Whey proteins: from milk to medicine 9780128121245, 0128121246

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Whey Proteins

Whey Proteins From Milk to Medicine

Edited by Hilton C. Deeth School of Agriculture and Food Sciences, The University of Queensland, St Lucia, QLD, Australia

Nidhi Bansal School of Agriculture and Food Sciences, The University of Queensland, St Lucia, QLD, Australia

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-812124-5 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisition Editor: Megan R. Ball Editorial Project Manager: Tasha Frank Production Project Manager: Denny Mansingh Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

List of Contributors

Ajmol Ali Massey University, Auckland, New Zealand Syamala Athira ICAR-National Dairy Research Institute, Karnal, Haryana, India Nidhi Bansal University of Queensland, Brisbane, QLD, Australia André Brodkorb Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Aoife Buggy Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland David C. Clark Bovina Mountain Consulting LLC, Englewood, FL, United States Thomas Croguennec Agrocampus Ouest, Rennes, France Hilton Deeth University of Queensland, Brisbane, QLD, Australia MaryAnne Drake North Carolina State University, Raleigh, NC, United States Kamil P. Drapala University College Cork, Cork, Ireland Mark A. Fenelon Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Rita M. Hickey Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Sean A. Hogan Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Thom Huppertz FrieslandCampina, Amersfoort, The Netherlands Romain Jeantet Agrocampus Ouest, Rennes, France Phil Kelly Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Rajesh Kumar ICAR-National Dairy Research Institute, Karnal, Haryana, India Veronique Lagrange Consultant, Washington, DC, United States Lotte B. Larsen Aarhus University, Aarhus, Denmark Thao T. Le Edith Cowan University, Joondalup, WA, Australia xvii

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List of Contributors

Cécile Le Floch-Fouéré Agrocampus Ouest, Rennes, France Sung-Je Lee Massey University, Auckland, New Zealand Naiyan Lu Jiangnan University, Wuxi, People’s Republic of China Bimlesh Mann ICAR-National Dairy Research Institute, Karnal, Haryana, India Noel McCarthy Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Eve M. Mulcahy University College Cork, Cork, Ireland Kerstin Müller Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany Daniel M. Mulvihill University College Cork, Cork, Ireland Eoin G. Murphy Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Eve-Anne Norwood Agrocampus Ouest, Rennes, France James A. O’Mahony University College Cork, Cork, Ireland Julian Price Milk Specialties Global, Eden Prairie, MN, United States Kay J. Rutherfurd-Markwick Massey University, Auckland, New Zealand Prabin Sarkar ICAR-National Dairy Research Institute, Karnal, Haryana, India Markus Schmid Albstadt-Sigmaringen University, Sigmaringen, Germany Pierre Schuck Agrocampus Ouest, Rennes, France Rajan Sharma ICAR-National Dairy Research Institute, Karnal, Haryana, India Ranjan Sharma OzScientific Pty Ltd, Hoppers Crossing, VIC, Australia Mark Stout North Carolina State University, Raleigh, NC, United States Todor Vasiljevic Victoria University, Melbourne, VIC, Australia Heni B. Wijayanti NIZO Food Research, Ede, The Netherlands Di Zhao South China University of Technology, Guangzhou Shi, China Peng Zhou Jiangnan University, Wuxi, People’s Republic of China Bogdan Zisu RMIT University, Bundoora, VIC, Australia

About the Editors

HILTON DEETH Hilton Deeth grew up on a dairy farm in Australia which initiated his lifelong interest in milk and dairy products. After completing a science degree and a PhD in organic chemistry at the University of Queensland, he worked as a research food scientist in the Queensland Department of Primary Industries (QDPI) for 23 years. His areas of research included quality aspects of milk, butter, and cheese, specializing in lipase and lipolysis. At the time of leaving QDPI to take up an academic position at the University of Queensland in 1995, he was Manager of Food Research and Development, supervising a range of projects on dairy, seafood, meat, and fruits and vegetables. At the University of Queensland, he taught subjects including dairy science, emerging food technologies, and food product development. He also supervised research projects on a range of topics and was advisor for more than 30 PhD and research Masters students, as well as several coursework Masters students. His main dairy research interests included UHT processing and products, denaturation of whey proteins, calcium-induced gels, quality aspects of milk, yogurt and milk powders, and new processing technologies. In 1996, he established a specialist Centre for UHT Processing and Products for the Australian dairy industry at the University of Queensland and directed the Centre until 2008. The research initiated in the Centre was continued in a dairy industry-funded Food Science Research Program at the University of Queensland which he managed until his retirement in 2011. He has published over 160 research papers and reviews, and 30 book chapters. Since retiring as Emeritus Professor of Food Science, he has remained involved in dairy science and technology as a consultant assisting dairy companies with product and process development, and trouble shooting, and also providing technical training in thermal processing and other dairy topics for companies in Australia and other countries. In 2017, he coauthored, with Mike Lewis from Reading University, the book High Temperature Processing of Milk and Milk Products.

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About the Editors

NIDHI BANSAL Dr. Nidhi Bansal has been working at the University of Queensland since 2010 and is a senior lecturer in Food Science and Technology. She completed her PhD from University College Cork, Ireland 10 years ago. After finishing her PhD, she worked at Dairy Products Technology Center, California Polytechnic State University, USA for 2 years before joining the UQ. She has been associated with research in the dairy field for over 14 years now. Her research is focused largely on examining both the fundamental and applied aspects of dairy science. Since 2011, she has been involved in supervising 23 PhD students at the UQ. Additionally, she has supervised over 30 coursework Masters and Honors students. Currently, she leads a research group working in the areas of texture and flavor modification, nonthermal processing, and shelf life extension of dairy products, and bioactive compounds in milk. She has published B70 research papers and book chapters and has edited two books so far. Dr. Bansal's research is aimed to translate directly into the dairy industry through her industry collaborations. Her teaching interactions spans from first- to third-year undergraduate students and Masters students. She teaches courses such as Principles of Food Preservation, Food Product Development, and Food Science at the UQ. Her teaching is guided by her belief that teacher and learner must work together towards a common goal of not only enhancing knowledge but also fostering personal development and growth.

Preface

This book describes the many aspects of whey proteins, the acid-soluble proteins in milk. For many years the value of these proteins was not fully appreciated as they were components of whey, which was considered to be a problematic waste by-product of cheesemaking. In addition, they tended to coagulate with heat and cause problems during heat processing of products containing them. However, in recent years, the value of whey proteins has been recognized in several ways including their physical functionality and their benefits in sports and exercise supplements and nutrition- and healthrelated products. Applications of whey proteins in both native and modified forms have been extensively researched and several whey protein products have been commercialized. These developments have coincided with the accumulation of a mass of data on the chemical and physical aspects of these proteins and their behavior under a wide range of processing and storage conditions. The book covers the basic properties of whey proteins, the production and properties of whey protein products, and their many industrial, nutritional, and therapeutic applications. It commences with an overview of four major and nine minor whey proteins. This includes their chemical and physical characteristics as well as some of their significant properties and applications. In addition to this overview in Chapter 1, further details on many of the whey proteins are discussed in other chapters. The second chapter reviews the history of the development of the various whey protein products together with the development of the membrane separation technologies and other processes which are crucial for the separation of the whey proteins from other components of whey or for their subsequent manipulation for various applications. This is followed by a complementary chapter on the technical aspects of the manufacture and properties of the various whey protein products. While it covers the more common products, whey protein concentrate (WPC) and whey protein isolate (WPI), it also discusses several modified products such as whey protein hydrolysates and microparticulated whey proteins.

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Chapter 4 deals with the practical aspect of changes that occur in whey protein products during storage. Knowledge of these changes and their consequences is essential to enable the dairy industry, and food industries in general, to take full advantage of the many beneficial properties of these products. An important aspect of gaining such knowledge is effective analysis of the products and the changes that occur. Such analyses, which include both chemical and physical testing, are covered in this chapter and in Chapter 5 which focuses on the many chemical analyses. Chapter 5 introduces some of the modern analytical methods such as proteomic analyses which are revolutionising the way dairy products are analyzed. Chapter 6 covers the most common chemical reactions whey proteins undergo during thermal processing. It focuses on the denaturation of the major whey protein, β-lactoglobulin, and the subsequent protein 2 protein interactions which can be beneficial or problematic depending or the circumstances. One of the outcomes of these changes is the formation of gels, which have now found several applications. Chapter 7 discusses a useful method of manipulating whey proteins to alter their functional properties. This is conjugation with reducing carbohydrates by the same mechanism as the well-known Maillard reaction. This reaction has considerably expanded the application of whey proteins as the nature of the conjugate can be specifically tailored through judicious choice of carbohydrate and reaction conditions. Importantly, the reaction can take place in either a liquid solution or a powder. Chapter 8 introduces several novel technologies and their effects on whey proteins. Technologies included are high pressure processing, shear processing, low-frequency, high-power ultrasound processing, pulsed electric field, and UV processing. The focus is on the effects of these technologies on protein structure and the functional properties of the whey proteins. This is exemplified in shear processing which has already found application in the production of microparticulated whey proteins. Chapters 9 11 cover various aspects of the application of whey proteins in relation to their use as food ingredients. Chapter 9 provides an overview of several of these applications including beverages, yogurt, bars, desserts, and cheese. It takes a practical approach and makes reference to several commercial whey protein products. The focus of Chapter 10 is the important aspect of flavor in relation to the use of whey protein products as food ingredients. Since flavor is a primary driver of product acceptance, it is a key attribute of whey protein products. The chapter addresses current research on whey protein flavors and the influence of processing and handling on flavor and flavor stability.

Preface

Chapter 11 reviews the development to date of a comparatively new application of whey proteins, their use in packaging, films, and coatings. This chapter illustrates the breadth of the uses of whey proteins. With the increasing concern about the pollution caused by used plastic material, alternatives such as whey protein-based materials have considerable potential. Chapter 12 is the first of six chapters which address various applications related to nutrition and health. It covers one of the major uses of whey proteins, infant formulas. This has been, and continues to be, important for the health and well-being of many infants. The chapter emphasizes the differences between the components of human and cows’ milks, and discusses the ways in which whey proteins are used to make infant formulas resemble human breast milk. It contains an informative comparative review of the whey proteins in human and cows’ milk. Chapter 13 reviews the development of nutrition bars based on whey proteins. These are products we have come to increasingly accept, but seldom appreciate the role played by whey proteins in their manufacture. It discusses the many challenges in producing such bars and the common quality issues such as hardening during storage. Given the nutritional benefits of whey proteins outlined in the following chapters, whey protein-based bars may be a very acceptable and convenient means for some people to increase their intake of these proteins. Chapter 14 covers an aspect of whey proteins which has been actively researched for some time. Hydrolysis catalyzed by proteolytic enzymes produces whey protein hydrolysates, as discussed in Chapters 2 and 3; however, amongst the hydrolysates are numerous peptides which have specific biological functionality and are now being increasingly sought after for several health-related applications. Chapter 15 provides an overview of several nutritive and therapeutic aspects of whey proteins. It discusses the possible roles of whey proteins in undernutrition, overnutrition, management of chronic disease, body composition, and nutrition of the aged. With the increasing proportion of elderly persons in the population and appreciation of the beneficial effects of whey proteins in the alleviation of age-related conditions such as sarcopenia, whey protein products and ingredients have the potential to become very valuable for this cohort. Chapter 16 reviews the studies on the role of whey proteins in sport and exercise. Whey protein-based supplements have become well-known amongst people who are actively engaged in sport and exercise in general and in body-building in particular. This is a major market for whey protein products produced by the dairy industry and by all accounts quite a lucrative one.

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The final Chapter 17, gives an up-to-date account of the use of whey proteins in functional foods, that is, foods which provide some physiological benefit to the consumer over and above pure sustenance. This chapter has a practical focus and introduces several commercial products. These products contain a range of whey protein products and fractions. Some contain enhanced levels of individual proteins such as α-lactalbumin, glycomacropeptide, lactoferrin, and immunoglobulins while others contain mixtures of whey proteins such as hydrolysates and individual native (undenatured) whey proteins including, for example, lactoperoxidase and lysozyme which have antibacterial activities in their native form. While the book covers a wide range of topics related to whey proteins, it does not claim it to be comprehensive in all aspects. However, in line with the title of the book Whey Proteins: From Milk to Medicine, the chapters present a good coverage of the various processes to which whey proteins are subjected in their journey from milk to ingredients in nutritional/medicinal products. Any reader of the book will appreciate the tremendous transformation of whey proteins from a waste product into a host of valuable commodities that has increasingly occurred over the last 50 years.

Abbreviations

1-DGE 2-DGE AA ACE ACP aw BCAA BHA BHT bLf BOD BSA BV CA CaCN CD CE CHO CIP CML CMP CN CPP CSS CV CVD CWPCP Cys d Da DAD DDS DE DH DM DSC DWP

one-dimensional gel electrophoresis two-dimensional gel electrophoresis amino acid angiotensin I-converting enzyme analogue cheese product water activity branched-chain amino acid butylated hydroxyanisole butylated hydroxytoluene bovine lactoferrin biochemical oxygen demand bovine serum albumin biological value cellulose acetate calcium caseinate circular dichroism capillary electrophoresis carbohydrate cleaning-in-place carboxymethyllysine caseinomacropeptide casein casein phosphopeptide corn sirup solids coefficient of variation cardiovascular disease casein whey protein concentrate precipitate cysteine day dalton diode array detector De Danske Sukkerfabrikker dextrose equivalent degree of hydrolysis dry matter differential scanning calorimetry demineralized whey powder

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Abbreviations

DWPC EAA ED EGF ELISA ES ESI EU FBP FDA FDM FGF FOSHU FTIR G0 GF GIT GMP GOS GSH h hLf HMF HMO HPLC HTST HWP HWPI ICAT IE IEC IF Ig IGF IL IMAC IPG iTRAQ KMS LAB LAL LBM LC Lf LP LWPC MALDI MD MF

denatured whey protein concentrate essential amino acid electrodialysis epidermal growth factor enzyme-linked immunosorbent assay emulsifying salts electrospray ionization European Union folate-binding protein Food & Drug Administration (US) fat in dry matter fibroblast growth factor foods for specific health use Fourier transfer infrared storage modulus gel filtration gastrointestinal tract glycomacropeptide galactooligosaccharides glutathione hour human lactoferrin hydroxymethyfurfural (5-hydroxymethyl-2-furfuraldehyde) human oligosaccharides high-performance liquid chromatography high-temperature, short-time hydrolyzed whey proteins hydrolyzed whey protein isolate isotope-coded affinity tag ion exchange ion exchange chromatography infant formula immunoglobulin insulin-like growth factor interleukin immobilized metal affinity chromatography immobilized pH gradient isobaric tagging for relative and absolute quantification koch membrane systems lactic acid bacteria lysinoalanine lean body mass liquid chromatography lactoferrin lactoperoxidase liquid whey protein concentrate matrix-assisted laser desorption ionization maltodextrin microfiltration

Abbreviations

min mo MPa MPB MPS Mr MR MRM MRP MS mTOR MW Mw MWCO MWP NaCN NEC NEM NF NPB NZ O/W OPA OPN PA PAGE PCA PCN PCP PCr PDCAAS PDWPC PEF PEG PES PG pI PKU PL PP3 PS PTM PVDF Q-TOF Ra Rcp RH RNA RO

minute month megapascal muscle protein breakdown muscle protein synthesis molecular mass maillard reaction multiple reaction monitoring maillard reaction product mass spectroscopy mammalian target of rapamysin molecular weight molecular weight molecular weight cut-off microparticulated whey proteins sodium caseinate necrotizing enterocolitis N-ethylmaleimide nanofiltration net protein balance New Zealand oil/water o-phthaldialdehyde osteopontin polyamide polyacrylamide gel electrophoresis principal component analysis phosphocasein processed cheese product phosphocreatine protein digestibility corrected amino acid score partially denatured whey protein concentrate pulsed electric field polyethylene glycol polyethersulfone propyl gallate isoelectric point phenylketonuria placebo proteose peptone 3 polysulfone posttranslational modification polyvinylidene difluoride quadrupoletime-of-flight resistance due to adsorption resistance due to gel formation relative humidity ribonucleic acid reverse osmosis

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Abbreviations

ROS Rp RPC RPE RTD s SCFA SDS-PAGE SEC -SH SMP SMUF SPC SPI SRM SSHE SW SWP TBARS TBHQ Td TFC Tg TGase TGF-β Th2 cells TMP TMR TNBS TNF TOF TT TTE Tv UF UHT UK VCF w/w WHO wk WP WPC WPH WPI WPPC XPS yr α-La β-Lg

reactive oxygen species resistance due to pore blocking reversed-phase chromatography rating of perceived exertion ready to drink second short-chain fatty acid sodium dodecylsulfate-polyacrylamide gel electrophoresis size-exclusion chromatography sulfhydryl group skim milk powder simulated milk ultrafiltrate soy protein concentrate, also Serum protein concentrate soy protein isolate selective reactive monitoring scraped-surface heat exchanger spiral wound sweet whey protein thiobarbituric acid reactive substances tert-butylhydroquinone denaturation temperature thin-film composite glass transition temperature transglutaminase transforming growth factor beta T helper cells 2 trans membrane pressure total mixed ration trinitrobenenesulfonic acid tumor necrosis factor time-of-flight time trial time to exhaustion critical temperature for viscosity increase ultrafiltration ultra-high temperature United Kingdom volume concentration factor weight/weight basis World Health Organisation week whey protein whey protein concentrate whey protein hydrolysate whey protein isolate whey protein phospholipid concentrate X-ray photoelectron spectroscopy years α-lactalbumin β-lactoglobulin

CHAPTER 1

Whey Proteins: An Overview Hilton Deeth and Nidhi Bansal University of Queensland, Brisbane, QLD, Australia

1.1

INTRODUCTION

The title of this book uses the term “whey proteins” but opinions differ on the use of this term. Some contend that the terms “serum proteins” and “soluble milk proteins” are more appropriate, as whey proteins literally refer to those in whey produced during cheese or casein manufacture. While this is a legitimate position, we have taken the view that the term “whey proteins” is very widely used and is understood to refer to the proteins not associated with the casein micelle or other milk particles. It is acknowledged, however, that the proteins in whey, particularly cheese whey, differ from those in milk serum as they include some starter bacteria proteins, bacterial metabolites, and glycomacropeptide (GMP), a product of rennet action on κ-casein. For this reason, the commercially available milk serum protein concentrate which is made from skim milk differs from whey protein concentrate which is made from whey (DeBoer, 2014) (see also Chapter 3). A further nomenclature issue is the use of “proteins” rather than “peptides.” Some scientists maintain that the term “peptides” is more inclusive and should be used instead of “proteins.” The position adopted here is that the term “protein” refers to intact gene products while “peptides” refer to fragments of proteins. As indicated below, some important “peptides” are mentioned briefly. It is therefore necessary to define which proteins are included and which are not. For this chapter, we have categorized the included proteins into major and minor whey proteins including enzymes. Proteins which are normally associated with the casein micelle, membrane structures (milk fat globule membrane and skim milk membranes), and cells (somatic and microbial) that become solubilized under some conditions are not 1 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00001-1 © 2019 Elsevier Inc. All rights reserved.

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considered here to be “whey proteins.” In addition, proteose peptones 5 and 8 which are fragments of caseins are not included. However, included for completeness under minor whey proteins is glycomacropetide derived from κ-casein by rennet action in cheesemaking, as it is an integral and substantial component of cheese whey proteins. A further justification for its inclusion is that milk naturally contains a low concentration of free GMP (Furlanetti & Prata, 2003). The proteins discussed in this chapter are all significant in some way and this significance is highlighted for each one. For many of these proteins, commercial products are now available. These include β-lactoglobulin, α-lactalbumin, lactoferrin, lactoperoxidase, osteopontin, and glycomacropetide. DeBoer (2014) provides useful “Information Sheets” on 50 dairy ingredients including these six individual whey proteins. All milk contains some soluble casein; at low temperature, the concentration of these proteins increases due to the increased solubilization of β-casein. For this chapter, these soluble caseins are not considered to be “whey proteins.” Several growth factors have been identified in milk and whey in small quantities. These are not included here although it is recognized that they represent important biological activity, such as wound healing, and that extracts with growth factor activity have been commercialized. They are briefly discussed in Chapter 12. An excellent review of growth factors in milk was published by Gauthier, Pouliot and Maubois (2006). In many cases there are important nutritional and health-related aspects associated with the proteins covered in this chapter. Some of these aspects are alluded to here; however, these are more fully discussed in Chapters 12, 15, 16, and 17 in this book. This chapter provides an overview of whey proteins. Space does not permit a detailed discussion of the nature and properties of each one. Besides, several excellent reviews have been published on these proteins in recent years and the reader is referred to these for detailed information. In particular, the Advanced Dairy Chemistry and Encyclopedia of Dairy Sciences series contain several chapters on whey protein products and individual whey proteins. These chapters and other pertinent reviews are referenced throughout this chapter. The whey proteins are discussed in this chapter under the broad headings of major and minor proteins, which largely reflects their abundance in bovine milk. Fig. 1.1 shows a reducing SDS-PAGE (Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis) electrophoretogram of the most abundant whey proteins.

1.2 Major Whey Proteins

FIGURE 1.1 Reduced SDS-PAGE gel of bovine whey proteins. 1 5 dialyzed whey (pH 4.6); 2 5 lactoferrin; 3 5 bovine serum albumin; 4 5 IgG, heavy chain (top) and light chain (bottom); 5 5 β-lactoglobulin; 6 5 α-lactalbumin; 7 5 dialyzed whey (pH 4.6); 8 5 protein standards. From Farrell et al., 2004.

1.2 1.2.1

MAJOR WHEY PROTEINS β-Lactoglobulin (β-Lg)

Most mammalian milks contain β-Lg as a major whey protein. β-Lg is the most abundant whey protein in milks of ruminants such as dairy cattle, goats, sheep, and water buffalo (Oftedal, 2013), but it is not found in milks of some species including some rodents such as mice, rats, and guinea pigs; some lagomorphs such as rabbits; some camelids such as dromedary camels and llamas; and some primates such as humans and chimpanzee (Sawyer, 2013). In bovine milk, β-Lg represents about 10%
12% of the total proteins in milk and B50% of the total whey proteins (Creamer, Loveday, & Sawyer, 2011; O’Mahony & Fox, 2013). The β-Lg content in milk shows high interspecies variability; it ranges from as low as 0.6 mg/mL in pigs to as high as 16.2 mg/mL (according to literature compiled by Sawyer, 2013). Within the milk of a species, the β-Lg concentration can vary depending on animal breed, stage of lactation, and environmental factors such as season and diet.

1.2.1.1 Characteristics β-Lg is a highly structured globular protein that belongs to the lipocalin family. It is synthesized in the mammary gland by the secretory epithelial cells. The complete amino acid sequence of β-Lg is now known for several species

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and has been mentioned in several publications, including for bovine (Farrell et al., 2004), ovine (Tulipano, Cocchi, & Caroli, 2012), caprine (Tulipano et al., 2012), and donkey (Cunsolo et al., 2017). Many of these sequences have been deposited in UniProt and can be freely accessed at http://www.uniprot.org. Variations in amino acid sequence in many ruminant milks have been illustrated and compared to bovine β-Lg B by Sawyer (2013), who has also compiled the complete amino acid sequences of β-Lg of several nonruminant milks and compared them to bovine β-Lg B. Several genetic polymorphs of β-Lg have been described in the literature and huge interspecies variations are seen. For example, 11 genetic variants (A
J, W) of bovine β-Lg were described by Farrell et al. (2004), with variants A and B being the most abundant. The number of genetic variants in bovine β-Lg have been updated to 13 in a recent publication (Martin, Bianchi, Cebo, & Miranda, 2013). In sheep milk, three genetic variants (A
C) of β-Lg have been identified that vary from each other in one or two amino acids (Selvaggi, Laudadio, Dario, & Tufarelli, 2014; Selvaggi, Laudadio, Dario, & Tufarelli, 2014). In goats’ milk, while several nucleotide substitutions have been reported, no variants containing amino acid changes have been identified so far (Selvaggi et al., 2014). The isoelectric point of bovine β-Lg variants A and B has been reported as 5.13, while their isoionic points are 5.35 and 5.41, respectively (Farrell et al., 2004).

1.2.1.2 Structure Bovine β-Lg contains 162 amino acid residues and has a molecular weight of B18,300 Da (Boland, 2011). Bovine β-Lg variants A and B contain two disulfide bonds at Cys66-Cys160 and Cys106-Cys119 and one free sulfhydryl group on Cys121 buried within the protein structure (McKenzie, Shaw, & Ralston, 1972; Papiz et al., 1986). However, the free sulfydryl group is absent in β-Lg of some species (O’Mahony & Fox, 2013). Several techniques, such as infrared spectroscopy, circular dichroism, nuclear magnetic resonance spectroscopy, and X-ray, have been used to study the structure of β-Lg. Bovine β-Lg contains 45% β-sheets, 8% α-helices and 47% random coils (Sawyer, 2013). The nine major β-sheets form a “calyx” or globulet-like structure (Creamer et al., 2011; Sawyer, 2013). This cavity can act as a binding site for several molecules and has a significant role to play in functions attributed to β-Lg. Structural differences have been found between genetic variants of bovine β-Lg (Dong et al., 1996; Oliveira et al., 2001) with some studies suggesting variant A to be more flexible than variant B (Dong et al., 1996). Under physiological conditions, ruminant β-Lg exists as a dimer of B36 kDa. The association/dissociation behavior of β-Lg has been a topic of extensive research for a long time (Sawyer, 2013 and references therein). The association/dissociation of β-Lg monomers is dependent on several factors

1.2 Major Whey Proteins

including pH, temperature, ionic strength and protein concentration (Brownlow et al., 1997). It also varies between species. Native bovine β-Lg dissociates into monomers of B18 kDa at pH ,3.5 and .7.5. Also, between pH 3.5 and 5.2 (particularly at BpH 4.6), genetic variant A of bovine β-Lg associates into octomers of B144 kDa (McKenzie & Sawyer, 1967; Townend, Weinberger, & Timasheff, 1960; Zimmerman, Barlow, & Klotz, 1970). The dissociation equilibrium and rate constants over the pH range of 2.5
7.5 for bovine β-Lg have been calculated (Mercadante et al., 2012). Dissociation of β-Lg into monomers is also seen at low ionic strength (Renard, Lefebvre, Griffin, & Griffin, 1998). Reversible dissociation and conformational changes in β-Lg are observed up to 60 C. Irreversible conformational changes in monomers appear between 60 C and 70 C, leading to formation of molten globules (de Wit, 2009). Above 70 C, the unfolded monomers aggregate to form β-Lg oligomers and, in milk, form complexes with other proteins such as κ-casein via sulfhydryl 2 disulfide interactions. These phenomena are covered in Chapter 6 of this book.

1.2.1.3 Function Numerous functions have been ascribed to β-Lg, but a consensus on its exact biological function has still not been reached. Some reports have suggested a primarily nutritional function to β-Lg as a source of amino acids (Bottomley, Evans, & Parkinson, 1990; Creamer et al., 2011; Oftedal, 2013). It contains a relatively large amount of branched-chain amino acids and sulfur amino acids. The high content of sulfur amino acids may lead to its participation in an active immune system (García-Garibay, Jiménez-Guzmán, & HernándezSánchez, 2008). However, due to its globular structure, β-Lg is resistant to proteolysis by enzymes and acid (Guo, Fox, Flynn, & Kindstedt, 1995). Hence, it has been suggested that β-Lg may have some more specific biological function. Due to the presence of the calyx in its structure, β-Lg has the capacity to bind with numerous hydrophobic and amphiphilic ligands. This binding capacity has been associated with a variety of functions of β-Lg. It is capable of binding vitamin A (retinol) and D and hence may act as a carrier for them (O’Mahony & Fox, 2013). Also, it has been associated with stimulating lipase activity due to its ability to bind fatty acids (O’Mahony & Fox, 2013). But β-Lg does not bind to vitamin A or fatty acids in pigs and horses (Oftedal, 2013). β-Lg also binds minerals and transports them across the intestinal walls (García-Garibay et al., 2008). A discrepancy in ascribing a specific biological function to β-Lg is that it is not homogenously distributed in all milks and is absent from milk of many species (Creamer et al., 2011; Oftedal, 2013). Another possibility that has been suggested is that ancestral β-Lg might had played an essential role

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during early stages of pregnancy and had been involved in fetal development, but now has evolved to have more of a nutritional role in milk (Kontopidis, Holt, & Sawyer, 2004; Sawyer, 2013; Kontopidis, Holt, & Sawyer, 2002; Cavaggioni, Pelosi, Edwards, & Sawyer, 2006). In addition to biological functionality, β-Lg has huge technological significance. Due to its prevalence as the major whey protein in bovine milk, the properties of β-Lg under various processing conditions govern the functionality of whey protein ingredients such as whey protein concentrates and isolates. The functional properties of β-Lg and its implications are discussed in several chapters throughout this book.

1.2.1.4 Significance As the most abundant whey protein, β-Lg has a major influence on the behavior of whey protein products. In particular, its denaturation by heat and its subsequent aggregation with itself and other proteins, and formation of gels, are of great significance in the dairy industry. Denaturation and aggregation are covered in Chapter 6 which also discusses several approaches which have been used to reduce the adverse effects of β-Lg denaturation during processing and to produce heat-stable whey protein powders. One interesting approach is the use of chaperone proteins such as caseins which associate with the whey proteins and reduce denaturation (Mounsey & O’Kennedy, 2010). Solutions of β-Lg gel on heating; however, the ability to gel and the strength of the gels formed depends on several factors including protein concentration (which must exceed a certain threshold level, B10% at pH 7 (Remondetto, Paquin, & Subirade, 2002)), temperature of heating (needs to be higher than a certain threshold value), pH (optimum is B6.5), presence of other proteins (e.g., firmer gels are formed if α-La is present), and the presence of mineral salts particularly ionic calcium salts. β-Lg can also form cold-set gels under certain conditions (Relkin, Launay, & Liu, 1998). The binding of β-Lg to calcium (up to three atoms of Ca per β-Lg molecule) (Jeyarajah & Allen, 1994; Pappas & Rothwell, 1991; Simons, Kosters, Visschers, & de Jongh, 2002) has significant practical effects. Calcium promotes β-Lg denaturation and aggregation (Ju & Kilara, 1998; Petit, Herbig, Moreau, & Delaplace, 2011), and influences the properties of β-Lg gels (Matsudomi, Rector, & Kinsella, 1991). It also increases the foaming capacity of β-Lg (˙Ibano˘glu & ˙Ibano˘glu, 1999). Calcium can induce gelation at pH values (e.g., 8) at which heat-induced gels do not normally form, and reduce the temperature at which β-Lg gels (Twomey, Keogh, Mehra, & Okennedy, 1997). β-Lg can also form cold-set gels after being preheated under conditions which do not cause thermal gelation, and salts of calcium and other metals (Remondetto et al., 2002;

1.2 Major Whey Proteins

Kuhn, Cavallieri, & da Cunha, 2010) are added. The ability of β-Lg to form gels with metal ions such as iron could have important nutritional implications. A significant issue for the dairy industry is the role of β-Lg in the formation of fouling deposits in heat exchangers, since denatured β-Lg is a major component of fouling deposits in the first stages of UHT heating of milk (at temperatures up to B100 C), so-called Type A deposits (De Jong, Bouman, & Van der Linden, 1992; Hiddink, Lalande, Maas, & Streuper, 1986; Deeth & Lewis, 2017). In fact, computer models of fouling are based on the kinetics of denaturation of β-Lg (Grijspeerdt, Mortier, De Block, & Van Renterghem, 2004). During heat processing of whey protein concentrate in the temperature range of 60
90 C, the amount of fouling deposit increases with the concentration of calcium. Khaldi et al. (2015) developed a predictive model in which the amount of fouling deposit formed during processing is a function of the calcium content, Reynolds number and the bulk fluid temperature.

1.2.2

α-Lactalbumin (α-La)

α-La is the second most abundant whey protein in bovine milk. Bovine milk contains about 1.2
1.5 g/L α-La (Farrell et al., 2004) which represents about 20% of the total whey proteins and about 3.5% of the total proteins in milk (O’Mahony & Fox, 2013). In the absence of β-Lg, α-La is the most abundant whey protein in human milk (Lönnerdal, 2003). The concentration of α-La in human milk varies due to several factors including genetic, environmental and dietary factors (Jackson et al., 2004; Santos & Ferreira, 2007). A multinational study analyzed 452 mature human milk samples from at least 50 women from nine different countries on five continents and estimated the mean α-La concentration to be 2.44 6 0.64 g/L (Jackson et al., 2004). α-La is present in milk of almost all mammalian species (Brew, 2013), except in some Otariidae species such as the Northern fur seal (Dosako et al., 1983). Unlike β-Lg whose concentration increases with stage of lactation, the concentration of α-La in bovine milk decreases at the end of lactation (Caffin, Poutrel, & Rainard, 1985). This decline in its concentration is positively correlated with the decline in lactose concentration in milk toward the end of lactation (Farrell et al., 2004). A similar trend was reported for human milk α-La; it was positively correlated with total nitrogen during lactation (Jackson et al., 2004).

1.2.2.1 Characteristics α-La is a small compact protein that has been classified as a calcium metalloprotein (Hiraoka, Segawa, Kuwajima, Sugai, & Murai, 1980). Calcium binding has been shown to be necessary for proper folding and disulfide bond

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formation of the native α-La. The molecular weight of bovine α-La has been calculated as 14,178 Da (Farrell et al., 2004). α-La has a very high content of the essential amino acids, 63.2% of the total amino acid content (Heine, Klein, & Reeds, 1991). It contains B1.9% sulfur and is a rich source of tryptophan (O’Mahony & Fox, 2013). Bovine α-La has an isoelectric point between pH 4.2 and 4.5 (Farrell et al., 2004). Initially two genetic variants (A and B) of bovine α-La were identified (Bhattacharya, Roychoudhury, Sinha, & Sen, 1963). Variant B had a Arg substitution for Gln at position 10 compared to variant A (Gordon, 1971). A third variant (variant C) was later identified in Bali cattle (Bell, Hopper, & McKenzie, 1981). Variant C was reported to have either an Asn for Asp or a Gln for Glu substitution relative to variant B; however, the precise position of substitution was not established (Bell et al., 1981). Most recently, a new variant (named D) has been identified with a Gln for His substitution at position 65 (Visker, Heck, van Valenberg, van Arendonk, & Bovenhuis, 2012). Compared to bovine milk, no genetic variants of α-La have been identified in goat milk, while two variants (A and B) have been shown for sheep milk (Martin, Cebo, & Miranda, 2013). Genetic polymorphism of α-La has also been studied in human milk, although to a lesser extent. Some studies have shown two variants of α-La in human milk (Chowanadisai et al., 2005; Santos & Ferreira, 2007). It was also found that the polymorphism was most prevalent in milk samples from Asia (Chowanadisai et al., 2005).

1.2.2.2 Structure The amino acid sequence of bovine α-La was published in 1970 (Brew, Castellino, Vanaman, & Hill, 1970). Mature bovine α-La contains 123 amino acids. α-La is synthesized in the mammary gland as a preprotein containing 142 amino acids; the first 19 amino acids work as a secretion signaling sequence (Gaye et al., 1987; Hurley & Schuler, 1987). Milk from other mammalian species have 121 to 140 amino acid residues (Brew, 2011). α-La does not contain any free sulfhydryl groups, but contains four intramolecular disulfide bonds (Brew, 2013). The structure of α-La contains about 26% α-helices, 14% β-sheets, and 60% unordered structures (Brew, 2003). α-La has a bilobal structure similar to lysozyme. One lobe consists of α-helices while β-sheets and unordered structures make up the other (Brew, 2013). The two lobes are separated by a cleft which is closed at one end (Brew, 2011). Calcium binding is crucial to the stability of α-La and plays an important role in its proper folding (Rao & Brew, 1989). The primary calcium binding site in α-La is located at the junction between the two lobes (Brew, 2011).

1.2 Major Whey Proteins

1.2.2.3 Function The key biological function of α-La is regulation of lactose synthesis and production of the aqueous phase of milk. The lactose concentration in milk is proportional to its α-La content. α-La forms a complex with β-1,4-galactosyltransferase-1 and modulates its affinity for glucose. Under physiological conditions, α-La reversibly binds to β4-galactosyltransferase-1 and increases its affinity for glucose by 1000-fold (Brew, 2011). This allows for efficient lactose production in the lumen of the Golgi apparatus. In recent years, another biological activity of α-La has been highlighted. A partially folded molten globule state of α-la has been identified which has shown apoptotic activity against cancerous cells (Svensson et al., 1999). This nonnative state of α-La is stabilized by its interaction with oleic acid and has been designated as human α-La made lethal to tumor cells (HAMLET) (Svensson, Hakansson, Mossberg, Linse, & Svanborg, 2000). Purified α-La from bovine, equine, porcine, and caprine milk can also form complexes with oleic acid and show biological activity similar to HAMLET (Pettersson, Mossberg, & Svanborg, 2006).

1.2.2.4 Significance In addition to its roles in lactose synthesis and other physiological functions discussed above, α-La is highly significant because it is the major whey protein in human milk. While it only represents 20%
25% of the whey proteins in bovine milk it represents over 40% of the whey proteins in human milk. In terms of its percentage of the total proteins, the difference between bovine and human milk is more marked: B3.5% compared with B20%. Given that human milk does not contain β-Lg, there is a challenge for the dairy industry to produce α-La-enriched products from bovine milk for inclusion in infant formula. This can be achieved in various ways including chromatography, membrane filtration, aggregation/precipitation, and enzyme hydrolysis (Kamau, Cheison, Chen, Liu, & Lu, 2010) (see Chapter 3 for further information on α-La enrichment and Chapter 12 for use of α-La in infant formula). α-La is the most heat-stable of the major whey proteins in milk. For example, in UHT milk where β-Lg is usually denatured by more than 90%, α-La is typically denatured by 50%
80% in indirect UHT plants and even less in direct plants (Tran, Datta, Lewis, & Deeth, 2008). The stability is due to the fact that it has two disulfide bonds, no free sulfhydryl groups and binds a calcium ion. The binding of α-La with calcium is very strong, much stronger than the binding of β-Lg with calcium. It binds calcium down to pH 4.0 whereas β-Lg binds calcium at pH . 5.0 (Patocka & Jelen, 1991). This binding with calcium has implications for heat stability and other functional

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properties such as foaming. In its native state, α-La is not fully saturated with calcium. This is evident in the findings of ˙Ibano˘glu and ˙Ibano˘glu (1999) that both removal of calcium from native α-La with EDTA and addition of calcium improved its foaming ability. α-La has high foaming capacity and can form foams from solutions with as little as 0.01
0.05 mg protein/mL (Bacon et al., 1988; ˙Ibano˘glu & ˙Ibano˘glu, 1999).

1.2.3

Serum Albumin

Serum albumin (SA) in milk of almost all species is present in much lower concentrations compared to β-Lg and α-La. Cows’ milk contains about 0.4 g/L bovine serum albumin (BSA), which amounts to 1.5% of total milk protein and about 8% of total whey proteins (Farrell et al., 2004). Marked increases in bovine BSA concentrations have been reported during mastitis (Harmon, Schanbacher, Ferguson, & Smith, 1976). Levieux and Ollier (1999) studied changes in BSA concentration during the early lactation period. They reported that the average concentration of BSA in colostrum of 60 Holstein-Friesian cows they studied was about 1.21 6 0.44 g/L which decreased abruptly from the second milking onwards. A similar abrupt decrease in SA concentration was reported in caprine milk (Levieux, Morgan, Geneix, Masle, & Bouvier, 2002). Camel milk has been shown to contain a much higher concentration of SA than bovine milk at equal whey proteins concentration (Merin et al., 2001). Similar to other milks, camel colostrum contains a high proportion of SA which is reduced by 3 days postpartum (Merin et al., 2001).

1.2.3.1 Characteristics The serum albumin in milk is physically and immunologically identical to blood SA which is a major blood protein. It is not synthesized in the mammary gland but transported into the milk via passive leakage from the bloodstream. It is believed that SA leaks into milk due to the deterioration of tight junctions between maternal epithelial cells (Wynn, Morgan, & Sheehy, 2011). Initially it was calculated that BSA contained 582 amino acids and had a molecular weight of 66,267.1 Da but in 1990 it was determined to contain 583 amino acids with a molecular weight of 66,465.8 Da (Hirayama, Akashi, Furuya, & Fukuhara, 1990). The complete amino acid sequences of several mammalian SAs are known, including for human and bovine (Carter & Ho, 1994). The sequence homologies are high among SAs. For example, the sequence identities between human SA and BSA is 76% (Peters, 1985). SA contains no phosphorus, has 17 disulfides, and 1 free sulfhydryl group. SAs contain a high percentage of charged amino acids and lower percentages of tryptophan, glycine, and methionine (Peters, 1985). SAs have an isoionic point of pH 5.13 and an isoelectric point between pH 4.7 and 4.9 (Farrell et al., 2004).

1.2 Major Whey Proteins

1.2.3.2 Structure The three-dimensional crystal structure of horse SA was identified in 1993 (Ho, Holowachuk, Norton, Twigg, & Carter, 1993). The Protein Data Bank also contains several structural entries for human SA. The structure of bovine and rabbit SA were identified in 2012 (Majorek et al., 2012) and added to the Protein Data Bank. SA is a highly structured but flexible nonglycoprotein. SAs have been identified as heart-shaped and comprising three helical domains (I, II, and III). These domains consist of nine disulfide loops formed by the 17 disulfides (Carter & Ho, 1994). Each helical domain is further subdivided into two subdomains, leading to six subdomains per molecule of SA.

1.2.3.3 Function SA is described as a multifunctional protein with extraordinary ligand binding capacity (Majorek et al., 2012). It is best known for its bonding of fatty acids but also binds flavor compounds and metal ions. The solubility of fatty acids bound in this way is markedly increased. BSA binds a range of fatty acids but Spector, John and Fletcher (1969) reported that the C16 and C18 acids bound more strongly than lauric acid (C12). SA also binds long-chain hydrocarbons but the binding is much weaker than that of fatty acids of equivalent chain length. This strongly indicates a key role of the carboxylate group in the binding. Because of its fatty acid binding properties, BSA is often used in lipase assays to absorb the released fatty acids and reduce product inhibition in the reaction. For this purpose, BSA is defatted using, for example, activated charcoal. BSA with bound fatty acids has increased stability to urea denaturation, heat denaturation, and tryptic hydrolysis. It also has improved emulsification properties. This is attributed to the reduction in surface hydrophobicity. Hence the binding of fatty acids could improve the properties of BSA as a food material (Saito, Ogasawara, Nasu, Monma, & Chikuni, 1995). BSA also binds calcium ions (Bthree per protein molecule).

1.2.3.4 Significance Of the proteins discussed in this chapter, SA, or more specifically BSA, has the most limited potential as an industrial product. BSA is extensively used in biochemical and biomedical research as a model SA but only relatively small quantities are used for this purpose. As pointed out in Chapter 17, BSA also has limited therapeutic potential; however, some studies have indicated it may have cancer-inhibiting properties and that peptides produced by hydrolysis may have beneficial bioactivities. The level of BSA in milk is elevated during a mastitic infection and hence can be used as an indicator of mastitis. However, as Sheldrake, McGregor and

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Hoare (1983) reported, it is not as good an indicator as somatic cell count which is used universally for this purpose. Cows’ milk protein allergy (CMPA) affects 2%
7.5% of children under 3 years of age. In general, these children are most allergic to β-lactoglobulin, α-lactalbumin, and casein but some are allergic to BSA (Rangel et al., 2016). According to Mousan and Kamat (2016), about 13%
20% of children with CMPA are allergic to BSA. However, some authors have reported BSA to be more significant. For example, Wroblewska and Kaliszewska (2012) concluded that the allergenicity was mainly correlated with BSA, lactoferrin, and α-casein.

1.2.4

Immunoglobulins

Immunoglobulins (Igs), or antibodies, in colostrum and milk are an important component of the defence system of the young animal against pathogenic bacteria and viruses and other toxins. They have been extensively studied in many species and reviewed by several authors (e.g., Hurley & Theil, 2013; Marnila & Korhonen, 2011). This section focuses on the Igs in bovine milk. Further information on Igs is provided in Chapter 12 in relation to infant formula and Chapter 17 in relation to functional foods.

1.2.4.1 Characteristics Igs are present in milk in various forms. The major forms are IgG, IgA, and IgM. IgG, the major form in bovine milk and colostrum, exists as IgG1 and IgG2. Each form has the same basic structure but whereas IgG exists in monomeric form with a molecular weight (MW) of B160 kDa, IgA exists as dimers (MW of B370 kDa), as well as tetramers, and IgM exists as a pentamer with a molecular weight of 900
1000 kDa. The levels of the four forms in bovine milk and colostrum are given in Table 1.1. Table 1.1 Immunoglobulins in Bovine Colostrum and Mature Milk (g/L) Immunoglobulin

Colostrum

Milk

Total IgG IgG1 IgG2 IgA IgM Total immunoglobulin

60 (20
200) 15
180 1
3 3.5 (1
6) 5 (3
9) Up to 200

0.47 (0.15
0.8) 0.35 (0.3
0.6) 0.02
0.12 0.05
0.4 0.04
1.0 0.6
1.0

From Marnila, P., & Korhonen, H. (2011). Immunoglobulins. In (2nd ed). J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Volume 2). San Diego, CA:Academic Press, pp 807
815.

1.2 Major Whey Proteins

While the immunoglobulins are classed as whey or serum proteins, some are also associated with other milk fractions. Some IgM is associated with the fat globule and casein, while some IgA and IgG2 is associated with the fat globule and casein, respectively. However, they are mostly present in the whey/ serum. Immunoglobulins are relatively stable to heat. Dominguez, Perez and Calvo (1997) reported that IgG was stable to HTST pasteurization and had a D81 Cvalue of 152 s and a z-value of 6.6 C. Their IgG was from colostrum of cows immunized with peroxidase; they determined the IgG level by competitive ELISA using antiperoxidase coated to a plate. However, Igs are not stable to UHT treatment. Fukumoto, Skura and Nakai (1994) isolated IgG from normal cows’ milk using immobilized metal affinity chromatography and, after microfiltering a solution of it through a 0.2 μm membrane, added it to UHT milk. This enabled them to test its stability during storage of the milk and also to determine its D- and z-values in the temperature range 62
80 C. They reported that the IgG (measured by radial immunodiffusion which is based on the protein’s antigen binding) did not decline during storage at 4 C, 25 C, or 35 C over 5 months. Furthermore, they found the D80 C to be 258 s and the z-value to be 4.3 C.

1.2.4.2 Structure The molecular structures of Igs are very complex. An outline is given here but the reader is referred to other publications for more detailed descriptions (e.g., Hurley & Theil, 2013). Schematic representations of the structures are shown in the review by Marnila and Korhonen (2011). The basic (monomeric) structure of the immunoglobulins, as in IgG, consists of two identical light chains (B23 kDa) and two identical heavy chains (B53 kDa). Both the heavy and light chains contain constant (CH and CL) and variable (VH and VL) sections. The light chains are linked by disulfide bonds and the heavy chains are linked to the light chains by disulfide bonds. The result of the linkages is a Y-shaped molecule. IgA also has a junction component, the so-called J chain (B15 kDa) which links the chains near the C-terminal region of the heavy chains. The five units of IgM are arranged in a cyclic arrangement and are held together by one J chain and disulfide bonds. The basic Ig structure has two identical antigen-binding regions. Each is formed by the N-terminal part of a heavy chain and the variable region of a light chain. Igs are glycoproteins with carbohydrates being linked to constant regions in the C-terminal part of the heavy chains. IgG1 and IgG2 have approximately three carbohydrate molecules per protein molecule, IgA has 6
10, and IgM

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has 10
12. The major carbohydrates are hexoses (mannose, galactose, glucose), hexosamines, fucose, and sialic acid.

1.2.4.3 Significance Their major significance is their role in the immune system of the young animal. As shown in Table 1.1, the levels in colostrum are much higher than those in mature milk. When a calf is born, it has virtually no blood Igs and relies on the colostrum to obtain passive immunity in the early stages of its life. Normally in the mammary gland the epithelium sheet between the milk and interstitial spaces prevents most of the immunoglobulins from entering the milk; however, prepartum, the epithelium allows a considerable amount of immunoglobulins to pass into the milk from the blood in readiness for supplying the immune-deficient calf with antibodies. Several studies have shown milk immunoglobulins are effective against rotovirus and other infections. Marnila and Korhonen (2011) summarized the results of eight such studies. The immunoglobulins can be obtained from the colostrum of cows which have been immunized with human rotovirus. Brüssow et al. (1987) reported that the antibody titer could be increased 100-fold by this technique while Kramski et al. (2012) reported that B500 g/L of IgG can be obtained from an immunized cow. It has been suggested such preparations could be used for inducing passive immunity to rotovirus gastroenteritis in children and that they could be included in infant formulae (Goldman, 1989). Tawfeek, Najim and Al-Mashikhi (2003) demonstrated the effectiveness of infant formula with added Igs in reducing diarrhea in children in Iraq. Milk immunoglobulins are now being incorporated into functional foods. As shown in Chapter 17, one commercial product is promoted on the basis of containing a high level of immunoglobulins (from colostrum) which reduce the risk of gastrointestinal disorders including travelers’ diarrhea. Igs in colostrum can be treated by high pressure processing at low pH and retain their activity (Carroll, 2008). Ready-to-drink acidified colostrum products produced in this way are manufactured commercially.

