Table 8.3. Means from consumer evaluation of beef patties extended with 30% TWP. Treatment

Tender-nessa

Juiciness

Texturea

Flavor

Acceptability

100% beef 30% TWPbase 30% TWPacid 30% TWP2+ Ca 30% TWPH2O 30% TSP

6.17A 6.67A 4.86D 5.77B,C 5.64C,B 5.23C,D

5.87A,B 6.19A 4.90C 5.57B 5.65A,B 4.78C

6.32A 6.16A 3.43D 4.60B,C 4.53C 5.19B

6.45A 6.28A 4.73C 5.51B 5.27B,C 4.00D

6.35A 6.32A 4.69B 5.14B 5.01B 4.23B

a Tenderness relates to initial bite and texture relates to mouthfeel during chewing. Patties were evaluated using a hedonic scale by panelists. All patties were adjusted to 20% fat. Statistics calculated with analysis of variance using SAS (Cary, NC). Within a column, mean values sharing a superscript letter are not different ( p > 0.05). Reprinted with permission from Hale et al. 2002.

12% carbohydrate. The TWP patties were compared to commercial soybased patties using a hedonic scale by a consumer panel (Table 8.4). The TWP patties were preferred by panelists over the commercial soy-based patties. There was no significant difference with respect to appearance and texture, but there were significant differences between the TWP and

Figure 8.3. Hydrated TWP containing 50% protein extruded with 0.2 N NaOH.

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197

Mean scores, rank sums, and difference rankings of meatless patties.

Description

A

B

C

Product

Commercial TVP garlic

TWP mushroom

TWP vegetable

Binding code Appearance

258 5.20A

314 5.31A

769 5.42A

Texture

4.35A

4.85A

5.01A

NS cr = 0.59

Flavor

2.95B

5.11A

4.60A

BC > A cr = 0.59

Aftertaste

3.24B

4.89A

4.71A

BC > A cr = 0.57

Overall acceptance

3.38B

5.00A

4.73A

BC > A cr = 0.58

Preference ranking

121B

193A

172A

BC > A cd = 29.8

NS cr = 0.60

Patties were evaluated using a hedonic scale by consumer panelists. Like superscripts on means within a row indicate no significant difference among the means (α = 0.05). NS = not significantly different; cr = least significant difference critical range. Acceptability means differing by the cr or more are different (α = 0.05). cd = critical difference. Rank sums differing by more than the critical difference are different (α = 0.05). Reprinted with permission from Journal of Food Science.

the commercial soy patty with respect to flavor, aftertaste, and overall acceptability. Future of Textured Whey as Meat Replacer/Analog The observed advantages of TWP over other textured protein products includes the absence of off flavors and the high nutritional amino acid composition of whey protein. Unfortunately, the disadvantage includes the higher cost of whey protein. There is a future for TWP as an extender/ analog if the products are marketed as value-added products due to the higher nutritional quality. Current research in the development of TWP has explored the production of TWP from various (>10) WPC sources and altering the formulation to include fiber to increase the nutritional profile of the TWP.

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References Akdogan, H. 1999. High moisture food extrusion. Int. J. Food Sci. Technol. 34:195–207. Allen, K.E, Carpenter, C.E., and Walsh, M.K. 2007. Influence of protein level on the physical and chemical properties of extruded-expanded whey products. Int. J. Food Sci. Technol. 42:953–960. Burgess, K.G., Downey, and Tuhoy, S. 1978. Making meat substitutes from Milk. Farm and Food Res. 9(3):54–55. Cheftel, J.C., Kitagawa, M., and Queguiner, C. 1992. New protein texturization processes by extrusion cooking at high moisture levels. Food Rev. Int. 8:235–275. Code of Federal Regulations, Title 7, part 210, Subpart F, Section II. Alternate Protein Products. http://www.access.gpo.gov/cgi-bin/cfrassemble.cgi?title=200607. Accessed February 11, 2008. Cuddy, M.E., and Zall, R.R. 1982. Performance of lipid-dried acid whey in extruded and baked products. Food Technol. 1:54–59. DeWit, J.N., and Klarenbeek, G. 1984. Effects of various heat treatments on structure and solubility of whey proteins. J. Dairy Sci. 67:2701–2710. Golbitz, P. 2006. Soyfoods: The U.S. market 2006. Soyatech.Com http://www. soyatech.com. Accessed October 1, 2006. Hale, A.B., Carptenter, C.E., and Walsh, M.K. 2002. Instrumental and consumer evaluation of beef patties extended with extrusion-textured whey proteins. J. Food Sci. 67(3):1267–1270. Harper, J.M. 1981. Extrusion of Foods, Vol. II. Boca Raton, FL: CRC Press. Harper, J.M. 1986. Extrusion texturization of foods. Food Technol. 3:70–75. Ker, Y.C., and Toledo, R.T. 1992. Influence of shear treatments on consistency and gelling properties of whey protein isolate suspensions. J. Food Sci. 57(1):82–90. Kitabatake, N., and Doi, E. 1992. Denaturation and texturization of food protein by extrusion cooking. In Food Extrusion Science and Technology, edited by J.L. Kokini, and M.V. Karve, pp. 361–371. New York: Marcel Dekker. Lee, G., Huff, H.E., and Hsich, F. 2005. Overall hat transfer coefficient between cooling die and extruded product. Am Soc Ag Eng. 48(1):1461–1469. Li-Chan, E. 1983. Heat induced changes in the proteins of whey protein concentrate. J Food Sci. 48:47–56. Lin, S., Huff, H.E., and Hsieh, F. 2000. Texture and chemical characteristics of soy protein meat analog extruded at high moisture. J. Food Sci. 65(2):264–269. Lin, S., Huff, H.E., and Hsieh, F. 2002. Extrusion process parameters, sensory characteristics, and structural properties of a high moisture soy protein meat analog. J. Food Sci. 67(3):1066–1072. Martinez-Serna, M.D., and Villota, R. 1992. Reactivity, functionality, and extrusion performance of native and chemically modified whey proteins. In Food Extrusion Science and Technology, edited by J.L. Kokini, and M.V. Karve, pp. 387–414. New York: Marcel Dekker. Noguchi, A. 1989. Extrusion cooking of high-moisture protein foods. In Extrusion Cooking, edited by C. Mercier, P. Linko, and J.M. Harper, pp. 343–372. St. Paul, MN: American Association of Cereal Chemists.

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Onwulata, C.I., Konstance, R.P., Cooke, P.H., and Farrell, H.M., Jr. 2003. Functionality of extrusion-texturized whey proteins. J Dairy Sci. 86:3775–3782. Onwulata, C.I., Isobe, S., Tomasula, P.M., and Cooke, P.H. 2006. Properties of whey protein isolates extruded under acidic and alkaline conditions. J. Dairy Sci. 89:71– 81. Queguiner, C., Dumay, E., Salou-Cavalier, C., and Cheftel, J.C. 1992. Microgoagulation of a whey protein isolate by extrusion cooking at acid pH. J. Food Sci. 57(3):610– 616. Ranasinghesagara, J., Hsieh, F.H., and Yao, G. 2005. An image processing method for quantifying fiber formation in meat analogs under high moisture extrusion. J. Food Sci. 70(8):E450–E454. Stanley, D.W. 1989. Protein reactions during extrusion processing. In Extrusion Cooking, edited by C. Mercier, P. Linko, and J.M. Harper, pp. 321–340. St. Paul, MN: Am Association of Cereal Chemists. Taylor, B.J., and Walsh, M.K. 2002. Development and sensory analysis of a textured whey protein meatless patty. J. Food Sci. 67(4):1555–1558. Tolstoguzov, V.B. 1993. Thermoplastic extrusion—the mechanism of the formation of extrudate structure and properties. J. Am. Oil Chem. Soc. 70(4):417–424. Tuohy, J.J. 1980a. Physical properties of textured whey protein I. Texture. Ir. J. Food Sci. Technol. 4:35–44. Tuohy, J.J. 1980b. Physical properties of textured whey protein II Bulk density, water binding capacity and protein solubility. Ir. J. Food Sci. Technol. 4:111–123. Walsh. M.K., and Carpenter, C.E. 2003. Textured whey protein product and method. U.S. Patent 6,607,777. Yao, G., Liu, K.S., and Hsieh, F. 2004. A new method for characterizing fiber formation in meat analogs during high-moisture extrusion. J. Food Sci. 69(7):E303–E307.

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Chapter 9 Whey Inclusions K.J. Burrington

Introduction A new generation of whey ingredients has been commercialized in recent years that have expanded the functionality and applications for conventional whey protein ingredients. Whey inclusions, often referred to as whey protein crisps, bring all the nutritional benefits of whey proteins to a food application in a puffed, crisp, and crunchy form. Extrusion processing of whey protein ingredients with carbohydrate ingredients yields a crisp product similar to a rice or soy crisp (Pszczola 2006a, b). Whey protein crisps can be made with a wide range of protein levels, and a variety of shapes, sizes, colors, and flavors, offering the product developer an ingredient which provides nutritional and organoleptic appeal.

Whey Inclusion Technology Whey extrusion techniques used to produce whey protein crisps were in the developmental stages as early as the late 1970s (Kosikowski 1979). Extrusion of whey proteins stretches and shears their globular structure into fibrous bundles creating texturized proteins (Onwulata et al. 2003). Several researchers over the years have investigated extrusion processing or texturization of whey proteins and their work is reviewed in the other chapters of this book (Marie K. Walsh, Charles I. Onwulata, and Lester O. Pordesimo). Extruded products have been produced using dried sweet whey (13% protein) and whey protein concentrates with 34–80% protein. Whey 201 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Figure 9.1. Grande Custom Ingredients Group.

proteins have been extruded with many different carbohydrate ingredients, such as corn meal, wheat starch, cornstarch, potato flour, rice flour, and barley flour, all typically used in cereal extrusion cooking (Onwulata et al. 1998; Onwulata and Tomasula 2004; Walsh, 2003). Fibers have also been extruded with whey proteins as an additional nutritional component incorporated in a protein crisp (Engleson et al. 2006). The commercial products available today typically use a whey protein concentrate or whey protein isolate produced from sweet whey and further concentrated by ultrafiltration, microfiltration, and diafiltration to 80% protein and 90% protein, respectively (on a dry basis). The carbohydrate ingredients coextruded with the whey protein ingredients are often cornstarch, rice flour, and tapioca starch. The protein content of the crisp products typically ranges from 25 to 80% protein (Mannie 2006). An example of some of the possibilities for shapes and sizes is shown in Figure 9.1.

Applications Bars The U.S. market for food bars, including sales of cereal/granola bars and nutrition bars, is expected to reach $6 billion in 2007. Cereal and

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granola bars account for 51% of the market with nutrition bars including high protein, sports/energy, diet/weight management, low carbohydrate, and others represent 49% (Wright 2005). Bars containing additional proteins from whey and other sources are traditionally very dense and chewy which is a characteristic of using high levels of protein in an intermediate moisture product. The development of protein crisps, with soy made available first and more recently whey protein, have provided a means to add protein while also contributing some crunch and textural variety. The appeal of adding protein crisps along with other nut, fruit, and confectionary inclusions in sports and nutrition bars have resulted in increased sales of these products as compared to a typical single-layer bar (Hazen 2005). A classic example of a bar that utilizes a crisp product is a rice crisp bar. The typical formulation which uses marshmallow, butter, and Rice KrispiesR provided by KelloggsR results in a bar that contains 1 g of protein per 22 g bar. Replacement of the Rice KrispiesR with a whey crisp containing 50% protein will increase the protein content to 6 g per bar and increase it to 10 g using an 80% protein whey crisp (calculations provided by Kathy Nelson-Wisconsin Center for Dairy Research), see Table 9.1 and Figure 9.2. Procedure 1. Combine the dry ingredients in the bowl of a large mixer. Mix at low speed for 2 min. 2. Add butter and vegetable oil to the dry ingredients and mix until evenly distributed. 3. Combine maltitol and glycerine, and add to the dry ingredients, mixing at low speed for 1 min. 4. Add water and mix at low speed for 1.5 min, or until the mixture comes together. 5. Sheet bars to 8 mm thickness and cut into 1 × 11/2 pieces. Place on parchment-lined pans so that they do not touch each other. 6. Bake at 400◦ F (204◦ C) for 10 min. Nutrition Information Serving size: 28 g Calories: 80

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Whey Processing, Functionality and Health Benefits Table 9.1. Baked cinnamon granola bars (provided by Wisconsin Center for Dairy Research). Ingredients Maltitol Water Almonds, ground Oat fiber Whole wheat flour Whey protein crisps Butter, unsalted Whey protein concentrate 60 Plum powder Brown rice crisp cereal Rolled oats, old-fashioned Rolled oats, quick Raisins Vegetable oil Flax seed, ground Glycerine Cinnamon Psyllium Salt Sodium bicarbonate Sucralose Total

(%) 18.43 14.91 8.13 7.59 7.18 6.23 5.42 5.08 4.88 4.07 4.07 4.06 3.66 2.71 1.35 0.65 0.54 0.54 0.30 0.18 0.02 100.00

Fat: 4 g Carbohydrate: 15 g Protein: 3 g

Snacks The snack category is full of examples of high fat and high carbohydrate foods but protein is not typically a focus. Puffed snacks, chips, crackers, pretzels, and snack mixes are some of the most common offerings. Whey crisps offer the best way to add protein and better nutrition to this food category (see Figure 9.3 and Tables 9.2 and 9.3).

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Figure 9.2. Grande Custom Ingredients Group.

Procedure 1. Place all the ingredients in a bowl and mix at low speed until ingredients come together to form a ball. 2. Sheet to 10 mm thickness, cut into small pieces (approximately 0.5 × 0.75 ) and place on a parchment-lined cookie sheet. 3. Bake 25 min at 325◦ F.

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Figure 9.3. Grande Custom Ingredients Group.

4. Cool on cookie sheet. 5. Store in air-tight containers. Nutrition Information Serving size: 30 g Calories: 120

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Table 9.2. High protein cheese cracker (provided by the Wisconsin Center for Dairy Research). Ingredients

(%)

All-purpose flour Extra-sharp cheddar cheese, grated Butter, unsalted Whey protein concentrate 60 Whey crisps–50% protein (smallest size) Whey permeate Water Cheese blend, cheddar type Cayenne pepper

28.36 21.13 19.56 9.78 7.82 5.87 5.09 2.35 0.04

Total

100.00

Fat: 8.0 g Carbohydrate: 9.0 g Protein: 6.0 g Procedure 1. Melt butter in a saucepan. 2. Remove from heat and add salt, onion powder, garlic salt, and Worcestershire sauce. 3. Stir until dissolved completely. Table 9.3. Whey protein snack crunchers (provided by Grande Custom Ingredients). Ingredients Butter Salt Onion powder Garlic salt Worcestershire sauce Grande WPCrisp C50004 Total

(%) 19.42 0.16 0.20 0.42 0.84 78.96 100.00

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4. Pour the mixture over WPCrisp and mix thoroughly. 5. Bake in a roasting pan at 225◦ F for 45 min stirring every 15 min. Nutritional Information Serving size: 28 g Fat: 6.0 g Carbohydrates: 9.0 g Protein: 11.0 g Breakfast Cereals The typical breakfast cereal contains high levels of carbohydrates. It is important to also include protein and a small amount of healthy fat for breakfast to stay full throughout the morning (Ohr 2005). Breakfast cereals that delay stomach emptying could improve satiety or a feeling of fullness and help control the amount a person eats throughout the rest of the day. Different foods provide different levels of satiety; for instance, oatmeal will provide a higher level of satiety than a high bran cereal. Adding protein to breakfast cereals will delay stomach emptying and lead to greater satiation. Adding volume to foods using air or water has also been shown to enhance their satiety effect (Camire and Blackmore 2007). Crisp products from rice, corn, and soy had their beginnings as breakfast cereals. Whey crisps can also provide an ingredient that has added volume and protein to a breakfast cereal. A granola-type cereal could gain over twice the amount of protein by the addition of a whey crisp product with 50% protein (Table 9.4 and Figure 9.4). Procedure 1. Mix all ingredients together and blend thoroughly. Table 9.4. High protein cereal (provided by Grande Custom Ingredients). Ingredients Granola cereal blend TM Grande WPCrisp C50003 Sliced almonds Dried cranberries Total

(%) 55.81 16.28 11.63 16.28 100.00

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Figure 9.4. Grande Custom Ingredients Group.

Nutritional Information: Serving size: 60 g Fat: 6.5 g Carbohydrates: 33.5 g Dietary Fiber: 4.0 g Protein: 11.0 g Toppings Toppings provide texture, flavor, nutrition, and visual appeal (Pszczola 2007a, b).

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Whey Processing, Functionality and Health Benefits Table 9.5. Whey protein salad topping (provided by Grande Custom Ingredients). Ingredients Garden style salad toppinga TM Grande WPCrisp Butter Salt Total

(%) 82.14 13.86 3.87 0.13 100.00

a Durkee Salad Sensations Garden Style salad topping used.

Crunchy toppings are often used for yogurt, frozen desserts, salads, and desserts. Using a whey protein crisp will add protein to a topping that is not typically high in protein. The salad topping formulation (Table 9.5) provides 50% more protein than a serving of the traditional salad topping that is used in the formulation. Procedure 1. Melt butter in a saucepan. 2. Add salt and stir until dissolved completely. TM 3. Pour the mixture over WPCrisp and mix thoroughly. 4. Bake in a roasting pan at 225◦ F for 45 min stirring every 15 min. 5. Combine with all the other ingredients. Nutritional Statement Serving size: 7 g Fat: 1.90 g Carbohydrates: 2.80 g Dietary Fiber: 0 g Protein: 1.50 g

Summary The possible food applications that could utilize a whey crisp inclusion are seemingly unlimited. Whey crisp technology has developed products that are in many shapes, sizes, and colors. Commercial ingredients are readily available and waiting to be used in potential new product

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introductions. As the nutrition information related to the importance of whey protein and health evolves, there will be even more interest in using whey protein as an inclusion.

References Camire, M.E., and Blackmore, M. 2007. Breakfast foods and satiety. Food Technol. 61(2):24–30. Engleson, J., Porter, M.A., Atwell, W.A., Baier, S.K., Elmore, D.L., Gilbertson, D.B., Aimutis, W.R., Jr., Sun, N., Muroski, A.R., Smith, S.A., Lendon, C.A., and May, T.L. 2006. Extruded ingredients for food products. United States Patent Application 20,050,208,180. Hazen, C. 2005. Food bars with customized appeal. Food Prod. Des. 9:32–57. Kosikowski, F.V. 1979. Whey utilization and whey products. J. Dairy Sci. 62:1149– 1160. Mannie, E. 2006. Customizing Crunch. Prepared Foods, October, p. 86. Ohr, L.M. 2005. Back to basics with breakfast. Food Technol. 59(6):167–172. Onwulata, C.I., Konstance, R.P., Smith, P.W., and Holsinger, V.H., 1998. Physical properties of extruded products as affected by cheese whey. J. Food Sci. 63(5):814–818. Onwulata, C.I., Konstance, R.P., Cooke, P.H., and Farrell, H.M., Jr. 2003. Functionality of extrusion-texturized whey proteins. J. Dairy Sci. 86:3775–3782. Onwulata, C., and Tomasula, P. 2004. Whey extrusion: A way forward. Food Technol. 58(7):50–54. Pszczola, D.E. 2006a. Ingredients—2005 annual meeting & food expo (review). Food Technol. 59(9):50–66. Pszczola, D.E. 2006b. Exploring new “tastes” in textures. Food Technol. 60(1):44–55. Pszczola, D.E. 2007a. New toppings rise to the challenge. Food Technol. 61(1):39–47. Pszczola, D.E. 2007b. Problem-solving with dairy. Food Technol. 61(2):47–57. Walsh, M.K., and Carpenter, C.E. 2003. Textured whey protein product and method. U.S. Patent 6,607,777. Wright, T. 2005. Bar market still booming. Nutraceuticals World 8(1):28–40.

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Chapter 10 Functional Foods Containing Whey Proteins B. Faryabi, S. Mohr, Charles I. Onwulata, and Steven J. Mulvaney

Overview It is well known that raw cranberries have high antioxidant activity, but due to their tartness are often consumed sweetened, for example, as sugar-infused dried cranberries or in blended juices. We observed that the sugar infusion syrup used to dehydrate raw cranberries turned bright red during the process. Therefore, it was hypothesized that this high solid cranberry infusion syrup would make an excellent raw material for the manufacture of natural cranberry-based functional jelly products. The objective of this work was to develop a prototype high solid natural jelled product from this cranberry infusion syrup with acceptable color, flavor, and texture as a convenient way of delivering potential health benefits of cranberry phytochemicals to a wide range of consumers. Texturized whey proteins (TWPs) were also added in a way that preserved the product’s texture and appearance to further enhance the nutritional profile of the product. The cranberry infusion syrup had a Brix of 68◦ , natural cranberry color, and tartness (pH ∼2.3) due to the presence of citric (0.04%), malic (0.04%), and quinic (0.06%) organic acids. ORAC analysis of the cranberry syrup showed that its antioxidant activity was 5,949 μmole/L Trolox equivalents. A “good” formulation for a product gelled with pectin had a degree of elasticity and firmness of 53% and 28.1 N, as compared to 52.8% and 27.80 N, respectively, for a commercial pectin high solid confectionery jelly. The ORAC value for the final product was 5 μmole/g Trolox equivalents. The product with and without texturized whey protein kept its natural color intact due to the low pH of the syrup throughout the boiling concentration process. This work demonstrates 213 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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the feasibility of using this natural cranberry infusion syrup with added TWPs to manufacture novel functional foods with textures similar to those already acceptable to consumers.

Introduction The confectionery industry is a large and diverse industry that includes sugar confectionery as a major subcategory. This subcategory includes numerous products such as toffees, caramels, and high solid (∼80%) jellies. In particular, the latter products come in many different shapes, colors, flavors, and textures. Texture in turn depends to a large degree on the gelling agent used. Common gelling agents used are agar-agar (fruit slices), pectin (fruit jellies), modified starch (fruit drops), and gelatin (gummies) (Edwards 2000). Traditional jellies are generally made of water, sugar, corn syrup, a gelling agent, and some artificial or natural flavors and colors (De Mars and Ziegler 2001). Many of these products, although often designed to simulate the appearance of real fruit, contain little or no natural fruit antioxidants, although some may include real fruit juice as a source of vitamin C. On the other hand, as consumers are becoming more and more health conscious, they are demanding that the food industry provide new food products with enhanced characteristics and associated health benefits (Clydesdale 2004; Hasler 2002). McHugh and Huxsoll (2000) also observed that many commercial products available today utilize only a small percentage of dried fruit, fruit juice concentrate, fruit powder, or fruit puree in the final product. They also suggested that a wider variety of fruit and/or vegetablebased products in convenient forms would help consumers attain the 2–4 servings of fruits as recommended in the USDA Dietary Guidelines for Americans. Their invention describes how restructured 100% fruit products were made by extruding either fruit puree concentrate or dried fruit concentrate using peach puree as an example. Textural properties could be adjusted by varying the level of added starch (and extrusion temperature to either gelatinize the starch or not) and the level of added corn syrup, liquid sugar, or fruit juice concentrate. These products could be selected by consumers as healthier alternatives to conventional confectionery products. Given the current interest in foods positioned for their health benefits, it was thought that further enhancing the “functional food” aspects of

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natural fruit-based jelled confections would be a good idea, especially given the proven popularity of the textures of many of these products that contain artificial fruit flavor and color. Though there is no universal definition for functional foods, in general they have been defined as “any modified food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains” (Hasler 2002). We chose cranberry as a model for incorporating real fruit antioxidants into a jelled confectionery product, because it has been shown that cranberries contain a high amount of antioxidant compounds in free form, suggesting that they will be present in good amount in cranberry juice extracts (Sun et al. 2002). The genesis of this research project was the observation that natural infusion fruit syrups were a byproduct of the osmotic drying process used to produce sweetened, soft and chewy dried fruits from fresh (frozen) cranberries, as well as some other tart berry fruits. The high sugar content and low pH of the byproduct cranberry infusion syrup combined with its obvious bright red natural color and natural flavor led to the idea of producing a jelled confection from the cranberry syrup. Pectin was a natural choice of gelling agent due to its requirements for gelation (∼80% soluble solids and pH 3.5 or lower for pectin-based confectionery jellies), which was consistent with the low pH and high sugar solids of the cranberry infusion syrup. A typical batch infusion process for producing dried fruit and a byproduct infusion syrup is shown in Figure 10.1.

Figure 10.1. Flow diagram of dried fruit production and byproduct infusion syrup.

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Fruit and 67% sugar solution are added to a tank at a ratio of about two parts syrup to one part fruit. After the allotted time, the syrup is drained off and about two parts of diluted sugar syrup (∼47◦ Brix) is obtained. This naturally colored infusion syrup is then available for the production of fruit jellies, or can be recycled back into the infusion process. Based on all the above observations, the overall objective of this work was to develop a prototype high solid pectin-based natural cranberry confectionery product that was also fortified with pre-denatured TWPs as an additional nutrient and/or texturizing agent.

Material and Methods Materials The materials used were classic AS 507 pectin (“Confectionery, Gum and Jelly Products” (www.herbstreith-fox.de, accessed February 12, 2008)), sucrose (Domino Foods, Inc., Baltimore, MD), 62 DE corn syrup (Corn Product, Inc., Bedford Park, IL), FCC sodium citrate (Roche Vitamins Inc., Parsippany, NJ), and citric acid (Roche Vitamins Inc., Parsippany, NJ) made up to 50% w/w solution in distilled water. Classic AS 507 pectin was supplied by Amcan Industries, Inc. (Elmsford, NY) and is a high-methoxyl (DE between 58 and 65%) apple pectin standardized with potassium sodium tartrate E 337, polyphosphate E 452, and dextrose. The cranberry infusion syrup was provided by Atwater Foods LLC. (Lyndonville, NY). It had natural cranberry color and tartness due to the presence of citric (0.04%), malic (0.04%), and quinic (0.06%) organic acids and an antioxidant activity of 5,949 μmole/L Trolox equivalents. The latter analyses were provided by Analytical Food Laboratories Inc. (Grand Prairie, TX) and Brunswick Laboratories (Wareham, MA), respectively. TWPs STWP and GTWP were made at the USDA, ERRC, laboratory (Wyndmoor, PA) using the following process conditions: whey protein isolate (PROVON 190) was purchased from Glanbia Ingredients. The compositions were as follows: WPC80, moisture 2.8%, protein 83.6%, fat 0.8, ash 3.3%, carbohydrate by difference; WLAC, moisture 5.5%, protein 89.9%, fat 3.8, and ash 0.5%, carbohydrate by difference; and WPI, moisture 2.8%, protein 89.6%, fat 25, ash 3.3%, carbohydrate by

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difference. A ZSK-30 twin-screw extruder (Krupp Werner Pfleiderer Co., Ramsey, NJ) with a smooth barrel was used. The extruder had nine zones, and the effective cooking zone temperatures were set to 100, 110, and 125◦ C, respectively, for zones 7, 8, and 9. Zones 1–3 were set to 35◦ C and zones 4–6 were set to 55◦ C to produce GTWP, and 70◦ C to produce STWP. Melt temperature was monitored behind the die. The die plate was fitted with two circular inserts of 3.18 mm diameter each. The screw elements were selected to provide low shear at 200 rpm. Feed was conveyed into the extruder with a series 6,300 digital feeder, type T-35 twin-screw volumetric feeder (K-tron Corp., Pitman, NJ). The feed screw speed was set at 600 rpm, corresponding to the rate of 3.50 kg/h. Water was added into the extruder at the rate of 1.0 L/h with an electromagnetic dosing pump (Milton Roy, Acton, MA). Samples were collected after 25 min of processing, dried in a laboratory oven at 12◦ C for 5 min, and stored at 4.4◦ C until analyzed. Preparation of Pectin Cranberry Jelly with 80 ◦ Brix (CJ-80) The procedure for making the first version of the cranberry pectin jelly was adapted from a formulation and procedure suggested in the brochure “Confectionery, Gum and Jelly Products” (accessed February 12, 2008, from the website www.herbstreith-fox.de) for a pectin-based high solid confectionery product. The formulation for this sample, denoted as CJ80 (cranberry jelly; 80% soluble solids), is shown in Table 10.1 for a batch size of 222.6 g and the process flow diagram for making the samples is shown in Figure 10.2. Table 10.1. Formulation of a “good” pectin-based cranberry jelly with final solids of 80◦ Brix (CJ-80 in text).

Ingredient Pectin Sucrose (sugar)

Quantity (g)

Percentage in recipe

Soluble solid in ingredient

Soluble solids in recipe (g)

5.1

2.3

1

5.1

49.5

22.2

1

49.5

0.48

79.2

0.5

1.5

Cranberry syrup

165

Citric acid (50% w/w solution)

3

75 1.3

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Figure 10.2. Flow chart for preparation of a “good” pectin-based cranberry jelly with 80◦ Brix (CJ-80 in text).

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Table 10.2. Formulation of a “good” pectin-based cranberry jelly fortified with texturized whey protein with final solids of 75◦ Brix (CJ-80-TWP in text). Ingredients Pectin Sucrose (sugar) Cranberry syrup Citric acid (50% w/w solution) STWP

Quantity (g)

Percentage in recipe

Soluble solid

Solids in recipe (g)

5.1 41 165 5.4

2.3 18.6 75 2.4

1 1 0.48 0.5

5.1 41 79.2 2.7

5

2.27

1

5

The final temperature at 80◦ soluble solids was 110–111◦ C and the pH was 2.9–3.2. The hot mix was then deposited into cylindrical molds, which were allowed to set covered at 25◦ C for 72 h before rheological testing. Preliminary work showed that this combination of storage time and molding method yielded reproducible rheological measurements that did not change with further aging. Some samples shrunk a little on cooling, so all the samples were trimmed to a uniform height of 21 mm before texture testing. Texturized Whey Protein Cranberry Jelly with 75◦ Brix (CJ-75-TWP) TWP powder was first milled to pass through a size of 60 mesh. The milled TWP powder and 2.5 g more citric acid were added to the cranberry jelly formulation after evaporation to 75◦ Brix (Table 10.2 and Figure 10.3). Apparently, the addition of TWP added buffering capacity to the formula. The mixture was stirred efficiently with a spatula for 50 s in order to disperse the TWP completely.

Physical Properties of Products Optical Fluorescence Millimeter-sized blocks of the internal parts of a pectin control gel sample and a pectin gel containing 5 g of added STWP were excised manually with a stainless steel razor blade, and digital images of the

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Figure 10.3. Flow chart for preparation of a pectin cranberry jelly with TWP and 75◦ Brix (CJ-75 –TWP in text).

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microscopic structure were taken using transmitted light from a 150 W halogen lamp in a model Intralux 5000-1 light source (Volpi Manufacturing USA Co., Inc., Auburn, NY) and epifluorescence from green excitation (540–546 nm) using a model MZ FLIII stereofluorescence microscope (Leica Microsystems, Inc., Bannockburn, IL). Color and Appearance of Final Products It was desired to maintain as natural a color as possible and to reduce any browning during evaporation. The effect on the translucency of the products was evaluated qualitatively. One sample from each batch was cut into a thin slice (3 mm) with a stainless steel razor and then a round piece was extracted out of each thin slice. The slices were placed over typed letters, which were written on white paper. The clearer the sample, the easier it was to see the letters. This is shown in Figure 10.4. Large Deformation Compression Testing Large deformation testing was conducted at room temperature (∼25◦ C) TM in a TA-XT2 Texture Analyzer (Texture Technologies, Scarsdale, NY) using a single compression–decompression cycle. The compression stainless steel plate measured 75 mm in diameter and samples were compressed to 25% of their original height, which was less than the fracture deformation. Crosshead speed was 0.2 mm/s followed immediately by decompression at the same crosshead speed. Results represent the average and standard deviation for three samples from a single batch. Analysis of Compression—Decompression Experiments Relative firmness of the product was characterized by the force at 25% compression, which in all cases was also the maximum force, that is, the sample did not appear to rupture or fracture up to 25% compression. The main objective of this work was to determine if a natural cranberry jelly (with and without added TWP) could be made with a texture similar to that of a commercially available pectin confectionery jelly. It is well known that many soft solid foods show nonideal elastic behaviors at large deformation, such as hysteresis between the compression and recovery curves. Thus, the degree of elasticity of the samples based on the recoverable work of compression was determined as the ratio of the

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(b)

(a)

(c) Figure 10.4. Color and transparency of a commercial pectin high solid confectionery product (PC; a); a typical cranberry jelly made using the infusion syrup (CP5; b); and a cranberry jelly including texturized whey protein (CP6; c). The soluble solid content of the cranberry jelly was 80◦ Brix, and that of the whey-containing cranberry jelly was 75◦ Brix.

work recovered and the total work of compression, and was abbreviated as DEw .

Results and Discussion Several formulations for pectin cranberry jellies were developed based on different total soluble solids and cranberry syrup content (Table 10.3).

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One “good” formulation for the prototype pectin cranberry jelly product consisted of pectin (2.3%), added sucrose (22.2%), cranberry syrup (75%), and additional citric acid solution (1.3%). No corn syrup was added. As shown in Table 10.3, the DEw and firmness for this prototype product (CJ-80) were 53% and 28.1 N, respectively. This compared to 52.8% and 27.8 N for a commercially available pectin high solid confection that was purchased in a local supermarket. Apparently, the addition of a citric acid solution prior to boiling helped achieve a texture similar to that of the commercial pectin high solid confectionary product for this pectin cranberry jelly formulation. According to the results from the sugar profile testing provided by Analytical Food Laboratories Inc. (Grand Prairie, TX), a partial explanation for this result might be that most of the sucrose in the cranberry infusion syrup was already inverted to fructose and glucose. The prototype product essentially kept its natural color intact throughout the

Table 10.3. The degree of elasticity DEW (%) and force (N) at 25% compression determined for a commercial pectin high solid confectionery product, cranberry-jelly with 80◦ Brix made using a 50% cranberry infusion syrup, cranberry-jelly with 80◦ Brix made using a 75% cranberry infusion syrup in formulation, CJ-80, and CJ-75-STWP. Maximum force (N)

DEw (%)

27.80 ± 0.04 a

52 ± 0.01 a

Pectin jelly 80 Brix using 50% cranberry infusion syrup in formulation

30 ± 0.1 b

54 ± 0.7 a

Pectin jelly 80◦ Brix using 75% cranberry infusion syrup in formulation

35 ± 0.05 c

54 ± 0.6 a

CJ-80; “good” pectin-based cranberry jelly; Pectin jelly 80◦ Brix made using 75% cranberry infusion syrup in formulation. Citric acid added to infusion syrup prior to boiling concentration

28.1 ± 0.04 a

53 ± 0.52 a

CJ-75-STWP

28.88 ± 1.2 a

53 ± 0.3 a

Sample Commercial pectin high solids confection ◦

Numbers with the same letters in the same column denote a statistically insignificant difference at 95% confidence.

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boiling concentration process and remained translucent (Figure 10.3). The ORAC value of the gel after evaporation to the desired solids content was 5 μmole/g Trolox equivalents, that is, much of the original antioxidant activity survived the evaporation process. The last step was to incorporate TWP into the prototype cranberry jelly, while also maintaining its color and texture as much as possible. The best results were obtained using STWP (CJ-75-STWP), which had DEw and firmness values of 53% and 25.9 N, respectively, as compared to 52.8% and 27.8 N for the commercial pectin high solid confectionery product (Table 10.3), while it also maintained translucency and cranberry natural color (Figure 10.4). These values for the whey-containing product, which were insignificantly different from those for the control commercial sample, were obtained by reducing the soluble solids to 75◦ Brix. Fluoresence Microscopy It seemed that adding STWP did not affect significantly the elasticity, as inferred from DEw , which suggested that TWP did not affect substantially the pectin gelation process upon cooling. Addition of TWP did cause an increase in the firmness. This last effect could be due to increased viscosity, or acting as a filler of the cosolute phase. Fluoresence microscopy images (Figure 10.5) of a pectin control gel sample and one with added STWP revealed that many irregular, polygonal autofluorescent particles ranging in size from 0.1 to 0.4 mm wide were suspended in a transparent matrix, presumably the pectin gel matrix. Images of the pectin jelly with no STWP in crosssection were transparent in white light and contained no autofluorescent particles when illuminated with green light.

Conclusions This work demonstrates the feasibility of obtaining high solid confectionery “functional foods” with acceptable “instrumental texture” from natural cranberry infusion syrups. Opportunities for incorporating predenatured TWP into this novel food as an additional nutrient source and/or texturizing agent were also demonstrated. Additionally, it could

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(a)

(b)

(c)

(d)

225

Figure 10.5. Optical fluorescence of a pectin control gel sample using transmitted light from a 150 W halogen lamp (a), epifluorescence from green excitation (540– 546 nm) (b), a pectin gel containing 5 g of added STWP using a 150 W halogen lamp (c), epifluorescence from green excitation (d).

be concluded that the elasticity of the pectin jelly was independent from its firmness, as added TWP was predenatured, and did not form a network of its own or interfere too much with the pectin gelation process.

References Clydesdale, F.M. 2004. Functional foods: Opportunities and challenges. Food Technol. 58(12):35–40. De Mars, L.L., and Ziegler, G.R. 2001. Texture and structure of gelatin/pectin-based gummy confections. Food Hydrocoll. 15(4):643–653.

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Edwards, W.P. 2000. The Science of Sugar Confectionery. Cambridge, UK: Royal Society of Chemistry. Hasler, C.M. 2002. Functional foods: Benefits, concerns and challenges—A position paper from the American Council on Science and Health. J. Nutr. 132(12):3772– 3781. McHugh, T.H., and Huxsoll, C.C. 2000. Restructured fruit and vegetable products and processing methods. U.S. Patent 6,027,758. Sun, J., Chu, Y.-F., Wu, X., and Liu, R.H. 2002. Antioxidant and antiproliferative activities of fruits. J. Agric. Food Chem. 50(25):7449–7454.