1.3

MINOR WHEY PROTEINS

The proteins included here are typically present in mature bovine milk in concentrations of ,B300 mg/L. The concentrations of some of the enzymes, in particular, are very low, e.g., ,1 mg/L. To facilitate comparison of the different minor proteins and enzymes, their characteristics are summarized in Tables 1.2 and 1.3, respectively.

Table 1.2 Summary of Minor Bovine Milk Whey Proteins

Protein

Molecular Weight/Number of Amino Acids

Concentration

Protein Type

Lactoferrin

80,000/689

20
500 mg/L

Glyscosylated metalloprotein

Stable to heating at pH 4.0 and 90 C for 5 min

8.7

Antibacterial; binds two atoms of iron per molecule

Osteopontin

29,283/262Migrates as 60,000 on electrophoresis due to glycosylation

18
22 mg/L

Glycosylated, phosphorylated protein

Stable to heating at 95 C for 30 min

4.3

Has some beneficial health effects

PP3

28,000/138

200
300 mg/L

Glycosylated, phosphorylated protein; several isoforms

Stable to heating at 95 C for 30 min

3.5, 4.0

Emulsifier; inhibitor of lipolysis

GMP

6755
10,000/ 64 Da (theoretical); 20,000
50,000 Da (apparent by gel filtration)

Low natural concentration in milk(B4 mg/L); 20
25 g/100 g protein in rennet cheese whey

Mixture of nonglycosylated, nonphosphorylated and glyscosylated and phosphorylated proteins

Heat stable

# 4.0

Contains no phenylalanine
suitable for phenylketonuria sufferers

Heat Stability

pI

Main Significance

Other Binds one HCO32 per iron atom; Isolated by cation exchange chromatography
often with lactoperoxidase; proteolytic products, e.g., lactoferricin are more antibacterial than parent protein Acidic protein; isolated by anion exchange chromatography; now manufactured commercially Hydrophobic protein; isolated by hydrophobic (e.g., phenylSepharose) or affinity (concanavalin ASepharose) chromatography Isolated by anioic ion exchange

Table 1.3 Summary of Bovine Milk Soluble (Whey) Enzymes

Protein

Molecular Weight (Da)/No. of Amino Acids

Concentration/ Activity in Milka

Protein Type

pH Opt

Heat Stability

Lactoperoxidase (EC 1.11.1.7)

78,030/612

30 mg/L/ 0.73 U/mL

Glycosylated metalloprotein

5
6

Survives HTST, inactivated at 78 C for 15 s

Lysozyme (EC 3.2.1.17)

18,000/154

Single polypeptide chain

7.5

Cathepsin D (EC 3.4.23.5) system

Mature cathepsin D:39,000/346; procathepsin: 45,000, 46,000/474; active pseudocathepsin, 43,000/ 473; heavy chain mature, 31,000/245 and light chain mature, 13,000
14,000/ 99 42,000 (a minor acid phosphatase of Mr 37,000 has been isolated from a lactoferrin-rich fraction)

, 3 mg/L
much lower than in human, horse & donkey milk/ 2.03 U/mL 0.4 mg/L

Glycoprotein, aspartic enzyme

3
4

75% survival at 80 C for 15 s; more stable at pH 3
4 than at $ 7 8% survives HTST, inactivated at 70 C for 10 min

0.13 U mL21

A basic glycoprotein

4

11
40 mg/L /32 U/mL

B is a glycoprotein, A contains no carbohydrate

7.0
7.5

Acid phosphatase (EC 3.1.3.2)

Ribonuclease I (A&B) (EC 3.1.27.5) (From Bingham & Zittle, 1962, 1964; see also Table 1.4) a

A: 13,700/B: 14,700 6 300/124

the enzyme activities are taken from Griffiths (1986).

80%
90% survives pasteurization, 63 C for 30 min; inactivated at 88 C for 10 min or 100 Cfor 1 min In milk: 40% loss at 80 C for 15 s; stable in acid whey pH 3.5, at 90 C for 20 min

Main Significance Forms antibacterial system with H2O2 and thiocynanate or halide; marker for highr-pasteurized milk Anibacterial, especially against Gram-positive bacteria

Other Contains a heme group; pI 9.6; isolated by cation exchange chromatography

pI 9.5; isolated by cation exchange, chromatography

Contributes to proteolysis in cheese through hydrolysis of caseins

Inactive procathepsin D is the major form in milk; isolated by pepstatinSepharose affinity chromatography;

Mastitis marker, marker for superpasteurized milk

pI 7.9; isolated by cation exchange resin;

No technological significance; important biological functions

pI B9.0; often isolated with Lf by cation ion exchange chromatography

1.3 Minor Whey Proteins

1.3.1

Lactoferrin

1.3.1.1 Characteristics Although a minor protein in bovine milk, lactoferrin has attracted considerable attention because of its antibacterial properties and many other bioactivities. It also occurs in several other biological fluids including tears, synovial fluid, and saliva. The biochemical properties and biological functions of lactoferrins, particularly those in bovine and human milk, have been reviewed by several authors including Korhonen and Marnila (2011), Lönnerdal and Suzuki (2013), and Jhaveri and Arya (2015). Lactoferrin occurs in bovine milk in low concentration (0.02
0.5 g/L); however, human milk contains a much higher concentration (1.5
2.0 g/L). Sheep milk lactoferrin has been reported to have 0.8 g/L while a wide range of levels has been recorded for buffalo and camel milk (0.03
3.4 g/L and 0.02
7.28 g/L, respectively) (Claeys et al., 2014). Higher levels are found in colostrum, mastitic milk, and fluid from the involuting mammary gland. Bovine colostrum contains an average of 1.5 g/L (range 0.2
5 g/L) and human colostrum contains up to 16 g/L. Milk from mastitic cows was reported to have a lactoferrin level of 1.2 g/L, compared with that of corresponding healthy cows of 0.09 g/L. The fluid from the involuting mammary gland can have concentrations of 20
50 g/L (Dionysius & Milne, 1997; Korhonen & Marnila, 2011). Lactoferrin is an iron-binding glycoprotein with a molecular weight of B80,000 Da. The bovine protein has 689 amino acids and the human protein has 691. It is similar to the iron-binding protein, transferrin, which is in blood and also milk; however, the iron-binding capacity of lactoferrin is much greater than that of transferrin which is an iron carrier in the blood. Lactoferrin consists of a single polypeptide chain arranged in two highly homologous lobes linked by an α-helix structure. Each lobe contains a ferric iron-binding site. It has 16 intramolecular disulfide bonds but no free sulfhydryl groups. Lactoferrin can exist in forms which are fully (holo-form) or partially saturated with iron, and the iron-free form, the apo-form. In its native form, it is partially saturated (6%
12% in the human protein and 5%
30% in the bovine protein). Iron can be removed from the protein by dialysis against 0.1 M citric acid followed by extensive dialysis against water. This form can be used to prepare lactoferrin saturated with iron or other metal ions such as zinc, cobalt, manganese and copper. The iron-saturated form can be produced by titrating the apo- or low-iron form with ferrous ammonium sulfate stabilized by ascorbic acid. The saturation level can be monitored by the absorbance at 450 nm as the iron content is positively correlated with the intensity of the red color. The apo-form is colorless and hence does not

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absorb at 450 nm. Naturally occurring lactoferrin with a low iron saturation level has a salmon color. The presence of bicarbonate greatly enhances the iron binding. Bicarbonate or carbonate ions are bound to lactoferrin in the ratio of 1:1 with the metal ion (Masson & Heremans, 1968; Dionysius, Grieve, & Milne, 1993). Bovine lactoferrin exists in two different glycosylated forms. Both forms are N-glycosylated at Asn233, Asn368, Asn476, and Asn545 but variant A is also N-glycosylated at Asn281. Variant A constitutes about 30% of colostrum lactoferrin and about 15% of mature milk lactoferrin (van Veen, Geerts, van Berkel, & Nuijens, 2004). The glycans are varied and complex and are a mixture of high-mannose, complex, and hybrid types; up to 59 different glycan structures have been identified. The glycans are more complex in colostrum and their composition varies from early to late lactation. The sialic acid, N-glycosylneuraminic acid, occurs more in early lactation milk while latelactation milk contains an abundance of oligomannose type glycans; the glycan Man8GlcNAc2 has been reported to account for 38% of these glycan structures (O'Riordan et al., 2014). Lactoferrin, like lactoperoxidase, is a basic protein with an isolectric point (pI) of 8.7; lactoperoxidase has an isoelectric point of 9.6. The theoretical pI of bovine lactoferrin based on its amino acid composition is 9.4. Hence at pH values lower that its pI, such as those in milk and other physiological situations, lactoferrin has a positive charge and can bind to negatively charged compounds such as heparin, lysozyme, and casein. Interestingly, lysozyme acts synergistically with lactoferrin in antibacterial activities. Because of their high isoelectric point, lactoferrin and lactoperoxidase can be readily isolated from whey by ion exchange chromatography (Dionysius, Herse, & Grieve, 1991). This produces a product containing both lactoferrin and lactoperoxidase which are difficult to separate. They even move together as one band on 1D-polyacrylamide gel electrophoresis (PAGE). They can however be separated using their different binding affinities to ion exchange resins. This can be achieved with a step-wise gradient elution protocol using 0.2 M NaCl and 0.5 M NaCl. Furthermore, high yields of lactoferrin but low yields of lactoperoxidase are obtained from ion exchange treatment of wheys with high conductivities, e.g., rennet and acid whey produced during production of rennet and acid casein. Whey with a conductivity of $15 mS yields a product which is almost entirely lactoferrin (Dionysius et al., 1991). It has been reported that lactoferrin has some ribonuclease activity (Ye, Wang, Liu, & Ng, 2000). While this may be the case, it should be noted that 2D PAGE electrophoresis of some commercially produced lactoferrins has shown the presence of the ribonuclease protein (J. Holland, Pers. Com.).

1.3 Minor Whey Proteins

Lactoferrin is relatively resistant to proteolytic enzymes with the ironsaturated form being more resistant than the apo-form. Furthermore, the form of glycosylation can affect proteolysis. For example, bovine lactoferrin variant B is 10 times more susceptible to trypsin proteolysis than the A variant and this has been attributed to the additional glysosylation site at Asn281 which protects the A form from trypsin attack at Lys282 (van Veen et al., 2004). Because of the resistance of ovine lactoferrin to proteolytic attack, it has been suggested that this property may enable bovine lactoferrin to remain active in the adult human gastrointestinal tract (Lönnerdal & Suzuki, 2013). However, bovine lactoferrin can be proteolyzed at low pH by pepsin or heat and these conditions have been used to prepare biologically active peptides from lactoferrin, sometimes called lactoferricins. Typically, a lactoferrin solution is reacted with pepsin at pH 2.5 at 37 C for 4 h. From such a digestion, Dionysius and Milne (1997) isolated three cationic antibacterial peptides which originated from the N-terminus of lactoferrin. Peptide I corresponded to residues 17 to 42, peptide II consisted of residues 1 to 16 and 43 to 48 linked by a disulfide bond, and peptide III corresponded to residues 1 to 48 cleaved between residues 42 and 43; the cleaved sections were disulfide-linked. Peptide I was almost identical to a previously reported peptide, lactoferricin. Interestingly, the peptides from lactoferrin show higher antimicrobial activity than the parent lactoferrin. The antimicrobial activity of these peptides is correlated with their positive charge (Pihlanto, 2011). Furthermore, these peptides do not contain iron, indicating the antimicrobial activity of lactoferrin is not solely related to its ironbinding activity as initially thought. Lactoferrin can be analyzed by HPLC on a cation exchange column such as a Mono S column (Dionysius et al., 1993). It can also be analyzed by ELISA methods using either polyclonal or monoclonal antibodies (Dupont, Croguennec, Brodkorb, & Kouaouci, 2013).

1.3.1.2 Significance Since the discovery of its multiple biological functionalities, lactoferrin has been isolated from milk and whey for use as a commercial product in various applications. A sizable commercial market for bovine lactoferrin has developed which is expected to increase in coming years. In most cases, the commercial product is a mixture of lactoferrin and lactoperoxidase which are both basic proteins with similar biological functionalities. According to one 2014 report (www.stuff.co.nz/business/farming/dairy/63702051/Tatua-dairycompany-punches-above-weight), lactoferrin sells for up to US$ 420,000/ tonne. Several dairy companies are key players in the lactoferrin market including Fonterra Group, Bega Cheese, FrieslandCampina (DMV), Milei

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Gmbh, Glanbia Nutritionals, Westland Milk, Tatua, Synlait Milk, WBC, Murray Goulburn, and Ingredia Nutritional (360 Market Updates, 2017). The targeted functionalities include antibacterial, iron absorption, antiinflammatory, intestinal flora protection, immune cell stimulation, and antioxidant. The major market segments for lactoferrin are food products, infant formula, sports and functional food (e.g., prebiotic drinks and immune supplements), pharmaceuticals, veterinary and feed specialties, and personal care products (O'Riordan et al., 2014; Ubic Consulting, 2015).

1.3.2

Proteose Peptone 3

1.3.2.1 Introduction The proteose peptone fraction of milk is defined as the proteins/peptides soluble in 12% trichloroacetic acid (TCA) after milk has been heated to 95 C for 30 min and acidified to pH 4.6. Heating followed by acidification precipitates caseins and the major whey proteins denatured by the heat treatment (O’Mahony & Fox, 2013). Proteose peptones account for 0.8
1.2 g/L of milk (Claeys et al. 2014; Sørensen & Petersen, 1993b). Proteose peptones are a mixture of proteins/peptides. They can be classified as those resulting from proteolysis of caseins and those indigenous to milk. The major products of proteolysis in this fraction are proteose peptone 5 (PP5), proteose peptone 8 (slow) (PP8s), and proteose peptone 8 (fast) (PP8f). These are, respectively, the following fragments of β-casein resulting from plasmin-catalyzed proteolysis: f1-105/107, f29-105/107, and f1-28. Other minor fractions such as f29-105 and f29-107 have been identified; several other fractions are present in low concentrations. As these peptides are casein proteolytic products they can be considered with the caseins (O’Mahony & Fox, 2013). They are not considered further in this chapter. The main proteose peptones which are indigenous to milk are proteose peptone 3 (PP3) and osteopontin. PP3 is discussed in this section and osteopontin is discussed in the next section.

1.3.2.2 Characteristics of PP3 Proteose peptone 3 (PP3), sometimes called lactophorin, is a heat-stable, phosphorylated glycoprotein. It is a major component of the total proteose peptone fraction of bovine, goat and yak milk accounting for B25% (Paquet, 1989; Sørensen & Petersen, 1993b), i.e., 0.2
0.3 g/L in bovine milk. It does not appear to have been found in human milk (Campagna, Mathot, Fleury, Girardet, & Gaillard, 2004). Bovine milk PP3 has 138 amino acid residues and an apparent molecular weight of 28,000 Da. It is phosphorylated at Ser29, Ser34, Ser40, and Ser46,

1.3 Minor Whey Proteins

O-glycosylated at Thr16 (50%) and Thr86, and N-glycosylated at Asn77 (Sørensen & Petersen, 1993a). Sørensen and Petersen (1993a) also reported a low-molecular-weight (18,000 Da) fraction (f54-135) which they suggested was formed by plasmin hydrolysis of PP3. Two glycosylation sites (Thr86 and Asn77) are located in this fragment. Lister et al. (1998) reported similar characteristics for caprine PP3 whose amino acid sequence has 88% identity with the bovine PP3 sequence. It has 136 amino acids. It is phosphorylated at Ser30 and Ser41, O-glycosylated at Thr16 (partial), and N-glycosylated at Asn78. As for bovine PP3, the glycans contained galactosamine and glucosamine. Girardet et al. (1995) made a detailed study of the N-linked glycans of PP3 and demonstrated their extreme complexity. The monosaccharides identified were fucose, galactose, mannose, N-acetylgalactosamine, N-acetyl glucosamine, and N-acetyl neuraminic acid. These were arranged in several different glycans with up to 10 monosaccharide components each. Coddeville et al. (1998) investigated the O-linked glycans and identified the following glycans linked to the Thr86: GalNac; Gal(β 1-3)GalNAc; and Gal(β 1-4)GlcNAc(β16)[Gal(β 1-3)]GalNAc. As a result of this glycosylation complexity, together with phosphorylation, PP3 is a mixture of several different isomers. This has been demonstrated by isoelectric focussing on a pH 2-5-10 gradient which resulted in the separation of numerous bands between pH 3 and 6; there were two major ones with calculated pI values of 3.5 and 4 (Paquet, 1989). A similar separation of isomers due to post-translational phosphorylation and glycosylation has been reported for κ-casein (Holland, Deeth, & Alewood, 2006). PP3 is a hydrophobic protein and this enables it to be separated by hydrophobic interaction chromatography on, for example, phenyl-Sepharose (Paquet, 1989) and epoxy-Sepharose (Sousa et al., 2008). It is sometimes called the hydrophobic component of the proteose peptones as its hydrophobicity is a feature used to separate it from the other PP components. As a result, it readily aggregates. On Sephacryl S 200 gel filtration at pH 8, PP3 eluted as the fraction with molecular weight of 270,000 Da (Paquet, 1989). Another technique used to separate PP3 from other PP components is affinity chromatography on concanavalin A-Sepharose (Coddeville et al., 1998; Girardet et al., 1995).

1.3.2.3 Significance of PP3 As indicated above, PP3 can be readily isolated from the proteose peptone fraction in milk using hydrophobic chromatography and/or affinity chromatography, such as on concanavalin A-Sepharose. Its level in milk at 0.2
0.3 g/L is much higher than either lactoferrin or osteopontin, discussed

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above, which are extracted and marketed commercially. A functionality which could have commercial application is its emulsification ability. The total proteose peptone fraction from milk has useful emulsification properties but PP3 produces more stable emulsions in ice-cream and in soybean oil emulsification (Innocente, Comparin, & Corradini, 2002; Innocente, Corradini, Blecker, & Paquot, 1998a; Innocente, Corradini, Blecker, & Paquot, 1998b). The optimum concentration in an emulsification trial with soybean oil was 10 g/L. Its emulsification activity was at least as good as that of β-Lg (Innocente et al., 1998b). PP3 has also been found to be an inhibitor of lipolysis. Several authors have reported that PP3 inhibits lipase action in assays of purified bovine milk lipoprotein lipase using an emulsified triglyceride substrate such as Intralipid, as well as spontaneous lipolysis in whole milk (Anderson, 1981; Cartier, Chilliard, & Paquet, 1990). Similar inhibition of yak milk lipoprotein lipase by yak milk PP3 has also been reported (He et al., 2012). In the trials using assays of purified lipoprotein lipase, the PP3 was incubated with the substrate prior to the assay being performed. Anderson (1981) showed that the PP3 had no effect on the in vitro activity of milk lipoprotein lipase and its stability and concluded that PP3 does not interact with lipase or a lipase activator and that the inhibition was due to interaction with the substrate. Other authors have come to a similar conclusion, including Girardet, Linden, Loye, Courthaudon and Lorient (1993) following a study of PP3 inhibition of pancreatic lipase activity in an assay with tributyrin as substrate. They concluded that PP3 preferentially adsorbs to the oil
water interface causing a decrease of interfacial tension and preventing the adsorption of lipase. This phenomenon led Campagna et al. (1998) to investigate which part of the PP3 molecule was most likely to adsorb to hydrophobic surfaces. They identified the f119-135 region of PP3 as the lipid-binding motif. Interestingly, Campagna et al. (2004) produced a 23amino acid peptide f113-135 (termed lactophoricin) which had a low level of antibacterial activity, especially against Gram-positive bacteria.

1.3.3

Osteopontin

1.3.3.1 Characteristics Osteopontin occurs in many tissues and secretions but the highest concentrations are found in milk. Its concentration in milk is 0.018
0.022 g/L. Human milk contains B0.138 g/L. (Christensen & Sørensen, 2016; Schack et al., 2009; Sørensen & Petersen, 1993b). A review on “Osteopontin
a molecule for all seasons” was published by Mazzali et al. (2002). It is an acidic, phosphorylated, glycoprotein with two major isoforms: one of 60,000 Da, which is considered to be bovine osteopontin, and a truncated form of 40,000 Da (Bissonnette, Dudemaine, Thibault, & Robitaille, 2012).

1.3 Minor Whey Proteins

The 60,000 Da form corresponds with the protein of the same molecular weight which Sørensen and Petersen (1993b) isolated from the proteose peptone fraction of milk, and identified as osteopontin. Bayless, Davis and Meininger (1997) also reported isolation of ostepontin from bovine milk with a molecular weight of 60,000 Da. However, the molecular weight based on the 262 amino acids present has been calculated as 29,283 Da (Wynn & Sheehy, 2013). Reviews of the structure, function, and nutritional potential, and of the biological roles of milk osteopontin have been recently published by Christensen and Sørensen (2016) and Jiang and Lonnerdal (2016), respectively. It has two heparin-binding sites and an Arg-Gly-Asp sequence which mediates cell attachment. Through its many negatively charged amino acids and phosphate groups it binds calcium and forms soluble osteopontin 2 calcium complexes. Milk osteopontin contains a large number of serines and threonines which may be glycosylated or phosphorylated. In bovine osteopontin, 27 serines and one threonine are phosphorylated; in human milk osteopontin, the phosphates are attached to 34 serines and two threonines (Jiang & Lönnerdal, 2016). Bovine osteopontin has three O-glycosylated threonine sites (Thr115, Thr124, and Thr129) while the human milk protein has these same three sites plus two additional threonine-linked oligosaccharides. The glycans of bovine osteopontin have a disialylated GalNAc-galactose core while the glycans in the human protein are large fucosylated Nacetyllactosamine units (Christensen & Sørensen, 2016).

1.3.3.2 Significance It has been reported to have numerous biological roles including in angiogenesis, apoptosis, inflammation, wound healing, tumor metastasis, immune function modulation, and, possibly, bone mineralization and growth (Wynn & Sheehy, 2013). It has a high affinity for lactoferrin (dissociation constant 1026) and may act synergistically with lactoferrin (Jiang & Lönnerdal, 2016). Oral administration of bovine osteopontin has been shown to produce “promising results” when fed to infants. It has also been shown to protect against alcohol-induced injury in mice (Christensen & Sørensen, 2016). Jiang and Lonnerdal (2016) concluded that human osteopontin is essential for the immunological, intestinal, and cognitive development of young infants and that addition of bovine milk osteopontin to infant formula may enable formula-fed infants to perform more like breast-fed infants. Osteopontin can be isolated from bovine whey by anion exchange chromatography. It is now commercially available (e.g., “Lacprodan OPN-10,” Arla

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Foods Ingredients, Viby, Denmark) and has been used in several animal and infant feeding trials (Christensen & Sørensen, 2016).

1.3.4

Glycomacropeptide

1.3.4.1 Characteristics Glycomacropeptide (GMP) is naturally present in bovine milk in low concentration (B4.3 mg/L) (Furlanetti & Prata, 2003) but is best known as a component of sweet cheese whey where it represents a substantial proportion (20%
25%) of the total whey protein, B1.2 g/L. It derives from κ-casein through the action of chymosin in the rennet used in cheesemaking. It has attracted considerable attention recently for its nutritional/health effects and its technological properties; these have been reviewed by Thomä-Worringer, Sorensen and Lopez-Fandiño (2006) while the chemical and functional aspects of GMP were reviewed by Neelima, Sharma, Rajput and Mann (2013). During cheesemaking, chymosin cleaves κ-casein at the Phe105 2 Met106 bond into a 105-amino acid N-terminal peptide, para-κ-casein, and a 64amino acid C-terminal peptide, GMP. The GMP concentration in sweet cheese whey is 9
15 times higher than the free GMP content of normal milk. Higher levels of the natural free GMP occur in colostrum and mastitic milk (Furlanetti & Prata, 2003). In addition, milk in which substantial growth of pseudomonads has occurred before heat treatment may also have elevated levels of GMP because some Pseudomonas proteases cleave κ-casein at or near the Phe105 2 Met106 bond, as evidenced by the presence of paraκ-casein (Miralles, Amigo, Ramos, & Recio, 2003). κ-casein is a very complex protein due to genetic variation and posttranslational phosphorylation, glycosylation, and disulfide linkages. For example, Holland et al. (2006) identified 17 isoforms in one genetic variant (from the milk of a cow homozygous for the B variant) of κ-casein, and since most milk contains at least two genetic variants, A and B, the total number of variants is at least twice this number. A total of 11 genetic variants have been reported and hence the total number of isoforms is large. Since phosphorylation occurs at Ser149, Ser 121 and Thr145, and glycosylation occurs at Thr131, Thr142, Thr 133, Thr 145, Thr121, and Thr165, all post-translational modifications occur in the C-terminal end of κ-casein and hence are present in GMP. In addition, the two amino acid substitutions between the major genetic variants, A and B, (Thr136 to Ile136, and Asp148 to Ala148) also occur in the C-terminal end and hence in GMP. A further factor which increases the complexity of κ-casein is variation in intra- and interprotein disulfide bonding between Cys11 and Cys88 (Holland, Deeth, &

1.3 Minor Whey Proteins

Alewood, 2008). However, as both of these amino acids are in the Nterminal end, they do not carry through to GMP. About half of the GMP molecules are glycosylated. Those without carbohydrates are sometimes referred to as caseinomacropeptide (CMP). The carbohydrates in GMP are all O-linked to threonine residues and are mostly composed of the monosaccharides galactose, N-acetylgalactosamine, and Nacetylneuraminic acid (sialic acid). Because of the presence of sialic acid, the level of soluble GMP is often determined by the level of this acid. For example, Furlanetti and Prata (2003) reported that the mean level of free naturally occurring GMP in milk was 3.3 mg/L of sialic acid compared with that of GMP released by rennet of 34.3 mg/L of sialic acid. The monosaccharides are arranged in glycans containing four (56%), three (36.9%; 18.5% branched, 18.4% linear trisaccharide,), two (6.3%), and one (0.8%) monosaccharides (Saito & Itoh, 1992). The amino acid composition is unique in that it contains very low levels of the aromatic amino acids. Their percentages of the total protein (compared with those of total whey protein) are: tyrosine, 0.1% (2.9%); phenylalanine 0.5% (3.1%); and trptophan 0.0% (2.4%). In addition, GMP has no cysteine whereas whey protein, as in WPI, has 2.3% Pellegrino, Masotti, Cattaneo, Hogenboom, & de Noni, 2013). The very low level of aromatic amino acids makes it an attractive protein for people suffering from phenylketonuria, a serious inherited disease in which phenylalanine cannot be metabolized. In some countries every baby at birth is tested for this metabolic disorder. GMP has a pI of # 4 and hence can be isolated from sweet whey with anion exchange chromatography. An interesting practical point is that due to the extremely low level of aromatic amino acids, it is not possible to monitor release of GMP by measuring absorbance at 280 nm. On the other hand, any absorbance at 280 nm is a measure of impurities present. The highly anionic nature of the glycosylated forms of GMP enables it to be separated from the nonglycosylated forms by formation of complexes with the polycationic polysaccharide, chitosan. Membrane technology has also been used to separate GMP from other whey proteins. However, this is challenging as the apparent molecular weight of GMP is pH-dependent due to self-association at higher pH. Although the theoretical molecular weight is between 7000 and 10,000 Da, at pH 7 the molecular weight according to gel filtration was 20,000 to 50,000 Da; at pH 3.5 it was 10,000 to 30,000 Da, depending on the degree of glycosylation. At pH 4.0, GMP permeates an ultrafiltration membrane while other whey proteins are retained (Kawasaki et al., 1993).

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FIGURE 1.2 Typical reversed phase-HPLC chromatogram of bovine GMP. 1, glycosylated GMP isoforms (genetic variants A & B); 2, nonglycosylated GMP (genetic variants A & B); 3, monoglycosylated GMP (genetic variant B); 4, nonglycosylated GMP (genetic variant B). From Thomä-Worringer et al., 2006, reproduced with permission from Elsevier.

Analysis of GMP is challenging because of its heterogeneous nature. No procedure gives a clear picture of all the components but HPLC gives a reasonable separation of some groups of components (Fig. 1.2) (Thomä-Worringer et al., 2006). It should be noted however, that since GMP is the only whey/ serum protein containing sialic acid (N-acetylneuraminic acid), analysis of this carbohydrate gives an indirect measure of GMP. A high correlation (r 5 0.996) has been found between values obtained from HPLC and those of sialic acid determined by the spectrophotometric method using the reaction with ninhydrin (Furlanetti & Prata, 2003).

1.3.4.2 Significance As indicated above, GMP constitutes a substantial proportion of the proteins in sweet whey and hence any economically viable application for it will greatly improve the financial returns from whey. Besides its possible beneficial application in the diet of phenylketonurics (Ney et al., 2016), GMP also has potential for several other biological roles including in oral hygiene and clinical nutrition (DeBoer, 2014; Thomä-Worringer et al., 2006). It also has physical functionality such as emulsification and foaming. It has good emulsifying capacity at alkaline pH but a minimum capacity at pH 4.5
5.5. A 10% GMP solution was shown to have good foamability, i.e., produced high overrun, but poor foam stability compared with egg white (Thomä-Worringer et al., 2006).

1.3 Minor Whey Proteins

1.3.5

Lactoperoxidase

1.3.5.1 Characteristics Lactoperoxidase (EC 1.11.1.7) (LPO) belongs to a family of peroxidase enzymes which are ubiquitous in both the plant and animal kingdoms. Lactoperoxidase occurs in milk and is present in the milk of virtually all species investigated. In bovine milk it is the second most abundant enzyme after xanthine oxidase, which is associated with the milk fat globule membrane; therefore, LPO is the most abundant soluble enzyme in bovine milk. Its concentration is around 0.03 g/L, or about 0.5% of the total whey proteins. However, the amount of LPO is often expressed in terms of its enzyme activity. Bovine milk is reported to have 1.2
19.4 U/mL, about 20 times the activity of the enzyme in human milk (Buys, 2011). This enzyme has attracted extensive attention since it was first reported in 1881, mainly because of its antibacterial properties. A database search performed during preparation of this chapter revealed some 1200 entries with the word lactoperoxidase in their titles over the last 50 years. It is not surprising then that it also been the subject of numerous reviews. Some relatively recent reviews are by Kussengrager & Hooijdonk (2000), FAO/WHO (2006), Buys (2011), O’Mahony, Fox and Kelly (2013), Jafary, Kashanian and Sharieat (2013), and Bafort, Parisi, Perraudin and Jijakli (2014). Lactoperoxidase has several defining characteristics. It is a glycoprotein with a single polypeptide chain of 612 amino acids giving it a molecular weight of 78,030 Da. It has four or five carbohydrate chains which make up 10% of its molecular weight. It has two principal forms, A and B, due to differences in deamidation of glutamine and asparagine residues; together with variation in the carbohydrate chains, it exists in a total of 10 variants. It contains a heme group, ferriprotoporphyrin IX, at the active site which is covalently bound through two ester groups to Glu375 and Asp275. Each LPO molecule has one iron atom; iron accounts for 0.07% of the proteins mass. The iron lends a green color to the protein which has an absorbance maximum at 412 nm. The A412:A280 ratio is B0.9. It also contains one calcium per iron atom which is responsible for its structural stability. At the pH of milk, B6.7, it is relatively heat-stable; it survives normal HTST pasteurization (72 C for 15 s) but is inactivated by heating at 78 C for 15 s. Inactivation of the LPO activity is very temperature-sensitive; Griffiths (1986) reported that it had a D70 C of 940 s and a z-value of 5.4 C. At low pH, # 5.3, it is much less stable and this is attributable to loss of calcium. However, LPO has maximum enzyme activity at pH 5
6. Because of the nature of the heat stability of LPO, the presence of LPO activity in heat-treated milk has been proposed as a test for distinguishing

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between HTST pasteurized milk, which should be LPO-positive, and “highpasteurized” (now referred to as extended shelf-life (ESL) milk) which should be LPO-negative. According to a former EU regulation (EU, 2005), an LPOnegative milk would need to be labeled as “high pasteurized.” While this is still a valid test, the EU regulation no longer applies. LPO can catalyze the oxidation of several substrates including unsaturated fatty acids, aromatic amines, phenol, aromatic acids, and leuco dyes. Its activity can be measured by its oxidation of the chromogenic substrate ABTS [2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)]. Typical assay conditions are phosphate buffer pH 5.5 and temperature 25 C. The increase in absorbance at 436 nm during the assay is a measure of the activity (Sigma, 1995). LPO is strongly cationic with a pI of 9.6. This property allows it to be readily isolated from whey by cation ion exchange. It is usually isolated with lactoferrin which is also cationic. A higher salt concentration is required to remove LPO from the ion exchange resin than to remove lactoferrin, thereby offering a means of separating the two proteins. Large quantities of LPO, some mixed with lactoferrin, also an antibacterial agent, are now produced commercially.

1.3.5.2 Significance The major significance of LPO is its antibacterial properties and this is the main use of the commercial LPO product. It is used in a wide range of products including toothpaste, mouthwashes, cosmetics, calf starters, and fish feed (DeBoer, 2014). It is also used as a bleaching agent for whey containing annatto (see Chapter 10). In conjunction with thiocyanate and hydrogen peroxide, LPO forms a powerful antibacterial system. It is 50
100 times more antibacterial than hydrogen peroxide alone (O’Mahony et al. 2013). Thiocyanate is present in raw bovine milk at a concentration of B24 mg/L, more than enough to activate the LPO system which requires B15 mg/L. However, bovine milk normally has a very low level of hydrogen peroxide. The LPO system can be activated considerably by additions of thiocyanate and hydrogen peroxide although incorporating a hydrogen peroxide generating system such as sodium percarbonate may be more effective than adding hydrogen peroxide itself. The oxidation products, for example, hypocyanous acid and hypothiocyanate, have strong antimicrobial activity due to oxidation of sulfhydryl groups and reduced nicotinamide nucleotides of bacterial cell walls (Reiter and Harnulv, 1982). The LPO system is active against a wide range of organisms: Gram-positive and Gram-negative bacteria, viruses, and fungi. It may be either bactericidal

1.3 Minor Whey Proteins

or bacteriostatic against spoilage and pathogenic bacteria in raw milk. Several authors have shown that activation of the LPO system extends the shelf-life of raw milk by inhibiting bacterial growth. This strategy has been suggested for use in countries where refrigeration is not widely used (FAO/WHO, 2006; IDF, 1988). Its use was demonstrated by Fweja, Lewis and Grandison (2008) who found that added iodide was a more effective electron donor than thiocyanate in the LPO system.

1.3.6

Lysozyme

1.3.6.1 Characteristics Lysozyme or muramidase (EC 3.2.1.17) is common in living organisms as a part of their defence mechanisms. It is usually regarded as lysosomal; however, the milk enzyme is isolated from whey and hence is soluble. Its mode of action is to break down peptidoglycans, which are components of bacterial cell walls, thereby lysing the cell. Biochemically, it catalyzes the hydrolysis of the β1-4 bonds between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan. Since Gram-positive bacteria have a high percentage (B90%) of peptidioclycan in their cell walls, they are more susceptible to lysozyme than Gram-negative bacteria with a lower percentage (5%
10%) of peptidoglycan. The action of lysozyme on Gram-positive bacteria is the basis of a common assay of the enzyme’s activity involving lysis of Micrococcus luteus (formerly Micrococcus lysodeikticus). In Gram-negative bacteria, the peptidoglycan is protected by an outer layer of lipopolysaccharide. If this barrier layer is disrupted, the bacteria become sensitive to lysozyme. For example, with E. coli, lysozyme acts synergistically with lactoferrin which alters the outer layer and makes it susceptible to the lysozyme. Thus, combinations of lysozyme and lactoferrin are more bacteriostatic than either protein alone (Severin & Wenshui, 2005). While the hydrolytic effect of lysozyme on the cell wall of bacteria is generally regarded as the primary mechanism for its bacteriostatic and bactericidal effects, there is now a body of evidence to indicate that a nonenzymic mechanism may also be involved (O’Mahony et al., 2013). The concentrations of lysozyme in bovine milk (,3 mg/L; range 0.7
6 mg/L) (Farkye & Bansal, 2011; Wynn & Sheehy, 2013) and buffalo milk (3.85 mg/L, Abd El-Aziz, 2006) are low compared with its concentration in human milk (100
120 mg/L) and much lower than the concentrations in the milk of some other animals, e.g., horse (790 mg/L) and donkey (1000 mg/L) (Farkye & Bansal, 2011). According to O’Mahony et al. (2013) lysozyme has been isolated from the milk of a wider range of species than any other milk enzyme. However, the levels in all milks are very low compared with the richest source of lysozyme, egg white, where the concentration is 3
5 g/L. Consequently, the source of commercial lysozyme is almost exclusively egg white. In recent

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years, recombinant human lysozyme has also been expressed in the milk of transgenic cows, goats, and other animals (e.g., Maga et al., 2006; Yang et al., 2011). Yang et al. (2011) reported that the concentration of human lysozyme produced in bovine milk was up to 26 mg/L. Bovine milk lysozyme is a single polypeptide of 154 amino acids and has a molecular weight of 18,000 Da while the human milk enzyme has 130 amino acids and molecular weight is 15,000 Da (Farkye & Bansal, 2011). The amino acid composition of bovine milk lysozyme is very different to that of human milk lysozyme and egg white lysozyme (Eitenmiller, Friend, & Shahani, 1976). However, the amino acid sequence of lysozyme is highly homologous with that of α-La which is known to modify the specificity of the enzyme UDP-galactosyltransferase in the biosynthesis of lactose. It is believed that, historically, lysozyme evolved to become α-lactalbumin (O’Mahony et al., 2013). Lysozyme is quite stable to heat at pH 3
4 but not at pH .7. However, most ( . 75%) of the lysozyme activity in bovine milk survives heating at 80 C for 15 s so HTST pasteurization has little effect on its activity. It is inactivated by mercaptoethanol through reduction of disulfide bonds; curiously however, it can be reactivated by oxidation and the oxidized enzyme has considerably more (B330%) activity than the native enzyme (Eitenmiller et al., 1976; Friend, Eitenmiller, & Shahani, 1972). Lysozyme has been isolated from human milk by affinity chromatography on heparin-Sepharose followed by gel filtration (Sephadex G-50) (BoesmanFinkelstein & Finkelstein, 1982), the lysozyme from buffalo milk was isolated using Sephadex G-50 followed by cation exchange chromatography on carboxymethyl cellulose (Hussain, Zahoor, Anjum, Shahid and Saeed (2015), while the bovine milk enzyme has been isolated using ion exchange (Amberlite IRC-50) followed by ammonium sulfate fractionation and gel filtration on Sephadex G-50 (Chandan, Parry, & Shahani, 1965). The ability to use cation exchange chromatography to isolate lysozyme is because it is a cationic protein with a pI of B9.5 (Chandan et al. 1965).

1.3.6.2 Significance Because of the low level of lysozyme in bovine milk and buffalo milk, and the uncertainty about whether the indigenous enzyme in these milks can provide any antibacterial protection for the calf or extend the shelf life of the milk, the interest in lysozyme in the dairy industry is not so much in the native enzyme but in the use of commercial (egg white) lysozyme. There are several global suppliers of lysozyme (Chemical Book, 2016). An example of its use in the dairy industry is in cheesemaking for inhibiting the growth of Clostridium tyrobutyricum. This sporeforming bacterium is not

1.3 Minor Whey Proteins

inactivated by pasteurization and causes a defect during maturation of cheeses such as Gouda, Edam, Emmental, Parmigiano Reggiano, and Grana Padana known as late gas blowing. As egg lysozyme may induce an allergenic response in some people, legislation in some countries, e.g., EC legislation (2003/89/EC, Annex IIIa amending Directive 2000/13/EC) requires its addition to be declared on the product label. In addition, the use of lysozyme may not be allowed for some cheeses, such as those having Protected Designation of Origin status. Therefore, in such circumstances, its presence needs to be monitored for regulatory purposes. To this end, a sensitive HPLC method which can detect lysozyme at levels down to 0.8 mg/L (Pellegrino & Tirelli, 2000) has been developed. Lysozyme can also be detected by enzyme-linked immunosorbent assay (ELISA) methods, and liquid chromatography-mass spectrometry (LCMS) techniques (O’Mahony et al., 2013). The lower incidence of gastrointestinal illnesses in breast-fed infants compared with formula-fed infants has been attributed, at least in part, to the human milk lysozyme. As a consequence, lysozyme has been added to some infant formulae (O’Mahony et al., 2013). One application of lysozyme in dairy processing which has attracted considerable research interest is in conjunction with some nonthermal technologies and other antibacterial agents. In this sense, lysozyme acts as a microbiological hurdle. It has been shown to increase the bactericidal effectiveness of high pressure processing, pulsed electric field technology and high-pressure homogenization with or without the presence of other antibacterial agents such as nisin, lactoferrin, or lactoperoxidase (Deeth, Datta, & Versteeg, 2013; Ross, Griffiths, Mittal, & Deeth, 2003).

1.3.7

Cathepsin D

1.3.7.1 Characteristics Cathepsin D (EC 3.4.23.5) is a lysosomal enzyme but occurs in whey in soluble form. It is an aspartic protease with a low pH optimum. Like its alkaline protease counterpart in milk, plasmin, cathepsin D exists in more than one form, with an inactive precursor of the enzyme, procathepsin D being in highest concentration. Procathepsin D is converted through an autoproteolytic process, with the loss of 18 amino acid residues, to pseudocathepsin D which in turn is converted to the mature cathepsin D by other proteases with the loss of a further 26 residues. Pseudocathepsin D and mature cathepsin D are active proteolytic enzymes. As a further level of complexity, mature cathepsin D exists in both single- and two-chained polypeptide forms in approximately equal amounts. Cathepsin D is also a glycoprotein (Hurley,

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Larsen, Kelly, & McSweeney, 2000b; Larsen, Benfeldt, Rasmussen, & Petersen, 1996). The total concentration of the cathepsin D in bovine milk is around 0.4 mg/ L in milk and 0.3 mg/L in whey which indicates that it is mostly in soluble form. Interestingly, the concentration of cathepsin D is similar to that of lysozyme, another lysosomal enzyme in bovine milk. Significantly, the concentration of both enzymes increases with an increase in somatic cell count in the milk. The molecular weights of the three major forms of cathepsin D were reported to be 45,000 and 46,000 Da (procathepsin D; the two isoforms are believed to differ in molecular weight through difference in glycosylation), 43,000 Da (pseudocathepsin D) and 39,000 (single-chain mature cathepsin D, 346 amino acid residues). In addition, the molecular weights of the two chains produced from the single-chain mature form were 31,000 Da (heavy chain from the N-terminus, f1-99) and 13,000
14000 (light chain from the C-terminus, f102-346) (Hurley et al., 2000b; Larsen et al., 1996). Bovine milk cathepsin D has been isolated using pepstatin-Sepharose affinity chromatography; pepstatin is an inhibitor of cathepsin D. However, human milk procathepsin has been isolated using concanavalin A-Sepharose affinity chromatography and detected in column fractions by Western Blotting analysis using antibodies raised to synthetic human cathepsin D and by a 14Cmethylated hemoglobin assay (Vĕtviˇcka et al., 1993). It is interesting that the hemoglobin assay was used as procathepsin is generally reported to be enzymically inactive. It is possible that the human milk procathepsin is enzymically active or the procathepsin D autoactivated to the active pseudocathepsin D during the assay incubation at pH 3-4. Larsen, Boisen and Peteresen (1993) reported this conversion could take place during incubation at pH 3.5-5 for 30
120 min; no mature cathepsin D was formed during the incubation. Cathepsin D has also been detected in ewes milk by a Western Blotting method. Procathepsin D and the heavy-chain form of mature cathepsin D, but not pseudocathepsin D or the light-chain mature form, were detected ˇ (Spehar et al., 2013). Both bovine pseudocathepsin D and cathepsin D are able to catalyze the hydrolysis of the four caseins and α-La, but preferentially αs1-casein. Fragments initially produced from the caseins, including glycomacropeptide from κ-casein, are similar to those produced by chymosin. β-Lg appears to be resistant to cathepsin D. Cathepsin D is also able to slowly coagulate milk (Larsen et al., 1996). Cathepsin D is partially inactivated by the cooking procedure used in the manufacture of some cheeses (55 C for 30 min) (55% inactivation) and

1.3 Minor Whey Proteins

largely inactivated by HTST pasteurization (92% inactivation). It is completely inactivated by heating at 70 C for 10 min. (Hayes et al., 2001; Kaminogawa & Yamauchi, 1972). Cathepsin D is resistant to high-pressure processing at room temperature. However, it is significantly inactivated by high pressure at 650 MPa at 40 C and at 450 MPa at 55 C (Moatsou et al. 2008).

1.3.7.2 Significance In the medical field, cathepsin D has been proposed as a marker for breast cancer although this is somewhat controversial. It is also reported to have an involvement is several disease states, including hypertension, AIDS, peptic ˇ ulcer disease, and prostate cancer (Hurley et al., 2000b; Spehar et al., 2013). However, the significance of the milk cathepsin D system in humans or other species is unclear. Cathepsin D appears to have no direct physiological function in bovine milk. However, because of its partial resistance to pasteurization, its action has been implicated in proteolysis in some cheeses, e.g., Swiss cheese (Cooney, Tiernan, Joyce, & Kelly, 2000) and quarg (Hurley, Larsen, Kelly, & McSweeney, 2000a). Therefore it is possible that it contributes to changes in the flavor and texture in these and other dairy products. In an analysis of UHT milk during storage, Gaucher, Molle, Gagnaire and Gaucheron (2008) identified five peptides which they attributed solely to cathepsin D proteolysis and another 10 which could have been due to proteolysis by either cathepsin D or another protease. Given the thermal stability characteristics of cathepsin D discussed above, it is curious that any of the enzyme would remain active in milk after being preheated at 82 C for 20 s and indirectly sterilized at 140 C for 4 s. This requires further investigation.

1.3.8

Acid Phosphatase

1.3.8.1 Characteristics Acid phosphatase or acid phosphomonoesterase (EC 3.1.3.2) occurs in more than one form in bovine milk. About 80% of the activity is associated with the skim phase and the remainder is associated with the milk fat globule membrane (MFGM). The enzymes in these two milk fractions have different properties and hence are accepted as different proteins. As this chapter only concerns soluble enzymes, the one associated with the MFGM is not considered further. A further complication is that acid phosphatase activity in milk with high somatic cell count, i.e., mastitic milk, is considerably elevated, four to 10 times; the activity arises from three enzymes, the indigenous skim milk enzyme and two of leukocyte origin. The leucocyte enzymes are also not considered further here.

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The acid phosphatase in skim milk, which is also in whey and is therefore soluble, is the focus here. It has a molecular weight of B42,000 Da and a pH optimum of B4.0. It is inhibited by fluoride, several metal ions, and oxidizing agents. Its activity is not affected by magnesium ions which activate its alkaline counterpart in milk. It is a basic glycoprotein with a pI of 7.9. Because of its basic nature, it can be isolated from milk or whey by cation exchange chromatography on a resin such as Amberlite IRC50. Yamaguchi, Miura, Baba and Akuzawa (2012) separated an acid phosphatase from a lactoferrin-rich fraction they isolated from skim milk by cation exchange chromatography on Amberlite FPC-3500 followed by gel filtration on Cellofine CM500 carboxymethyl cellulose. It has a molecular weight of 37,000 Da and is optimally active at pH 3
4 and at 60 C. The authors proposed that the enzyme was a previously undocumented member of the mammalian purple acid phosphatase family. It catalyzes the dephosphorylation of phosphoproteins such as the phosphocaseins and is therefore considered a phosphoprotein phosphatase. However, it has broad specificity and is also able to act on low-molecular-weight phosphates. Like alkaline phosphatase, it can be assayed using p-nitrophenol phosphate or phenolphthalein phosphate. It can also be assayed using the Fluorophos Test System. Balci and Wilbey (1999) used this method successfully for measuring the survival of acid phosphatase in bovine milk after heat treatment in the range 75 to 88 C for 15 s. Griffiths (1986) reported the acid phosphatase activity in bovine milk to be 0.13 U/mL (range, 0.004
0.37 U/mL).

1.3.8.2 Significance Dephosphorylation of the phosphocaseins occurs during cheese ripening. This can increase proteolysis, as phosphopetides are resistant to proteolysis, and aid flavor and texture development (Andrews & Alichanidis, 1975a; Fox, Uniacke, McSweeney, & O’Mahony, 2015). Two acid phosphatases contribute to the dephosphorylation, the indigenous milk enzyme and the enzyme from the cheese starter bacteria (Akuzawa & Fox 2004). However, the phosphatase action in cheese is due largely to the milk enzyme with the starter enzyme being of minor importance (Andrews & Alichanidis, 1975a). The acid phosphatase activity in milk increases four- to 10-fold during mastitis (Mullen, 1950). It could therefore be used as a marker for mastitis along with lysozyme and NAGase. These enzymes and two of the acid phosphatase isoenzymes in milk from mastitic cows originate from the leucocyte lysosomes (Andrews & Alichanidis, 1975b). Acid phosphatase is reasonably heat-stable. It survives pasteurization (63 C for 30 min) (80%
90%) and requires a heat treatment of 88 C for 10 min

1.3 Minor Whey Proteins

or 100 C for 1 min for complete inactivation (Mullen, 1950). As a result of this, it has also been proposed as an indicator enzyme to distinguish HTST pasteurized milk from superpasteurized (ESL) milk. The former should contain acid phosphatase activity whereas the latter should not. However, lactoperoxidase, as indicated in Section 1.3.4 (Griffiths, 1986), and mannosidase (Zehetner, Bareuther, Henle, & Klostermeyer, 1996) have also been proposed for this purpose.