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Chapter 11 Whey Protein Hydrogels and Nanoparticles for Encapsulation and Controlled Delivery of Bioactive Compounds Sundaram Gunasekaran

Introduction Whey proteins (WPs) are highly nutritional protein source; indeed, WPs are considered the best protein source based on their high biological value. WPs help improve the blood level of glutathione, an antioxidant essential for a healthy immune system. Because of this and other salubrious effects on human health, WPs are popularly used as protein supplements in various health foods. Furthermore, the physicochemical properties of WPs suggest that they may be suitable for other novel food and nonfood applications. For example, whey protein gels may be used as hydrogels for controlled delivery of biologically active substances (Gunasekaran et al. 2006b). A hydrogel can be defined as a threedimensional network that exhibits an ability to swell in water, maintains its overall physical shape, and retains a significant fraction of water within its structure. There is a wide variety of hydrogels made from natural and synthetic polymers. One of the unique properties of hydrogels is that they could swell, shrink, bend, or probably degrade (i.e., undergo “phase transition”) when subjected to small changes in environmental factors such as pH, temperature, electric field, ionic strength, salt type, solvent, stress, light, pressure, sound, and chemical substance. Such systems are known as stimuli sensitive or smart hydrogels. These unique properties make hydrogels one of the important “intelligent” materials, 227 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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extremely useful for myriad of applications: catalysts, enzymes, and cell immobilization; autonomous actuation; biosensing; dewatering, decontaminating, and concentrating; and self-regulated controlled delivery of bioactive compounds. WPs can also be formed into nanoparticles—matrix systems of a dense polymeric network of under 100 μm in size in which an active molecule may be dispersed throughout (Nakache et al. 2000). Since nanoparticles are submicron and subcellular in size, they have versatile advantages for targeted, site-specific delivery purposes (Vinagradov et al. 2002) as they can penetrate circulating systems and target sites. The nanoparticles offer the feasibility to entrap drugs or bioactive compounds within but not chemically bound to them. Various biocompatible and biodegradable biopolymers have been used in the formation of nanoparticles to maximize delivery efficiency and increase the desirable benefits (Coester et al. 2000; Kreuter 1994; Rhaese et al. 2003). Albumin nanoparticles have been extensively investigated with respect to their preparation methods and release properties (Langer and Peppas 1983; Lindenbaum et al. 1959; Vural et al. 1990). Human serum albumin (HSA) and bovine serum albumin (BSA) have also been used as natural matrix materials for delivery devices (Brannon-Peppas and Peppas 1991).

Hydrogels Hydrogels could simulate the structure and function of natural gels in living systems such as swelling. The high water content and soft and rubbery consistently give hydrogels a strong, superficial resemblance to living soft issue, which also contribute to their high biocompatibility by minimizing mechanical (friction) irritation to surrounding cells and tissue. Low interfacial tension, which exists between the surface of hydrogel and surrounding aqueous solution, reduces the tendency of the proteins in body fluids to adsorb and to unfold upon adsorption. Minimal protein interaction is important for the biological acceptance of foreign materials since the denaturation of proteins may trigger the mechanism for initiating thrombosis or for other biological rejection mechanisms. The expandable nature of hydrogels and the permeability of hydrogel structure to small molecules can be used for controlled release of certain immobilized, biologically active molecules. Therefore,

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hydrogels represent a group of very promising materials with many applications of controlled release. Various hydrogel systems have been developed as controlled drug release carriers using water-soluble, biodegradable polymeric materials (Gudeman and Peppas 1995; Gunasekaran et al. 2006a; Kim and Park 1998; Wang and Gunasekaran 2006; Wang et al. 2004; Wen and Stevenson 1993) including synthetic or natural polymers. Among the natural polymers used to develop pH-sensitive hydrogels are alginates (Kim and Park 1998) and chitosan (Deyao et al. 1993; Wang et al. 2004). The latter is usually cross-linked with other polymers such as polyvinyl alcohol (Wang et al. 2004) or polyether (Deyao et al. 1993) using glutaraldehyde to produce semi-interpenetrating networks. Kim and Park (1998) reported that pH-sensitive hydrogels can be prepared from egg albumin simply through heat-induced gelation. They investigated the effect of gel preparation conditions, particularly the initial pH of the protein solution, on the swelling of dried albumin gel in phosphate buffer solutions. The albumin hydrogels exhibited pH-sensitive swelling behavior; the degree of swelling is low around the protein isoelectric point (pI ≈ pH 4) and increased with pH. Formation of pH-Sensitive Hydrogels The characteristic of pH-sensitive hydrogel is cross-linked polycations or polyanions, which is shown in Figure 11.1. The high density of charged groups present in pH-sensitive hydrogels could be pendant weak acidic or basic groups, such as carboxylic acids and primary amines, or strong acids and bases, such as sulfonic acids and quaternary ammonium salts. Depending on the specific ionizable groups of the hydrogel, the hydrogel could be either ionized or unionized in response to changes in surrounding environment pH; therefore, it undergoes either taking up water (swelling) or releasing water (shrinking) (Figure 11.2). This causes a significant change in hydrogel volume, and thus the physical properties of the gel. The ordinary ionic groups, which are commonly present in pH-sensitive hydrogels, are listed in Table 11.1. In addition to physical gelation of proteins and certain polysaccharides, chemical cross-linking can lead to the formation of pHsensitive hydrogels. The use of chemical cross-linking agent is important not only for cross-linking the macromolecules, therefore forming

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Figure 11.1. The cationic pH-sensitive hydrogel.

three-dimensional network, but also for introducing the desired charge groups as well as immobilizing bioactive substances, such as catalysts, enzymes, cells (White and Kennedy 1980), and drugs (Heller 1988). These hydrogels will not dissolve in water or other organic solvents unless the covalent bonds are cleaved. The two common ways to prepare

Solutions of different pH Figure 11.2. Schematic of pH-sensitive swelling and shrinking of a hydrogel.

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231

Common ionic groups presented in pH-sensitive hydrogels.

Monomer

pH-sensitive groups

Anionic Acrylic acid −COO− Mannuronic acid or guluronic acid of algin

References Kopecek et al. (1971), Kou et al. (1988), Ricka and Tanaka (1984), and Vacik and Kopecek (1975)

Sodium stryenesulfonate Sulfonate galactan of carrageenan

−SO− 3

Michaels and Morelos (1955)

Cationic Aminoethyl methacrylate Glucosamine of chitosan

−NH+ 3

Kang et al. (1993)

N ,N -dimethylaminoethyl methacrylate

−N(CH3 )2 H+ Siegel (1990) and Siegel and Firestone (1988)

Vinylbenzyl −N(CH3 )+ 3 trimethylammonium chloride

(Michaels 1954; Michaels and Morelos 1955)

such chemical gels are as follows: (1) Copolymerizing water-soluble monomers in the presence of small amount of coreactable anionic or cationic monomers. For example, copolymeric gel containing hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) are crosslinked by free radical polymerization using ethylene glycol dimethacrylate as the cross-linker (Khare and Peppas 1995). The commonly used water-soluble monomers are acrylic acid, MAA, acrylamide, N alkylacrylamide, methacrylate, vinylpyrrolidone, methyl methacrylate, HEMA, and vinylpyridine. The size of the cross-linking agent can vary widely from small molecule, such as N , N -methylenebisacrylamide to macromolecules such as proteins. (2) Cross-linking functional groups of the existing cationic or anionic water-soluble polymers to form threedimensional structure. The cross-linking agents used usually have two reactive sites or groups, which join reaction sites of the ionizable polymers either to form “loops” or to connect two different molecules to form “bridges.”

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Cross-linking of polymers is one form of chemical modification, which carries the same form of reaction as their corresponding low molecular homologs, except its reaction rate and maximum conversion may differ significantly from those of low molecular homologs (Odian 1991). The efficiency of cross-linking depends on the specific groups to be cross-linked. While it would be ideal if the reagents possessed high selectivity for certain functional groups, the lack of specificity may be more important in the cross-linking of macromolecules possessing more than one type of functional groups. According to the functionality of the cross-linker, it could be divided into three classes: homobifunctional, heterobifunctional, and zero length (Wong 1991). Homobifunctional cross-linking agents contain two identical functional groups that react with the same functional groups of the polymer. Heterobifunctional agents contain two different reactive sites and hence react with different functional groups of the polymer. Zero-length cross-linking agents link polymers without the addition of extrinsic compounds. Many zero-length cross-linking agents condense carboxyl groups with primary amine (−NH2 ), hydroxyl (−OH), carboxylic (−COOH), and thio groups (−SH) to form amide, ester, or thioester bonds. For example, excess of primary amine groups or carboxylic group of macromolecules is essential for a pH-sensitive hydrogel not only because they introduce these net charges of formed hydrogel matrix but also provide these functionality sites, to which the cross-linking agents easily react. For amino groups, the common cross-linking agents are N , N carbonyldiimidazole, bisoxirane, divinylsulfone, carbon disulfide, and urea-formaldehyde working for polymer molecules containing hydroxyl (−OH) or amino groups (−NH2 ) (Amiya and Tanaka 1987; Patwardhan and Das 1983; White and Kennedy 1980). The diisocyanates, diisothiocyanates (Plotz 1977), and bifunctional acrylazides such as tartryl diazide, and bisepoxides (Pishko et al. 1991), and formaldehyde and glutaraldehyde (Odian 1991). Glutaraldehyde is probably the most widely used homobifunctional cross-linking agent for amino groups, which forms linkage resistant to extremes of pH and temperature (Sturgeon 1988). A glutaraldehyde cross-linked gelatin layer was used to enhance the biocompatibility of biomaterials for the artificial heart (Emoto et al. 1990). Also, it was used to cross-link gelatin microsphere hydrogels for the delivery of low-molecular-weight-drug phenytoin (Chibata et al. 1987; Geysen

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et al. 1984; Mattiasson 1983; Netti 2000; Raymond et al. 1990), high– molecular-weight drug interferon (Tabata and Ikada 1987), and anticancer agents (Yan et al. 1991). Albumin hydrogel-containing urease was also prepared by using cross-linker glutaraldehyde for enzymemediated drug delivery as well as for delivery of variety of other drugs (Morimoto and Fujimoto 1985; Sheu and Sokoloski 1991). There are relatively few cross-linking agents that directly react with carboxyl groups. The epoxy groups react with both amine groups and carboxyl groups (Imamura et al. 1989). Polyepoxides were used to crosslink carboxyl groups of collagen, and bioprostheses such as porcine aortic leaflets or canine carotid arteries (Nojiri et al. 1987). The treatment with polyepoxy compound resulted in pronounced improvement in the biocompatibility of the bioprostheses. In the presence of diamines, polymers containing carboxyl groups can be cross-linked by forming amide bonds using many reagents such as carbodiimide, Woodward’s reagent K, ethylchloroformate, and carbonyldiimidazole (Odian 1991). These cross-linking agents induce amide bonds by removing atoms from the carboxyl and amine groups. Since no extrinsic spacer is added by cross-linking agents, they are known as zero-length cross-linking agents. Chondroitin sulfate was also cross-linked with diaminododecane in the presence of dicyclohexylcarbodiimide. The cross-linked chondroitin sulfate was used for colon drug delivery due to its degradability by the bacteria of the large intestine (Rubinstein et al. 1992). Swelling of pH-Sensitive Hydrogels The pH-sensitive hydrogel is built from ionizable monomer unit, which carries ionogenic groups. These groups, as well as the ions they form, tend to be surrounded by polar solvent molecules. Monomers with such groups are thus soluble in polar solvents. The dissolution process is driven by the tendency for affiliating these ionogenic groups and ions for the polar solvents like water. The coiled and packed chains of the macromolecules unfold to make room for solvent molecules, that is, a conformation change of the composition polymer, for example, from a coiled and aggregated state to a stretched and expanded state due to the osmotic pressure difference caused by pH change. As a result, the water molecules transfer into hydrogel from outer environment and participate in those conformational changes. Therefore, the water content of the hydrogel increases and hence the hydrogel volume. As a result, the

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ionic gels swell significantly but do not dissolve because they are still interconnected by the cross-linkages. Swelling of hydrogel is a thermodynamic equilibrium process, which is a balance of opposing forces. The tendency of the polar and ionic constituents of the gels to surround themselves with solvent and thus to stretch the gel matrix is met with an increasing resistance by the retracting force of the cross-linked gel matrix. The repulsion force of the same charges along the polymer chain will also contribute to an increase in swelling of the hydrogel. Equilibrium swelling is finally attained when the elastic forces of the matrix balance the dissolution tendency. Various factors governing the dissolution tendency of pH-sensitive hydrogel are summarized as follows: Nature of Solvent As mentioned above, ionogenic groups are necessary for the swelling of pH-sensitive hydrogels. Therefore, as a basic rule, the polar solvents are better swelling agents than the nonpolar solvents since they interact more strongly with the ions and polar groups in the hydrogel. Hydrogels swell better in aqueous solutions compared to in nonpolar organic solutions, which instead often lead to shrinking of hydrogels. Nature of the Polymer’s Ionic Groups The greater the affinity of the ionic groups for the polar solvent, the more strongly does the gel swell. In particular, hydrogels swell more strongly when their fixed ionic groups are completely ionized. So, the apparent acid or base dissociation constant (pK a ) of an ionic hydrogel is the determining factor here. A basic or cationic hydrogel will be ionized at pH < pK a , but unionized at pH > pK a ; thus, the equilibrium degree of swelling increases at low pH. An acidic or anionic hydrogel exhibits the opposite effect—swelling when pH > pK a and shrinking when pH < pK a (Brannon-Peppas and Peppas 1991). Degrees of Cross-Linking The ability of hydrogels to swell is inversely proportional to the extent of cross-links present in their network (Gregor et al. 1951). A large number of cross-links make the network very rigid; therefore, it is difficult for the polymer chains to expand or relax. In the earlier macroscopic models proposed by Gregor, the molecular “springs” are harder, while

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in other so-called molecular models the chains are shorter and, hence, the loss in configurational entropy accompanying a given expansion is greater. Ion Capacity or Concentration An ionic gel with higher ion concentration and high capacity has the higher tendency for pore liquid to dilute itself (higher osmotic pressure difference due to the higher counter-ion concentration attracted by ionic groups) and the resulting swelling are more pronounced than those with a low ion concentration/low capacity (Lindenbaum et al. 1959). Nature of the Counterion The effect of counter ions on swelling equilibrium is somewhat complex. According to the earlier research on ionic exchangers, in moderately and highly cross-linked gels, in which most of the solvent is present in the form of solvation shells, the size and solvation tendency of counterions are the most important (Gregor 1948, 1951). The gels swell when a counterion is replaced by another, which in its solvated state occupies more room. The hydrated ionic volume of the following ions can be relatively compared: Cs+ < Rb+ < K+ < Na+ < Li+ . However, in a highly cross-linked gel, solvation may remain incomplete. The sequence may be partly or completely reversed according to the hydrated ionic volume since the Li+ is the smallest and Cs+ is the largest ion when not solvated. In weakly cross-linked gels, which contain relatively large amount of “free” solvent, that is, solvent not in the form of solvation shells, the valence of the counterions is the most important factor for swelling (Calmon 1952, 1953). The tendency (osmotic pressure difference) to take up free solvent (water) depends on the total number of counterions attracted to the gel. This number is cut in half when univalent counterions are replaced by bivalent ones. The osmotic pressure difference in the Gregor’s models or the free energy of mixing in the molecular models becomes correspondingly smaller. Ionic size and solvation effects in these highly swollen gels are relatively unimportant. Thus, weakly cross-linked gels swell less when the valence of the counterion is high, and usually the opposite holds in moderately and highly cross-linked gels since the polyvalent ions are usually more strongly hydrated.

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Nature of Ion Pair The hydrogel’s ability to swell decreases when counterions and fixed groups associated to form complexes. Such localization of the counterions reduces, in the Gregor’s models, the tendency to form solvation shells and the osmotic activity depressed and, in molecular models, it reduces the free energy of mixing. For example, weak-acid gels swell less in H+ form than in alkali-ion forms. Weak-base gels swell less in free base than in chloride form, whereas the opposite usually holds for strong-acid and strong-base gels (Katchalsky 1954). Concentration of the Swelling Solution Gels that are equilibrated with electrolyte solutions swell more strongly when the external swelling medium concentration is low (Pepper et al. 1952). Any increase in external ion concentration will minimize the osmotic pressure difference between the interior and exterior of the gel or, in molecular models, the free energy of mixing. Thus, the “driving force” for solvent uptake becomes smaller. Thermodynamics of Swelling According to the model of Gregor (Gregor 1948, 1951), a gel matrix is a network of elastic springs. When the gel swells, the network stretches and exerts a pressure on the internal “pore-liquid” molecules resulting in a higher pressure (Ppore ) than the external swelling medium pressure (P). The pressure difference between the pore solvent and the outer swelling medium is the “swelling pressure” (Psw ). Psw = Ppore − P

(11.1)

and Psw =

−RT ln aw vw

(11.2)

where ν w is partial molar volume of the solvent, and aw is solvent activity in the gel. The swelling pressure is the result of contraction forces of the elastic matrix. The contraction forces increase when the matrix expands, and according to Equation (11.2), the solvent activity decreases. Once the solvent activity in the gel is equal to its activity in the swelling medium, or the osmotic pressure (driving force of swelling)

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is balanced by swelling pressure, the swelling reaches equilibrium. If the solvation is one of the most important factors determining swelling and the chains of the gel matrix behave like ideal elastic springs, Gregor found that the swelling pressure of a given gel is a linear function of its equilibrium volume of the gel (Ve ). Ve = a Psw + b

(11.3)

where a and b are empirical constants for the gel and are independent of the ionic form and the relative humidity. The constant a reflects the elastic properties and is large for a highly cross-linked gel. The constant b is the volume of the unstrained gel and is essentially independent of degree of cross-linking. The Gregor’s model is purely mechanical; it does not consider the single ion as a discrete particle, and the elasticity of the springs represents the matrix network structure. In contrast, the model proposed by Rice and Nagasawa (1961) is based on the considerations of a molecular scale. The elasticity of the matrix is not mechanical, but rather is due to an increase in total entropy, which accompanies the configuration of coiling polymer chains. Swelling equilibrium is attained when the free energy of gel system is at its minimum. The free energy change (F) involved in the swelling process may be written as follows: F = FM + Fel + FI

(11.4)

where FM is the free energy change of mixing polymer chain with the solvent, Fel is the free energy change of ion-pair formation associated with the change in configuration of the network. FI is the contribution coming from the electrostatic interactions and from the free energy of permutation of ionized and nonionized groups. From Flory’s polymer solution theory, FM = kT (n 1 ln v1 + χ1 ln v2 )

(11.5)

where n 1 is the number of the solvent molecule, χ 1 is the interaction parameter of solvent with polymer network, v1 and v2 are the volume fractions of solvent and polymer network, respectively, that is, v1 = 1 − v2 .

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If αs represents the linear deformation factor, then by the condition of isotropy αx = α y = αz = αs , we can write Fel as follows    kT ve  2 Fel = 3αs − 3 − ln α3s (11.6) 2 where ve is the effective number of chains in the network. For nonionic networks, Flory (1953) derived the following simplified equation for swelling:   1 − 2Mc −1 (1/2 − χ1 ) 5/3 ∼ qm = (v Mc ) (11.7) M v1 where qm is the equilibrium or maximum swelling ratio, v is the specific volume of polymer, Mc is the molecular weight per cross-linking unit, M is the molecular weight of polymer repeat unit, and χ1 is the interaction parameter of polymer and solvent. These simple relationships offer a clear insight into the dependence of the equilibrium swelling ratio on the quality of the solvent, expressed by χ 1 , and on the extent of cross-linking. Because of the nature of simplifications (such as ignoring the connection between monomer units, assuming the polymer chains are homogenously distributed and the size of monomer is equal to the size of the solvent molecule), this equation can be applied only to the networks of very low degree of cross-linking in good solvents. For ionic network, in order to calculate the third term in Equation (11.4), it is assumed that the ionogenic groups are uniformly distributed, occupying sites in a spatial lattice. Electrostatic interactions between the nearest neighbors are calculated using the Debye–Huckel theory for various “microscopic” states of the gel, that is, for various possible distributions of the ion pairs. A partition function is then formed by combining all microscopic states according to their free energy in essentially the same way as in the statistical lattice theories of liquid (Hirschfelder et al. 1954). In addition to polymer–solvent mixing and polymer elasticity contributions, network ionization is another major factor contributing to the swelling of ionic hydrogels. An increase in ionization in the polymer network increases its hydrophilicity, and therefore leads to a higher equilibrium as well as faster swelling. The charge density of the polymer network affects the pH sensitivity of the gel. Anionic or acidic hydrogels

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containing carboxylic acid groups swell at pH higher than the gel pK a due to ionization. The gel is in an unionized state at pH lower than the gel pK a and therefore does not swell to a large extent. Opposite behavior is observed in the case of basic or cationic gels containing amine groups and transitional pH depends again on the gel pK a . In the case of an ampholyte, that is, a gel containing both acidic and basic groups, an isoelectric pH is the deciding factor. A polyampholyte is in the ionized form at a lower acidic pH and at a higher alkaline pH, and therefore the gel is highly swollen in those regions. Near the isoelectric pH, the gel is in a moderately swollen state. Proteins are examples of polyampholytes. The interaction between each protein molecule denatured on heating is mainly governed by the surface net charge and the hydrophobic area exposed by heating of the protein molecules. The former usually arises due to electrostatic repulsion, and attractive force originates from the hydrophobic interaction. It is expected that the structure of heat-induced protein gel can be controlled by adjusting either surface net charge or hydrophobicity. Therefore, it is possible to design the structure of heat-induced protein gels as drug carriers that have a pH-sensitive property, and this means that we can prepare the drug carriers having different structures (pH sensitivities) with protein. To do this, it is necessary to control the interaction forces between protein molecules. Between attractive and repulsive forces, the latter can be readily controlled by controlling the surface net charge of protein molecules. The easiest way to control the surface net charge is by changing the pH of protein solutions.

Kinetics of Swelling As mentioned above, hydrogels are widely used in drug release systems. For swelling-controlled drug release systems, swelling kinetics are the most important because they determine the rate of drug release from the hydrogel. Diffusion-controlled swelling shows first-order kinetics, and for other types of swelling mechanism, complex behavior is observed. There have been extensive theoretical and experimental investigations on swelling kinetics. Two simple kinetic models and a semiempirical model are widely used that are described below to highlight the essential aspects of swelling dynamics.

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First-Order Kinetics (Schott 1992) According to the Fick’s second law of diffusion, ∂ 2C ∂C (11.8) =D 2 ∂t ∂r where r is the radial distance of the diffusant from its initial position, D is the diffusion coefficient, C is the diffusant concentration, and t is the diffusion time. For the simplest situation, one-dimensional swelling of slabs (films or tablets), when suitable boundary conditions are applied, we can get the following solution of Equation (11.8) (Barrer 1951; Jost 1960):    ∞ W∞ − Wt 8  (2n + 1)π 2 1 Dt (11.9) = 2 exp − W∞ π n=0 (2n + 1)2 H where W and W∞ are mass of the hydrogel sample at any time t and ∞, respectively, H is thickness of the hydrogel tablet, and n is integral number. For long swelling time, the terms in n ≥ 1, as well as ln(8/π 2 ), can be neglected. Equation (11.9) then simplifies to   W∞ π 2 Dt ∼ ln (11.10) = W∞ − Wt H2 Letting K = π 2 D/H 2 , we obtain Wt = 1 − exp(−K t) W∞

(11.11)

In the above derivation, it is assumed that the thickness of the slab or film and the diffusion rate do not change so that K is a constant. This is not true, however, for extensive swelling. Obviously, H increases; meanwhile, D increases because the local viscosity in the swelling polymer gel decreases as the influx of solvent lowers the solids content. As long as the increase in D matches the increase in H 2 reasonably well, K remains nearly constant and the swelling seems to obey the first-order kinetics. When the increase in H 2 exceeds the increase in D to a point where the variation in K is large, the deviation from the first-order kinetics becomes significant. For long periods, swelling increases and levelsoff to an equilibrium value and swelling kinetics results are analyzed

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according to a second-order kinetic model (Gonzalez et al. 1999; Schott 1992; Vazquez et al. 1997). Second-Order Kinetics The rate of swelling is assumed to be directly proportional to two quantities: first, to the relative or fractional amount of swelling capacity still available at time t, [(W∞ − Wt )/W∞ ]; second, to the internal specific boundary area Sint enclosing those sites of the polymer network that have not yet interacted with water at time t but will be hydrated and swell in due course. This yields the following equation:   W∞ − Wt dW (11.12) = K1 Sint dt W∞ where Sint is the specific surface envelope that surrounds all sites where interchain hydrogen bonds are located. A value of Sint might be estimated by applying geometric considerations. As swelling proceeds and interchain hydrogen bonds are being ruptured, Sint decreases commensurately: Sint is directly proportional to the number of interchain hydrogen bonds that are still intact and, therefore, also to the relative swelling capacity still available.   W∞ − Wt Sint = K 2 (11.13) W∞ Combining Equations 11.12 and 11.13 results in the following secondorder equation:   W∞ − Wt 2 dW = K (W∞ − Wt )2 (11.14) = K1 K2 dt W∞ Integrating Equation (11.14) and rearranging, we obtain the following: t (11.15) Wt = A + t/W∞ 2 where A = 1/K W∞ .

Semiempirical Model The kinetics of swelling could also be treated from the sorption point of view. However, the contours of time versus penetrant uptake curve deviates more often from what is predicted by the classical Fickian model. In these cases, the sorption process is not a passive diffusion of

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the solvent molecules into the void spaces of the network but includes a concomitant relaxation of the network segments resulting from the advancing solvent front, which leads to plasticization of the material and a large increase in volume. The generalized semiempirical equation is (Peppas 1987; Rathna et al. 1994; Valencia and Pierola 2002; Windle 1983) as follows: Wt = K tn W∞

for

Wt < 0.6 W∞

(11.16)

where K is a characteristic constant of the system, which is a function of the hydrogel tablet geometry and the diffusion constant. This equation has been used to distinguish three types of sorption behavior, with the value of n as the monitoring index (Lucht and Peppas 1987; Windle 1983). For a perfect Fickian process, where the rate of solvent penetration is slow, in comparison to the chain relaxation rate, and hence being the rate-determining step, the value of n is close to 0.5, which is called case I sorption. When the mobility of the penetrant is substantially faster than the chain relaxation rate, the solvent uptake is directly proportional to the time and this is called case II sorption. When the rate of penetrant mobility and segmental relaxation are comparable, the value of n ranges between 0.5 and 1.0, and these are classified as the anomalous case. Controlled Drug Release from Hydrogels There are many challenges to create safe, economical, and efficient means of providing for our health and well-being. In almost every case, the solution lies in the development of creative systems for responding to and/or controlling biological factors such as temperature, pH, and chemical species in the living body (Cowsar 1974). In the last few decades, many kinds of newly synthesized drugs have been developed that have shown good results in treating various diseases. However, these drugs, including existing ones, also have unexpected side effects and may be very expensive. Controlled drug release technology may be a key to solve these problems. Controlled drug delivery can be used to achieve the following: r

Sustained constant concentration of therapeutically active compound in the blood with minimum fluctuations. r Predictable and reproducible release rates over a long period.

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Protection of bioactive compounds having very short half-life. Elimination of side effects, waste of drug, and frequent dosing. Optimized therapy and better patient compliance. Solution of the drug stability problem. A typical drug delivery system consists of a polymer carrier in which the drug is uniformly distributed or dispersed.

Understanding the drug release kinetics is critical in the design of a drug delivery system. The administration routes of hydrogel-based formulations include transdermal, oral, nasal, or parenteral (Kuu et al. 1992). With the advent of controlled release formulations for bioactive compounds such as proteins and polypeptides, there has been significant interest in the development of stimuli-responsive drug delivery systems utilizing hydrogels. Drug delivery systems can be classified based on the mechanism controlling the drug release as follows (Langer and Peppas 1983): 1. Diffusion-controlled systems: reservoir (membrane systems) and matrix (monolithic systems). 2. Chemically controlled systems: bioerodible and biodegradable systems and pendent chain systems. 3. Solvent-activated systems: osmotic-controlled systems, swellingcontrolled release systems, and modulated-release systems. Swelling-Controlled Systems When a glassy polymer comes into contact with an aqueous solution, it begins to imbibe water. This water uptake can lead to considerable swelling of the polymer. Due to the swelling action, the drug, which is dispersed in the polymer, begins to diffuse out. Thus, drug release depends on three simultaneous rate processes: water diffusion into the polymer, polymer chain relaxation, and drug diffusion out of the polymer. The continued swelling of the matrix causes the drug to diffuse out at a faster rate. The overall drug release rate is controlled by the rate of swelling of the polymer network. Depending on the rate of water diffusion and macromolecular chain relaxation, the time dependence of the rate of drug release can be determined. Usually in a swelling-controlled release system, an initially glassy polymer is used as a carrier for the drug. pH-sensitive hydrogels have

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been found to be appropriate carriers as swelling-controlled release devices. The ability to control the dynamics of swelling by changing the pH or ionic strength of the external swelling medium provides various opportunities for stimuli-responsive drug delivery. For example, oral drug delivery systems based on pH-sensitive hydrogels can be used to release drugs at a specific site of the gastrointestinal tract when the pH falls within a certain range (Beltran et al. 1990). More complex systems based on pH sensitivity are glucose-sensitive drug delivery systems (Choi et al. 1992; Ito et al. 1989). With the advent of novel methods for preparation of new potent drugs and proteins, there is an increased interest in the development of site-specific modulated drug delivery systems (Banga and Chien 1988; Lee 1991; Pitt 1990; Zhou and Po 1991a, b). Ionic hydrogels are one type of potential carriers for these solutes. Bioadhesive, thermosensitive, photosensitive, electrically sensitive, pH-sensitive, or enzyme-digestible ionic hydrogels (Gehrke and Lee 1990) are widely used in such applications. In a swelling-controlled system, a three-component system consisting of a polymer, water, and a dispersed solute or drug molecule is present. The rate of drug release can be adjusted by modifying the polymer morphology, for example, hydrophilic–hydrophobic balance, degree of ionization, degree of crosslinking, or by controlling the solute-partitioning characteristics of the polymer–water system, or by manipulating the properties of external swelling medium, for example, pH, ionic strength, buffer composition, or temperature. Dynamics of Drug Release by Swelling-Controlled Systems The dynamic swelling behavior of hydrogels is controlled by the structure of the polymeric network and polymer–solvent interactions. When a drug is incorporated into a glassy polymer network, water transport controls the associated drug release as shown in Figure 11.3. Both glassy and rubbery networks have characteristic swelling kinetics. This principle forms the basis for swelling-controlled release devices. Drug release by swelling-controlled mechanisms is related to drug diffusion from and through the initially glassy polymer matrix, under countercurrent diffusion of water or biological fluids into the polymer matrix. The drug is originally dispersed or dissolved in a swollen gel. As the solvent is evaporated, a solvent-free glassy polymeric matrix is obtained, with bioactive agent dispersed in it. This system forms a typical swellingcontrolled release system. As the dissolution medium (e.g., water, saline,

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Glass polymer-containing drug molecule, D

Glass-to-rubbery transition

Swelling front disappears

Rubbery hydrogel

Figure 11.3. Schematic of a swelling-controlled drug release system.

and biological fluid) penetrates into the polymer matrix, the solventfree polymer starts swelling. If the polymer is thermodynamically compatible with the dissolution medium, the matrix will become rubbery, because of the reduction in glass transition temperature of the matrix below the temperature of the release medium. This penetration leads to

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Figure 11.4. Schematic diagram of a dynamically swelling thin polymer disk in a swelling agent. Here, v1 is the velocity of the glassy–robbery interface and v2 is the velocity of the swelling interface.

a considerable increase in the macromolecular mobility due to plasticization of the network and therefore to considerable volume expansion of the network. Two fronts (interfaces) are characteristic of this swelling behavior: a front separating the glassy from the rubbery core, called the “swelling interface,” which moves inward toward the glassy core at a velocity v as shown in Figure 11.4, and a front separating the expanding rubbery core from the pure dissolution medium (polymer interface), which moves outward. For planar geometry, the glassy core constrains swelling to one dimension, normal to the front. This constraint causes a compressive stress inside a glassy core and a tensile stress in a rubbery core. Once these two advancing swelling interface fronts meet at the center, the glassy core vanishes and the polymer becomes rubbery. Thus, the swelling constraint disappears, and swelling proceeds in three dimensions (Ritger and Peppas 1987a). Depending on the dynamics of polymer swelling and the relative mobility of drug and water, Fickian or non-Fickian drug transport may be observed. The relative importance of water diffusion and polymer relaxation can be described by the Deborah number (De ) (Ritger and

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Peppas 1987b), defined as the ratio of a characteristic relaxation time (τ ) to a characteristic diffusion time (θ ). τ De = (11.17) θ Here, θ = L 2 /Dwp , where L is the characteristic length of the controlled release device and Dwp is the water diffusion coefficient. When De  1, relaxation is much faster than diffusion and Fickian diffusion is observed. This occurs well above glass transition temperature (Tg ), where gels are rubbery and the drug diffusion coefficient (Dip ) is generally a strong function of concentration. Fickian diffusion is also observed for De  1, corresponding to diffusion in a glassy polymer well below Tg , where polymer relaxation is so slow that its structure is unchanged by the diffusion process. When De ≈ 1, relaxation and diffusion are coupled, leading to a complex transport behavior, known as “anomalous” or non-Fickian transport (Lee 1988). In Fickian diffusion, the rate of water absorption shows a linear increase as a function of the square root of time. Fickian diffusion is observed when the time scale of the macromolecular relaxation is either effectively infinite or zero compared to the time required to establish a concentration profile in the polymer sample. These two limits signify the elastic and viscous Fickian diffusion limits, respectively. In non-Fickian or anomalous transport, both diffusion as well as macromolecular relaxation time scales are similar and both control the overall rate of penetrant absorption. Case II transport is the limit when relaxation predominates. Zero-order, time-independent case II kinetics are characterized by a liner mass uptake with time. The types of transport based on the exponent n are listed in Table 11.2. The amount of drug released from a thin slab at time t (Mt ) with respect to the total amount of drug released (M∞ ) can be expressed Table 11.2. 1988).

Transport mechanisms of a penetrant through a polymer slab (Lee

Exponent n

Type of transport

Time dependence

0.45 ∼ 0.5 0.45 ∼ 0.5 < n < 0.89 ∼ 1.0 0.89 ∼ 1.0 n > 0.89 ∼ 1.0

Fickian diffusion Non-Fickian (anomalous) Case II transport Super case II transport

t −0.5 t n−1 Time-independent t n−1

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in terms of the semiempirical Equation (11.16) presented above (Frish 1980; Ritger and Peppas 1987a, b). Mt = K tn (11.18) M∞ The value of n determines the dependence of the release rate on time. The relationship between n and the drug transport mechanism through a polymer slab is shown in Table 11.2. Time-independent drug release is described by value of n = 1. The constant K incorporates characteristics of the macromolecular network/drug system and the dissolution medium. The above equation is applicable to one-dimensional, isotropic, isothermal water transport in a thin polymer slab under perfect sink conditions. An alternative model has been suggested by Berens and Hopfenberg (1978). Mt = K 1 t + K 2 t 0.5 (11.19) M∞ This expression may be used to analyze Fickian, non-Fickian, or case II transport. It is important to note that with either model (Equations (11.18) and (11.19)), these exponential expressions are only strictly valid for the first 60% of the release. Manipulation of the drug release rate is very important for medical practice since in some circumstances slow drug release is needed, and in certain others fast drug release is preferred. Example Applications of Drug Release from pH-Sensitive Hydrogels pH-sensitive hydrogels are in many respects eminently suited for use as a base material for “biologically active” biomaterials. Biologically active molecules, such as antibiotics, anticoagulants, antibodies, drug antagonists, contraceptives, and estrousinducers, can be used in conjunction with hydrogel. pH-sensitive hydrogels usually have a large number of polar reactable sites on which other molecules may be attached or immobilized by a variety of chemical techniques. Besides their own biological and physiological properties, the biologically active molecules can be entrapped within the cross-linked network structure of hydrogels. Since hydrophilic pH-sensitive hydrogels may interact less strongly than more hydrophobic materials with the biologically active molecules, which are immobilized to or within them, thus leaving a large fraction of the molecules active. Also, due to their high biocompatibility, pH-sensitive hydrogels

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can be left in contact with blood or tissue for extended periods without causing any unpleasant reaction, making them useful for devices to be used in long-term treatment of various conditions. The pH condition of the surrounding environment changes the chemical or physical properties of the gel, which is usually indicated as the significant volume change of the hydrogel. For example, the early pH-sensitive hydrogel investigated was based on chemical cross-linked acrylic acid and MAA network (Katchalsky and Michaeli 1955). It was observed that the swelling ratio of this hydrogel changes in response to the changes in the surrounding pH value. When the medium pH was increased, the gel swelled significantly. Sheppard et al. (1993) prepared pH-sensitive hydrogel from 2-HEMA and N , N -dimethylaminoethyl methacrylate by redox-initiated free-radical solution polymerization. Tetraethylene diacrylate was used as the cross-linker, and water was used as the solvent. The gels had maximum swelling rate at low pH (4.0–6.0) and with water content 65%, and the swelling rate was independent of that pH range but increased with the N ,N -dimethylaminoethyl methacrylate content. The fact that polyelectrolyte gels change properties in response to different pHs could be exploited in the development of novel drug delivery systems. Small molecules (drugs, enzyme substrates) can diffuse through hydrogels. The rate of permeation can be controlled by altering the swelling ability of hydrogel controlled by copolymerizing the hydrogel in varying ratios with different monomers or adjusting cross-linking degree. For instance, the basic poly(methyl methacrylate-co-N , N diethylaminoethyl methacrylate) hydrogel was developed for gastrointestinal tract delivery of foul-tasting drugs (Siegel and Firestone 1988). The pH-sensitive hydrogel based on the N -isopropylacrylamide, acrylic acid, and vinyl-terminated polydimethylsiloxane was fabricated for in vitro release of indomethacin, which usually causes severe gastric irritation. It was found that there was no drug released at pH 1.4, 37◦ C in 24 h, whereas at pH 7.4, 37◦ C, more than 90% drug was released during 5 h (Dong and Hoffman 1991). Formulated pH-sensitive gelatin coated with polyacrylic polymer, and surfactant, sodium laurate and cetyl alcohol in arachis oil, was also investigated for encapsulating insulin for enteric drug delivery. It was found that in vitro release rate of insulin was dependent on the surrounding media pH (Touitou and Rubinstein 1986). The controlled release properties of antifungal drug terbinafine hydrochloride was studied using pH-sensitive hydrogel synthesized

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using poly(acrylamide/maleic acid). In vitro drug release behavior of the hydrogel in different buffer solutions was affected by the solution pH and maleic acid content of hydrogel (Sen et al. 2000). Semiinterpenetrating polymer network of chitosan and polyvinyl pyrrolidone (PVP) hydrogel was proposed as the potential candidate for controlled release of antibiotic amoxicillin in acidic environment (Risbud et al. 2000). Several examples of biomedical applications for immobilized enzymes are presented in Table 11.3. Other Applications of pH-Sensitive Hydrogels Besides the controlled release of small-molecular-weight pharmaceuticals, hydrogels have also been studied for the release of large-molecularweight protein and peptide-based pharmaceuticals (Peppas et al. 2000). Polymethacrylic acid grafted with polyethylene glycol (PEG) pHsensitive hydrogel was tested as oral drug delivery carrier for peptide drug salmon calcitonin (Torres-Lugo and Peppas 1999). In addition to the delivery of pharmaceuticals using pH-sensitive hydrogel, immobilization of special enzymes (Spagna et al. 1998) or catalysts, chelating agent for purifying water (Guibal et al. 1998), living cells and even DNA fragments could be carefully designed using pH-sensitive hydrogels. For example, pH-sensitive hydrogels have been studied for potential use as artificial organ and muscle. A synthetic cationic articular cartilage material for use in a synthetic joint was constructed by simultaneous radiation cross-linking polyvinyl alcohol (PVA) and grafting of a cationic monomer to the PVA chains, allyltrimethyl-ammonium bromide and 2-hydroxy3-methacryloxypropyltrimethylammonium chloride. Such materials strongly adsorb negatively charged hyaluronic acid and produce a viscous layer for better lubrication (Bary and Merrill 1973). Artificial muscle synthesized by polyvinyl alcohol (PVA) containing both polyacrylic acids (PAA) and polyallylamines (PalAm) has bees investigated (Suzuki and Hirasa 1993). By repeatedly freezing and thawing the mixed aqueous solutions of PVA, PAA, and PalAm, it was turned into a rubberlike elastic hydrogel with many pores in the range from 1 to 3 μm because of the slow freezing process. This structure was very stable below 50◦ C for years and exhibited rapid volume change corresponding to the change in surrounding solution pH or the change of the surrounding solvent type like from water to acetone, which could generate force as high as that of frog’s muscles.