1.3.9

Ribonucleases

1.3.9.1 Characteristics Ribonucleases (RNases) are phosphodiesterases that catalyze the hydrolysis of ribonucleic acid (RNA) on the 30 side of pyrimidine nucleotides. They cleave 30 -phosphomononucleotides and 30 -phosphooligonucleotides ending in C-P or U-P. They are very widespread in living organisms and play various roles including cleaning up cellular RNA which is no longer required. They occur in a wide range of tissues and secretions including milk. They have been isolated from the milk of many different animals, apart from cows, including human (Dalaly, Eitenmiller, Friend, & Shahani, 1980), goat (Gupta & Mathur, 1989), buffalo (Azza, Ahmed, & Khorshid, 1977), and rat (Liu & Owens, 1983). The first RNase to be sequenced was the one from bovine pancreas which has become a reference RNase. Bovine milk contains a relatively high concentration of RNase: 11
40 mg/L; 32 U/L, considerably higher (B3
100 times) than the level in human milk (Bingham & Zittle, 1962; Griffiths, 1986; Farkye & Bansal, 2011; Meyer, Capeless, Kunin, & Meyer, 1986). There have been several papers describing various RNases in bovine milk. However, the reported RNases form a rather confusing picture. Table 1.4 summarizes the information on the various RNases which have been reported. Unfortunately, some of the papers make no reference to previously reported RNases from bovine milk and hence it is difficult to correlate the findings in these reports. The following is a brief summary of the reports; at this time it is not possible to ascertain with certainty the number and nature of the RNases present. Research to clarify the situation is clearly warranted. RNase was first described in some detail by Bingham and Zittle (1962, 1964; Bingham & Kalan, 1967). They identified two major forms, which they designated A and B, in the ratio of 3.9:1. The B form is a glycoprotein with 5.17% mannose and 4.2% hexosamines (Bingham & Kalan, 1967) while the A form contains no carbohydrate. They have identical amino acid compositions. The bovine milk RNase-A has been shown to be very similar to, if not identical with, pancreatic RNase-A while the milk RNase-B differs from the pancreatic RNase-B in its hexosamine composition. The similarity of the RNases-A was

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based on physical and enzymic characteristics (Bingham & Zittle, 1964) and serological identity (Coulson & Stevens, 1964). The pancreatic enzyme (and hence the milk enzyme) is generally designated RNase I (EC 3.1.27.5, Web, 1992; EC 3.1.2.75, Ako & Nipo, 2012) although the terms RNase-A and RNase-B are still used. Both the milk and pancreatic enzymes have been obtained in crystalline form. It appears that milk isoform A and pancreatic form B are the major components of the respective crystals (Groves, 1966; Plummer, & Hirs, 1963). Bingham and Kalan (1967) confirmed the presence of RNase-A and -B and also reported the presence of two other RNase peaks in their ion exchange chromatography profile which they labeled RNase-C and RNase-D; they did not characterize C and D. Later Meyer, Kunin, Maddalena & Meyer (1987) reported RNase-A and -B as isoforms of what they classified as RNase II, together with a minor RNase which they designated RNase II-I. The ratio of the three enzymes was 70:30:1. They considered RNase II-I to be unique as it was unable to catalyze hydrolysis of polycytidylate (Poly C). The RNases discussed above have molecular weights of about 14,000 Da (A, 13,700; B, 14,700 6 300). They have a high pI of B9 and are optimally active at pH 7.0
7.5. They are usually assayed against yeast transfer RNA. They are quite heat stable, losing only about 40% activity at 80 C for 15 s (in milk) while in acid whey at pH 3.5, they are stable to heating at 90 C for 20 min. Ye and Ng (2000) reported the presence of “lactoribonuclease” in what they claimed to be the “first report of the presence of an RNase resembling bovine pancreatic RNase in bovine milk.” Given the above discussion, this claim can clearly not be supported. The authors made no reference to the reports discussed above and the report by Ye, Cheng and Ng (1999) of lactogenin, a secretory RNase. While it cannot be definitively confirmed, it appears that “lactoribonuclease” is the same enzyme as previously reported by Bingham and Zittle (1962, 1964), Bingham and Kalan (1967), Meyer et al. (1987), Groves (1966), Ye et al. (1999), and later by Knight et al. (2014). Because RNase is a strongly basic protein, it is commonly isolated from milk by cation exchange chromatography. However, other major whey proteins, particularly lactoperoxidase and lactoferrin, and some minor proteins such as growth factors (Dyer et al., 2008) are also very basic and are isolated from milk or whey in the same way. Both lactoperoxidase and lactoferrin, both separately and in mixed form, are produced commercially and these preparations often have RNase activity. Some preparations such as lactermin (Francis, Regester, Webb, & Ballard, 1995) contain growth factors. Groves (1966) separated RNase from a lactoperoxidase preparation by gel filtration on Sephadex G-200.

1.3 Minor Whey Proteins

Ye et al. (2000) reported RNase activity in a lactoferrin preparation which they attributed to the lactoferrin molecule. They maintained that the RNase activity was unlikely to be due to contamination with RNase or to binding of RNase with lactoferrin (lactoferrin is known to bind RNase). The activity had a pH optimum of B7.5 in common with other reported RNase activities in bovine milk. They assayed their RNase activity using yeast transfer RNA but also determined that their lactoferrin had RNase activity on poly C (polycytidylate) but none on poly A (polyadenine), poly G (polyguanine), or poly U (polyuridylic acid). Di Liddo et al. (2010) reported the isolation and characterization of an RNase which they designated RNase-4. They made their designation based on the substrate specificity of their preparation against simple dinucleotides: more reactive against UpG than CpG. They stated that the marked preference of their RNase for uridine over cytidine contrasted with the more general pyrimidine specificity of other ribonucleases such as RNase-1, -2, -3, and angiogenin. The enzyme was isolated by a method common to that used for other RNases from milk, namely cation exchange chromatography, in this case on CM-Sepharose FF at pH 7.7, followed by a clean-up procedure, in this case high-resolution ion exchange chromatography on a Mono-S column. The authors made no reference to any other reports of RNase in bovine milk and so it is not possible to identify this RNase with any of the others reported. However, they did compare some biological activities of their enzyme with those of a commercially available RNase-A, presumably of bovine pancreatic origin, and found significant differences. The RNase-4 had a positive proliferative effect on normal cell lines while RNase-A had an inhibitory effect (this was attributed to the higher binding capacity of RNase4 with RNase inhibitor than of RNase-A) and RNase-4 induced a higher in vitro angiogenic effect than RNase-A. The cytotoxic effects of the enzymes on carcinoma cell lines were similar. These results indicate RNase-4 differs from RNase-A and if the RNase-A used is the same as bovine milk RNase-A, then the reported bovine milk RNase-4 differs from the RNases discussed above. The RNase family also includes angiogenins, three of which have been reported to be in bovine milk, angiogenin-1 and lactogenin (Ye et al., 1999), and angiogenin-1 and -2 (Strydom, Bond, & Vallee, 1997). Strydom et al. (1997) published a diagram showing the phylogenetic relationship between the RNases and angiogenins. An angiogenin is a protein which promotes angiogenesis which is the induction of new blood vessel growth. In addition, an angiogenin has RNase activity which is necessary for angiogenic activity (Strydom et al., 1997). Ye et al. (1999) considered the protein they designated lactogenin to be different from the previously reported angiogenin-2 and to be similar to bovine liver ribonuclease (RNase BL-4), an RNase-4.

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Therefore it is possible that lactogenin is the same protein as Di Liddo et al. (2010) later reported as RNase-4 as discussed above. Knight et al. (2014) reported isolation of RNase-5, also known as angiogenin, from whey using cation exchange chromatography (SP Sepharose resin). Their crude preparation from the ion exchange contained only about 10% RNase, the remainder being lactoperoxidase, lactoferrin, immunoglobulins, and some unidentified whey proteins. They increased the concentration of RNase to .95% (according to SDS-PAGE) by cation-exchange HPLC followed by chromatography on Mono S and Sephacryl S-100 columns. They showed that their RNase preparation had RNase activity (using the

Table 1.4 Summary of Reported Bovine Milk Ribonucleases (RNases) Designation

Molecular Weight

Other

Reference Bingham & Zittle (1962, 1964) Groves (1966) Bingham & Kalan (1967) Meyer et al. (1987)

RNase-A & -B (ratio 4:1)

13,700 & B14,700

See Table 1.3

RNase-A plus 2 minor unidentified proteins RNase-A and B, plus C and D RNase II isoforms A and B plus RNase II-1 (ratio 70:30:1)

NA

RNase-A crystallized

NA

C and D not characterized

NA

Angiogenin 2

20,000; 123 amino acids

Lactogenin (a secretory RNase)

17,000

Lactoribonuclease

17,000

RNase activity due to lactoferrin

78,000

RNase-4

NA

RNase-5 (angiogenin)

14,500

Unlike A & B isoforms, RNase II-1 was unable to catalyze hydrolysis of polycytidylate (Poly C). Cows’ milk contains 40 mg/L RNase II; human milk contains 1% of ribonuclease II in cows’ milk 4 disulfide bonds; angiogenic activity assessed by chick-embryo (CAM) assay ; very low ribonuclease activity on yeast tRNA; separated from angiogenin 1 & a RNase on Mono-S column but the RNase was not characterized Similar to bovine liver RNase BL4 More active towards poly C than poly U pH opt 7.5; similar to but not identical to pancreatic RNase (no reference to other reports of milk RNase) pH opt 7.5; assayed against yeast tRNA at 37 C; Lf exerted RNase activity on poly C but not poly A, poly G, and poly U. pI .9.5; specificity of UpG was higher than that of CpG; used purification method of Ye et al. (1999) pI 10.5; separated from Lf, LP and Igs; stimulates muscle cell differentiation in vitro; increased nuscle mass in mice (no reference to other reports of milk RNase)

NA, not available.

Strydom et al. (1997)

Ye et al. (1999) Ye & Ng (2000) Ye et al. (2000) Di Liddo et al. (2010) Knight et al. (2014)

References

commercial Ambion RNaseAlert QC system based on use of a fluorogenic RNA substrate), and angiogenin activity (stimulating muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis, and promoting muscle weight gain and grip strength in mice fed a diet supplemented with milk-derived RNase5 preparations). Although their preparation method was similar to those used by other researchers for isolating RNase and angiogenins, Knight et al. (2014) make no reference to any other paper on these proteins in milk. Presumably, RNase-5 bears a close relationship with one or more of the previously described angiogenins/RNases.

1.3.9.2 Significance Milk RNases, including angiogenins, appear to have no technological significance in milk but have considerable potential for commercial applications as ingredients with various biological activities, including antiviral activity (Modak & Marcus, 1977). However, clarification of the proteins involved and of their respective biological activities is required to take full advantage of the opportunities they offer.

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446. Ako, H., & Nipo, W. K. (2012). Enzyme classification and nomenclature. In F. Toldrá, L. M. L. Nollet, & B. K. Simpson (Eds.), Food biochemistry and food processing (2nd ed, pp. 109
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85). New York, NY: Springer. O’Mahony, J. A., Fox, P. F., & Kelly, A. L. (2013). Indigenous enzymes of milk. In P. L. H. McSweeney, & P. F. Fox (Eds.), Advanced dairy chemistry. Vol. 1a Proteins: Basic aspects (pp. 337
385). New York, NY: Springer. O'Riordan, N., Gerlach, J. Q., Kilcoyne, M., O'Callaghan, J., Kane, M., Hickey, R. M., & Joshi, L. (2014). Profiling temporal changes in bovine milk lactoferrin glycosylation using lectin microarrays. Food Chemistry, 165, 388
396. Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao, R., . . . Kraulis, P. J. (1986). The structure of β-lactoglobulin and its similarity to plasma retinolbinding protein. Nature, 324(6095), 383
385. Pappas, C. P., & Rothwell, J. (1991). The effects of heating, alone or in the presence of calcium or lactose, on calcium-binding to milk-proteins. Food Chemistry, 42, 183
201. Paquet, D. (1989). [The proteose-peptone fraction of milk - Review.]. Lait, 69, 1
21. Patocka, G., & Jelen, P. (1991). Calcium association with isolated whey proteins. Canadian Institute of Food Science and Technology Journal, 24, 218
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Pellegrino, L., & Tirelli, A. (2000). A sensitive HPLC method to detect hen’s egg white lysozyme in milk and milk products. International Dairy Journal, 10, 435
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538). New York, NY: Springer. Peters, T. (1985). Serum albumin. In C. B. Anfinsen, J. T. Edsall, & F. M. Richards (Eds.), Advances in protein chemistry (Vol. 37, pp. 161
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5810. Pettersson, J., Mossberg, A. K., & Svanborg, C. (2006). α-Lactalbumin species variation, HAMLET formation, and tumor cell death. Biochemical & Biophysical Research Communications, 345, 260
270. Pihlanto, A. (2011). Bioactive peptides. In (2nd edJ. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 3San Diego, CA: Academic Press. Plummer, T. H., & Hirs, C. H. W. (1963). Isolation of ribonuclease b, a glycoprotein, from bovine pancreatic juice. Journal of Biological Chemistry, 238, 1396
1401. Relkin, P., Launay, B., & Liu, T. X. (1998). Heat- and cold-setting gels of β -lactoglobulin solutions. A DSC and TEM study. Thermochimica Acta, 308, 69
74. Remondetto, G. E., Paquin, P., & Subirade, M. (2002). Cold gelation of β-lactoglobulin in the presence of iron. Journal of Food Science, 67, 586
595. Rangel, A. H. D., Sales, D. C., Urbano, S. A., Galvao, J. G. B., de Andrade, J. C., & Macedo, C. D. (2016). Lactose intolerance and cow's milk protein allergy. Food Science & Technology, 36, 179
187. Rao, K. R., & Brew, K. (1989). Calcium regulates folding and disulfide-bond formation in α-lactalbumin. Biochemical & Biophysical Research Communications, 163, 1390
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109.

CHAPTER 2

History of the Development and Application of Whey Protein Products Julian Price Milk Specialties Global, Eden Prairie, MN, United States

2.1

INTRODUCTION

Whey is produced during the manufacture of cheese or casein. It is the liquid that remains after the casein is removed by coagulation with either rennet or acid. It contains about half of the total solids (B6.5% by weight) of milk, including around 20% of the total milk protein. Of the solids in whey, lactose accounts for B75% and crude protein B13%. The nitrogen-containing components include soluble proteins, collectively known as the whey proteins, which are mostly denatured by heat treatments greater than B65 C, and nonprotein nitrogen such as small peptides, ammonia, and urea. The major whey proteins in both their native and heated forms are highly valued for both their physical and physiological functionalities. The other solids in whey are minerals, organic acids, milk fat (rich in phospholipids), and several interesting minor components. Cheese was originally produced on a small local scale as a way of preserving milk; the whey was largely considered a waste product. It was mostly used as an animal feed but also for producing some food products such as fermented whey drinks. Its nutritional and health-giving properties were also recognized in some cultures. However, when large-scale production of cheese commenced, utilization of the large quantities of whey became a problem. For this book it is instructive to recall the origins of industrial modern whey processing, and to appreciate what has driven the development of products and processes in the intervening years. One technology that completely transformed industrial processing of whey in general, and whey protein in particular, was cross-flow membrane filtration. Developed in the early 1970s, it is now used in some form in almost every whey processing operation. The drivers for the development of whey protein products differed significantly in the United States, Europe, and New Zealand (NZ), the regions Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00002-3 © 2019 Elsevier Inc. All rights reserved.

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where most of the development occurred. In the mid-western United States, in the late 1960s, the primary driver was concern over environmental pollution from the dumping of huge volumes of whey. For example, in Wisconsin in 1950 there were around 150 cheese plants, mostly collecting around 25,000 100,000 L of milk per day, and a large number of these disposed of their whey directly into their local river. Subsequently, many of these cheese plants amalgamated into larger factories, resulting in a reduction of the number of individual plants by about half by 1975. This exacerbated the pollution problems with some rivers becoming akin to open sewers. This prompted the authorities in many states to place a maximum biochemical oxygen demand (BOD) of 20 ppm on dairy wastewater, regardless of where it was disposed. This meant it was no longer possible to dispose of whey without treatment; this provided the first real driver to establish a whey processing industry. One response adopted by many small cheese plants was to dry the whey using roller dryers to produce what became known as “popcorn whey.” This was a very inefficient process for drying whey which contains only 6.5% solids. Furthermore, the product was of low value, being sold for pig feed at around US$250 per tonne in the 1960s. In the early 1970s the cost of natural gas rose, resulting in production of “popcorn whey” becoming uneconomic; however, it continued for some time as it enabled cheese manufacturing to continue. It was recognized that much of the cost of producing the dried whey could be saved if an economical means of preconcentrating whey before roller drying, other than by thermal evaporation, could be developed. This was the trigger for the introduction of membrane filtration into the dairy industry. Between 1975 and 1985 approximately 100 reverse osmosis (RO) plants were installed in the United States, and treated over 50% of all cheese whey production. Through the 1980s with continued consolidation of cheese manufacturing, roller dryers were replaced by evaporators and spray dryers. However, RO systems were commonly used for preconcentrating whey before evaporation, or to reduce volumes of whey and hence the cost of transporting the whey from small plants to factories with evaporators and dryers. In the early 1970s a second fundamental membrane process, ultrafiltration (UF), was introduced. Toward the end of the 20th century, the other two fundamental membrane processes, nanofiltration (NF) and microfiltration (MF), began to be used for whey processing. Concomitantly, the trend to develop added-value products intensified. The drivers for the introduction of membrane filtration into the European dairy industry were somewhat different from those in the United States. Initially, there was less pressure to reduce BOD in the wastewater although the industry was aware this could happen in the future. This provided the European industry time to focus more on added-value products. This resulted

2.2 Membrane Development—1960s to 1980s

in less emphasis on RO and more on UF. Denmark was the first European country to use UF on cheese whey; a major incentive for this was an agreement with a German pharmaceutical company to produce a lactose-rich product as a fermentation substrate. In New Zealand, the development of whey processing was driven by an unusual combination of political changes on the other side of the world and an approach by a global beverage company (MacGibbon, 2014) (see Section 2.3.2.3). From its beginnings in the 1970s, membrane processing has become an integral part of virtually all industrial whey processing. The rest of this chapter covers the development of the key processes used, and how and why they have become so important in developing added-value whey protein ingredients, together with some of the challenges involved. The chapter concludes with a discussion of further developments of whey protein-based ingredients and products, as well as some of the challenges yet to be met.

2.2 2.2.1

MEMBRANE DEVELOPMENT—1960S TO 1980S Cellulose Acetate

In the mid 20th century, the availability of water, especially to increasing populations in arid regions, became a major concern. Water which was unsuitable for drinking became the focus of the attention. This led to consideration of seawater, which until then could be converted to drinking water only by costly and high energy-consuming thermal evaporation processes. Research on using membranes to desalinate seawater commenced in the 1950s. The early research on the newly named RO process focused on the use of cellulose acetate (CA) membranes. CA was used for photographic film and the process of converting it into an asymmetric filtration membrane involved a chemical modification using magnesium perchlorate and acetone. One advantage of CA was that it was possible to make a membrane with fairly controlled pore size in the 10,000 to 100,000 molecular weight range. In the late 1960s, it was recognized that filtration in this UF range could be of interest to the dairy industry for concentration or separation of macromolecules such as proteins. Other advantages of CA were that it was a low-cost material and that it was hydrophilic, making it relatively resistant to fouling, an important consideration in dairy applications. While CA membranes proved suitable for purifying brackish and surface water, there were difficulties in their application in the dairy industry. They were found to have pressure, temperature, and pH limitations, and to be

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susceptible to attack by microorganisms. For example, their pH range is 3 8, and their maximum operating/cleaning temperature is 40 C. Their introduction was a very important milestone in membrane technology but their widespread application was relatively short lived.

2.2.2

Polysulfone and Polyethersulfone

Polysulfones (PS) are thermoplastic polymers with the following chemical structure:

PS membranes were introduced by Union Carbide in 1965 and showed extremely high thermal and chemical stability. PS was found to be a more suitable membrane material than CA for desalination of seawater. PS membranes could be manufactured with reproducible properties and controllable pore size down to 40 nm; the first US patent was issued in 1971. PS membranes dominated this market for more than a decade in several formats, e.g., the DuPont fine hollow fiber concept. UF-PS membranes were invented after it was observed that some experiments with PS produced “leaky” membranes. These membranes were introduced into the dairy industry by several companies but principally by Rhone Poulenc in France. They were well suited to food and dairy applications where their high chemical and thermal stabilities were more important than for desalination applications because of the regular cleaning required. With a normal operating pH range of 2 11 and temperatures of up to 55 C, PS therefore displaced CA as the preferred membrane material. Interestingly, PS is stable beyond 55 C, but this limit was set for the applications for which the membrane was used. The robust characteristics of PS contributed considerably to the rapid adoption of PS, although it was largely superseded a few years later by polyethersulfone (PES), a chemically similar material, as the preferred material for membranes used for separation of whey proteins. A major reason for this was that PES is more hydrophilic than PS, and so more resistant to fouling in dairy applications. PES has the structure:

2.3 Systems and Applications Development

2.2.3

Polyamide

In the mid 1970s, John Cadotte of North Star Research in St Paul, Minnesota, invented a new RO membrane made of polyamide (PA). PA has characteristics superior to those of CA and aryl compounds such as PS for RO applications. The main advantages of PA membranes were a relatively high capacity, very high salt rejection, and good temperature- and pHresistance. However, they are not resistant to oxidizing agents such as chlorine, which makes them more difficult and expensive to clean than PS membranes. PA is used in thin-film composite (TFC) membranes constructed in two-layer designs, although three-layer designs have also been developed. These membranes consist of a PS membrane as support for a very thin layer of PA, which is polymerized in situ on the PS membrane. TFC membranes quickly became the RO membrane of choice in virtually all applications, including whey processing.

2.3

SYSTEMS AND APPLICATIONS DEVELOPMENT

In today’s dairy industry almost every cheese plant has an RO or UF system using the very familiar spiral-wound format. The following discussion shows how the industry evolved to this position.

2.3.1

Reverse Osmosis

In addition to RO (and UF) membranes being extremely thin, and hence having to be supported, RO presents the additional challenge of operating at much higher pressure compared to UF. The early pilot work on RO membranes in food applications used a plate-and-frame or tubular format which could accommodate such pressures. The first publication relating to the application of RO in food systems, including whey, was carried out on a plate-and-frame device, known as the Wurstack, by a group at the USDA’s Western Regional Laboratory at Albany, California, in 1965. Although several companies investigated the plate-and-frame assembly, only one (De Danske Sukkerfabrikker, DDS—see below) successfully commercialized it in dairy applications. At the end of the 1960s, another configuration, the tubular design, was generally considered to be a superior format for RO in food applications, as it was relatively simple from an engineering point of view and conducive to hygienic design. Several companies had been developing tubular systems, most notably American Standard and the Havens Company of San Diego, California, whose owner Glenn G. Havens is generally considered the

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inventor of tubular glass-fiber RO systems. In the United Kingdom, Babcock & Wilcox and Patterson Candy International (PCI) were two main players developing RO food applications. Babcock & Wilcox used porous glass fiber to support the membrane, whereas PCI used a polyester-backed membrane supported in perforated stainless steel tubes. In the 1970s the only two successful suppliers of commercial RO systems using CA membranes into the dairy industry were the Danish Sugar Company, or DDS, using a plate-and-frame system with circular membrane sheets, and PCI, using their tubular design. Both companies supplied over 40 systems into the United States between 1975 and 1985. However, both systems were relatively expensive and had unique problems. The plates in the DDS system began to fail after a few years and were prohibitively expensive to replace, leaving many dairies that had bought these systems in financial stress. The tubular membranes were very expensive to replace and had an extremely high energy consumption. FilmTec, which introduced the TFC RO membrane to the dairy industry around 1980, was already a manufacturer of spiral membranes for the water industry. As spiral membranes were being introduced to dairies for UF applications, they were tried for RO as well. The capital cost of systems was greatly reduced, replacement membrane costs and energy consumption were significantly lower compared with the plate-and-frame, and tubular systems. Spiral RO quickly became the standard and tubular and plate-and-frame designs changed from being the only options to being obsolete in a few years.

2.3.2

Ultrafiltration

2.3.2.1 In the United States—1970 85 The company Abcor Inc. commenced work on UF in the late 1960s and in 1970 manufactured a system for a project in New Zealand (see Section 2.3.2.3). Abcor’s initial design involved a CA membrane supported on a porous plastic tube, similar in concept to the RO tubular designs but operating at lower pressure. A truly remarkable development occurred in 1971 which was largely due to the efforts of one individual. It began at the unlikely place of Pollock, South Dakota, where a former dairy farmer called Frank Thomas, originally from Ontario, Wisconsin, born in 1923, had purchased the former Dakota Cheese Company. After attending a seminar at the University of Wisconsin on the potential for UF in the dairy industry, he came up with the idea of processing whey by UF to make calf milk replacer in which the protein level would be increased from 12% to 35% by removing the small-molecular-weight components, lactose, and minerals.

2.3 Systems and Applications Development

Thomas developed his process using spiral UF membranes housed in 4-in. (100 mm) dairy tubing for protein recovery. The first plant was commissioned in 1972 and had an output of around 90 kg of protein per day; a photograph, from the local news, of this plant appears in Fig. 2.1. A second system followed at Lynn Dairies at Granton in central Wisconsin, with a plant being about three-times the size of the first plant at Pollock. Thomas’s early UF plants were built by Ladish Tri-Clover, a name still familiar to many in today’s dairy industry. The plants were difficult to run and because the membranes were made of CA they proved even more difficult to clean. As mentioned in Section 2.2.1, the pH range for CA membranes is 3 8 and the highest allowable temperature is 40 C. Thomas used his success at Lynn Dairy to start Thomas Technical Services Inc. (TTS) which sold UF systems for whey protein concentrate (WPC) production. Several other larger companies became aware of the potential of UF for separating whey protein and began entering the market. Abcor changed

FIGURE 2.1 Frank Thomas’ first UF system in Pollock; Rushmore Ads/News, June 28th, 1972. Courtesy of Thomas Technical Services Inc., Neillsville, WI.

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its default design from tubular to spiral-wound as they realized the cost advantage. They supplied their customers with highly engineered spiralwound systems and soon passed TTS as the dominant spiral system supplier. Nevertheless, Thomas’s design was the basis for most UF systems now used in the dairy industry. The introduction of PS-, and particularly PES-, UF membranes allowed spiral systems to be easily cleaned at pH in the range 2 11 and at temperatures up to 55 C. This was a critical event which enabled spiral-wound technology to become successful. DDS marketed their system toward difficult applications such as 75% and 80% WPCs and for UF of milk for cheese making. They were very successful in these markets through to the mid to late 1980s, when the lower-cost spiral systems had developed sufficiently to displace their plate-and-frame format as the preferred technology. An alternative system was the “leaf” UF system marketed by competitor Dorr Oliver which was basically similar to the DDS plate-and-frame system, except that with the Dorr Oliver system membrane cartridges were replaced while with the DDS system individual flat membrane sheets were replaced. While the cost of the membranes was relatively low with the DDS system, the labor cost to change possibly hundreds of membrane sheets was very high; with the Dorr Oliver system the membrane cost was higher but replacing the membranes was cheaper as it involved simply sliding in new cartridges. Romicon introduced a hollow-fiber UF design to the dairy industry and installed several systems. Their big advantage was their ability to be cleaned by running the cleaning solution in the reverse direction though the membranes to easily remove the fouling. If this was tried on the other system designs, the membranes would be destroyed. In the United States in the 1970s, there was a relatively slow uptake of UF compared to RO. Apart from the difficulties of operating and cleaning these plants, UF, unlike RO left another liquid by-product in the form of UF permeate, which contained the majority of the solids of the original whey. Therefore UF contributed very little toward solving the whey pollution problem, and small dairies tended to dispose of their UF permeate in the same way as they had disposed of their whey. This restricted the uptake of UF by the larger dairies, where this option was not available, until other solutions were found.

2.3.2.2 In Europe—1975 85 UF in Europe started later than in the United States but quickly took the lead in terms of producing high-protein WPCs; for instance WPC80 1 was produced in Denmark as early as 1978, which was several years earlier than similar products were routinely produced in the United States. It is also the

2.3 Systems and Applications Development

main reason why the adoption of spirals as the dominant technology happened later in Europe than in the United States. DDS was among the first to use PS-UF membranes, which were readily applicable to their plate-and-frame equipment. The initial research and development effort took place in a tight cooperation with Mejeriselskabet Danmark (MD Foods, now Arla) in Denmark. After a few years of research and development, a “pilot plant,” but of full commercial size, was installed in the MD cheese operation in Holstebro, Denmark. This UF plant was in operation in early 1974. Pasilac (then owned by DDS) designed and installed DDS plate-and-frame UF systems using their M35 membrane and was prominent in the market in Europe, as well as dominating the market in Australia and New Zealand for around a decade from the mid 1970s. The DDS/Pasilac systems delivered most of the high-protein WPC plants built over this period. They were certainly ahead of their competitive rivals in this regard. Fig. 2.2 shows a WPC75 plant at Danmark Protein from approximately 1980. In Ireland, Abcor’s tubular design employing CA membranes first appeared in the mid-1970s. The first whey UF installation to run commercially was the Carbery plant at Ballineen in County Cork, which ran from 1975. The UF permeate from this installation was successfully utilized for fermentation into alcohol. WPC and alcohol production have run continuously since

FIGURE 2.2 Multistage high-protein UF system comprising many M35 DDS plate-and-frame modules at Danmark Protein (now Arla) at Nr. Vium Mejeri, Videbæk, Jutland. Photograph courtesy of Bjarne Nicolaisen/Arla Food Ingredients.

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1975, making the site one of the longest continuous commercial producers of WPC in the world. They added whey protein isolate (WPI) and protein hydrolysates to their manufacturing portfolio from the late 1990s. Another Abcor plant, which actually predated the Carbery plant, was installed in Ireland at Avonmore’s Ballyragget site. This installation was used to manufacture WPC35, WPC50, and WPC75 for a limited period. However, the installation was never a commercial success. It was probably Carbery’s innovative utilization of the lactose-rich UF permeate stream that allowed them to make a commercial success of their UF installation, whereas Avonmore did not have any added-value use for its permeate at this time. Avonmore did not use membrane processes again commercially in dairy applications until 1989 and did not use UF again commercially until 1995. Also playing a minor but significant role in this period in Europe was Rhone Poulenc from France, who introduced a plate-and-frame system with a design similar to a plate heat exchanger. Another player was the UK company, PCI, which has the distinction of installing the first whey UF plant in the United Kingdom, at the Milk Marketing Board site at Aspatria in the county of Cumberland (now Cumbria). This was a UF system in which the membrane was deployed in tubular format as shown in Fig. 2.3. The plant was commissioned in

FIGURE 2.3 A PCI tubular UF system, similar to that installed at MMB in Aspatria, processing whey in Northern France. Photograph courtesy of PCI Membranes, Filtration Group.

2.3 Systems and Applications Development

1979, and was designed to make WPC76 for the Japanese market. The significance of 76 was that the protein level needed to be .75% for import into Japan to be classified as an ingredient rather than final product and thus benefit from greatly preferential import tariffs. The brand name for this ingredient was “Lacteen.” This project was only made possible by the pioneering work carried out over the preceding 10 years in New Zealand; this is covered in some detail in the following section.

2.3.2.3 In New Zealand—1960 90 In the late 1960s in NZ, two events combined, with the result that, within a few years, the NZ dairy industry was undoubtedly the world leader in the technology and knowledge to produce added-value whey protein products. During the 1960s, whey utilization in NZ was reasonably advanced for that time, as there was already large-scale casein production. Lactose, whey powder, demineralized whey powder, and lactalbumin (a whey protein extract, produced via heat denaturation and precipitation) were being produced but a significant proportion of whey was still being fed to pigs, irrigated on pasture or discharged to waterways, the last causing environmental damage as in other countries. However, in NZ this was not the main driver for change, due to the sparsely populated nature of the country. The advent of refrigerated shipping had enabled NZ to develop a substantial dairy export trade to the former “Mother Country” of the United Kingdom, which remained their largest export market until the 1970s, when Britain joined the Common Market, now known as the European Union (EU). The United Kingdom had first applied to join the Common Market in 1961 and it was clear that the NZ reliance on the UK market needed to be reduced. This prompted a large government commitment to research and development in the NZ dairy industry. Coincidentally, in 1969 the New Zealand Dairy Board (NZDB) was approached by a large multinational beverage company, now known to be Coca Cola, seeking to source a supply of soluble protein to fortify an acidic beverage product they were trying to develop. The company had a clear set of requirements for the ingredient, in that it needed to be clear in solution at pH 3.5 in the presence of phosphate or citrate in a carbonated beverage and not have any adverse effect on the overall flavor. The company had identified that whey protein had potentially the characteristics they were seeking. This was immensely challenging, as it represented the perfect storm of innovation as we recognize it today, in that this was a request for a brand new product, for a brand new application, using a technology or technologies that had not yet been identified for the purpose.

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Following a screening of alternative technologies considered to have the potential for extracting the whey protein from acid casein whey in a soluble form, the team fairly rapidly identified UF as the one with the greatest potential to form the basis of the extraction process. A tubular plant using CA membranes was acquired from Abcor in 1970, designated the Abcor UF300S, the 300 being the active filtration area in square feet (approximately 27 m2). This was installed at the NZ Dairy Research Institute (NZDRI) in Palmerston North, a full 2 years earlier than Frank Thomas commissioned his first spiral system in Pollock, USA. Work on this plant proved invaluable, not only to the NZDRI but also to Abcor, in that many technical challenges associated with system design were met and applied to future systems. This system proved capable of producing the degree of protein concentration required for the application, but UF alone could not produce a clear protein extract from whey. Consequently, development effort was also required to clarify the whey. In conjunction with centrifugation, diatomaceous earth filtration was found to provide the clarity required in the UF concentrate prior to spray drying and a product meeting the requirements requested by Coca Cola was successfully produced on a pilot scale. In order to scale up production, a much larger plant was acquired from Abcor and installed at the NZ Co-operative Dairy Co. in Waitakaruru, Waikato. The team learned to make the product successfully on a potentially commercial scale, incorporating discoveries and overcoming technical problems that are now taken for granted, e.g., the effect of silicon-based antifoams and the development of cleaning regimes involving proteolytic enzymes. During the early 1970s, NZDRI also evaluated alternative UF formats, with pilot plants purchased in plate-and-frame, flat leaf, and hollow-fiber formats, though spirals followed considerably later. The advantages of PS over CA were also evaluated. So, when whey UF was in its infancy in the United States and Europe, NZDRI had successfully developed a potential “gamechanger” for nutritional beverage applications, along with possessing the most comprehensive knowledge of whey UF in the world at that time. The potential for the whey protein required by Coca Cola was considered to potentially outstrip the ability of the NZ dairy industry to supply it, which may have contributed to what happened next, as before any commercial product launch, the corporation changed direction in that it took an alternative approach to delivering nutritional beverages and it took another 30 years before clear low-pH beverages fortified with whey protein would be commercially available (see Chapter 9: Whey Protein Ingredient Applications). Thus, despite the technical superiority of the NZ dairy industry, it had no market for its novel whey protein products at that time. The commitment to

2.3 Systems and Applications Development

development continued from the mid 1970s, to find new uses and customers for WPC. At the leading edge of the technology was the WPC product at .75% protein, mainly for the emerging market for functional proteins in Japan. Acid casein whey, the main starting material, happened to be particularly suitable for these applications and eventually the NZDB sales team found a successful application in the pumping of hams, as an alternative to egg white. The development activity that followed the original product development largely revolved around developing an understanding of the functional properties of whey proteins, such as solubility, foaming, whipping, emulsification, and gelation—the understanding in New Zealand of these aspects led the world at this time. In addition to the Abcor system whey UF plant there was another large commercial WPC operation in New Zealand at that time. In March 1970, The Lactose Company of NZ Ltd commenced commercial production of a WPC with a protein level of 53% 58%, named Prolac, which was spray-dried and marketed as an egg white replacer for bakery applications. The plant was in Edendale, Southland in the remote south of New Zealand’s South Island, more than 600 miles southwest of Palmerston North where the development work with Abcor took place. The Lactose Company had evaluated three US technologies for ultrafiltration, namely Havens, Abcor, and Door Oliver. The initial commercial plant utilized Havens modules, but by 1972 Door Oliver became the preferred design and the Havens modules were systematically replaced as new Door Oliver modules were acquired. Prolac production continued in Edendale until the operation was relocated and expanded to a purpose built facility at Kapuni, Taranaki in the North Island in 1977. In 1979, DDS supplied a large, seven-stage system for this operation to add capacity to the relocated Door Oliver system. Pasilac/DDS became the dominant supplier of commercial UF equipment for the next few years from the late 1970s. Their first installation in New Zealand was in 1978, processing whey at the sulfuric acid casein plant at Te Aroha Thames Valley, Waikato, making WPC55. This was the first of at least a dozen similar plants supplied by Pasilac to Australia and New Zealand over the next few years. These included a three-line DDS plant at Rangitaiki Plains Dairy Company (later Bay Milk Products) at Edgecumbe, Bay of Plenty, in around 1979, also making WPC55 from B1 ML/day of sulfuric acid casein whey, followed by a similar installation at Longburn, near Palmerston North in 1980/81 processing lactic acid casein whey. These operations at this time were not commercially successful, as there was insufficient market for the WPC, so the NZDB acquired the assets, paid the dairies to operate the plants and took responsibility for the sale of the product. Over a number of years as markets developed so did the processes, with an incremental increase from WPC55 to WPC75, WPC80, and finally WPC82.

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The next plant built in the early 1980s was at Hautapu, Waikato, processing sweet whey from both cheese and rennet casein. This was the last significant installation before the first big spiral system was built at Kiwi Dairies’ factory in Hawera, Taranaki around 1990. The Hawera installation was capable of processing around 2 ML/day of cheese whey, as well as 1.5 ML/day of lactic casein whey. Further large spiral systems followed at Clandeboye, Canterbury in 1996 and Lichfield, Waikato in 1998. As an interesting aside, the Kiwi Dairies installation was not the first major spiral UF owned by the NZ dairy industry. A large spiral system designed by Abcor and built by APV Crawley in the United Kingdom, with about 2500 m2 of membrane area, was ordered for the Tirau site in Waikato at the start of the 1980s. However the Tirau dairy was actually built and commissioned as a lactalbumin plant rather than a WPC operation and the spiral plant remained in storage until it was finally scrapped about 10 years later. The decision to produce lactalbumin rather than WPC certainly appears to have been related to lack of market demand for WPC. However, it is interesting that the plant was never installed anywhere else rather than being scrapped.

2.3.2.4 Domination of the Spiral-Wound Membrane—From the Mid 1980s By the early 1980s, most new whey UF systems deployed PS or PES membranes and RO systems were moving toward the new TFC membrane. However, there were many formats of membrane equipment in use, and each had its own advantages and disadvantages. The DDS plate-and-frame UF system introduced in the 1970s focused on engineering to achieve the absolute highest membrane performance per unit of applied area through supplying a very high shear rate over the membrane surface. The result was the need for much less membrane surface to achieve the same or better results than spiral-wound systems were capable of delivering but the capital cost of the plate-and-frame system was very high. DDS marketed their system toward difficult applications such as 75% and 80% WPC and for UF of milk for cheese making. They were very successful in these markets, as at the time they had relatively little real competition in these technically more challenging applications. Abcor/KMS may claim to have been close to DDS in technical capability but out-sold by DDS; however, what is beyond doubt is that DDS was dominant by the end of the 1970s. Of the other plate-and-frame formats, Dorr Oliver and Rhone Poulenc had failed to gain a significant foothold, while the Romicon hollow-fiber design faded after a short time as individual membrane fibers frequently broke, resulting in protein losses. The membrane cartridges were expensive and it became common to glue a toothpick or similar into the ends of the damaged

2.3 Systems and Applications Development

fiber to allow the cartridge to continue to be used. The damaged fibers were found by running water into the permeate side and seeing which fiber was allowing a high volume of water passage. There were obvious limitations with the early spiral systems as pioneered by Thomas Technical Services. They were difficult to clean, as alluded to earlier, due to the restrictive processing and cleaning conditions associated with CA membranes. The fairly basic engineering of the systems meant that the hydraulic conditions within the plant also made cleaning inefficient, with flow rate across the membrane surface being below that required for efficient cleaning. This, together with the inherent characteristics of whey compared to water, from which the technology was borrowed, meant that those early systems could make products like WPC35 reasonably reliably, but higher protein levels could not be achieved. It became obvious that the spiral format was by far the most economical for filtration membranes, so that, if certain challenges could be overcome, it could compete with and displace the other formats in more challenging applications. The first of these, already mentioned, was the availability of PS/ PES membranes in the spiral-wound format. Desalination Systems Inc. (Desal) in California was among the pioneers. They had previous success with spiral-wound elements in other applications, such as water purification, and when they decided to enter the dairy industry, this was their membrane configuration of choice. There were several challenges of adapting water elements for dairy use, which mainly centered around utilizing more robust materials to enable the elements to stand up to the repeated operation and cleaning cycles required of a dairy application. They also needed to accommodate the higher and varied viscosities, as whey protein was increased to higher levels, required to compete with the plate-and-frame and other more highly engineered formats. Other innovations followed to fulfill the requirements of the hygiene standards required of membrane elements used, to make the new WPCs acceptable in food applications. In the case of Desal, this was their patented Durasan outer wrap, which they used to promote the use of spiral-wound elements in the dairy industry. As well as these innovations in the manufacture of spiral-wound elements, there was scope for improving the engineering of the membrane systems. Abcor/KMS had built their early tubular systems to run in batch mode, but hygiene problems were common and they realized the need to reduce product time-in-process. By the time they built their tubular plants in Ireland, they had already designed and incorporated features that are taken for granted in today’s spiral systems, such as a single feed pump feeding multiple (typically five) stages in series, each with their own circulation pump (without interstage valving) and a “floating” baseline pressure, as well as the use of a volume ratio

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controller to control the process, rather than using a refractometer. These revolutionary features were adopted on Abcor/KMS’s early spiral systems, greatly enhancing the hydraulics for both production and cleaning when compared to systems built by TTS. They continued to improve engineering and system designs to facilitate the building and operation of larger, more sophisticated, systems. For example, they had worked out how to make WPC at .75% protein through the incorporation of diafiltration by the time the ill-fated APVbuilt system was delivered into New Zealand (B1980). All other membrane filtration formats succumbed to the spiral design, with the exception of certain niche applications, as the frontiers of what spiralwound membranes could achieve were pushed back. From a purely technical aspect, the spiral design remains, relatively speaking, low-tech. It still requires much higher membrane areas for the same processes when compared to the other systems but the capital and operating costs are so much lower that a spiral system can be delivered at a much lower cost and the replacement membranes are also much less expensive. It is important to remember that, at that time, virtually all applications ran on hot (unchilled) whey, typically at 40 50 C. The advantages of running hot were that the cost of cooling the whey was avoided and the lower viscosity of the hot feed streams meant that permeate fluxes were much higher, so reducing the size of the system and minimizing the capital cost. However, as system and process complexity increased, the downsides of hot processing became more and more apparent. These were rapid fouling of the membranes in certain applications, the realization that membrane life was much shorter and the growing appreciation of the microbial risks involved. Nevertheless, this was such an important step in the evolution of dairy processing that spiral-wound membrane systems now feature in virtually all significant whey processing operations worldwide.

2.3.3

Nanofiltration

From the early 1980s there was a growing appreciation that the salt content of certain whey streams was limiting their utilization in added-value applications, either for reasons of taste or nutrition. For demineralized whey powders for infant formula, ion exchange (IE) and electrodialysis (ED) were already being used but these technologies were prohibitively expensive for other applications. Hence a more economic way to remove minerals, particularly sodium, potassium, and chloride, from dairy streams such as acid whey, delactosed whey, or salt whey, was sought. At least two technologies were trialled in industrial-scale plants, one of which endured while the other did not. The one that did not last was a membrane

2.3 Systems and Applications Development

process called “Counter Diffusion,” first applied for demineralizing cane molasses in the Australian sugar industry in the early 1980s. It involved a membrane consisting of immobilized organic crystals providing a porous but highly selective barrier held within hollow fiber modules, with the feed material on one side of the membrane and water on the other, so that salt diffused across the membrane from feed to water. At least one plant was installed for whey in Australia, and another in Avonmore’s Ballyragget site in Ireland in 1989, the technology being applied to the desalting of delactosed whey. The Australian plant operated for about 3 years, before the equipment supplier went out of business. By this time the alternative new technology for salt reduction was gaining critical mass. In 1984 the first of a new type of membrane was tested for the first time to complete the family of pressure driven membrane separations as we know them today. This NF membrane application was piloted at the Mid-American Dairymen’s plant in Winsted, Minnesota—shown in Fig. 2.4. The application was to demineralize Cheddar cheese salt whey by removing the sodium chloride with a reduction of the normal sweet whey limits. The membrane was the first attempt by FilmTec to develop a TFC RO membrane for desalination of seawater but the problem was it did not reject salt! It was called the FT 40. A subsequent conversation ensued between FilmTec’s Director of

FIGURE 2.4 The first NF/UO for salt whey, at Mid-American Dairymen’s plant in Winsted MN in 1985. Photograph courtesy of George Hutson.

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Research, Bob Peterson, and George Hutson, the owner of a growing Minneapolis-based company specializing in building spiral membrane systems for the dairy industry called Filtration Engineering Inc. (now part of Tetra Pak). Hutson was asked by Peterson whether such a membrane could have any useful application, to which he replied “Will it reject lactose?” The response was along the lines of “I have no idea, let’s try it.” It did reject lactose and a new application was born, based on a membrane that showed repeatable characteristics that were not those intended when it was originally manufactured. There was disagreement as to what to call the membrane and its application; the industry in general adopted “nanofiltration” (from the original “leaky RO”) whereas Hutson and Filtration Engineering registered the name “Ultra Osmosis” (UO) and continued to use that name for many years before finally accepting the term nanofiltration following FE’s acquisition by Tetra Pak. The first system was installed in Winsted in 1985 with six FilmTec 3838 NF membranes. The process successfully removed monovalent ions such as sodium, potassium, and chloride, while leaving lactose, polyvalent ions, such as calcium, and of course protein in the concentrate. Shortly after this Gene Sorenson, then President of FilmTec, said they did not want to continue manufacturing the membrane and Hutson took the idea to Desalination Systems, as the patent for manufacture was publicly owned. Don Bray, the president of Desal agreed to manufacture it. Desal achieved the desired characteristics at the first attempt and the membrane was termed the DK, a term which was still recognized 30 years later (The D stood for Don (Bray) and the K for his lab assistant Karen). The first major installation was sold in 1988 to Avonmore Dairies in Ireland to demineralize hydrochloric acid (HCl) casein whey. The research was led by Paddy O’Donovan with the objective of removing the majority of the chlorides and neutralizing the whey to pH 5.6 to make a product analogous to sweet whey. The application was a success, and Filtration Engineering installed the first system at Carrick-on-Suir in 1989 with the second system at Shannonside the same year. Hence the UO/NF process changed the entire HCl casein whey process in Ireland, and many applications subsequently incorporated UF as well, to turn acid casein whey into high-value WPC. NF has since been widely adopted in desalting of salt whey, as well as in several other applications where total solids concentration with partial demineralization is beneficial to the application. The Carrick-on-Suir plant was eventually relocated to Avonmore’s Ballyragget site, where it was converted to run as a UF system making WPC35 in 1995. This was replaced by a new UF system commissioned in 1996, supplied by Separation Technology Inc. (SeparaTech), a small Minnesota company supplying membrane filtration systems for food and dairy applications.

2.3 Systems and Applications Development

2.3.4 Ion Exchange Technology and Microfiltration—in the Development of Whey Protein Isolate The development of WPI can be traced back to some IE resin work at Bath University in South West England in the 1970s. This included resin development work and utilizing whey as a substrate from which to extract proteins. The Bio-Isolates group from Swansea built a pilot scale plant in South Wales, before a larger plant was installed at Mitchelstown Co-operative Creamery, Ireland (which became part of DairyGold Co-op in 1989), in the late 1970s to scale up the work that was being done at Bath in conjunction with BioIsolates. Mark Davis of Davisco Cheese, Le Sueur, Minnesota, became aware of this work at about this time and the first commercial plant specifically designed to extract whey proteins via IE was built as a joint venture in Le Sueur in 1982 83. The process involved a cation exchange process on acidified whey, in a stirred reaction tank, at a pH value below the isoelectric point of the major whey proteins, so that these proteins were positively charged. This was followed by washing to remove lactose and other nonprotein materials then elution from the resin by raising the pH with an alkali, e.g., NaOH, to release the protein. The process created a new dairy ingredient category, as the product eluted from the resin was essentially free of fat and lactose and so was close to pure whey protein. Following concentration (using UF, which also removed any excess ions), the product was spray-dried to a powder with typically around 95% protein on a dry basis. The new ingredient was called BiPRO, and it opened up new added-value applications for whey protein ingredients, initially due largely to its unique functionality, which was attributed to a combination of various aspects of its composition. BiPRO was a commercial success; the factory was expanded in the late 1980s and Davisco continued to develop a range of WPIs and derivatives over the subsequent decades, through to the company’s acquisition by the Canadian cooperative Agropur in 2014. Bio-Isolates went public as a publicly listed company, after which Davisco took over the company in about 1992, acquiring all the associated technology and patents and adding further manufacturing capacity in the late 1990s. Soon after the introduction of BiPRO, work was progressing, mainly in Europe, to manufacture an analogous product using membrane filtration. This required the development of another filtration process that was new to the dairy industry and was capable of removing the residual milk fat and other colloidal material from whey. This enabled UF to be used on the clarified whey stream to give a composition similar to that achieved in BiPRO. This process was, of course, MF. All the initial work was done with ceramic membranes, which were available with proven tightly controlled pore sizes in the region of 0.1 0.5 μm. This

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included development work at the Institut National de la Recherche Agronomique (INRA) in France, as well at Avonmore Foods (now Glanbia) at Moorepark in Ireland, in conjunction with Tetra Pak in a project led by Paddy O’Donovan. At the successful conclusion of this project, a commercial system was built at Avonmore’s plant at Richfield, Idaho, in 1989 90 using Pall membranes and this remains the only ceramic MF system for commercial production of WPI in the United States. For several years ceramic microfiltration remained the only filtration technique available for the defatting of whey but as with the previous examples of RO and UF, the high capital cost of ceramic systems was all the incentive required to induce the pioneers of spiral system designs to find a more costeffective alternative. At SeparaTech Inc., the decision was made by the owner Randy Willardsen to investigate the use of polymeric membranes in a spiral configuration for comparison with ceramic MF for WPI production. An initial screening of different membrane types indicated that polyvinylidene difluoride (PVDF) MF membranes with a nominal pore size of 0.2 μm, which were at the time being used for clarification of corn syrup, could successfully be used to clarify whey or preconcentrated WPC ahead of a final UF step in the production of WPI. The initial supplier of membranes for this application was Advanced Membrane Technologies Inc. (AMT) of San Diego, California, closely followed by Synder Filtration of Vacaville, California. SeparaTech sold the first plant using this design to Land O Lakes in Perham, Minnesota, in 1995. This first plant needed expansion and some reengineering to meet process guarantees but did produce a high quality WPI; the plant became the prototype for essentially all spiral MF-WPI produced to the present day. Two further installations followed in 1996 at the Protient sites in Juda, Wisconsin, and Mountain Lake, Minnesota, followed by a fourth plant installed at Volac International Ltd. in Wales in 1997. This was Europe’s first WPI system, built before there was any market for WPI in nutritional applications other than perhaps infant formula in that continent. The pore size of the PVDF membranes adopted for MF of whey proteins was, and remains, of the same order as with the ceramic membranes they were competing against, though the size distribution was far less precise. Consequently, one of the main challenges was to achieve an acceptable rate of protein passage through the membrane without allowing the smallest of the fat globules present to either pass into the permeate or to lodge in the pores, thereby fouling the membrane. Related to this issue was the limitation on the maximum feed pressure that could be applied to the membrane, which is in the region of 0.15 MPa (1.5 bar, 22 psi). As a pressure drop of around 0.1 MPa is required across each spiral element to achieve adequate cross-flow velocity, such systems were limited to one membrane element per

2.3 Systems and Applications Development

housing and also the trans-membrane pressure was very variable across the membrane. This meant that even with a great degree of control of the hydraulic flow within a spiral MF system making WPI, the resultant performance was much less efficient or consistent than the ceramic systems they were competing against. Nevertheless, the spiral MF system utilizing PVDF membranes was a fraction of the capital cost of the ceramic systems, which more than compensated for the much greater membrane area required for the same duty, so once again the development of spiral applications had displaced other more capitalintensive filtration alternatives. Two other ceramic systems were built for WPI manufacture, one in New Zealand and another at Carbery in Ireland in 1997 98 but these proved to be the last and the Carbery system was replaced with a spiral system built by Complete Filtration Resources (CFR) of Marshfield, Wisconsin, in 2010 11.

2.3.4.1 Optimizing Spiral MF Conditions for WPI Production Initially the spiral MF applications were generally run at around 50 C. However, as this author learned at a very early stage in 1998, the initial high flux was extremely short lived on this process. PVDF MF fouling tends to be prohibitively rapid at these temperatures, so reengineering of systems to run at ,20 C quickly followed. The trend toward cold processing continued through the 1990s, such that today most whey protein membrane filtration systems are run cold. The balance between high temperature, high flux rate, rapid fouling and cold processing, low flux, long run time, better microbial quality is just one example of the frequently occurring issues relating to the processing of whey and whey protein products. There are invariably multiple theoretical ways to achieve the same goal and the art of achieving the desired goal effectively is the successful management of the compromises and trade-offs that need to be made.

2.3.4.2 By-Products of WPI Production Another issue typical of whey protein processing which becomes a little more complex in the manufacturing of WPI using MF is the issue of byproducts (or coproducts). This is a perennial issue in cheese and whey processing, going back to the early days where finding a whey solution became paramount for cheese manufacturers. Then, when UF became established, the majority of the solids again ended up in the by-product, i.e., the UF permeate. Thus solutions had to be found to accommodate this before WPC could really be commercialized. MF used in production of WPI extends this even further, as it generates yet another coproduct, which in this case is the MF retentate, a very interesting but challenging

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product, containing all the residual milk fat, colloidal material, and an elevated proportion of the larger whey proteins such as the immunoglobulins. This retentate is now available as a commercial product. It is marketed as Whey Protein Phospholipid Concentrate or High-Fat Whey Protein Concentrate. It typically contains about 65% protein, 20% fat, and 8% ash (American Dairy Products Institute, https://www.adpi.org/Portals/ 0/Standards/WheyProteinPhospholipidConcentrate_book.pdf).

2.4

KEY DEVELOPMENTS SINCE 1990

The following section is by no means a tour de force of all developments in whey protein processing and applications in the last 27 years, but rather it is intended to give a flavor of the most significant developments of recent years.

2.4.1 Agglomeration/Instantization and Whey Protein in Sports Nutrition Until the early 1990s most WPC manufactured was WPC34/35 produced on relatively simple spray dryers, typically as a fine powder and utilized as animal feed or in industrial food applications. With a low-fat, relatively lowprotein product such as this, reconstitution characteristics in water tended to be reasonable and end-use requirements were not particularly demanding. However, from the late 1980s, there was a growing understanding of the potential role proteins could perform in recovery after arduous exercise, as in sports. At this time the proteins that were already in this embryonic market were egg white and caseinate. Whey protein began to attract a lot of attention in this regard for a number of reasons including the growing body of research regarding not only its nutritional properties but also its bioactive properties. The principal pioneer of this work at this time was David Jenkins, who was able to combine seeing and understanding the need with engineering experience to turn the whey protein into an easy-to-use form and the business acumen to make a success of the enterprise. That company, and the product it produced, was Designer Protein.

2.4.1.1 Designer Protein—The First Instantized Whey Protein Powder As a chemical engineer, three-time UK Olympian and Olympic Silver medallist at Munich in 1972, David Jenkins had long been fascinated with all manner of performance enhancement for sport and life. By the late 1980s his focus was how to best improve recovery in sport. An extensive paper based on the multiyear research done by Professor Saris on the Tour de France

2.4 Key Developments Since 1990

competitors was highly influential. His recovery formula proposals included protein, and not just “carbs” (carbohydrates), common sense today—but a game-changer 40 years ago. Also influential were postoperative surgery formulae, all of which had combinations of proteins, carbohydrates, fats, vitamins, and minerals. Adopting this approach, Jenkins developed a formula and prototype for an instantized product in 1987, called ProOptibol, the first recovery optimizer, which began selling in health food stores in Southern California and Hawaii in early 1988 and became very popular among cyclists and triathletes (see Fig. 2.5). ProOptibol contained 33% protein, 60% carbohydrate, MCT (medium-chain triglyceride) oil, vitamins, and minerals and was made at Owatonna Riverbrands of Owatonna MN, a subsidiary of Innovative Food Processors owned and founded by the late Dr. Gene Sanders. Sanders had been a professor of food engineering at the University of Minnesota. He had set up his own instantizing/agglomeration plant south of Minneapolis and had started by agglomerating maltodextrins for Grain Processing Inc. This was a batch process involving the use of hot air to fluidize a quantity of powder and raising the moisture level by spraying in water to make the powder particles stick together via collisions into agglomerates, followed by redrying. This process is known as rewet agglomeration. Through careful management of process parameters, it is possible to accurately control the particle size profile of the final product, and it is also possible to incorporate surfactants such as lecithin with the spray, such that almost any soluble but difficult-to-mix powder can be transformed into an easy mixing version. The original whey protein source was WPC75 from Golden Cheese in Corona, which made product primarily for the Japanese market. In 1991 this changed to WPC80 to align with changes in the Japanese import tariffs. This proved a significant benefit as the increase in protein level resulted in much lower lactose levels that would be better tolerated by lactose intolerant individuals when ingested. Working with Gene Sanders and his research and development team in late 1992, Jenkins was eventually successful making small (5 kg) batches of instantized whey protein. Without any added carbohydrates this proved quite a challenge, as the protein powders behaved very differently from the carbohydrates that Sanders had become used to agglomerating. The process was then successfully scaled up to run in Sanders’ larger fluid-bed agglomerators, processing .300 kg at a time. Once they had established the ideal agglomerated particle size distribution and specifications for optimal dissolution in cold water or milk, they had developed the first instantized protein powder in the United States—it was easily mixed with a spoon with no blender required. The first flavor was vanilla praline; chocolate and strawberry

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FIGURE 2.5 Advertisement for ProOptibol 1991, endorsed by elite triathlete Paula Newby-Fraser. Courtesy of David Jenkins.

2.4 Key Developments Since 1990

followed. The products were branded Designer Protein. The commercial launch was in July 1993 at the National Health Food Show in Las Vegas. An advertising campaign in bodybuilding magazines followed. By 1994 it was the number one selling protein brand in America and grew at 100% a year through 1998 (Fig. 2.6). As the 1990s progressed, the demand for high-protein powders was fuelled by changes in the sports nutrition market, particularly by those, such as bodybuilders and weightlifters, wanting to rapidly build their muscle mass, coinciding with the period when the use of anabolic steroids was becoming unacceptable (see also Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated and Chapter 16: Sports and Exercise Supplements).

2.4.1.2 The Growth of Instantized Whey Protein Powders Thus the demand for high-protein powders was dramatically increasing, accompanied by the new requirement for powders to be easy mixing, as the users would be simply mixing the powders into water, milk, or juice. At the same time, the higher-protein whey powders, especially fat-free WPIs, were inherently more difficult to mix as fine powders than the lower-protein versions. This led to an explosion in demand for agglomerated protein powders, first applied in the United States, and around 5 years ahead of the situation in Europe. This demand is still increasing as the understanding of the nutritional benefits of protein in general and whey protein in particular reaches a wider audience. More academic studies were showing the benefits of whey protein, not only in promoting the gain of muscle mass but even more favorable evidence was emerging regarding the importance of protein in maintaining general health (see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins). As the demand for agglomeration of whey protein powders took off, an opportunity emerged for a more efficient, higher-capacity (i.e., lower cost) method of agglomeration to expand the availability of instant whey protein powders. As it happened, this technology already existed and was conveniently concentrated in the Midwestern United States. This technology was analogous to the batch rewet agglomeration system used by Gene Sanders in Owatonna, but was a continuous version. The following Wisconsin companies were all involved in the instantizing of WPC or WPI from around the late 1990s: I I I I I

Main Street Ingredients, La Crosse Maple Island, Medford Lake Country Foods, Oconomowoc Century Foods, Sparta Marron Foods, Durand

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FIGURE 2.6 Designer Protein—the only five-times winner of the American Taste Award—hence the Gold Medal and Splash from 1998. Courtesy of David Jenkins.

2.4 Key Developments Since 1990

The process of continuous rewet agglomeration started with the pioneering research of David Peebles at the beginning of the 1950s, and instantized nonfat dry milk was first marketed in 1954. Soon it replaced the existing spray-dried products on the retail market. The basic Peebles instantizer is shown in Fig. 2.7; commercial rewet agglomeration processes for highprotein WPC and WPI were based on this technology, commencing commercially around 1997. Even though these continuous agglomeration processes dominated the agglomeration market for several years, and many thousands of tonnes of protein powders are still instantized in this way, the batch system was never entirely displaced. Vision Process started a batch agglomeration business in 2003 in Litchfield MN, and subsequently expanded and moved to Owatonna. The tolling costs were higher than the continuous alternative, but the greater control possible in a batch system allowed them to compete in terms of quality and consistency. This business is now part of Kerry Foods. As recently as 2014, Marron Foods also added a batch system to their existing continuous facility.

FIGURE 2.7 Peebles instantizer, as first marketed in 1954. rGEA Group 2018.

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2.4.1.3 Continued Development—Removing the Need for Rewet Agglomeration One obvious disadvantages of rewet agglomeration was the cost associated with shipping the product to the contract processor, the cost of the agglomeration (including 1% 2% processing losses), and the cost of financing the additional time and inventory to permit the agglomeration to take place. Consequently, the designers and manufacturers of spray dryers started working in earnest to find a way to spray dry, agglomerate, and instantize in a continuous process. Spray dryers with integral agglomeration and instantization for WPC80 and WPI began to appear in the early 2000s, the designs being variations on a theme. The agglomeration was achieved by the use of multiple atomization nozzles with overlapping sprays, with fines return to the wet zone below the nozzles, giving some flexibility to vary the particle size and density via manipulation of variables such as lance position and spray cone angle. The fines would be recovered from chamber and fluid bed exhaust air, with lecithin sprayed at the base of the chamber or in the first section of the fluid bed, prior to final drying and cooling, as illustrated in Fig. 2.8. An example of the microstructure of such a powder is shown in Fig. 2.9. Depending on the design and adjustment of the system, particularly the location of the introduction of the fines in relation to the atomization nozzles, different agglomerate structures can result. These can influence final powder properties, primarily wetting and dispersing characteristics, but also bulk density and mechanical stability.

2.4.2 Whey Protein Fractionation and Whey Protein Hydrolysates for Infant Formula The history of infant formula development goes back to the 19th century but our specific interest starts in 1962, with the first products brought to market reflecting the realization that the casein to whey protein ratio in bovine milk was very different to that in human milk; also, the total protein content in breast milk is much lower than in bovine milk (see also Chapter 12: Whey Proteins in Infant Formula). These newly developed formula milks contained demineralized whey added to the skim milk to standardize the protein content and bring the casein to whey ratio essentially to the 40:60 found in human milk. The production of whey powders demineralized by up to 90% by a combination of IE and ED developed as a result of this from the late 1970s, with Euroserum in France producing around one-third of the world’s demineralized whey powders. However, as the science developed, it became apparent that the whey protein

FIGURE 2.8 Schematic diagram of an agglomerating spray dryer. rGEA Group 2018.

FIGURE 2.9 A photomicrograph of an agglomerated dairy powder. rGEA Group 2018.

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component in breast milk and that in formula milk were very different. For instance β-lactoglobulin (β-Lg), a protein making up around half of the protein present in demineralized whey, is not present in human milk. Further to this, β-Lg has been shown to be an inducer of allergy in early infancy, due to the presence of antigenic sites on the peptide sequence of the protein. The majority of the proteins in human milk are contributed by α-lactalbumin (α-La) and lactoferrin. As a consequence, with the quantity of infant formula manufactured worldwide increasing rapidly, not only has the overall quantity of whey protein being used in infant formula increased greatly but also so has the desire to “humanize” infant formula made from bovine milk and to address the allergenicity issue. This has driven several technological developments in whey protein processing, primarily whey protein fractionation and protein hydrolysis.

2.4.2.1 Whey Protein Fractionation Pure (or at least highly concentrated) whey protein fractions including α-La have been produced commercially via IE chromatography for over 20 years, e.g., by Davisco. These separations utilize charge affinity characteristics of the different whey proteins for particular ligands within a resin matrix. Essentially, any whey protein separation is possible using this technology but, as is so frequently the case in whey protein processing, the tail tends to wag the dog in that the production of a new (potentially) high-value derivative results in the majority of the (in this case already valuable) starting material as a by-product needing to be found a market. With α-La only comprising about 20% of total bovine whey protein, the value needed to be derived from the added-value component must be great enough to cover the cost of extraction, assuming that the value of the by-product is at least the same as the starting material. Of the minor whey proteins, lactoferrin has received the main attention, together with lactoperoxidase. They tend to be grouped together because they have similar isoelectric points, and very much higher than those of the major whey proteins. Hence they tend to be extracted together in chromatographic processes as, at a pH close to neutral, they both carry a net positive charge while the remainder of the proteins present are all negatively charged (see Chapter 1 for further information on lactoperoxidase and lactoferrin). Lactoferrin is currently manufactured in New Zealand by Fonterra, Synlait, and Tatua, in Australia by Murray Goulburn, Bega Bioingredients, Warrnambool Cheese & Butter, and Beston Global, as well as in Europe by Friesland Campina and in the United States by Glanbia. Lactoferrin is considered a bioactive component in milk. In the EU, bioactive components come under the novel foods legislation and may therefore only

2.4 Key Developments Since 1990

be marketed once safety has been demonstrated. In November 2012, the European Commission published its decision approving lactoferrin produced by Friesland Campina as a novel food, meaning that lactoferrin can now be used in a variety of foods throughout the EU, including infant formula. For the economics to work for the production of purified minor whey components, the minor premium component has to command a very high price, to cover both the cost of extraction and potentially some reduction in the value of the coproduct, which is often produced in great excess of the target compound. The ideal situation, of course, is where all the components produced have a premium value in relation to the starting material. To this end, there have been attempts to market a “whey protein refinery” capable of splitting the whey protein into its major constituent components, e.g., high purity α-La, β-Lg-depleted WPI, high purity β-Lg, immunoglobulin-enriched WPI, glycomacropeptide (GMP), lactoferrin, and lactoperoxidase. Upfront Chromatography in Denmark sold a system into Dairy Farmers Co-op in Queensland, Australia, in 2002 based on their “Rhobust” expanded bed adsorption technology but this does not appear to have been followed up with any further major systems. The original system was relocated to Adelaide in South Australia, but is not believed to be heavily utilized at the present time. Another example is “Continuous SEParation” (CSEP) Technology, a so-called system approach to continuous simulated moving bed chromatography. This technology was intended to provide the benefits of conventional chromatography (specificity, reproducibility, and mildness) while addressing some of the perceived shortcomings, e.g., cost, throughput, flexibility, productivity, and complexity. A commercial system was installed at Murray Goulburn’s plant at Leongatha in Australia in around 2004. It was reported to still be in use as recently as 2017, but the technology was not adopted elsewhere. There have been attempts to separate α-La from β-Lg via membrane filtration, though the similarity in molecular size makes this difficult and no such process has yet been commercialized. There are other commercial processes by which the concentration of α-La relative to the other whey proteins has been altered to make the whey protein more attractive to manufacturers of infant formula. One such product has been manufactured in California by Hilmar Ingredients. Their α-La enrichment process (for which they have a patent) involves pH and mineral manipulation, followed by centrifugal separation and concentration of the α-La-enriched fraction by UF. The other fraction can be returned for incorporation in WPI or WPC80. While the technology exists to produce whey protein ingredients capable of making infant formula which more closely resemble human milk, e.g., by increasing proteins such as α-La and lactoferrin while depleting levels of

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β-Lg, the adoption of such ingredients appears to have been retarded by the economics of using them. However, not surprisingly, they do seem to have found their way into the “high end” formulae for preterm infants and for normal term infants for the first 4 6 months; this is where their use is likely to grow (see also Chapter 12).

2.4.2.2 Whey Protein Hydrolysis One hundred percent whey-protein partially hydrolyzed formulae (PHF-W) became available in the late 1980s. Whereas greater (or less well controlled) hydrolysis tended to result in a bitter flavor from short-chain peptides, this type of formula had a taste comparable to standard intact protein formulae, which led to better acceptance than other hydrolysates. In addition, PHF-W are commercialized in the United States as routine infant formulas, at prices broadly comparable to other routine formulae. The following statement has been approved by the Food and Drug Administration in the United States: For infants who are not exclusively breastfed, emerging clinical research in healthy infants with family history of allergy shows that feeding a 100% Whey-Protein Partially Hydrolysed formula may reduce the risk of common food allergy symptoms, particularly allergic skin rash, when used instead of whole-protein cow’s-milk formula from the initiation of formula feeding.

Several infant formula manufacturers have developed their own proprietary enzymes and processes for producing their hydrolyzed whey protein products. Whey protein hydrolysates have also found niche applications in other areas, e.g., sports nutrition and some clinical applications, where products are used for both their claimed nutritional and functional attributes compared to their nonhydrolyzed analogues. Therefore many producers of high-protein whey products also offer hydrolysates for such applications as well as for infant formula. In the United States, they are produced by Davisco, Hilmar, Leprino Foods, and Milk Specialties Global, while in Europe the producers would include Carbery in Ireland, Arla in Denmark, and Kerry Foods in The Netherlands and Ireland (see also Chapter 14: Bioactive peptides for information on whey protein hydrolysis and bioactive peptides).

2.5 WHEY PROTEINS AND ADVANCES IN CHEESE MAKING TECHNOLOGY Over the last 25 years, considerable effort has gone into finding ways to increase the efficiency of cheese making without adversely affecting quality.

2.5 Whey Proteins and Advances in Cheese Making Technology

This can generally be divided into two areas, which to a degree overlap. These are: I I

making more cheese per vat of milk processed (increased output); and making more cheese for a given volume of milk processed (increasing yield).

2.5.1

Increasing Cheese Output

From the 1980s, developments in milk UF paralleling those for WPC production have led to the use of this technique to increase the total milk protein concentration (typically standardized to a constant protein-to-fat ratio) being increasingly widely adopted for increasing the productivity of existing and newly built cheese making operations. This is beyond the scope of this review, other than as a background to a potentially superior alternative that has implications for the arrival of a new type of WPC or WPI. UF of cheese milk has been predicted to become widely, if not universally, adopted but it is actually limited by what happens to the whey proteins. As UF concentrates the casein, it also concentrates the whey proteins, resulting in a greater proportion of those proteins ending up in the cheese. This does increase cheese yield, but, above a certain level, the native whey proteins adversely affect the cheese quality, with both textural and flavor changes becoming increasingly noticeable. Increasing levels of calcium associated with the reduced whey drainage also plays a role in adversely affecting the cheese texture. Therefore, UF of cheese milk has tended to be limited to a very modest level, particularly to eliminate seasonal variations in milk composition, so that the composition of the milk going into the vat is consistently around the highest “naturally occurring” level all year (or season) round. As the understanding of the limitations of UF in this application became more widely understood from the late 1990s onwards, research shifted to MF as a potential alternative. The likes of Jean-Louis Maubois at INRA in France and David Barbano at Cornell University in Ithaca, NY, in the United States demonstrated that these limitations on protein standardization could be overcome by using MF instead of UF, as the native whey proteins passed into the permeate so that the cheese milk could be standardized to a casein-to-fat ratio rather than protein-to-fat. This technique was slow to gain traction for a combination of reasons: ceramic MF was very expensive and not widely understood in the dairy industry, plus there are some legislative restrictions in the United States. The ability to make more cheese per vat is indisputable and most of the arguments over cheese quality seem to have been won. However, the situation over yield

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is much less clear-cut than with UF as no, or very little, whey protein is captured in the cheese and around 1% 2% of the casein is lost into the permeate as free casein not trapped into micelles. As with whey UF and MF, further work has reduced the cost, largely by working out how to use cold spiral rather than hot ceramic MF to effect an efficient separation and the number of plants microfiltering milk is increasing. Lloyd Metzger at the University of South Dakota has been prominent in this work. The principal reason to include MF of milk in this discussion is that the coproduct of the casein concentrate is the MF milk permeate, containing the whey proteins. This whey stream is very interesting, in that it differs considerably from typical cheese or acid whey streams. The industry has not yet adopted a universal name for the product, and those which have been put forward include “milk serum,” “ideal whey,” “native whey,” and “virgin whey.” Its interesting characteristics compared to more traditional wheys include: I

I I

I

It is free from all additives associated with the cheese making process, such as annatto, rennet, and cultures, and its pH is essentially the same as that of fresh milk. It is fat free. It is free from GMP, the portion of κ-casein that is cleaved during the renneting process, which makes up  20% of the protein in cheese whey. The reported casein loss into this fraction also implies that around 4% 8% of the protein would be made up of monomeric αs- and β-casein. These proteins do appear in electrophoretograms of one commercially available product analyzed by the author.

These attributes make milk MF permeate suitable for the production of protein isolates with significantly different attributes to traditional WPIs made by either IE, MF, or UF processes. For instance, the absence of cheese making “debris” (particularly annatto in some countries), together with the relatively higher concentration of α-La, enhances its suitability for use in infant formula. Such ingredients are becoming commercially available, e.g., from Sachsenmilch in Germany, Ingredia and Lactalis in France, as well as Leprino Foods in the United States. These products are generally being marketed as nutritionally superior to WPI, which is not without its challenge with WPI having been promoted as “the ideal protein for human nutrition.” For example, Lactalis has been marketing their version under the brand name Pronativ, with the emphasis on improved recovery times after strenuous exercise.

2.5 Whey Proteins and Advances in Cheese Making Technology

2.5.2

Increasing Cheese Yield

As indicated earlier, there is an obvious desire to capture some or all of the whey proteins in cheese without adversely affecting quality, which has not proven possible to a great degree to date through membrane filtration on its own. Limited success has been achieved through steps such as pasteurizing the milk at a temperature high enough to denature the whey protein, often with salts such as calcium chloride to decrease whey protein solubility at elevated temperatures. UF of cheese milk has had success in some particular areas. The most spectacular example was undoubtedly in the manufacture of Feta cheese, with the UF process taking the retentate up to the dry matter level of the final cheese curd, thereby capturing 100% of the whey proteins in the cheese. This technique was pioneered by Arla in Denmark using bovine milk, and was commercially very successful, with production in Denmark peaking at an estimated 100,000 tonnes per year. However, since 2002, when the EU awarded Feta PDO (protected designation of origin) status, this product can no longer be marketed within the EU as Feta. Another technique which has found widespread application, especially in the manufacture of pizza cheese, is microparticulation (further information on microparticulation appears in Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated). The generally used process is to take a WPC at around 60% protein and subject it to a combination of a heat, to denature and precipitate the protein, and a mechanical process to break the protein particles into a fairly tight size distribution in the 1 10 μm range. These particles are similar in size to fat globules in milk and so when the microparticulated WPC is added back into the cheese milk, the particles become trapped in the cheese curd. SPX (formerly APV) of Silkeborg in Denmark produces equipment which uses scraped surface heat exchangers to do the heating and provide the shearing action Fig. 2.10. Other equipment manufacturers such as GEA, Tetra Pak, and Alpma produce equipment along broadly similar lines. The use of such “recycled” whey protein streams does have some legislative restrictions, but these have largely been overcome via classification as “secondary starter.” Such cheese yield extension techniques have generally been applied to relatively high-moisture cheeses. So far it has not proven possible to incorporate a significant amount of whey protein into hard cheeses, such as cheddar, without adversely affecting the sensory characteristics.

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FIGURE 2.10 Microparticulation plant, for producing LeanCreme, from liquid WPC. Courtesy of SPX Flow.

2.6 CURRENT WHEY PROTEIN PRODUCTS AND THEIR APPLICATIONS The spectrum of whey protein products that have emerged over the last 40 years are, more or less in their time order of appearance and increasing value, as follows: I I I I I I I I I I

whole whey powder demineralized whey powder delactosed whey powder WPC34/35 WPC60/65 WPC75/80/85 WPI whey protein hydrolysates individual protein fractions native WPI

However, within the above categories the properties of the product depend on the type of whey being processed and also on the specifics of the process used to make the product, which can influence the functionality to make the product work in specific applications. As far as the range of applications is concerned, these are divided into five broad categories: 1. Filler. 2. Cheese yield extender (as discussed earlier).

2.6 Current Whey Protein Products and Their Applications

3. Functional ingredient in food applications (gelling, water binding, emulsification, foaming, heat stability. and fat replacement). 4. Nutritional applications (animal feed, sports, lifestyle, clinical. and infant) 5. Applications using the bioactivity of whey proteins.

2.6.1

Filler

The “Filler” classification includes some long-standing relatively low-value applications for whey powder such as biscuits, bakery, bread, and confectionery. For many of these applications, the whey protein portion of the ingredient serves little function, so these will not be dwelt upon, other than to say that over time more of these of applications have moved to using whey permeate powder, possibly with a degree of demineralization if there is any sensitivity over salt levels. This change has contributed to the freeing up of the protein fraction for higher value applications. Another example of the use of whey powder as a filler is processed cheese, where it is mixed with “real” cheese and emulsifiers, to create a “cheese product” from which the fat does not separate when it is heated.

2.6.2

Physically Functional Applications

The most obvious example of where whey type and/or differences in manufacturing process are crucial is in delivering a specific physical functionality in the WPC or WPI. The functional properties typically of interest to food manufacturers would be required in conjunction with some heating or cooking process. The attributes which may be varied to influence functionality would typically be the mineral profile and the pH to produce a WPC or WPI with good gelation and water-binding characteristics, e.g., to hold water in a cooked meat product. In this case one would look to produce a product with a low level of calcium, a pH above 7, and possibly the addition of a mineral-sequestering agent such as disodium phosphate or sodium polyphosphate. For such a product, acid casein whey is a better starting material than sweet whey, as it is free from GMP; the latter is particularly heat stable and forms  20% of the total protein in sweet whey WPC and reduces the functionality. Acid whey is high in calcium in comparison to sweet whey but at pH 4.4 very little is bound to the protein so essentially it is all removed in the UF permeate when a high-protein WPC is produced. Then the final step would be to neutralize the WPC to the desired pH with NaOH or KOH. There is so much more sweet whey produced than acid whey, so manufacturers have tried to recreate this functionality starting from sweet whey, some with more success than others. This is incidentally why (apart from the fact that it appeared first) BiPRO is much more widely used for its functional attributes than MF-WPI. BiPRO does not contain GMP, and is also naturally

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very low in calcium, whereas MF-WPI would typically contain  0.4% calcium. The absence of fat in a WPI compared to a WPC also generally aids the functionality. Before the market in sports nutrition developed, the largest market for BiPRO was in applications utilizing its physical functionality, e.g., in products such as surimi in Japan. The distinction between physically functional and nutritionally functional applications is obviously blurred, and all the more so with high-protein WPCs. The most obvious example is dairy products, where there is a useful distinction between generally more “traditional” uses of whey protein ingredients where the primary driver for their use is to cheapen a formulation, e.g., whey powder, WPC, or demineralized delactosed whey replacing milk protein in an ice cream or yoghurt, to where the objective is protein fortification, with whey protein being the protein of choice.

2.6.3

Nutritionally Functional Applications

WPCs developed specifically for nutritional purposes and to replace other protein sources were used in animal nutrition before human nutrition, with the arguable exception of infant formula, which has already been discussed. Volac International in the United Kingdom pioneered the use of low-heat-processed WPC in calf milk replacer from 1990, with feeding trials consistently showing enhanced performance in comparison to skim milk-based formulations. However, the lamb milk formula remained a skim-based formula, as it was deemed to be the most sensitive and to carry the highest perceived risk to change. This formulation changed to WPC for the 1993 season; it again outperformed the skim-based formulation and it has remained the UK brand leader ever since. As Volac’s WPI business developed from the late 1990s, the whey protein source for their milk replacers gradually became more fortified by Procream, the whey MF retentate by-product of WPI manufacture. This synergy further benefited the nutritional quality of the milk replacers Volac produces, with the Procream disproportionately rich in immunoglobulins (among the larger whey proteins) and phospholipids from milk fat globule membrane and skim milk membrane material, which are concentrated with the small fat globules remaining after whey separation in the MF process. Undoubtedly the biggest growth area for whey proteins in nutritional applications over the past decade and more is in a market that barely existed when Volac was launching their whey protein-based calf milk replacer. It was not until the 1990s that significant quantities of WPC80 were finding their way into high protein powders for bodybuilders and power athletes in the United States, with WPI later in the decade. Nutrition bars containing whey protein appeared just before the millennium, which typically incorporate a blend of dairy proteins (see Chapter 13: Whey Protein-Based Nutrition Bars

2.6 Current Whey Protein Products and Their Applications

for further information on high-protein bars and Chapter 16 on sports nutrition). Market development in Europe was around 5 years behind the United States but with the sector expanding from the “hard-core” to elite athletes through to recreational athletes, supply has struggled to keep up with demand, with over 30,000 tonnes per year of whey protein currently used in these applications in Europe. A large majority of this is consumed by males under the age of 35, whose motivation has been to consume whey protein in a convenient form and, to a certain extent, have not been too bothered about the sensory experience of consuming the product, in the belief that it is delivering tangible results, i.e., increased muscle mass. As the market for whey protein-based or fortified products expands beyond the recreational athlete to the general health and wellness sectors, the marketing message has needed to evolve, and the taste and convenience of the products needed to improve. Success in these markets will only be achieved by high quality ready-toconsume products. These will most likely be beverages, but could also include products such as snack bars or extruded products. Euromonitor reported global RTD (ready-to-drink) launches with a “high source of protein” claim grew 24% a year from 2010 to 2015, with whey protein being the dominant source of protein. Whey protein-fortified RTDs tend to fall into two categories: those formulated at a pH of ,3.5 and those at a more neutral pH. The more acidic drinks tend to be clear fruit flavors, where the high net positive charge results in the protein remaining soluble following a high heat treatment. This general type of formulation has been around commercially for approximately 15 years, but the high astringency of the protein at low pH was a big challenge to mass market appeal. However, the palatability of such drinks has improved dramatically in recent times, together with the application of novel formats. The most interesting of these is probably Fizzique, a carbonated version with 20 g of protein in a 12-oz (350 mL) slim-line can, launched in 2017 (Fig. 2.11). The technical challenge for the low-acid/neutral whey protein-based RTDs is very different from that of the high-acid examples. Applying the heat treatment required for food safety will render the whey protein insoluble, so the developer has to develop a formulation and a process which can cope with the fouling effects of denaturing whey protein, as well as a product which is both stable and has the desired sensory characteristics. These products tend to be similar to drinking yogurts or smoothies, and another widely seen example is whey protein-fortified coffees. One example of a technique already mentioned to modify whey protein for use in such products is microparticulation, where the whey protein is “predenatured” but in a controlled manner that is tailored to meet the requirements of the end product. Arla in Denmark has launched a number of ingredients and retail products in this

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FIGURE 2.11 Fizzique carbonated protein water. Courtesy of David Jenkins.

2.7 Further Developments

space, and Volac in the United Kingdom launched a retail brand to market its Upbeat product, a whey protein smoothie containing fruit and fruit juice which won the Best Dairy Drink at the 2014 World Dairy Innovation Awards. The market for clinical nutrition is much smaller though developing, with applications such as WPI-based preparations for postoperative recovery, or more highly specialized applications such as preparations based on purified GMP for patients suffering from phenylketonuria, as GMP is the only known dietary protein that in its pure form contains no phenylalanine (see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins for further information on nutritive and therapeutic aspects and Chapter 17 on functional foods).

2.6.4

Bioactivity of Whey Proteins

All the proteins and peptides in whey demonstrate biological activity in their native form, which is not surprising given the millions of years of evolutionary development of mammalian milk. Whey has been used in certain medical treatments over several centuries, but obviously without the understanding of the physiology involved. The multitude of physiological effects of the different components has been widely studied over the past 20 years or so, and some high-value niche applications are developing. Bioactivity is too wide a subject to cover in detail in this chapter. For a review of the bioactivity of the major whey proteins, as well as minor components present at levels down to parts per billion, see the review by Smithers (2008) (see also Chapter 14: Bioactive Peptides, Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins, and Chapter 17 for information on bioactive peptides, nutritive and therapeutic aspects, and the use of whey proteins in functional foods, respectively).

2.7

FURTHER DEVELOPMENTS

Today the technology exists to make WPCs, isolates, hydrolysates, and fractions that find their way into a wide spectrum of applications. This concluding section is an attempt to predict where the likely growth areas will be, as far as the uses of whey protein products over the next 5 10 years and where market developments, technological developments, or other primary factors may be the essential drivers for these developments. For instance, the use of whey protein for infant formula will continue to grow but it will be technologically and compositionally “more of the same” unless novel ways are found to enable the incorporation of the components which will further “humanize” the products more economically than is currently the case.

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2.7.1

New Markets

Scientific research continues to reinforce our knowledge relating to the benefits in a healthy diet of protein in general and whey protein in particular. When aligned with the realization that the average diet is deficient in protein compared to the other macronutrients, i.e., carbohydrates and fats, comes the belief that there is massive potential to expand the marketing of whey protein products for their nutritional benefits, from the under 35 male to the wider population. As well as postexercise recovery, whey protein can help to maintain a healthy lifestyle in areas such as weight management, minimizing sarcopenia (the tendency to lose muscle mass and function as we age), and the maintenance of bone density through aiding calcium absorption (see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins). This will really be a continuation of the developments outlined in Section 2.6.3, with more and more whey protein-fortified analogues of common food and beverage groups with which we are familiar today. Alongside the introduction of high-quality retail products rich in whey protein, we can also expect to see the introduction of ingredients specifically developed to enable protein fortification of prepared foods at the expense of the other macronutrients, e.g., in soups, sauces, breakfast cereals, or analogue creams with the fat partially or completely replaced by whey protein. These ingredients will be delivering both a functional and nutritional benefit, and so will add cost to the final products, so there will inevitably be the question of affordability.

2.7.2

New Technologies or Processes

The technologies already exist to essentially take whey apart into its constituent components, so it is difficult to see a revolution or eureka development in the next decade in this area. There may be incremental improvements to existing processes—one example could be that if minor whey proteins such as lactoferrin were to find wider application in infant formula then a hybrid between IE and membrane processing might emerge. Affinity microfiltration membranes could combine a traditional MF separation process with the charge on the membrane simultaneously and reversibly bind the target protein. Such a process could potentially make the isolation of lactoferrin more economical, which would make it easier to include it at meaningful levels in infant formula. Novel applications of existing technologies may be applied to target the minor proteins found in milk. Osteopontin is a good example. An ingredient recently commercialized by Arla Food Ingredients contains osteopontin at nearly 90% purity (Lacprodan OPN-10). Osteopontin occurs at far higher concentrations in human milk than in bovine milk, and it has become highly

2.7 Further Developments

valued as another ingredient to humanize infant formula. The Arla process involves a combination of ultrafiltration and microfiltration, together with managing the solubility of the target and other proteins to allow its effective purification. This example is likely to be repeated with the isolation and commercialization of other minor proteins/peptides, as well as other components such as milk fat globule membrane (MFGM) material and milk oligosaccharides such as sialyllactose. There is an area where, if the appropriate technology can be developed and introduced, there would be a massive impact on whey protein processing worldwide. This is the “holy grail” of cold pasteurization. Whey processing is dominated by the continual need to make compromises between process temperature, the wonderful medium for bacterial growth which is WPC, and the heat-sensitive nature of the product, making pasteurization difficult and higher heat treatments all but impossible. Work is going on in several areas, e.g., high pressure, pulsed electric fields, and various frequencies in the electromagnetic spectrum, but to date, these technologies have found limited commercial application in the dairy industry.

2.7.3

Novel Products

There has been some interesting work done over the past decade and more on using whey proteins to form edible films and coatings, and as novel biodegradable packaging materials with tailored barrier properties. However, industrial implementation of this new technology remains reliant on further research before we see significant commercial adoption (see Chapter 11: Whey Protein-Based Packaging Films and Coatings for information of the use of whey proteins in films and coatings). Another promising area of research is aimed at the controlled production of whey protein nanoparticles—submicron-sized structures capable of encapsulation of sensitive compounds such as aroma and bioactive ingredients for delivery within beverages or other food systems.

2.7.4

New Areas of Application

The final area for new development concerns the application of the bioactivity of whey proteins in specific nutritional, therapeutic, or pharmaceutical applications. The production techniques generally exist to isolate all the characterized components, so continuing science-led advances in the understanding of the bioactivity should produce market-led demand for some extremely high-value (though low-volume) products for new applications based on whey protein fractions. The burdens of regulatory acceptance of functional claims are obviously a challenge, which will probably mean

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that novel applications in this area are fairly slow to develop. Nevertheless there appears to be some very compelling evidence of potential to develop products in this area and once again Smithers (2008) provides a good summary in his review (see also Chapter 17 on functional food products). One example not covered in the Smithers (2008) paper that also demonstrates the issues around claims and their regulation is some remarkable wound-healing effects attributed to the antimicrobial effects of lactoferrin, in the treatment of chronic open wounds with biofilms by Randall Wolcott at the Southwest Regional Wound Care Center in Lubbock, Texas. In a study by Sun, Scot, Smith, Rhoads, and Wolcott (2008), a group of 190 patients (over a period from 2002 to 2006) classified with a high risk of amputation was treated with lactoferrin mixed with xylitol applied to the wound site. This treatment had a far higher success rate, in terms of wound closure and saving of the limb, than had been reported in similar studies elsewhere. However, despite more than a decade of (apparently) greatly increased success in the treatment of this debilitating chronic problem, particularly of the elderly, the Center is very restricted in the claims it can make for the treatment. (Therapeutic and nutritive aspects of whey proteins are covered in Chapter 16).

2.8

CONCLUSIONS

This chapter is not intended to be a definitive account of all the significant events in whey protein processing over the past 40 years or so but to give a flavor of the events over this time and link many of the developments within the context of when, why, and how they came about. It is also intended as a tribute to the scientists, engineers, and entrepreneurs who made it happen.

Acknowledgments There are a number of people who were “there at the time” and played very significant roles in the development of whey processing on an industrial scale, who gave generously of their time, their recollections, and their archive materials. I take full responsibility for any mistakes, but those errors and omissions would have been far greater without the valuable contributions from the following people: Ken Burgess, Mark Chilton, Mitch Davis, Bill Eykamp, George Hutson, David Jenkins, Kevin Marshall, Bjarne Nicolaisen, John Joe O’Flynn, Dan O’Shea, Randy Thomas, Dan Twomey, Dave van der Werff, Jorgen Wagner, Randy Willardsen. Also acknowledged is the Society of Dairy Technology in the United Kingdom (www.sdt.org) who published an article by the author in 2013 which formed the basis of this chapter.

References MacGibbon, J. (Ed.), (2014). Whey to go. Whey protein concentrate: A New Zealand success story. Martinborough: Ngaio Press.

Further Reading

SDT. (2012). Dairy technology handbook. Society of Dairy Technology. Available from www. sdt.org. Smithers, G. W. (2008). Whey and whey proteins 2 From ‘gutter-to-gold’. International Dairy Journal, 18, 695 704. Sun, Y., Scot, E. D., Smith, E., Rhoads, D. D., & Wolcott, R. (2008). Biofilms in chronic wounds. Wound Repair and Regeneration, 16, 805 813.

Further Reading Bylund, G. (Ed.), (2003). Dairy processing handbook (2nd ed.). Lund: Tetra Pak Processing Systems. Cheryan, M. (1997). Ultrafiltration and microfiltration handbook (2nd ed). Lancaster PA: Technomic Publishing Co. Deeth, H. C., Datta, N., & Versteeg, C. (2013). Non thermal technologies. In G. Smithers, & M. A. Augustin (Eds.), Advances in dairy ingredients. Chichester: Wiley-Blackwell Publishing. Grandison, A. S., & Lewis, M. J. (1996). Separation processes in the food and biotechnology industries: Principles and applications. Cambridge: Woodhead Publishing. Law, B. A., & Tamime, A. Y. (Eds.), (2010). Technology of cheesemaking (2nd ed.). Oxford: WileyBlackwell. (Society of Dairy Technology Series). Tamime, A. Y. (Ed.), (2013). Membrane processing: Dairy and beverage applications. Oxford: WileyBlackwell. (Society of Dairy Technology Series). Wong, W. K. (Ed.), (2001). Membrane separations in biotechnology (2nd ed.). New York: Marcel Dekker.

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Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated Phil Kelly Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

3.1 WHEY PROTEIN CONCENTRATES AND ISOLATE BY ULTRAFILTRATION Within the last quarter of the 20th century, the widespread adoption of pressure-driven membrane processes such as ultrafiltration (UF) represented a major technological evolution within the dairy industry in facilitating the recovery of whey proteins in a near native form from whey. By 2007, UF installed membrane area in the dairy industry globally amounted to 350,000 m2 (Gésan-Guiziou, 2007). UF membranes span a wide range of molecular weight cutoffs (MWCOs) within the membrane separation spectrum. However, whey protein separation for the most part in the dairy industry is accomplished using 10,000 Dalton (10 kDa) MWCO UF membranes with some processors now incorporating even tighter 5 kDa elements as concentration advances in multistage plants. The level of protein enrichment, ranging from 35% (whey protein concentrate35, WPC35) to B80% (WPC80) is directly dependent on the volume concentration factor (VCF) applied. For WPC35, a VCF of 4.5
7.0 is required, while for WPC60, VCF should reach 13
20. Combined with diafiltration, whey UF (VCF: 30
35) can lead to WPC enrichment of 75%
85%. The proteins retained (retentates) by the UF membranes are further processed and spray-dried to produce whey protein concentrate (WPC)-based ingredients. WPC as defined by the American Dairy Products Institute (ADPI) is obtained by the removal of sufficient nonprotein constituents from whey so that the finished dry product contains $ 25% protein (ADPI, 2016). The proximate compositions of WPC35, WPC80, and whey protein isolate (WPI) are compared with demineralized whey (Demin 90) in Table 3.1. While Demin 90 falls outside the scope of the WPC product definition, the proportionality of 97 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00003-5 © 2019 Elsevier Inc. All rights reserved.

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Table 3.1 Proximate Composition of Commercially Traded Whey Protein Concentrates (WPCs) Containing 35% and 80% Total Protein, Whey Protein Isolate (WPI), and 90% Demineralized Whey (Demin 90) Composition (%)

Demin 90a

WPC35b

WPC80c

WPId

Protein (dry basis) Fat Ash Lactose Moisture

13.0 1.0 0.9 83.0 2.5

35 4 6 60 5

80.0 8.0 3.5 3.0 6.0

90.0 1.0 4.0 1.0 6.0

a

https://www.domo.nl/app/uploads/sites/2/2015/06/PDS-Deminal%C2%AE-90_EU.pdf. https://carbery.com/wp-content/uploads/Carbelac-35-Technical-Specification.pdf. c https://carbery.com/wp-content/uploads/Carbelac-80-Technical-Specification.pdf. d https://carbery.com/wp-content/uploads/Isolac-Technical-Specification1.pdf. b

its 13% protein relative to its 83% lactose content is a much demanded ingredient for infant milk formulation. Process-facilitated reductions in lactose and ash contents are commensurate with the attainment of higher protein content during manufacture of WPC80. The achievement of 90% or more protein on a dry basis requires interventions to reduce the 8% fat content associated with WPC80 (Table 3.1). Successful adoption of the measures outlined below to reduce the residual lipid content to # 1.0% will bring its own reward in terms of upgraded reclassification from whey protein “concentrate” to “isolate” status, i.e., WPI. A well-defined market is now established for WPCs with protein contents ranging from 35% to B80%. Such a wide protein range reflects the diversity of end uses and market opportunities. At the lower protein end of the range, WPC35 competes economically and substitutes on an equal protein basis, where opportune, with commodity-produced skim milk powder (SMP). Higher-protein WPCs, on the other hand, are preferred by formulators wishing to achieve a desired ingredient functionality or minimize the amount of nonprotein WPC components added. The extent to which whey protein retentates are enriched during UF processing is influenced by the VCF, and beyond a certain point, the amount of wash water (diafiltration) used to promote further permeation of nonprotein constituents from whey. Residual fat in cheese whey, even after application of a centrifugal cream separation step, is a factor which usually limits the upper protein concentration of WPC to ca. 80% due to its retention by UF membranes. The extensive concentration taking place during WPC80 manufacture usually amounts to 7%
8% fat retention in the powder. Furthermore, this residual fat is prone to oxidation and is known to negatively impair the protein ingredient’s sensory attributes. For a period, the attainment of higher protein concentrations was only

3.1 Whey Protein Concentrates and Isolate by Ultrafiltration

possible using ion exchange resin systems—the most significant of which industrially was the Vistec process which later underwent a name change to Bio-Isolates. Thus, it was possible to produce whey protein powders with protein concentrations .90%, i.e., WPI. Meanwhile, the issue of residual fat and UF was confronted during the 1980s with the development of a flocculation type whey pretreatment—later to be known as the “thermocalcic process” (Fauquant, Vieco, Brule, & Maubois, 1985) which relied on calcium addition (depending on cheese type) to achieve a threshold concentration of 1.2 g/L followed by pH adjustment under moderate heating conditions. Light flocs of fat-containing aggregates were formed which could be separated by first generation cross-flow ceramic MF membranes introduced to dairy processes. The resulting clear whey yielded better flux rates, and achieved WPIgrade protein levels during subsequent UF. Later, direct processing of whey by cross-flow MF became mainstream as it was demonstrated to adequately remove residual lipids without recourse to the use of the thermocalcic pretreatment. Over time, more affordable spiral-wound organic MF membranes were developed for this defatting duty which when combined with UF is now a widely used industrial processing platform for the manufacture of WPI. Market demand for high added value WPI continues to grow, arising from the increasing body of scientific evidence to support claims regarding its contribution to performance and recovery nutrition. Global cheese manufacture represents the greatest resource of whey available for processing and protein recovery. However, cognisance needs to be taken of the diversity of cheese types and its impact on whey quality. At one extreme, “sweet whey,” so called because of its relatively high pH 6.6, is released during the manufacture of semihard cheese varieties such as Gouda, while at the other extreme, acid-gel-based cheeses such as Cottage cheese give rise to whey with pH values close to the isoelectric point (pH 4.6) (Fig. 3.1). Thus, given such extremes in pH, whey composition will differ and UF membrane separation performance is also likely to be affected. Wheys from noncheese sources are generated during the manufacture of casein: (1) acid casein whey results from the isoelectric precipitation (pH 4.6) of skim milk (SM); or (2) rennet casein whey from chymosininduced clotting of milk at near-neutral pH. Like Cottage cheese whey, acid casein whey contains higher concentrations of minerals due to the solubilization of colloidal salts, principally calcium and phosphate. Additionally, there are salts contributed by the acidifying agent, such as chloride where HCl is used as a precipitant or lactate resulting from fermentation by lactic acid bacteria. (The reader is referred to Morr and Ha (1993) for more detailed information on compositional and quality differences of wheys used for WPC and WPI manufacture.) Nanofiltration (NF) is frequently used to preconcentrate and partially demineralize these acid wheys before further processing.