Drug delivery systems, receptor Biosensor Biosensor Bioreactors, artificial organs, biosensor Peptide synthesis DNA probe assays and peptide synthesis

Immobilized hormones

Immobilized neurotransmitters

Immobilized cells and organelles

Immobilized amino acid

DNA and RNA

Glucose sensor-artificial pancreas

Glucose oxidase

Immobilized drugs

Artificial kidney, Biosensor

Urease

Immunoassays, therapeutics and diagnostics, biosensors

Membrane oxygenator

Carbonic anhydrase, catalase

Immobilized antibodies and antigen

Leukemia treatmnent, biosensor

Asparaginase, glutaminase

Removal of airborne infection

Nonthrombogenic surface

Streptokinase

DNase and RNase

Nonthrombogenic surface

Urokinase

Blood alcohol electrode

Nonthrombogenic surface

Brinolase

251

Netti (2000)

Geysen et al. (1984)

Chibata et al. 1987), Mattiasson (1983), Zaborsky (1973)

Venter (1982)

Kaetsu (1996) and Venter (1982)

Venter (1982) and Wingard (1983)

Line and Becker (1975) and Miyata and Uragami (1999)

Horak et al. (2001), Kirwan (1974), and Miyamoto et al. (1998)

Guilbault and Lubrano (1974)

Guilbault and Lubrano (1971) and Podual et al. (2000)

Chang (1972) Eremeev and Kukhtin (1997)

Broun et al. (1971) and Podual et al. (2000)

Durso et al. (1994) and Moser et al. (1995)

Vakkalanka et al. (1996)

Kusserow and Larrow (1972) and Nakayama et al. (1999)

Nguyen and Wilkes (1974)

References

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Alcohol oxidase

Applications

Biomedical applications of immobilized biologically active species.

Immobilized enzymes

Table 11.3.

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Gudeman and Peppas (1995) developed ionic polyelectrolyte (PVA– PAA) hydrogel, which exhibited both volume and structural changes over the pH range of 3.0–6.0. Kang et al. (1993) investigated another pH-sensitive hydrogel using biopolymers like chitosan and synthetic polyether. The swelling of this hydrogel was affected by the concentration of chitosan acetic solution, the amount of cross-linking agent, and polyether. This hydrogel could also undergo the abrupt changes in volume by changing medium pH between 1.0 and 13.0. The pH-sensitive hydrogels have also been used as reactive matrix membranes in different sensors (Hoffman 1991) since these special hydrogels possess many advantageous properties, such as rapid and selective diffusion of the analyte, which is necessary for effective biosensors. Hydrogels can be made tough and flexible with a desirable refractive index (Davis et al. 1991). Especially, the ability of sensitive hydrogels in solutions to reversibly swell and shrink with small changes in environmental conditions can be used to prepare special separation and purification devices (Marchetti and Cussler 1989; Vasheghani-Farahani et al. 1992). Sensitive hydrogels, especially temperature- and pH-sensitive hydrogels, have been used to concentrate dilute aqueous solutions of macromolecular solutes including proteins, polypeptides, and enzymes, with no adverse effect on the enzyme activity (Gehrke et al. 1986). Gehrke et al. (1986) investigated linear PEGs and dextran gel by increasing the molecular weight and concentration of the PEG-favored adsorbing ovalbumin, BSA, cytochrome C, and hemoglobin. When needed, these proteins could be quantitatively recovered by immersing the gel into PEG-free solution. Separation of bioactive proteins produced by fermentation in recombinant DNA technology is one of most critical steps, which remains as a major hurdle for the wide application of this technology. Separating products by direct adsorption to absorbents is always attractive due to its convenience, cost-effectiveness, and operability in mild conditions, but the absorbents tend to become fouled by colloidal contaminants and large macromolecules. This problem can be overcome by immobilizing diethylaminoethyl cellulose-triacryl absorbents into hydrogels such as agarose and calcium alginate gel (Nigam et al. 1988). It was found that immobilized absorbent could very effectively adsorb more than 95% of the β-lactase in the crude homogenate. Moreover, the immobilized adsorbents do not contact with contaminants, and the separation becomes easier and more effective than solids preparation.

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The advantages and limitations of using sensitive hydrogels including pH-sensitive hydrogel for bioseparation especially for various protein molecules have been discussed by Kim (1998). Whey Protein Hydrogels The main whey protein component, β-lactoglobulin, is the principal gelling agent; it is a globular protein of 162-amino acid residues, with a monomer molecular weight of 18.3 kDa with two disulfide bridges, between residues 106 and 119 and between 66 and160, and a free thiol group (SH) at 121. These characteristics provide a potential for intermolecular and intramolecular disulfide link interchange during conformational changes associated with pH alterations, heat, or pressure treatment. Strong or weak heat-induced gels with high or low waterholding capacity may be prepared from whey protein solutions simply by adjusting several of the gelation variables. Thus, it is possible to design heat-induced whey protein gels with good pH sensitivity, tailored permeability, and mechanical properties that can be used as drug carriers. Based on these, Gunasekaran et al. (2006a, b) prepared pH-sensitive hydrogels using whey protein concentrate (WPC) powder (82.5% protein). The advantages of using whey protein-based gels as potential devices for controlled release of pharmaceutics is that they are completely biodegradable and there is no need for any chemical cross-linking agents for their preparation. These are two of the major requirements for wide use of hydrogels not only in the pharmaceutical area but also in many food and bioprocessing applications. Effect of Swelling Medium pH Gunasekaran et al. (2006a, b) prepared 10 mm diameter, 2-mm-thick WPC gel tablets and studied their pH sensitivity at a range of pHs from 1.8 to 11.4 in phosphate buffer solutions of 0.2 M ionic strength at 37.5 ± 0.5◦ C. Swelling ratio (SR) was calculated from wet gel (m w ) and dry gel (m d ) mass measurements as follows: SR =

mw − md md

(11.20)

The swelling kinetics of 15% WPC hydrogel denatured at pH 10.0 are shown in Figure 11.5a. The SR of WPC hydrogels is sensitive to the swelling medium pH; the higher the swelling medium pH, the faster

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(a)

Figure 11.5. (a) The swelling kinetics of 15% whey protein hydrogels prepared at pH 10.0 at different swelling media pH. (b) Fitting of swelling kinetics of 15% whey protein prepared at pH 10.0 at different swelling media pHs by the power law, SR/SR∞ = K t n ; the values of n are shown in the figure.

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the swelling. At pH 10.0, the gels reached the equilibrium SR value in about 50 min, while at pH 1.8, it took almost twice as long. The kinetics of swelling may be understood by considering several simultaneous effects. The contours of time versus penetrant uptake curves deviate more often from the classical Fickian model. In these cases, the sorption process is not a passive diffusion of the solvent molecules into the void spaces of the network but includes a concomitant relaxation of the network segments resulting from the advancing solvent front, which leads to plasticization of the material and a large increase in volume. In these cases the generalized semiempirical equation, similar to Equation (11.16), has been successfully used to describe the swelling kinetics (Harogoppad and Aminabhavi 1991; Rathna et al. 1994; Valencia and Pierola 2002) as follows: SRt = K tn SR∞

(11.21)

where K is a characteristic constant of the system, which is a function of the geometry of the hydrogel tablet and the diffusion constant. Equation (11.21) is valid when SRt /SR∞ < 0.6. Based on the value of the exponent n, this equation has been used to distinguish different types of sorption behavior (Table 11.2). Thus, the relative importance of solvent diffusion and polymer matrix relaxation effects can be analyzed by examining the exponent n of the power law. As shown in Figure 11.5b, at pH 10.0, n is 0.51 and the process may be considered diffusion-controlled case I sorption, while at pH 1.8 and pH 7.6, n is 0.56 and 0.65, respectively, and it may be the case of anomalous sorption. This kind of kinetic behavior can be understood considering the network structure of the WPC hydrogel. At pH 10.0, the polymer chain relaxation reduces greatly because of strong electrostatic repulsion among negative charges at the surface of the gel microstructure, so that water diffusion is faster than relaxation of polymer chain and swelling turns out to be diffusion-controlled. On the other hand, when the swelling medium pH is 7.6 most of the net negative charges were neutralized by the positive charges from the swelling medium, so smaller amount of net charges existed in the hydrogel. As a result, the electrostatic repulsion strongly decreases and the polymer chain relaxation increases to be comparable with water diffusion, resulting in an anomalous sorption mechanism.

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Hydrogel swelling is also governed by ionization of negatively charged groups. When the swelling medium pH = 10.0, the number of negatively charged groups is the most, so the equilibrium SR (=5.5) is the highest because of the strong electrostatic repulsion. When the swelling medium pH = 7.6, the proton from the swelling medium neutralizes most of the negatively charged groups, so the equilibrium SR (=3.8) is lower due to the reduced electrostatic repulsion. When the swelling medium pH = 1.8, all negatively charged groups are neutralized; instead, there would be some positive amine groups. Because the amine groups in the hydrogel are fewer than the carboxyl groups, the net charges in this case are few, so that the equilibrium SR (=2.2) is very low. The equilibrium swelling ratio reached the minimum when the swelling medium pH is close to the pI of the whey protein (∼5.4). This is because the net charge of whey protein molecules is at a minimum at pI, which means low electrostatic repulsion between chains in thermally denatured whey protein. Low electrostatic repulsive force resulted in low equilibrium swelling ratio. However, as the pH differs from pI, the net charge of whey protein molecules increases (positive charge below pI and negative charge above pI). The extent of swelling ratio increasing at acidic swelling medium was very small, because very few free amino groups exist at protein chains and so the positive charges are very limited. On the other hand, there are a lot of negatively charged groups in the protein chains, so the gels would contain a lot of net charges when the swelling medium of high pH value is used, which results in increased equilibrium swelling ratio. Effect of WPC Concentration and WPC Denaturation pH The equilibrium swelling ratios of hydrogels at different whey protein concentrations (12, 15, and 18%) versus pH values of swelling media are shown in Figure 11.6a. At all swelling media pH values, 12% WPC hydrogel took up the most water while 18% WPC gels took up the least. This was explained by the Flory’s swelling theory (Flory 1953). At higher concentration the density of protein network is high, and because of this, the equilibrium SR should decrease with increasing protein concentration. The equilibrium swelling ratios of 15% WPC hydrogels prepared at different denaturation pH values versus pH of swelling medium are shown in Figure 11.6b. At all swelling medium pHs, there is a general

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(a)

Figure 11.6. Equilibrium swelling ratio (SR) of WPC gels (a) of different concentrations (12%, 15%, 18%) prepared at pH 10.0 and (b) of protein preparation pH (5.1, 5.7, 6.2, 6.8, 7.2, and 10.0) at 15% WPC concentration.

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trend that the higher the gelation pH, the higher the equilibrium SR. Because the structure of thermally denatured protein gels depends on pH of the protein solution, the higher the pH, the more surface charges, the higher electrostatic repulsive force, and higher equilibrium SR. Consequently, SR reached the minimum when swelling medium pH was close to the pI of the whey protein (=5.4). This is because the net charge of whey protein molecules is at a minimum at pI, which means low electrostatic repulsion between chains in thermally denatured whey protein. Low electrostatic repulsive force resulted in low equilibrium SR. The extent of increase in SR at acidic swelling medium was very small, because there are very few free amino groups that exist at protein chains and so the positive charges are very limited. On the other hand, there are a lot of negatively charged groups in the protein chains, so the gels would contain a lot of net charges when the swelling medium of high pH value is used, which results in increased equilibrium SR. The linear regressions of equilibrium SR of WPC denatured at various pHs (5.1, 5.7, 6.2, 6.8, 7.2, and 10.0) are presented in Figure 11.7a. The slope of these lines represents the pH sensitivity, which is plotted against pH of 15% WPC solution used for gel preparation in Figure 11.7b. The WPC hydrogels denatured at higher pH showed higher pH sensitivity. The gels denatured at higher pH value have higher surface net charges or negative charges, so that electrostatic repulsion between the charges led to the higher equilibrium SR and higher pH sensitivity. From Figure 11.7b, we can observe another fact: the sensitivity changes a lot in the pH range of 5.0–7.0. This can be explained by the buffer theory, because protein solution can be regarded as a buffer solution. From Henderson–Hasselbach formula, pH = pK a − log

acid salt

(11.22)

Differentiating the above equation and simplifying, we get d(pH) =

C × d(salt) 2.303 × (salt) × (acid)

(11.23)

or 2.303 × (salt) × (acid) d(salt) = d(pH) C

(11.24)

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Figure 11.7. Equilibrium swelling ratio (SR) of 15% WPC gel (a) prepared at pH 10.0 with different swelling medium pHs and (b) pH sensitivity of 15% whey protein hydrogel versus the preparation pH. The pH sensitivity was defined as the slope (d(SR)/d(pH)) of the first-order regression shown in (a).

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where C is the sum of (salt) and (acid) or total concentration of solution. It is easy to see that d(salt)/d(pH) reaches maximum when (salt) = (acid) = C/2, and d(salt) 2.303 × C = d(pH) 4

(11.25)

In our system, the salt is COO− when pH is higher than pI and NH+ 3 when pH is lower than pI, respectively. As we discussed above, the swelling is due to the electrostatic repulsion between net charges, so swelling sensitivity should be proportional to the ratio of charge density to pH, or d(salt)/d(pH). Therefore, from Equations (11.24) and (11.25) it was explained why the swelling sensitivity changed greatly around pI. Moreover, from Figure 11.7b, it can be observed that swelling pH sensitivity changed more when pH > pI than when pH < pI. According to Equation (11.25), this is simply because there are less NH+ 3 than − COO in this system, so the ratio of salt concentration change to the pH − change is smaller when NH+ 3 dominates than when COO dominates in the system. Cyclical Swelling From Figures 11.6a and 11.6b, we know that equilibrium swelling ratio is the highest in pH 11.4 solution and the lowest in pH 5.8 solution. The cyclical swelling between these two swelling media is shown in Figure 11.8. When the fully swollen hydrogels in pH 11.4 swelling medium were put into the pH 5.8 swelling medium, the gel contracted rapidly. As mentioned above, the reason the gels had the highest swelling ratio at pH 11.4 swelling medium is because when the external solution diffuses into the gel, due to the high pH, most of negatively charged groups are ionized, and thus the osmotic pressure is higher, or electrostatic repulsion is stronger, and the gel will swell to the greatest extent. While this gel with a lot of COO− group inside is put into pH 5.8 swelling medium, most of COO− groups will be protonated to form COOH, so the net charges inside of the gel decreases, and then there is not enough electrostatic repulsion force to maintain the high osmotic pressure, which results in the contraction of the gel. Another observation from this experiment is that contraction is faster than swelling, which is easier to understand. For swelling, it requires the solvent or water to diffuse into the gel while the gel is at a glassy state, and the polymer chain

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Figure 11.8. Cyclical swelling experiment of WPC gels (15%, preparation pH 10.0) between pH 5.8 and 11.4.

relaxation also takes time. On the other hand, for contraction, water can very easily diffuse out since polymer is at a rubbery state. Controlled Drug Release from WPC Hydrogel Controlled drug release from WPC hydrogel was investigated using caffeine as model drug. WPC hydrogel tablets were prepared with encapsulated caffeine at 1:20 drug/WPC mass ratio. Caffeine was chosen as the model drug because of the following desirable properties: its UV absorbance is easy to detect; it is thermally stable at 80◦ C, the gelling temperature of WPC; it is readily water soluble; and it does not interact with WPC. Furthermore, caffeine-encapsulated WPC gels were alginate coated by placing the tablets in 1% sodium alginate solution for 2 min (Kikuchi et al. 1999). The gels were then cured in a 0.1 M CaCl2 solution for 15 and 30 min to gel alginate on the surface. Additional layers of alginate coating were applied by repeating the procedure twice (for two alginate layers) or four times (for four alginate layers), as needed. The thickness of each alginate layer was about 37.5 ± 1.0 μm. The alginate-coated gels were washed twice using deionized water and dried in a desiccator. The alginate coating is desirable in case of protein gels

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Figure 11.9. Effect of release medium pH value on the in vitro release profile of caffeine from WPC hydrogel tablet.

to prevent gel hydrolysis by proteolytic enzymes in the stomach (e.g., pepsin). The in vitro drug release tests were carried out using pH 7.5 phosphate buffer as the dissolution medium at 37.5 ± 0.5◦ C. Caffeine release profiles from the 15% WPC hydrogel in release media pH of 7.6 and 1.8 are shown in Figure 11.9. At pH 7.6 caffeine release is substantially faster than at pH 1.8. The slower release at pH 1.8 is due to fewer net charges and electrostatic repulsion. This is consistent with pH-sensitive swelling behavior. The n values determined (per Equation (11.18)) were 0.5 at pH 7.6 and 0.47 at pH 1.8. These n values suggest that the release at both pHs was diffusion-controlled. Effect of Alginate Coating on Swelling and Drug Release Alginate can form very stable gel in the presence of Ca2+ , and it is widely used for coating of polymer matrices used in controlled drug

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Figure 11.10. Swelling ratio (SR) of alginate-coated WPC gel and WPC gel (15%, preparation pH 10.0) at swelling medium of pH 7.5.

delivery system (Kikuchi et al. 1999). It is well known that alginate coating lowers the diffusion of solvent and encapsulated drug release. Figure 11.10 shows such an effect of alginate coating on swelling of 15% WPC gel prepared at pH 10.0. The equilibrium SR and the rate of swelling decreased dramatically after coating whey protein gel with alginate compared with that of the gel without coating. It is well known that alginate gel formed through calcium ion bridges is very rigid and does not swell easily (Papageorgiou et al. 1994). Furthermore, n value determined (per Equation (11.18)) for the alginate-coated gel was 0.44 (Figure 11.11). Thus, we could say that alginate coating not only lowers the diffusion rate but also alters the swelling kinetics from anomalous sorption (n = 0.65 before alginate coating) to diffusion-controlled sorption. The caffeine release profile from 15% WPC hydrogel prepared at pH 10.0 with alginate coating is shown in Figure 11.12. The release profile from whey protein gel without alginate coating is also shown for comparison. It is obvious that the caffeine release rate is reduced significantly by alginate coating, which is consistent with the results of the swelling study. The Ca2+ -induced alginate gel is very strong, rigid, and hard to swell, so the diffusion through this coating is the rate-limiting

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−0.1 Log(SR/SR∞)

18:58

y = 0.65x − 1.05 R 2 = 0.9916

− −0.2 − −0.3 − −0.4

y = 0.44x − 1.10 R 2 = 0.9993

−0.5 −0.6 −0.7 Log(t, min)

Figure 11.11. Fitting of swelling kinetics of 15% whey protein with or without alginate coating prepared at pH 10.0 swelling medium with pH 7.5 by the power law, SR/SR∞ = K t n ; the values of n are shown in the figure.

step for swelling and drug release. The release was prolonged by additional alginate layers on the hydrogel surface. The release profile of the sample with four alginate layer coating is interesting in that not only the release rate was significantly lower, but also the release kinetics changed to zero order. Similar results have been reported by others (Giunchedi et al. 2000; Ritger and Peppas 1987a). The curing time of alginate gel seems to have no significant effect on the drug release behavior indicating that alginate coating completely cured within 15 min. Nanoparticles Nanoparticles are defined as sub-100-nm size particles of a dense polymeric network in which an active molecule may be dispersed (Nakache et al. 2000). Therefore, nanoparticles enable entrapping drugs or bioactive compounds within but not chemically binding them. Because of their submicron and subcellular size, nanoparticles are well-suited for targeted, site-specific delivery purposes (Vinagradov et al. 2002) as they can penetrate circulating systems and target sites.

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Figure 11.12. In vitro release profiles (pH 7.5) of caffeine from WPC gel tablet (), one layer alginate-coated tablets at different surface gelation time (15 min (- - - -) and 30 min (- - - r- - -)), and two layers alginate-coated tablets at different surface gelation time (15 min (- - -  - - -) and 30 min (- - - - -)).

Various biocompatible and biodegradable biopolymers have been used in the formation of nanoparticles to maximize delivery efficiency and increase the desirable benefits (Coester et al. 2000; Kreuter 1994; Rhaese et al. 2003). In particular, albumin nanoparticles have been extensively investigated as potential nanoparticles systems (Langer and Peppas 1983; Lin et al. 1993; Vural et al. 1990). HSA and BSA have been used as natural matrix materials for delivery devices (Brannon-Peppas and Peppas 1991). The delivery of protein particles in the body is mainly influenced by particle size and surface characteristics (Moghimi et al. 2001). Oral delivery systems confront problems such as their breakdown or major irritation caused by harsh environments of the digestive system in the body (Allemann et al. 1998). Desirable delivery system should pass through the stomach and ultimately release loaded materials in target sites. Bovine β-lactoglobulin (BLG) is the major component and the primary gelling agent of whey proteins. It is a small (18.3 kDa) globular

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protein with two disulfide bonds and one free thiol group, which is inaccessible to solvent at or below neutral pH (Papiz et al. 1986). BLG is known to be stable at low pH and highly resistant to proteolytic degradation in the stomach. Because it can maintain a stable globular conformation, BLG is resistant to peptic and chymotryptic digestion (Reddy et al. 1988). Emulsion and desolvation methods have been used for nanoparticle formation of proteins such as HSA, BSA, and vicilin (Arnedo et al. 2002; Arshady 1990; Ezpeleta et al. 1996; Langer and Peppas 1983; Lin et al. 1993; Roser and Kissel 1993; Santhi et al. 2000, 2002). When using the emulsion method, it is difficult to remove the oil phase and to obtain a narrow-size distribution of the particles formed. However, the desolvation process has been successfully used to prepare HSA nanoparticles of around 100 nm diameter (Lin et al. 1993). During particle formation, protein solutions undergo conformational changes with various properties depending on the type of protein, concentration, cross-linking methods, and environmental conditions, especially pH (Langer and Peppas 1983; Lin et al. 1993). The conformational changes in a protein, that is, the unfolding of protein structure, expose its interactive sites such as disulfide bonds and thiol groups (Kinsella and Whitehead 1989b). Subsequently, cross-linking leads to the formation of a network that allows particles to entrap bioactive compounds. For manufacturing particles with an appropriate size distribution and surface properties, a balance between attractive and repulsive forces is necessary. During the particle formation, unfolding of a globular protein makes its disulfide bonds, thiol groups, and hydrophobic regions exposed to exterior, which increases intramolecular cross-linking but decreases hydrophobic interaction (Clark et al. 1981; Ezpeleta et al. 1996; Harwalkar and Kalab 1985). Thus, size and surface properties of protein particles depend on the number of disulfide bonds and thiol groups, degree of unfolding, electrostatic repulsion among protein molecules, pH, and ionic strength. Whey Protein Nanoparticles Ko and Gunasekaran (2006) hypothesize that increasing unfolding and decreasing hydrophobic interaction of protein molecules are important for preparing nanoparticles of desirable size. Thus, small molecular weight, highly unfolding, and less hydrophobic protein is preferred. Since BLG is smaller and less hydrophobic than BSA, it is a good

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candidate for preparing nanoparticles. In addition, the optimization of manufacturing procedures to increase unfolding and decrease hydrophobic interaction of protein molecules, for example, pH far from pI, and preheating of protein solution to increase protein unfolding are important. Preparation of BLG Nanoparticles BLG nanoparticles were prepared by a desolvation method (Langer and Peppas 1983; Loo et al. 2004; Marty et al. 1978; Weber et al. 2000). Two percent (w/v) solution of BLG in 10 mM NaCl at pH 9.0 was stirred on a 500 rpm magnetic stirrer at room temperature, and acetone, a desolvating agent, was added at 1 mL/min rate until the solution became just turbid. The rate of acetone addition was controlled carefully since it also influences the resulting particle size (Langer and Peppas 1983). The amount of acetone addition for BLG formation was 22.5 mL. At the end of acetone addition the solution pH was 8.1. After the desolvation process, 0.01 mL of a 4% glutaraldehyde-ethanol solution was added to induce particle cross-linking and stirred continuously at room temperature for 3 h. The nanoparticles formed were purified by five cycles of centrifugation and dispersion. For each centrifugation step, BLG solution was centrifuged at 25,000 g for 30 min. After centrifugation BLG pellets were redispersed to the original volume of acetone solution at pH 9.0 to prevent particle aggregation among the particles. Each redispersion step was performed in an ultrasonication bath. The excess cross-linking agent was removed from the particles by purification steps. The resulting nanoparticles were stored in absolute ethanol at 4◦ C. In order to decrease the size of the nanoparticles, the BLG solution prepared as above was heated in a water bath at 60◦ C for 30 min before the desolvation process. During acetone addition, solution pH lowered but was subsequently readjusted to 9.0. Particle Size and Zeta Potential Average size, distribution, and zeta potential of BLG nanoparticles were determined by photon correlation spectroscopy using a commercial particle size analyzer. Figure 11.13 shows the distribution of BLG nanoparticles formed without preheating and pH adjustment. For the BLG nanoparticles, the peak of the distribution was at 127 ± 4 nm. The number average diameter of BLG was 131 ± 8 nm, respectively.

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Figure 11.13. Particle size distribution of BLG (average size, 131 ± 8 nm; size at peak, 127 ± 4 nm; half-bandwidth of 80% distribution, 36 ± 10 nm) nanoparticles prepared without preheating and pH adjustment.

From the particles size distribution data, the size range around the peak that contains 80% of the particles was calculated. One-half of this 80% particle bandwidth was used as a measure of particle size dispersion. The half width of 80% particle bandwidth for BLG nanoparticles was 36 ± 10 nm. Figure 11.14 shows the zeta potential of BLG nanoparticles decreased with increasing pH. The surface of BLG is charged positively at acidic condition and negatively at neutral and basic conditions with the transition occurs at its pI. The particles size of BLG can be explained by its surface charge and surface hydrophobicity. BLG is characterized by a high content of charged amino acids (Brown 1975; Papiz et al. 1986). At basic pH, the size of the protein aggregates as well as the void spaces within a particle generally decreases (Schmidt 1981). In addition, proteins are generally more unfolded at basic pH, which exposes more reactive sites for cross-linking (Kinsella and Whitehead 1989a). The unfolding of

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Figure 11.14. Zeta potential of BLG nanoparticles.

the BLG molecule at basic pH increases thiol-disulfide interchange reaction, which may enhance particle formation but inhibit the formation of large aggregates. Our BLG nanoparticles were manufactured at pH 9.0 whose molecules would have negative charge. This condition resulted in small BLG particles charged negatively on their surface since coacervate precipitation was suppressed at pH 9.0. Another critical factor, the surface hydrophobicity dictates the propensity to bind nonpolar amino acid groups to a hydrophobic part of its surface. Hydrophobic interactions between hydrophobic regions of unfolded polypeptide chains lead to their aggregation resulting in size increment (Ismond et al. 1988). At basic pH, protein unfolding results in the change of the protein secondary structure. As pH increases, β-sheet formation in a protein increases due to an increase in hydrogen bonding (Krimm and Bandekar 1986). At basic pH, a thiol group or previously hidden hydrophobic groups in BLG becomes exposed and the thioldisulfide interchange reaction is accelerated. The degree of unfolding is related to the amino acid composition (Birdi 1976). The effective hydrophobicity of BLG is 12.2. Thus, small hydrophobic interactions of BLG suppressed the aggregation of the molecules and then resulted in smaller particles. Figure 11.15 shows the size distribution of BLG nanoparticles formed after preheating to 60◦ C and the pH readjusted to 9.0. The average

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Figure 11.15. Particle size distribution of BLG (average size, 59 ± 5 nm; size at peak, 50 ± 4 nm; half-bandwidth of 80% distribution, 27 ± 5 nm) nanoparticles prepared with preheating at 60◦ C and pH adjustment at 9.0.

particle size was 59 ± 5 nm with majority of the particles of size 50 ± 4 nm. In addition, the half-bandwidth of 80% of particles was narrower (27 ± 5 nm) than what was obtained without preheating and pH readjustment. This is a substantial improvement in both lowering the average particle size and improving the particle size uniformity. Preheating makes protein molecules unfolded so that the hydrophobic interactions between them are suppressed, which reduces self-aggregation. Further, by maintaining pH 9.0 we have generated high repulsive forces between BLG molecules and increased their unfolding. Therefore, we think that optimizing preheating and pH adjustment may be the key in preparing uniform BLG nanoparticles of sub-100-nm size range. Size and morphology of BLG nanoparticles were measured using atomic force microscopy (AFM), which scans topological shape of a specimen without any artefact. AFM is an alternative method to determine the size of particles but observes details of individual particles while PCS measures average size of large group of particles. An AFM system was used under tapping mode to measure size and topological

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shape of BLG nanoparticles. The BLG nanoparticles prepared were spread onto a mica surface and dried in air. The topography and error signal of the samples were generated by recording the vertical movements on the sample during scanning. The AFM scan area was 1 × 1 μm2 and 2 × 2 μm2 , respectively, for BLG nanoparticles prepared with and without preheating. In both cases, the resolution was set at 348 × 348 pixels. The particles on the images were analyzed to determine their size using commercial image analyzer software. The average particle size was determined as four times the average hydraulic radius. Hydraulic radius is defined as the ratio of particle area to particle perimeter. The AFM (error signal) images of BLG nanoparticles formed before and after pH adjustment are shown in Figures 11.16a and 11.16b. The average sizes of BLG nanoparticles before and after pH adjustment measured from AFM micrographs were 127 ± 50 nm and 51 ± 18 nm, respectively. These results were in agreement with those obtained from PCS.

In Vitro Degradation of BLG Nanoparticles To determine the degradation stability of BLG nanoparticles, in vitro degradation was performed at 37◦ C in pH 7.4 phosphate-buffered saline (PBS) according to published procedures (Gopferich 1996) under acidic and neutral conditions. For the acidic condition, 30 mL of 0.1 M PBS solution adjusted to pH 2.0 was used with and without 0.6 mL of a 0.1% pepsin solution. For the neutral condition, 30 mL of PBS at pH 7.4 was used with or without 1,000 enzyme unit/mL of a trypsin. The degradation plots of the BLG nanoparticles at acidic and neutral conditions are shown in Figures 11.17a and 11.17b, respectively. All the degradation curves exhibited a typical rapid initial decrease in absorbance followed by a fairly stable tail region. By linearizing the initial and final regions, the degradation time (Dt ) was determined at the intersection of the two linear segments (Figure 11.18). The average Dt values are listed in Table 11.4. BLG particles were relatively stable in the acidic environment (pH 2.0) (Figure 11.17a). At the acidic pH far from pI, BLG unfolds only partially (Kinsella and Whitehead 1989a). As degree of unfolding increases, it is easier for the degradative factors such as invasion of water and proteolytic enzymes to attack. The degradation rate increased substantially when pepsin was added, The Dt value of BLG nanoparticles was 7.3 h.

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Figure 11.16. (a) AFM (i) topography, (ii) error signal image, and (iii) its particle analysis of BLG nanoparticles without preheating and pH adjustment. Average particle size was 127 ± 50 nm. (b) AFM (i) topography and (ii) error signal image of BLG nanoparticles with preheating to 60◦ C and pH adjustment at 9.0. Average particle size is 51 ± 18 nm.

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Figure 11.17. Degradation of BLG nanoparticles with () or without () pepsin at pH 2.0 (a) and with or without trypsin in pH 7.4 PBS (b).

At neutral pH of 7.4, BLG nanoparticles was highly stable (Figure 11.17b). Only, less than 20% of initial amount was degraded over 4d. Addition of trypsin accelerated the degradation. For BLG particles Dt was 15 h. At neutral pH, trypsin is expected to attack specific sites on the surface and in the interior of the protein particles. The number of

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Figure 11.18. Determination of degradation time (Dt ) using linear fits to the initial and tail regions (shown by dotted lines) of the absorbance versus time curves. Dt is the time at which the two straight lines intersect.

susceptible peptide bonds is likely important in determining the rate and extent of degradation. For trypsin, it has been reported that the main site at which hydrolysis occurs is the carboxyl group of basic amino acids, such as lysine and arginine (Magee et al. 1995). BLG has 15 lysine and 3 arginine residues whereas BSA has 59 lysine and 23 residues (Brown 1975; Carter and Ho 1994; Papiz et al. 1986). The portion of lysine and arginine residues of BLG is 9.3 and 1.9%, respectively (Kinsella Table 11.4.

Degradation time (Dt , h) of BLG nanoparticles.

Acidic condition (pH 2.0)

Neutral condition (pH 7.4)

No enzyme

Pepsin

No enzyme

Trypsin

22.1 ± 12.4

7.3 ± 0.3

—a

15.4 ± 1.4

a Could

not be determined.

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and Whitehead 1989a). At pH 7.4 PBS-containing trypsin, the BLG particles were degraded slowly. The proteolytic enzyme activities on the surface of the BLG particles were less since the amount of basic amino acids was limited. Several factors such as particle preparation technique, degradation environments, enzyme activity, surface area, porosity, tortuosity, and size can affect the degradation on the matrix of protein nanoparticles. The development of a dense cross-linking matrix for nanoparticles offers resistance against the proteolytic degradation since it is difficult for the enzymes to penetrate into the particles. For BLG nanoparticles manufactured under the similar processing condition, they showed better resistance against enzyme degradation under both neutral and acidic environments. Only 11.2% basic amino acid residues (lysine + arginine) retarded the hydrolysis of the BLG nanoparticles. Thus, we could attribute the resistance of the BLG nanoparticles against the enzyme attack to its dense structure and small portion of basic amino acid composition. Summary Whey proteins can be used as hydrogels and/or nanoparticles systems for encapsulation and controlled release of bioactive compounds. Whey protein hydrogels exhibit pH-sensitive swelling ability especially at pH above the isoelectric point. The release kinetics of the hydrogels parallel that of their swelling ability. The release properties can be conveniently altered by appropriately coating with sodium alginate. Nanoparticles of sub-100-nm size can be prepared from β-BLG, the primary gelling constituent of the whey proteins. The average particle size can be lowered by preheating the BLG solution to 60◦ C. Preheating can also improve particle size uniformity. The BLG nanoparticles are more stable at neutral conditions than at acidic conditions with and without proteolytic enzymes. References Allemann, E., Jean-Christophe, L., and Gurny, R. 1998. Polymeric nano- and microparticles for the oral delivery of peptides and peptidomimetics. Adv. Drug Deliv. Rev. 34:171–189.

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Chapter 12 Whey Proteins and Peptides in Human Health P.E. Morris and R.J. FitzGerald

Introduction Whey proteins are an excellent source of dietary nitrogen and essential amino acids. They also act as technofunctional ingredients in many formulated food systems due to their good solubility, surface activity, and gelling properties. In addition to their “classical” nutritional and technofunctional attributes, whey proteins and their associated peptides display significant functional food ingredient potential. Current evidence for the potential of whey proteins and peptides to have health benefits beyond basic nutrition, that is, to act as functional foods/food ingredients, arises from a number of sources. These include the ability of whey proteins, whey protein hydrolysates (WPHs), and their associated peptides to beneficially impact (a) in vitro biomarkers associated with a particular disease state or condition, (b) human cell culture studies, (c) in vivo studies with small animals, and (d) human trials/studies. Currently, a limited number of human studies demonstrating the beneficial health effects of whey proteins/peptides are in existence. However, the results of such studies, which are generally time-consuming and expensive, are a perquisite in order to generate scientifically validated health claims. Scientific data demonstrating positive health effects from human studies are ultimately required by all stakeholders, that is, producers, processors, legislators and, last but not least, consumers. In some cases specific biological activities in humans have been directly linked to particular whey protein sequences. In other instances, potentially beneficial effects have been observed following the presentation of intact whey proteins or their associated peptides to mammalian cells in culture. Furthermore, beneficial animal and 285 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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human effects have been observed following oral ingestion of intact whey proteins and WPHs. The beneficial health effects observed following ingestion of intact whey proteins is presumably in the main derived from peptides released via the action of gastrointestinal proteinase and peptidase activities. It is also conceivable that propeptide sequences within WPHs may be further processed to release biologically active entities during gastrointestinal transit. This raises the issue of peptide bioavailability, which is central to the ultimate manifestation of any biological/physiological effect. Therefore, in order for a given peptide to have a biological effect endogenously, it is essential that this peptide be resistant to degradation during gastrointestinal transit and that it can be transported across the gut mucosa while being resistant to epithelial peptidases. Furthermore, the peptide must then reach its target site/organ while being resistant to serum peptidase activities (FitzGerald and Meisel 2003). Peptides acting at gut level on the other hand presumably only need to survive the gastrointestinal transit process. It is therefore worth noting that intestinal, gut mucosal, and serum proteinase/peptidase activities can play a central role in protein/peptide processing in order to release peptide sequences responsible for mediating/triggering a particular physiological response. Interpretation of the current data in relation to the beneficial/potentially beneficial health effects of whey proteins/peptides must take cognisance of these possibilities. This chapter will outline developments with respect to the beneficial/ potentially beneficial human health effects of whey proteins and their associated peptide sequences. In particular, evidence/emerging evidence for the role of whey proteins/peptides as hypotensive, anticancer, immunomodulatory, opioid agonist and antagonist, mineral binding, antimicrobial, gut health enhancing, hypocholesterolemic, insulinotrophic and psychomodulatory agents will be described.

Hypotensive Peptides Hypertension, or high blood pressure (BP), is a controllable risk factor in the development of a range of cardiovascular disease states. It is well recognized that the risk of developing heart disease and stroke significantly increases at systolic/diastolic(SBP/DBP) values above 115/75 mm Hg (National Heart, Blood and Lung Institute 2003). Hypertension

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is estimated to affect ∼25% of the global population (Health, National Centre for Health Statistics 2002). It is reported that a 5 mm reduction in DBP can reduce the risk of cardiovascular disease by ∼16% (Collins et al. 1990; MacMahon et al. 1990). Current pharmaceutical strategies for the control of BP include the use of calcium channel blockers, vasodilators, diuretics, and angiotensin-converting enzyme (ACE) inhibitors. However, numerous side effects are associated with their use, for example, synthetic ACE inhibitors are associated with symptoms such as hypotension, increased potassium levels, reduced renal function, fetal abnormalities, skin rashes, and cough (Agostoni and Cicardi 2001; Ames 1983; Nakamura 1987; Sesoko and Kaneko 1985). Therefore, naturally derived food components with the ability to lower BP have significant potential as ingredients in health-promoting functional foods for human consumption. Numerous peptide sequences derived from food protein sources have been reported to inhibit ACE, an activity which plays a central role in BP control (Ariyoshi 1993; Meisel et al. 2006). The area of milk protein-derived ACE inhibitory peptides has been extensively reviewed (FitzGerald and Meisel 1999; FitzGerald and Meisel 2003; FitzGerald et al. 2004; Gobbetti et al. 2002; Korhonen and Pihlanto-Lepp¨al¨a 2006; Meisel et al. 2006; Murray and FitzGerald 2007; Pihlanto-Lepp¨al¨a 2001; Vermeirssen et al. 2004 for reviews). Whey proteins contain peptide sequences within their primary structures, which have the ability to inhibit ACE (FitzGerald and Meisel 1999). The characteristics of some potent bovine whey protein-derived ACE inhibitory peptides, or lactokinins, are summarized in Table 12.1. It is seen from this Table that lactokinins having different inhibitory potencies (IC50 values) are encrypted within the primary structure of the individual whey proteins. Various endoproteinases (pepsin, trypsin, chymotrypsin, and proteinase K) have been used to release these peptides from the intact proteins. Furthermore, fermentation with yogurt starter cultures followed by pepsin and trypsin hydrolysis has been employed in the release ACE inhibitory peptides from whey protein (Pihlanto-Lepp¨al¨a et al. 1998). A number of studies have been performed on the hypotensive effects of whey-derived peptides in spontaneously hypertensive rat (SHR). Significant decreases in BP have been observed following administration of the peptides, and Table 12.2 summarizes the results of some of these studies. While α-lactorphin, α-LA f(50–53), displays ACE inhibitory activity (Mullally et al. 1996), its BP-lowering effect following

Fragment f(104–108) f(99–108) f(52–53) f(50–52) f(105–110) f(50–53) f(18–19 / 50–51) f(50–53) — f(46–48) f(60–61) f(122–124)

α-Lactalbumin WLAHK VGINYWLAHK LF YGL LAHKAL YGLF YG YGLF

β-Lactoglobulin LRP LKP KW LVR

288 β-LG β-LG β-LG β-LG

0.27 0.32 1.63 14

77 327 349.1 409 621 733 >1,000 1.26c

IC50 (mM) (mg/mL)b

— — — —

Trypsin Trypsin Synthesis Pepsin, trypsin, chymotrypsin and ultrafiltration Fermentation, pepsin, and trypsin Pepsin and trypsin Synthesis —

Preparation

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α-LA α-LA α-LA α-LA α-LA α-LA/α-lactorphin α-LA α-LA/α-lactorphin

Source protein/name

Potent ACE inhibitory peptides derived from bovine whey proteins.

Peptide sequencea

Table 12.1.

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— — f(18–20)

Lactotransferrin LRP RP

β2 -Microglobulin GKP β2 -m/cheese whey

Lactotransferrin Lactotransferrin

BSA BSA/cheese whey

β-LG β-LG β-LG Cheese whey/β-LG β-LG/β-lactorphin β-LG

352

0.27 180

3 315

42.6 122.1 130 141 171.8 180

Proteinase K

— —

Synthesis Proteinase K

Trypsin Synthesis — Proteinase K Synthesis —

Adapted from Meisel et al. (2006). a One-letter code. b IC , concentration of material mediating a 50% inhibition of ACE activity. 50 c mg/mL. α-LA, α-lactalbumin; β-LG, β-lactoglobulin; BSA, bovine serum albumin; β2 -m, β2 -microglobulin; —, not determined.

f(208–216) f(221–222)

f(142–148) f(102–103) f(150–151) f(78–80) f(102–105) —

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Bovine serum albumin ALKAWSVAR FP

ALPMHIR YL SF IPA YLLF RP

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Table 12.2. Bovine whey protein-derived peptides displaying hypotensive effects in hypertensive rats. Peptide

Fragment

Sequencea

ICb50 (μM)

Maximum decrease in SBPc (mm Hg)

α-LAd β-LGe BSAf β2 -mg

50–53 78–80 221–222 18–20

YGLF IPA FP GKP

733 141 315 352

−23 −31 −27 −26

Adapted from FitzGerald et al. (2004). a One-letter amino acid code. b Concentration of peptide mediating 50% inhibition of ACE activity. c Systolic blood pressure (mean value). d α-Lactalbumin. e β-Lactoglobulin. f Bovine serum albumin. g β -Microglobulin. 2

intravenous administration to SHR was via interaction with opioid receptors, and α-lactorphins activity was abolished by naloxone, an opioid receptor antagonist (Nurminen et al. 2000; Sipola et al. 2002). A WPH generated with a bacterial proteinase preparation mediated a dose-dependent decrease in SBP in SHR (Costa et al. 2005). Mean SBP changed from 188.5 ± 9.3 (control 0.15 M NaOH) to 163.8 ± 5.9 (with WPH) mm Hg following intraperitoneal administration of the hydrolysate at a dose of 1 g/kg bodyweight 2 h after administration. A WPH with high ACE inhibitory activity generated with the same bacterial proteinase activity mediated a significant decrease in SBP following intraperitoneal administration. However, the hypotensive effect was not observed following oral ingestion of the WPH (Costa et al. 2007). These results indicate the role of gastrointestinal proteinase/peptidases activities in hydrolysate processing to peptides, which do not display hypotensive effects in SHR. A limited number of human studies have been performed on the hypotensive effects of WPHs and their associated peptides. Oral ingestion of synthetic Ala.Leu.Pro.Met.His.Ile.Arg (corresponding to α-LG f(142–148) Table 12.1) had no effect on BP in two human volunteers. In vitro incubation experiments showed that this lactokinin was degraded by serum peptidase activities indicating that even if this peptide was transferred across the gut, it was not sufficiently resistant to peptidase

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degradation to mediate an hypotensive effect (Walsh et al. 2004). A tryptic digest of whey protein isolate was reported to significantly reduce BP in comparison to a control containing unhydrolyzed whey protein isolate when consumed at 20 g/day by prehypertensive or stage 1 prehypertensive human volunteers. This pilot study involving 30 human volunteers with SBP/DBP levels ≥120/80 and ≤155/95 mm Hg reported that the hypotensive effect was observed 1 week after starting the trial and the effect was evident over the 6 weeks duration of the trial. Mean reductions in SBP and DBP were 8.0 ± 3.2 ( p < 0.5) and 5.5 ± 2.1 ( p < 0.5) mm Hg, respectively, in comparison to the control. Furthermore, it was reported that the treatment group had significantly lower ( p < 0.05) total and low-density lipoproteins cholesterol levels. White blood cell counts were increased ( p < 0.05) and high sensitively Creactive protein levels were reduced ( p < 0.05) during the course of the study (Pins and Keenak 2006). This was the first report of a hypotensive effect for WPH consumption in human volunteers. The mechanisms by which these effects were brought about were not elucidated. A randomized, placebo-controlled, double-blind human trial by Lee et al. (2007) reported no significant effects of whey peptides on BP in comparison to a control group. This trial involved 54 hypertensive volunteers who consumed whey peptides in the form of acid-reduced mineral whey powder. The volunteers ingested 125 mL/day of a drink containing 2.6% protein equivalent over a 12-week period. Furthermore, no significant effect on selected inflammation markers including C-reactive protein or metabolic variables including serum lipids was reported. The reason for the discrepancy between these two studies may include differences in study design, whey peptide preparation, and consumption levels of the test samples. Further long-term fully powered human studies are necessary to elucidate the potential hypotensive effects of WPHs.

Anticancer Properties Preventative screening and a healthy lifestyle including dietary measures are recognized approaches to help avoid certain forms of cancer. A diet low in fiber, the intake of red meat, and an imbalance of omega-3 and omega-6 lipids may contribute to an increased risk of certain types of cancer such as colorectal and prostate cancers (Divisi et al. 2006;

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Donaldson 2004; McIntosh et al. 1998). The role of milk intake is still controversial with some studies suggesting that it is a risk factor while others indicate that consumption has a protective role against some cancers. For example, Chan et al. (2005) and Colli and Colli (2006) indicated that dairy product consumption may increase the risk of prostate cancer. Alternatively, results from studies by Jain (1998), Moorman and Terry (2004), and Parodi (2005) report no significant association of dairy product consumption with cancers of the breast, bladder, lung, ovary, or pancreas. A pooled analysis of 10 cohort studies also reported that a milk and calcium-rich diet was associated with lower risks of colorectal cancer (Cho et al. 2004). A number of individual milk-derived components have demonstrated anticancer potential in both in vitro and in vivo studies. A number of studies have evaluated the role of whey protein in the prevention and/or treatment of several types of cancer. Bounous et al. (1991) first demonstrated the role of dietary whey protein concentrate (WPC) in the successful treatment of colon cancer. They reported inhibition of tumor incidence and a reduction of tumor burden in mice consuming WPC. Whey protein has potent antioxidant activity (Walzem et al. 2002) due to a high content of cysteine, which is a substrate for the biosynthesis of glutathione (GSH). Bounous and Molson (2003) reported that the anticancer properties of whey proteins were due to an elevation in tissue GSH levels that may in turn stimulate an immune response. In a study by McIntosh et al. (1995), rats fed a diet of whey protein (20 g protein/100 g bodyweight) exhibited improved protection against intestinal tumors in comparison to controls fed soy and red meat at the same protein concentration. It was also found that whey-fed rats had higher GSH concentration in several tissues (liver, spleen, colon, and tumor) and lowest amount of fecal fat in comparison to the controls. It has been reported that high fat levels in fecal matter may increase colon concentrations of cytotoxic lipids and thus represent a risk factor in colon carcinogenesis (Sesink et al. 2000). Xiao et al. (2006) demonstrated that rats fed a diet containing 20% (w/w) whey protein hydrolysate (WPH 917) had fewer instances of colon aberrant crypt foci, putative precursors of colon cancer, at 6 and 23 weeks compared to those fed with equivalent amounts of casein. However, colon tumor incidences were not dissimilar in rats fed on both diets. In a study by Hakkak et al. (2000), female rats were fed a diet containing 14% (w/w) whey protein for 50 days prior to injection with

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80 mg/kg 7,12-dimethylbenz-[α]-anthracene (DMBA), a chemical carcinogen used widely to produce mammary adenocarcinoma. The animals fed whey had approximately 50% less incidences of mammary cancers compared to casein-fed controls and one-third less tumors than soy-fed rats after a lifetime consumption (120 days). These results thus clearly indicated the potential of whey protein in reducing the risk of breast cancer development. Similar results were observed in the reduction of colon carcinogenesis in DMBA-treated or azoxymethane-treated rats following consumption of a whey-rich diet (8–32% whey over a 4-week period prior to injection with the chemical carcinogen) when compared to red meat or soybean diets (Belobrajdic et al. 2003; McIntosh and Le Leu 2001). A 50% reduction in the numbers of colon aberrant crypt foci in rats fed a combination of soy protein and β-lactoglobulin (β-LG) (5% (w/w) total protein) or soy protein and lactoferrin (LF 5% (w/w) total protein) 4 weeks prior to DMBA injection compared to controls fed soy protein alone (McIntosh et al. 1998). The tumor suppressor p53 becomes activated in response to oxidative stress and DNA damage, and initiates several pathways that ultimately arrest proliferation and prevent the generation of genetically altered cells (Halliwell 2007; Stiewe 2007). A high frequency of breast, colorectal, liver, lung, and ovarian cancers is attributed to mutations in p53 (Fleischmann et al. 2003; Lasky and Silbergeld 1996). Dave et al. (2006) used Tp53 gene expression in a rat model to determine the modulatory effects of a WPH in comparison to casein. On completion of the study (50 days), the mammary glands of rats fed WPH had lower levels of activated Tp53 indicating that they were more protected from DNA damage than the casein-fed rats. Serum from WPH-fed rats also had greater apoptotic activity and higher levels of monocyte chemoattractant protein (MCP-1) in comparison to the rats fed casein. Twenty human volunteers with a range of stage IV tumors were given a combination of 40 g/day IMUPlusTM (a nondenatured whey protein) with various amounts of transfer factor plus ascorbic acid, Agaricus blazeii murill teas, the medicinal herb Andrographis paniculata and a mix of vitamins, minerals, antioxidants, and immune-enhancing natural products (See et al. 2002). All the 16 survivors in the 6-month trial were reported to have significantly higher natural killer function, TNF-α levels, elevated hemoglobin, hematocrit, and GSH levels along with an improved quality of life.

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The whey protein bovine serum albumin (BSA) may display anticancer activity. The growth of MCF-7, an estrogen-responsive human breast cancer cell line, was inhibited by several commercial BSA preparations during in vitro cell culture (Laursen et al. 1990). BSA inhibited MCF-7 cell proliferation in a concentration-dependent manner similar to that of the positive control, newborn calf serum. BSA (1.5% w/v) also significantly inhibited activity of the mutagen, 4-nitroquinoline 1oxide, during an in vitro study on Chinese hamster cells (Bosselaers et al. 1994). It was concluded that BSA may protect mammalian cells against certain genotoxic compounds, although the mechanism(s) is unclear. The anticarcinogenic capability of α-lactalbumin (α-LA) has also been described. Bovine α-LA mediated antiproliferative effects in human colon adenocarcinoma cell lines (Caco-2 and HT-29 cells) during a 5-day dose-dependent growth study (Sternhagen and Allen 2001). Low concentrations of α-LA (10–25 μg/mL) stimulated cell growth during the first 3–4 days, but subsequently proliferation of the colon tumor cells ceased and viable cell numbers decreased dramatically. This suggested a delayed induction of apoptosis by α-LA. The ability of β1,4galactosyltransferases from human ovarian cancer, lymphoma spleen, and ovarian cancer sera to transfer galactose to N -acetylglucosamine were shown to be inhibited by bovine α-LA (Chandrasekaran et al. 2001). Human α-lactalbumin made lethal to tumor cell (HAMLET) induces apoptosis-like effects in tumor cells but differentiates most normal cells (Fast et al. 2005; Hallgren et al. 2006; Svensson et al. 2000). The HAMLET complex was prepared by first changing the conformation of α-LA from the native to a partially unfolded state by ethylenediaminetetraacetic acid (EDTA) treatment. The partially unfolded α-LA was then stabilized via binding of oleic acid to the exposed hydrophobic regions of the α-LA molecule (Svensson et al. 2000). On binding to the tumor cell surface, HAMLET is reported to travel through the cytoplasm to the nucleus. Therein it disrupts chromatin structure and causes DNA fragmentation while interacting with mitochondria, causing the release of cytochrome c and activation of the caspase cascade (Hakansson et al. 1999; Kohler et al. 2001). The ability of bovine αlactalbumin made lethal to tumor cells (BAMLETs) to successfully induce apoptosis in L1210 tumor cells with no apparent difference in rate of apoptosis between HAMLET and BAMLET was also reported by Svensson et al. (2003). The in vivo therapeutic effect of HAMLET

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has also been studied. HAMLET (0.7 mM) was administered into invasively growing human glioblastoma tumors in mice over a 2-month period (Fischer et al. 2004). Reduced intracranial tumor volume and delayed onset of pressure symptoms were reported in the tumor-bearing rats while no toxic side effects were observed. Daily topical application of HAMLET (one drop of 0.7 mM HAMLET) to skin papillomas over a 2-month period reduced lesion volume by more than 75% in 20 human participants (Gustafsson et al. 2004). Two years subsequently all lesions had completely disappeared in 83% of the patients treated with HAMLET, again with no adverse reactions reported. No in vivo trials have been performed on BAMLET to date, but as it possesses equivalent cytotoxity as HAMLET, it has potential as a novel antitumor agent. Several physiological roles have been reported for LF, an iron-binding glycoprotein found in whey. In rat models, LF treatment significantly reduced chemically induced carcinogenesis and/or metastasis in esophagus, tongue, lung, liver, colon, and bladder tumors (Tsuda et al. 2002; Ward et al. 2005). The anticancer properties of LF have been attributed to prevention of oxidant-induced carcinogenesis by free iron. LF can bind iron locally in a number of tissues (Gill and Cross 2000; Weinberg 2006). Stimulation of natural killer cells, interleukin-18 (IL-18), and other cytokines along with the regulation of cell proliferation and/or apoptosis has been linked to LFs observed anticancer activity (Matsuda et al. 2007; Norrby et al. 2001; Varadhachary et al. 2004; Wang et al. 2000a,b). The effects of a high LF-containing WPC on the cytotoxicity of the potential anticancer drug, baicalein, was characterized in vitro using the human hepatoma cell line, HepG2 (Tsai et al. 2000). The LF preparation alone did not have any effect; however, when used in conjunction with baicalein, it was shown that cell cytotoxicity was significantly improved with nearly 13 times more cells undergoing apoptosis than cells grown in baicalein alone. This clearly demonstrated that the LF-containing whey preparation may function as an adjuvant in cancer treatment. Different modes of administration (oral versus intravenously) appear to have limited effect on the anticancer efficacy of LF. However, oral administration is the predominant means of intake as the intraperitoneal route may lead to allergic reactions since LF is a high-molecular-mass glycoprotein molecule (Iigo et al. 2004; Kuhara et al. 2006; Tsuda et al. 2002). A human trial was performed on seven patients with metastatic carcinomas with a high concentration LF-containing WPC (Kennedy

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et al. 1995). At the end of the study, two patients displayed signs of tumor regression while two others exhibited tumor stabilization, indicating the benefit of food supplementation in the fight against cancer. An LF-containing whey preparation (10 g twice daily) has been successfully used in a clinical trial involving 24 cystic fibrosis patients. The rationale of this trial was to increase GSH levels leading to downregulation of the inflammatory response and thereby potentially neutralizing the adverse effects of oxidative stress in cystic fibrosis sufferers (Grey et al. 2003). Bovine lactoferricin LfcinB, f(17–41), is released by pepsin hydrolysis of LF (Bellamy et al. 1992a,b). A number of mouse and human model studies have demonstrated that LfcinB and related peptides are present in the gastrointestinal tract following the ingestion of LF (Kuwata et al. 1998, 2001). LfcinB demonstrated a cytotoxic effect against a panel of human neuroblastoma cell lines (Kelly, IMR-32, SK-N-DZ, SHEP-1, and SH-SY-5Y) with IC50 values ranging from 15.5 to 60 μM (Eliassen et al. 2006). Experiments using flow cytometry indicated that LfcinB caused necrosis in the neuroblastoma cells by destabilizing the cytoplasmic membrane. This causes the cells to lose membrane integrity, which may in turn allow LfcinB to gain access to and disrupt the mitochondria of the cancer cells. LfcinB (50 μg/mL) was also found to be a potent inducer of apoptosis in cultures of THP-1 human monocytic leukemia cells in a dose- and time-dependent manner (Yoo et al. 1997). Conversely, native LF was unable to induce apoptosis in THP-1 leukemia cells even at tenfold higher concentrations than LfcinB. This indicated that the apoptosisinducing activity was exclusive to LfcinB within this cell line. Eliassen et al. (2002) also reported cytotoxic activity by both LfcinB and LF against murine tumor cell lines and experimental tumors. LfcinB caused membrane disruption and eventual cell lysis, while the cytotoxicity of LF was not via this mechanism. It has since been shown that LfcinB selectively induces caspase-dependent apoptosis in human carcinoma cell lines (Furlong et al. 2006). On the other hand, in human breast carcinoma and head and in neck cancer cell lines, the inhibitory effect of LF occurs at the G1 to S transition in the cell cycle that may be controlled, in part, by changes in Akt phosphorylation (Cornish et al. 2006; Xiao et al. 2004). In vivo rat studies have also demonstrated that the apoptotic activity of LF may be linked with the expression of Fas receptors and other apoptosis-related molecules (Fujita et al. 2004).

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Human myeloid leukemic cells (HL-60) were exposed to bovine LF (bLF) and proteolytic hydrolysates of bLF in order to determine the effects on cell growth. Peptic hydrolysates of bLF showed a greater growth-suppressive effect than tryptic hydrolysates or native bLF (Roy et al. 2002). Four peptides with antiproliferative activity against human leukemia HL-60 cells were purified from the pepsin hydrolysates using ion-exchange, reverse-phase, and gel-filtration chromatography. Peptide 1, LF f(17–38) exhibited the greatest inhibition of cell proliferation compared to the three other peptides (Table 12.3). Inhibition of human leukemia HL-60 cells by the pepsin hydrolysates was shown to be due to induction of apoptosis by peptide 2 LF f(1–16)-(45–48), peptide 3 LF f(1–15)-(45–46), and peptide 4 LF f(1–13), whereas peptide 1 caused necrosis. Mader et al. (2005) further demonstrated the apoptic nature of both synthetic and pepsin-generated LfcinB (both at 200 μg/mL) against human leukemia and carcinoma cell lines (colon, breast, and ovary) was by causing DNA fragmentation. It was also established that LfcinB was noncytotoxic to primary cultures of normal human T lymphocytes, fibroblasts, and endothelial cells. LfcinB was found to inhibit tumor-mediated angiogenesis in mice models. It furthermore mediated an antiproliferative effect against proangiogenic factor-induced human umbilical vein endothelial cells (Mader et al. 2006). It was found that LfcinB competitively inhibited the binding of basic fibroblast growth factor and vascular endothelial growth factor to their respective receptors thereby preventing receptor-stimulated angiogenesis. Eliassen et al. (2003) produced a number of synthetic LfcinB derivatives by replacing the cysteine residue at position 3. Furthermore, the two tryptophan residues in position 6 and 8 were substituted with the aromatic amino acids, β-(2,5,7-tri-tert-butyl-indol-3-yl) alanine (Tbt), β-[2-(2,2,5,7,8-pentamethyl-chroman-6-sulfonyl)-indol3-yl]alanine (Tpc), β-(4,4 -biphenyl)alanine (Bip), and βdiphenylalanine (Dip) the structures of which are illustrated in Figure 12.1. These modifications improved LfcinB’s anticancer properties against three human tumor cell lines (MT-1, RMS, and HT-29) and normal human cell lines (HUV-EC-C and MRC-5), as shown in Table 12.4. The [Tbt6,8 ]-LfcinB and [Tpc6,8 ]-LfcinB, in which both tryptophan residues were replaced by Tbt and Tpc, respectively, were at least twice as active against all tumor cell lines tested (IC50 values ranging from 14.7 to 48.1 μM) than the peptide derivatives with only

298 17–38 (1–16)-(45–48) (1–15)-(45–46) 1–13

Adapted from Roy et al. (2002). ND, not determined.

Peptide 1 Peptide 2 Peptide 3 Peptide 4

Fragment LF FKCRRWQWRMKKLGAPSITCVR APRKNVRWCTISQPEW–CIRA APRKNVRWCTISQPE–CI APRKNVRWCTISQ

N-terminal sequence 2,753.88 2,430.13 2,017.92 1,558.73

Molecular mass (Da)

2.22 11.9 ND 22.1

IC50 μM

6.1 28.9 ND 34.5

IC50 μg/mL

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Table 12.3. Amino acid sequence of the cell proliferation inhibitors from pepsin-digested lactoferrin and the concentration of inhibitor needed to inhibit cell proliferation in human leukemia HL-60 cells by 50%.

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Figure 12.1. Structure of side chains of the aromatic amino acid residues. Tpc, β[2-(2,2,5,7,8-pentamethyl-chroman-6-sulfonyl)-indol-3-yl]alanine; Tbt, β-(2,5,7-tritert-butyl-indol-3-yl)alanine; Dip, β-diphenylalanine; Bip, β-(4,4 -biphenyl)alanine. (Taken from Eliassen et al. (2003).)

one of the two tryptophan residues replaced. These derivatives also displayed a higher anticancer activity than the unmodified LfcinB (IC50 >500 μM), as shown in Table 12.4. The replacement of cysteine at position 3 with Tpc and Tbt resulted in similar, and in some cases better, anticancer activity to [Tbt6,8 ]-LfcinB and [Tpc6,8 ]-LfcinB. The introduction of N-terminal moieties such as Fmoc, dodecyl, and adamantanoyl to LfcinB also resulted in improved activity against the tumor cells tested with IC50 values ranging from 36.7 to 165 μM. The replacement of Cys3, Gln7, and Gly14 with alanine residues in the derivative [A3,7,14 Tbt6,8 ]-LfcinB resulted in an increase in anticancer activity when compared to [A3,7,14 ]-LfcinB. However, this derivative had comparable activity to [Tbt6,8 ]-LfcinB and [Tpc6,8 ]-LfcinB. The inhibition of liver and lung metastasis during a mouse model study following subcutaneous administration of the iron-depleted form of LF, apo-LF (1 mg/mouse), and LfcinB (0.5 mg/mouse) 1 day after

FKCRRWQWRMKKLGA FK(Tpc)RRWQWRMKKLGA FKCRR(Tpc)QWRMKKLGA FKCRRWQ(Tpc)RMKKLGA FKCRR(Tpc)Q(Tpc)RMKKLGA FK(Tbt)RRWQWRMKKLGA FKCRR(Tbt)QWRMKKLGA FKCRRWQ(Tbt)RMKKLGA FKCRR(Tbt)Q(Tbt)RMKKLGA FK(A)RRW(A)WRMKKL(A)A FK(A)RR(Tbt)(A)WRMKKL(A)A FK(A)RRW(A)(Tbt)RMKKL(A)A FK(A)RR(Tbt)(A)(Tbt)RMKKL(A)A Fmoc-FKCRRWQWRMKKLGA Dodecyl-FKCRRWQWRMKKLGA Adamantanoyl-FKCRRWQWRMKKLGA

Lactoferricin (Lfcin) (Tpc3 )Lfcin (Tpc6 )Lfcin (Tpc8 )Lfcin (Tpc6,8 )Lfcin (Tbt3 )Lfcin (Tbt6 )Lfcin (Tbt8 )Lfcin (Tbt6,8 )Lfcin (A3,7,14 )Lfcin (A3,7,14 Tbt6 )Lfcin (A3,7,14 Tbt8 )Lfcin (A3,7,14 Tbt6,8 )Lfcin Fmoc-Lfcin Dodecyl-Lfcin Adamantanoyl-Lfcin

>500 21.8 57.2 50.1 29.9 21.1 49.2 53.5 19.4 >500 10.3 37.4 30.8 72.6 36.7 72.3

>500 23.5 67.2 52.9 20.9 35.5 46.2 41.3 16.1 >500 11 26 19.9 50.3 42.2 86.8

>500 24.5 95.8 53.2 40.4 25.7 67 63 44 >500 22.3 43.1 24.1 45.9 64.5 90.5

>500 33.3 176.9 120.2 48.1 53.4 151.1 127.6 39.5 >500 23.5 27.8 31 70.8 54.5 165

>500 32 135.1 90.1 18.7 33.3 96.2 63.4 14.7 >500 21.6 21.6 35.5 63.7 66.1 107.8

MT-1 RMS HT-29 HUV-EC-C MRC-5 IC50 (μM) IC50 (μM) IC50 (μM) IC50 (μM) IC50 (μM)

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300

Modified from Eliassen et al. (2003). Cell lines: MT-1 mammary carcinoma; RMS human melanoma cells; HT-29 colorectal adenocarcinoma cells; HUV-EC-C normal human umbilical vein endothelial cells; MRC-5 embryonic fibroblast cell line.

Peptide sequence

Name

Cell culture

Table 12.4. Antitumoral effects of lactoferricin derivatives containing aromatic amino acids in positions 3, 6, and 8 and lipophilic moieties at the N-terminal.

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tumor inoculation was demonstrated by Yoo et al. (1998). LfcinB inhibited angiogenesis and suppressed tumor growth up to day 8 after tumor inoculation. Apo-LF significantly suppressed tumor growth up to 21 days after a single administration. It was also reported that holo-LF (iron-saturated LF) and human apo-LF at 1 mg/mouse did not produce the anticancer effects under identical conditions. Oral administration of LF at 0.2% and 2% (w/w) and LfcinB at 0.1% (w/w) to rats that had previously been injected with azoxymethane to promote carcinogenesis resulted in 25, 15, and 10% decreased incidence, respectively, of colon adenocarcinomas during a 36-week feeding study (Tsuda et al. 1998). The majority of evidence suggesting that whey proteins have anticancer properties has been obtained from in vitro studies using carcinoma cell lines or in vivo studies using animal models. Valuable information can be gained from these studies but care should be taken when extrapolating from these results as to their potential cancer protective effects in humans. More long-term multicenter human clinical trials using composite whey and/or individual whey proteins/peptides are required to conclusively determine their efficacy in the treatment/ prevention of cancer.

Immunomodulatory Properties The immune system plays a central role in host protection against many pathogenic microbes and various disorders including cancers, allergic, and autoimmune diseases (Fleisher and Bleesing 2000). Chronic inflammatory diseases such as ulcerative colitis, irritable bowel syndrome, and Crohn’s disease (Berrebi et al. 2003; Peluso et al. 2006) are currently of particular interest in the area of immunosuppressive and immunomodulatory research. It is therefore desirable to seek out products that can modulate immune function to establish which foodstuffs may be of benefit to human health. In vitro models allow whey proteins to be incorporated into cell culture systems to assess their effect on different parameters such as cellular proliferation. Particular interest has been paid to lymphocytes including B lymphocytes that produce antibodies and T lymphocytes that control the antigen-specific immune response including tissue-damaging inflammatory reactions in digestive tract diseases (Cross and Gill 2000; Gauthier et al. 2006). These in vitro studies allow for a wide range of

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experimental variables to be measured but results from such studies merely demonstrate the potential of the test component. A number of whey proteins (WPC, α-LA, β-LG, LF) have been cited for their immunomodulatory effects on the body’s immune system. This relates to either activation or suppression of specific functions with particular interest in their modulation of lymphocyte proliferation in both in vitro and in vivo test models (Gill et al. 2000; Kelleher and Lonnerdal 2001; Yalcin 2006). However, limited clinical data exist evaluating the immunomodulatory effects of peptides produced by enzymatic hydrolysis of whey proteins. Cross and Gill (1999) tested a modified whey protein concentrate (mWPC) for its immunosuppressive activity in vitro. The mWPC was prepared using low temperature cation exchange to extract the majority of the whey proteins to give a WPC product that contained 70% protein comprising of 35% glycomacropeptide (GMP), 17% α-LA, 16% β-LG, 13% γ-globulin, and 1% serum albumin. mWPC (0.4 mg/mL) suppressed the lymphocyte activation process in murine erythocytes by reducing T and B lymphocyte proliferative responses to mitogens in a dose-dependent manner. However, mWPC showed no suppressive effect against IL-2-sustained proliferation of mitogen-activated T-cell blasts at the same concentration. The inclusion of 0.4 mg/mL mWPC also suppressed IFN-γ and IL-4 secretion in splenic lymphocyte cells. Further demonstration of bovine whey proteins ability to suppress lymphocyte function was presented in studies by Barta et al. (1991) and Torre and Oliver (1989). These authors reported that the degree of inhibition of lymphocyte blastogenesis by whey proteins was dose-dependent. It should be noted that the observed immunomodulatory response of cultured cells to whole whey proteins may be the cumulative response to any number of peptides derived from the intact proteins with different suppressive and immunoenhancing activities. Lothian et al. (2006) studied a whey-based oral supplement (HMS90) given twice daily (10 g) for 1 month to children with atopic asthma (a Th2 cytokine disease) in an effort to improve lung function and to decrease serum IgE levels. While, serum IgE levels decreased following supplementation, no significant changes in lymphocyte GSH levels or in lung function tests were found for the group overall. This study did however demonstrate a moderate impact of whey protein supplementation on cytokine response in atopic asthma but further long-term studies are necessary to confirm these findings.

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Orally administered proteins are subjected to degradation in the gastrointestinal tract by digestive enzymes (e.g., pepsin, trypsin, and chymotrypsin) and by intestinal bacteria (Gauthier et al. 2006; Gill et al. 2000; Lonnerdal 2003; Meisel and Bockelmann 1999). Prioult et al. (2004) reported that a number of immunomodulatory peptides were released from a β-LG tryptic-chymotryptic hydrolysate following fermentation with Lactobacillus paracasei, which was originally isolated from feces of a healthy infant. The peptide fragments released (sequence information not given) were reported to repress lymphocyte proliferation and to upregulate the immunosuppressant interleukin-10 (IL-10) at 1 and 20 μg/mL. In the first few months of a neonate’s life, it is important for the immune system to develop oral tolerance to molecules especially when milk proteins represent the exclusive protein supply for the newborn. Prioult et al. (2004) indicated that immunomodulatory peptides produced by intestinal bacteria may induce oral tolerance to β-LG by modulating the immune responses described above. β-LG has also been reported to mediate an immune response in an indirect manner. β-LG can bind retinoic acid, a vitamin A precursor (Guimont et al. 1997; Kontopidis et al. 2002), which in turn may participate in modulation of the gut immune system. Elitsur et al. (1997) reported that retinoic acid can stimulate proliferation of human colonic lamina propria lymphocytes in an in vitro study of the human gut mucosal immune system. Furthermore, Iwata et al. (2004) found that picomolar concentrations of retinoic acid can activate T-cells in vitro. Two synthetic peptides corresponding to α-LA f(50–51) (Tyr-Gly) and f(18–20) (Tyr-Gly-Gly) were reported to enhance proliferation and protein synthesis of ConA-stimulated human peripheral blood lymphocytes in vitro. Maximal stimulation was achieved at 10−4 mol/L with Tyr-Gly and 10−8 mol/L with Tyr-Gly-Gly (Kayser and Meisel 1996). Yanaihara et al. (2000) demonstrated the effect of LF on the proliferation of human endometrial stroma cells in comparison to estradiol and epidermal growth factor in vitro. LF at concentrations of 10, 100, and 1,000 ng/mL increased the rate of cell proliferation moderately in cells cultured without fetal bovine serum in comparison to controls. Cell proliferation increased fivefold (at 1,000 ng/mL LF) in endometrial stroma cells cultured with 2% fetal bovine serum. The effect of LF on cell proliferation at a concentration of 100 ng/mL was comparable to that of 10 nmol/L estradiol, but less than that of 10 mg/mL epidermal growth factor.