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Whole milk

Cream Skim milk Rennet casein

Acid casein

Acid gel cheeses

Semihard cheeses

Acid whey Sweet whey MF Micellar casein

Milk microfiltrate “ideal” whey

FIGURE 3.1 General classification of whey types (acid, sweet, and “ideal”) associated with the manufacture of diverse cheeses, casein, and native micellar casein-based ingredients.

Selective permeation of mainly monovalent ions by NF helps to bring their overall total mineral content into line with that of cheese wheys (Kelly & Kelly, 1995). Follow-on concentration to 35%
42% total protein by UF with some process adaptation succeeds in producing a high-gelling WPC35. Hence, considerable functionality is attained at modest whey protein concentration by virtue of technical innovation created around the higher calcium content of WPC originating from acid whey. MF SM permeate represents another source of whey that is regarded as “ideal” because it does not contain any residual fat, glycomacropeptide, or low-molecular-weight components released during the early stages of cheesemaking. The successful adaptation of cross-flow MF for novel dairy applications based on ceramic membranes in the 1990s made the harvesting of casein micelles in their native state from SM a commercial reality with the additional bonus of permeating a high-quality whey that contributes to improved flux during subsequent UF.

3.1.1

Membrane Performance

Permeate flux (J, expressed as L/m2 per hour) through a membrane is proportional to the applied trans-membrane pressure (TMP) as described by D’Arcy’s law (Kelly, 2011): J5

TMP μp :Rm

3.1 Whey Protein Concentrates and Isolate by Ultrafiltration

where Rm is the membrane hydraulic resistance, μρ is permeate viscosity, and TMP is the pressure drop between the retentate and permeate sides of a membrane at a particular point according to the equation: TMP 5

P1 1 P2 2 P3 2

where P1, P2, and P3 are feed inlet, feed outlet, and permeate outlet pressures, respectively. Membrane area (m2) is an obvious factor affecting performance—the relationship here is that the larger the surface area, the greater the permeate volume. This performance dimension is already accounted for in the permeate flux rate expression: L/m2 per hour. Membrane resistance (Rm) stems from the materials used in membrane construction, their crosssectional microstructure as depicted by the nature of the surface, and supporting structural layers, surface charge, and thickness. However, this is not the end of the story as product fouling of membranes during operation adversely affects performance not alone in terms of flux decline but also in terms of distorting membrane selectivity. Product fouling (Rf) adds another layer of resistance to that of the membrane (Rm) and the effectiveness of applied pressure is diminished by the increasing osmotic effects (ΔΠ) as macromolecular solutes become more concentrated. Hence, the following adaptation has been made to D’Arcy’s Law: J5

TMP 2 ΔL μρ ðRm 1 Rf Þ

Rf, in turn, represents the combined resistance of the various forms of fouling taking place: adsorption (Ra), pore blocking (Rp), precipitation/gel layer formation (Rg), and concentration polarization (Rcp), so that a predominance of such factors may override performance as follows (Kelly, 2011): J5

TMP Rm 1 Ra 1 Rp 1 Rg 1 Rcp

In UF, a surface layer (also called dynamic membrane, gel layer, filter cake) forms on the membrane surface and dominates the subsequent behavior of the membrane, while during MF both pore plugging (Rp) and surface layer (Rg) formation occur. In the case of protein-containing solutions such as milk or whey, steric effects lead to the selection of MF membranes with large pore sizes and the risk of severe pore plugging that will eventually result in the formation of a surface layer. Once a surface layer has formed on an MF membrane, its selectivity is dominated by the surface regardless of the original pore size of the membrane. From the moment that product commences circulation in a membrane plant, product constituents accumulate/concentrate on the membrane surface—hence, the term concentration polarization (Rcp) which gives rise to a concentration gradient of retained components

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that mediates subsequent separation. Generally, protein deposition and mineral (calcium phosphate) precipitation are major fouling agents during membrane processing of dairy products. Other product constituents such as lipid or fat material and peptides may contribute to a lesser extent. Product concentration is more intense along the interface of a membrane as the driving pressure on the product side is opposed by the membrane’s selectivity and behavior of rejected solutes. The phenomenon is characterized by a drop off in permeate flux through a membrane to a constant value irrespective of increasing TMP during RO or UF. Such fouling may become irreversible should the concentration of rejected component(s) increase to the point that deposition occurs on the membrane. The film model is considered to provide a reasonably good prediction of Rcp during UF protein processing. In this case, it is assumed that the concentration boundary layer resides within a thin laminar film at the membrane surface and that all mass transfer takes place by diffusion perpendicular to it. However, it does not apply that readily to MF because small particles rejected by the membrane do not move away in a predictable manner.

3.1.1.1 UF Processing Parameters Improved temperature and pH tolerance made polymeric membranes very appealing when first introduced to dairy UF given the limitations of first generation cellulose acetate-based membranes that had upper operating temperature limits of 37 C and poor tolerance of cleaning protocols that deviated from near-neutral pH conditions. Thus, for several decades, whey processors experienced the benefits of optimum flux rates while ultrafiltering whey at 50 C. While permeate flux at 50 C is about twice as high as that at 10 C (Gésan-Guiziou, 2007), more exacting market demands arose for WPCs with higher microbiological specification in the case of spore-forming and thermoduric bacteria, thus making it imperative for UF to operate at temperatures ,15 C. This technological change became commercially viable as improved production methods reduced the costs of spiral-wound membranes and made the specification of extra filtration area more affordable in order to compensate for reduced flux performance at lower operating temperature. D’Souza and Wiley (2013) identified that operating parameters such as temperature (T), pressure (ΔP), cross-flow velocity (V), feed concentration (C), and pH had a significant effect on permeate flux and rejection of total solids, but not on protein retention during UF of reconstituted sweet whey using a Koch 5838 K131-NYT polysulfone membrane. Cross-flow velocity and pH were the only parameters that did not influence rejection of lactose. Increasing the feed concentration resulted in a greater retention of total solids at higher cross-flow velocities. Factors V, pH, and interactions T 3 C, V 3 C, V 3 pH, and C 3 pH had a significant effect on ash rejection. A decrease in

3.1 Whey Protein Concentrates and Isolate by Ultrafiltration

ash rejection at pH 5 as cross-flow velocity increased was attributed to calcium solubilization as pH decreases, while higher shear rates at the membrane surface would be more effective in removing deposited materials (D’Souza & Wiley, 2013). An industrial simulation study examining TMP (150 and 300 kPa) and feed protein concentration (0.9% and 10%) on resistance reduction and flux recovery following cleaning (cleaning-in-place, CIP) during UF with polysulfone membranes revealed that the decreased filtration performance during the first days of usage may be related to build-up of internal fouling (Berg et al., 2014). Efforts at recovering flux by means of CIP, on the other hand, were impaired following filtration at high protein concentration.

3.1.1.2 Effect of Pretreatment—Original Milk and Wheys Changes in cheese production processes may have a significant effect on subsequent whey composition and functionality, particularly if cheesemilk is subjected to high heat treatment at the outset (Outinen, Rantamäki, & Heino, 2010). The quantity of nonprotein nitrogen in whey total protein was elevated by the high heat treatment of the original milk and reduced during subsequent UF. Changes were also noted in concentrate solids during processing, but there were no significant differences in ingredient physicochemical properties (degree of denaturation), rheology (viscosity), and functionality (water-binding capacity, emulsifying capacity, and emulsion stability) (Outinen et al., 2010). All wheys arising from curd production processes require a clarification step to remove curd fines followed by whey cream separation to recover milk fat losses that originate in cheese vats during milk coagulation, cutting, and stirring (note that this cream separation step is preliminary to the more extensive delipidation measures that required MF intervention as outlined earlier in this chapter). A pretreatment protocol (comprising salt addition, pH and temperature modification, and centrifugation) that removes calcium and phosphate salts from acid whey without altering the profile of valuable proteins enhances significantly the permeate flux of whey when cross-flow ultrafiltered through a 50 kDa tubular ceramic membrane both in total recycle and continuous diafiltration mode (Almécija, Guadix, Martinez-Ferez, González-Tello, & Guadix, 2009). Flux was improved by a factor of 3.0
3.5 when operated at a TMP of up to 2 bar with respect to the original whey, which could be explained by a resistance-in-series model. With respect to the behavior of pretreated whey under continuous diafiltration, the final volume of permeate collected after 4 h operation was tripled by comparison with the unpretreated whey (Almécija et al., 2009). Acid whey, prepared by fermenting a reconstituted ultra-low-heat SMP was clarified by centrifugation before UF at 50 C to a volume reduction ratio of 10 using either 10 or 30 kDa membranes. The

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clarification step removed bovine serum albumin (BSA) and immunoglobulins (Igs) to a lesser extent (Konrad, Kleinschmidt, & Faber, 2012). Flux improvement at ,pH 3.9 was directly dependent on pH lowering, while the severe flux decline occurring above this pH caused the process to virtually stop at pH 4.6. The largest flux (B34 6 5 kg/h per m2), obtained using a 30 kDa membrane at a TMP of 2 bar and 50 C, was comparable with that for sweet whey. The removal of heat-denatured whey protein aggregates originating from cheesemaking processes by upstream MF increased UF flux rates on sweet and acid whey by B2 3 and 3 3 , respectively (Steinhauer, Schwing, Krauß, & Kulozik, 2015). Significant calcium retention was observed during UF of sweet whey which suggests that membrane fouling for both whey types seem to follow different molecular mechanisms. UF, using a 20 kDa MWCO ceramic membrane, of fresh or microfiltered (0.5 μm pore size ceramic membrane) whey at 20 C gave higher flux rates and lower total filtration resistance than when pasteurized and UF processed at 50 C, at which temperature it was believed that the calcium present may have contributed to protein deposition on the membrane (Barukˇci´c, Boˇzani´c, & Kulozik, 2015).

3.1.2 Membrane Separation Operation and Protein Selectivity 3.1.2.1 Continuous Operation and Control Modeling the operability of continuous ultrafiltration plants used for the production of whey protein concentrates revealed that a standard 12-loop commercial ultrafiltration plant is appropriate for the production of the desired concentrate specifications (Yee, Wiley, & Bao, 2007). A particular challenge is to sustain sufficient retentate flow rate across the membrane as the protein concentration increases and volume is reduced during the advanced stages of UF processing (Fig. 3.2). The studies also indicate that mid-run washing of the plant is necessary in order to reduce the effects of long-term membrane fouling. Although it becomes more difficult to achieve the desired product flow rate as protein concentration in the feed increases, proper knowledge of process variations makes it possible to compensate for fluctuations in whey composition during continuous operation in order to maintain the desired WPC compositional specification. The compressive rheology of fouling layers during UF forms the basis of a model proposed by Davey et al. (2004) to predict the performance of filtration processes. The model, developed from data generated by centrifuge tests and combined with the pressure drop of the clean membrane, can be used to optimize cycle times, pressures, and other UF operating parameters. Even though the permeate recycle ratio can be used to control the specifications of the retentate, a high retentate recycle ratio used in typical whey UF processing

3.1 Whey Protein Concentrates and Isolate by Ultrafiltration

Whey feed

Diafiltration water Bypass

Loop 1

Loop 2

Loop 3

Loop 4

Loop 9

Loop 5

Permeate Spray Dryer WPC/WPI

Retentate

FIGURE 3.2 Typical multistage spiral-wound ultrafiltration plant configuration for WPC and WPI manufacture with up to nine individual recirculation loops. Diagram shows points where diafiltration water is introduced between the individual stages including bypass options.

places a limit on how fast the effects of fluctuations in feed flow rate and composition can be mitigated (Yee, Alexiadis, Bao, & Wiley, 2009). Since a high retentate recycle ratio is required to improve the productivity of the whey UF process, a trade-off must be obtained between the productivity and the time required to mitigate the effects of fluctuations in feed flow rate and composition. Sen, Roy, Bhattacharya, Banerjee, and Bhattacharya (2011) developed a mathematical model based on an artificial neural network in conjunction with the mass balance model related to membrane separation. This combined approach for mathematical formulation was called a knowledge-based hybrid neural network. The authors intended to evaluate such a mathematical model in the membrane separation process to predict the percentage purity of the whey protein at each stage of diafiltration. Having differentiated the permeation process into two periods according to the dominant factors controlling filtration resistance, i.e., initial pore blockage followed by the dominance of cake deposition, Karasu et al. (2010) developed a combined cross-flow model to represent the whole UF process which compared favorably with experimental UF data. Increasing resistance during whey UF when using regenerated cellulose acetate membranes was predominantly attributed to concentration polarization, while the contribution of membrane fouling to total resistance was regarded as negligible (Ábel, László Kiss, & Beszédes, 2015). However, the authors found that ultrasonically-assisted UF succeeded in reducing resistance contributed by the concentration polarization layer.

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3.1.2.2 Diafiltration Different strategies were tested involving (1) a change of the volumetricconcentration factor (VCF), (2) diafiltration (DF) water volume, and (3) number of DF steps during the concentration and purification of whey proteins by UF (Baldasso, Barros, & Tessaro, 2011). Pushing to a higher VCF ensures that UF will achieve WPCs with .70% by weight (dry basis) of protein (Baldasso et al., 2011). In addition, discontinuous DF was more effective when performed a greater number of times with smaller volumes. Recently, model-based predictive control has been developed to improve the performance of UF membrane systems used in industrial whey protein production processes (Bahadır Saltık, Özkan, Jacobs, & van der Padt, 2017). Parameter estimation is based on experimental data captured from a whey processing plant. The model takes account of flux performance deterioration from the point where the initial resistance due to the membrane increases as a fouling layer forms during operation. The dynamic model is interrogated for its predictive capabilities in order to identify optimal operation strategies.

3.1.3

Membrane and Process Developments

3.1.3.1 Charged Membranes Functionalization of polyethersulfone (PES) membranes was accomplished by polymerization of styrene in the membrane pores followed by sulfuric acid treatment of the resulting polystyrene grafts. The charged membrane was calculated to possess five times better selectivity than the raw membrane at pH 7.2 based on data from single protein transmission experiments. The enhanced selectivity of the tailor-made membrane was manifested by increased retention of β-lactoglobulin (β-Lg) due to a reduction in molecular sieving combined with electrostatic repulsion between negatively charged β-Lg and the negatively charged membrane (Cowan & Ritchie, 2007). Widerpore membrane could be deployed for the same WPC separation duty as electrostatic repulsion predominated over molecular-based sieving. Adding a positive charge to ultrafiltration membranes and adjusting solution pH promotes the permeation of proteins such as glycomacropeptide which have little or no charge, and retain proteins having a positive charge (Bhushan & Etzel, 2009). The authors were able to support a claimed selectivity increase in excess of 600% over uncharged membranes using a stagnant film model study that related the observed sieving coefficient to membrane parameters such as the flux, mass transfer coefficient, and membrane Peclet number. Negatively-charged UF 100 kDa regenerated cellulose membranes had an 85% higher flux than unmodified 10 kDa membranes and equivalent protein retention during evaluation of laboratory-scale flat sheet and pilot-scale spiral-wound membranes (Arunkumar, Molitor, & Etzel, 2016).

3.1 Whey Protein Concentrates and Isolate by Ultrafiltration

3.1.3.2 Nanolayered Membrane Nanolayer technology was used to create a hydrophilic filler consisting of MgAl double hydroxide (Mg-Al NLDH) on top of a high-flux PVDF-based UF membrane (Arefi-Oskoui, Vatanpour, & Khataee, 2016). Pure water flux was significantly increased from 473.8 L/m2 h for the PVDF only to 702.2 L/m2 h for the nanolayered PVDF membrane. The modified membrane had a better antifouling property during processing of WPC. A polysulfide-amide (PSA) copolymer was blended with PES in order to improve the hydrophilicity of the resulting asymmetric membrane (Jalali, Shockravi, Vatanpour, & Hajibeygi, 2016). However, its porosity was not comparable with a membrane fabricated from a polymer blend based on polyvinyl pyrrolidone (PVP)/PES membrane. By combining the better of the membrane polymers, the authors developed a membrane with good solution flux and antifouling properties using 1% PVP and 1% PSA. The potential of forward osmosis (FO) for whey protein concentration was explored by Wang, Wang, Li, and Tang (2017) using high-performance inhouse fabricated hollow-fiber membranes. Their investigation focused on the effects of various operating conditions (cross-flow velocity, draw solution concentration) on maximum attainable concentration. Rapid formation of a polarized or gel foulant layer at the membrane surface as protein concentration in the feed increased was responsible for early flux decline. Membrane performance was restored during cleaning routines after processing. The lower operating hydraulic pressures used in FO makes this novel membrane separation technology appealing as a sustainable, lower-energy process technology for protein recovery while at the same time enhancing product quality by lowering the impact of induced-pressure/temperature changes. Microfiltered whey permeate was used as feedstock during subsequent UF with a wide-pore polyvinylidene fluoride UF membrane (50 kDa MWCO) operated at TMP of 207 kPa and overall flux of 49.46 L/m2 h to selectively concentrate α-lactalbumin (α-La) and generate an ingredient with an α-La purity of 0.63, α-La:β-Lg ratio of 1.41 and α-La yield of 21.27% (Marella, Muthukumarappan, & Metzger, 2011). Tangential flow UF of SM using a 100 kDa MWCO regenerated cellulose membrane (Purosep 7000, SmartFlow Technologies) at an operating temperature of 26 C and TMP of 186 kPa produced a permeate containing .80% α-La (Holland, Kackmar, & Corredig, 2012).

3.1.3.3 Protein Cross-Linking Transglutaminase (TGase)-induced cross-linking of cheese whey protein prior to UF improved recovery rate by 15%
20%, decreased lactose rejection rate by 10%, and increased relative permeate flux by 30%
40% compared to the

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non-TGase-treated sample (Wen-Qiong, Lan-Wei, Xue, & Yi, 2015). The overall performance improvement was attributed to the positive effects of enzyme-catalyzed increased particle size and decreased zeta potential on total resistance and fouling layer cake resistance. Traditionally, a colored variant of Cheddar cheese manufacture, namely “Red Cheddar,” has relied on the addition of annatto dye at the outset to the cheese milk. A certain degree of partitioning of this colorant occurs between the cheese curd and whey at the draining stage of manufacture. In recent years, residual levels of annatto in whey have become undesirable in processed ingredients sourced by processors of specialist nutritional products such as infant formula. Hence, various technological approaches are being explored to either accomplish 100% entrapment of annatto in the cheese curd or eliminate the residual annatto in whey using bleaching or other methods. Bleaching whey, with or without added annatto, improved UF and DF flux performance during preparation of WPC80 using spiral-wound membranes (10 kDa MWCO) configured to operate in batch recirculation mode at 50 C (Adams, Zulewska, & Barbano, 2013). While the mean flux was similar during UF processing of Cheddar cheese whey and MF permeate of SM, some differences were evident during subsequent DF where mean flux WPC80 (cheese whey) was greater than mean flux WPC80 (MF permeate). In terms of bleaching agent efficacy, hydrogen peroxide generally produced higher flux rates than benzoyl peroxide, but the authors were unable to explain the phenomenon other than to confirm that the performance improvements were consistent with a reduction in fouling conditions. The shear effects of a rotating disk UF are particularly effective in controlling concentration polarization. The pH of microfiltered casein whey along with membrane rotation and TMP had a strong effect on UF flux and rejection when the authors (Sarkar, Ghosh, Dutta, Sen, & Bhattacharjeek, 2009) adopted a two-membrane separation strategy, i.e., a 30 kDa followed by a 5 kDa MWCO. The higher flux rate at low pH during UF module rotation was explained in terms of prevailing monomer
dimer equilibrium of β-Lg, as well as being due to conformational changes of protein molecules with respect to their isoelectric points. An α-La-rich fraction in the permeate stream from the more open membrane was readily separated from lactose in the 5 kDa MWCO membrane. It is suggested that a novel technology such as ultrasound may be deployed in a process intensification mode during whey pretreatment to improve UF separation efficacy and prevent blockage of the nozzle orifice(s) of a spray dryer atomizing device (Gajendragadkar & Gogate, 2016). It is also claimed that there are additional benefits in terms of improvements to the heat stability of the whey proteins and quality of the resulting WPC powder.

3.2 Whey Protein Fractions by Membrane-Based Separations

WPCs produced from the MF permeates (0.2 μm pore size ceramic membrane) of SM and buttermilk (BM) were comparable in terms of amount of whey protein and phospholipids (PLs) present—the main differences being among the concentrations of individual PLs (Svanborg, Johansen, Abrahamsen, & Skeie, 2015). No difference in protein solubility was observed at pH values of 4.6 and 7.0, and the overrun was the same for BMWPC and SMWPC; however, the BMWPC made a less stable foam than SMWPC. In terms of membrane module design, spacers reduce concentration polarization and, thus, are the main fouling control factor. Flux rate improvements by a factor of 7 or more are possible (Da Costa, Fane, & Wiley, 1993). The authors also noted that membrane cleaning was not significantly affected by the presence of spacers, or their characteristics. Narong and James (2008) created particulates in whey using a salt-based coagulant and examined the effects of surfactant addition and pH change during UF with a ceramic zirconia membrane. The best permeate flux was found at pH 6.2 when both particles and membrane had significant negative zeta-potentials. In contrast, the best reduction in total organic carbon (TOC) was observed when the zeta-potential of both membranes and particles were positive, but very close to zero.

3.1.4 UF Spiral-Wound Membrane Manufacturers and the State of the Art A snapshot of commercially available spiral-wound UF membranes compiled from technical information outlined in manufacturers’ websites is presented in Table 3.2 with a view to understanding the current state of the art in terms of technological capability on offer to whey processors. The list is not exhaustive, but intended to provide the reader with an opportunity to contrast the current industrial perspective with research developments outlined in the preceding section.

3.2 WHEY PROTEIN FRACTIONS BY MEMBRANE-BASED SEPARATIONS Industrial chromatography is considered expensive and usually resorted to for the fractionation of proteins when other potentially cheaper options are less successful. Selective precipitation of some whey protein fractions is possible, but subsequent separation can be problematic due to the weak precipitate/aggregates formed. Considerable efforts have been underway for some time at adapting membranes to fractionate whey proteins. Similarities in the molecular size of many of the individual protein fractions present challenges given that UF membranes operate on the principle of molecular sieving. In

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Table 3.2 Salient Features of Spiral-Wound Ultrafiltration (UF) Membranes Offered by Major Manufacturers for Whey Protein Concentration Membrane Manufacturer

Featured UF Spiral-Wound Membranes for Whey Applications

Sanitary Compliance

Synder Filtration http://synderfiltration.com/ ultrafiltration/membranes/

25 years’ experience of servicing the dairy industry. Recommended UF membranes VT (3 kDa), MT (5 kDa) and ST (10 kDa) Innovation in feed spacer geometry in addition to spacer thickness for highsolids whey protein concentration. The use of open channel ribbed spacers reduced pressure drop by 37% across the length of the element compared to standard diamond spacers With over 40 years of experience in dairy membrane applications, KMS claim that their Dairy-Pro range have improved blister resistance, are more energy efficient and have longer operating life than their previous KMS elements UF element lines are represented by their HFK-131 and HFK-328 membranes. PES construction Dairy UF-PE manufactured with polyester support material. MWCO 5
20 kDa Dairy UF-pHT high pH and temperature tolerance. PS or PES cast on polypropylene backing material MWCO 2
20 kDa S-Series PES membrane with polyester support in 5, 10, and 20 kDa MWCO S-Series PES membrane with polypropylene support in 5 and 10 kDa MWCO for greater temperature and chemical tolerance ( . pH 10) Hydranautics highlight the automated rolling of the full-fit membrane modules in its DairyUF 5K and 10K series elements (PES with PP backing) which are characterized by superior glue line adhesion, strength and blister resistant properties. Tighter rolling and caging enable greater flow rates and structural reliability Hydrophilic PES asymmetric membranes developed for whey proteins: 10 kDa: TUF 10K—whey processing at all concentrations TUF 10K HR—very high protein retention; application at high concentration end of WPC and WPI plants 5 kDa: TUF 5K
very high retention membrane. TUF 5K HR—low flux membrane with maximum retention for special applications TurboClean elements feature a patented sanitary hard-shell design—claimed to deliver better system performance due to 60% less bypass flow—more feed flows across the membrane; more effective membrane cleaning due to higher cross-flow velocity NADIR UP005 (PES) 5 kDa MWCO offers higher protein rejection. UP010 10 kDa and UP020 20 kDa membranes also available in standard and pHT design

Compliance with FDA standards listed in CFR Title 21, 3-A Sanitary Standards No. 45-02, and USDA Sanitary Standards

Koch Membrane Systems (KMS) www. kochmembrane.com/Process-Separations/ Dairy/Dairy-Pro-Sanitary-Elements.aspx

Alfa Laval www.alfalaval.com/products/ separation/membranes/spiral-membranes/ufspiral/ Parker www.parker.com/sanitarymembranes

Hydranautics http://membranes.com/solutions/ products/process/dairyuf/

Toray www.toraywater.com/products/ specialty/index.html

Microdyn Nadir www.microdyn-nadir.com

Microdyn-Nadir US, Inc. www.trisep.com/ turboclean/

PES, polyethersulfone; pHT, pH- and temperature-tolerant; PP, polypropylene; PS, polysulfone.

USDA, FDA and 3 A, EU, and Halal compliant

All materials in compliance with EU Regulations (EC) 1935/2004 and FDA regulations (CFR) Title 21

Conform to 3-A, FDA/CFR Title 21 & USDA standards Certified EU1935/2004EC & Plastics Regulation 10/2011 All elements meet FDA regulations CFR Title 21 ABS 3A Sanitary Standards for Cross flow Membrane Modules, Number 45-01, Section C

Toray’s TUF UF membranes are USDA accepted and 3A/FDA compliant

3.2 Whey Protein Fractions by Membrane-Based Separations

the knowledge generated to date, it is evident that membrane systems can be adapted to enrich one or more fractions relative to another. In other words, the final ingredient may still be classified as a WPC that contains a particular enriched fraction compared to a pure protein/isolate produced by chromatography. However, hybrid systems are occasionally encountered where a combination of membranes, chromatography, and phase separation techniques are used. Muller, Chaufer, Merin, and Daufin (2003) examined the prepurification of α-La from acid casein whey using a range of ceramic membranes (Carbosep (0.14 μm pore size and 150 kg/mol (kDa); 20.6 nm); Céram 300, 220, and 150 kg/mol (kDa)). Industrially-sourced WPCs, derived from hydrochloric acid whey were in a variety of conditions (1) liquid—frozen and used after thawing; (2) liquid subjected to MF clarification (0.1 μm), then frozen and thawed before use; and (3) powder—reconstituted before use, and clarified by pH 5.0 adjustment/denatured protein precipitation and decantation. Based on the outcome of their studies, the authors could only proffer an operational guideline based on the selection of ceramic UF MMCO (from 150 to 300 kg/mol (kDa)) because of the complexities surrounding the physicochemical characteristics of the whey protein sources (initial α-La purity and concentration; heat denaturation of proteins) and irreversible membrane fouling, mainly due to the β-Lg fraction. By way of example, better performance was obtained by processing liquid WPC—α-La purity in the permeate was increased from 0.25 in the initial feed to B0.44 with a yield of around 0.53 using Céram membrane 300 kg/mol. Stationary UF flux was B64 L/h per m2 at a volume reduction ratio of 9. Enhanced α-La purity (0.44) and satisfactory yield (0.53) was achieved with a continuous UF concentration mode (VCF 5 9) owing to a proper selection of average membrane pore size (no BSA and IgG transmission) and of operating conditions so as to limit fouling and ensure a satisfactory transmission of α-La while limiting transmission of β-Lg (Muller et al., 2003). Enhanced performances could certainly result from adapting processing conditions during the preparation of the whey protein source with the major objective of preserving native properties of whey proteins. A two-stage tangential flow UF system, involving 100 and 30 kDa membranes in series, was demonstrated for the fractionation of both α-La and β-Lg from WPI (Cheang & Zydney, 2004). Optimal separation required careful adjustment of buffer concentration and filtration velocity for each UF stage. α-La purification was greater than 10-fold at 90% yield. Good partitioning of higher-molecular-weight constituents (Ig and BSA) from α-La and β-Lg was achieved by processing whey at pH 5.0
5.2 on a 0.1 μm SCT Ceraver ceramic membrane fitted to a Tetra Pak Alcross MFS-19 and

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operated with uniform TMP control (Mehra & Kelly, 2004). Exploitation of electrostatic properties through pH-mediation of the whey proteins in conjunction with membrane pore size played a key role in manipulating the hydrodynamic radius of individual protein fractions in order to facilitate better separation during both MF and DF. This may also explain the large differential between the 0.1 μm pore size MF membrane and the molecular size of immunoglobulins. In a second stage separation, Mehra and Kelly (2004) subjected the aforementioned MF whey permeate to UF using three different Koch hollow-fiber membranes (30, 50, and 100 kDa MWCO) in an effort to fractionate β-Lg and α-La. With rejection values of 0.98 and 0.78 for β-Lg and α-La, respectively, greater enrichment of α-La (α-La: β-Lg 5 2.5) in the UF permeate was obtained using the 30 kDa membrane. Reduced α-La retention was observed with the larger UF MWCO membranes, but the simultaneous increase in β-Lg permeation denied the opportunity for greater α-La enrichment Two-stage UF with 30 and 10 kDa flat-disk membrane in a stirred rotatingdisk module revealed that membrane rotation was key to enhancing flux and highly efficient in reducing concentration polarization (Bhattacharjee, Ghosh, Datta, & Bhattacharjee, 2006). In the first stage UF with a 30 kDa membrane, most of the BSA, lactoferrin (Lf), and Igs were rejected, and in the other stage with 10 kDa membrane, α-La and β-Lg were rejected to give clear permeate with low protein concentration. At feed pH 2.8 and membrane rotation at 300 rpm or more, higher fluxes were observed within the TMP range of 4
5 kg/cm2 (390
490 kPa). A 75% purity β-Lg (on total protein basis) was obtained in the final stage, i.e., in 10 kDa retentate at a TMP of 4 kg/cm2 (390 kPa) using a stirred rotating-disc module with 600 rpm membrane rotation speed (Bhattacharjee, Ghosh, et al., 2006). A β-Lg fraction with 87.6% purity (on total protein basis) was obtained in the filtrate of ion-exchange membrane chromatography (IEMC) during fractionation of WPC using two-stage UF with 30 and 10 kg/mol (kDa) MWCO flat-disk membranes in a stirred rotating disk module followed by IEMC using Vivapure Q Mini-H column (Bhattacharjee, Bhattacharjee, & Datta, 2006). Prior to UF, centrifugation, MF, and a four-stage discontinuous DF were carried out to obtain WPC from raw casein whey. DF using 5 kDa PES membrane with VCR 2 in each stage was employed to selectively enrich whey proteins. A 36% higher flux was obtained with a feed pH of 2.8 rather than 5.6 at a fixed TMP of 5.0 3 105 Pa. The rotating disk membrane was particularly effective in enhancing flux by 33% (observed at 1 min with membrane disc rotating at 300 rpm) due to reduction in concentration polarization, the main limiting phenomenon for flux decline. A suitable loading buffer pH was investigated during IEMC runs to facilitate transport of β-Lg over α-La through the strong anion-exchanger membrane.

3.2 Whey Protein Fractions by Membrane-Based Separations

The effect of working pH was evaluated in terms of flux
time profiles and the retentate and permeate yields of α-La, β-Lg, BSA, IgG, and Lf during UF of clarified whey with a 300 kDa MWCO tubular ceramic membrane in a continuous diafiltration mode (Almécija, Ibáñez, Guadix, & Guadix, 2007). For α-La and β-Lg, the sum of retentate and permeate yields was around 100% in all cases, which indicates that no loss of these proteins occurred. After four diavolumes, retentate yield for α-La ranged from 43% at pH 9 to 100% at pH 4, while for β-Lg, was from 67% at pH 3 to 100% at pH 4. In contrast, BSA, IgG, and Lf were mostly retained, with improvements up to 60% in purity at pH 9 with respect to the original whey. The highest α-La enrichment was obtained using a hydrophilic UF 30 kDa regenerated cellulose membrane along with a transmission increase from 10% to 26% when the solution temperature was raised from 25 C to 40 C (Metsämuuronen & Nyström, 2009). A 13
15-fold α-La:β-Lg ratio was achieved in comparison to initial whey in a single step at pH 6.5. Final α-La purity from all protein based products was only ca. 21% due to the high amount of small protein fragments. The selectivity of the 100 kDa MWCO membrane decreased with temperature probably due to dissociation of β-Lg. The α-La transmission of the most hydrophobic membrane decreased steadily with time and TMP, but simultaneously its selectivity increased due to pore narrowing and higher retention of β-Lg. Two-phase aqueous extraction based on the partitioning behavior of α-La and β-Lg in polyethylene glycol (PEG) and sodium polyacrylate, respectively enabled satisfactory purification and yields to be obtained (Pereira Alcântara et al., 2014). Simultaneous partitioning of α-La to the PEG rich phase and β-Lg to the salt-rich bottom phase from acid whey using PEG 1000
trisodium citrate systems succeeded in separating α-La with 89% recovery and purity of 96% in the top phase, while that of β-Lg in the bottom phase was 96% and 76%, respectively (Kalaivani & Regupathi, 2015). A combination of 20 3 UF concentrated Cheddar cheese whey and immobilized metal affinity chromatography (IMAC) succeeded in isolating IgG with 52% purity using glycine as the eluting buffer (Fukumoto, Li-Chan, Kwan, & Nakai, 1994). The authors found that IMAC gave higher recovery and purity of IgG from concentrated whey than ion-exchange chromatography.

3.2.1

Absorption Processes

A strategy to simultaneously purify Lf and IgG from two integrated expanded bed absorption processes while generating WPC by UF from their eluent was proposed (Du et al., 2014). The two sequential expanded beds separated Lf on the first absorption bed (cationic exchanger, Fastline SP) and IgG in the

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second (mixed-mode resin, Streamline Direct CST-1). Three integration strategies were compared to improve the separation efficiency, especially for the purification of IgG in the second expanded bed. The authors succeeded in recovering 64% of IgG with a purity of 84% depending on the process strategy adopted.

3.3

MICROPARTICULATED WHEY PROTEIN

Interest in whey protein microparticulation has been stimulated ever since the invention of the Simplesse concept in 1979 by scientists Yamamoto and Davis at the Canadian Beer company, John Labatt Ltd. The underlying principle behind the invention exploits protein chemistry concepts associated with heat-induced protein denaturation and aggregation that are very close to every process technologist’s interests when manufacturing dairy products. However, it was the unique combination of heat and shear that the inventors applied to the proteins in order to create a microdispersion of small particles that possessed the creaminess and richness sensation of fat. An additional feature that the inventors observed was the ability of these microparticles to “roll over” each other in the colloids formed after microparticulation. Fat replacement on an equivalent basis reduced the calorie loading from 9.0 cal/g for regular fat to 1.0
1.3 cal/g for Simplesse while maintaining a rich creaminess texture in the reduced-fat product. Following Labatt’s licensing of the technology to NutraSweet in 1984, the concept was assigned the “Simplesse” trademark in 1988. Subsequently, the product was the subject of approval by the US Food and Drugs Administration (FDA)—initially for use in frozen desserts such as ice cream, and later in 1991 for use in salad dressings, butter, and baked goods. There are some limitations to the versatility of Simplesse since the ingredient is protein-based and does not lend itself to thermal processing without undergoing major structural change and loss of textural function. Hence, cooking should be avoided in favor of applications that employ cold formulations (ice cream, yogurt, cheese spread, salad dressings, margarine, mayonnaise, coffee creamer, soups, and sauces) which at most may only require moderate heating. Where ingredient declaration is concerned, the emphasis is on the form of Simplesse used in the final product, e.g., a microparticulated combination of egg white and milk proteins may be referred to in the ingredient listing as “egg and milk protein.” Microparticulation of both egg and whey protein combinations were featured in early Simplesse prototypes, but gradually the microparticulated whey protein (MWP) form became established in its own right. Currently, CP Kelco (a former NutraSweet subsidiary), promotes Simplesse to food processors as a “microparticulated WPC” in dry powder form suitable for label declaration as dairy proteins.

3.3 Microparticulated Whey Protein

The thermomechanical coagulation of a WPC at acid pH (US patent 4,734,287, 1988, J. Labatt Ltd.) or that of egg white proteins on the surface of casein micelles at neutral pH (International Application WO 89/05,587, 1989, NutraSweet Co.) provides in both cases spherical particles with a diameter ranging from 0.1 to 2.0 μm. Size, shape, and uniformity of the microparticles were illustrated in light micrographs generated by Singer and Dunn (1990). Cheftel and Dumay (1993) reviewed the technology landscape following the Labatt invention and discovered that other techniques used to generate fat analogue from whey included (1) precipitation of soluble proteins at their isoelectric point (European Application 0,400,714, 1990, NutraSweet Co.); (2) precipitation from ethanol solutions (International Application WO 90/03,123, 1990, Enzytech Inc.); (3) formation of complexes between electrically charged proteins and polysaccharides embedded in modified starch (US Patent 4,308,294, 1981, General Foods Corp.); or (4) fragmented under high shear forces (European Application 0,340,035, 1989, Kraft Co.) to create particles in the size range 0.5
15 μm. The properties of the protein aggregates generated following heat- and sheartreatment of WPCs with different lactose contents in a scraped-surface heat exchanger (SSHE) at various temperatures were correlated with the denaturation kinetics of β-Lg and the different mechanisms of unfolding and aggregation (Spiegel, 1999). Lactose appears to interfere with protein unfolding at temperatures ,85 C resulting in the formation of a loose porous structure with larger particle size. Smaller aggregates were generated during heating at 85
95 C, which became more dense and compact when heating was extended to .100 C. At these temperatures, particle size was independent of lactose concentration. Substitution of lactose with inulin caused greater whey protein denaturation during microparticulation (Tobin et al., 2010). The increased denaturation levels were correlated with reductions in lactose and calcium content of the microparticulated solutions, which increased aggregate size and solution viscosity post-processing. Decreasing pH from 6.7 to 4.5 retarded the denaturation rate of β-Lg at 80 C, and formed small particles (,5 μm) under shear between pH 4.0 and 5.5. The denaturation rate of β-Lg at 80 C is considerably retarded as the pH-value decreases from pH 6.7 to 4.5 (Spiegel & Huss, 2002). Aggregates which are produced under shear between pH 4.0 and 5.5 reveal a smaller particle size (,5 μm) due to low reactivity of the thiol groups and the small net charge of the proteins in this pH-range. The large rubber-like particles formed as a result of calcium reduction were attributed to fragmentation of a fine-stranded gel rather than aggregate behavior. Cheftel and Dumay (1993) utilized twin-screw extrusion technology as a thermomechanical means of accomplishing whey protein microparticulation. With a twin-screw extruder configured with a long barrel and operating with

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feed water content B77%; protein content B20%; barrel temperature 85
100 C; screw rotation speed 100
200 rpm; and feed rate 20 kg/h, whey protein microparticulates were achieved with .50% of particles having a diameter within 6
11 μm on a volume basis (Cheftel & Dumay, 1993). The resulting semi-solid spreads displayed high nitrogen solubility: 43%
47% and 69%
70% for the acid (pH 3.5
3.9) and neutral (pH 6.5
6.7) products, respectively. Calcium caseinate was used by the authors to inhibit extensive protein aggregation. The MWPs prepared by extrusion dispersed easily at a 5%
10% level, developed desirable “creaminess,” and withstood further heat processing when incorporated in food formulations. Microparticulation of whey proteins at low concentration (2%, w/v), was examined in a pilot plant tubular heat exchanger (Kerche, Weterings, & Beyrer, 2016). In terms of design for scale-up, the authors recommended that modeling of flow dynamics by shear rates should be replaced by a more complex description that took into consideration temperature- and sheardependent dynamic viscosity of liquids and the shape of tubes. This leads to flow dynamics based on Reynolds numbers and friction factors.

3.3.1 Effect of Spray Drying on Microparticulate Functionality Simplesse was originally introduced to the market in liquid form as there were reservations at the time that its unique fat substituting properties may be compromised during further processing and transformation into the dried ingredient form. Gradually, over time dried versions of the ingredient became available. Some insights into the effects induced by spray drying of solutions of 10% (w/w) SSHE MWPs generated from native (ratio of α-La:βLg, 20:70) or isolated forms with denaturation degree $ 90% have been elucidated (Toro-Sierra, Schumann, & Kulozik, 2013). A slight increase in powder particle size independent of inlet and outlet drying temperatures was observed, but reversed after dissolution in water. In terms of microstructure, the spray-dried powder particles were spherical in shape but displayed morphological differences depending on the protein composition of the solutions. Spray-dried, denatured MWP produced at pH 3 (SD-MWP-pH3) had approximately two orders of magnitude reduction in particle size compared with those produced conventionally at neutral pH with high colloidal stability (Dissanayake, Liyanaarachchi, & Vasiljevic, 2012). SD-MWP-pH3 exhibited enhanced heat stability along with better emulsifying properties and a capacity to create comparatively strong cold acid-set gels. Microparticulated WPC emulsions showed significantly enhanced heat stability compared with standard WPC emulsions provided that the microparticulation process was conducted in a manner to ensure that the protein particles have fewer active sites available for further aggregation during secondary heat treatment

3.3 Microparticulated Whey Protein

(retorting) (Çakır-Fuller, 2015). Other functionality improvements in WP microaggregates could be generated by combining heat and high hydrodynamic pressure at low pH. The remarkably good solubility of dry Simplesse 100 as a result of microparticulation involving a considerable degree of thermal treatment prompted Lieske and Konrad (1994) to investigate this exceptional functionality using analytical techniques such as gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). They elucidated that the benign effect on MWP was related to two molecular events: (1) optimal unfolding of protein molecules, and (2) stabilization of the unfolded status by carbohydrate. Both effects were considered to favor noncovalent bonds, which contribute to the outstanding physicofunctional and nutritive properties of microparticles.

3.3.2

Examples of Commercial Initiatives

In a case study example, whey protein microparticulation has been adapted by Fonterra for the production of a WPC80 with enhanced heat stability (Sure Protein WPC550 from NZMP) (www.foodnavigator-usa.com/ Promotional-Features/Way-forward-with-whey-protein). The process appears to have been redesigned to limit particle interaction through reduction of available thiol groups and, thus prevent formation of larger aggregates. This functional whey protein is claimed to be stable under high-temperature heating (i.e., retorting or UHT) and may be added as an ingredient at high concentrations with no adverse effects during processing. Maintaining small protein particle size in the range 0.1
3.0 μm ensures creaminess while promoting good suspendability in beverage applications to obtain a long shelflife, with no sedimentation. It is claimed that the microparticulated WPC possesses the same nutritional benefits of a standard WPC. This concurs with the findings of Erdman (1990) who used three analytical approaches to monitor changes in amino acid and protein value following microparticulation: amino acid analysis, protein efficiency ratio bioassay, and both one- and two-dimensional SDS-PAGE. The APV LeanCreme process development was initiated in 2003 following SPX Flow Technology’s engagement with the early process development work undertaken by the research team at the Technical University of Munich (TUM), Weihenstephan, Germany. The key process innovation introduced during this technology transfer stage was to combine both heat and shear into a single-step operation using SSHEs in order to provide better control over the quality of MWP. Much of TUM’s early development work was corroborated during performance evaluation of the early prototypes of the LeanCreme process, e.g., optimized operating temperature of 85 C, more rapid β-Lg denaturation occurring in the presence of reduced lactose

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content—the corollary of which is to use higher temperature when processing WPC30 rather than WPC80. WPC calcium content was found to be important especially during microparticulation using the LeanCreme process as low Ca (typical of rennet casein whey) leads to fouling, loss of color and viscosity, and pressure rises. The company’s LeanCreme reference process refers to MWPC generated from a WPC60 substrate following ultrafiltration of sweet whey with a pH between 6.3 and 6.6. SPX cautions that particle size analyzers from various instrument manufacturers produce differing results for MWPs because of the unique mathematical calculations and theories employed in each case. Hence, it is recommended that the “Fraunhofer” theory should be used in conjunction with particle size analyzers. One typical index, 3 50 value, represents an expression of 50% quantile and, thereby, one type of mean particle size. Another is the D[4,3] value representing the volume-based mean particle size. Comparing the 3 50 and D[4,3] values of 3.51 and 3.98 μm, respectively, in each of these cases illustrates the effects of data harmonizing brought about by the “Fraunhofer” theory. SPX advise against overreliance on instrumentation based on the MIE theory for LeanCreme particle size measurement as the MIE requires estimation of two values—the so-called “refractive index” and the “absorption index” which are not commonly known for LeanCreme particles. GEA has developed a simple unit process for microparticulation of whey proteins, namely MICRO FORMULA for microparticulation (http://www.gea. com/en/products/Micro%20Formula%20for%20Microparticulation.jsp). The MICRO FORMULA unit is claimed to generate particles similar to milk fat and has a number of novel design features, e.g., no exposure to heat transfer surfaces in excess of denaturation temperatures, standard plate heat exchangers without recourse to the use of SSHE or tubular heat exchangers, and long production runs of 18
20 h between CIP.

3.4

CONCLUSION

Polymeric UF membranes in SW configuration have proven to be the most widely adopted and cost-effective separation process for the preparation of WPC retentates over a wide protein concentration range of approximately 25%
80% protein. PS and PES membranes have been consistently used for whey protein recovery applications since the 1970s. Design improvements have been focused for the most part on optimal configuration of multistage UF plants along with judicious use of diafiltration water in order to minimize membrane fouling. Alternative and effective approaches such as the use of high shear across the surface of membranes using rotating membrane discs

References

have not detracted the industry from its commitment to SW-based membrane systems. Instead, membrane manufacturers are constantly upgrading their SW module designs in order to maintain higher cross-flow velocities by reducing the amount of feed bypass within the membrane modules. The introduction of a technological step-change in the 1990s involving pretreatment of whey by cross-flow MF in order to remove residual lipids in advance of subsequent concentration by UF helped to elevate protein concentration to isolate status, i.e., $ 90% on a dry basis. While the initial innovation in whey defatting was achieved using inorganic membranes, organic MF membranes using different polymers in the active separation layer were developed and readily adopted by whey processors. The timing of these technological developments coincided with the emergence of new markets for performance nutrition ingredients and products. While PS- and PES-based membranes continue to dominate in UF plants manufacturing WPCs and WPIs, researchers continue to be active in developing new membrane filtration materials involving different mixtures of polymers and use of nanotechnology to create active membrane surface layers. All the indications are that the next generation of membranes under development will facilitate large-scale production of selectively-enriched fractions of WPCs in the near future. Microparticulation is proving to be a useful extension to WPC manufacturing processes in order to generate additional functionality, e.g., enhancing the colloidal and thermal stability of WPC80 retentates by microparticulating at low pH and high hydrodynamic pressure conditions. UF whey retentates of differing protein contents have been utilized as feedstocks for microparticulation by different researchers. Such process integration is now possible given the availability of dedicated microparticulation plant that enables the dairy processor to pursue ongoing ingredient innovation.

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2047. ADPI. (2016). Whey protein concentrate (WPC) standard. Elmhurst, IL: American Dairy Products Institute. Available from https://www.adpi.org/Portals/0/Standards/WPCStandard_book.pdf, downloaded 12.11.17. Almécija, M. C., Guadix, A., Martinez-Ferez, A., González-Tello, P., & Guadix, E. M. (2009). A flux enhancing pretreatment for the ultrafiltration of acid whey. Desalination, 245, 737
742. Almécija, M. C., Ibáñez, R., Guadix, A., & Guadix, E. (2007). Effect of pH on the fractionation of whey proteins with a ceramic ultrafiltration membrane. Journal of Membrane Science, 288, 28
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386. Barukˇci´c, I., Boˇzani´c, R., & Kulozik, U. (2015). Influence of process temperature and microfiltration pre-treatment on flux and fouling intensity during cross-flow ultrafiltration of sweet whey using ceramic membranes. International Dairy Journal, 51, 1
7. Berg, T. H. A., Knudsen, J. C., Ipsen, R., van der Berg, F. W. J., Holst, H. H., & Tolkach, A. (2014). Investigation of consecutive fouling and cleaning cycles of ultrafiltration membranes used for whey processing. International Journal of Food Engineering, 10, 367
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108. Bhushan, S., & Etzel, M. R. (2009). Charged ultrafiltration membrane increase the selectivity of whey protein separations. Journal of Food Science, 74, E131
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50. Cheang, B., & Zydney, A. L. (2004). A two-stage ultrafiltration process for fractionation of whey protein isolate. Journal of Membrane Science, 231, 159
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5607. Jalali, A., Shockravi, A., Vatanpour, V., & Hajibeygi, M. (2016). Preparation and characterization of novel microporous ultrafiltration PES membranes using synthesized hydrophilic polysulfide-amide copolymer as an additive in the casting solution. Microporous and Mesoporous Materials, 228, 1
13. Kalaivani, S., & Regupathi, I. (2015). Synergistic extraction of α-Lactalbumin and β-Lactoglobulin from acid whey using aqueous biphasic system: Process evaluation and optimization. Separation and Purification Technology, 146, 301
310. Karasu, K., Yoshikawa, S., Ookkawara, S., Ogawa, K., Kentish, E., & Stevens, G. W. (2010). A combined model for the prediction of the permeation flux during the cross-flow ultrafiltration of a whey suspension, . Journal of Membrane Science (361, pp. 71
77). Kelly, J., & Kelly, P. M. (1995). Desalination of acid-casein whey by nanofiltration. International Dairy Journal, 5, 291
303. Kelly, P. M. (2011). Membrane-based fractionation
Milk protein products. In (2nd ed.) J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (Vol. 3) San Diego, CA: Academic Press. Kerche, F., Weterings, M., & Beyrer, M. (2016). The effect of temperature and shear upon technological properties of whey protein concentrate: Aggregation in a tubular heat exchanger. International Dairy Journal, 60, 32
38. Konrad, G., Kleinschmidt, T., & Faber, W. (2012). Ultrafiltration flux of acid whey obtained by lactic acid fermentation. International Dairy Journal, 22, 73
77. Lieske, B., & Konrad, G. (1994). Microparticulation of whey protein: Related factors affecting the solubility. Zeitschrift fuer Lebensmittel Unters Forschung, 199, 289
293. Marella, C., Muthukumarappan, K., & Metzger, L. E. (2011). Evaluation of commercially available, wide-pore ultrafiltration membranes for production of α-lactalbumin-enriched whey protein concentrate. Journal of Dairy Science, 94, 1165
1175. Mehra, R. & Kelly, P. M. (2004). Whey protein fractionation using cascade membrane filtration. In Proceedings of IDF World Dairy Summit 2003 symposium ‘Advances in fractionation and separation: Processes for novel dairy applications’ (pp. 40
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137.

CHAPTER 4

Changes in Whey Protein Powders During Storage Eve-Anne Norwood, Thomas Croguennec, Cécile Le Floch-Fouéré, Pierre Schuck and Romain Jeantet Agrocampus Ouest, Rennes, France

4.1

INTRODUCTION

The global market for dairy powders has undergone two major transitions over the past four decades, firstly by integrating scientific and technological knowledge, and secondly due to the growing demand from the emerging markets. The use of separation techniques such as membrane filtration (e.g., microfiltration and ultrafiltration) allowed access to a new range of dry products with higher added value, including concentrates and isolates of specific whey protein fractions (Schuck, 2002). By the production of the latter, the dairy sector turned toward the nutritional control of these powders as well as their technological functionalities. Whey protein powders are widely used as dry food ingredients due to their high nutritional values and are produced on a large scale. Whey proteins now appear to be the one of the most valuable components of bovine milk. Besides their nutritional aspects, they possess interesting functional properties such as solubility, emulsification, foaming, and gelling. Milk goes through a number of processing steps including preheating, evaporation, and filtration before drying to obtain whey protein powders of specific characteristics (e.g., composition). These steps involve high temperature, shear, and removal of water from whey solids, which may cause physicochemical changes in whey components such as protein, lactose, minerals, and fat (Fox, 2003). The quality of fresh whey protein powders can be evaluated through their solubility at pH 4.6, which is a method of quantification of the native whey proteins (nondenatured) content. It gives an indirect indication of the extent of whey proteins denaturation and aggregation as well as the severity of heat treatments that have been used during whey protein powder production. This constitutes an important parameter as whey protein functional properties are dependent on the production history. 123 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00004-7 © 2019 Elsevier Inc. All rights reserved.

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The stability during storage is a primary requirement of powdered dairy products. The dry state is the way that most dairy products are stored since dry products are considered stable in terms of physicochemical reactions. Moreover, it makes dried dairy products easy to handle and requires low transportation and storage costs. However, whey protein powders sent overseas are usually subjected to high temperature and humidity during delivery (Leinberger, 2006) and stored without refrigeration, again exposing the product to high temperature and humidity conditions. The shelf life of whey protein concentrate (WPC) powders under these conditions must be known to prevent the product from being rejected by industrial costumers (Tunick et al., 2016). Stability over storage is mainly influenced by water activity, temperature, and time as they play an important role in the stabilization of physical, chemical, and textural characteristics of food systems (Thomas, Scher, & Desobry, 2004). Despite many years of scientific and technological research, and even though the microbiological quality of the powders is now perfectly controlled, a lack of knowledge and fundamental understanding still remains on the physicochemical changes in WPC and whey protein isolate (WPI) powders over storage whereas the uses of these dry ingredients are expected to strongly increase in the next 5 years (Potier, 2015). The agingrelated changes include the relationship between the aging of whey protein powders and its consequences on the functional properties. Changes in protein structure are believed to result from the storage conditions—temperature, humidity—to which the powders are subjected with regard to their respective stability thresholds (water activity aw, glass transition temperature Tg). Protein stability is usually associated with its ability to maintain primary and secondary structures as well as the aggregation rate. The conversion of native proteins into denatured proteins alters their functionality (Fasman, 1989) and thus the overall powder functionalities. Besides protein denaturation, there are many other factors that may cause physicochemical changes in whey protein powders which lead to a decrease in native protein content, color change, and changes in other functional properties (Burgain et al., 2016; Le, Bhandari, Holland, & Deeth, 2011; Norwood, Chevallier, et al., 2016; Tunick et al., 2016). This chapter aims to list exhaustively the physicochemical changes at macroscopic, mesoscopic, and molecular scales that are thought to be responsible for changes in the functional properties of whey protein powders over storage. Understanding the changes occurring during storage of whey protein powders is crucial to guarantee the preservation of their properties on long-term storage. This will eventually help the manufacturers to improve their competitiveness in a challenging market by reducing added value losses consecutive to uncontrolled changes in powder functionalities.

4.2 Physicochemical Changes During Storage

4.2 4.2.1

PHYSICOCHEMICAL CHANGES DURING STORAGE Powder Stability and Molecular Mobility

Dairy powders are predominantly in an amorphous glassy state defined as an out-of-equilibrium state and, as such, are considered to be stable as long as they are stored below their Tg (Struik, 1977). The latter is specifically the property of an amorphous material (Bhandari & Howes, 1999) and relates to the molecular mobility within the powder. When the temperature crosses Tg (by an increase in temperature and/or aw), the amorphous glassy state (high viscosity) changes to a rubbery state (low viscosity) accelerating molecular mobility. As a result, reactants move toward each other, free to interact, causing physicochemical and functional changes in the powders (Roos, 2002). Variations in the molecular mobility including internal molecular motions and molecular migration or diffusion are supposed to rule dairy powders stability over storage (Fan & Roos, 2016). Therefore, they affect protein structures and thereby facilitate intermolecular interactions (Abdul-Fattah, Kalonia, & Pikal, 2007). However, keeping dairy powders under Tg is not sufficient by itself to ensure their physical and chemical stability during longterm storage (Roudaut, Simatos, Champion, Contreras-Lopez, & Le Meste, 2004; Zylberman & Pilosof, 2002). In terms of free energy landscape, protein powders might still undergo a progressive relaxation process toward a more stable state. Changes in powders occurring during the relaxation process were called physical aging by Schokker et al. (2011). The level of physical aging is facilitated by increasing temperature and aw, as water acts as a plasticizer and promotes relaxation processes (Struik, 1978). Relaxation enthalpy is specific to the molecule and is local, including restricted long-range cooperative motions and internal molecular motions—not involving surrounding molecules—in the glassy state (Fan & Roos, 2016). It also concerns translational molecular motions of molecules or segments of molecules when the glassy material is above Tg (Liu, Bhandari, & Zhou, 2006). Nevertheless, the physicochemical stability of dairy powders depends on powder composition and time, and is related to viscosity changes that may occur even below Tg. Indeed, some chemical reactions such as the Maillard reaction can develop in the amorphous state, mainly because these reactions are directly related to molecular diffusion (Champion, Le Meste, & Simatos, 2000; Schebor, Buera, Karel, & Chirife, 1999). In this case, the chemical reactions are only detected after long storage times, since the diffusion velocities are very low below Tg. Above Tg, physical degradations, such as crystallization of lactose, particle collapse, or powder caking are able to occur (Bhandari & Howes, 1999; Champion et al., 2000; Chung et al., 2000; Hennigs, Kockel, & Langrish, 2001). Powder storage is not as well mastered as other manufacturing steps. Stored powders can be brought close to and even above their Tg. As it has

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been reported by Leinberger (2006), they can be subjected to temperature and humidity up to 50
C and 80%, respectively, during shipment. Indeed, mid Tg of whey proteins powders around aw of 0.3 usually vary from 48 to 84
C according to their protein content from low to high, respectively (Bhandari & Roos, 2016). The WLF equation (Williams, Landel, & Ferry, 1955), which allows the calculation of the viscosity variation from the difference between storage temperature and Tg, also makes it possible to determine the relaxation time of these changes as a function of TTg (Thomas, 2004): logτ 2 logτ g 5

2 C1 3 ðT 2 Tg Þ C2 1 ðT 2 Tg Þ

ð4:1Þ

where τ (s): relaxation time of a physical or chemical modification at temperature T; τ g (s): relaxation time of a physical or chemical modification at temperature Tg; T (K): storage temperature; Tg (K): powder’s glass transition temperature; C1 and C2: constants.

4.2.2

Modifications at the Particle Scale

4.2.2.1 Color of Stored Powder The color of dairy powders constitutes an essential criterion for their end use properties as it reflects overall changes occurring in powders. Due to their high protein content and their close-to-neutral pH, whey protein powders constitute a sensitive medium for the occurrence of the Maillard reaction. Lactose, as a reducing sugar, reacts with free amino groups of proteins, peptides, and free amino acids and can thus negatively influence powder properties and alter the powder quality (loss of lysine residue, development of a brown color, protein cross-linking) (Van Boekel, 1998). Thus, whey protein powders that incorporate amorphous lactose are susceptible to nonenzymatic browning through the Maillard reaction, because of the formation of melanoidins (Van Boekel, 1998). The color measurements are often run in the L*a*b* space, where L* indicates lightness, and a* and b* are the chromaticity coordinates. Maskan (2001) proposed a formula combining the L*a*b* data to assess purity of the brown color of food which was called the “browning index” (BI): BI 5

100ðx 2 0:31Þ 0:17

ð4:2Þ

where x5

a 1 1:75L 5:645L 1 a 2 3:012b

ð4:3Þ

This index is an indicator of Maillard reaction extent, as demonstrated by Martinez-Alvarenga et al. (2014). These authors followed the color change of

4.2 Physicochemical Changes During Storage

a WPI powder with a varying amine:carbonyl ratio, subjected to high temperature (θ $ 50
C) and high humidity ($50% RH). They were able to establish that the powder increasingly browned as a function of heating time. In the 1980s, some studies reported that WPC powders browned under accelerated storage conditions (Labuza & Saltmarch, 1982; Li-Chan, 1983). This browning was correlated with the decrease of residual lactose in the powder and also with the decrease in protein quality with a loss of lysine availability. More recently, Norwood, Le Floch-Fouéré, et al. (2016) showed that WPI powders developed color changes with storage time and temperature. During storage at 20
C, changes in the browning index were very slight or nil over 15 months, whereas powders stored at 40
C or 60
C browned from as early as 15 days during storage. Similar results were reported by Tunick et al. (2016), who claimed that all samples became yellower with storage, according to the analysis of the b* value, but none of them held at room temperature exceeded a b* value of 16.8 during 18 months of storage. The yellow appearance was more marked in samples stored at 35
C.

4.2.2.2 Particle Microstructure The surface structure of powder particles is an important factor influencing the functional properties of powders. Sadek et al. (2015) reported that drying of WPI droplets led to a smooth, semispherical particle shape, which is related to the specific mechanical properties of whey proteins. After storage, the particle surface of WPI powder remained mainly smooth but cracks and broken structures were observed by SEM (Fig. 4.1; Burgain et al., 2016) meaning that the particle structure was affected by storage conditions. Interestingly, the adhesion maps of the aged WPI powder appeared to be heterogeneous and presented large hydrophobic patches. As these patches correlate with heterogeneous surface morphology, it is believed that a chemical modification on particle surface occurred upon storage at high temperature (Burgain et al., 2016).

4.2.2.3 Particle Surface Chemistry Considering surface particle composition, results differ from one dairy powder to another. Given their properties (e.g., surface activity), a segregation of the components into the particle occurs during the drying process resulting in a component gradient between the surface and the core (Gaiani et al. 2006; Kim, Chen, & Pearce, 2002; Nijdam & Langrish, 2006). Indeed, both proteins and fat accumulate at the particle surface during spray drying, whereas lactose content is reported to be higher in the core of the particle (Nijdam & Langrish, 2006). Similarly, lipids and proteins were found to be mainly located at the particle surface of micellar caseins powders, whereas lactose was located in the core of the particle (Gaiani et al., 2010).

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FIGURE 4.1 SEM images of (A) fresh WPI powder and (B) aged WPI powder. The bottom images are magnifications of the corresponding top ones (Burgain et al., 2016).

In addition, not all whey protein powder particles have the same surface component concentration due to variation in powder composition. For WPI powder, no lipids were found on the powder surface (Burgain et al., 2016), as they have already been retained during the microfiltration step implemented for obtaining the WPI concentrate. Many analytical methods can provide information on surface chemical composition; among them, XPS (X-Ray Photoelectron Spectroscopy) and ToF-SIMS (Time of FlightSecondary Ion Mass Spectrometry) stand out as the most relevant and most widely used techniques (Kingshott, Andersson, McArthur, & Griesser, 2011). These were used in the study of Burgain et al. (2016) in order to characterize the surface composition of WPI particles after storage. In this case, XPS analysis revealed no difference in surface composition between the fresh and the aged WPI powders. However, results from ToF-SIMS showed that the particle surface of aged WPI powders presented less amino acid than that of fresh powder. In particular, the decrease was detected on the crack areas in addition to a higher hydrophobicity. The changes in surface morphology during storage were thus connected to surface chemical modifications, which were related to the formation of Maillard reaction products and/or denaturation of proteins (Havea, 2006; Patel, Modi, Patel, & Aparnathi, 2013).

4.2 Physicochemical Changes During Storage

4.2.2.4 Effect of Particle Size In the literature, it has been shown that powder particle surface plays an important role in the properties and aging of powders (Gaiani et al., 2010). Indeed, as the particle surface area per mass fraction unit increases as particle size decreases, a greater interface area is provided for reaction/interaction to take place, potentially resulting in more structural and functional changes. As a consequence, the local changes that occurred on the WPI powder surface observed in the study of Burgain et al. (2016) were thought to increase with decreasing WPI particle size. Moreover, when particle size decreases, more contact points between particles are available for interparticle interactions (Scholl & Schmidt, 2014). Consequently, modulating the particle size of WPI powders was considered as an interesting tool to control structural and functional changes in WPI powders during storage. The study of Norwood (2017) showed that no difference in structural or functional evolution rate were observed between four WPI powders differing in particle size upon storage (ranging from 37 to 74 μm). Indeed, WPI powders are composed of at least 90% of proteins and small amounts of lactose, minerals, and fat. This could be due to limiting lactose concentration at the particle surface to induce Maillard reaction, as lactose could be hypothesized to be mostly located in the core of the particle. Maillard reaction would thus likely start in the core of the particle and then progress through a series of reactions toward the particle surface. In this view, the particle surface would not be the starting point of the Maillard reaction but only an indicator of its extent, through browning index evolution. Another hypothesis was that the component distribution is more homogeneous in the particle due to high viscosity of the concentrate decreasing the mobility of the components and spray drying conditions. It is known that high feed solid content and spray drying kinetics greatly affect the component distribution within the particle, this being reduced at high viscosity and under fast drying kinetics (Kim, Chen, & Pearce, 2009). This would eventually lead to a relative homogenous spread of the Maillard reaction in the whole particle and as a consequence, explain why the particle size has no effect upon aging. Nevertheless, even though the variation of the particle size didn’t affect the changes observed during storage of WPI powder, this parameter still remains to be tested according to the powder composition and with a wider range of particle size.

4.2.2.5 Caking and Flow Properties Storage of whey protein powders provokes clumping and caking that is more pronounced in powders stored above room temperature (Tunick et al., 2016). Caking depends on powder composition as fat melts at elevated temperature and lactose may crystallize or partially dissolve in the presence of moisture, both forming bridges between particles (Fitzpatrick, Barry, et al., 2007).

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Indeed, caking would primarily be due to the formation of noncovalent bonds from fat in the powder (Özkan, Walisinghe, & Chen, 2002). In addition, amorphous lactose drives moisture uptake of powder during storage. Powders having a large amount of amorphous lactose are more susceptible to moisture uptake (hygroscopic powder), which can be enhanced by proteins also adsorbing water. Water adsorption leads to a decrease in powder glass transition temperature, an increase in lactose crystallization kinetics and a release of water from lactose crystallization in the powder. This ends up with the formation of bridges between particles responsible for powder caking phenomenon (Fitzpatrick, Barry, et al., 2007; Fitzpatrick, Hodnett, et al., 2007; Fitzpatrick, Iqbal, Delaney, Twomey, & Keogh, 2004). However, caking is not frequently reported for whey protein powders as it is for other types of dairy powders due to their low fat and amorphous lactose content. It also appeared that the particle size is an important factor as small particles would increase the powder cohesiveness and caking (Rennie, Chen, Hargreaves, & Mackereth, 1999). The smaller the particles, the more contact points between the particles; thereby the cohesion of the powder increases which decreases its flow properties (Fitzpatrick et al., 2004).

4.2.3

Protein Related Changes

4.2.3.1 Modifications of Protein Structure Instability of amino acids under heating. Several amino acids in protein powders can undergo deamidation, transamidification, dehydration, β-elimination reaction, and isomerization at high temperature (Geiger & Clarke, 1987; Gerrard, 2002; Tomizawa, Yamada, Wada, & Imoto, 1995). Such modifications can still occur during storage of whey protein powders and would be worth being studied. Recent studies reported deamidation and lactosylation of β-lactoglobulin (β-Lg) during storage of WPC with protein content of at least 80% of the dry matter (WPC80) and WPI (Le et al., 2011; Norwood, Chevallier, et al., 2016). Secondary/tertiary structures. Gulzar, Bouhallab, and Croguennec (2011) showed that secondary and tertiary structures evolve after dry heating of β-Lg at 100
C for 24 hours and under controlled pH. In particular, these authors observed that at acidic pH (2.5), the rigid structure of tryptophan is partially lost. Changes in the secondary structures of the proteins were detected at acidic pH and neutral pH. Other authors, such as Havea (2006), also showed changes in the structure of proteins with an increased exposure of hydrophobic residues by denaturation during storage. However, these studies did not agree on the changes in protein structures. Norwood, Le Floch-Fouéré, et al. (2016) showed that the protein is lactosylated during storage without any change in its secondary structure, which was analyzed by circular dichroism.

4.2 Physicochemical Changes During Storage

FIGURE 4.2 Chemical structures of (A) N-terminal pyroglutamic acid resulting from the cyclization of the N-terminal glutamic acid of α-La, (B) a cyclic imide resulting from the cyclization of an internal aspartyl residue (Gulzar et al., 2013).

Cyclization. Aside from the formation of covalent aggregates, a significant proportion of the nonaggregated whey proteins were converted into nonnative forms during dry heating treatment (Gulzar, Bouhallab, Jardin, Briard-Bion, & Croguennec, 2013). Mass spectrometry analysis revealed a change in the molecular weight of the whey proteins attributed to a loss of one or two water molecules per protein. The loss of water molecules was observed for 73% of nonaggregated α-lactalbumin (α-La) and only 18% of nonaggregated β-Lg molecules. For α-La, water molecule loss was attributed to the formation of pyroglutamic acid from the N-terminal glutamic acid and to the formation of an internal cyclic imide at position Asp 64 (Fig. 4.2).

4.2.3.2 Protein Crosslinking This part has been extensively studied among storage of casein-rich powders but needs further study in the field of whey proteins powder. Still, some studies reported that protein cross-linking could occur in whey protein powders. Morr and Ha (1993) showed that, during storage of WPC powders, whey proteins partially denature and gradually polymerize. It is important to note that temperature and time of storage have a positive effect on aggregation and polymerization from 25
C onwards (Morr & Ha, 1993). In this regard, Gulzar, Bouhallab, Jeantet, Schuck, and Croguennec (2011) observed a decrease in the amount of native whey proteins in parallel with formation of aggregates after a dry heat treatment of WPI powder at 100
C for 24 hours at pH 2.5, 4.5, and 6.5, the aggregate size increasing with pH. At acidic pH,

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aggregates are soluble and covalent bonds between proteins are only made of disulfide bridges while at higher pH, aggregates are partly soluble and covalently linked by disulfide bonds and other types of bonds that still have to be determined. Many types of protein cross-linking other than disulfide bridges can occur such as xenobiotic cross-linking arising from β-elimination and condensation reactions and leading to protein aggregation through lysinealanine and histidinealanine bonds (Friedman, 1999). Al-Saadi, Easa, and Deeth (2013) also reported that lysinoalanine—a cross-link derived from condensation of lysine and dehydroalanine residues—could occur during heat treatment of milk proteins. However, these reactions have not been demonstrated yet in whey protein powders during storage even if some studies have already invoked their presence (Norwood et al., 2017). Guyomarc’h et al. (2014) reported a study on denaturation and aggregation of whey proteins in three types of powder—one commercial WPI (WPIC) and two model WPI powders composed of pure proteins, one without added lactose (WPIM 2 L) and the other one with lactose (WPIM 1 L) in an equivalent amount as measured in WPIC. It was observed that the presence of lactose (WPIM 1 L) had a dramatic effect on formation of aggregates at pH 6.5; this suggests that some covalent intermolecular bonds formed in the WPIC at pH 6.5 involving some degradation products of Maillard reaction. Conversely in WPIM 1 L powder at acidic pH (pH 2.5), the condensation reaction between lactose molecules and whey proteins is a limiting step preventing further aggregation.

4.2.4

Lactose Related Changes

4.2.4.1 Maillard Reaction and Storage Conditions In whey protein powders, as in all kinds of high-protein dairy powders, Maillard reaction is strongly dependent on the storage conditions— temperature, water activity, and time—and the powder composition—lactose content. Within these factors, temperature and lactose content have the most significant impact on the extent of Maillard reaction (Martinez-Alvarenga et al., 2014; Norwood, Chevallier, et al., 2016; Norwood et al., 2017). Effect of temperature. The temperature influences the reaction kinetics as reported by Norwood, Chevallier, et al. (2016). These authors evidenced two successive steps in the progress of the Maillard reaction, lactosylation and protein aggregation, differently affected by storage temperatures. As shown in Fig. 4.3, changes in the browning index were very slight or nil during storage at 20
C over 15 months, in accordance with the relatively small changes in protein lactosylation and no noticeable protein aggregation. Powders stored at 40
C for up to 15 months first presented a sharp increase in protein

4.2 Physicochemical Changes During Storage

FIGURE 4.3 Correlation between lactosylation and aggregation for fresh powder (empty diamond ) and powders stored at 20
C (circle), at 40
C (triangle), and at 60
C (square) from 15 days to 15 months (from white to black).

lactosylation (Fig. 4.3A), which then decreased toward protein aggregation (Fig. 4.3B). The decrease in lactosylation after 3 months storage also matched the concomitant increase in browning. The authors suggested that, with storage time, free lactose molecules should be bound to protein, while a few of these that were already bound were further degraded into aggregates with a concomitant darkening of powder from the first month of storage. These results were in agreement with other studies showing that storage at high temperatures induces an increase in the amount of Maillard reaction products (Ipsen & Hansen, 1988; Kieseker & Clarke, 1984). Even if WPI powders are extensively diafiltered before spray drying, the very small amount of lactose still remaining in an amorphous state (in the range of 11.5 w/w% corresponding to a lactose/protein molar ratio of 0.550.8) plays an important role in the aging of whey protein powders according to the storage temperature. Aggregation from cross-linking might also be due to heat-induced denaturation of proteins in view of the increase in free thiol groups exposed on the protein surface according to the storage temperature (Norwood, Le Floch-Fouéré, et al., 2016). In this scheme, the storage temperature would act as a dry heat treatment which could affect the exposure of buried thiol groups with partial unfolding of β-Lg, which initiates thiol or disulfide interchange reactions leading to protein aggregation (Busti, Gatti, & Delorenzi, 2006; Hoffmann & van Mil, 1997). Effect of humidity. Le et al. (2011) also showed that the Maillard reaction in WPC80 was very dependent on powder relative humidity. More generally,

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the Maillard reaction in dairy powders proceeds maximally when aw is between 0.50 and 0.80 (Martinez-Alvarenga et al., 2014; Thomas, Scher, Desobry-Banon, & Desobry, 2004). Above this range, the dilution of reactants and high water content lead to a reaction limitation because of the mass action law. Effect of lactose content. Based on the results obtained in the study of Norwood et al. (2017), a small variation of lactose content was shown to have a significant effect on aging of whey protein powders. In the early stages of storage (up to 3 months storage at 40
C or during the first weeks at 60
C), protein lactosylation was observed as the first step of powder aging. For a lactose content of 0.1 w/w%, the level of protein lactosylation seemed to remain constant over time during storage at 40
C and quickly decrease at 60
C without reaching 10% of lactosylated proteins. For higher lactose contents ($2.6 w/w%), quantification of the level of protein lactosylation was not possible, probably due to the rapid degradation of lactosylated proteins (Guyomarc’h, Warin, Donald Muir & Leaver, 2000; Van Boekel, 1998). The authors stated that for identical storage conditions, the difference in lactosylation rate may be found in the lactose/protein molar ratio, which varied from 0.06 to 12 for the whey protein powders considered in the reported study, that were WPI with low lactose content and WPC, respectively. The Maillard reaction proceeded very rapidly and to a much larger extent for powders with more than one lactose molecule per protein than for the powders containing less than one molecule of lactose per protein. For the WPI powder with 1.28 w/w % lactose, over 70% of the proteins could be theoretically lactosylated, considering that one protein molecule binds no more than one molecule of lactose, but only a maximum of 30% was reached in the study of Norwood et al. (2017). The most plausible explanation relates to the number of lactose molecule bound per protein, which could be more than one (Morgan et al., 1998). Furthermore, the higher the lactose content, the more the protein aggregated as shown in the case of WPC powder (16 w/w% lactose) for which the extent of the Maillard reaction led to a high browning index and covalent aggregation of all proteins present in the medium in accordance with previous studies (Kim, Saltmarch, & Labuza, 1981). With storage time, aggregates grew and were stabilized by more and more covalent bonds resistant to reducing agents, up to their insolubilization. On the other hand, reduced lactose content (#0.1 w/w%) significantly limited the rate of protein aggregation and browning by reducing the level of lactosylated proteins. These results evidenced that the aging of whey protein powders was mostly due to the Maillard reaction and that the step of lactosylation was essential for protein aggregation. The absence of lactose in whey protein powders also allowed the observation of lactose-independent aging mechanisms to take place. Studies on dry

4.2 Physicochemical Changes During Storage

heating of purified whey proteins showed that the changes were not the same with and without lactose (Gulzar et al., 2013; Guyomarc’h et al., 2014; Norwood et al., 2017). Dry heating in the absence of lactose caused changes in nonaggregated proteins. A high proportion of nonaggregated α-La was converted into a non-native form (Gulzar et al., 2013) as indicated previously (see Section 4.2.3.1). Zhou and Labuza (2007) also showed that the absence of lactose did not lead to greater structural changes in the WPI powder compared to pure β-Lg powder during storage, although the conditions applied (storage at 45
C for 2 weeks) appeared too mild to assess any differences between powders in views of our previous results.

4.2.4.2 Effect on the Protein Structure The first step of Maillard reaction—protein lactosylation—does not lead to whey protein conformational changes (Hiller & Lorenzen, 2010; Norwood, Le Floch-Fouéré, et al., 2016). Indeed, the compact globular conformation of whey proteins would limit changes induced by the sugar binding. In the course of the reaction, polymers of heterogeneous size would be formed concomitantly to the disappearance of monomers present at first and the decrease in the soluble fraction of nitrogen. Hiller and Lorenzen (2010) also reported that several studies suggested a loss of organized tertiary protein structures upon covalent binding of sugar molecules. Sugar binding would induce a steric stress favoring protein unfolding by a decrease in intra-/intermolecular interactions (Nacka, Chobert, Burova, Léonil, & Haertlé, 1998; Wooster & Augustin, 2007). Temperature- (Broersen, Voragen, Hamer, & de Jongh, 2004) and humidity-dependent (Morgan et al., 1999) losses of tertiary structure of proteins were also observed. For Báez, Busti, Verdini, and Delorenzi (2013), glycation did not result in changes in the secondary structure of proteins, but led to very slight changes in tertiary structure. This structural change results in an exposure of tryptophan after partial protein unfolding as well as an appearance of a small amount of aggregates after glycation. However, according to these authors, conformational changes are more likely to be due to heat treatment than protein glycation.

4.2.4.3 Change of State: Crystallization Literature shows that there is no or only little lactose crystallization in whey protein powders. First of all, lactose crystallization is very unlikely to occur due to their low lactose concentration, far from saturation conditions. Moreover, lactose crystallization in milk powder has been shown to be delayed in the presence of high-molecular-weight polymers, such as proteins, resulting in increased Tg of the system (Karmas, Pilar Buera, & Karel, 1992). Whey proteins are a particular case. They are thought to act as stabilizers over storage delaying or inhibiting lactose crystallization compared to other dairy constituents (Foster, Bronlund, & Tony Paterson, 2005; Le et al., 2011).

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For example, Shrestha, Howes, Adhikari, Wood, and Bhandari (2007) showed that no crystallization occurred for lactose and hydrolyzed WPI mixtures (1:4) kept at awB1.0. Three hypotheses have been raised to explain this particular behavior. The first one is related to a difference in miscibility that would lead to phase separation of protein and lactose (Haque & Roos, 2004). On the other hand, Thomas, Scher, and Desobry (2004) proposed that the presence of β-Lg is responsible for the delay in lactose crystallization in freeze-dried powders (two different powders were used with protein:lactose ratios of 10:90 and 40:60). The spatial organization of β-Lg would account for this effect, since β-Lg might be located on the surface of powder particles and might thus limit the access of water to amorphous lactose. Finally, the last hypothesis refers to existing molecular interaction between whey proteins and lactose, which would impede crystallization. This would explain why lactose crystallization was delayed in WPC35 (protein content of at least 35% of the dry matter)—compared to SMP—and totally inhibited in WPC60. Lactose molecules would interact with polar groups of globular proteins through hydrogen bonding. Moreover, the competition between whey protein 2 lactose interactions and lactose 2 lactose interactions (nucleation) would reduce the propensity of lactose to crystallize with whey protein 2 lactose hydrogen bonding being more intense in whey protein powders.

4.2.5

Impact on Functional Properties

4.2.5.1 Solubility No solubility loss was evidenced during 3-month storage of whey protein powders (Hsu & Fennema, 1989; Le et al., 2011).

4.2.5.2 Heat-Induced Aggregation Properties Heat-induced aggregation is generally considered a two-stage mechanism. First, proteins expose some initially buried hydrophobic groups by unfolding. Then, they assemble into small oligomers, which further assemble into larger aggregates through hydrogen and hydrophobic interactions; subsequently, the structure of the aggregates is stabilized through covalent disulfide bridges (Schmitt et al., 2010). In recent studies, it has been shown that heat-induced aggregation properties were affected by storage conditions. Norwood, Chevallier, et al. (2016) highlighted a decrease in the size of heat-induced aggregates prepared at pH 5.8, 6.2, and 6.6 as a function of storage time up to 12 months at 40
C, when no changes were evidenced for storage at 20
C. Moreover, the decrease in size was concomitant with changes occurring in their shape, between strands and microgels. These changes were discussed according to the structural changes observed during storage. Numerous studies have shown that

4.2 Physicochemical Changes During Storage

protein glycation modifies the process of protein heat-induced aggregation. Liu and Zhong (2013) reported that glycation of WPI with lactose enabled transparent dispersion after heating 7% w/v WPI solutions at 88
C for 2 minutes, whereas a WPI control solution formed turbid solutions. The transparent solution was composed of particles smaller than 14 nm. Mulsow, Jacob, and Henle (2008) also stated that covalent attachment of lactose to the whey proteins significantly improved the heat-stability of whey proteins. A first hypothesis was based on clustering of oligomers upon heating, which would rapidly reach a critical charge density, thus limiting aggregation, and smaller aggregates would form preferentially. However, Broersen et al. (2007) stated that the inhibition effect of glycosylation on heat-induced aggregation could not be explained by this mechanism. Thereby, they proposed that inhibition of aggregate growth would result from an increase in heat-induced aggregation kinetics. Indeed, glycosylation is believed to facilitate protein denaturation when the proteins are heated at their denaturation temperature or above in the preliminary stage of aggregation (Broersen et al., 2007; Sun et al., 2011). Lastly, Mulsow et al. (2008) hypothesized that the increase in hydrophilicity resulting from lactose molecule binding to the protein may weaken the hydrophobic interactions and thus hinder aggregation. By varying the lactose content in whey protein powders, Norwood et al. (2017) demonstrated that heat induced-aggregation was affected by the Maillard reaction products resulting from the degradation of lactosylated proteins and not solely from the protein lactosylation. In these conditions, proteins would be less flexible and thus lose part of their ability to form aggregates inhibiting protein aggregation (Da Silva Pinto et al., 2012; Lee, Park, Paik, & Choi, 2009). It is possible that the glycated protein powders considered in the previous stated works also contained some Maillard reaction products. For example, a WPI dispersion exhibited a dark color in Liu and Zhong (2013) and an elevated furosine content in the study of Mulsow et al. (2008). Moreover, Broersen et al. (2007) might have overestimated protein glycation using the o-phthaldialdehyde (OPA) method, which refers to the blocking of free amine groups by reducing sugar. Indeed, this method of quantification provides information on glycated proteins and their degradation products, but is not able to differentiate one from the other. On the other side, as oligomers are formed from native proteins (Jones, Adamcik, Handschin, Bolisetty, & Mezzenga, 2010; Phan-Xuan et al., 2013), changes observed at 40
C would result from the reduction in amount of native proteins as some of them were already involved in the aggregation process over storage. A steady state is thus reached when the solution no longer contains native monomers. At pH 6.2 and 6.6, shape changes were explained by the decrease in the amount of native protein (Lee et al., 2009). The extensive binding of lactose and Maillard reaction products to whey

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proteins (mainly on amino groups) stored at 40
C could also increase their net charge. Consequently, clustering of oligomers upon heating might rapidly reach a critical charge density, thus limiting aggregation, and strands would form preferentially.

4.2.5.3 Foaming Properties No or minor changes in the foaming properties were shown at low or high protein content in whey protein powders during storage (Hsu & Fennema, 1989; Norwood, Le Floch-Fouéré, et al., 2016). Only severe storage conditions led to more stable foams by decreasing the drainage rate with a concomitant decrease in the air phase volume. This was supposed to result from an increase in the relative percentage of whey protein polymers in the solution, as the coexistence of aggregated proteins and nonaggregated proteins has been assessed to be a key parameter for foaming properties (Foegeding, Luck, & Davis, 2006). Two mechanisms were proposed. The first displayed a forced adsorption of protein aggregates during foam formation, which would strengthen the viscoelasticity of the bubble interface. The presence of more hydrophobic denatured proteins was also considered as a decisive factor for the formation and stabilization of protein foams due to the rapid formation of viscoelastic films stabilizing bubbles against disproportionation (Báez et al., 2013; Moro, Báez, Busti, Ballerini, & Delorenzi, 2011). The second mechanism, which is believed to be additional, is based on the aggregates’ inability to adsorb at the air bubble interface. Indeed, they would be trapped in the film during foam formation and structure themselves into layers difficult to remove from the film (Sethumadhavan, Nikolov, & Wasan, 2001a, 2001b). The foam stability would then be improved by the repulsive forces structuring the aqueous film (Sethumadhavan et al., 2001b). This kind of aggregates is also subject to cross-linking between the interfaces to form a gel-like network which prevents the film becoming thinner, thereby preventing the interfacial layers of air bubbles to come closer and finally helping to retain the solution in the film (Rullier, Novales, & Axelos, 2008; SaintJalmes, Peugeot, Ferraz, & Langevin, 2005). Moreover, these aggregates can be located in the plateau borders, playing a role of steric clutter impeding drainage. It is assumed that shape and size of aggregates are crucial to the stability of foams (Fameau & Salonen, 2014; Rullier, Axelos, Langevin, & Novales, 2010; Zhu & Damodaran, 1994). However, the size of aggregates formed during storage is too heterogeneous and their shapes couldn’t be studied to confirm this hypothesis (Norwood, Le Floch-Fouéré, et al., 2016). As the Maillard reaction seems to be one of the major causes of structural changes during storage, foaming properties can also be discussed according to its occurrence. Martinez-Alvarenga et al. (2014) noted an improvement in

4.2 Physicochemical Changes During Storage

foamability after glycation at high temperature (#50
C), that they explained by the surfactant ability to encapsulate air. This evolution in foamability was also explained by heat-induced protein denaturation rather than actual glycation (Báez et al., 2013). On the other hand, it is believed that Maillard reaction products improved foam stability (Hiller & Lorenzen, 2010). These changes would be due to Maillard reaction products causing steric protection against coalescence (Dunlap & Côté, 2005), or because they are found in the interfacial film which would be thicker and more viscoelastic (Ganzevles, Cohen Stuart, van Vliet, & de Jongh, 2006; Wooster & Augustin, 2007). For Báez et al. (2013), aggregates formed by the Maillard reaction (β-Lg 1 glucose) altered foam stability by decreasing the rigidity and viscoelasticity of the interfacial film. However, it is worth noting that the foaming process largely differed between these studies, which could explain the difference in the observed results. Indeed the bubbling apparatus or Aero-Latte stirrer was used in the previous stated studies, generating different types of foam and thus involving two different stabilizing mechanisms.

4.2.5.4 Emulsifying Properties Changes in emulsifying properties have been also reported. Hiller and Lorenzen (2010) observed a low emulsifying activity of whey proteins 2 glucose mixtures due to the formation of Maillard reaction products of high molecular weight causing a delay in the adsorption and deployment at the interface. However, other authors including French and Harper (2003), who studied β-Lg 2 lactose mixture, reported no difference. Emulsion stability also decreased for mixtures of WPI with glucose or lactose. The decrease was explained by the fact that Maillard reaction products would cover an insufficient area of the interface and/or Maillard reaction products of high molecular weight would remain at the interface of several lipid droplets in the meantime, acting as connector and thereby promoting coalescence.

4.2.5.5 Interfacial Properties It appeared that surface tension of protein solutions was unaffected by the Maillard reaction (Fechner, Knoth, Scherze, & Muschiolik, 2007; Hiller & Lorenzen, 2010). However, all authors did not share this assumption. Baeza, Carrera Sanchez, Pilosof, and Rodríguez Patino (2005) claimed that Maillard reaction products increased the surface tension of protein solutions. These differences could be due to the type of sugar involved in the Maillard reaction, as sugar properties and ability to induce protein structural change after binding differ. At mild storage conditions, protein lactosylation occurred with time and was thought to increase the diffusion coefficient of WPI after rehydration without

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affecting the concentration at which the surface tension differs from 0 (Γ0), or the amplitude of interaction at the interface (θ; Norwood, 2017). Indeed, hydrophilic polysaccharides promote interfacial adsorption and dilatation of milk protein (Baeza et al., 2005; Hiller & Lorenzen, 2010). Baeza et al. (2005) showed a cooperative behavior between the hydrophilic polysaccharide and the protein leading to a significant increase of surface pressure of adsorbed films. However, lactosylated protein tended to aggregate thus increasing in molar mass, which was believed to be the cause of the decrease in the diffusion coefficient (D) with storage time. Contrary to small protein aggregates, larger aggregates, coming from an extensive Maillard reaction, are not able to adsorb at the air 2 water interface due to their size and the lack of surface activity (Rullier et al., 2008; Rullier, Axelos, Langevin, & Novales, 2009). However, at severe storage conditions (6 months at 60
C) with more than 65% aggregated proteins in the dry state, the amount of native protein was too low to adsorb at the air 2 water interface, or aggregated proteins were too slow to adsorb within the time of experiments. It is interesting to note that for shorter storage times at 60
C, Γ0 and θ were not or only slightly affected (Norwood, 2017).

4.3 MEASURES TO OVERCOME CHANGES DURING STORAGE 4.3.1

Monitoring Powders Evolution

4.3.1.1 Maillard Reaction One of the easiest changes to monitor in order to evaluate powder aging is browning. However, it has been shown that browning only indicates that the powder is already in an advanced state of aging with significant changes in its functional properties (Norwood, Le Floch-Fouéré, et al., 2016; Norwood et al., 2017). Indeed, the brown color comes from the degradation of lactosylated proteins into Maillard reaction products, which have been proven to affect functional properties of whey protein powder ingredients. Twenty years ago, De Block, Merchiers, and Van Renterghem (1998) suggested the use of capillary electrophoresis (CE) of the whey protein fraction of milk powder to monitor the effects of storage. This method was based on the change in the isoelectric point of whey proteins over storage, which was mainly related to lysine damage. Consequently, CE allowed the determination of the ratio of native to total β-Lg in order to monitor the storage conditions of milk powders. This method could also be used for evaluating the primary changes occurring during the storage of whey protein powders. Leiva, Naranjo, and Malec (2017) proposed several other indicators to monitor the Maillard reaction of whey proteins under adverse storage conditions.

4.3 Measures to Overcome Changes During Storage

The OPA method appeared to be a good indicator of the early stage of the Maillard reaction, monitoring the loss of accessible lysine. However, CE was considered to be a more sensitive method for evaluating the extent of native protein glycosylation than the OPA method to monitor the loss of lysine. The authors also stated that if the advanced stages of the Maillard reaction were not reached, the determination of total 5-hydroxymethyl-2-furfuraldehyde (HMF), related to the estimation of the Amadori product, would have correlated well with reactive lysine content. Finally, the measurement of free HMF and color were claimed not to be suitable at the storage conditions used in this study for assessing the occurrence of the intermediate and final stages, respectively. According to Charissou, Ait-Ameur, and Birlouez-Aragon (2007), carboxymethyllysine (CML) quantification in food samples using gas chromatography-mass spectrometry is an accurate method to evaluate the intermediary state of the Maillard reaction. Indeed, CML is considered to be a useful marker of protein damage in severely heated foods. The authors also compared their results with enzyme-linked immunosorbent assay, which was performed on infant formulas, and found satisfactory results in powdered but not in liquid formulas. However, caution has to be taken regarding the monitoring of the advanced states of the Maillard reaction as previous studies have already shown that these structural changes affect functionalities of whey protein ingredients such as heat induced aggregation and, to a lesser extent, foaming properties (Norwood, Chevallier, et al., 2016; Norwood, Le Floch-Fouéré, et al., 2016). In this regard, the monitoring of protein lactosylation or the reduction in free lactose content in the powder seems to be more appropriate in order to avoid functional changes over storage. Mass spectrometry coupled with electrospray ionization provided satisfactory results regarding the monitoring of β-Lg lactosylation (Fenaille, CamposGiménez, Guy, Schmitt, & Morgan, 2003; Morgan et al., 1998). This technique allows the determination of the average number of lactose linked per β-Lg monomer and the distribution of glycoforms as described by Morgan, Léonil, Mollé, and Bouhallab (1997). Its cost makes it very rarely used in the industry for routine control tests but it is used to a greater extent in research laboratories. Other recent research studies were based on the identification and quantification of lactosylated protein during storage (Le, Deeth, Bhandari, Alewood, & Holland, 2013; Stephani et al., 2017). Le et al. (2013) quantified whey protein lactosylation in milk powders using multiple reaction monitoring (MRM), which is a mass spectrometry-based quantification method. The MRM method stemmed from knowledge of peptide fragmentation. Indeed, lactosylated peptides are represented by the neutral losses of 162 and 216 Da referring to the cleavage of galactose and the formation of furylium ion, respectively, and thus selected as MRM transitions. The peak areas of these two fragmentation ions were then taken to quantify protein lactosylation. This study showed a good correlation between normalized

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lactosylation levels and furosine content, which proved the feasibility of this method. The extent of the Maillard reaction has also been monitored by Fourier Transform Infrared spectroscopy (Wnorowski & Yaylayan, 2003).

4.3.1.2 Lactose State Raman spectroscopy was presented as a tool to identify WPC modification during storage (Stephani et al., 2017). Combined with chemometrics, this method is capable of identifying lactose related changes during production and storage by studying the spectral changes in bands located near 2900 cm21 and between 1200 and 1800 cm21, which are attributed to the vibrational modes of crystalline lactose. One of the main advantages of this method is that it doesn’t involve analytical conditions that may interfere with the chemical environment of other constituents.

4.3.2

Actions on the Key Influencing Factors

4.3.2.1 Extrinsic Factors Humidity and temperature are the most important and controllable factors for delaying whey protein powder changes during storage. Powders should have a low water activity and be kept at temperatures below or equal to 20
C to be stored without significant functional changes over a year or more. Relative humidity was shown to be less influential than temperature on flavor and functional properties (Tunick et al., 2016). Moreover, powder packaging has to be carefully considered since humidity sorption would accelerate powder aging (Stapelfeldt, Nielsen, & Skibsted, 1997). Even though Tunick et al. (2016) showed that thicker bag liners didn’t enhance shelf life of whey protein powders, dairy companies should still consider thicker plastic liners bags because shipments could be handled improperly during loading and unloading resulting in tearing of normal bags. Alternatively, powder packaging under modified-atmosphere may help prevent color and clump formation. However, this approach has not been explored with WPC powder.

4.3.2.2 Intrinsic Factors Several intrinsic factors could be further tested in order to improve whey protein powders over storage: particle size and lactose content. Particle size. Variation of particle size in the range of 3774 μm did not seem to greatly influence the aging-related changes of WPI powder (Norwood, 2017). Nevertheless, this is still a parameter to be considered according to the powder characteristics (e.g., powder composition, wider range of particle size).

4.3 Measures to Overcome Changes During Storage

Lactose content. The presence of lactose, albeit at low concentration (,2%) in WPI powders, is believed to have a strong effect on the ingredient stability during storage according to previous reports (Norwood, Chevallier, et al., 2016; Norwood, Le Floch-Fouéré, et al., 2016). Reducing its content would thus extend the shelf life of whey protein powders. Indeed, Norwood et al. (2017) showed that a low lactose content (#0.1%) significantly limited the rate of protein aggregation and browning of whey proteins in WPI powders during storage by reducing the level of lactosylated proteins, and causing less change in functional properties. This also proved that the aging of whey protein powders was mostly due to the Maillard reaction and the step of lactosylation was essential for the aggregation. Even if the absence of lactose does not prevent lactose-independent aging reactions in the powders (Gulzar et al., 2013; Guyomarc’h et al., 2014), the effect of these reactions on whey protein functional properties seems limited. Therefore, controlling the lactose content in whey protein powders is essential to control whey protein powder properties during storage. The control of the lactose content before and after spray drying in whey protein powders is of major importance for their longterm preservation, particularly under harsh storage conditions. Increasing the diafiltration of lactose before spray drying would be an issue for food industries so as to have a very stable whey protein powder during storage.