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Interleukin-6 (IL-6) is a multifunctional cytokine that is commonly associated with the spread of gastric cancer (Lin et al. 2007). It is also involved in the communication of inflammatory information to the central nervous system (Oka et al. 2007). Both bLF and hLF demonstrated an IL-6 inhibition capability in a lipopolysaccharide-stimulated monocytic cell line (THP-1) suggesting an anti-inflammatory role for LF (Mattsby-Baltzer et al. 1996). It was also reported that LfcinB was more effective as an IL-6 suppressor in comparison to the intact protein (Mattsby-Baltzer et al. 1996). Haversen et al. (2002) have shown that LF can inhibit IL-6 cytokine production in a human monocytic cell line via NF-κB activation. A peptic digest of LF containing LfcinB significantly enhanced immunoglobulin (IgM, IgG, and IgA) production in cultured murine splenocytes and IgA production in Peyer’s patch cells (Miyauchi et al. 1997). It was also reported that mice orally immunized with cholera toxin and fed a diet supplemented with the LF pepsin hydrolysate had significantly greater anti-CT IgA levels in the intestine contents and bile than mice fed a control diet. Cyclophosphamide is a commonly used drug in the treatment of human cancer and autoimmune diseases including multiple sclerosis (La Mantia et al. 2007). The disadvantages of using cyclophosphamide is that humoral and cellular immune responses are markedly impaired (Artym et al. 2003; Hadden 2003). Oral administration of LF to cyclophosphamide-treated mice led to partial reconstitution of the humoral response with an elevation of T- and B-cell content (Artym et al. 2005). LF was also reported to demonstrate similar immune-restorative properties in a study with immunocompromised mice caused with treatment of methotrexate—a drug involved in the treatment of psoriasis, certain cancers, and inflammatory diseases such as rheumatoid arthritis (Artym et al. 2004). Results obtained from a number of studies also indicate that LF may have a therapeutic value in the treatment of autoimmune disorders. “Autoimmune” New Zealand black mice were treated with bLF during a dose-dependent study, which resulted in decreased frequencies of hemolytic anemia (Zimecki et al. 1995). Further in vitro studies using peritoneal cells incubated with LF resulted in a decreased number of cells recognizing Hb antigen on autologous erythrocytes (Zimecki et al. 1995). LF has also demonstrated the ability to inhibit autoimmune conditions including experimentally induced colitis (Togawa et al. 2002),

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encephalomyelitis (Zimecki et al. 2007), and autoimmune and septic arthritis in mice and in rat models (Guillen et al. 2000). During an in vitro study by Wong et al. (1997a,b), both LF and lactoperoxidase were separately found to inhibit proliferation and interferon-γ production in ovine blood lymphocytes, in response to mitogenic stimulation. However, the combined use of LF and lactoperoxidase or combinations with WPC significantly reduced the immune response to the mitogen. Wong et al. (1997a,b) also demonstrated that α-LA had a stimulatory effect on IL-1β production in vitro. α-LA consumption also mediated immunoenhancing effects in mice especially in comparison to casein, soy, and wheat proteins (Bounous and Kongshavn 1985; Bounous et al. 1983). It has been reported that milk growth factor, which has complete N-terminus homology with bovine TGF-β2, can suppress human T lymphocyte function including cytokine-stimulated cell proliferation in vitro (Gill et al. 2000). GMP, κ-casein f(106–169), is a highly biologically active peptide that has the ability to modulate immune function (Brody 2000; Li and Mine 2004). GMP blocked the action of interleukin IL-1 by binding to its receptor in the mouse monocyte/macrophage cell line, P388D1 (Monnai and Otani 1997). Furthermore, it suppressed IgG antibody production in newborn mice in vivo (Monnai et al. 1998). GMP was also reported to be an immunoenhancer by increasing cell proliferation and phagocytic activities in a human macrophage-like cell line U937 (Li and Mine 2004). The immunomodulatory properties of bovine IgG have also been reported. Bovine IgG suppressed human lymphocyte proliferative responses to B- and T-cell mitogens at a dose of 0.3 mg/mL (Kulczycki et al. 1987). Bovine milk-derived growth factor at 1 ng/mL was also described as a potent suppressor of human T-lymphocyte functions including mitogen- and IL-2-stimulated cellular proliferation and recall proliferative responses to tetanus toxoid antigen (Stoeck et al. 1989). The complement-derived anaphylatoxin peptides C3a and C5a are generally thought to be important inflammatory mediators with antianalgesic and antiamnesic properties in host defense (Drouin et al. 2001; Jinsmaa et al. 2000; Wust et al. 2006). Chiba and Yoshikawa (1991) first characterized the multifunctional bioactive peptide, albutensin A from BSA. Takahasi et al. (1998) reported that the tryptic fragment, albutensin

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A, had affinities for complement C3a (IC50 110 ± 6.8 μM) and C5a (IC50 2 mM) receptors and induced ileum contraction in guinea pig. Ohinata et al. (2002) also reported that administration of albutensin A (3–50 nmol/mouse) decreased food intake after central or peripheral administration and delayed gastric emptying, which was mediated through C3a receptor. It was suggested that C3a purposely reduces food intake during an inflammatory state. De Noni and Floris (2007) have reported an optimized method of tryptic digestion of serum albumins at acidic pH to achieve albutensins with high yield and purity that could potentially be consumed by humans. The use of albutensin A in the management of obesity was proposed as this peptide displays satiety-inducing properties. Albutensin A was also reported to have ACE inhibitory activity (Chiba and Yoshikawa 1991).

Opioid Agonist and Antagonist Activities Opioid peptides are defined as peptides that have affinity for opiate receptors and produce opiate-like effects, which can be inhibited by naloxone (Meisel 1998). Endogenous opioid peptides such as enkephalins are small molecules that are naturally produced in the central nervous system and in various glands throughout the body (Chaturvedi et al. 2000; Janecka et al. 2004). Dependent on their location, opioid receptors demonstrate a number of regulatory functions and can interact with both endogenous and exogenous opioid ligands (Teschemacher and Scheffler 1993). Systems such as the spinal cord, adrenal gland, and the digestive tract contain μ- and δ-receptors while the pituitary gland and hypothalamus possess μ-, δ-, and ε-receptors (Dziuba et al. 1999; Teschemacher et al. 1994). The μ-receptors are involved in neuroendocrine function and are linked with pain sensation and analgesia (Zakharova and Vasilenko 2001). Typical opioid peptides all originate from three precursor proteins: proopiomelanocortin (endorphins), proenkephalin (enkephalins), and prodynorphin (dynorphins) (Teschemacher et al. 1997), which possess the same N-terminal sequence, Tyr-Gly-Gly-Phe, which is the fragment that interacts with the receptors (Janecka et al. 2004; Teschemacher 2003). Opioid peptides derived from a variety of precursor proteins including milk proteins are termed “atypical opioid peptides” as they differ in amino acid sequences and only the N-terminal tyrosine is conserved (Teschemacher and Scheffler 1993).

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Enzymatic fragmentation of whey proteins has yielded biofunctional peptides that behave like opioid receptor ligands in vitro and in vivo. Opioid agonists have been found in α-LA, β-LG, and BSA, whereas opioid antagonists have been isolated from LF (see Table 12.5). Peptides from both α-LA and β-LG contain sequences in their primary structure similar to typical opioid peptide sequences, whereas serorphin BSA f(399–404) (Tani et al. 1994) can be classed as an atypical opioid peptide with a dissimilar amino sequence (Table 12.5). The opioid antagonist peptide, LfcinB, exhibits no similarity to endogenous opioid peptide amino acid sequences nor does it contain a tyrosine residue at the N-terminal (Meisel and FitzGerald 2000). Both α-lactorphin, α-LA f(50–53), and β-lactorphin, β-LG f(102– 105), have demonstrated opioid properties with pharmacological activity at micromolar concentrations as shown in Table 12.5 (Antila et al. 1991). These peptides also act as ACE inhibitors (Mullally et al. 1996). α-Lactorphin can be released from α-LA using pepsin whereas βlactorphin is produced by the digestion of β-LG with pepsin followed by trypsin or by trypsin and chymotrypsin (Antila et al. 1991; Sipola et al. 2002). α- and β-Lactorphins were identified as μ-type receptor ligands (Paakkari et al. 1994). Along with receptor binding, α-lactorphin exerted an opioid effect ex vivo by the inhibition of smooth muscle contraction in guinea pig ileum (Antila et al. 1991; Paakkari et al. 1994). The effect of α-lactorphin on guinea pig ileum was antagonized by the addition of naloxone. Binding of β-lactorphin to the opioid receptors was similar to that of α-lactorphin. However, β-lactorphin exerts a stimulatory effect on smooth muscle, which was not antagonized by naloxone. Digestion of β-LG with chymotrypsin yields β-lactotensin f(146– 149) which demonstrated similar pharmacological activity to βlactorphin as both peptides induced ileum contraction that was antagonized neither by naloxone nor by atropine (Pihlanto-Lepp¨al¨a et al. 1997). A pancreatic digest of α-LA was found to increase mucin discharge that was mediated by opioid receptor activation in a rat model system (Claustre et al. 2002). Mucins are the predominant components of the mucus gel within the intestine, and alterations of mucin secretion/expression could be involved in several illnesses such as cancer and intestinal inflammatory diseases (Aksoy and Akinci 2004). In vitro and in vivo studies by Ushida et al. (2007) demonstrate the potential gastroprotective activity of α-LA through the ability of α-LA to stimulate mucin production and secretion in gastric ulcer models.

308

c

b

a

—

—

ALKAWSVAR —

Albutensin A

Serorphin

Antagonist

Agonist

Agonist

NDc

NDc

Hayashida et al. (2004) and Tsuchiya et al. (2006)

Stimulation Takahasi et al. (1998)

3

Tani et al. (1994)

Stimulation PihlantoLepp¨al¨a et al. (1997)

NDc

NDc

Stimulation Antila et al. (1991)

38 ± 7b

Agonist

85

Antila et al. (1991)

Inhibition

67 ± 13b

Agonist

β-Lactotensin Agonist

GPI: Effect on the contractions of guinea pig ileum in vitro. Morphine exhibited an IC50 value of 23 ± 12 μM, which inhibited the GPI effect. ND, not determined.

Lactoferrin

208–216

YGFQNA

HIRL

146–149

β-Lactorphin

α-Lactorphin

References

Opioid effect IC50 (μM) GPI effecta

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Bovine serum albumin 399–404

YLLF

102–105

β-Lactoglobulin

YGLF

50–53

α-Lactalbumin

Fragment Peptide sequence Name

Examples of opioid peptides derived from bovine whey proteins.

Precursor protein

Table 12.5.

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Intravenous administration of synthetic α-lactorphin to SHRs and to normotensive Wistar Kyoto rats lowered both systolic and diastolic BP in a dose-dependent manner without affecting heart rate (Nurminen et al. 2000). It was reported that the BP-lowering effect of α-lactorphin was due to interaction with opioid receptors as the effect was reversed following the administration of naloxone, a specific opioid receptor antagonist. Further studies have also found that α-lactorphin and β-lactorphin improved vascular relaxation in SHRs ex vivo (Sipola et al. 2002). LfcinB exhibits some similarity to casein opioid antagonists with selective μ-opioid receptor antagonist activity with moderate potency (Teschemacher et al. 1997). Other studies have demonstrated that the opioid antagonist activity of LF is linked to κ- as well as μ-receptors (Takeuchi et al. 2003; Tani et al. 1990). The κ-opioid receptor is also involved in analgesia during the developmental period (Barr et al. 1986). Takeuchi et al. (2003) reported that intraperitoneal injection of bovine LF in 5–18 days old rat pups evoked an opioid-mediated suppression of anxiety and reduced physical distress during a maternal separation study. This was postulated to be due to nitric oxide inducing an opioiddependent antistress effect. Other recent studies by Hayashida et al. (2003) and Tsuchiya et al. (2006) reported that bovine LF enhances analgesia induced by morphine and potentially hindered the development of tolerance to morphine in mice. The coadministration of LF and morphine could potentially be of benefit to patients with severe pain who are developing resistance to morphine. The above studies demonstrate that LfcinB could be of physiological importance as a natural analgesic substance administered either on its own or in combination with other painkilling drugs. The concentrations of opioid peptides in bovine milk proteins may be adequate to generate positive results in vitro. However, it is unlikely that equivalent peptide concentrations studied in vitro would be produced as a result of proteolysis during in vivo digestion of milk (Pihlanto-Lepp¨al¨a 2001). Further studies are required to assess whether oral ingestion of these bioactive peptides can exert similar results in human clinical trials.

Mineral Binding Properties Foods rich in minerals and elements are important in human health (Fraga 2005; Lukaski 2004). A growing number of foods are fortified

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with micronutrients, including infant foods, beverages, sweets, cereals, and dairy products, to reduce the risk of suboptimal intake of important vitamins and minerals (Wagner et al. 2005). The minerals can be added as salts or in combination with metal-binding peptides (Meisel 2005; Vyas and Tong 2004), where milk-derived components are deemed as acceptable ingredients for dietetic food when compared with some artificial additives. Whey proteins capable of functioning as carriers for different minerals include α-LA, β-LG, BSA, LF, and the immunoglobulins (Vegarud et al. 2000). Enzymatic hydrolates of α-LA have the ability to bind calcium, copper, iron, magnesium manganese, phosphorus, and zinc (Etcheverry et al. 2004; Kelleher et al. 2003; Pellegrini 2003; Permyakov and Berliner 2000; van Dael et al. 2005; Vegarud et al. 2000). β-LG has affinities for calcium, cadmium, copper, iron, magnesium, manganese, and zinc (Dufour et al. 1994; Mata et al. 1996; Simons et al. 2002; Vegarud et al. 2000). Hydrolysis of β-LG with chymotrypsin yielded peptides having a greater affinity for iron with higher scavenging capacity than the intact protein (Elias et al. 2006). Both β-LG and serum albumin have also been identified as mercury-binding proteins (Kontopidis et al. 2004; Mata et al. 1997; Sundberg et al. 1999). Serum albumin also has zinc-binding capabilities (Davidsson et al. 1996; Singh et al. 1989). Maintenance of bodily iron homeostasis is essential as excessive iron can be detrimental, promoting microbial growth (Radtke and O’Riordan 2006) and cellular damage via free radicals (Puntarulo 2005). LF has been shown to play a major role in iron regulation in mammals (Bullen et al. 2005; Lambert et al. 2005). In its native state, LF is only 8– 30% iron-saturated. This allows for the chelation of iron resulting in bacteriostatic and antioxidative effects (Ha and Zemel 2003; Walzem et al. 2002). Furthermore, it has been reported that pepsin and trypsin hydrolyates of LF bind iron at higher affinities than the intact protein (Kawakami et al. 1993; Wakabayashi et al. 2003). In a study by Davidsson et al. (1994), it was demonstrated that human infants fed LFcontaining breast milk had lower levels of iron absorption in comparison to infants fed the same milk from which LF had been removed. This illustrated the role of LF in sequestering free iron in the digestive tract. Furthermore, these results outlined the importance of LF in neonatal primary defense against pathogenic microorganisms. Iron deficiency is one of the major nutritional problems in the world especially in infants, children, and in women of childbearing age. Ironsaturated LF has demonstrated potential for use as a safe iron supplement

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(Uchida et al. 2006). Following a single oral dose (2.5 g iron/kg bodyweight), the hemoglobin content in rats fed iron-saturated LF was significantly higher than controls fed with ferrous sulfate, which is the most frequently used iron supplement. Similar results were observed in a human trial involving administration of iron-saturated LF to pregnant women at different stages of pregnancy (Paesano et al. 2006). After 30 days oral administration of iron-saturated LF (100 mg of 30% ironsaturated LF twice a day), hemoglobin and total serum iron values were higher than those supplemented with ferrous sulfate (520 mg once a day). Another advantage to the use of iron-saturated LF observed in the human trial was that it did not produce any of the common side effects of iron supplements such as stomach pain, cramps, and constipation in comparison to ferrous sulfate.

Antimicrobial Activity Intact whey contains several components with broad antimicrobial activity. Furthermore, antimicrobial peptides may be generated from whey protein by proteolysis during gastrointestinal transit (Chatterton et al. 2006; Clare et al. 2003; Floris et al. 2003; Gauthier et al. 2006; Meisel 1997; Pellegrini 2003; Yalcin 2006 for reviews). The advantages of whey protein-derived antimicrobial peptides are that they are derived from a safe substance and they may be produced by naturally occurring enzyme activation. Antibacterial Effects Bruck et al. (2003) reported a significant decrease in cell numbers of the infant fecal microorganisms, Escherichia coli 2348/69 (O127:H6) and Salmonella serotype typhimurium (DSMZ 5569), during a continuous culture model containing GMP and α-LA. After a 6-day period the infant formula supplemented with α-LA (68% w/w) inhibited growth of the E. coli strain whereas GMP at the same concentration inhibited both the E. coli and Salmonella strains. Antibacterial peptides have been identified in α-LA enzymatic hydrolysates. Peptide fragments LDT1 f(1–5) and LDT2 f(17–31SS109–114) were produced by tryptic digestion of α-LA while peptide LDC f(61–68S-S75–80) was released by chymotryptic hydrolysis as summarized in Table 12.6 (Pellegrini et al. 1999). These α-LA peptides

IPAVFK VAGTWY

VLVLDTDYK

AASDISLL DAQSAPLR VLVLDTRYKK

15–20

92–100

25–40 92–101

CKDDQNPH– ISCDKF

(61–68)S– S(75–80) 78–83

GYGGVSL PEWVCTTF– ALCSEK

(17–31)S–Sa (109–114)

312 Trypsin Modified

P*92 −101

Trypsin

Trypsin

Trypsin

Chymotrypsin

Trypsin

Trypsin

Preparation

LGDT4

LGDT3

LGDT2

LGDT1

LDC

LDT2

LDT1

Name

B. subtilis, M. luteus, S. epidermidis, S. lentus, S. zooepidemicus, E. coli, B. bronchiseptica

B. subtilis, S. lentus,S. zooepidemicus

B. subtilis, M. luteus, S. aureus, S. epidermidis, S. lentus

B. subtilis, M. luteus, S. aureus, S. epidermidis, S. lentus

B. subtilis, S. lentus,S. zooepidemicus

K. pneumoniae, B. subtilis, S. lentus

S. aureus, K. pneumoniae, S. epidermidis, S. lentus, M. luteus, S. zooepidemicus, B. subtilis,

S. epidermidis, S. lentus, B. bronchoseptica, S. zooepidemicus, B. subtilis M. luteus

Sensitive microorganisms

Pellegrini et al. (2001)

Pellegrini et al. (1999)

References

April 4, 2008

β-Lactoglobulin

EQLTK

1–5

α-Lactalbumin

Peptide sequence

Fragment

Examples of antimicrobial peptides derived from bovine whey proteins.

Precurs or protein

Table 12.6.

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Lactoferrin

313 APRKNVRW CTISQPEW– LECIRA APRKNVRW CTISQPEW FKCRRWQW RMKKLGAPS ITCVRRAFA – LECIRA

(1–42)S–S (43–48)

FKCRRWQW RMKKLGAPS ITCVRRAF/A

Peptide 3

Peptide 2

Lactoferricin

Pepsin

Pepsin

Pepsin

L. monocytogenes, E. coli, B. cereus, Ps. fluorescens, S. aureus, S. salford

L. monocytogenes, E. coli, B. cereus, Ps. fluorescens, S. aureus, S. salford

E. coli, K. pneumoniae, Ps. aeruginosa, S. aureus, S. bovis, S. mutans, L. monocytogenes, S. enteritidis, P. vulgaris, Ps. fluorescens, S. epidermidis, S. haemolyticus, S. hominus, E. faecalis, L. lactis, L. casei, C. diphtheriae, C. renale, B. subtilis, B. cereus, B. natto, B. circulans, C. ammoniagenes, Cl. perfingens, Cl. paraputrificum, C. albicans

(cont.)

Dionysius and Milne (1997)

Bellamy et al. (1992a, b), Hoek et al. (1997), and Ueta et al. (2000)

April 4, 2008

(1–16)S–S (43–48)

17–41/42

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Peptide sequence APRKNVR WCTISQPEW– FKCRR WQWRM KKLGAPS ITCVRRAF ALECIRA FKCRRWQWR

KKLGAPSIT CVRRAFA

APRKNVR WCTISQPEW– CIRA APRKNV RWCTI– FKCRRWQW RMKKLGAP SITCVRRAF ALECIR FKCRRWQW RMKKLG

Fragment (1–16)S–S (17–48)

17–25

27–42

(1–16)S–S (45–48) (1–11)S–S (17–47)

17–30

(continued)

Precurs or protein

Table 12.6.

314 Peptide LFb 17–30

Peptide 4

Peptide 2

Synthetic

Pepsin

Pepsin

CNBr cleaved LfcinB

CNBr cleaved LfcinB

Chymosin

Preparation

S. aureus, S. mutans, S. sobrinus, S. salivarius, E. coli, K. pneumoniae, P. intermedia, P. gingivalis, F. nucleatum, C. albicans

Micrococcus flavus

Micrococcus flavus

L. monocytogenes, E. coli, B. cereus, Ps. fluorescens, S. aureus, S. salford

Groenink et al. (1999) and van der Kraan et al. (2004)

Recio and Visser (1999)

Hoek et al. (1997)

E. coli

L. monocytogenes, E. coli, B. cereus, Ps. fluorescens, S. aureus, S. salford

References

Sensitive microorganisms

April 4, 2008

Subfragment 2

Subfragment 1

Peptide 3

Name

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GAPSITCVRRAF RRWQWR RWQWRM DLIWKLLS KAQEKFG KNKSR

30–41

20–25

20–26

265–284

315

a S–S

= disulfide bridge.

GLIWKLL SKAQEKF GKNKSR DGIWKL LSKAQEK FGKNKSR DLIGKL LSKAQEKFGK NKSR DLIWGLLS KAQEKFGKNKSR DLIWKL LSKAQGKF GKNKSR

FKCRRWQWRM FKARRWQWRM

17–26

actoferrampin D265G L266G W268G K269G E276G

Lactoferrampin

Peptide 5

Peptide 4

Peptide 3

Peptide 2 Peptide 2

Peptide LFb 19–37

Synthetic

Synthetic

Synthetic

Synthetic

Synthetic

Synthetic

Synthetic

Comparable candidacidal activity to lactoferrampin

C. albicans, B. subtilis, E. coli, Ps. aeruginosa

C. albicans

C. albicans

C. albicans

C. albicans

S. aureus, S. mutans, S. salivarius, P. intermedia, P. gingivalis, F. nucleatum

van der Kraan et al. (2005)

van der Kraan et al. (2004)

Ueta et al. (2000)

April 4, 2008

265–284

CRRWQWRM KKLGAPSITCV

19–37

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possess strong activity against gram-positive bacteria but weak activity against the gram-negative strains tested. Both LDT2 and LDC are composed of two peptide fragments covalently linked by disulfide bridges. Both fragments are necessary for bactericidal activity. The individual peptide fragments of LDT2 and LDC do not possess any bactericidal activity. Tryptic digestion of β-LG yielded four peptide fragments: LGDT1 f(78–83), LGDT2 f(15–20), LGDT3 f(92–100), and LGDT4 f(25–40) that are reported to be bacteriocidal for gram-positive bacteria as summarized in Table 12.6 (Pellegrini et al. 2001). LGDT3 was subsequently modified by replacing the negatively charged Asp98 with positively charged Arg and addition of positively charged lysine residue at the Cterminus (VLVLDTRYKK). The antimicrobial activity spectrum of the modified peptide was extended to include the gram-negative bacteria E. coli and Bordetella bronchiseptica. However, a reduction in activity against gram-positive species was observed. Pihlanto-Lepp¨al¨a et al. (1999) reported that intact α-LA and β-LG did not inhibit growth of E. coli JM103 at 0.1 g/mL. Conversely, α-LA hydrolysates generated by digestion with trypsin or pepsin and β-LG hydrolyzed with AlcalaseTM , trypsin, or pepsin had antibacterial activity against the E. coli strain at the same concentration. The α-LA and β-LG hydrolysates (0.025 g/mL) also exhibited a bacteriostatic effect against E. coli after 8 h of growth. Nevertheless, the concentration of the α-LA and β-LG hydrolysates necessary to cause a bacteriostatic effect was far higher than reported for an LF hydrolysate produced by heat treatment or by LfcinB. Saito et al. (1991) reported that 10 μg/mL of an LF hydrolysate, produced by heat treatment under acidic conditions, was required to exhibit antibacterial activity against E. coli 0–111. On the other hand, LfcinB inhibited the growth of both gram-positive and gram-negative organisms including E. coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas. aeruginosa, Campylobacter jejuni, Staphlococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes, and Clostridium perfringens at concentrations between 0.3 and 150 μg/mL dependent on the strain and the culture medium used (Table 12.6, Bellamy et al. 1992a,b). Inhibition of Candida albicans by LfcinB (in the range of 18–150 μg/mL) was also reported (Bellamy et al. 1993). LfcinB possessed stronger anticandidal activity than intact LF (Muller et al. 1999).

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Although the mechanism(s) involved the bacteriostatic activity of LF are not definitively elucidated, it is commonly recognized that its activity is linked to iron sequesteration from the bacterial growth medium (Arnold et al. 1981; Ling and Schryvers 2006; Tomita et al. 1994a,b for review). It has also been reported that LF demonstrates direct bactericidal activity in gram-negative organisms by binding to the lipid A part of bacterial lipopolysaccharide, with an associated increase in membrane permeability (Yamauchi et al. 1993). LF can enhance the antibacterial activity of lysozyme (Ellison and Giehl 1991). Transmission electron microscopy results show that E. coli cells cultured with LF and lysozyme become enlarged and hypodense. This suggested that the antibacterial activity was caused via alteration of osmotic balance. Bellamy et al. (1992a,b) later described that the N-terminal region of LF was accredited to its membrane-associated bactericidal activity. LfcinB is derived from the N-terminal region of LF, and its bactericidal activity is independent of iron-binding (Hoek et al. 1997). The bactericidal activity of LfcinB has been correlated with the net positive charge of the peptide that is, it contains a high proportion of basic amino acid residues (Bellamy et al. 1993; Gifford et al. 2005 for review; Meisel and Bockelmann 1999). The bactericidal sequence of LfcinB was found to consist mainly of a loop of 18 amino acid residues formed by a disulfide bond between cysteine residues 19 and 36 of bovine LF or residues 20 and 37 in human LF (Tomita et al. 1994a,b). The antimicrobial role of LF is of particular interest to intestinal function and in the prevention of gastroenteric diseases through control of intestinal microflora. While LF exhibits bactericidal activity against pathogens such as coliforms, it also provides probiotic support for beneficial microorganisms such as Bifidobacteria and Lactobacilli (Baldi et al. 2005; Yamauchi et al. 2006). LF-derived peptides, in particular LfcinB, exhibit an antimicrobial activity that is more potent than that of intact LF. As shown in Table 12.6, LF-derived peptides exhibit antibacterial activity against a number of gram-positive and gram-negative bacteria and yeasts and filamentous fungi (Hoek et al. 1997; Tomita et al. 1991; Wakabayashi et al. 2003; Yamauchi et al. 1993 for review). In the study by Ueta et al. (2001) six LF peptides consisting of 6–25 amino acid residues were released following pepsin digestion, summarized in Table 12.6. Peptide 2, bLF f(17–26), has stronger anticandidal activity than the intact protein and LfcinB (peptide 1). Peptides 1, 3, 4, and 5 and LF suppressed iron uptake by Candida cells while iron

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uptake was not inhibited by peptide 2. Further studies with peptide 2 have reported that it prolongs the survival of mice infected with a lethal dose of C. albicans (Tanida et al. 2001). Lactoferrampin, Lfampin f(265–284), exhibited bactericidal activity against C. albicans, Bacillus subtilis, E. coli, and Ps. aeruginosa, as shown in Table 12.6 (van der Kraan et al. 2004). Lfampin exhibited substantially higher candidacidal activity than intact LF. The concentration of Lfampin, which causes death in 50% of C. albicans (LC50 value), was 4.3 μg/mL compared to 578 μg/mL LF. A glycine substitution scan was used to identify residues in Lfampin that are required for its candidacidal activity (van der Kraan et al. 2005). Some substitutions of positively charged residues led to a considerable reduction in candidacidal activity, while other glycine substitutions had comparable LC50 values (Table 12.6). Helicobacter pylori is known as the causative agent in the majority of duodenal ulcers and is believed to be responsible for 50–60% of all gastric carcinomas (Collins et al. 2006; Dzieniszewski and Jarosz 2006). H. pylori infections are difficult to treat due to the location of the bacteria and its ability to readily develop antibiotic resistance. A number of studies have reported that daily administration of LF positively suppresses gut colonization of H. pylori in infected subjects. In a large multicentered human trial, 402 H. pylori-positive volunteers were assigned to either a triple therapy of esomeprazole (20 mg), clarithromycin (500 mg), and tinidazole (500 mg) twice daily for 7 days or LF (200 mg) twice daily for 7 days. This was followed by the triple therapy or the triple therapy plus LF combination (Di Mario et al. 2003, 2006). The eradication rate of H. pylori in infected patients was 77% with the triple therapy, 73% with LF, and 90% in the group treated with a combination of LF and the triple therapy. Okuda et al. (2005) reported on the efficiency of LF to suppress H. pylori infection during a 12-week trial containing 59 subjects. Twice daily oral administration of LF (200 mg) was reported to reduce the colonization density of H. pylori although complete eradication was not achieved. Group A streptococci (GAS) are common pathogens considered to be the principal contributing agent of dental caries and oral infections (Berlutti et al. 2004). It was reported that 1 mg/mL LF significantly reduced the in vitro invasion of cultured epithelial cells (isolated from patients suffering from pharyngitis) by GAS (Ajello et al. 2002). The positive results from the in vitro study led to a trial in children with

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pharyngitis, which demonstrated the effectiveness of LF as an antimicrobial agent when used in combination with the traditional antibiotic erythromycin. Fifteen days prior to tonsillectomy, erythromycin (500 mg) and LF gargles (100 mg) were taken three times daily. This resulted in lower numbers of intracellular GAS in tonsil specimens from children treated with the LF–erythromycin combination compared to treatment with erythromycin alone. Murdock and Matthews (2002) examined the antimicrobial activities of intact LF and a peptic digest of LF (0.125–8 μg/mL) against the foodborne pathogens L. monocytogenes and E. coli O157:H7 in ultra-high temperature (UHT) milk. Following adjustment of the UHT milk to pH 4, the LF pepsin-digest reduced E. coli O157:H7 and L. monocytogenes population by approximately 2 log cycles, while at pH 7 the LF pepsindigest did not inhibit L. monocytogenes. The results indicated that the pepsin-digest of LF can reduce the population of pathogenic bacteria in a dairy product, under the appropriate conditions. Milk naturally contains specific immunoglobulins against certain pathogenic bacteria. Immunization of cows against defined pathogens can also lead to the production of specific immunoglobulins (Korhonen et al. 2000 for review). A milk immunoglobulin concentrate, prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes, has been used to successfully protect humans against E. coli infection (Tacket et al. 1988). The addition of bovine-specific antibodies to an LGG-fermented milk product and to UHT toddler’s milk inhibited streptococcal growth over a long-term storage period (Wei et al. 2002). This study demonstrated that the inclusion of specific immunoglobulins in a food product may extend the shelf life of the product while also helping in the prevention of dental caries and oral infections. Antiviral Effects LF, α-LA, and β-LG have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) (Chatterton et al. 2006; Marshall 2004; Ng et al. 2001; Wang et al. 2000a,b for reviews). LF strongly inhibited HIV-1 reverse transcriptase activity but only slightly inhibited HIV-1 protease and integrase, whereas α-LA and β-LG inhibited HIV-1 protease and integrase but did not inhibit HIV-1 reverse transcriptase (Ng et al. 2001).

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HIV infection is associated with intracellular GSH, where low concentrations of GSH allow the virus to multiply while high levels slow down viral replication (Micke et al. 2001; Yalcin 2006). As described previously, whey proteins can increase GSH levels in the body, and on this basis whey protein has been orally administered to both adults with advanced HIV-infection and children with rapidly progressive AIDS. Two separate double-blind clinical trials were performed with whey protein in combination with antiretroviral therapy in infected adults and children. These results showed an increase in erythrocyte GSH levels in the whey protein-supplemented group (Micke et al. 2002; Moreno et al. 2006). In conclusion, WPC supplementation can stimulate GSH synthesis and, possibly, encourage a more efficient immune response to viral infections. A number of studies have reported on the chemical modification of βLG by hydroxyphthalic anhydride, yielding a product designated 3HPβ-LG, a potent HIV-1 inhibitor that also exhibits activity against herpes simplex virus types 1 and 2 (Kokuba et al. 1998; Neurath et al. 1998). These results indicate that modified whey proteins, in particular 3HPβ-LG, may be potential agents for preventing transmission of genital herpesvirus infections as well as the spread of HIV. Some interesting results have also been described using bovine immunoglobulins in the treatment of HIV and its associated symptoms. An immunoglobulin preparation (10 g/day) with high antibacterial antibody titers was well tolerated and highly effective in the treatment of severe diarrhea in AIDS patients (Stephan et al. 1990). Rump et al. (1992) also reported significant clinical benefits in HIV-infected and immunodeficiency patients with chronic diarrhea when treated orally with 10 g/day immunoglobulins from bovine colostrum for 10 days.

Gastrointestinal Health As discussed previously, some whey peptides can prevent the growth and proliferation of undesirable and pathogenic organisms while some demonstrate probiotic functions (Kilara and Panyam 2003; Yalcin 2006 for reviews). Bifidobacteria and Lactobacilli are probiotic bacteria that may positively alter intestinal microflora, boost immune function, promote good digestion, and increase resistance to infection (Bengmark 2000; Chow 2002). Prebiotics are defined as food substances intended

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to promote the growth or activity of certain bacteria in the gut (Yalcin 2006). α-LA was reported to be a potent growth promoter with a high activity for stimulating the growth of Bifidobacterium infantis and Bifidobacterium breve, however, it did not promote the growth of two strains of Bifidobacterium bifidum (Petschow and Talbott 1991). GMP can inhibit the adhesion of H. pylori and rotavirus to the cell membrane by binding to pathogen receptor sites (Kawakami 1997). Bouhallab et al. (1993) reported that GMP strongly stimulated growth of Lactococcus lactis subsp. lactis CNRZ 1076 in reconstituted skim milk, while Idota et al. (1994) described that glycomacropeptide promoted the growth of B. breve, B. bifidum, B. infantis as well as L. lactis. Petschow et al. (1999) demonstrated the ability of LF to promote the growth of the probiotic bacteria B. infantis, B. bifidum, and B. breve in vitro. Similar results by Kim et al. (2004) reported that both apoLF and holo-LF promoted growth in the B. breve, B. infantis, and B. bifidum species whereas only holo-LF promoted growth in Lactobacillus acidophilus. These results demonstrate the ability of LF to promote the growth of Bifidobacterium species and exhibit a prebiotic-type activity.

Hypocholesterolemic Effects High total serum cholesterol levels are associated with higher risks of coronary disease. It is widely accepted that the supplementation of a natural lipid-lowering agent in combination with diet and exercise would be preferential to the use of cholesterol-lowering medications. Kontopidis et al. (2004) and Wang et al. (1997) reported that β-LG had the ability to bind cholesterol. Cholesterol binding takes place at the central cavity of β-LG at pH 7.3. Furthermore, LF was reported to significantly inhibit the accumulation of cellular cholesteryl esters in macrophages by acting as a scavenger in an in vitro study (Kajikawa et al. 1994). Nagaoka et al. (2001) reported on the hypocholesterolemic action of lactostatin, β-LG f(71–75), in human cell lines (Caco-2 cells) and in rat studies. Cholesterol uptake was 40% lower in the Caco-2 cells treated with 1 mg of lactostatin compared to cells treated with the same concentration of a tryptic casein hydrolysate. The incorporation of cholesterol in intestine, serum, and liver in the rat models was significantly

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lower when treated with lactostatin (1 mg) compared to a tryptic casein hydrolysate and the anticholesterol drug β-sitosterol. This group subsequently reported that lactostatin (a) regulated the phosphorylation of extracellular signal-regulated kinase and (b) intracellular Ca2+ concentration involved in the calcium channel-related MAPK signaling pathway in HepG2 cells (human hepatoblastoma cells). This regulatory pathway in turn results in greater cholesterol degradation via lactostatin when compared to the action of β-sitosterol (Morikawa et al. 2007). β-Lactotensin, β-LG f(146–149), exhibited hypocholesterolemic activity in mice 90 min after intraperitoneal administration for 2 days at a dose of 30 mg/kg or on oral administration at 100 mg/kg (Yamauchi et al. 2003). Intraperitoneal administration of β-lactotensin was more effective in reducing total serum cholesterol. A 22.7% decrease in cholesterol levels was obtained following intraperitoneal administration compared to a 13.8% reduction following oral administration. Whey peptides, particularly lactostatin and β-lactotensin, show promise as naturally derived molecules for the development of nutraceuticals and functional foods to prevent and decrease hypercholesterolemia and atherosclerosis in vivo.

Insulinotropic Effects Whey proteins contain more essential amino acids and branched-chain amino acids than most other food proteins, and as a consequence are associated with the modulation of insulin responses in humans (Etzel 2004 for review; Nilsson et al. 2004; Pfeuffer and Schrezenmeir 2007). Individual amino acids such as phenylalanine, lysine, leucine, and arginine have potent insulin stimulation properties and are present in moderate quantities in the individual whey proteins (Marcelli-Tourvieille et al. 2006; McLeod 2004; Siminialayi and Emem-Chioma 2006). Additionally branched-chain amino acids from whey including isoleucine, leucine, and valine have been linked with postprandial stimulation of insulin and increased plasma amino acid levels (Matthews 2005; Nilsson et al. 2004). Calbet and MacLean (2002) reported a two- to fourfold increase in insulin secretion in six human test subjects following administration of a whey hydrolysate (0.25 g/kg body mass) after 30 min compared to the response obtained with a glucose solution (25 g/L) and cow’s milk. The insulin response was correlated to the increase

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in plasma levels of leucine, isoleucine, valine, phenylalanine, and arginine. According to Frid et al. (2005), supplementation of meals having a high glycemic index with whey proteins increased insulin secretion and improved blood glucose control in type 2 diabetic subjects. In a study containing 14 subjects, whey powder (27.6 g) was supplemented at breakfast and lunch on day 1 and was exchanged for lean ham (96 g) and lactose (5.3 g) on day 2. Four hours after breakfast and 3 h after lunch, the levels of insulin and glucose-dependent insulinotropic polypeptide were higher when meals were supplemented with whey protein. A threefold increase in insulin response was reported following the administration of glucose with whey (75 mg) in mice that was superior to the 1.5-fold increase with 34 mg oleic acid (Gunnarsson et al. 2006). The whey protein also increased GLP-1 secretion but did not affect glucose-dependent insulinotropic polypeptide secretion. The insulinotropic effect of whey protein is not necessarily observed in longer-term intervention studies. Insulin-resistant rats were fed a high-protein diet for 6 weeks containing either 80 or 320 g protein/kg WPC or meat protein (Belobrajdic et al. 2004). WPC reduced plasma insulin concentration by 40% and the insulin glucose ratio, a measure of insulin resistance, was lower in rats fed WPC than in rats fed red meat. It can be concluded that whey proteins and their associated peptides may serve as exogenous regulators of incretin hormones with beneficial influences in humans especially those affected by diabetes.

Memory and Stress An imbalance in brain serotonin levels is a possible factor manifesting the negative effects of chronic stress, fatigue, and delirium (Castell et al. 1999; van der Mast and Fekkes 2006). Under stressful conditions, serotonin and tryptophan (the precursor of serotonin) levels are exhausted to below functional needs (Markus et al. 1999). The whey protein α-LA has a high (5.3% w/w) tryptophan content compared to other whey proteins (Etzel 2004), and therefore a number of studies have been performed to evaluate its potential use to improve cognitive performance.

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Markus et al. (2002) reported the effects of α-LA on high and low stress-vulnerable subjects during a double-blind study. Within a 2-h period, two subjects (one high stress vulnerable and one low stress vulnerable) were administered 20 g of whey protein or sodium caseinate (control diet) in two hot chocolate drinks. Cognitive performance tests were performed 90 min after the second ingestion of the drink. The ratio of plasma tryptophan to other large neutral amino acids (Trp/LNAA) was higher in the α-LA diet compared to the control diet. Improved cognitive performances in memory tests were observed in subjects in the high stress-vulnerable group on the α-LA diet but not in the low stress-vulnerable subjects. In another study by Schmitt et al. (2005), 16 women experiencing premenstrual symptoms (mood swings, irritability, anxiety, breast tenderness, bloating, and cognitive complaints such as poor concentration, confusion, and forgetfulness) underwent a clinical trial on 2 premenstrual days between days 22 and 28 of the menstrual cycle. On both premenstrual test days, the participants received a low-protein breakfast, snack, and lunch where two chocolate drinks (200 mL) were served, each containing either 20 g of a whey preparation rich in α-LA or 15.5 g casein (control condition). Cognitive performance tests were performed on the participants 2 h after ingestion of the second chocolate drink. The α-LA preparation partially improved long-term memory for abstract figures, but not for words, with significantly faster response times during the premenstrual phase. Booij et al. (2006) reported on a clinical trial, which assessed if α-LA could improve the memory of depression patients. The 23 remitted depression subjects and 20 controls underwent the same diet as described by Markus et al. (2002) with profile of mood state (POMS) tests and cognitive performance tests performed 1.75 h after ingestion of a second drink. The plasma Trp/LNAA ratio improved significantly (71.5%) when compared to the control diet. α-LA improved recognition and speed of retrieval from short- and long-term abstract visual memory and simple motor performance in both recovered depression patients and healthy individuals while mood was unaffected. β-Lactotensin, β-LG f(146–149), was reported to mediate an antistress property in vivo (Yamauchi et al. 2006). β-Lactotensin (10 or 30 mg/kg) was administered intraperitoneally to mice following 1 h of acute restraint-induced stress where a significant decrease in stress-related behaviors were observed. In fear-conditioning tests, β-lactotensin also

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reduced the freezing responses/symptoms compared to that of the controls. Antianxiety and antistress drugs can cause adverse side effects in some patients; therefore, the results described above for α-LA and βlactotensin are promising in the search for novel therapies in this area.

Conclusion The topics covered in this chapter demonstrate the different beneficial biological activities of intact whey proteins and their peptides. In some cases the benefits of the active peptides were demonstrated in human trials. A major bottleneck to the widespread utilization of intact whey proteins and their associated peptides as functional food ingredients/ nutraceuticals is the lack of data from clinically validated appropriately powered human trials. Once this issue has been addressed, we are likely to see major developments by the food and healthcare sectors in the widespread application of whey proteins and their associated peptides as functional food ingredients and nutraceuticals.

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Petschow, B.W., and Talbott, R.D. 1991. Response of Bifidobacterium species to growth promoters in human and cow milk. Pediatr. Res. 29(2):208–213. Petschow, B.W., Talbott, R.D., and Batema, R.P. 1999. Ability of lactoferrin to promote the growth of Bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level. J. Med. Microbiol. 48(6):541–549. Pfeuffer, M., and Schrezenmeir, J. 2007. Milk and the metabolic syndrome. Obes. Rev. 8(2):109–118. Pihlanto-Lepp¨al¨a, A. 2001. Bioactive peptides derived from bovine whey proteins: Opioid and ACE-inhibitory peptides. Trends Food Sci. Technol. 11(9–10):347–356. Pihlanto-Lepp¨al¨a, A., Paakkari, I., Rinta-Koski, M., and Antila, P. 1997. Bioactive peptides derived from in vitro proteolysis of bovine beta-lactoglobulin and its effect on smooth muscle. J. Dairy Res. 64(1):149–155. Pihlanto-Lepp¨al¨a, A., Rokka, T., and Korhonen, H. 1998. Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. Int. Dairy. J. 8:325– 331. Pihlanto-Lepp¨al¨a, A., Marnila, P., Hubert, L., Rokka, T., Korhonen, H.J., and Karp, M. 1999. The effect of α-lactalbumin and β-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103. J. Appl. Microbiol. 87(4):540–545. Pins, J.J., and Keenak, J.M. 2006. Effects of whey peptides on cardiovascular disease risk factors. J. Clin. Nutr. 8(11):775–782. Prioult, G., Pecquet, S., and Fliss, I. 2004. Stimulation of interleukin-10 production by acidic β-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei NCC2461 peptidases. Clin. Diagn. Lab. Immunol. 11(2):266–271. Puntarulo, S. 2005. Iron, oxidative stress and human health. Mol. Aspects Med. 26(4– 5):299–312. Radtke, A.L., and O’Riordan MX. 2006. Intracellular innate resistance to bacterial pathogens. Cell Microbiol. 8(11):1720–1729. Recio, I., and Visser, S. 1999. Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane. J. Chromato. A. 831(2):191–201. Roy, M.K., Kuwabara, Y., Hara, K., Watanabe, Y., and Tamai, Y. 2002. Peptides from the n-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells. J. Dairy Sci. 85(9):2065–2074. Rump, J.A., Arndt, R., Arnold, A., Bendick, C., Dichtelmuller, H., Franke, M., Helm, E.B., Jager, H., Kampmann, B., Kolb, P., Kreuz, W., Lissner, R., Meigel, W., Ostendorf, P., Peter, H.H., Plettenberg, A., Schedel, I., Stellbrink, H.W., and Stephan, W. 1992. Treatment of diarrhoea in human immunodeficiency virus-infected patients with immunoglobulins from bovine colostrum. Clin. Investig. 70(7):588–594. Saito, H., Miyakawa, H., Tamura, Y., Shimamura, S., and Tomita, M. 1991. Potent bactericidal activity of bovine lactoferrin hydrolysate produced by heat treatment at acidic pH. J. Dairy Sci. 74(11):3724–3730. Schmitt, J.A., Jorissen, B.L., Dye, L., Markus, C.R., Deutz, N.E., and Riedel, W.J. 2005. Memory function in women with premenstrual complaints and the effect of serotonergic stimulation by acute administration of an α-lactalbumin protein. J. Psychopharmacol. 19(4):375–384.

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Chapter 13 Current and Emerging Role of Whey Protein on Muscle Accretion Peter J. Huth, Tia M. Rains, Yifan Yang, and Stuart M. Phillips

Introduction Protein is essential for many aspects of human physiology and serves a key structural role by providing amino acid substrates to support protein synthesis and anabolism in virtually all tissues of the body particularly skeletal muscle, the body’s largest reservoir of protein. Although the level and quality of protein in the diet for enhancing muscle mass, strength, and metabolic functions for athletes has long been recognized, it is now becoming appreciated that increasing and/or maintaining muscle mass is important for the general population to help prevent or manage disorders such as obesity, type 2 diabetes, osteoporosis, and loss of muscle mass due to aging (sarcopenia). In the context of the prevalence of overweight and obesity, diets higher in protein and lower in carbohydrate (CHO) have been implicated as an alternative dietary approach to more efficient and long-term weight loss (Krieger et al. 2006). A growing body of evidence has emerged to support the relationship between dietary protein essential amino acids and their direct effect in stimulating muscle protein synthesis and accretion. Dairy proteins, specifically casein and whey, are excellent natural sources of essential amino acids that have been shown to induce synthesis and new muscle accretion following exercise. The goal of this review is to (1) examine the evidence on the role of dietary protein, especially whole dairy protein, casein, and whey on

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normal muscle metabolism, acute changes in muscle protein synthesis (MPS), and longer-term muscle protein accretion (i.e., hypertrophy); (2) characterize the independent and synergistic stimulatory role of exercise and protein; and (3) evaluate the influence of protein types on MPS and muscle protein accretion. It is notable that the focus on acute changes in MPS and breakdown is not simply to gain mechanistic insight into how dairy proteins affect protein metabolism, which is in and of itself a worthy experimental goal. However, the proposal is that acute changes in protein turnover are ultimately predictive of longer-term adaptive changes in skeletal muscle (Phillips 2004).

General Muscle Protein Metabolism There is substantial evidence on the role of dietary protein in supporting growth and maintenance of body proteins, and a detailed discussion is beyond the scope of this review. Readers are referred to an excellent authoritative report by the Institute of Medicine (Institute of Medicine of the National Academies 2002). Rather, this section provides an overview on the current state of the science. Whole-body protein balance, also known as protein turnover, is determined by the algebraic difference between protein synthesis and protein breakdown (Figure 13.1). Despite the large mass of skeletal muscle, protein turnover of this tissue compartment makes only a modest contribution to whole-body protein turnover at approximately 25–30% because skeletal muscle proteins turnover relatively slowly compared to other tissues (Wolfe 2002). The majority of whole-body protein synthesis and breakdown is composed of turnover of proteins in the splanchnic area (representing both intestinal plus hepatic compartments). Like skeletal muscle, and virtually all other tissue compartments, the process of protein synthesis in the splanchnic area is dependent on adequate concentrations of essential amino acids (EAA). In instances where any one EAA intake is low or absent, amino acid becomes limiting and protein synthesis is attenuated, whereas protein breakdown is stimulated and amino acids are oxidized and excreted (Tome and Bos 2000). This process occurs in both children and adults and is the basis for the recommendation for consumption of high-quality protein within the diet. In adults, where dietary protein intake is adequate, approximately 250 g/day of protein is synthesized and degraded (Institute of Medicine

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Figure 13.1. Schematic of protein turnover and various metabolic fates of amino acids in tissues. Note that in skeletal muscle the essential amino acid leucine is markedly stimulatory for protein synthesis (see text for details).

of the National Academies 2002). In growing infants and children, the rate of protein synthesis and net protein balance is much greater than that for adults due to net new tissue protein that is being laid down to support growth. Total body protein turnover is regulated by several factors that impact protein synthesis, protein breakdown, or both. For example, increasing amino acid availability stimulates overall protein synthesis with a small effect on breakdown. Conversely, in adult humans insulin has a modest stimulatory effect on synthesis and a strong suppressing effect on breakdown, resulting in a net positive impact on protein turnover (Rennie et al. 2004). However, feeding a protein-free diet in the presence of enhanced insulin does not significantly stimulate protein synthesis suggesting that insulin imparts a permissive rather than stimulatory effect in the presence of amino acid availability and provides an anabolic environment for muscle protein synthesis (Fujita et al. 2006; Prod’homme et al. 2004). Other hormones also impart an anabolic effect on total body protein turnover. For example, in growing children, growth hormone (GH) plays a major role in linear growth and skeletal maturation. In conjunction with GH, insulinlike growth factor (IGF-1) stimulates amino acid uptake and incorporation into protein in both the liver and skeletal muscle (Shils 1999). The anabolic roles of all these hormones are dependent on the consumption of adequate amounts of

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high-quality protein providing essential amino acids within the diet of growing children (Graham et al. 1981). Within skeletal muscle specifically, increased availability of essential amino acids initiates a cascade of events that stimulate protein synthesis leading to a positive net protein balance (i.e., synthesis exceeding breakdown) (Tome and Bos 2000). Following a mixed meal containing a source of high-quality protein, there is an influx of amino acids into the circulation. Amino acids are then actively transported into muscle, particularly the branched-chain amino acids (BCAA), leucine, isoleucine, and valine, which are not extensively first pass cleared by the liver and constitute a disproportionately high (∼20%) of all amino acids within muscle tissue. Essential amino acids, including the BCAA, have been shown to directly stimulate muscle protein synthesis particularly in the presence of insulin (Layman 2002; Matthews 2005; Platell et al. 2000; Prod’homme et al. 2004; Smith et al. 1998; Wolfe 2002; Yoshizawa 2004). At the same time the postprandial hyperaminoacidemia is present, and protein breakdown is inhibited both as a result of the postmeal rise in insulin levels (Shils 1999) and to some degree by the rise in amino acids, likely the BCAA, themselves. Overall, there is a net positive effect of dietary protein intake on muscle protein accretion following a meal. However, in healthy adults under sedentary conditions, the effects of dietary protein intake on increased protein synthesis are transient (Tome and Bos 2000). As levels of amino acids drop during the postmeal period, protein synthesis slows and protein breakdown is increased to balance protein synthesis with protein degradation. While net protein balance is neutral in adults, that is, they do not have a net expansion or growth of any tissue protein pool, there is a net positive protein balance in children. This growth is driven to a large extent by the presence of growth-related hormones (e.g., GH, IGF) necessary to promote enhanced rates of linear growth (Institute of Medicine of the National Academies 2002). There is also substantial evidence that resistance or strength training exercise synergistically enhances the effects of essential amino acids on MPS (Borsheim et al. 2002; Miller et al. 2003; Rasmussen and Phillips 2003; Rennie and Tipton 2000; Tipton and Wolfe 2001). The mechanisms by which MPS is influenced following a meal are multiple; however, current consensus opinion is that the main locus of control is at the initiation of protein translation pathways within the muscle cell. Although all the exact underlying mechanisms are not

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Current and Emerging Role of Whey Protein on Muscle Accretion 349 completely understood, there is growing evidence that specific amino acids and, to a lesser degree, insulin impact the phosphatidylinositol 3-kinase/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway, which controls the translational machinery that promotes the formation of a translation-competent ribosome, initiation of protein synthesis, and elongation of proteins within the muscle cell (Gautsch et al. 1998; Kimball and Jefferson 2001; Lynch et al. 2003; Nair and Short 2005; Peyrollier et al. 2000; Prod’homme et al. 2004). Within this pathway, Akt, eukaryotic initiation factor 4-E binding protein (eIF4E-BP) and S6 kinase 1 are key intermediates. Essential amino acids, especially leucine, have been shown to simulate phosphorylation of mTOR, 4EBP1, and S6 kinase, leading to the formation of the eIF4F complex, a key complex in the initiation of protein translation; for a comprehensive review of this pathway see Proud (2007). Insulin alone has also been shown to stimulate phosphorylation of protein kinase B and S6 kinase, which results in a small overall effect on protein synthesis (Greiwe et al. 2001; Prod’homme et al. 2004). In fasted rats, activation of these key proteins is reduced in muscle, but subsequently restored in response to feeding a protein-containing meal (Balage et al. 2001) and muscle protein synthesis is subsequently enhanced. Leucine alone is able to reproduce the effects of a mixture of essential amino acids on translation initiation and also stimulates MPS, an effect that is further enhanced in the presence of insulin, at least in rodents (Anthony et al. 2000, 2002; Balage et al. 2001; Prod’homme et al. 2004). Leucine has also been shown to stimulate protein synthesis in human muscle at rest, after exercise, and in other animal species, suggesting that this specific BCAA may be a key regulator of protein synthesis in all mammals (Baar and Esser 1999; Escobar et al. 2005; Garlick 2005; Greiwe et al. 2001; Layman 2002; O’Connor et al. 2003; Smith et al. 1998).

Effects of Milk Protein, Whey, and Casein on Protein Accretion In light of evidence showing that specific EAA can regulate protein synthesis, it has been hypothesized that different dietary protein sources may produce variable effects on protein balance, given differences in both amino acid composition and digestibility of intact proteins. Dairy proteins, specifically casein and whey, contain all the EAA in ratios that are similar to that of body protein and are especially rich in the BCAA

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particularly leucine, which has been shown to independently stimulate MPS and activate key regulatory signaling proteins (see above). Accumulating evidence from human trials indicates that dairy protein in the form of whey, casein, fluid milk, or extracted milk protein stimulates whole body and/or muscle synthesis resulting in a net accretion of new protein. The following section reviews the human trials that have specifically utilized milk proteins to assess their effects on protein anabolism either alone, in combination with other macronutrients, or compared to other protein sources.

Protein Sources and Absorption Rates Studies have examined how the rate of absorption of amino acids (AAs) from different intact protein sources might affect whole-body protein synthesis, breakdown, and oxidation following a meal. Boirie et al. (1997) evaluated these parameters using whey protein or casein as models for “fast” and “slow” proteins, respectively. Whey protein is an acid-soluble protein, whereas casein clots when exposed to acidity of the stomach, which delays its gastric emptying and thus may result in a slower release of AA (Mah´e et al. 1996). In a crossover design, 16 healthy subjects were randomly assigned to ingest either 30 g whey protein or 43 g casein protein (equal leucine to whey treatment) and whole-body protein utilization and metabolism as measured by leucine kinetics was determined over a 7-h period. Despite the higher nitrogen content in the casein treatment, circulating amino acid concentrations increased less with 43 g casein than with 30 g whey initially (∼3 h posttreatment), but the effect was transient in the whey protein group. Casein produced a lower, but sustained increase in circulating amino acids. Circulating leucine values mirrored this finding, with casein producing a lower, but sustained increase over the 7-h timeframe ( p < 0.05). Total protein synthesis was stimulated by 68 and 31% by whey protein and casein, respectively, levels that tended to be statistically different ( p > 0.05). Protein breakdown was not altered after whey protein, but was inhibited by 34% with casein ( p < 0.05). Total leucine oxidation over 7 h was 373 and 272 μmol/kg in the whey and casein, respectively ( p < 0.05). This was the first evidence to show that while both proteins promote net whole-body protein synthesis, slower digesting casein favored a greater inhibition of protein breakdown and whole-body net protein balance as

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Current and Emerging Role of Whey Protein on Muscle Accretion 351 compared to whey under resting conditions. What cannot be determined from this investigation, however, is to what specific tissue the ingested amino acids were directed for synthesis and in what tissue was breakdown suppressed. It seems more than likely that the bulk of the leucine oxidation would have occurred in skeletal muscle as the main site of the rate-limiting enzyme for leucine oxidation branched-chain oxoacid dehydrogenase. In a similar study, Dangin et al. (2001) evaluated the importance of protein digestion rate of “fast” whey and “slow” casein on net protein balance when consumed as either a large bolus or in small repeated doses. Subjects consumed 30-g protein meals: a single meal of intact casein, a single meal of free amino acid mimicking casein composition, a single meal of intact whey proteins, or small repeated meals of whey proteins mimicking a slower digestion rate. Results showed that whey produced a higher but transient increase in circulating amino acids as compared to casein, an effect that was also seen in the free amino acid treatment. While whole-body protein synthesis, as measured by leucine kinetics, increased in all subjects, the whey protein and free amino acid group increased protein synthesis twofold higher than casein or the small whey meals ( p < 0.001). However, there was also a more rapid and higher peak value of leucine oxidation after the faster digesting treatments (whey and free amino acids) versus baseline ( p < 0.001). Overall, there was a greater net effect on leucine retention after the casein and small whey meals as compared to the amino acid treatment and whey protein bolus ( p < 0.05). Thus, while both dairy proteins (casein and whey) stimulate protein anabolism, sustained levels of essential amino acids in the plasma after casein or small repeated intakes of whey favor greater net protein anabolism.

Influence of Resistance Exercise on Muscle Protein Anabolism A number of excellent reviews have been written on this topic for readers who are interested in a more detailed discussion (Phillips 2004; Phillips et al. 2005; Rennie 2001; Rennie et al. 2004; Wolfe 2006). Briefly, MPS and muscle protein breakdown (MPB) vary in magnitude based on the type and intensity of exercise. Studies have consistently demonstrated that MPS is the primary variable stimulated by exercise that can range from 20 to 100% over basal resting levels of synthesis after intense

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resistance exercise (Biolo et al. 1995; Chesley et al. 1992; Phillips et al. 1997, 1999; Yarasheski et al. 1993). However, MPB is also stimulated with resistance exercise when performed in the fasted state, but to a lesser degree ranging from 10 to 25% such that the balance between MPS and MPB is typically not positive (Biolo et al. 1995). The fasted-state exercise-induced increase in MPB, however, is completely suppressed with the consumption of AA (Biolo et al. 1997) or CHO (Borsheim et al. 2004a,b; Chow et al. 2006; Fujita et al. 2006). Thus, it is only when adequate nutrition is provided, especially protein that resistance exercise results in a net anabolic state that stimulates protein accretion. Interaction of Dietary Protein and Resistance Exercise Numerous short-term human studies have been conducted using crystalline AA in various doses, with and without CHO and under resting and exercise conditions, to elucidate the impact of these variables on muscle protein anabolism and accretion (Borsheim et al. 2002, 2004a,b; Miller et al. 2003; Rasmussen et al. 2000; Tipton et al. 1999, 2001; Volpi et al. 1998, 2003). However, AAs are typically provided in meals as intact proteins and not crystalline AA. This raises the question as to whether results of studies using AA accurately reflect the situation of intact proteins. For example, in an initial study using crystalline EAA, Tipton et al. (2001) demonstrated that net amino acid uptake by skeletal muscle was greater when free EAA plus CHO were ingested before resistance exercise rather than following exercise. In a subsequent study using intact whey protein, however, although net MPS switched from negative to positive following ingestion of whey protein at either time, there was no difference of AA uptake between pre- and postexercise. Thus, the response of net muscle protein balance to timing of intact protein ingestion does not respond the same as that of the combination of EAA and CHO (Tipton et al. 2007). As discussed elsewhere, the digestion rate of protein is clearly different for intact dietary proteins than AA and is an independent factor that regulates MPS. In the case of dairy proteins, short-term studies using intact milk proteins either as fluid milk or whey and casein have all been shown to stimulate muscle protein accretion and new muscle after resistance exercise. For example, studies have been conducted to compare the impact of intact casein and whey protein on muscle accretion after resistance exercise (Tipton et al. 2004). In this study, healthy, untrained subjects

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Current and Emerging Role of Whey Protein on Muscle Accretion 353 were randomly assigned to one of three treatment beverages after a 25-min bout of resistance exercise. One hour after a high-intensity exercise protocol, subjects consumed 20 g casein, 20 g whey, or artificially flavored colored water and net muscle protein balance was determined by amino acid balance across the leg. Results showed that despite a peak leucine, net balance was over 2.5× greater for whey protein, both casein and whey-stimulated muscle protein synthesis following exercise as compared to control ( p < 0.01). There were no differences between the proteins, suggesting that both dairy proteins promote positive effects on muscle protein synthesis. These results provide support for the thesis that acute ingestion of either casein or whey after exercise stimulates muscle protein anabolism to a similar degree, despite differences in circulating amino acid levels. Interaction of Dairy Protein and Carbohydrate Intake It may be expected that energy intake will impact muscle protein synthesis. Early studies in humans indicated that regardless of the amount of nitrogen intake, nitrogen balance improved as energy intake increased (Calloway and Spector 1954). Although the mechanism by which energy affects nitrogen retention is unclear, it is unlikely that there is a direct effect of glucose or fatty acids on muscle protein synthesis since there is adequate substrate in the basal state to produce the ATP necessary for protein synthesis (Waterlow et al. 1978). Rather, the effect of CHO is more likely due to its stimulation of insulin and the resulting anabolic interaction with AA leading to protein synthesis. In a study to assess the impact of CHO and fat on muscle protein synthesis under resting conditions, Fouillet et al. (2001) tested the effect of milk protein delivered alone or simultaneously with carbohydrate or milk fat. Healthy subjects were assigned to receive either (1) 30 g milk protein, (2) 30 g milk protein plus 100 g sucrose, or (3) 30 g milk protein plus 43 g milk fat. As expected, plasma insulin levels were significantly increased following ingestion of milk protein plus sucrose ( p < 0.05). Protein synthesis estimates yielded incorporations of 43, 37.5, and 33% of ingested dietary nitrogen in the peripheral (i.e., nonsplanchnic) compartment with milk protein, milk protein plus fat, and milk protein plus sucrose, respectively. Conversely, dietary nitrogen incorporation into the splanchnic proteins was modeled to yield incorporations of 18, 24, and 35% of ingested nitrogen after milk protein, milk protein plus fat, and

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milk protein plus sucrose, respectively. These results provide support for the concept that protein, and specifically milk protein, can stimulate both splanchnic and peripheral protein synthesis, which would likely include muscle synthesis under nonexercising conditions, and that addition of other macronutrients may impact the region-specific utilization of dietary nitrogen. The effect of exercise on the interaction of protein and CHO on protein synthesis was evaluated by Borsheim et al. (2004a,b). After a 20-min bout of resistance exercise, subjects were provided two dietary treatments that were consumed 1 h postexercise (1) 17.5 g whey protein plus 77 g maltodextrin (CHO) and 5 g of EAA or (2) 100 g maltodextrin. Using isotopic tracer techniques that measure phenylalanine uptake into the leg muscle, net muscle protein synthesis was measured up to 4 h after the treatments. Muscle protein synthesis increased after the whey/CHO/AA beverage versus the CHO control during the first hour following ingestion of the treatments ( p = 0.042). There were no effects of the treatments on MPB. These results are consistent with previous work showing that the combined effect on net muscle protein synthesis of CHO and AA given together after resistance exercise is roughly equivalent to the sum of the independent effects of either given alone (Miller et al. 2003). Taken together, these results support the notion of a synergistic interaction between protein and CHO and that a mixture of whey protein, AA, and carbohydrate can stimulate muscle protein synthesis to a greater extent than isoenergetic CHO alone.

Influence of Protein Types on Synthesis and Accretion Accumulating evidence from studies that have compared the effects of different protein types on protein accretion has shown that different proteins have different impacts on protein kinetics, muscle protein synthesis, and accretion in different tissues (e.g., gastrointestinal, liver, and muscle) as well as whole-body lean mass accretion. This information may have implications for choosing protein sources in an attempt to optimize muscle protein metabolism and accretion for athletes who consume protein for a competitive advantage. Additionally, different protein sources differ in their effects on protein synthesis based on age. For example, whey and casein do not appear to differ in their effects on protein synthesis in young individuals (Tipton et al. 2004), whereas in

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Current and Emerging Role of Whey Protein on Muscle Accretion 355 older adults the evidence suggests that whey protein has a greater effect on stimulating protein synthesis than casein (Dangin et al. 2003). The objective of this section is to examine the acute and chronic effects of dairy protein, casein, whey, and soy proteins on muscle protein synthesis and accretion. Acute Studies Numerous short-term studies involving dairy and soy protein have been conducted in humans to evaluate the impact of dairy and soy protein on muscle protein synthesis and accretion, and all have shown that these proteins are able to support muscle accretion. Fouillet et al. (2002) used a compartmental model of protein kinetics to quantify protein balance within specific tissues (splanchnic and peripheral) using milk and soy protein. Healthy subjects consumed either 30 g of milk protein or soy protein plus 100 g sucrose under rested, fasting conditions. Over an 8-h period following consumption of test proteins, stable isotope kinetics were used to measure whole body and splanchnic protein utilization, and a model was applied to predict protein synthesis rates in various body compartments. Results showed that there was delayed appearance and a different pattern of release of amino acids into the plasma after the milk protein than after the soy protein. The result was a more rapid absorption and assimilation of dietary nitrogen from soy as compared to milk protein into splanchnic proteins and urea. There was also lower whole-body retention of soy protein compared with milk protein (72% vs 80%) because of higher splanchnic oxidation of amino acids and differences in the digestibility of the proteins ( p < 0.05). Within the specific compartments, soy protein promoted higher protein synthesis in the splanchnic region versus milk protein, with 30 and 23% of dietary nitrogen being incorporated into splanchnic proteins, respectively ( p < 0.03), whereas the opposite effect was seen in the peripheral tissues whereby milk protein promoted higher protein synthesis than soy (32 and 24% of dietary nitrogen incorporated in peripheral proteins, p < 0.03). The exact peripheral site of protein deposition could not be ascertained in this study; however, it is tempting to speculate that it was skeletal muscle into which greater AA uptake occurred following milk protein ingestion. These results reinforce that dietary protein can stimulate protein synthesis, but also suggest a differential metabolic utilization of dietary proteins within

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specific compartments of the body such that milk protein resulted in greater net protein anabolism in peripheral tissues compared to soy. Bos et al. (2003) have also provided evidence that the nitrogen from milk protein is utilized differently than the nitrogen from soy protein. In this study, a randomized, intervention trial, young healthy adults (∼28 year old; BMI = 21.4) were fed a 700 kcal mixed meal containing 15% protein (∼25 g) from either milk or soy protein after an overnight fast. The metabolic fate of nitrogen from the protein meals was followed for up to 8 h following the test meal, and whole-body protein utilization and metabolism were measured. The kinetics of dietary amino acids showed that for all amino acids except proline, soy protein resulted in greater peripheral appearance and higher peak AA concentration as compared to the milk protein, an effect consistent with a faster digestion of soy protein (bovine milk is 4:1 casein/whey protein by composition). This was associated with a faster transfer of dietary nitrogen into urea in the soy group (peak at 3 h vs 4.75 h in the milk group, p < 0.005). Total nitrogen retention from the ingested proteins was 74.7% in the milk protein group and 69.9% in the soy group. There was also a higher incorporation of dietary nitrogen from soy into serum proteins ( p = 0.02), suggesting that dietary nitrogen from milk protein would be incorporated into peripheral (i.e., nonsplanchinic) tissues, including skeletal muscle. These data further reinforce that there are differences in the utilization of nitrogen from dietary soy versus milk protein such that the amino acids from soy are digested more rapidly, are directed toward deamination pathways and urea production, and are incorporated into blood-borne proteins more readily than milk proteins. Thus, milk protein ingestion results in higher net protein anabolism in peripheral tissues, which may result from slower amino acid kinetics into circulation or the amino acid composition, particularly the higher level of BCAAs found in milk. In a follow-up study using the same design, Morens et al. (2003) assessed whether a diet adequate in protein (1 g/kg body wt./day) or high in protein (2 g/kg body wt./day) could cause differences between milk and soy protein on whole-body protein anabolism in healthy subjects under nonexercised conditions. Results showed that subjects consuming the higher protein diet had higher baseline values of urea and serum proteins as compared to the lower (but adequate) protein group. Regardless of the protein level in the diet, the protein source of the test meal impacted incorporation of dietary nitrogen into plasma proteins such that soy led to 7.6–8.0% incorporation versus 7.0–7.2% after the milk

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Current and Emerging Role of Whey Protein on Muscle Accretion 357 (nonsignificant). The production of urea was increased in the subjects on the high protein diet, but protein type also influenced urea production with significantly more urea produced in the first 2 h following ingestion of soy compared to the milk protein ( p < 0.05). The protein type also significantly influenced the amount of nitrogen retained. On the adequate protein diet, 74.4 and 70.7% of dietary nitrogen were retained in the milk and soy groups, respectively ( p < 0.0001), compared to 70.6 and 61.2% retention from milk and soy, respectively, on the high protein diet ( p < 0.0001). Thus, at normal protein intakes, milk protein results in greater stimulation of net whole-body protein synthesis versus soy protein. The ability of dietary protein to stimulate net whole-body protein anabolism was lower after adaptation to a higher protein diet, but this decrease was much more pronounced for soy than for milk protein. Taken together, results from these and other studies have consistently demonstrated the following: 1. Proteins, such as soy and whey, which are digested rapidly, lead to a large but transient increase in circulating AA. 2. These proteins markedly stimulate MPS and have been referred to as “fast” proteins. Conversely, casein is a “slow” protein because its slower digestion characteristic promotes a slower, more moderate, and longer-lasting rise in circulating AA and has only a modest effect on MPS but a substantial effect on suppressing MPB (Boirie et al. 1997). 3. Metabolism of ingested soy protein, as opposed to milk protein, promotes AA retention in the splanchnic bed and not in the peripheral tissue such as skeletal muscle. Based on these considerations, Wilkinson et al. (2006) recently hypothesized that in order to promote an optimal environment for MPS and muscle accretion, a supply of both “fast” and “slow” proteins may be required in conjunction with resistance exercise and would be superior to an environment with only “fast” protein sources such as soy or whey. Bovine milk is a source of both “fast” and “slow” proteins, which contains, respectively, 20% whey and 80% casein by weight of total protein. This hypothesis was tested in a randomized controlled trial that examined the effects of consuming nonfat fluid milk (500 mL, 745 kJ, 18.2 g protein) or a soy beverage matched for protein, energy, and macronutrient levels on muscle protein synthesis and net muscle accretion after

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Figure 13.2. Two-pool model-derived mean values for the positive area under the curve (AUC) for chemical NB of phenylalanine across the leg after consumption of a nonfat milk-protein beverage or an isonitrogenous, isoenergetic, macronutrientmatched (750 kJ, 18.2 g protein, 1.5 g fat, and 23 g carbohydrate) soy-protein beverage after a bout of resistance exercise. Values are means ±SE (N = 8). From Wilkinson et al. (2006) with permission.

resistance exercise (Wilkinson et al. 2006). Results showed that both soy and milk resulted in a positive protein balance. However, milk ingestion resulted in an overall greater net muscle protein balance (Figure 13.2, p < 0.05). The rate of MPS was also greater after milk consumption than after soy consumption (Figure 13.3, p = 0.05). Thus, in this short-term study, milk-based protein promoted greater muscle

Figure 13.3. Mean fractional synthetic rate (FSR) of muscle proteins during resistance exercise (exercise) and 3 h after resistance exercise with the consumption of a nonfat milk-protein beverage or an isonitrogenous, isoenergetic, macronutrient-matched (750 kJ, 18.2 g protein, 1.5 g fat, and 23 g carbohydrate) soy-protein beverage (3 h recovery). Values are means ± SE (N = 8). From Wilkinson et al. (2006) with permission.

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Current and Emerging Role of Whey Protein on Muscle Accretion 359 protein accretion than soy protein after resistance exercise and suggests that the chronic consumption of milk proteins may support a more rapid muscle mass accrual. Chronic Studies Since the period of exercise-induced MPS is approximately 48 h long (Phillips et al. 1997), acute studies in which proteins are ingested in close temporal proximity before or after exercise would appear to be predictive of long-term muscle growth only to the degree that the response of MPS and net muscle protein balance are affected during the acute postexercise period. Thus, evidence suggests that the optimal period during which protein should be consumed appears to be within an hour prior to or after resistance exercise (Andersen et al. 2005; Cribb and Hayes 2006; Esmarck et al. 2001; Holm et al. 2006; Phillips et al. 1997; Wilkinson et al. 2006). Indeed, results from chronic studies have shown that individuals ingesting protein in the postexercise period gain greater lean mass compared to those receiving nothing or CHO only (Andersen et al. 2005; Cribb and Hayes 2006; Esmarck et al. 2001; Hartman et al. 2007; Holm et al. 2006). For example, the acute differences seen in MPS and muscle accretion between fluid milk and soy protein have recently been shown to be maintained in a longer-term 12-week study (Hartman et al. 2007). In this study, young men were randomly assigned to one of three groups: control (receiving only carbohydrate postexercise), soy (receiving an energy matched, to control, soy drink with 18 g of soy protein and 26 g of maltodextrin), or milk (receiving 500 mL of low fat milk, isonitrogenous and macronutrient ratio matched with the soy group and isoenergetic with both the control and soy groups). The subjects trained for 5 days/week for 12 weeks. The a priori hypothesis of this work was based on the acute findings of Wilkinson et al. (2006) which are detailed above, but essentially the concept was that milk drinkers should gain more muscle than soy drinkers and both should gain more muscle than the control group. The beverage treatments were only administered around the 2 h prior to and 2 h after the resistance exercise workout with the proposal that it is this time which is most important in determining the gains in muscle mass with resistance training programs (Andersen et al. 2005; Cribb and Hayes 2006; Esmarck et al. 2001; Holm et al. 2006). The results were in agreement with the hypothesized changes, whereby the milk drinkers gained on average 4.0 kg of lean

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mass, whereas the soy drinkers gained only 2.9 kg and the control group even less at 2.4 kg. Of interest, is that the milk drinkers lost 0.8 kg of body fat, whereas the control group lost 0.5 kg, and the soy drinkers 0.2 kg, although the “loss” in the soy drinkers was not significantly different from zero. Thus, these data provide support for the idea that milk proteins as fluid milk per se provide an advantage over soy proteins when it comes to gaining lean mass and losing body fat in response to intense resistance exercise training. The generalizability of these data to other populations who may stand to benefit from such a body composition change, such as the obese or those with type 2 diabetes, remains to be determined. Clearly, however, incremental increases in lean mass that are coincident with declines in fat mass would represent a significant positive change for those suffering from these and other chronic disease conditions. A number of other studies have been conducted in which milk proteins, oftentimes with other constituents such as creatine, crystalline amino acids, and carbohydrate, have been compared to soy proteins or simply with energy as carbohydrate (Brown et al. 2004; Burke, et al. 2001; Candow et al. 2006; Chromiak et al. 2004; Cribb and Hayes 2006; Cribb et al. 2007; Demling and DeSanti 2000; Kerksick et al. 2006; Maesta, et al. 2007; Rankin et al. 2004). Figure 13.4 shows the mean exercise-induced gains in lean body mass from supplemental protein sources reported by these studies compared to the median change in lean body mass in all the studies. The gains in lean body mass with milk proteins, and whey in particular, exceed the median response. By contrast, gains supported by soy protein and carbohydrate are consistently lower than the median reported gain. These data represent findings from more than 300 subjects with a variety in training backgrounds in a number of environments and using a number of different training protocols. The common link between these studies is that all had, as their independent goal, the maximal muscle gain supported by differing nutritional interventions including at least one milk-based protein-consuming group. Thus, when considered together, Figure 13.4 highlights a large body of data demonstrating (1) the superiority of milk proteins (whey and casein) in synergistically interacting with exercise to support exercise-induced lean mass gains greater than those seen with soy or an energy-matched CHO placebo; (2) that dietary energy per se (usually exclusively in the form of CHO) does not induce gains in lean mass that are comparable to those seen when milk-based proteins are fed; and (3) the observation that dietary energy, primarily as CHO, induces a markedly lower gain

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Current and Emerging Role of Whey Protein on Muscle Accretion 361 in lean mass compared to milk, whey, casein, or soy-based proteins is evidence supporting the fact that the insulin response in and of itself is not going to have a large impact on muscle protein accretion compared to the provision of protein, and in particular milk-based proteins.

Conclusions A large body of data shows that ingestion of milk proteins is able to stimulate protein synthesis at both a whole-body and muscle level. In

Figure 13.4. Resistance training-induced changes in lean mass in studies of subjects receiving supplemental protein sources. A total of 11 studies are incorporated (Brown et al. 2004; Burke et al. 2001; Candow et al. 2006; Chromiak et al. 2004; Cribb and Hayes 2006; Cribb et al. 2007; Demling and DeSanti 2000; Hartman et al. 2007; Kerksick et al. 2006; Maesta et al. 2007; Rankin et al. 2004). N = 306 subjects for all studies (247 men and 43 women) are incorporated into the figure with protein supplements of either fluid milk (3 studies, N = 42 total subjects), whey protein (8 studies, N = 91 total subjects), casein protein (2 studies, N = 20 total subjects), isolated soy protein (4 studies, N = 65 total subjects), or carbohydrate (8 studies, N = 78 total subjects). Studies in which other components were included in the supplement (i.e., creatine or crystalline amino acids) are omitted from this analysis unless these compounds were present in all supplements, in addition to the protein source itself. All studies were at least 8 weeks in duration and up to as long as 16 weeks (mean 11.2 weeks). Mean gains in muscle mass as a result of resistance training and protein supplementation were as follows (means ± SD): milk = 2.7 ± 1.3 kg (range 1.9–3.9 kg); whey = 2.9 ± 1.6 kg (range 0.2–5 kg); casein = 2.4 ± 2.3 (range 0.8–4.1 kg); soy = 1.4 ± 0.6 (range 1.1–2.0 kg); and carbohydrate (CHO)/placebo = 0.9 ± 0.6 kg (range 0.3–1.8 kg). The dashed line represents the median change in lean body mass in all the studies. Values are means ± 95% confidence limits.