4.3.3

Prediction of Whey Protein Powder Changes

This section is aimed at providing monitoring indicators to industry in order to anticipate or predict powder behavior during storage and/or transportation. To date, only Norwood (2017) established a relationship between structural and functional changes in WPI powders and storage conditions. Two hypotheses have been proposed: (1) WPI powders are subject to a catching up phenomenon, or (2) WPI powders follow different paths depending on the storage conditions. Results suggested the evolution of WPI powders followed a catching up behavior during aging at different temperatures (Norwood, Chevallier, et al., 2016). Changes in protein lactosylation at 20
C over 15 months of storage were likely to catch up to the level of protein lactosylation reached after 3 months at 40
C. More concretely, Norwood (2017) showed that several storage conditions proved to be equivalent (Fig. 4.4) by correlating the level of aggregated proteins with browning index, both being representative of aging of WPI powders. Indeed, the points corresponding to WPI powders stored for 18 months at 20
C and 40
C were superimposed on those of powders stored 15 days at 40
C and 60
C, respectively (Fig. 4.4, Box A and B, respectively). Furthermore, powder storage conditions corresponding to 3, 6, and 9 days at 60
C were similar regarding the above aging indexes to around 3 months,

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80 70

6 months

60

3 months

Aggregated proteins (%)

CHAPTER 4:

A

6 15 days

5 4

18 months

3 2 1 0

50

15

40

16 17 18 19 Browning Index (A.U.)

20

1 month 12 months

30

15 days

15 months 9 months

6 days

20 A

10

B

18 months

9days

Aggregated proteins (%)

Aggregated proteins(%)

144

3 days

18 months

6 months

15 days 3 months 1 month

35

B

33 15 days

31 29 27

18 months

25

0 0

10

20

30

40

50

60

70

80

50

51 52 53 54 Browning Index (A.U.)

55

Browning Index (A.U.)

FIGURE 4.4 Correlation between the amount of aggregated proteins and the browning index for WPI powders stored at 20
C (black symbols), at 40
C (gray symbols), and at 60
C (white symbols). Box A represents the correspondence of changes occurring in WPI powders stored 18 months at 20
C and those stored for 15 days at 40
C; Box B represents the correspondence of changes occurring in WPI powders stored for 18 months at 40
C and those stored for 15 days at 60
C.

69 months, and around 12 months storage at 40
C, respectively. Finally, the results including all storage temperatures and times showed a positive correlation (r 5 0.94) between the BI and the amount of aggregated proteins. Norwood (2017) performed a principal component analysis (PCA; Figs. 4.5 and 4.6) to draw the aging trajectory of WPI powder while taking into account the different structural modifications (e.g., protein lactosylation and aggregation, browning) and powder functional properties (e.g., heat induced aggregation) occurring upon storage. Fig. 4.6 shows the complexity of WPI powder aging, which is composed of three interdependent stages. To date, this has never been clearly stated in the literature. The first stage of WPI powder aging is represented by the cluster 2 of the PCA (represented by number 1 on Fig. 4.6). It is characterized by the increase in protein lactosylation without affecting the aggregation state of either the protein—remaining in a monomeric form—or the functional properties. The second stage of aging involves the consumption of lactosylated proteins previously formed into aggregated products (represented by number 2 on Fig. 4.6). The presence of aggregated entities in the powder significantly

4.3 Measures to Overcome Changes During Storage

FIGURE 4.5 Correlation circle of the principal components analysis.

impact on the heat-induced aggregation properties of the whey proteins after rehydration. The third and final stage reflects the progress of the Maillard reaction with total consumption of the lactosylated proteins, a high rate in aggregated proteins and a pronounced browning of the powder (represented by number 3 on Fig. 4.6). Ultimately, these changes are accompanied by a change in foaming and interfacial properties, in addition to the heat-induced aggregation properties as previously mentioned. This stage is observed for very old WPI powders which have undergone high temperature episodes. It is now well known that high-protein dairy powders represent a usedeferred strategy on a high-value market. Indeed, the market share allocated to these products has continuously increased in recent years thanks to their change of status from coproducts to ingredients themselves and by being involved in the production of many food and/or dairy products such as infant formula. Securing specifications of high-protein dairy powders proposed on the current market is therefore an issue of major importance for the international dairy industry.

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FIGURE 4.6 Drawing of the aging path of WPI powder resulting from the principal component analysis.

According to the results presented above, keys are given for monitoring the evolution of WPI powders in order to better anticipate their use after storage. From a practical point of view, a control of the free lactose content in the fresh whey protein powder could actually be helpful to predict rapidly the storage stability of the powders; the lower the free lactose content, the more stable the whey protein powders. Any decrease in the free lactose content in the powder indicates a change in the structural and/or functional properties of the powders during storage. In the absence of visible browning, the powder preserves its functional properties. These powders would either be stored at moderate temperatures (below or equal to 20
C) or for very short time at temperature slightly over 20
C. In contrast, a decrease in the free lactose content accompanied by a browning of the powder informs the powders underwent one or more periods of time at temperatures $ 40
C. Thus, characterizing the average free lactose content and the browning index of a WPI powder just after being produced, after shipments or immediately before use clearly constitute rapid and easy control points for the evaluation of the extent of powder aging. Norwood (2017) also estimated theoretical correspondences between powders stored at 40
C and 60
C for periods of time below 3 days at 60
C by considering the browning index only (Table 4.1).

4.4 Conclusions

Table 4.1 Correspondences of the Browning Index at 40
C Related to an Accelerated Storage at 60
C Storage Time at 40
C (days)

Storage Time at 60
C (days)

0 15 30 60 90

0 0.5 1.2 1.3 2.5

These results allow the prediction of WPI powder aging by accelerated storage tests defined as several days at 60
C and these under identical conditions of aw to those currently used industrially (aw of B0.23). Such correspondences constitute the framework for the building of accelerated storage stability tests for whey protein powders and their subsequent classification regarding their storage stability.

4.4

CONCLUSIONS

The shelf life of whey protein powders depends on their composition and storage conditions. Protein structural changes appear to be minor and difficult to characterize, although the complexity of whey protein powders is reduced because of their high protein content and low lactose and mineral contents. The changes concern essentially the powder aspects. Regarding protein structure, the molecular mobility is thought to be responsible for mesoscopic changes (e.g., color), for local molecular changes (e.g., protein cyclization), and also for interaction reactions with other components in the medium (e.g., polymerization reactions). These structural modifications are influenced by storage conditions including temperature, time, and moisture content. Moreover, it has been demonstrated that lactose plays a crucial role in the aging of whey protein powders even though its content is low and would thus be thought not to be a key player in the aging process of WPC powders. The higher the lactose content in whey protein powders, the higher the rate of functional changes. A low lactose content (such as in WPI) will still lead to structural changes, but with no great change in the functional properties. Regarding the lactose:protein molar ratio, the threshold is located around 1, knowing that under this value, the Maillard reactionrelated changes considerably decrease. Besides, temperature, time, and moisture content are crucial parameters to take into account in order to control the quality of powders, especially in regard to the Maillard reaction.

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Understanding the underlying mechanisms of structural changes helps to avoid or at least minimize usability issues after long-term storage. From an industrial point of view, one measure to overcome the aging-related evolutions of whey protein powders would be to monitor the Maillard reaction and the lactose state during storage. Besides monitoring, one would deal with the key influencing factors, which have been divided into two subclasses: extrinsic (e.g., temperature, humidity) and intrinsic (e.g., particle size, lactose content) factors. Finally, the extent of aging of whey protein powders can also be predicted using a simple correlation between browning index and protein aggregation.

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Morgan, F., Mollé, D., Henry, G., Vénien, A., Léonil, J., Peltre, G., . . . Bouhallab, S. (1999). Glycation of bovine β-lactoglobulin: Effect on the protein structure. International Journal of Food Science & Technology, 34(56), 429435. Moro, A., Báez, G. D., Busti, P. A., Ballerini, G. A., & Delorenzi, N. J. (2011). Effects of heat-treated β-lactoglobulin and its aggregates on foaming properties. Food Hydrocolloids, 25(5), 10091015. Morr, C. V., & Ha, E. Y. W. (1993). Whey protein concentrates and isolates: Processing and functional properties. Critical Reviews in Food Science & Nutrition, 33(6), 431476. Mulsow, B. B., Jacob, M., & Henle, T. (2008). Studies on the impact of glycation on the denaturation of whey proteins. European Food Research & Technology, 228(4), 643649. Nacka, F., Chobert, J. M., Burova, T., Léonil, J., & Haertlé, T. (1998). Induction of new physicochemical and functional properties by the glycosylation of whey proteins. Journal of Protein Chemistry, 17(5), 495503. Nijdam, J. J., & Langrish, T. A. G. (2006). The effect of surface composition on the functional properties of milk powders. Journal of Food Engineering, 77(4), 919925. Norwood, E.-A. (2017). Molecular and functional changes occurring during ageing of whey protein isolate powders. PhD thesis. Rennes: Agrocampus-Ouest. Norwood, E.-A., Chevallier, M., Floch-Fouéré, C. L., Schuck, P., Jeantet, R., & Croguennec, T. (2016). Heat-induced aggregation properties of whey proteins as affected by storage conditions of whey protein isolate powders. Food & Bioprocess Technology, 9(6), 9931001. Norwood, E.-A., Le Floch-Fouéré, C., Briard-Bion, V., Schuck, P., Croguennec, T., & Jeantet, R. (2016). Structural markers of the evolution of whey protein isolate powder during aging and effects on foaming properties. Journal of Dairy Science, 99(7), 52655272. Norwood, E.-A., Pezennec, S., Burgain, J., Briard-Bion, V., Schuck, P., Croguennec, T., . . . Le Floch-Fouéré, C. (2017). Crucial role of remaining lactose in whey protein isolate powders during storage. Journal of Food Engineering, 195, 206216. Özkan, N., Walisinghe, N., & Chen, X. D. (2002). Characterization of stickiness and cake formation in whole and skim milk powders. Journal of Food Engineering, 55(4), 293303. Patel, K. N., Modi, R., Patel, H., & Aparnathi, K. (2013). Browning, its chemistry and implications in dairy products: A review. Indo-American Journal of Agricultural and Veterinary Sciences, 1, 112. Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., Schmitt, C., & Bovetto, L. (2013). Tuning the structure of protein particles and gels with calcium or sodium ions. Biomacromolecules, 14(6), 19801989. Potier, M. (2015). The global market for infant formulas: A key market. Gira compilations, estimates and forecasts based on interviews, dairy reports and articles. Rennie, P. R., Chen, X. D., Hargreaves, C., & Mackereth, A. R. (1999). A study of the cohesion of dairy powders. Journal of Food Engineering, 39(3), 277284. Roos, Y. H. (2002). Importance of glass transition and water activity to spray drying and stability of dairy powders. Le Lait, 82(4), 475484. Roudaut, G., Simatos, D., Champion, D., Contreras-Lopez, E., & Le Meste, M. (2004). Molecular mobility around the glass transition temperature: A mini review. Innovative Food Science & Emerging Technologies, 5(2), 127134. Rullier, B., Axelos, M. A. V., Langevin, D., & Novales, B. (2009). β-Lactoglobulin aggregates in foam films: Correlation between foam films and foaming properties. Journal of Colloid & Interface Science, 336(2), 750755. Rullier, B., Axelos, M. A. V., Langevin, D., & Novales, B. (2010). β-Lactoglobulin aggregates in foam films: Effect of the concentration and size of the protein aggregates. Journal of Colloid & Interface Science, 343(1), 330337.

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Rullier, B., Novales, B., & Axelos, M. A. V. (2008). Effect of protein aggregates on foaming properties of β-lactoglobulin. Colloids & Surfaces A: Physicochemical & Engineering Aspects, 330 (23), 96102. Sadek, C., Pauchard, L., Schuck, P., Fallourd, Y., Pradeau, N., Le Floch-Fouéré, C., & Jeantet, R. (2015). Mechanical properties of milk protein skin layers after drying: Understanding the mechanisms of particle formation from whey protein isolate and native phosphocaseinate. Food Hydrocolloids, 48, 816. Saint-Jalmes, A., Peugeot, M.-L., Ferraz, H., & Langevin, D. (2005). Differences between protein and surfactant foams: Microscopic properties, stability and coarsening. Colloids & Surfaces A: Physicochemical & Engineering Aspects, 263(13), 219225. Schebor, C., Buera, M. del P., Karel, M., & Chirife, J. (1999). Color formation due to nonenzymatic browning in amorphous, glassy, anhydrous, model systems. Food Chemistry, 65(4), 427432. Schmitt, C., Moitzi, C., Bovay, C., Rouvet, M., Bovetto, L., Donato, L., . . . Stradner, A. (2010). Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment. Soft Matter, 6(19), 48764884. Schokker, E. P., Church, J. S., Mata, J. P., Gilbert, E. P., Puvanenthiran, A., & Udabage, P. (2011). Reconstitution properties of micellar casein powder: Effects of composition and storage. International Dairy Journal, 21(11), 877886. Scholl, S. K., & Schmidt, S. J. (2014). Determining the physical stability and watersolid interactions responsible for caking during storage of alpha-anhydrous glucose. Journal of Food Measurement & Characterization, 8(4), 326335. Schuck, P. (2002). Spray drying of dairy products: State of the art. Le Lait, 82, 375382. Retrieved from ,http://cat.inist.fr/?aModele 5 afficheN&cpsidt 5 14033923.. Sethumadhavan, G. N., Nikolov, A., & Wasan, D. (2001a). Film stratification in the presence of colloidal particles. Langmuir, 17(7), 20592062. Sethumadhavan, G. N., Nikolov, A. D., & Wasan, D. (2001b). Stability of liquid films containing monodisperse colloidal particles. Journal of Colloid & Interface Science, 240(1), 105112. Shrestha, A., Howes, T., Adhikari, B., Wood, B., & Bhandari, B. (2007). Effect of protein concentration on the surface composition, water sorption and glass transition temperature of spraydried skim milk powders. Food Chemistry, 104(4), 14361444. Stapelfeldt, H., Nielsen, B. R., & Skibsted, L. H. (1997). Effect of heat treatment, water activity and storage temperature on the oxidative stability of whole milk powder. International Dairy Journal, 7(5), 331339. Stephani, R., Oliveira, K., de, S., de Almeida, C. E. R., Perrone, Í. T., de Carvalho, A. F., . . . Almeida, M. R. (2017). Raman spectroscopy as a tool to identify modification of whey protein concentrate (WPC) during shelf life. Food Packaging & Shelf Life, 11, 19. Struik, L. C. E. (1977). Physical aging in plastics and other glassy materials. Polymer Engineering & Science, 17(3), 165173. Struik, L. C. E. (1978). Orientation effects and cooling stresses in amorphous polymers. Polymer Engineering & Science, 18(10), 799811. Sun, W.-W., Yu, S.-J., Yang, X.-Q., Wang, J.-M., Zhang, J.-B., Zhang, Y., & Zheng, E.-L. (2011). Study on the rheological properties of heat-induced whey protein isolatedextran conjugate gel. Food Research International, 44(10), 32593263. Thomas, M. E. C. (2004). Influence de l’activité de l’eau sur les interactions lactose / β-lactoglobuline de poudres laitières modèles lyophilisées. Vandoeuvre-les-Nancy: INPL. Retrieved from ,http:// www.theses.fr/2004INPL065N..

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Thomas, M. E. C., Scher, J., & Desobry, S. (2004). Lactose/β-lactoglobulin interaction during storage of model whey powders. Journal of Dairy Science, 87(5), 11581166. Thomas, M. E. C., Scher, J., Desobry-Banon, S., & Desobry, S. (2004). Milk powders ageing: Effect on physical and functional properties. Critical Reviews in Food Science & Nutrition, 44 (5), 297322. Tomizawa, H., Yamada, H., Wada, K., & Imoto, T. (1995). Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions. Journal of Biochemistry, 117(3), 635640. Tunick, M. H., Thomas-Gahring, A., Van Hekken, D. L., Iandola, S. K., Singh, M., Qi, P. X., . . . Tomasula, P. M. (2016). Physical and chemical changes in whey protein concentrate stored at elevated temperature and humidity. Journal of Dairy Science, 99(3), 23722383. Van Boekel, M. A. J. S. (1998). Effect of heating on Maillard reactions in milk. Food Chemistry, 62 (4), 403414. Williams, M. L., Landel, R. F., & Ferry, J. D. (1955). The temperature dependence of relaxation mechanisms in amorphous polymers and other glass-forming liquids. Journal of the American Chemical Society, 77(14), 37013707. Wnorowski, A., & Yaylayan, V. A. (2003). Monitoring carbonyl-amine reaction between pyruvic acid and α-amino alcohols by FTIR spectroscopy. A possible route to Amadori products. Journal of Agricultural & Food Chemistry, 51(22), 65376543. Wooster, T. J., & Augustin, M. A. (2007). Rheology of whey proteindextran conjugate films at the air/water interface. Food Hydrocolloids, 21(7), 10721080. Zhou, P., & Labuza, T. P. (2007). Effect of water content on glass transition and protein aggregation of whey protein powders during short-term storage. Food Biophysics, 2(23), 108116. Zhu, H., & Damodaran, S. (1994). Heat-induced conformational changes in whey protein isolate and its relation to foaming properties. Journal of Agricultural & Food Chemistry, 42(4), 846855. Zylberman, V., & Pilosof, A. M. R. (2002). Relationship between the glass transition, molecular structure and functional stability of hydrolyzed soy proteins. In H. Levine (Ed.), Relationship between the glass transition, molecular structure and functional stability of hydrolyzed soy proteins (pp. 158168). Retrieved from ,http://pubs.rsc.org/en/content/chapter/ bk9780854048663-00158/978-0-85404-866-3..

CHAPTER 5

Analytical Methods for Measuring or Detecting Whey Proteins Thao T. Le1, Di Zhao2 and Lotte B. Larsen3

1

Edith Cowan University, Joondalup, WA, Australia, 2South China University of Technology, Guangzhou Shi, China, 3Aarhus University, Aarhus, Denmark

5.1

INTRODUCTION

The term “whey proteins” refers to a series of globular proteins, which are more soluble than caseins in milk. Whey proteins largely originate from whey, the coproduct of cheese-making in the dairy industry; this whey is designated sweet whey. Another whey type is acid whey from acid precipitation of the caseins and from manufacture of acid cheeses and Greek-style yogurt. Whey proteins also comprise the serum fraction of milk from which the proteins are produced by ultracentrifugation of skimmed milk, where casein micelles are sedimented. The whey proteins include β-lactoglobulin (β-Lg) (50%
55%), α-lactalbumin (α-La) (20%
25%), bovine serum albumin (BSA) (5%
10%), immunoglobulins (Ig), lactoferrin (Lf) and lactoperoxidase (LP), and several minor fractions, whose detection will depend on the used method, separation, sensitivity, and specificity (Marshall, 2004). Whey from rennet cheesemaking also includes glycomacropeptide (GMP) (up to 20% w/w) which is released into the whey when rennet (chymosin) cleaves κ-casein (κ-CN) into soluble (GMP) and insoluble (para-κ-CN) (Neelima, Rajput, & Mann, 2013). The whey proteins also include a number of enzymes (Fox & Kelly, 2006a, 2006b) (see Chapter 1 for an overview of whey proteins). Whey proteins are commonly available in the market as whey protein concentrate (29%
89% protein), whey protein isolate (90% protein), and whey protein hydrolysate (semidigested protein) (Patel, 2015). Whey protein provides an essential amino acid profile, which fulfills all the qualitative and quantitative requirements established by Food Agriculture Organization (FAO) and Word Health Organization (Millward, Layman, Tome, & Schaafsma, 2008; Sindayikengera & Xia, 2006). Whey proteins have a wide application as a nutritional ingredient in food and beverages and, in Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00005-9 © 2019 Elsevier Inc. All rights reserved.

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particular, are an excellent choice for body builders, elite athletes, and those whose health is compromised (Bawa, 2007; Buckley et al., 2010; Ha & Zemel, 2003; Tipton et al., 2004) (see also Chapters 9 and 16). Additionally, whey proteins serve as a crucial ingredient for food products to impart functional characteristics, including emulsification, thermal stabilization, foam formation, and gelation (Jambrak, Mason, Lelas, Herceg, & Herceg, 2008; Sobhaninia, Nasirpour, Shahedi, & Golkar, 2017; Spotti et al., 2014). Ig and other minor whey fractions such as Lf, LP, and insulin-like growth factor have been found to possess passive immunity, anticancer, or antimicrobial bioactivity (Farnaud & Evans, 2003; Gill & Cross, 2000; Meisel, 2005). GMP also has both biological (immunoregulatory, prebiotic, antimicrobial) and functional (foaming, emulsifying) properties (Brody, 2000; Neelima et al., 2013). Moreover, bioactive peptides from hydrolysates of whey protein have been extensively documented. For example, several studies found that the peptides such as β-lactophorin, α-lactophorin, and lactoferricin exhibit angiotensin-converting-enzyme (ACE) inhibition, ileum stimulation, and antimicrobial bioactivity (Korhonen, 2006; Meisel, 2005; Pan, Cao, Guo, & Zhao, 2012). Recently, micro-, submicro- and nanocapsulated whey proteins or their hydrolysates have been developed to convey hydrophobic molecules such as β-carotene, anthocyanins, and avocado oil (Bae & Lee, 2008; Betz & Kulozik, 2011; Wang, et al., 2015). (See Chapter 14 for further information on bioactive peptides.) All this information indicates extensive applications of whey proteins in food, medical, and nanoscience, transforming the “gutter” product in cheesemaking into a “gold” product (Smithers, 2008). Therefore, establishing a series of suitable analytical methods for detecting whey proteins qualitatively and quantitatively raises special interest. Traditional methods including gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), Western or immune blotting, capillary electrophoresis (CE), and liquid chromatography (LC) have been widely used for protein identification (Block et al., 2003; Bordin, Raposo, de la Calle, & Rodriguez, 2001; Larsen, Benfeldt, Rasmussen, & Petersen, 1996). Furthermore, as some whey proteins are enzymes they have also been detected by activity assays, which are often relatively more sensitive than the abovementioned traditional detection methods for proteins. One-dimensional gel electrophoresis (1-DGE) is very commonly used but it lacks sufficient resolution to resolve complex biological matrices. 2-DGE has excellent resolving power but identification of individual spots still requires mass spectrometry (MS) for unambiguous identification; however, in some cases when well-known samples are analyzed and the 2-DGE method is well-established in the laboratory, it is possible to identify spots on the gels by their position by referring to earlier published studies. ELISA is highly specific for particular proteins, but its quantification still presents

5.1 Introduction

some challenges. If a specific antibody is available, Western blotting may be a good choice, as it provides both a unique detection of a protein and an estimate of its molecular mass, derived from its mobility in the gel. The specificity and usefulness of enzyme-based assays depend on the available substrates and the actual assay conditions such as pH and salt concentration which need to be optimized. In addition to the major forms of the whey proteins, genetic variation and posttranslational modifications (PTM) such as phosphorylation, glycosylation, oxidation, disulfide bond formation, deamidation, and proteolysis challenge the traditional analytical methods of protein detection. Until now, three α-La genetic variants (A, B, C) and 11 β-Lg variants (A, B, C, D, E, F, G, H, I, J, W) have been described (Caroli, Chessa, & Erhardt, 2009; Farrell, et al., 2004). Heat treatments during food processing result in further modifications of whey proteins, including glycation at lysine (Lys) and arginine (Arg) residues, oxidation at methionine (Met), disulfide bond formation between cysteine (Cys) residues and between Cys and cystine (Cys2) residues via thiol-disulfide bond interaction, and deamidation at asparagine (Asn) and glutamine (Gln) residues (Arena, et al., 2010, 2011; Le, Deeth, & Larsen, 2017). These modifications change the molecular weight, isoelectric point (pI), structure, and properties of the protein, thus, depending on analysis principle, the traditional analytical methods may face difficulties in identifying and characterizing the modified protein. Notably, cross-linked structures (e.g., methylglyoxal-derived lysine dimer (MOLD), glyoxal-derived lysine dimer (GOLD), pentosidine and lysinoalanine) resulting from heat treatments and Maillard reaction (MR) products induce aggregation of proteins and thus present even more obstacles in whey protein identification (Glomb & Lang, 2001; Le, Bhandari, Holland, & Deeth, 2011; Lederer & Klaiber, 1999). Proteomics, with the combined application of separation methods, advanced MS, enzymatic hydrolysis, and establishment of databases with protein sequences including commonly-encountered modifications, has recently been developed to overcome the limitation of traditional methods (Arena et al., 2010, 2011; Le et al., 2016; Milkovska-Stamenova & Hoffmann, 2016). Proteomics is an efficient way of identifying and quantifying proteins, including their genetic variants and posttranslational-modifications. Peptide fragments of whey proteins, which are difficult to sequence by traditional methods due to their small size, can also be efficiently analyzed by highthroughput MS methods (Le et al., 2016). In this chapter, we present an overview of analytical methods, including their pros and cons, that have been applied to both native and modified whey proteins. Immunologic, gel-based, chromatographic, and proteomics methods are

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included with the aim of providing information to enable the reader to choose the appropriate tool for the identification and quantification of specific whey proteins or mixtures thereof.

5.2 PROCESS-INDUCED MOLECULAR CHANGES AFFECTING THE ANALYSIS OF WHEY PROTEINS Whey proteins account for B20% of the total milk protein and include the major proteins, α-La, β-Lg, BSA, and Ig, and a range of minor proteins (Table 5.1). The quality of whey proteins is considered to be of the highest standard due largely to the presence of bioactive peptides, essential and branched-chain amino acids, such as leucine, isoleucine, and valine, which play a role in metabolism and glucose homoeostasis (Patel, 2015) (see Chapters 14
17 for further information on the nutritive and therapeutic aspects of whey proteins). In addition, sulfur amino acids such as Cys and Met provide whey proteins with great antioxidant potential (Smithers, 2008). The presence of multiple Cys and Cys2 residues in whey protein sequences leads to the native forms being denatured and aggregated during thermal processing (Wijayanti, Bansal, & Deeth, 2014); in this chapter whey proteins are classified into two subgroups: native and denatured/modified. The secondary and tertiary structures of native whey proteins greatly depend on the number and positions of Cys residues, and their participation in S
S bond formation (inter- and intramolecular). Therefore whey protein denaturation/aggregation is related to the interaction of free S
H and S
S bonds of proteins containing Cys and Cys2 residues (e.g., β-Lg, α-La, BSA, κ-CN, and αS2-casein (αS2-CN)) (Foegeding, Davis, Doucet, & McGuffey, 2002; Wijayanti et al., 2014). During heat treatment of milk, β-Lg may be linked with κ-CN and αS2-CN via thiol
disulfide exchange reactions, thereby increasing heat stability and inhibiting the precipitation of heat-denatured β-Lg (Singh, 1991). Unlike the intrinsic disordered structure of β-CN and αs1CN due to numerous proline residues and no disulfide bonds (Creamer, Richardson, & Parry, 1981; Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014), the rigid globular structure of α-La and β-Lg could make them more resistant to proteolysis (Qin et al., 1998; Takagi, Teshima, Okunuki, & Sawada, 2003). In summary, knowledge of whey protein denaturation/aggregation processes is extremely useful for minimizing potential negative practical consequences and for improving functional properties of whey protein ingredients in many food applications. Depending on the analytical method or detection system used, these molecular changes will affect their detection and need to be taken into account, e.g., in the MS-based methods.

Table 5.1 Characteristics of Whey Proteins and Methods Suitable for Their Measurement or Detection

Protein

Proportion of Total Whey Protein (%, w/w)

Concentration in Whey (mg/L)

Molecular Weight (kDa)

Isoelectric Point

References

β-Lg

50
55

3000

18.3

4.9
5.4

Farrell et al. (2004)

α-La

20
25

1200

14.2

4.8

Farrell et al. (2004)

IgG, IgA, IgM BSA

10 1
6

100
1000 100
400

146.0
970.0 66.2

5.5
8.3 4.8

Farrell et al. (2004) Farrell et al. (2004)

GMP Lf

0
15b 2
3

0
900 150

7.0 80.0

, 3.8 8.8

Farrell et al. (2004) Turner and Thompson (2007)

LP

0.5

30c

78

9.6

Kussendrager and van Hooijdonk (2000)

Osteopontin

0.3

18c

60
75

3.5

Plasminogen/ plasmin

0.006

0.3

85/50

6/7

Bissonnette, Dudemaine, Thibault, and Robitaille (2012), Christensen and Sørensen (2016) Benfeldt et al. (1995)

Procathepsin D/cathepsin D

0.005

0.3

45/39

6.6/6.4

Larsen et al. (1996)

Lysozyme

0.002

0.13c

18

11

Carlsson and Björck (1987)

Examples of Methods of Measurement or Detectiona 1 and 2-DGE, ELISA, Western blotting, column chromatography/A280, N-terminal sequencing, LC-MS, MALDI TOF MS, LC-MS 1 and 2-DGE, ELISA, Western blotting, column chromatography/A280, N-terminal sequencing, LC-MS, MALDI TOF MS, LC-MS 1 and 2-DGE, MALDI TOF MS, LC-MS 1 and 2-DGE, ELISA, Western blotting, column chromatography/A280, N-terminal sequencing, LC-MS, MALDI TOF MS, LC-MS 1 and 2-DGE, LC-MS, LC-MS 1 and 2-DGE, ELISA, column chromatography/A280, N-terminal sequencing, MALDI TOF MS, LC-MS 1 and 2-DGE, column chromatography/ A280, N-terminal sequencing, MALDI TOF MS 1 and 2-DGE, Western blotting, column chromatography/A280, N-terminal sequencing, MALDI TOF MS

ELISA, Western blotting, activity assays, chromatography/A280, N-terminal sequencing ELISA, Western blotting, activity assays, chromatography/A280, N-terminal sequencing Activity assays

a Not exhaustive, but showing examples of detection methods used to illustrate connection between protein characteristics, concentration, and possible detection or measurement method. b Only present in cheese whey, not in acid whey or ultracentrifuged milk serum. c Concentration in milk.

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Whey proteins in milk or whey products are easily chemically modified due to the components of these products (especially protein and lactose) and the thermal treatments used to extend their shelf-life. Nonenzymatic lactosylation (the reaction of lactose with the ε-NH2 in Lys and/or the guanidyl group of Arg) of proteins, the first stage of the MR, is one of the most dominant chemical reactions of whey proteins, both in heat-treated liquid milk and in powder products (Table 5.2). Furosine, which is not present in milk and is formed by acid hydrolysis during analysis, is well-known as an indicator for the first-stage MR (Erbersdobler & Somoza, 2007). However, advanced MS not only detects lactosylation occurring in the protein, but also characterizes lactosylated sites, allowing the direct measurement of products (lactosylated peptides/proteins) of the MR (Section 2.3.4). MR occurs more rapidly in the dry state (i.e., in powders) than in the liquid state of milk products; this could be due to the proximity of the reactants. Table 5.2 illustrates the extent of lactosylation occurring in pasteurization, ultrahigh-temperature (UHT), and spray-drying processes; much higher degrees of lactosylation occur in the last two processes. The lactosylation of Lys blocks trypsin hydrolysis of milk proteins (Dalsgaard, Nielsen, & Larsen, 2007; Deng, Wierenga, Schols, Sforza, & Gruppen, 2017). Lactosylation may also reduce the release of biologically active peptides such as the antimicrobial peptides, ACE inhibitor β-lactophorin, and opioid peptides derived from α-La or β-Lg (Siciliano, Mazzeo, Arena, Renzone, & Scaloni, 2013). Proteins with advanced glycation are more resistant to proteolysis, which may result in increased allergenicity of whey protein hydrolysate where formation of such advanced glycations have occurred (Rahaman, Vasiljevic, & Ramchandran, 2016). Overall, many studies have shown the effects of MR and its adducts on the digestibility of milk proteins, although most were investigated in model experiments and few were tested in animal or clinical trials. Whey proteins can also be modified by deamidation. Amide groups are converted to carboxyl groups causing an increased negative charge on the protein, thus reducing its pI. The rate of deamidation depends on pH, neighboring amino acid residues, and the higher order structure of the unfolded protein (Sarioglu, Lottspeich, Walk, Jung, & Eckerskorn, 2000). The most common sites for deamidation within protein sequences in order of abundance are Asn-Gly . Asn-Ser .Asn-Ala (NG . NS . NA) occurring on the asparagine (Asn). Bovine β-Lg has five Asn residues, but only one N-S motif (N119-S120) in variant A or two likely deamidation sites (N63-G64 and N119-S120 motifs) in variant B (Holland, Gupta, Deeth, & Alewood, 2012). Deamidation has been shown to occur at position N63 in both variants A and B of β-Lg in acidic whey protein isolate drinks during storage, as assessed by 2-DGE and matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS (Le et al., 2016). In addition, iso-Asp derived from

5.3 Analytical Methods

Table 5.2 Lactosylation Sites of Whey Proteins (K 5 lysine) Whey Protein

UHT Milk

Milk Powder

K ,K ,K ,K ,K ,K , K100, K135, and K138 residues of β-Lg No lactosylation sites of α-La were indentified No lactosylation sites of BSA were indentified

All K residues in β-Lg

All K residues in β-Lg

All K residues in α-La except for K94 and K108 All K residues of BSA except for K180, K187, K239, and K535

Lf

K243, K313, K522, and K608 residues of Lf

LP

K299 residues of LP

All K residues of LTF except for K221, K269, K273, K329, and K633 K173, K308, K428, K479, and K556 residues of LP

K58, K114 and K122 residues in α-La All K residues of BSA except for K159, K242, K362, K388, K556, and K563 All K residues of LTF except for K28, K151, K243, K263, K452, and K454 K308, K428, K479, and K563 residues of LP

β-Lg α-La BSA

Pasteurized Milk 47

60

75

77

83

91

From Arena, S., Renzone, G., Novi, G., Scaloni, A., 2011. Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures. Proteomics, 71, 2453
2475.

Asn deamidation could lead to the loss of bioactivity of a protein and change its susceptibility to proteolysis (Girardet et al., 2004). Indications of acidinduced hydrolysis of whey proteins, in combination with deamidation, have also been reported (Le et al., 2016). Here, the heat-induced deamidation leads to an increased amount of Asp and Glu residues produced from Asn and Gln, respectively, forming the basis of acid-induced hydrolysis of α-La, β-Lg, and caseinomacropeptide (CMP). Moreover, enzymatic hydrolysis has been applied to produce whey protein hydrolysate, which has significant nutritional and functional properties (see also Chapters 9 and 14). The product has high heat stability, bioactive activity, and reduced allergenicity (Foegeding et al., 2002). Thus, investigation of the functional consequences of protein modifications can lead to many positive outcomes, as well as introducing novel whey protein ingredients onto the market. Furthermore, it underlines the necessity of sensitive detection and quantification methods for both native and modified whey proteins.

5.3 5.3.1

ANALYTICAL METHODS Immunological Techniques

The immunological methods include ELISA, Western blotting, immunoprecipitation, as well as more advanced combination methods, e.g., immunoprecipitation with MS detection. ELISA is a widespread method, normally applied in a sandwich detection of analyte protein between two antibodies,

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which may be similar or different, dependent on epitope recognition. If a specific antibody is available, ELISA is a very reliable method for detecting and quantifying proteins, including low-abundance proteins in whey, such as enzymes. For example, ELISA using polyclonal antibodies was used to determine levels of the proteolytic enzymes cathepsin D and plasmin, and their pro-forms (zymogens or proenzymes) in whey (Larsen et al., 1995, 1996) (Table 5.1). Immunoblotting or Western blotting is another versatile method, again given specific antibodies are available. Whereas by ELISA it is difficult to ascertain if cross-reactions occur due to the nonspecificity of the used antibody, by Western blotting the molecular masses can be obtained, thereby giving additional useful information on cross-reactivity and presence of multiple forms as well as the mass of the parent epitope. Commercial antibodies may be available for some bovine proteins, or eventually for human counterparts, but then it is necessary to check for cross-reactivity, as in some instances the structural differences are too large for epitopes to be recognized. For example, minor milk proteins, including enzymes, may be purified originally from a bovine source, e.g., plasminogen from bovine milk (Benfeldt, Larsen, Rasmussen, Andreasen, & Petersen, 1995), or, alternatively, a bovine commercial protein may be available which can then be further purified by preparative chromatography, and then used for immunization to produce antibodies for immunological analyses, e.g., cathepsins D and B (Larsen et al., 1996; Magboul, Larsen, McSweeney, & Kelly, 2001). In Western blotting, the steps are first electrophoresis, in one or two dimensions, followed by electroblotting onto membranes, where polyvinylidene difluoride membranes are commonly used, due to their low nonspecific binding and stability. After blotting, the membranes are washed and incubated with primary and secondary antibodies, where the primary antibody is the recognizing one, and the secondary antibody recognizes the primary antibody and is linked to a reporter enzyme that enables detection after enzymatic reaction to detect the reactive electrophoretic bands. This method has been used for the detection of various molecular forms of proteolytic enzymes in whey, such as the various forms of cathepsin D; procathepsin D, pseudocathepsin D, mature single-chained and mature two-chained cathepsin D (Larsen & Petersen, 1995), due to the high resolution power of the electrophoresis coupled with the specificity of the antibody. Western blotting is thus very powerful for discrimination of different molecular forms of the target protein; e.g., to follow activation by proteolytic cleavage such as during purification. Immunoprecipitation, eventually in combination with MS detection of precipitates, is an alternative method for detection, especially of minor whey

5.3 Analytical Methods

proteins or protein complexes when suitable antibodies are available. This has been used for the study of proteins complexing with Lf (Sokolov, Zakahrova, Kostevich, Samygina, & Vasilyev, 2014), and for identification of prolactin-binding protein in human milk (Kline & Clevenger, 2001). It has been an especially valuable tool for studying protein complexes in biological processes, but, in combination with MS, may have greater potential, e.g., in the study of heat-induced protein complexes.

5.3.2

Activity Based/Enzymatic Assays

More than 60 enzymes or enzymatic activities have been detected in bovine milk, several of which are present in the whey fraction (Fox & Kelly, 2006a, 2006b). The number is increasing as the limit of detection and methods are refined to reveal new enzymatic components of milk and whey, e.g., by proteomic methods. Traditionally enzymatic methods have been, and still are, powerful and versatile methods for the detection of enzymatic activities in milk and milk products. Furthermore, analytically, these methods are very useful for following the progress of enzymes during their isolation or fractionation and other processes. The enzymatic methods reflect the enzyme class, and furthermore, in contrast to methods such as Western blotting, are dependent on the presence of enzymatically active molecules, and not only on their presence as proteins. Therefore, combinations of different methods are very useful. However, detection of molecules in whey solely by their enzymatic activity does not provide unequivocal identification; this contrasts with, e.g., N-terminal sequencing and MS-based methods, which provide unique molecular identifications. Among the enzymes present in milk, the proteolytic, lipolytic, and oxidative are the most important (Fox & Kelly, 2006a, 2006b). The proteolytic enzymes are detected by their activity on either protein or peptide substrates, the latter normally being linked to chromophores or fluorophores which enables the detection of the products after hydrolysis; alternatively, the reaction products can be detected by high-performance liquid chromatography (HPLC). An important and well-known whey enzyme is LP (Table 5.1), one of the most abundant enzymes of bovine whey, which is assessed by its oxidative activity. LP activity of the milk samples can be analyzed by a 2,20 azino-di[3ethylbenzthiazoline-6-sulphonic acid] (ABTS) method (Turner & Thompson, 2007). LP contains one iron atom per molecule (0.07% w/w) (Kussendrager & van Hooijdonk, 2000), and gives a reddish color during purification, allowing its migration during isolation to be monitored by preparative chromatography (Fig. 5.1) (see also Chapter 1).

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2.6

α-LA + BSA

LP LF 1.4

β-LG

0.6 0.4

0.10

1.0

M AmAc

1.8

UNITS

2.2

A280 (—)

164

0.2 0.6

0.05

0.0

0.2 0

50

100

150

200 Fractions

0.00 250

FIGURE 5.1 Ammonium sulphate precipitated whey proteins separated on a column of DEAE-Sepharose. The proteins were separated by a gradient of ammonium acetate, pH 5.0. They were monitored by absorbance at 280 nm (A280). LP, Lf, α-La, BSA, and β-Lg designate lactoperoxidase, lactoferrin, α-lactalbumin, bovine serum albumin, and β-lactoglobulin, respectively. Simultaneously, cathepsin D was monitored in units of TCA-soluble peptides released from hemoglobin in an activity assay (L. B. Larsen, unpublished results; Larsen et al., 1993).

Lysozyme, being a widespread enzyme in biological secretions, is normally assessed by its enzymatic activity against peptidoglycans of Gram-positive bacteria (Carlsson & Björck, 1987). Its activity can be determined by a turbidometric method based on the decrease in turbidity at 600 nm. Lysozyme specifically catalyzes the cleavage of the glycosidic linkage between the C-1 of N-acetylmuramic acid and the C-4 of N-acetyl glucosamine in the peptidoglycan component of bacterial cell walls (Fox & Kelly, 2006b), causing leakage of the cell’s interior components (lysis) (see also Chapter 1). The proteolytic enzymes in whey comprise an (increasing) list of proteases, including endopeptidases (proteinases), as well as some exopeptidases (amino- or carboxypeptidases), some of which have not been fully identified. (Kelly, O’Flaherty, & Fox, 2006). The major proteolytic system in bovine milk is the plasmin/plasminogen system (Bastian & Brown, 1996). While most plasmin and plasminogen are associated with the casein micelle, a small amount occurs in the milk serum and even more in whey, especially

5.3 Analytical Methods

when produced by acid precipitation of casein (Benfeldt et al., 1995; Hayes & Nielsen, 2000) (see also Chapter 1). A recent improved spectrophotometric method for the determination of plasmin and plasminogen in turbid samples of milk or dairy products extracts, like cheese, have been reported (Rauh et al., 2014). Previous methods for measurement of plasmin and plasminogen-derived activities were widely based on measurements in skimmed milk, and therefore have included a centrifugation step to remove the cream or have not specified the fat content of the analyzed samples. Proteolytic enzymes that have been identified in milk by enzymatic assays include the cathepsin D/procathepsin D system (Larsen, Boisen, & Petersen, 1993) (see also Chapter 1). Both synthetic peptide substrates and protein substrates have been used (Larsen et al., 1993; Larsen et al., 2006), with hemoglobin being a very useful substrate for cathepsin D, mimicking its natural substrate in organisms. Fig. 5.1 shows the activity profile of indigenous cathepsin D, in units of TCA-soluble peptides released from bovine hemoglobin at pH 3.5, during separation of whey proteins by preparative DEAE anion exchange chromatography. The cathepsin D activity is mainly present in the first peak, coeluting with Lf and LP (L. B. Larsen, unpublished result; Larsen et al., 1993). This illustrates the usefulness of monitoring enzyme activity of minor whey proteins during preparative purification, and the use of enzymatic activity units for the quantification of enzyme level.

5.3.3

Gel Electrophoresis

Proteins can be separated by electrophoresis solely on the basis of their molecular weight (Mw) (one-dimensional electrophoresis, 1-DGE) or on both Mw and their pI (two-dimensional electrophoresis, 2-DGE). In the first dimension of 2-DGE, immobilized pH gradient (IPG) strips are usually used. The introduction of IPG strips (Görg, Obermaier, Boguth, & Weiss, 1999) increased the use of 2-DGE as a large amount of sample is able to be absorbed on IPG strips, a prerequisite for efficient identification of lowabundance proteins by MS (Garbis, Lubec, & Fountoulakis, 2005). The use of wide pH gradients up to pH 12 allows longer separation distances and simplification of electrophoresis procedures (Görg et al., 1999). The combined technique of isoelectric focusing at pH 3
10 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has allowed the separation of genetic variants of casein and whey proteins as well as isoforms caused by PTM such as phosphorylation and glycosylation (Holland, Deeth, & Alewood, 2004, 2006). By separating proteins in both dimensions, the problem of resolving proteins of similar Mw or pI is overcome; this is not possible in 1-DGE, as shown in Fig. 5.2, where separation of whey proteins by both 1- and 2-DGE are shown. 2-DGE has also been used to analyze

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FIGURE 5.2 1D PAGE (A) and 2D (B) PAGE separation of proteins in raw, sweet whey with added chymosin. (A) Sweet whey proteins were analyzed by 1D PAGE (nonreducing) and stained with Coomassie Brillant Blue. (B) The same sample was analyzed by 2D PAGE, under reducing conditions, and stained with silver staining. Protein bands or spots were identified by MALDI-TOFMS (L. B. Larsen, unpublished data). FABH: Fatty acid binding protein; NPC-2: Niemann Pick protein C2, Ig HC, Immunoglobulin G heavy chain, IG LC, Immunoglobulin G light chain.

differences between types of whey prepared from raw milk; sweet whey prepared by addition of chymosin to raw skimmed milk, acidic whey prepared by isoelectric precipitation of the caseins, as well as milk serum prepared by ultracentrifugation and recovery of the supernatant (Jensen, Poulsen, Møller, Stensballe, & Larsen, 2012). The 2-DGE analysis revealed several differences between the whey types; a proteomic approach using MS methodologies of the separated spots was used to identify the range of spots, which differed between the types, including CN cleavage products present in the sweet whey. Normally, 2-DGE is carried out using reducing agents in both dimensions, but it is possible to combine reducing and nonreducing conditions in the two dimensions. This was applied for visualization of disulfide-linked complexes present in sweet whey made from low-pasteurized milk (Larsen, Wedholm-Pallas, Lindmark-Månsson, & Andrén, 2010) and of changes in milk introduced as a result of heat treatment at 90oC for up to 3 min % (Chevalier, Hirtz, Sommerer, & Kelly, 2009). In addition, 2-DGE coupled with MS, a major separation technique in proteomics, reveals more characteristics of proteins in gel spots. For example, the C-terminal truncated forms of β-Lg in whey from cows’ milk have been determined by 2D-PAGE coupled with MALDI-TOF-MS (Zappacosta, Di Luccia, Ledda, & Addeo, 1998).

5.3 Analytical Methods

Le et al. (2016) applied 2-DGE with LC-ESI-MS/MS to separate multiple isoforms of GMP present in acidic whey drinks. Le, Deeth, Bhandari, Alewood, and Holland (2012) were able to quantify lactosylation levels of whey proteins in stored milk powder by applying image analysis of 2D gels. 2D gels coupled with LC-MS/MS have been used for monitoring host-defense-related response in bovine milk during mastitis (Smolenski, Broadhurst, Stelwagen, Haigh, & Wheeler, 2014), where the quantitative response was validated by Western blotting. Thus, 2-DGE is a good technique for separating, detecting, and relatively quantifying protein isoforms such as genetic variants and PTM (Manso, Léonil, Jan, & Gagnaire, 2005).

5.3.4

Capillary Electrophoresis

Compared with traditional electrophoresis, CE analysis is faster and require less solvent and manpower. Protein samples are separated in capillaries in which less heat is produced; the higher resolution than in traditional electrophoresis has been attributed to this difference (Patrick & Lagu, 2001). However, one major drawback of using CE for protein analysis is the adsorption of proteins onto the capillary wall, mainly due to interactions between negatively changed silanol groups of the silica surface of the capillary and positively charged protein regions. To reduce these undesirable interactions, coated capillaries are often used together with polymeric additives in the separation buffer (Cifuentes, De Frutos, Santos, & Diez-Masa, 1993; Recio & Olieman, 1996). Another obvious difference between CE and traditional electrophoresis is that a detector such as UV, diode array detector (DAD), or mass spectrometer can be coupled to the CE analysis of proteins (Patrick & Lagu, 2001). Therefore, CE is usually regarded as an alternative to column separation (see also Section 2.3.5), and not to gel electrophoresis. Regarding whey proteins, several CE methods have been reported to separate, and in some cases, quantify the major components (β-Lg, α-La, BSA, and Ig) (Cartoni, Coccioli, Jasionowska, & Masci, 1999; Herrero-Martínez, Simó-Alfonso, Ramis-Ramos, Gelfi, & Righetti, 2000; Miralles, Ramos, & Amigo, 2003). In addition, CE is an efficient way of quantifying changes in individual whey proteins, e.g., by MR, which changes the pI of whey proteins and thus can be distinguished by CE (De, Merchiers, & Rvan, 1998), or degree of denaturation of whey proteins as a result of heat treatment (Ardö et al., 1999). Proteolysis of whey proteins detected by CE using a coated fused-silica capillary column was reported by Miralles et al. (2003). Moreover, a sensitive analysis for β-Lg and α-La was developed using immunoaffinity CE (with antibody magnetic beads inside) combined with MALDI TOF MS (Gasilova, Gassner, & Girault, 2012).

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5.3.5

Liquid Chromatography

LC has been the method of choice for separating and measuring whey proteins, including their various isoforms due to genetic variation, posttranslational modification, and processing, in complex matrices such as raw milk, whey streams, and powders. The application of LC in whey protein separation and analysis can be divided into preparative and analytical chromatography.