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the muscle, this stimulation is accompanied by phosphorylation of key signaling proteins in the muscle, including Akt, mTOR, and S6 kinase. Against a background of resistance exercise, which is by itself stimulatory for MPS, there is a synergistic stimulation of MPS such that the acute response is a positive net balance and acute protein retention. Again, milk proteins have been shown, in the acute setting, to stimulate MPS and result in a positive net balance. Other data suggest that milk proteins are more effective than soy proteins in stimulating MPS. By contrast, soy protein ingestion appears to result in a greater stimulation of splanchnic protein synthesis, urea production, and blood protein synthesis. Ultimately, the theoretical framework for how protein accretion takes place would predict that the acute differences in the efficacy of milk as opposed to soy proteins would favor a greater hypertrophic response, which is in fact what occurs. Moreover, when data from a number of chronic resistance training studies are compiled (Figure 13.4), it appears that milk proteins are more effective than soy in inducing muscle protein accretion and certainly more effective than simply energy as CHO. Future work will undoubtedly begin to delineate how these findings, from mostly younger healthy individuals, can be extended to older persons or persons with chronic diseases that are localized or at least influenced by skeletal muscle including obesity, metabolic syndrome, and type 2 diabetes.

References Andersen, L.L., Tufekovic, G., Zebis, M.K., Crameri, R.M., Verlaan, G., Kjaer, M., Suetta, C., Magnusson, P., and Aagaard, P. 2005. The effect of resistance training combined with timed ingestion of protein on muscle fiber size and muscle strength. Metabolism 54:151–156. Anthony, J.C., Yoshizawa, F., Anthony, T.G., Vary, T.C., Jefferson, L.S., and Kimball, S.R. 2000. Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J. Nutr. 130:2413–2419. Anthony, J.C., Lang, C.H., Crozier, S.J., Anthony, T.G., MacLean, D.A., Kimball, S.R., and Jefferson, L.S. 2002. Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine. Am. J. Physiol. Endocrinol. Metab. 282:E1092–E1101. Baar, K., and Esser, K. 1999. Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am. J. Physiol. 276:C120–C127. Balage, M., Sinaud, S., Prod’homme, M., Dardevet, D., Vary, T.C., Kimball, S.R., Jefferson, L.S., and Grizard, J. 2001. Amino acids and insulin are both required

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Current and Emerging Role of Whey Protein on Muscle Accretion 363 to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 281:E565–E574. Biolo, G., Maggi, S.P., Williams, B.D., Tipton, K., and Wolfe, R.R. 1995. Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. Am. J. Physiol. 268:E514–E520. Biolo, G., Tipton, K.D., Klein, S., and Wolfe, R.R. 1997. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am. J. Physiol. 273:E122–E129. Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrere, B. 1997. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc. Natl. Acad. Sci. USA 94:14930–14935. Borsheim, E., Tipton, K.D., Wolf, S.E., and Wolfe, R.R. 2002. Essential amino acids and muscle protein recovery from resistance exercise. Am. J. Physiol. Endocrinol. Metab. 283:E648–E657. Borsheim, E., Aarsland, A., and Wolfe, R.R. 2004a. Effect of an amino acid, protein, and carbohydrate mixture on net muscle protein balance after resistance exercise. Int. J. Sport. Nutr. Exerc. Metab. 14:255–271. Borsheim, E., Cree, M.G., Tipton, K.D., Elliott, T.A., Aarsland, A., and Wolfe, R.R. 2004b. Effect of carbohydrate intake on net muscle protein synthesis during recovery from resistance exercise. J. Appl. Physiol. 96:674–678. Bos, C., Metges, C.C., Gaudichon, C., Petzke, K.J., Pueyo, M.E., Morens, C., Everwand, J., Benamouzig, R., and Tome, D. 2003. Postprandial kinetics of dietary amino acids are the main determinant of their metabolism after soy or milk protein ingestion in humans. J. Nutr. 133:1308–1315. Brown, E.C., DiSilvestro, R.A., Babaknia, A., and Devor, S.T. 2004. Soy versus whey protein bars: Effects on exercise training impact on lean body mass and antioxidant status. Nutr. J. 3:22–26. Burke, D.G., Chilibeck, P.D., Davidson, K.S., Candow, D.G., Farthing, J., and SmithPalmer, T. 2001. The effect of whey protein supplementation with and without creatine monohydrate combined with resistance training on lean tissue mass and muscle strength. Int. J. Sport Nutr. Exerc. Metab. 11 (3):349–364. Calloway, D.H., and Spector, H. 1954. Nitrogen balance as related to caloric and protein intake in active young men. Am. J. Clin. Nutr. 2:405–411. Candow, D.G., Burke, N.C., Smith-Plamer, T., and Burke, D.G. 2006. Effect of whey and soy protein supplementation combined with resistance training in young adults. Int. J. Sport Nutr. Exerc. Metab. 16(3):233–244. Chesley, A., MacDougall, J.D., Tarnopolsky, M.A., Atkinson, S.A., and Smith, K. 1992. Changes in human muscle protein synthesis after resistance exercise. J. Appl. Physiol. 73:1383–1388. Chow, L.S., Albright, R.C., Bigelow, M.L., Toffolo, G., Cobelli, C., and Nair, K.S. 2006. Mechanism of insulin’s anabolic effect on muscle: Measurements of muscle protein synthesis and breakdown using aminoacyl-tRNA and other surrogate measures. Am. J. Physiol. Endocrinol. Metab. 291:E729–E736. Chromiak, J.A., Smedley, B., Carpenter, W., Brown, R., Koh, Y.S., Lamberth, J.G., Joe, L.A., Abadie, B.R., and Altorfer, G. 2004. Effect of a 10-week strength training

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program and recovery drink on body composition, muscular strength and endurance, and anaerobic power and capacity. Nutrition 20(5):420–427. Cribb, P.J., and Hayes, A. 2006. Effects of supplement timing and resistance exercise on skeletal muscle hypertrophy. Med. Sci. Sports Exerc. 38:1918–1925. Cribb, P.J., Williams, A.D., Stathis, C.G., Carey, M.F., and Hayes, A. 2007. Effects of whey isolate, creatine, and resistance training on muscle hypertrophy. Med. Sci. Sports Exerc. 39(2):298–307. Dangin, M., Boirie, Y., Garcia-Rodenas, C., Gachon, P., Fauquant, J., Callier, P., Ballevre, O., and Beaufrere, B. 2001. The digestion rate of protein is an independent regulating factor of postprandial protein retention. Am. J. Physiol. Endocrinol. Metab. 280: E340–E348. Dangin, M., Guillet, C., Garcia-Rodenas, C., Gachon, P., Bouteloup-Demange, C., Reiffers-Magnani, K., Fauquant, J., Ball`evre, O., and Beaufr`ere, B. 2003. The rate of protein digestion affects protein gain differently during aging in humans. J. Physiol. 549(2):635–644. Demling, R.H., and DeSanti, L. 2000. Effect of a hypocaloric diet, increased protein intake and resistance training on lean mass gains and fat mass loss in overweight police officers. Ann. Nutr. Metab. 44(1):21–29. Escobar, J., Frank, J.W., Suryawan, A., Nguyen, H.V., Kimball, S.R., Jefferson, L.S., and Davis, T.A. 2005. Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation. Am. J. Physiol. 288:E914–E921. Esmarck, B., Andersen, J.L., Olsen, S., Richter, E.A., Mizuno, M., and Kjaer, M. 2001. Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans. J. Physiol. 535:301–311. Fouillet, H., Gaudichon, C., Mariotti, F., Bos, C., Huneau, J.F., and Tome, D. (2001) Energy nutrients modulate the splanchnic sequestration of dietary nitrogen in humans: a compartmental analysis. Am. J. Physiol. Endocrinol. Metab. 281:E248– E260. Fouillet, H., Mariotti, F., Gaudichon, C., Bos, C., and Tome, D. 2002. Peripheral and splanchnic metabolism of dietary nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling. J. Nutr. 132: 125–133. Fujita, S., Rasmussen, B.B., Cadenas, J.G., Grady, J.J., and Volpi, E. 2006. Effect of insulin on human skeletal muscle protein synthesis is modulated by insulininduced changes in muscle blood flow and amino acid availability. Am. J. Physiol. Endocrinol. Metab. 291:E745–E754. Garlick, P.J. 2005. The role of leucine in the regulation of protein metabolism. J. Nutr. 135:1553S–1556S. Gautsch, T.A., Anthony, J.C., Kimball, S.R., Paul, G.L., Layman, D.K., and Jefferson, L.S. 1998. Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise. Am. J. Physiol. 274:C406–C414. Graham, G.G., Creed, H.M., MacLean, W.C., Kallman, C.H., Rabold, J., and Mellits, E.D. 1981. Determinants of growth among poor children: Nutrient intake-achieved growth relationships. Am. J. Clin. Nutr. 34:539–554.

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O’Connor, P.M., Kimball, S.R., Suryawan, A., Bush, J.A., Nguyen, H.V., Jefferson, L.S., and Davis, T.A. 2003. Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs. Am. J. Physiol. Endocrinol. Metab. 285:E40– E53. Peyrollier, K., Hajduch, E., Blair, A.S., Hyde, R., and Hundal, H.S. 2000. Lleucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: Evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucineinduced up-regulation of system A amino acid transport. Biochem. J. 350(2): 361–368. Phillips, S.M. 2004. Protein requirements and supplementation in strength sports. Nutrition 20:689–695. Phillips, S.M., Tipton, K.D., Aarsland, A., Wolf, S.E., and Wolfe, R.R. 1997. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am. J. Physiol. 273:E99–E107. Phillips, S.M., Tipton, K.D., Ferrando A.A., and Wolfe, R.R. 1999. Resistance training reduces the acute exercise-induced increase in muscle protein turnover. Am. J. Physiol. 276:E118–E124. Phillips, S.M., Hartman, J.W., and Wilkinson, S.B. 2005. Dietary protein to support anabolism with resistance exercise in young men. J. Am. Coll. Nutr. 24:134S– 139S. Platell, C., Kong, S.E., McCauley, R., and Hall, J.C. 2000. Branched-chain amino acids. J. Gastroenterol Hepatol. 15(7):706–717. Prod’homme, M., Rieu, I., Balage, M., Dardevet, D., and Grizard, J. 2004. Insulin and amino acids both strongly participate to the regulation of protein metabolism. Curr. Opin. Clin. Nutr. Metab. Care 7:71–77. Proud, C.G. 2007. Signalling to translation: How signal transduction pathways control the protein synthetic machinery. Biochem. J. 403(2):217–234. Rankin, J.W., Goldman, L.P., Puglisi, M.J., Nickols-Richardson, S.M., Earthman, C.P., and Gwazdauskas, F.C. 2004. Effect of post-exercise supplement consumption on adaptations to resistance training. J. Am. Coll. Nutr. 23(4):322–330. Rasmussen, B.B., and Phillips, S.M. 2003. Contractile and nutritional regulation of human muscle growth. Exerc. Sport Sci. Rev. 31:127–131. Rasmussen, B.B., Tipton, K.D., Miller, S.L., Wolf, S.E., and Wolfe, R.R. 2000. An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise. J. Appl. Physiol. 88:386–392. Rennie, M.J. 2001. Control of muscle protein synthesis as a result of contractile activity and amino acid availability: Implications for protein requirements. Int. J. Sport Nutr. Exerc. Metab. 11(Suppl):S170–S176. Rennie, M.J., and Tipton, K.D. 2000. Protein and amino acid metabolism during and after exercise and the effects of nutrition. Annu. Rev. Nutr. 20:457–483. Rennie, M.J., Wackerhag, E.H., Spangenburg, E.E., and Booth, F.W. 2004. Control of the size of the human muscle mass. Annu. Rev. Physiol. 66:799–828. Shils, M. 1999. Modern Nutrition in Health and Disease. Philadelphia: Lippincott Williams & Wilkins.

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Current and Emerging Role of Whey Protein on Muscle Accretion 367 Smith, K., Reynolds, N., Downie, S., Patel, A., and Rennie, M.J. 1998. Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein. Am. J. Physiol. 275:E73–E78. Tipton, K.D., and Wolfe, R.R. 2001. Exercise, protein metabolism, and muscle growth. Int. J. Sport Nutr. Exerc. Metab. 11(1):109–132. Tipton, K.D., Ferrando, A.A., Phillips, S.M., Doyle, D., Jr., and Wolfe, R.R. 1999. Postexercise net protein synthesis in human muscle from orally administered amino acids. Am. J. Physiol. 276:E628–E634. Tipton, K.D., Rasmussen, B.B., Miller, S.L., Wolf, S.E., Owens-Stovall, S.K., Petrini, B.E., and Wolfe, R.R. 2001. Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise. Am. J. Physiol. Endocrinol. Metab. 281:E197–E206. Tipton, K.D., Elliott, T.A., Cree, M.G., Wolf, S.E., Sanford, A.P., and Wolfe, R.R. 2004. Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise. Med. Sci. Sports Exerc. 36:2073–2081. Tipton, K.D., Elliott, T.A., Cree, M.G., Aarsland, A.A., Sanford, A.P., and Wolfe, R.R. 2007. Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise. Am. J. Physiol. Endocrinol. Metab. 292:E71–E76. Tome, D., and Bos, C. 2000. Dietary protein and nitrogen utilization. J. Nutr. 130:1868S–1873S. Volpi, E., Ferrando, A.A., Yeckel, C.W., Tipton, K.D., and Wolfe, R.R. 1998. Exogenous amino acids stimulate net muscle protein synthesis in the elderly. J. Clin. Invest. 101:2000–2007. Volpi, E., Kobayashi, H., Sheffield-Moore, M., Mittendorfer, B., and Wolfe, R.R. 2003. Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am. J. Clin. Nutr. 78:250–258. Waterlow, J.C., Golden, M.H., Garlick, P.J. 1978. Protein turnover in man measured with 15N: Comparison of end products and dose regimes. Am. J. Physiol. 235(2):E165– E174. Wilkinson, S.B., Tarnopolsky, M.A., MacDonald, M.J., Macdonald, J.R., Armstrong, D., and Phillips, S.M. 2006. Consumption of fluid skim milk promotes greater muscle protein accretion following resistance exercise than an isonitrogenous and isoenergetic soy protein beverage. Am. J. Clin. Nutr. 85:1031–1040. Wolfe, R.R. 2002. Regulation of muscle protein by amino acids. J. Nutr. 132:3219S– 3224S. Wolfe, R.R. 2006. Skeletal muscle protein metabolism and resistance exercise. J. Nutr. 136:525S–528S. Yarasheski, K.E., Zachwieja, J.J., and Bier, D.M. 1993. Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women. Am. J. Physiol. 265:E210–E214. Yoshizawa, F. 2004. Regulation of protein synthesis by branched-chain amino acids in vivo. Biochem. Biophys. Res.Commun. 313:417–422.

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Chapter 14 Milk Whey Processes: Current and Future Trends Charles I. Onwulata

Separation Technologies Once upon a time, whey was a nuisance, but with time, science and technology, what used to be a problem has been turned into a gold mine. With the growth whey has experienced in the last 25 years, and given the steadily increasing worldwide demand, more growth and utilization of whey proteins are expected. Whey processing and application today are yielding a wealth of quality products that are increasingly seen as ingredients in formulations that have recognized positive health benefits. The health benefits are expanding as more studies are reported in scientific journals. This chapter looks mostly at cutting-edge processes, discusses potential applications, and makes projections on emerging technologies and processing techniques. Major advances made in processing whey into various components using membrane technologies such as ultrafiltration and ion exchange have produced purer protein fractions with defined specific functionality. Particular whey fractions, β-lactoglobulins (β-Lg) or α-lactalbumins (α-La), and enriched fractions containing lactoferrins or glycomacropeptides make possible the many reported food health functionalities and broadens applications for whey proteins (Johnson and Lucey, 2006). New processing techniques are displacing older methods, leading to products with better nutrient and application profiles. For example, using the right pore sizes in microfilters, processors can produce separate protein fractions from skim milk, bypassing the cheese-making step. It is possible to produce

369 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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two streams: one enriched in casein micelles and the other in serum proteins avoiding rennet byproducts of the cheese-making process (Johnson and Lucey, 2006). In economic terms, given the price range of newly enriched fractions possible up to $20.00/lb for 98% protein isolates, compared to the early 2007 bulk cheese price of $1.35, many of the fractions from milk whey are more profitable, making cheeses the byproduct of milk processing. Whey fractionation and modifications have been covered extensively in other chapters in this book. Also, new techniques for protein modification and product application have been presented. But looking into the future on the basis of the current technological trends one sees processing for the sake of improving functional quality attributes, purifying fractions, and improving functionality for use of whey proteins in nontraditional places, as the future. Many sophisticated technologies for purifying whey has resulted in an array of commercial products including whey protein concentrates (WPC) and whey protein isolates of varying protein contents, allowing for increased purity within the two classes. The most recent advancement has come from various membrane filtration techniques, which further fractionate whey into individual components. The individual components include beta-lactoglobulins (β-Lg), alpha Lactalbumins (α-La), immunoglobulins (Ig), lactoferrins (LF), lactoperoxides (LP), and glycomacropeptides (GMP). Other techniques for more purified fractions may combine both a physical process such as filtration and an enzymatic pretreatment such as hydrolysis and/or ion exchange chromatography (Korhonen, 2002). Membrane Separation Membrane separation is used widely in milk and whey processing to separate whole milk, concentrate whey proteins after cheese making, and then, to fractionate the whey proteins into specific components. Membrane technology has many benefits and is particularly beneficial to dairy processing because it operates at near-room temperatures and does not damage the proteins or degrade its nutrients. Membrane processing of dairy products has grown in sophistication over the years from its earliest use simply to concentrate and partially separate proteins to now where it can be used to isolate, extract, and concentrate any single health-benefiting component. Progress in membrane processing has been driven by the improvement in flow of materials across nanometersized very fine membrane barriers. Product qualities have improved and

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the number of applications as well. The membrane filtration process has advanced from particulate filtration (PF), microfiltration (MF), ultrafiltration (UF), reverse osmosis (RO), to nanofiltration (NF) (Kelly, 2003). Although the operating principles of membrane processing by size exclusion are generally similar, MF and UF act as passive sieves, while RO and NF are active sieves using more than size exclusion for particle separation (Cheryan, 1998). In concentrating and separating processes, colloidal fluids are mechanically forced through a separating membrane where the particles are separated by size and/or shape. The separating membrane is usually a semipermeable barrier that separates the colloidal fluid with particulates into two streams: permeate, water, and the retentate, containing more of the particulates (Goff, 1995). Permeate of fluid milk separated by ultrafiltration contains water and lactose, and the retentate will contain water, fat, protein, lactose, and minerals. If whey is ultrafiltered, the proteins are concentrated in the retentate while lactose, minerals, and vitamins are in the permeate. As seen previously in the chapter on whey production and utilization, whey products are made with a combination of any of the five main processes with the following different end (retentate) product targets: UF-whey proteins, MF-casein, NF-lactose, and RO-minerals. Whey protein concentrates are mainly produced by UF and MF for commercial applications; whey protein isolates are produced by adsorption onto ion-exchange beads, followed by washing, elution of the adsorbed protein, cleaning, and regeneration of the beads. Recent processes developed were aimed at concentrating selected whey proteins through ion-exchange chromatography for subunits such as lactoglobulins and lactoferrins. They include direct continuous, sequential separation of whey proteins by chromatography. First, whey proteins are adsorbed on a suitable separation medium packed in a chromatographic column, and sequentially, individual fractions are eluted: Ig, β-Lg, α-La, bovine serum albumin (BSA), and LF. Further improvement of this chromatography process produces “clear” whey protein isolates. The clear whey protein isolate contains β-Lg, α-La, and Ig, but no lactose (Mozaffar et al., 2000). High Hydrostatic Pressure High pressure processing of milk was reported in the literature over 100 years ago; high pressure (HP) was used to induce changes in the proteins of bovine milk (Hite, 1899). In the intervening years, not much

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was done in HP processing of milk. Lately, a number of articles have appeared describing various applications of HP technology to milk products including whey, but no HP-processed milk product is known to be on the market. High-pressure processing has been shown to have marginal influence on the nutritional characteristics of milk, hydrolysis, or stability of vitamins. Through continuing research, some understanding of the difficulties of the process and benefits of the application in maintaining product quality are emerging. It has been found that HP treatment of milk affects the proteins, casein micelles, and whey proteins. HP processing at pressures greater than 100 MPa denatures β-Lg and α-La, increasing their association within the serum phase milk fat globule membrane, making separation into various fractions difficult (Huppertz et al., 2005a). Pressures up to 300 MPa were shown to have reversible effects on β-Lg, and pressures up to 600 MPa did not produce Maillard browning. Denaturation of proteins resulting from HP processing has no lasting effect on digestibility (Messens et al., 2003). The effects of HP treatment in milk and whey was very much pressure dependent; however, denaturation of α-La and β-Lg increased with increasing pressure. HP-induced denaturation of α-La and β-Lg in dairy systems was described earlier. It was suggested that β-Lg was denatured more by HP α-La in milk, but less so in whey. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of α-La and β-Lg significantly. HP-denatured β-Lg was associated more with casein micelles; this association provided opportunities for changing the properties of products made from HP-treated milk (Huppertz et al., 2004). Similar findings were reported as the effects of high pressure on some properties of buffalo milk (Huppertz et al., 2005b). It was shown that the casein micelle size decreased below 100 MPa and increased in pressures ranging from 600 to 800 MPa. Some other milk property changes were increasing calcium precipitation with increasing pressure up to 600 MPa, increased lightness value. Pulsed Electric Field Pulsed electric field (PEF), an emerging nonthermal process mostly applied to acid foods such as fruit juices to kill microorganisms, has also been used to process milk. In PEF processing, pulsed electric fields of alternating currents ranging from 10 to 100 kV are applied in less than 20 μs, creating high voltage fields up to 50 kV/cm. The high voltage field

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ranging from 35 to 50 kV/cm created within microseconds in the process chamber kills microorganisms and ruptures spores in the medium. If treatment is repeated in a multiple sequence the kill efficiency increases resulting in 5–9log reduction (Gaudreau et al., 2005). PEF has the advantage over the conventional thermal process of retaining flavor, color, texture, and nutrients of the raw material because it requires minimal additional heating. Microorganisms are destroyed through electroporation, a process that ruptures their shell disintegrating the whole organism. PEF-treated milk does not have the cooked flavor of the thermally processed milk. Floury et al. (2006) have shown limited effects using continuous square wave pulses with an electric field width of 45–55 kV/cm/250 ns, processing of raw skim milk with PEF under nonthermal conditions (T < 50◦ C). Under the stated conditions only 1.4log reduction of total microflora was achieved. Raw milk treated with PEF at 40 kV/cm preserved its quality at 4◦ C for 2 weeks. In a separate study, microorganisms resistant to PEF processing were identified, including Xanthomas malthophililia and Corynebacterium spp. Odriozola-Serrano et al. (2006) have shown microbiological stability of whole milk to be only 5 days and no proteolysis and lipolysis were observed in 7 days. However, they show that PEF was similar to thermal processes in denaturing whey proteins. The effect of PEF on the milk was decreased viscosity and coagulation properties, with some dose-dependent effects. It was speculated that the proteins, casein, and whey, were affected and resulted in changes in coagulation properties (Floury et al., 2006). PEF whey could be available in the future if cheeses are made from PEF-treated milk. In a recent study, Yu and Ngadi (2006), reported that cheddar-type cheese curds made from PEF-treated raw milk have similar proteolytic profiles to cheeses made from thermally pasteurized milk. The flavor profiles, measured by HPLC, showed PEF-treated profiles to be similar to raw milk. The conclusion was that PEF-treated milk would have the same flavor as raw milk. It is speculated that the whey proteins from such a process would be less denatured, and might provide better immunological benefits. Ultrasound Ultrasound processing, sonication, has been applied to milk products for varying applications. Early attempts to use low-frequency ultrasound

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for processing raw milk was done at the USDA, ARS Eastern Regional Research Laboratory, Eastern Utilization Research and Development Division, at Wyndmoor, Pennsylvania, in the mid-1960s (Huhtanen, 1968). Ultrasound was used to break bacterial “clump” which was masking total bacterial count in milk. For this particular problem, sonication at elevated temperatures improved the desired isolation of bacteria in raw milk (Huhtanen, 1966). The treatment of milk with lowfrequency sonication increased the total bacterial counts, but the heat produced by ultrasonic treatment did not account entirely for its effect. The ultrasonic effect was related to the energy output of the generator and to the energy absorbed by the treated materials. There was synergistic effect between heat and ultrasound in improving the bacterial count. A continuous-flow ultrasonic treatment could be a promising technique for milk processing provided there is an advantage for its use over proven technologies, an example might be using less energy to homogenize milk. The properties of cheese made with sonicated milk and the subsequent whey product depend on the effects of sonication on the proteins. For example, sonication of milk had the effect of homogenizing whole milk. Particle sizes of sonicated fat globules were similar to traditional pasteurizer when processed at 180 W for 10 min, while at 450 W for 10 min even much smaller particle sizes were obtained at the higher power (Ertugay et al., 2003). High-amplitude ultrasound was found to be very effective for inactivation of lactoperoxidase and alkaline phosphatase enzymes in milk; through longer exposure times were needed. Sonication and rising temperature had a synergist effect on enzyme inactivation (Ertugay et al., 2002). Recent research on sonication of milk has shown inactivation of bacteria in milk by continuous flow ultrasonic treatment, but the increase in temperature resulting from the sonication process reduced process effectiveness (Villamiel and de Jong, 2000a). Villamiel and de Jong (2000b) reported no effects on enzyme activity when ultrasound was applied without heat generation; the highest denaturation of enzymes and whey proteins was found in sonicated samples showing temperature increases. The effect of ultrasound processing on protein is synergistic in the presence of heat, increasing the denaturation of whey proteins α-La and β-Lg, but caseins are not affected because the highest temperatures reported were below 76◦ C. Increased homogenization efficiency

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was evidenced by a substantial reduction in fat globule size and a better particle distribution. The continuous flow high-intensity sonication process shows an important first step for future processing of milk components for food applications using ultrasound (Villamiel and de Jong, 2000a). In a dairy product application, yogurt made with ultrasound processed milk showed better fat distribution and smaller sizes as a result of improved homogenization (Wu et al., 2000). It was found that longer exposure times improved the ultrasonic homogenization effect and that sonication after inoculation improved fermentation efficiency, increasing sonication amplitude before inoculation improved water holding capacity and viscosity, and reduced syneresis. Thus, early application of ultrasound processing of milk to manufacture yogurt demonstrates the efficacy of this process. Ultrasonication can be combined with moderately elevated temperature to produce milk products with new properties. Microwave Microwave processing for pasteurized milk products was researched earlier using available microwave units, but was plagued by inconsistent heating. Recent advancement in tubular design along with focused microwave heaters makes possible milk pasteurization using microwave technology (Sierra et al., 1999). The interest is in developing a continuous microwave process that is comparable to the current hightemperature short-time (HTST) processes used for pasteurizing milk. The idea that a continuous microwave process can be used effectively to pasteurize milk has been demonstrated, but with varying results. Villamiel et al. (1996) treated raw cow and goat milks using a continuous flow microwave unit operated at temperatures ranging from about 70 to 100◦ C. The effect of microwave heat treatment on milk was low degree of whey protein denaturation, indicating that whey proteins from microwave pasteurization will retain similar properties as the HTST products. Sierra et al. (1999) showed that continuous flow microwave treatment of milk did not affect vitamin B1 , whereas in conventional HTST treatment carried out with the plate heat exchanger there was loss of vitamin B1. Continuous flow microwave treatment of milk can be compared favorably with conventional heating because more of the vitamin

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B1 was retained in the in-line holding process. This positive beneficial effect was attributed to shorter process time with microwave. In subsequent study, Sierra and Vidal-Valverde (2000) showed that milk heated in a continuous microwave system to 90◦ C without a holding phase, retained all vitamins B1 and B2 . But, when a holding time of 30–60 s was added, there was a small (<5%) loss of only vitamin B1 . Comparing a continuous microwave treatment of skim milk with an conventional ultrahigh temperature (UHT) process, similar effects on the proteins as measured by the initial thiol content are seen (Clare et al., 2005). Current UHT methods cause cooked-flavor and nutrient degradation through long exposure to heat. Microwave processing seems better than HTST in retaining flavor. Microwave processing affected other attributes of milk quality viscosity and color, minimally (Clare et al., 2005). Simunovic et al. (2005) reported that the flavor quality of microwave-treated products was superior to that of products made using conventional processes. These studies show that microwave-processed fluid milk or cheese whey may provide a functionally superior whey product with better attributes. The attributes such as improved flavor may enhance the quality of whey powders that have sometimes been characterized as having strong “stale” or astringent flavors (GallardoEscamilla et al., 2005).

Protein Modifying Processes Thermal processing is the current method of modifying whey proteins to create different food functionalities, and as a result, different products. Thermal denaturation of the two major protein fractions in whey protein isolates, β-Lg (50%) and α-La (22%), takes place mostly between 50 and 75◦ C and is accompanied by unfolding and unmasking of the SH groups (Walstra et al., 1999). When whey proteins are denatured, they become insoluble and aggregate, but are ultimately degraded by prolonged heating above 140◦ C. There are a number of other nonthermal processes that are used to modify the structure or texture of whey proteins, affecting their functionality by attenuating or amplifying particular effects, for example, enzymatic hydrolysis is used to modify emulsion properties; extrusion is used to impart texture, and other physical processes such as carbon dioxide precipitation can be used to enhance solubility.

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Modifications can be achieved either by chemical and enzymatic means, or by physical processing (Kilara and Panyam, 2003). Whey proteins are modified to enhance their functionality in foods, such as to improve gelation properties, thermal stability, foaming, or emulsification; the result is that whey protein ingredients can be tailored for different applications in a range of foods (Schmidt et al., 1984). Modifications of whey proteins based on enzymatic hydrolysis or heat-induced polymerization such as through extrusion processing greatly increase the number of products in which whey proteins can be used. Extrusion processing of whey proteins is modification by heat and shear, which produces a heat and acid stable physically cross-linked product with extended functional applications in snacks, meats, and sports drinks (Faryabi et al., 2008; Onwulata and Tomasula, 2004; Walsh and Carpenter, 2008). Ordinarily, the unmodified spray-dried powdery whey powders cannot be used for these applications because they are not suitable when used in amounts greater than 5% in products such as cakes or breads. Extrusion-modified whey proteins are essentially denatured by the two-step process of unfolding and aggregation, and are stabilized by hydrophobic interactions and disulfide bonds, as described by Schmidt et al. (1984). The new state, the meta-stable unfolded molecular conformation, has been described by Qi et al. (2001), as a molten globular state, an intermediate protein folding state characterized by native-like secondary structures with limited fluctuating tertiary structures (Farrell et al., 2002). Extrusion processing optimizes the conditions for whey protein denaturation and development of enhanced specific functional attributes such as sugar-tolerance in sugar-rich jelly-type products (Faryabi et al., 2008). Modified whey may offer health-benefiting functionality. Kilara and Panyam, (2003) showed that peptides derived from milk protein exert digestive and metabolic effects and influence the immune system. These biological effects may play an important role in the development of medical foods that treat or mitigate the effects of diseases. Proteins are allergens to susceptible people and therefore it is possible that products derived from modification of proteins may also be allergens. Some of the peptides in milk proteins are capable of affecting biological functions of an organism. These effects can be antimicrobial or probiotic in nature, that is, they can prevent the growth and proliferation of undesirable and pathogenic organisms, or promote the growth of desirable bacteria in the digestive tract of humans and animals.

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Enzymatic Modification The physical states and chemical properties of food proteins such as their hydrophobicity and molecular structure influence their functions as proteins. Enzymatic hydrolysis alters this state. In the process there may be improvement in nutritional quality, functionality and protein properties such as solubility, emulsification, foaming, and gelation, but the product may become bitter (Kilara and Panyam, 2003). Whey protein can be modified by controlled enzymatic proteolysis to generate whey protein hydrolyzates with low molecular weight peptides. Nieuwenhuizen et al. (2004), showed that the use of transglutaminase from Streptoverticillium mobaraense (MTGase) alters the physical properties of β-Lg and improved texture. The modified β-Lg lowered the interfacial tension in an emulsion, and improved its foaming property. The use of alcalase for enzymatic hydrolysis of whey protein isolates enhanced gelation via hydrophobic interactions. Over 120 small molecular mass peptide aggregates less than 2,000 Da were present. Hydrophobic interactions within the aggregates were enhanced improving gel formation in enzyme-treated whey protein isolates (Doucet et al., 2003). Enzymatic hydrolysis modifies whey proteins enhancing their functions and increases their values. Some of the biological applications for whey protein hydrolysates include producing bioactive peptide; altering functionalities such as gelation, foaming, or emulsification by allowing specific size mass and ration of peptides; and reducing allergenicity (Doucet et al., 2003). This positive functionality attribute is achieved through enzyme selection that dictates the type, size, and aggregation of peptides produced. Enzymes with known specificity have been used to hydrolyze whey proteins for antigenic responses; for example, trypsin, neutrase, and corolase were used to hydrolyze WPC containing 80% protein (Svenning et al., 2000). β-Lg was fractionated into peptides of high- and low-molecular-weight fractions possessing residual antibody binding activity depending on the degree of hydrolysis, type of enzyme used, and heat treatment regimes (Svenning et al., 2000). Taste of processed whey, including enzymatically modified whey, is sometimes perceived as bitter or astringent. The origin of the bitterness is assumed to be bitter peptides (Kilara and Panyam, 2003; Sano et al., 2005). It is assumed that acidic conditions (pH < 4.0) produces astringent taste sensation in whey protein isolates and modified whey protein.

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A threshold value of astringency of whey protein isolates and modified whey protein isolates ranged from 0.7 to 1.5 mg/mL. Astringency was shown to increase with protein concentration under acidic conditions, the causes being aggregation and precipitation of protein molecules in the mouth (Sano et al., 2005). Astringency is reported common in whey protein-containing drinks made at pH 3.0 or less. It is assumed that astringency is caused by polyphenols forming complexes with prolinerich proteins from saliva, producing large aggregates. Another view is that astringency is related to the pH, and the cause is a complex formed between positively charged whey proteins and proline-rich proteins from saliva (Foegeding, 2005). Chemical Modification For different purposes other than food use, many chemical agents can be used to modify proteins (Means and Feeney, 1990), but for food applications only compounds Generally Regarded As Safe (GRAS) are used. Milk is chemically modified by rennet-enzyme action or by direct acidification to coagulate and precipitate casein and fat complex which is pressed to form cheese and the serum, whey. Chemical modification of milk proteins involves significant changes in primary structures mainly through reactions involving the sulfide residues (–S–S–) of the amino acids. Specific reactions involving hydrophobic interactions, sulfhydryl linkages, and disulfide bonds are the means used to create many beneficial linkages that result in the varied functional properties which are the basis for milk-based products including whey (Walstra et al., 1999). For example, chemical modification mediated by disulfide bond formation results in aggregated proteins forming gels. In cold-set gels of whey proteins at ambient temperatures, the net electric charge of the aggregates and low pH induces intramolecular aggregation. Change in surface charge induced using succinic acid is termed succinylation. Succinylation of β-Lg starts this process as primary amines are crosslinked resulting in lowering of pH, but no disulfide bonds are formed between aggregates (Alting et al., 2002). Another chemical process that precipitates soluble whey fraction by lowering pH is sulfitolysis (Kananen et al., 2000). In sulfitolysis, the disulfide bonds are split using sodium sulfites, producing a sulfhydryl and sulfonated cysteine derivative Thiols (Bailey and Cole 1959). Changes in disulfide bonds increase protein flexibility and consequently the functional properties (Taylor

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et al., 2006). In one application, sulfonation of whey protein concentrate with sodium sulfites increased unfolding of the proteins, enhancing protein–starch interactions during the extrusion cooking process, although the functional effect was lost in expandability of the puffed product (Taylor et al., 2006). Functional properties of whey proteinderived products such as emulsification, digestibility, interfacial, and foaming properties are improved by sulfonation. Physical Modifications Extrusion Texturization Extrusion cooking is a short-time shear processing used to modify food structure, imparting texture. Physically modifying whey proteins by extrusion for an intended food application is relatively new. Ongoing research at USDA-ARS, Eastern Regional Research Center, and Utah State University has contributed to this knowledge as snacks and meat analog products with enhanced textural properties are produced and marketed. Extrusion processing is used to modify the physical texture, texturization, of whey proteins, expanding their potential use in snack foods and meat products. With texturized whey proteins (TWP), producers are formulating high-protein snacks with unique textures. For example, in beef patties application, TWP was used to replace meat up to 50% without noticeable differences in texture (Hale et al., 2002). In snack applications, expanded products containing up to 30% whey protein isolates were demonstrated (Onwulata et al., 1998). Texturization changes the folding of globular proteins improving interaction with other ingredients, the basis for creating new functional ingredients (Onwulata et al., 2006). Depending on the pH, the effect of texturization varies; for instance, alkaline conditions increases insolubility and pasting properties of whey protein isolates, while acidic conditions increase solubility. Subtle structural changes occur under acidic conditions but are more pronounced under alkaline conditions. In general, alkaline conditions increases denaturation of extruded whey protein isolates (WPI) resulting in stringy texturized meaty fibrous products, which could be used in meat applications (Hale et al., 2002; Onwulata et al., 2006). In the extruder, whey proteins can be modified using chemicals, heat, or shear. Chemical treatment alone alters the reactive groups of the amino acids, resulting in changes in the noncovalent forces that influence conformation, such as van der Waals forces, electrostatic

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interactions, hydrophobic interactions, and hydrogen bonding. Heating and shear alter the conformational structure of the whey protein through partial denaturation of the protein, thereby exposing groups that are normally concealed in the native protein. Combining the effects of chemical, heat, and shear results in the physical modification of whey proteins opens opportunity for wider use of dairy ingredients bringing along with it improved functionality and health benefits. Carbon Dioxide Precipitation Another physical means of modifying whey proteins is using supercritical CO2 precipitation. This process was developed for expanded use of enriched proteins in new food and nonfood applications (Tomasula et al., 1998). Whey proteins are fractionated into enriched (α-La) and β-Lg as described earlier (Tomasula and Yee, 2001). Tomasula et al. (1998) showed that maximal amounts of α-La were obtained from processing a 7% (w/w) whey protein concentrate solution at 64◦ C and at pressures ranging from 3 to 6 mPa. The CO2 precipitation lowered pH of whey protein concentrates to pH 4.5 below 5.5 mPa resulting in two protein-enriched fractions: 65% α-La and 80% β-Lg. So the ratios of the two main whey protein fractions can be selectively enriched, for different applications depending on their respective functionalities. Supercritical CO2 precipitation partially denatured α-La fraction, but not the β-Lg. The gelling properties of the β-Lg fractions were improved as the mild processing temperatures minimized heat damage to the proteins, enhancing solubility.

Functionality Physicochemical attributes that make proteins useful in foods are called functional properties, or functionality (Kilara and Vaghela, 2000). The physical functionality of proteins represents those properties that influence their usefulness in foods based on transient structural states. Functionality benefits derived from whey proteins are mostly from the properties of the amino acids, their constitution, composition, and conformation. The primary structure, the sequence of amino acids, and the different side chains produce these functional differences. The primary functional attributes are the water properties such as solubility, insolubility, water-holding capacity, swelling, and dispersibility which play a

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role in product thickness and viscosity. Protein solubility is influenced by temperature and pH, and as a fundamental property, affects other protein properties. Interactions of whey proteins with water may result in an increase in viscosity if the proteins are soluble or partially denatured. Other resultant physicochemical functions such as foaming or gelation are derived from the water properties (Cheftel et al., 1985). Changes in protein functionality parameters reflected in decreased solubility result from increased hydrophobicity upon modification by heating. Physical processes modify whey proteins faster at elevated temperatures. For example, in food applications, elevated temperatures ranging from 35 to 85◦ C affect the aggregation and denaturation of proteins, and in turn, affect functioning of whey proteins in foods. The heat-induced denaturation and aggregation of whey proteins are likely from disulfide-bonded strands formed by the aggregation of α-La and β-Lg, catalyzed by heat-induced unfolding of serum proteins, forming aggregates of different sizes. These aggregates are cross-linked by intermolecular disulfide bonds and by noncovalent interactions forming a β-Lg disulfide-bonded network. Structural stability of functional whey proteins is maintained by the unfolding of α-La and β-Lg over the hydrophobic residues and by the presence of thiol and disulfide groups. Heat processes result in the exchange of disulfide bridges, which produces the changes in functionality, beginning with solubility, then others like foaming. Thermal denaturation unmasks the sulfhydryl groups during unfolding of the protein molecule, and depending on the pH, the mode of intermolecular or intramolecular reassociation produces different functionality (Linden and Lorient, 1999). Foaming ability follows solubility and is also temperature dependent. Temperatures greater than 50◦ C favored foaming, while 4◦ C or below decreased foaming. This temperature dependence of foaming is attributable to the effect of heat on β-Lg (Kilara and Vaghela, 2000). Processing induces changes in the quantitative structure-functionality attributes that contribute to the functionality of proteins. Physical/Structural Functionality The functional and nutritional properties of whey proteins are related to their conformational structures and their biological and health benefits are derived from these. New process techniques that preserve the native structures improve biological functionality. For example, bioactive

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peptides produced by partial enzymatic hydrolysis or through selective precipitation and ion exchange chromatography, function to improve gut health (de Wit, 1997). Other peptides function as health promoting ingredients in foods; for example, anti-hypertensive peptides were derived from whey protein hydrosylates (Korhonen, 2002). The functional bioactivity of peptides or highly purified whey fractions comes from using functionality-specific processes under optimal conditions that preserve the native biological structures. These processes maintain maximum bioactivity during processing and through food formulation, handling, consumption, and delivery to targeted sites in the body, where their health benefiting function is needed (Korhonen, 2002). Functionality at the targeted sites is still difficult to assess as the tools needed to quantify changes in structures in formulated foods and in the body are rudimentary. Human studies needed to confirm delivery of targeted whey components and their efficacy upon consumption are just beginning. Biological and Nutritional Functionality The primary composition and sequence of amino acids in protein determine its nutritional functionality; its highly ordered secondary and tertiary structure produces the biological functions. The sequences of protein side chains, its three-dimensional folded structure, determine biological functionality (Fox and McSweeney, 2003). Whey proteins have a high-quality score due to the balance of its 162 amino acids, and a relatively high ratio of leucine and other branched chain amino acids (isoleucine, valine), that play distinct roles in human protein metabolism, particularly in muscle protein synthesis. Amino acid composition of whey protein is proportionally similar to that of skeletal muscle. The physiological advantages of whey protein include rapid absorption rates, initiation of synthesis by leucine, and abundance of other proteins as substrates for protein synthesis (Ha and Zemel, 2003). Other components of whey protein, the sulfur-containing amino acids cysteine and methionine, provide immune enhancing functions through intracellular conversion to glutathione. Biological active components of whey β-Lg, α-La, lactoferrins, glycomacropeptides, and immunoglobulins function as antioxidants, antihypertensive, antitumor, hypolidemic, antiviral, antibacterial, and chelating agents. β-Lg has been shown to suppress human lymphocyte

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proliferation. It functions as a carrier of small hydrophobic molecules, retinoic acid, and modulates lymphatic response (Marshall, 2004). Whey proteins have been shown to decrease the accumulation of body fat, accelerating weight and fat loss, and increasing feeling of satiety. The mechanism by which whey protein accomplishes its adiposity function is mediated by calcium (Ha and Zemel, 2003). Some other biological functions are antioxidant properties, and promoting growth of indigenous microflora; these are accomplished through the action of lactoferrins. Lactoferrins are polypeptides with two binding sites for ferric ions. The role of lactoferrins is to sequester iron, interact with microbial cell wall components, and cellular receptors through its highly positively charged N-terminus (Nuijens et al., 2005). Immunoglobulins modulate immune functions, and act as antibacterial agents. Lactoperoxidases (LP) inhibit bacterial growth by catalyzing thiocynates and other halides, and by reduction of hydrogen peroxides. GMP and bovine serum albumin (BSA) are available amino acids (Ha and Zemel, 2003). Health Benefits of Modified Whey Proteins The health benefits of whey are being documented through a series of studies including in vitro animal and human, which show use of whey proteins for clinical indications such as cancer, hepatitis, human immunodeficiency virus (HIV) disease, cardiovascular disease (CVD), and obesity. Other health benefits include antitumor and antiarcinogenic responses due to glutathione modulation which increases the relevant amount of glutathione in tissues, stimulating immunity and detoxifying potential carcinogens. It is thought also that the iron binding capacity of lactoferrins reduces oxidative damage caused by unbound iron in tissues (Marshall, 2004). Harper (2003) collated reported nutritional benefits of whey proteins and their roles in promoting health. Reports of the health benefits of whey proteins include enhanced antioxidative protection through increasing glutathione activity in cells and plasma. Enhanced antioxidative effects have been reported for whole whey protein products, which were found to protect against cancer, HIV infections, stroke, and Alzheimer’s disease (Harper, 2003). Cross and Gill (1999) showed that enzymatically modified whey protein concentrate modulated immune function in vitro by suppressing T and B lymphocyte growth responses

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to mitogens in a dose-dependent fashion. Enzymatically modified whey suppressed cytokine secretion and the formation of activated (CD25+) T cell blasts, inhibiting the lymphocyte activation process. Individual components of whey proteins such as Ig, LF, LP, and the GMP enhance protection against cellular oxidative stresses benefiting cardiovascular systems, antiinflammatory agents. These benefits are mediated by sulfur amino acids (cysteine), which enhance glutathione in the body and protects against oxidative stress (Ha and Zemel, 2003). Crystallizing the benefits of whey are new discoveries about its usefulness in enhancing antibody, lymphocytes, macrophages, humeral immune responses, and increasing antiviral activity, which resulted in an increase in the plasma glutathione in patients with advanced HIV infections. In others, whey protein improved muscle strength. Also, whey minerals including calcium have been reported as useful in managing obesity and weight control. More on the health benefits of whey is covered in Chapter 12. Future Applications Whey proteins, modified whey proteins, and whey components will be useful as nutritional or other supplements, but more important in health maintenance and healing. It is contemplated that new processes for purifying and modifying whey products will be developed, increasing the possible number of products that can be made. Whey proteins can easily fit into new products such as beverages, confectionery items (e.g., candies), convenience foods, desserts, baked goods, sauces, infant food and formulae, geriatric foods, animal feeds, and as drug constituents, and plastics. References Alting, A.C., De Jong, H.H.J., Visschers, R.W., and Simons, J.F.A. 2002. Physical and chemical interactions in cold gelation of food proteins. J. Agric. Food. Chem. 50:4682–4689. Bailey, J.L., and Cole, R.D. 1959. Studies on the reaction of sulfite with proteins. J. Biol. Chem. 234:1733–1739. Cheftel, J.C., Cuq, J.-L., and Lorient, D. 1985. Amino acids, peptides, and proteins. In Food Chemistry, 2nd edition, edited by O.R. Fennema, pp. 245–369. New York: Marcel Dekker.

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Cheryan, M. 1998. Performance and engineering models. In Ultrafiltration and Microfiltration Handbook. Lancaster, PA: Technomic Publishing. Clare, D.A., Bang, W.S., Cartwright, G., Drake, M.A., Coronnel, P., and Simunovic, J. 2005. Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage. J. Dairy Sci. 88(12):4172–4182. Cross, M.L., Gill, H.S. 1999. Modulation of immune function by a modified bovine whey protein concentrate. Immunol. Cell Biol. 77(4):345–350. de Wit, J.N. 1997. Nutritional and functional characteristics of whey proteins in food products. J. Dairy Sci. 81:597–608. Doucet, D., Otter, D.E., Gauthier, S.F., and Foegeding, E.A. 2003. Enzyme-induced gelation of extensively hydrolyzed whey proteins by alcalase: Peptide identification and determination of enzyme specificity. J. Agric. Food Chem. 51(21):6300– 6308. Ertugay, M.F., Sengul, M., and Sengul, M. 2002. Effect of ultrasound treatment on milk homogenization and particle size distribution of fat. Turk. J. Vet. Anim. Sci. 28(2004):303–308. Ertugay, M.F., Yuksel, Y., and Sengul, M. 2003. The effect of ultrasound on lactoperoxidase and alkaline phosphatase enzymes from milk. Milchwissenchaft 58(11– 12):593–595. Farrell, H.M., Jr., Qi, P.X., Brown, E.M., Cooke, P.H., Tunick, M.H., Wickham, E.D., and Unruh, J.J. 2002. Molten globule structures in milk proteins: Implications for potential new structure-function relationships. J. Dairy Sci. 85:459–471. Faryabi, B., Mohr, S., Onwulata, C., and Mulvaney, S. 2008. Functional foods containing whey proteins. In Whey Processing: Functionality and Health Benefits. Ames, IA: Blackwell Publishing. Floury, J., Grosset, N., Leconte, N., Pasco, M., Madec, M., and Jeantet, R. 2006. Continuous raw skim milk processing by pulsed electric field at non-lethal temperature: Effect on microbial sinactivation and functional properties. Le Lait 86:43–57. Foegeding, E.A. 2005. Astringency of whey proteins. 2005 Institute of Food Technologists Annual Meeting, July 15–20, New Orleans, LA. Fox, P.F., and McSweeney, P. 2003. Advanced Dairy Chemistry. Vol. 1: Proteins, Part A, 3rd ed. New York: Plenum Press. Gallardo-Escamilla, F.J., Kelly, A.K., and Delahunty, C.M. 2005. Sensory characteristics and related volatile flavor compound profiles of different types of whey. J. Dairy Sci. 88:2689–2699. Gaudreau, M., Hawkey, T., Petry, J., and Kempkes, M. 2005. Solid-state power systems for pulsed electric field (PEF) processing. IEEE Pulsed Power Conference, June 13–17, 2005, Monterey, CA. Goff, H.D. 1995. Dairy science and technology education series. http://www.foodsci. uoguelph.ca/dairyedu/home.html. Accessed February 14, 2008. Ha, E., and Zemel, M.B. 2003. Functional properties of whey, whey components, and essential amino acids: Mechanisms underlying health benefits for active people (Review). J. Nutr. Biochem. 14:251–258.

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Hale, A.B., Carpenter, C.E., and Walsh, M.K. 2002. Instrumental and consumer evaluation of beef patties extended with extrusion-texturized whey proteins. J. Food Sci. 67(3):1267–1270. Harper, J. 2003. Biological properties of whey components. http://www.fst.ohiostate.edu/People/HARPER/Functional-foods/Biological%20Functions%20of%20 Dairy%20Foods.htm. Accessed February 14, 2008. Hite, B.H. 1899. The effect pressure in the preservation of milk. Bull. West Virginia Univ. Agric. Exp. Stn. 58:15. Huppertz T., Fox, P.F., de Kruif, K.G., and Kelly, A.L. 2005a. High pressure-induced changes in bovine milk proteins: a review.Biochim. Biophys. Acta 1764(3):593–598. Huppertz, T., Fox, P.F., and Kelly, A.L. 2004 High pressure-induced denaturation of alpha-lactalbumin and beta-lactoglobulin in bovine milk and whey: A possible mechanism. J. Dairy Res. 71(4):489–495. Huppertz, T., Zobrist, M.R., Uniacke, T., Upadhyay, V., Fox, P.F., and Kelly, A.L. 2005b. Effects of high pressure on some constituents and properties of buffalo milk. J. Dairy Res. 72(2):226–233. Huhtanen, C.N. 1966. Effect of ultrasound on disaggregation of milk bacteria. J. Dairy Sci. 49:1008–1010. Huhtanen, C.N. 1968. Effect of low-frequency ultrasound and elevated temperatures on isolation of bacteria from raw milk. Appl. Microbiol. 16(3):470–475. Johnson, M.E., and Lucey, J.A. 2006. Major technological advances and trends in cheese. J. Dairy Sci. 89:1174–1178. Kananen, A., Savolainen, J., Makinen, J., Perttila, U., Myllykoski, L., and PihlantoLeppala, A. 2000. Influence of chemical modification of whey protein conformation on hydrolysis with pepsin and trypsin. Int. Dairy J. 10(10):691–697. Korhonen, H. 2002. Technology options for new nutritional concepts. Int. J. Dairy Technol. 55(2):79–88. Kelly, P.M. 2003. Membrane separation. In: Encyclopedia of Dairy Sciences, edited by H. Roginski., J.W. Fuquay, and P.F. Fox, Vol. 3, pp. 1777–1786. New York: Academic Press. Kilara, A., and Panyam, D. 2003. Peptides from milk proteins and their properties. Crit. Rev. Food Sci. Nutr. 43(6):607–633. Kilara, A., and Vaghela, M.N. 2000. Whey Proteins. In: Proteins in Food Processing, edited by R.Y. Yada, pp 72–99. Boca Raton, FL: CRC Press. Linden, G., and Lorient, D. 1999. The exploitation of by-products. In New Ingredients in Food Processing. edited by G. Linden, and D. Lorient, pp. 184–210. United Kingdom: Woodhead Publishing. Marshall, K. 2004. Therapeutic applications of whey protein. Altern. Med. Rev. 9(2):136–156. Means, G.E., and Feeney, R.E. 1990. Chemical modifications of proteins: History and applications. Bioconjug. Chem. 1:2–12. Messens, W., Van Camp, J., and Dewettnick, K. 2003. High-pressure processing to improve dairy product quality. In Dairy Processing–Improving Quality, edited by G. Smit. Boca Raton, FL: CRC Press.

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Mozaffar, Z., Ahmed, S.H., Saxena, V., and Miranda, Q.R. 2000. Sequential separation of whey. U.S. Patent 5,756,680. Nieuwenhuizen, W.F., Dekker, H.L., Groneveld, T., De Koster, C.G., and De Jong, G.A.H. 2004. Transglutaminase-mediated modification of glutamine and lysine residues in native bovine β-lactoglobulin. Biotechnol. Bioeng. 85(3):248–258. Nuijens, J.H. van Berkel, P.H.C., and Schanbacher, F.L. 2005. Structure and biological actions of lactoferrins. J. Mammary Gland Biol. Neoplasia. 1(3):285–295. Odriozola-Serrano, I., Bendicho-Porta, S., and Martin-Belloso, O. 2006. Comparative study on shelf life of whole milk processed by high-intensity pulsed electric field or heat treatment. J. Dairy Sci. 89:905–911. Onwulata, C.I., Isobe, S., Tomasula, P.M., and Cooke, P.H. 2006. Properties of whey protein isolates extruded under acidic and alkaline conditions. J. Dairy Sci. 89:71– 81. Onwulata, C.I., Konstance, R.P., Smith, P.W., and Holsinger, V.H. 1998. Physical properties of extruded products as affected by cheese whey. J. Food Sci. 63(5):814–818. Onwulata, C.I. Tomasula, P.M. 2004. Whey texturization: A way forward. Food Technol. 58(7):50–54. Qi, P.X., Brown, E.M., and Farrell, H.M., Jr. 2001. “New views” on structure-function relationships in milk proteins. Trends Food Sci. Technol. 12:339–346. Sano, H., Egashira, T., Kinekawa, Y., and Kitabatake, N. 2005. Astringency of bovine milk whey protein. J. Dairy Sci. 88:2312–2317. Schmidt, R.H., Packard, V.S., and Morris, H.A. 1984. Effect of processing on whey protein functionality. J. Dairy Sci. 67:2723–2733. Sierra, I., Vidal-Valverde, C., and Olano, A. 1999. The effects of continuous flow microwave treatment and conventional heating on the nutritional value of milk as shown by influence on vitamin B1 retention. Eur. Food Res. Technol. 209(5):352– 354. Sierra, I., and Vidal-Valverde, C. 2000. Influence of heating conditions in continuousflow microwave or tubular heat exchange systems on the vitamin B1 and B2 content of milk. Lait 80(2000):601–608. Simunovic, J., Coronel, P., and Clare, D. 2005. Application of microwave processing to extend shelf life of fluid milk. J. Dairy Sci. 88 (suppl. 1):78. Svenning, C., Brynhildsvold, J., Molland, T., Langsrud, T., and Elisabeth Vegarud, G. 2000. Antigenic response of whey proteins and genetic variants of β-lactoglobulin— the effect of proteolysis and processing. Int. Dairy J. 10(10):699–711. Taylor, D.P., Carpenter, C.E., and Walsh, M.K. 2006. Influence of sulfonation on the properties of expanded extrudates containing 32% whey protein. J. Food Sci. 71(2):E17–E24. Tomasula, P.M., Parris, N., Boswell, R.T., and Moten, R. 1998. Preparation of enriched fractions of α-lactalbumin and β-lactoglobulin from cheese whey using carbon dioxide. J. Food Proc. Preserv. 22:463–476. Tomasula, P.M. and Yee, W.C. 2001. Enriched fractions of alpha-lactalbumin (α -La) and beta lactoglobulin (β-Lg) from whey protein concentrate using carbon dioxide. Functional properties in aqueous solution. J. Food Process. Preserv. 25:267– 282.

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Appendix Acronyms

Alpha lactalbumin Angiotensin-converting enzyme Beta lactoglobulin Bovine serum albumin Branched-chain amino acids Capillary electrophoresis Capillary gradient electrophoresis Capillary zone electrophoresis Diafiltration Discontinuous diafiltration Electrodialysis Essential amino acids Glycomacropeptides Immunoglobulins Lactoferrin Leucine Microfiltration Molecular weight cutoff Nanofiltration Reverse osmosis Ultrafiltration

α-LA ACE β-LG BSA BCAA CE CGE CZE DF DDF ED EAA GMP Ig LF Leu MF MWCO NF RO UF

391 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Index

2005 International Whey Conference, 171 4-E binding protein (eIF4E-BP), 351 A α-LA and β-LG fractionation of, 22–5 α-lactalbumin (α-LA), 7, 140, 296–7, 371 effects on stress-vulnerable subjects, 326 enzymatic hydrolates of, 312 as a growth promoter, 323 inhibitory activity of HIV1, 321 monomer, 18 α-lactorphin, α-LA f(50–53), 289–90, 309 in SHR administration, 311 Accretion influence of protein types on, 356–63 ACE. See Angiotensin-converting enzyme Acid whey powder, 9 Acid whey, 1–2 Air drying rate, 137–8 All-trans-retinal, 30 American Society for Testing and Materials (ASTM), 146 Amino acids absorption rates, 352–3 Amorphous sucrose, 152 Analytical Food Laboratories Inc., 218 Angiotensin-converting enzyme (ACE), 172, 289 Antibacterial peptides, 313–21, 314t–317t Antimicrobial carriers, 160–3 Antioxidant carriers, 159–60

Appearance of pectin cranberry jellies, 223, 224f Aroma and oil permeability, 151 Aromatic amino acid residues, 301f B β-lactoglobulin (β-LG), 7, 140, 371 aggregation-role of thiol groups, 49–50 denaturation of, 47 combination with α-LA, 51 genetic variants, 51 denaturation/aggregation effect of heating on, 47–8 effect of ionic strength and ionic species, 48–9 effect of pH on heat-induced, 48 kinetic model, 50–51 health benefits/uses of, 56–7 health promoting bioactivities, 56 heat-induced gelation, 52 inhibitory activity of HIV1, 321 monomer, 18 nutritional benefits, 56 physico-chemical properties of, 46–54 polymerization of, 141 properties of, 16–17 rich fractions, 54 fractionation processes, 55–6 separation/fractionation of, 42–6 stabilized emulsions, 80 β-lactoglobulin (β-LG) gels, 52 β-lactoglobulin (β-LG) heating curves (upward curves), 105, 106f β-lactoglobulin (β-LG) variant A amino acid sequence of, 103f

393 Whey Processing, Functionality and Health Benefits Editors Charles I. Onwulata and Peter J. Huth © 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-80903-8

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Index

β-lactotensin, β-LG f(146–149), 309 antistress properties of, 326–7 hypocholesterolemic activity of, 324 β-lactorphin (β-LG) tryptic digestion of, 318 β-lactorphin β-LG f(102–105), 309 β-LG tryptic-chymotryptic hydrolysate, 305 B. bifidum, 323 B. breve, 323 B. infantis, 323 Bacillus subtilis, 320 Bacteriocin nisin, 162 Baicalein, 297 Baked cinnamon granola bars, 206t Bars, 204–6 BCAA. See Branched-chain amino acids Beeswax whey protein film, 149–50 Bifidobacteria, 319, 322, 323 Bioaffinity separation, 30 Bipolar membrane electroacidification, 31 BLG nanoparticles in vitro degradation of, 273–7, 274f, 275f preparation of, 269 Bodily iron homeostasis, 312 Bovine IgG, 307 Bovine lactoferricin LfcinB, 298–9 Bovine serum albumin (BSA), 8 Bovine whey protein, composition of, 102, 102f Bovine whey protein-derived peptides, 292t Bovine whey, 65 Bovine β-lactoglobulin (BLG), 267–8 Bovine α-lactalbumin made lethal to tumor cell (BAMLET), 296–7 Brain serotonin levels imbalance in, 325 Branched chain amino acid (BCAA), 173 Branched-chain amino acids (BCAA), 350–1 Breakfast cereals, 210–1, 211f Bridging flocculation, 93–4 Brunswick Laboratories, 218 BSA. See Bovine serum albumin Butylated hydroxyanisole (BHA) antioxidants, 159

Butylated hydroxytoluene (BHT) antioxidants, 159 Byproduct infusion syrup production of, 217–8, 217f C C. albicans, 320 Candida cells, 319–20 Carbon dioxide precipitation for whey protein modification, 383 Caseinomacropeptide. See Glycomacropeptide Carboxymethyl cellulose (CMC), 30–1 Casein effect on protein accretion, 351–2 twin-screw extrusion of, 178 Center for Science in the Public Interest, 159–60 Cheese making, 1 Cheese whey, 22–3 Chemical agents for whey protein modification, 381–2 Coacervation, 30–1 Coalescence, 71–2 Code of Federal Regulations (CFR), 187–8 Cold denaturation, 85, 181 Cold extrusion cooking, 181 Cold extrusion, 180–2 Cold gelation/cold-set gelation, 107 Color of pectin cranberry jellies, 223 Commercial products composed of whey proteins, 204 Complement-derived anaphylatoxin peptides, 307–8 Complexation, 29–30 Compression-depression analysis of pectin cranberry jellies, 223–4 Confectionery products, finishing of, 155–6 Constant rate period (CRP), 137 Cooking extrusion, 177–80 Cooling die, 188–90 Corn zein, 135 Counterions in hydrogels, 237 Covalent crosslinking, 191

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Index Cranberry infusion syrup, 215 Creaming, 69 Crisp products composed of whey proteins, 204 Critical flocculation concentration (CFC), 92–3 Cross-linking in hydrogels, 236–7 of whey proteins, 141 Cyclical swelling, 262–3 Cyclophosphamide for human cancer treatment, 306 Cysteines, 46 D Dairy protein and carbohydrate intake interaction, 355–6 Denatured whey proteins, 28–9 Depletion flocculation, 92–3 Diafiltration (DF), 5, 23 Dietary protein and resistance exercise interaction, 354–5 Dipping, 139 Dried fruit production of, 217, 217f Droplet aggregation, 70–3. See also Whey protein emulsifiers Droplet flocculation, 70–1 E EAA. See Essential amino acids ED. See Electrodialysis Edible coatings, 136 formation of, 138–9 Edible film drying, 137 Edible films solvent casting of, 137–8 Edible shellac, 155 Egg white, 55 Eggs, 154t shelf-life improvement, 155 Electrically charged biopolymers, 93–5 Electrodialysis, 4–5, 26 Electrophoresis, 26–7 Emulsion appearance, 76–8 Emulsion formation, 66–8 Emulsion premix

395

droplets of, 66–7 Emulsion rheology, 74–6 Emulsion stability, 68–74 Emulsion-based whey protein–lipid films, 149 Emulsions bulk physicochemical properties of, 74–8 Enzymatic hydrolysis for whey protein modification, 380–1 Enzyme-induced aggregation, 22 Escherichia coli 2348/69 (O127:H6), 313 Escherichia coli O157:H7, 321 Escherichia coli (E. coli), 320 Essential amino acids (EAA), 348, 351, 353 Excess emulsifier homogenization of, 83–4 External plasticizers, 142 Extruded sheets, 152–3 Extruded whey protein films properties of, 152–3 Extruded-expanded whey protein products, 190–1 Extruder, 143 Extrusion edible films, 138 physiochemical and configuration parameters, 189 texturization of, 382–83 whey protein films, 142–3 F Fabricated snacks, 171 Falling rate period (FRP), 137 Film stretching, 81–2 Film tearing, 82 Fine-stranded gels, 110 Flavor carriers, 159 Flocculated emulsions, 76 Flory’s polymer solution theory, 239 Fluidized-bed coating, 139 Fluoresence microscopy of pectin cranberry jellies, 226 Freeze-drying, 86 Fruits and vegetables freshness retaining packaging, 156–7

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Index

G

I

GAS. See Group A streptococci Gelation of milk interaction of β-LG and κ-casein, 52–4 Gels enzymatic crosslinking, 114–6 large strain properties of, 108–10 Globular proteins surface denaturation of, 79 Glutathione (GSH), 294, 322 Glycemic index, 172–3 Glycomacropeptide (GMP) 16–8, 31–2 GMP κ-casein f(106–169) peptide, 307 Gravitational separation, 69–70 Group A streptococci (GAS), 320–1 GSH. See Glutathione

Immunoglobulins (Igs), 32 Infrared drying rate, 138 Infusion fruit syrups, 217 Insufficient emulsifier homogenization, 83 mechanical stresses, 81 Insulinlike growth factor (IGF-1), 349 Interleukin-6 (IL-6), 306 Internal plasticizers, 142 Ion-capacity of hydrogels, 237 Ion-exchange chromatography, 27–8 β-LG-enriched fractions, 44–5 Ionic surfactants, 90 Ion-induced conformational changes, 48–9 Ion pairs in hydrogels, 238 Iron deficiency, 312–3

H Heated milk, 53 Heat-treated skim milk, 19 Helicobacter pylori, 320 High blood pressure. See Hypertension High hydrostatic pressure processing, 373–4 High protein cheese cracker, 209t Higher collision efficiency, 92 High-performance liquid chromatography (HPLC), 27 HIV. See Human immunodeficiency virus Homogenization, 65–6 Homogenizer, 66 Human immunodeficiency virus, 10, 322, 386 Human immunodeficiency virus type 1 (HIV1), 321 Human immunodeficiency virus type 2 (HIV2), 57 Human myeloid leukemic cells (HL-60), 299 Human α-lactalbumin made lethal to tumor cell (HAMLET), 296–7 Hydrogels, 230–1 controlled drug release from, 244–55 equilibrium swelling ratios, 258–62, 251f, 261f Hypertension, 288–93 Hypotensive peptides, 289–93

L L. monocytogenes, 321 Lactalbumin fraction, 7 Lactobacilli, 319, 322 Lactoperoxidase systems (LPOSs), 161–2 Lactoperoxidase, 8 Large deformation compression testing of pectin cranberry jellies, 223, 224f Lactoferrin (LF), 9, 32–3, 161, 295, 321 anticancer efficacy of, 297–8 anticandidal activity, 318 antimicrobial activity, 319 antitumoral effects on, 302t inhibitory activity against HIV 1, 321 peptic digest of, 306 stroma cell proliferation, 305 LF containing WPC effect on baicalein, 297 LF. See Lactoferrin Liquid whey processing, 2, 3f R Listerine pocket packs , 159 Long-tube multiple-effect evaporator, 3 Lower collision frequency, 92 Lysozyme, 161

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Index

397

M

O

Meat extenders, 188 Meat products protection of, 156 Membrane filtration, 4, 25–31 Membrane separation, 45–6, 372–3 Metastasis inhibition of, 301–3 Microwave drying, 138 Microwave processing of milk products, 377–8 Milk proteins, 362 effect on protein accretion, 351–2 ingestion of, 363 Milk whey processes, 371–87 Milk anticancer potential, 294 Minor whey proteins separation of, 31–3 Molecular weight cutoff (MWCO), 5–6 MPS. See Muscle protein synthesis Multiple-effect evaporator, 3 Multiple-layer formation, 94–5 Multivalent counter-ions, 87 Muscle protein anabolism influence of resistance exercise on, 353–6 Muscle protein metabolism, 348 Muscle protein synthesis (MPS), 348, 350, 352, 364 exercise-induced, 361–3

Oil-in-water (O/W) emulsions, 70 Opioid peptides, 308 derived from bovine whey proteins, 310t Optical fluorescence of pectin cranberry jellies, 221–3, 227f Osmotic drying, 217 Ostwald ripening, 73 Oxygen permeability, 146–8 Oxygen scavenger sachets, 159

N Nanoparticles, 266–77 formed through WPs, 229–30 Nonflocculated emulsions, 76 Nonionic surfactants, 90 Nonprotein components removal of, 22–3 Nutmeat, 153 Nutrition information high protein cheese cracker, 208–9 whey crisp bars, 205–6 whey protein salad topping, 212 whey protein snack cruncher, 210 Nuts and peanuts packaging of, 153–4

P Panning, 138–9 Partial coalescence, 72 Particle size of BLG nanoparticles, 269–73, 270f, 272f Particulate gel formation, 107 Pectin cranberry jelly, 218–21, 224–5 PEF. See Pulsed electric field Pepsin digested LF, 300t Peptic hydrolysis, 29 Peptides as a source of health, 287–88 Phase inversion, 73–4 pH-sensitive hydrogels drug release of, 250–55, 253t formation of, 231–35 swelling of, 235–44 Plasticizer efficiency, 144 Polyacrylamide gel electrophoresis (PAGE), 26 Potassium sorbate, 162 Potent ACE inhibitory enzymes, 290t–2t Preparation of BLG nanoparticles, 269 pectin cranberry jelly with TWP, 222t pectin cranberry jelly, 219–21, 220f, 221t Primary homogenization, 66 Procedure high protein cereal, 210, 210t high protein cheese cracker, 207–8, 209t whey crisp bars, 205 whey protein salad topping, 212 whey protein snack cruncher, 209–10, 209t

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398

Index

Protein chain cross-linking, 141 Protein conformational changes, 78–9 Protein gelation, 9 Protein sources absorption rates, 352–3 Protein texturization, 176–7 Protein–Ca2+ –protein, 48 Proteins impact on accretion, 356–61 impact on muscle protein synthesis, 356–61 Protein-stabilized oil-in-water emulsions, 86 change in relative velocity, 75–6, 75f droplet coalescence, 81 environmental stresses, 78–87 ageing, 78–9 aqueous phase, composition and properties of, 87–95 biopolymers, 92 chilling and freezing, 84–5 drying, 86–7 homogenization, 82–4 mechanical stresses, 81–2 pH and ionic strength, 87–9 sugars and polyols, 89–90 surfactants, 90–2 thermal processing, 79–81 influence of whey proteins optical properties, 78 rheological properties, 76 Proteolytic hydrolysis, 45 Ps. aeruginosa, 320 Pulsed electric field (PEF), 374–5 R Roller drying, 3 S S6 kinase 1, 351 Salmonella serotype typhimurium (DSMZ 5569), 313 Secondary homogenization, 66 Selective precipitation, 43–4 Shellac, 155 SHR. See Spontaneously hypertensive rat Small molecule surfactants, 90–1

Snacks, 206–10 Solvent-cast whey protein films, 140 appearance properties, 151–2 oil-barrier properties, 151 oxygen-barrier properties, 147 permeability properties, 146–51 tensile properties, 143–6 Spontaneously hypertensive rat (SHR), 289, 292 Spray coating, 139 Spray-drying, 86 Stokes’ law, 69–70 Sugar confectionery, 216 Sugar infusion syrup, 215 Supercritical fluid extrusion processing (SCFX), 175 Sweet whey 1-typical composition and physical properties, 15–16t Swelling alginate coating effects, 264–6 Swelling medium pH on whey protein hydrogels, 255–8, 256f Swelling solution concentration of, 238 Swelling-controlled systems, 245 drug release dynamics of, 246–50, 247f Swelling influence of protein types on, 356–63 kinetics, 241–4 thermodynamics, 238–41 T Texture, 216 Textured soy protein (TSP), 187 Textured whey protein (TWP), 192–4 as meat extenders/analogs, 197–9 Texturized proteins, 177 Texturized vegetable protein analogs, 187 Texturized whey pectin cranberry jelly, 221 Texturized whey proteins (TWPs), 180, 215–8 in prototype cranberry jelly, 226 for whey protein modification, 378–83 Thermally induced/heat induced gelation, 107 Thermoplastic protein extrusion, 188–9

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Index Toppings, 211–12 Total body protein turnover, 349 Transglutaminase, 141 Tumor suppressor p53, 295 Twin-screw (TS) extruders, 188 Twin-screw extrusion, 174–5 Two-phase partition, 28–9 TWP See Texturized whey proteins U U.S. foodservice regulations, 173 U.S. school lunch program, 173, 187–8 Ultrafiltration, 23 Ultra-osmosis (NF), 6 Ultrasound processing of milk products, 375–7 V Volatile organic compounds (VOCs), 155 W Water vapor permeability, 148–51 Whey crisps applications of, 204–13 in bars, 205, 206t, 207f Whey extrusion techniques, 203–4 Whey ingredients, 203 Whey peptides, 322–4 Whey processors, 4 Whey protein aggregates, 21–2 Whey protein bovine serum albumin, 296 Whey protein coating, applications of, 153–8 Whey protein concentrate (WPC), 6 Whey protein concentrates, 372 Whey protein denaturation, 19–20 changes in pH, 21 Whey protein emulsifiers, 67. See also Droplet aggregation Whey protein films, 139–43 active packaging, 158–63 network, 142 solvent casting of, 140–2 utilization of, 16–18 Whey protein gels, 107–10 Whey protein hydrogels, 255–66

399

Whey protein hydrolysates (WPH), 287, 292 Whey protein isolate (WPI), 6 Whey protein nanoparticles, 268–9 Whey protein salad topping, 212t Whey protein snack cruncher, 209t Whey protein stability, 104–5 Whey proteins, 203–4, 229, 277 acidic environment, 101–3 anticancer potential, 294–5 athletes’ protein choice, 173 biological and nutritional functionality of, 385–6 cross-linking of, 141–2 emulsion and foam properties, connection between, 120–2 enzymatic fragmentation of, 309 fibrous-textured, 192–9 liquid source, 194–5 protein concentration, 196–7 protein solubility, 195–6 foaming properties and effect of ph, 122–7 functional and nutritional properties of, 384–5 health benefits of, 386–7 heat-and-ph-treatment of characteristics of, 102t denaturation behaviour, 19–21 emulsifying properties of, 116–9 functional properties of, 103t gelation properties of, 105–10 gelation under acidic conditions, 111–4 isolation and fractionation, 24–5 structure and stability, effect of acidic conditions, 103–5 textural properties of, 113–4 immunomodulatory effects of, 303–8 in functional foods, 215–21 in source of health, 287–8 insulinotropic effects of, 324–5 isoelectric precipitation, 24 isolation through addition of salts, 23–4 lipid films, 149 physical and chemical properties of, 18–22

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400 Whey proteins (cont.) physical functionality of, 383–84 polymerization via thermal treatment, 119 processing, 174–6 protein efficiency ratio, 172 role in muscle accretion, 347–63 size, 18–9 stabilized emulsions, 65 stabilized emulsions, 79–80 with antimicrobial activity, 313–22 Whey separation by UF, 5 Whey texturization work, 179 Whey, 1–2 applications, 9–10 concentrating, 2–4 drying, 4 effect on protein accretion, 351–52 functionality, 8–9 mineral concentration, 20–21 modern techniques, 4–6

Index nutritional aspects, 7–8 WPC concentration effect on hydrogels, 258–62 WPC denaturation pH effect on hydrogels, 258–62 WPC gel tablet in vitro release profiles, 267f WPC hydrogel controlled drug release from, 263–4 WPC80, 179–80 WPH. See Whey protein hydrolysates WPI-coated paper packaging use, 157–8 WPs. See Whey proteins Y Young’s modulus/elastic modulus, 143 Z Zeta potential of BLG nanoparticles, 269–73, 271f