5.3.5.1 Preparative Chromatography Numerous separation techniques and/or devices such as chromatography, precipitation, centrifugation, and membrane filtration have been applied to isolate, separate, and purify whey proteins from complex milk matrices, allowing a high quality whey product to be produced. However, whey proteins are prone to denaturation during precipitation and membrane separation processes. Ultrafiltration is unable to completely remove lactose and hence is also unable to be used for the isolation of pure proteins (Gerberding & Byers, 1998). In contrast, LC provides a promising alternative with high efficiency and a small degree of whey protein denaturation. Ion exchange chromatography (IEC) is among the most commonly used LC techniques in the fractionation of whey proteins (Fig. 5.1), and exploits the differences in pI of the individual whey proteins (Table 5.1). The pI of a protein is used to predict the protein’s behavior during IEC separation, based on the assumption that a protein will not be retained at its pI, but retained by anion exchange resins at pH above its pI and by cation exchange resins below its pI (Ali, Aboul-Enein, Singh, Singh, & Sharma, 2010). Therefore, pH can be easily adjusted to alter the surface charges according to the separation requirements. The pIs of β-Lg and α-La (two major fractions) are 4.9
5.4 and 4.8, respectively, and are therefore in the acid range. Thus, anionexchange chromatography is commonly used at neutral pH to obtain purified β-Lg and α-La. Both weak (DEAE) and strong (Mono Q) anion-exchange materials have been utilized in the purification of β-Lg and α-La (Santos, Teixeira, & Rodrigues, 2012; Stojadinovic et al., 2012). Application of strong cation-exchange (HiTrap SP) chromatography was reported in the purification of these two proteins at acidic pH, but the possibility of denaturation and acid hydrolysis should be considered during this process (El-Sayed & Chase, 2010). In addition, GMP (pI ,3.8) was reported to be separated by anion-exchange chromatography (DEAE-Sephacel) from sweet whey at pH 2.0
4.5 (Nakano & Ozimek, 2000). Lf and LP, representing high-value whey fractions, are mostly purified using cation-exchange chromatography, due to their high pIs (8.8 and 9.6) as shown in Table 5.1. Carboxymethyl, sulfopropyl, and methacrylate copolymer cation-exchange chromatography have also been used for the isolation of these minor whey protein fractions (Fee & Chand, 2006; Hahn, Schulz, Schaupp, & Jungbauer, 1998). A combined

5.3 Analytical Methods

cation- and anion-exchange chromatography procedure has been used to separate a range of whey proteins (Pedersen, Mollerup, Hansen, & Jungbauer, 2003; Voswinkel & Kulozik, 2011; Ye, Yoshida, & Ng, 2000). In this process, chromatography using a strong anion-exchanger (Q-MA) was carried out at pH 7.0 to separate BSA and β-Lg in the first step, followed by use of a strong anion-exchanger to isolate Ig, Lf and LP; eventually α-La was collected in the elution after these two processes (Voswinkel & Kulozik, 2011). Gel permeation chromatography (GPC), also called size-exclusion chromatography (SEC) or gel filtration (GF), and reversed-phased chromatography (RPC) which separate proteins by their molecular size/weight and hydrophobicity, respectively, are also used for whey protein fractionation. Due to the varying molecular weights of whey proteins as shown in Table 5.1, GPC can efficiently separate those whey fractions with significant differences in mass (Liang, Chen, Chen, & Chen, 2006). It is a particularly useful tool when analyzing proteins in their native forms is important, as the buffers used for GPC are normally nondenaturing and do not contain organic solvents. Where the separation power of GPC is insufficient, combinations with other kinds of chromatography are advisable. A combination of IEC and GPC was reported for the isolation of α-La (14.2 kDa), β-Lg (18.3 kDa), and BSA (66.3 kDa) (Neyestani, Djalali, & Pezeshki, 2003). With regard to RPC, it has been shown that the most hydrophobic protein from whey is α-La, followed by BSA, β-Lg, and Lf, obtained by using a hydrophobic (octyl) column (Santos, Teixeira, & Rodrigues, 2011). Currently, whey proteins are a coproduct in the production of cheese and casein/caseinate. Considering the nutritional and biological significance of whey protein fractions, centralized fractionation of whey proteins on an industrial scale represents an economically attractive supplement to the principal dairy products. For this to occur, a “lab to industry” transformation, with extensive upgrade in LC equipment is required. A combination of LC and other separation methods such as precipitation, centrifugation, and membrane separation technologies can both improve efficiency and reduce the cost of fractionation of whey proteins. Notably, ion exchange membrane chromatography and simulated moving-bed technology have been applied in the fractionation of whey proteins (Andersson & Mattiasson, 2006; Bhattacharjee, Bhattacharjee, & Datta, 2006; Goodall, Grandison, Jauregi, & Price, 2008). Compared with column chromatography, IEC provides higher separation efficiency, and is more suitable for industrial up-scaling (Drioli & Romano, 2001). Kim, Choi, and Row (2003) reported the successful separation of α-La, BSA, and β-Lg using an anion-exchange membrane. Introduction of simulated moving-bed technology was found to raise productivity by 48%, to increase target protein concentration 6.5 times, and to reduce consumption of buffer 4.3 times, in the separation of Lf and LP (Andersson & Mattiasson, 2006).

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5.3.5.2 Analytical Chromatography HPLC alone: HPLC is the most widely used separation method and is wellknown for its capability in whey protein analysis. Several separation modes such as RP, IEC, and SEC can be used in HPLC to analyze proteins of any Mw, or with strongly acidic or basic properties (O’donnellO’Donnell, Holland, Deeth, & Alewood, 2004). IEC and SEC are mainly used for preparative whey protein isolation as discussed above. However, RP has been widely applied in detection and quantification of whey proteins. In the normal (not RP) mode, HPLC separates proteins by their polarity; under high pressure the proteins are adsorbed onto a stationary phase of a column packed with small polar particles (e.g., silica). Polar parts of the proteins interact with the polar surface of the column and the most polar proteins are eluted latest by the mobile phase. However, in the RP mode, as its name indicates, the order of elution of proteins is reversed because a stationary phase of low- or nonpolar material, such as compounds with a long hydrocarbon chain (e.g., C18), is applied to the column surface. This makes the column material more hydrophobic, resulting in shorter retention times for the more polar proteins, while the hydrophobic molecules, are retained longer, enabling their separation by manipulation of buffer gradients. RP-HPLC was firstly recommended by Parris and Baginski (1991) for the assessment of the degree of whey protein denaturation (mainly α-La and β-Lg). Various studies have been carried out to quantify whey proteins in raw or pasteurized milk and in sweet whey using RP-HPLC (Elgar et al., 2000). Depending on the whey proteins of interest, different columns, such as C8 or C18, have been used to enable the separation and detection of α-La, β-Lg, including its genetic variants A and B, BSA, IgG, the proteose peptone (PP) fraction, and CMP. Using both C8 and C18 columns, Leonil et al. (1995, 1997) were able to separate CMP, PP, α-La, β-Lg A, and β-Lg B. With MS, they identified two glyco-CMP variants and PP5 (β-CN-5-phosphate 1
105/ 107) originating from β-CN A1 and A2. However, none of the above studies allowed quantification of BSA, α-La, and β-Lg or CMP because their LC peaks were not sufficiently resolved from the solvent front. Elgar et al. (2000) successfully developed an RP-HPLC method to simultaneously measure α-La and β-Lg, PP, CMP, BSA, and IgG. Using a Resource RP-HPLC column based on an underivatized polystyrene-divinyl benzene matrix with great chemical and pH stability, they were able to quantify these proteins present in bovine whey, as well as in whey protein concentrate (WPC) and in whey protein isolate (WPI) powders. The described method was also used to analyze Lf in whey samples (Palmano & Elgar, 2002). RP-HPLC in combination with MS for protein or peptide analyses: Compared with other techniques such as gel electrophoresis, HPLC is more sensitive, has better dynamic range, is robust and easily automated, not only for a complete

5.4 Proteomics Approaches

separation of major whey proteins, but also for peptides originating from them (Elgar et al., 2000; O’donnellO’Donnell et al., 2004), even though a very pronounced advantage of gel electrophoresis is its great resolving power. However, a further advantage of RP-HPLC is its use in combination with electrospray ionization (ESI)-MS, which can overcome challenges in the identification of hydrophobic, low-molecular-mass proteins, which are undetectable on gel electrophoresis (Garbis et al., 2005). Whey protein denaturation has been assessed by applying LC in combination with ESI-Q-TOF. The degree of denaturation of the major whey proteins, α-La and β-Lg variants A and B as the percentage of soluble whey proteins at pH 4.5 was determined in samples subjected to different heat treatments (Akkerman et al., 2016). Typically for peptide analysis, proteins are digested with proteases (e.g., trypsin), which produce a mixture of peptides, before entering into the HPLC. The peptide mixture is subsequently analyzed by LC-MS using a C18 reversed phase, capillary column and an ESI—known as one-dimensional (1D) LC-MS. Lactosylation sites of β-Lg were firstly characterized by this technique (Morgan et al., 1998). By combining different separation modes such as two-dimensional (strong cation exchange/RP) or three-dimensional (strong cation exchange/avidin/RP) chromatography, it is theoretically possible to detect low-abundance-proteins, increase peak capacity, and overcome the low resolution of 1D LC methods (Aebersold & Mann, 2003). The application of 2D LC using a strong cation exchanger in the first dimension and reversed phase in the second dimension has resolved highly complex peptide samples (Link et al., 1999), and has been used to fractionate bovine milk proteins in combination with ESI-MS/MS and MALDI-MS/MS (Mollé et al., 2009).

5.4 5.4.1

PROTEOMICS APPROACHES What Is Proteomics?

The concept of proteome analysis is defined as the separation, identification and quantification of the entire protein complement expressed by a genome, a cell or a tissue (Wasinger et al., 1995; Wilkins et al., 1996). Proteomics using two major techniques, namely 2-DGE (Patterson & Aebersold, 1995) and MS (Aebersold & Mann, 2003), has become a powerful method for the analysis of complex mixtures of proteins. HPLC is an alternative separation technique for proteomic studies, especially in separation and identification of low-molecular-weight proteins and peptides (Garbis et al., 2005) (Fig. 5.3). The terms “gel-based” or “gel-free” proteomics are used in relation to the applied separation techniques, 2-DGE or HPLC; proteomics approaches can also be “bottom-up” or “top-down,” which basically identify proteins from their protease (e.g., trypsin) digests or as a whole via a mass spectrometer, respectively. From the data generated by the MS, the protein is

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FIGURE 5.3 General procedure of MS-based proteomics analysis of milk proteins. Adapted from Le, T.T., Deeth, H.C., Larsen, L.B., 2017. Proteomics of major bovine milk proteins: novel insights. Int. Dairy J., 67, 2
15.

either sequenced de novo by manual mass analyses of the spectra or processed automatically via sequence search engines such as SEQUEST, Mascot, Phenyx, X!Tandem, and OMSSA. These algorithms are developed based on the correlation between experimental and theoretical MS/MS data; the latter being generated from in silico digestion of protein databases such as UniProt/Swiss-Prot (Deutsch, Lam, & Aebersold, 2008). Since gel electrophoresis and LC methods for separation and detection of whey proteins/peptides have already been mentioned (Sections 5.2.3 and 5.2.5), MS as the primarily used tool in proteomics will be discussed more in this section. MS allows the determination of the molecular mass of proteins or peptides based on the mass to charge ratio (m/z) of ions in the gas phase. Three main components are included in a mass spectrometer: an ion source to form ions and transfer them into a gas phase, a mass analyzer to measure the m/z of these ions, and a detector to record the number of ions

5.4 Proteomics Approaches

at each m/z value. In proteomics, the two commonly used ion sources are electrospray ionization (ESI) or MALDI. There are four basic analyzers: the ion trap, TOF, quadrupole, and Fourier transform ion cyclotron resonance (FTICR); their combinations make powerful hybrid mass analyzers such as quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF-TOF), triple quadrupole, and quadruple ion trap (Q-IT). Typically, MALDI ionization is coupled with a TOF mass analyzer, whereas ESI ionization is used as the ionization source associated with either TOF, quadrupole, ion trap, or hybrid mass analyzers (Trauger, Webb, & Siuzdak, 2002). Because MALDI and ESI differ in the way in which the proteins or peptides are ionized, each has certain advantages (Wisniewski, 2008). Complex mixtures, and to some extent salts, are compatible with MALDI, while LC and CE are easily coupled with ESI. However, ESI produces multiple-charge states for large peptides, while only singly charged ions are produced with MALDI, resulting in less complex data analysis.

5.4.2 Proteomics in Whey Protein Analysis: Qualitative Versus Quantitative Walking through the history of proteomics in milk and whey protein studies, it can be easily seen that the improvement of separation techniques and the development of hybrid mass analyzers with high resolution and accuracy has allowed many breakthroughs in protein analyses both at qualitative and quantitative levels (Manso et al., 2005). This can also be illustrated from single-stage mass spectrometers measuring only the molecular mass of polypeptides to multistage MS fragmenting certain selected ions after the initial mass determination, providing more detailed sequence information. Vincent et al. (2015) identified a whole set of milk proteins extracted from bulk milk of Holstein-Friesian and Jersey cows; 186 different protein accessions were found, including all major casein and whey proteins, as well as lowabundance proteins such as minor glycoproteins, Ig, enzymes, antibodies, and antigens by using nanoLC-ESI-MS/MS. The capabilities of mass spectrometers are not limited to determining molecular masses and amino acid sequences, but also extend to identifying the site of attached groups and types of PTMs (Domon & Aebersold, 2006a, 2006b). Lactosylation sites on whey proteins in processed milk products have been characterized using MALDI-TOF-MS and nanoLC-ESI-MS/MS (Arena et al., 2010). Furthermore, MS-based methods can be used to predict the reactive Lys sites in whey protein sequences of varying matrices. Lys60, Lys75/77/83, and Lys135 in β-Lg were reported to be preferentially attached to lactose of milk samples with moderate heating, while lactosylated Lys47 and Lys60 could be detected in severely heat-treated samples (e.g., evaporated milk) (Meltretter, Wüst, & Pischetsrieder, 2014) (Table 5.2). Site-specific modifications can be therefore

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used as thermal markers in product quality control. In addition to “bottomup” techniques applied in the above studies, different forms (genetic variants, phosphorylation, and O-glycosylation) of κ-CN GMP have been simultaneously detected by using ESI-FTICR-MS for “top-down” analysis (Guerrero, Lerno, Barile, & Lebrilla, 2015). This enabled the identification of various glycoforms, particularly glycosylated diphosphorylated forms of GMP, that had not been reported before. Advanced MS techniques with isotope-coded affinity tag (ICAT) technology and isobaric tagging for relative and absolute quantification (iTRAQ) have overcome limitations of 2-DGE due to its difficult automation, poor dynamic range, and limited sensitivity. The iTRAQ technique is more widely used than ICAT as it can detect proteins without cysteine residues. The changes in the milk proteome of individual cows in response to lipopolysacharide were also quantified by iTRAQ (Danielsen et al., 2010). In addition, chemical labeling via dimethylation of peptides has recently been applied in milk studies (Zhang et al., 2015a, 2015b, 2015c). The incorporation of isotopic dimethyl labels into digested milk samples showed quantitative differences in the proteome of milk in the first 9 days after calving, overlactation, or during mastitis. In an analogous fashion, iTRAQ and dimethyl labeling rely on labeling primary amines (N-terminal amine or Lys side-chain); thus they have limitations for proteins containing PTMs such as lactosylation. To date, multiple reaction monitoring (MRM), also known as selective reaction monitoring (SRM), has been shown to be the most reliable MS-based method for protein/peptide quantification in milk research. A triple quadrupole monitors both precursor and product ions of selected peptides from proteins of interest; the targeted peptide is filtered through quadrupole 1 (Q1), enters Q2 for fragmentation, and one or more fragmented ions are then selected and detected in Q3 (Cox, Zhong, Duchoslav, Sakuma, & McDermott, 2005) (Fig. 5.4). Depending on whether stable-isotope-labeled synthetic peptides are used or not, absolute or relative quantification data

FIGURE 5.4 General scheme of a triple quadrupole instrument. The first quadrupole (Q1) acts as a filter where only ions of a specific m/z are allowed to pass through the second quadrupole. In the third quadrupole (Q3) one of the fragments (obtained from Q2) with specific m/z passes to the detector. Adapted from Le, T.T., Deeth, H.C., Larsen, L.B., 2017. Proteomics of major bovine milk proteins: novel insights. Int. Dairy J., 67, 2
15.

5.5 Conclusions and Outlook

can be produced. A MRM assay was developed by Lutter, Parisod, and Weymuth (2010) to detect and quantify trace amounts of four major milk proteins, αS2-CN, β-CN, κ-CN, and β-Lg, in different food products. The amount of α-La in infant formula and whey protein concentration has also been quantified by MRM (Zhang et al., 2012). Due to its high selectivity, MRM was introduced by Le, Deeth, Bhandari, Alewood, and Holland (2013) to relatively quantify lactosylated peptides from whey proteins in powdered milk. In a further step in MRM quantification of PTMed proteins, Meltretter, Wüst, and Pischetsrieder (2013, 2014) successfully semiquantified sitespecific PTMed β-Lg in heated milk and dairy products, allowing the detection of thermal markers in milk processing. Fifty-two modifications with 19 different structures at 26 binding sites were detected including N-terminal and lysine- or arginine-bound glycation or glycoxidation products, oxidation products of methionine, tryptophan, cysteine and the N-terminus, and finally, the hydrolysis product of asparagine. Cross-linked proteins derived via the MR (e.g., GOLD and pentosidine) as well as the glycoxidation product Nε-carboxymethyllysine (CML) have been quantified in a model system of glycated casein micelles and sodium caseinate by Moeckel, Duerasch, Weiz, Ruck, and Henle (2016). MRM is a remarkable tool and is an efficient MS method that could replace traditional assays such as ELISA for quantification of proteins, particularly low-abundance and PTMed proteins, including enzymes.

5.5

CONCLUSIONS AND OUTLOOK

Various analytical techniques have been listed and discussed in this chapter, including most common traditional methods as well as new methods, which could be chosen for whey protein analyses. Whether or not a method is the best one to apply depends on the availability of laboratory facilities and the costs of both instruments, such as nanoLC and high resolution MS, and standards (e.g., antibodies, heavy-isotope labeled peptides). Advanced mass spectrometers are currently the best instruments to use for protein identification and quantification because of their high sensitivity and resolution; however the running costs are relatively high. In addition, each method has its own pros and cons. For example, challenges of 2-DGE are in resolution and automation; image analysis of gels is good for relative quantification, but greatly dependent on staining, and has relatively low dynamic range. MS is very reliable but has its difficulties including determining trace levels of peptides and proteins in the presence of high-abundance proteins, the high cost of synthetic peptides and the difficult synthesis of PTM peptides, inefficient ionization of PTMed proteins, and insufficient databases for various PTM types. Traditionally, whey has been defined as the fraction left after cheese- or

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caseinate-making. However, the increasing value and potential of whey proteins and their derived peptides as food ingredients point towards a future where whey/serum is prepared for its own sake from milk by filtration processes without coagulation of proteins by acid or rennet. This will be accompanied by development of new products involving whey proteins and peptides therefrom, which will rely heavily on efficient, sensitive, and accurate analytical methods.

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1233. Santos, M. J., Teixeira, J. A., & Rodrigues, L. R. (2011). Fractionation and recovery of whey proteins by hydrophobic interaction chromatography. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 879, 475
479. Santos, M. J., Teixeira, J. A., & Rodrigues, L. R. (2012). Fractionation of the major whey proteins and isolation of beta-lactoglobulin variants by anion exchange chromatography. Separation and Purification Technology, 90, 133
139. Sarioglu, H., Lottspeich, F., Walk, T., Jung, G., & Eckerskorn, C. (2000). Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins. Electrophoresis, 21, 2209
2218. Siciliano, R. A., Mazzeo, M. F., Arena, S., Renzone, G., & Scaloni, A. (2013). Mass spectrometry for the analysis of protein lactosylation in milk products. Food Research International, 54, 988
1000. Sindayikengera, S., & Xia, W. S. (2006). Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex. Journal of Zhejiang University, Science, B, 7, 90
98. Singh, H. (1991). Modification of food proteins by covalent crosslinking. Trends in Food Science & Technology, 2, 196
200. Smithers, G. W. (2008). Whey and whey proteins 2 from ‘gutter-to-gold’. International Dairy Journal, 18, 695
704. Smolenski, G. A., Broadhurst, M. K., Stelwagen, K., Haigh, B. J., & Wheeler, T. T. (2014). Host defence related responses in bovine milk during an experimentally induced Streptococcus uberis infection. Proteome Science, 12, 1
15, Article19. Sobhaninia, M., Nasirpour, A., Shahedi, M., & Golkar, A. (2017). Oil-in-water emulsions stabilized by whey protein aggregates: Effect of aggregate size, pH of aggregation and emulsion pH. Journal of Dispersion Science and Technology, 38, 1366
1373. Sokolov, A. V., Zakahrova, E. T., Kostevich, V. A., Samygina, V. R., & Vasilyev, V. B. (2014). Lactoferrin, myeloperoxidase, and ceruloplasmin: Complementary gearwheels cranking physiological and pathological processes. BioMetals, 27, 815
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References

Spotti, M. J., Martinez, M. J., Pilosof, A. M. R., Candioti, M., Rubiolo, A. C., & Carrara, C. R. (2014). Rheological properties of whey protein and dextran conjugates at different reaction times. Food Hydrocolloids, 38, 76
84. Stojadinovic, M., Burazer, L., Ercili-Cura, D., Sancho, A., Buchert, J., Cirkovic-Velickovic, T., & Stanic-Vucinic, D. (2012). One-step method for isolation and purification of native betalactoglobulin from bovine whey. Journal of the Science of Food & Agriculture, 92, 1432
1440. Takagi, K., Teshima, R., Okunuki, H., & Sawada, J. (2003). Comparative study of in vitro digestibility of food proteins and effect of preheating on the digestion. Biological & Pharmaceutical Bulletin, 26, 969
973. Tipton, K. D., Elliott, T. A., Cree, M. G., Wolf, S. E., Sanford, A. P., & Wolfe, R. R. (2004). Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise. Medicine & Science in Sports & Exercise, 36, 2073
2081. Trauger, S. A., Webb, W., & Siuzdak, G. (2002). Peptide and protein analysis with mass spectrometry. Spectroscopy (Amsterdam, Netherlands), 16, 15
28. Turner, S. A., & Thompson, N. A. (2007). Variation of lactoferrin and lactoperoxidase in bovine milk and the impact of level of pasture intake. New Zealand Journal of Agricultural Research, 50, 33
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24. Voswinkel, L., & Kulozik, U. (2011). Fractionation of whey proteins by means of membrane adsorption chromatography. 11th International Congress on Engineering & Food (ICEF11) Procedia Food Science, 1, 900
907. Wang, S. S., Chen, X. D., Shi, M. X., Zhao, L. H., Li, W., Chen, Y. Y., . . . Li, Y. (2015). Absorption of whey protein isolated (WPI)-stabilized beta-Carotene carotene emulsions by oppositely charged oxidized starch microgels. Food Research International, 67, 315
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5373. Zhang, L., Boeren, S., van Hooijdonk, T., Vervoort, J., & Hettinga, K. (2015c). A proteomic perspective on the changes in milk proteins due to high somatic cell count. Journal of Dairy Science, 98, 5339
5351.

CHAPTER 6

Thermal Denaturation, Aggregation, and Methods of Prevention 1

Heni B. Wijayanti1, André Brodkorb2, Sean A. Hogan2 and Eoin G. Murphy2

NIZO Food Research, Ede, The Netherlands, 2Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

6.1

INTRODUCTION

Whey proteins in bovine milk are soluble proteins that are separated from casein during cheese or casein manufacturing (de la Fuente, Hemar, Tamehana, Munro, & Singh, 2002). The major whey protein is β-lactoglobulin (β-Lg), which accounts for B50% of total whey protein, with the remainder including α-lactalbumin (α-La, B20%), bovine serum albumin (BSA, B8%), immunoglobulins (Igs, B12%), and minor protein/ peptide components including lactoferrin, lactoperoxidase, lysozyme, and growth factors (Qi & Onwulata, 2011; Raikos, 2010) (see Chapter 1 for descriptions of the major and minor whey proteins). In contrast to caseins, whey proteins have higher levels of secondary and tertiary structure, and are thus more susceptible to denaturation by thermal processing. In brief, denaturation occurs when the folded structure of a globular protein unfolds; this leads to interactions between the unfolded proteins, and between the unfolded proteins and other milk proteins, forming protein aggregates. The denaturation/aggregation of whey proteins is an important factor affecting their functional properties such as emulsifying, thickening, gelling, and foaming (Boye, Ma, & Ismail, 2004; Poon, Clarke, & Schultz, 2001). Furthermore, it may lead to fouling of heat exchangers during processing (Bansal & Chen, 2006; Fryer, Belmar-Beiny, & Schreier, 1995), impaired renneting properties of heat-treated milk (Fox, McSweeney, Cogan, & Guinee, 2004), and formation of cooked flavor (Al-Attabi, D’Arcy, & Deeth, 2014; Zabbia, Buys, & De Kock, 2012). Therefore, the stability of whey proteins during thermal processing and ways of improving their stability continue to be important areas in dairy research. 185 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00006-0 © 2019 Elsevier Inc. All rights reserved.

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This chapter summarizes the latest theoretical approaches toward a comprehensive understanding of thermal denaturation and aggregation reactions of whey proteins, where primary attention is given to the fundamental aspect of interactions occurring during the reactions. Also discussed is how this knowledge can be implemented to improve the heat stability of whey proteins and promote their applications in the food industry. The first part of the chapter reviews current theory on the denaturation and aggregation reactions and how the molecular properties of β-Lg, as the major protein in whey, influence the reaction kinetics and thermodynamics. The second and third parts of this chapter cover the behavior of whey proteins during thermal processing and how thermal processing affects their functionality. In the last part, recent novel approaches to control the denaturation and aggregation of whey proteins are discussed.

6.2 THEORETICAL APPROACH OF WHEY THERMAL DENATURATION AND AGGREGATION 6.2.1 The Influence of Molecular Properties of β-Lg on Denaturation and Aggregation β-Lg is the major whey protein in milk and exists as a noncovalent dimer (in solution of neutral pH and concentration .50 mM) with a molecular weight of B36 kDa (de Wit, 2009; Hambling, McAlpine, & Sawyer, 1992). There are nine different genetic variants (A
H) of bovine β-Lg, the most common of which are A, B, and C. These variants have different heat stabilities (Croguennec, O’Kennedy, & Mehra, 2004; Manderson, Hardman, & Creamer, 1998). The dimer conformation in β-Lg is stable at pH 5.5
7.5 and at temperatures ,40 C, but starts to dissociate into two identical monomers at either pH ,3.5 or pH .7.5 and at temperatures .40 C (Fox & McSweeney, 1998; Townend, Herskovi, & Timashef, 1969). Each monomer contains two disulfide (
S
S
) bridges (Cys66-Cys160 and Cys106-Cys119) and one sulfhydryl (
SH) (Cys121) group that is hidden within the protein structure (McKenzie, Shaw, & Ralston, 1972; Papiz et al., 1986) (Fig. 6.1A). The secondary structure of β-Lg is composed of nine antiparallel β-sheets (labeled from A to I) and one C-terminal α-helix (Townend et al., 1969; Wu, Perez, Puyol, & Sawyer, 1999) (Fig. 6.1B). The secondary and tertiary structures are stabilized by the presence of hydrophobic-, ionic-, and hydrogen-bond interactions between the peptide chains (de Wit, 2009), as well as two
S
S
bridges, one of which is located on the internal folded conformation (Tolkach & Kulozik, 2007) (Fig. 6.1A). The two
S
S
bridges and the free
SH group play important roles in the reversibility of β-Lg denaturation (Kitabatake, Wada, & Fujita, 2001).

6.2 Theoretical Approach of Whey Thermal Denaturation and Aggregation

FIGURE 6.1 An illustration of β-Lg molecular structure showing the labeling of β-sheets (A) and cysteine (cys) positions (B) as balls-and-sticks. Image generated from PDB file using RasMol (version 2.7.4.2 ).

Heat-induced denaturation and aggregation of globular whey proteins are generally caused by the exposure of the internal free
SH group and hydrophobic amino acids due to unfolding (Considine, Patel, Anema, Singh, & Creamer, 2007; Creighton, 1997). The pathways involved in the unfolding phenomenon of whey proteins are complex and often assumed to be driven by β-Lg due to its highest concentration in whey. The unfolding may be reversible, reforming its native configuration, or be irreversibly involved in aggregation reactions with other (partially) unfolded proteins (Mulvihill & Donovan, 1987; Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth, 2014). Roefs and de Kruif (1994) proposed a pathway for heat-induced denaturation and aggregation of β-Lg involving the following steps: 1. Initiation. Dissociation of dimer into two monomers that unfold, leading to exposure of the free
SH group. The free
SH group of the monomer then becomes reactive and involved in reaction step 2. 2. Propagation. Reaction between the reactive monomer and available
S
S
bonds via
SH/
S
S
interchange reactions, which ultimately forms a new
SH group; this step can be repeated many times. 3. Termination. The reaction stops as soon as
SH groups on reactive intermediates are all used to form polymers of higher-molecular-weight aggregates. Nevertheless, their model excludes the possible involvement of noncovalent interactions and is limited to β-Lg heated at 65
70 C and under conditions of neutral pH. Others have attempted to improve the Roefs and de Kruif

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model and proposed the essential involvement of noncovalent interactions during aggregation of β-Lg (Apenten, Khokhar, & Galani, 2002; Considine et al., 2007; Croguennec et al., 2004; Mehalebi, Nicolai, & Durand, 2008; Nicolai, Britten, & Schmitt, 2011; Oldfield, Singh, Taylor, & Pearce, 1998; Verheul, Roefs, & de Kruif, 1998). This new model adopted the following steps and is illustrated in Fig. 6.2: 1. Dissociation. β-Lg dimers dissociate into two identical monomers upon heating. 2. Unfolding. The dissociation is followed by unfolding of the native structure forming reactive monomers with exposed free
SH group and hydrophobic residues, which participate in the aggregation steps if further heat is applied. 3. Aggregations via
SH/S
S
interchange reactions and/or noncovalent reactions. The reactive monomers interact to produce oligomers via
S
S
bonds and/or noncovalent interactions. Temperatures between 65 C and 85 C favor aggregate formation via noncovalent interactions, whereas temperatures above 85 C favor the involvement of
SH/
S
S
interchange reactions.

FIGURE 6.2 Pathways during heat-induced denaturation and aggregation of β-Lg.

6.2 Theoretical Approach of Whey Thermal Denaturation and Aggregation

4. Polymerization/formation of higher-molecular-weight aggregates via
SH/
S
S
interchange reactions. Oligomers form larger aggregates via covalent interactions. Ultimately, the completion of aggregation reactions is indicated by formation of larger networks or gels. The above model is complex, but clearly demonstrates that denaturation and aggregation depend on the molecular properties (exposed hydrophobic side chains and
SH group,
S
S
bridges) of the protein, in this case, β-Lg. The extent of β-Lg unfolding is influenced by the exposure of hydrophobic amino acids, whereas the extent of aggregation relates to the
S
S
bridges and electrostatic barrier (Delahaije, Gruppen, Giuseppin, & Wierenga, 2015; Delahaije, Wierenga, Giuseppin, & Gruppen, 2015; Gimel, Durand, & Nicolai, 1994; Pouzot, Durand, & Nicolai, 2004). Changes in the molecular properties of β-Lg have been investigated extensively and are reported to be driven by several factors, including temperature, protein concentration, ionic strength, pH. and the presence of other whey proteins or casein (Anema & McKenna, 1996; Baussay, Bon, Nicolai, Durand, & Busnel, 2004; Delahaije, Gruppen, Giuseppin, & Wierenga 2015; Delahaije, Wierenga, Giuseppin, & Gruppen 2015; Dissanayake, Ramchandran, Donkor, & Vasiljevic, 2013; Donato & Guyomarc’h, 2009; Erabit, Flick, & Alvarez, 2013; Iametti, Cairoli, Degregori, & Bonomi, 1995; Iametti, DeGregori, Vecchio, & Bonomi, 1996; Jeyarajah & Allen, 1994; Mills, 1976; Petit et al., 2016; Qi, Brownlow, Holt, & Sellers, 1995; Sava, Van der Plancken, Claeys, & Hendrickx, 2005; Wolz & Kulozik, 2015). Table 6.1 summarizes the major findings related to changes in the molecular properties of β-Lg caused by various factors. The denaturation of β-Lg is reversible at low protein concentrations and upon heating at temperatures ,60 C. However, at higher protein concentrations or at temperatures .70 C, the denaturation process is followed by irreversible aggregation reactions (Simons, Kosters, Visschers, & de Jongh, 2002). The denaturation and aggregation mechanism of β-Lg has also been reported to be pH-dependent (Table 6.1). Denaturation is promoted with increasing pH, especially between pH 6.25
7.0 (Gotham, Fryer, & Pritchard, 1992) and pH 7.4
8.5 (Dunnill & Green, 1966). At pH , 7.0, the amount of
SH group available for inter-/ intramolecular
S
S
bonds is increased, which promotes the formation of a partially denatured intermediate (Gotham et al., 1992). It has been reported that pH also influences the size of the resulting aggregates, where at pH 6.9 small aggregates are formed due to increasing reactivity of free
SH groups while secondary aggregates of very large particle size are formed at lower pH (pH ,6.4) (Hoffmann, Roefs, Verheul, van Mil, & DeKruif, 1996). According to these authors, the secondary large aggregates are formed via noncovalent cross-linking of smaller aggregates; they precipitate at pH 6.2. The findings by Zuniga, Tolkach, Kulozik, and Aguilera (2010) confirmed

189

Table 6.1 Summary of Reports on Various Factors that Influence the Molecular Properties of β-Lg

References Effect of pH Dunnill and Green (1966)

Experimental Conditions Titration of β-Lg with pchloromercuribenzoate, pH 2.4
10

Molecular Properties Changes G

Exposed hydrophobicity

Illustrations/Notes

N

N

Gotham et al. (1992)

β-Lg 60
130 mg/mL, pH 6.6

G G

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges

N

N

N

Hoffmann et al. (1996)

β-Lg 50 g/L, pH 6.2
8.0

G G

Donato et al. (2009)

Demineralized β-Lg 1.0% (w/w), heated at 70 C and 85 C, pH 5.7 and 5.9

G G

G

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges Exposed hydrophobicity Intermolecular disulfide bridges Charge

pH 6.7

pH>6.7

pH 6.6

pH 4-8

pH 6.5

R : Slow Reaction

R

pH 7.4

N: Native R: Reversible denaturation SH* : Reactive sufhydryl

SH* : Fast Reaction & 4x faster at pH 8.5

U : Induced by inter-molecular disulfide bonds U : Denaturation temperature increases with increasing pH U : Reaction fast, but aggregation rate decreases

N: Native U: Unfolded denatured

Zuniga et al. (2010)

β-Lg 5% (w/v), pH 6.0; 6.4; 6.8, 80 C

G G

G

Effect of Temperature Mills (1976)

β-Lg AB 0.043% in 0.1 M KCl and 0.02 M phosphate, pH 6.4
6.5, 15
90 C and cooled back to 20 C

G G

Exposed hydrophobicity Intermolecular disulfide bridges Charge

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges

β-Lg 2.5 mg/mL in 50 mM sodium phosphate buffer, pH 6.8

G G

T:20°C pH 6.5

Hydrophobic + Interior – Completely + Exposed

Iametti et al. (1996)

19 β-Lg (Trp )

β-Lg (Trp61)

T:50°C pH 6.5 T:80-90°C pH 6.5

T:>70-90°C pH 6.5 T:90°C pH 6.5

+ Partially Exposed –

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges

Continued

Table 6.1 Summary of Reports on Various Factors that Influence the Molecular Properties of β-Lg Continued

References

Experimental Conditions

de Wit (2009)

Review on heat-induced β-Lg up to 150 C, pH .6.8

Molecular Properties Changes

G

Exposed hydrophobicity Intermolecular disulfide bridges Charge

β-Lg 2 g/L in 10 mM sodium phosphate buffer, pH 7.0

G

Charge

β-Lg 10
40% (w/w), pH 6.7, 70
95 C

G

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges

G G

Delahaije, Gruppen, et al. (2015), Delahaije, Wierenga, et al. (2015) Wolz and Kulozik (2015)

G

Effect of Protein Concentration Roefs and de Kruif β-Lg AB 2
95 g/L, pH (1994) 6.75
6.95

G G

G

Exposed hydrophobicity Intermolecular disulfide bridges Charge

Illustrations/Notes

Ionic strength and pH affects the aggregate size or structure by decreasing the electrostatic repulsion between proteins or protein aggregate

N

U

A

70-80°C

85-95°C

: Reaction accelerates as increasing protein concentration

N : Native U : Unfolded State A : Aggregated State : Limited temperature

Electrostatic & Hydrophobic Interactions

N

T:65°C 2g/L

U

SH*

T:65°C

83.2-92.6g/L

-SH/disulfide interchange reactions

P N : Native U : Unfolded P : Polymerization SH*: Reactive sulfhydryl

Qi et al. (1995)

Iametti et al. (1995)

β-Lg A 2
120 mg/mL in 70 mM phosphate buffer at pH 6.7 or pH 8.05

G

β-Lg 3.8, 8.0, 16.0 mg/mL, pH 6.8

G

G

G

Exposed hydrophobicity Intermolecular disulfide bridges

Exposed hydrophobicity Intermolecular disulfide bridges

Possible pathways, depending on the concentrations:

Reaction accelerates A : dependent on protein concentration

T:75°C

N

U

3.8-16mg/mL

Reaction accelerates A : independent on protein concentration

T:85°C

N

U

N : Native U : Unfolded State A : Aggregated State

Hoffmann et al. (1996)

β-Lg 10
100 g/L, 60
75 C, pH 7.0

G G

Exposed hydrophobicity Inter-/ intramolecular disulfide bridges

Particle Size : –

pH 7.0 65-70°C

Reaction accelerates with + : increasing protein content 10-100g/L

Continued

Table 6.1 Summary of Reports on Various Factors that Influence the Molecular Properties of β-Lg Continued

References Dissanayake et al. (2013)

Experimental Conditions β-Lg 10%, 17.5%, and 25% (w/ w), pH 4.0
6.0, heated at 20
140 C

Molecular Properties Changes G G

G

Exposed hydrophobicity Intermolecular disulfide bridges Charge

Illustrations/Notes N

pH 5

U

10-25%, w/w Protein Content

Denaturation Rate – –

Protein Content

A :

B-Lg mostly stable at pH 4.0 and 25% (w/w) protein

+ + Denaturation Rate

N : Native U : Unfolded State A : Aggregated State

Effect of Salt/Ionic Strength Xiong et al. (1993) β-Lg 1.2 mg/mL, 25
96 C, NaCl 0.02
1.0 M and CaCl2 0.005
0.2 M, pH 5.50
6.50 Jeyarajah and Allen (1994)

β-Lg 20 mg/mL, CaCl2 1
15 mM, 65 C or 80 C for 15 min, pH 2
11

Simons et al. (2002)

β-Lg 20 mg/mL, pH 7.2, 70 C for 2 h, CaCl2 0
50 mM

G G

G G

G G

Charge Intermolecular disulfide bridges Charge Intermolecular disulfide bridges

To facilitate protein aggregation, a small amount CaCl2 should be added and pH of solutions need to be adjusted to # pH 6.0

Charge Intermolecular disulfide bridges

Ca21-induced aggregation of β-Lg by forming complex with the denatured/ unfolded parts of the protein and bind site specific to carboxylates with a threshold affinity

Baussay et al. (2004)

Petit et al. (2011)

β-Lg 0
100 g/L, pH 7.0, NH3COOH 0.03M-0.4 M

G

β-Lg 53 g/L, CaCl2 0, 66, 132 and 264 mg/kg

G

G

G G

Erabit et al. (2013)

Petit et al. (2016)

β-Lg 6%, 5.1
7.1 mM CaCl2, 85 C for 60 s

β-Lg 3.2
4.5 g/L, 64.5
98 C

G G

G

Charge Inter-/ intramolecular disulfide bridges

Charge Exposed hydrophobicity Intermolecular disulfide bridges Charge Inter-/ intramolecular disulfide bridges Charge

Addition of Ca up to 132 mg/kg increases kinetic rate and aggregation by lowering the electrostatic repulsion between the negatively charged β-Lg reactive
SH group, bridging β-Lg proteins or by ion-specific conformational change. A threshold effect was observed at addition of Ca above 132 mg/kg, where no further significant increase in aggregation rate was found Increasing concentration of CaCl2 increases aggregation of β-Lg monomers by reducing intermolecular repulsions. Larger particles aggregates were promoted at 7.1 mM CaCl2 Aggregation of β-Lg is promoted at low β-Lg concentration (4.5 g/L), in the presence of other whey proteins (3 g/L), high lactose (43 g/L), calcium (631 mg/L), and minerals (5.2 g/L), but had higher protective effect on β-Lg unfolding. This is because inorganic phosphates or chelates formed stable complexes with Ca, reducing the concentration of free ionic Ca21 and thereby reducing its interaction with β-Lg

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that larger aggregate formation is promoted at lower pH (,6.4) with particle size of B100 nm, whereas at higher pH (.6.0), smaller aggregates (B40 nm) tend to form due to increased reactivity of the -SH group. A stable aggregate can be formed upon heating at 85 C at pH 5.7
5.9 (Donato, Schmitt, Bovetto, & Rouvet, 2009). These authors further stated that aggregation is promoted due to a decrease in repulsion between protein particles, and limited unfolding. As the heating proceeds from 70 C to 85 C, covalent (
S
S
bonds) and noncovalent (electrostatic and hydrophobic) interactions are favored and play a major role in the stability of the aggregates (Schmitt et al., 2009). The addition of calcium has been reported to induce extensive aggregation reactions (Petit, Herbig, Moreau, & Delaplace, 2011), but the mechanism is still poorly understood at a molecular level. Nevertheless, it is believed that Ca21 ions trigger aggregation of β-Lg by forming intermolecular protein 2 Ca21 2 protein cross-linking or intramolecular electrostatic shielding of the negatively charged groups. Simons et al. (2002) proposed that the protein has specific calcium-binding carboxylate groups on its unfolded surface with a threshold affinity for calcium for the formation of such complexes. In contrast, it was postulated that Ca21 ions trigger aggregation of β-Lg by reducing the net negative charge of the protein and not by its involvement on the formation of intermolecular cross-linking (Hongsprabhas, Barbut, & Marangoni, 1999; Xiong, Dawson, & Wan, 1993). The strength of electrostatic interactions also influences the type of aggregates formed (Adamcik et al., 2010; Ako, Nicolai, Durand, & Brotons, 2009; Bromley, Krebs, & Donald, 2006). According to these authors, linear aggregates are formed at pH values far from the isoelectric point (pI) and at low ionic strength, but more densely branched aggregates are formed at high ionic strength or pH close to the pI. Stable spherical particles of β-Lg (100
300 nm) can be formed at pH up to 7.5 in the presence of Ca21 ions (Phan-Xuan et al., 2014). Their results suggest the importance of net charge density of the native proteins for the formation of spherical particles, which can be controlled either by pH or adding Ca21 ions. A more detailed discussion on how ionic strength influences the structure of β-Lg aggregates is provided in Section 6.3.4.

6.2.2 Kinetics and Thermodynamics of β-Lg Unfolding and Aggregation The kinetics of unfolding, denaturation, and aggregation of β-Lg have been widely studied for over 50 years. As discussed, Sawyer (1968) observed that sequential covalent and noncovalent interactions are the backbone of the aggregation mechanism. The early models, based on results using starch gel

6.2 Theoretical Approach of Whey Thermal Denaturation and Aggregation

electrophoresis and sedimentation velocity techniques, were later confirmed by Hoffmann and van Mil (1997). One of the most widely accepted models is the aforementioned polymerization/aggregation mechanism by Roefs and de Kruif (1994), involving activation, propagation, and termination. The kinetic and thermodynamic parameters of the initial unfolding step have been widely studied using calorimetric (differential scanning calorimetry, DSC) and spectroscopic techniques such as fluorescence, circular dichroism, and nuclear magnetic resonance (NMR). Electrophoretic mobility (using sodium dodecyl sulfate 2 polyacrylamide gel electrophoresis, SDS-PAGE), particle size measurement (dynamic light scattering and multiangle light scattering), and DSC can provide information about the aggregation step (Beaulieu, Corredig, Turgeon, Wicker, & Doublier, 2005; Fitzsimons, Mulvihill, & Morris, 2007; Singh, Ye, & Havea, 2000); however, accurate quantification can be limited. To date, one of the recognized methods for estimating the kinetic parameters (reaction velocity, reaction order, and reaction rate constant) of the whole denaturation and aggregation reaction is to follow the disappearance of native β-Lg over time. This may be achieved by isoelectric precipitation of the nonnative and aggregated material from heated samples and subsequent quantification of the native β-Lg by chromatographic methods such as size exclusion (Roefs & de Kruif, 1994) and reversed-phase high performance liquid chromatography (HPLC) (Kehoe, Wang, Morris, & Brodkorb, 2011). The process can be summarized by the following equation: dC 5 2 kCn dt

ð6:1Þ

where C is the concentration of native protein, k is the reaction rate constant, and n the reaction order. Integration of Eq. (6.1) gives: 

Ct C0

12n 5 1 1 ðn 2 1Þkt

for n 6¼ 1

ð6:2Þ

where Ct is the native protein concentration at time t and C0 is the initial protein concentration. Further rearrangement (natural log) gives:  1 Ct ðn21Þkt 12n 5 11 C0 C12n 0

ð6:3Þ

Logging decay data such as this equalizes the data per unit time and reduces error when solving the equations and determining the rate constant k and the reaction order with respect to concentration. Using this model it was predicted that the reaction order for the above mechanism would be 1.5; this value had been previously determined as the order of β-Lg denaturation (Dannenberg & Kessler, 1988). Roefs and de Kruif (1994) slowed the denaturation by lowering the heating temperature to 65 C over a long time

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(48 hours); they found that experimental data also fitted the above model. The unfolding step is a first-order (n 5 1) reaction whereas the aggregation process is a second-order (n 5 2) process. The overall reaction order becomes 1.0 if the unfolding step is rate limiting; it becomes 2.0 if the aggregation process is the rate-limiting step. Reaction conditions can be adapted to making unfolding the rate-limiting step: by lowering the heating temperature, bringing the pH close to the pI, or increasing the salt concentration. Conversely, to bring the reaction order closer to 2 the aggregation step has to become rate limiting. This will occur at pH values away from the pI, at low ionic strength, and high heating temperatures. Kehoe et al. (2011) presented some quantitative data on β-Lg present as native monomers, stable intermediates monomers, and aggregated protein (Fig. 6.3) as a function of concentration and heating time at 78 C. The concentration of nonnative monomers increased initially with heating time but then began to decrease as the concentration of aggregates increased.

FIGURE 6.3 Distribution of protein in 2% and 9% β-Lg at pH 7.0. The black regions represent the proportion of native β-Lg as measured by RPHPLC. The gray regions represent nonnative monomeric β-Lg (the difference in monomeric by GPC and native by RP-HPLC). The clear region was the quantity which was aggregated (measured by GPC). Reprinted from Kehoe, J. J., Wang, L., Morris, E. R., & Brodkorb, A. (2011). Formation of non-native β-lactoglobulin during heat-induced denaturation. Food Biophysics, 6, 487
496 with kind permission from Springer and Copyright Clearance Center.

6.3 Behavior of Whey Proteins During Thermal Processing

6.3 BEHAVIOR OF WHEY PROTEINS DURING THERMAL PROCESSING 6.3.1 Heat-Induced Changes in Whey Proteins Other Than β-Lg Since heat-induced denaturation/aggregation of β-Lg will be extensively discussed in Section 6.5.1.2, the other major whey proteins are the main focus to this section. α-La is the second-most abundant naturally occurring bovine whey protein. It exists as a monomeric, calcium-binding protein of approximately 14 kDa molecular mass (Brew, 2003). Various authors have reported that the denaturation temperature (Td) of α-La is lower than that of β-Lg (Bernal & Jelen, 1985; Klarenbeek, 1984; Murphy, Fenelon, Roos, & Hogan, 2014). For example, an early study by Ruegg, Moor, and Blanc (1977) reported a Td of 65 C for α-La compared to 73 C for β-Lg. More recent work by Boye and Alli (2000) demonstrated that the Td of α-La is strongly affected by its affinity for binding calcium; at pH 7, the Td of apo α-La (calcium free) was 35 C compared to 64 C for holo α-La (calcium bound). Thermally induced denaturation of α-La is largely reversible provided heating is limited to temperatures below 80 C. Chaplin and Lyster (1986) demonstrated that when α-La solutions were heated to 77 C for 15 minutes, more than 90% of the protein returned to a native state upon cooling; in comparison, only 40% of the protein regained its native structure when heated to 95 C for 15 minutes. Hong and Creamer (2002) found that the onset temperature of irreversible aggregation was lower in apo α-La solutions compared to holo α-La, emphasizing the importance of calcium binding in the behavior on heating. Reversibility of denaturation at lower temperatures is related to the lack of a free
SH group in the native protein. During heating at higher temperatures, one or more of α-La’s four
S
S
bonds may be disrupted and release highly reactive free
SH groups, which can initiate and propagate irreversible aggregation in much the same way as the naturally occurring free
SH in β-Lg (Chaplin & Lyster, 1986). BSA has been widely studied for its gelation properties. It has been reported to form gels at a rate up to 100 times faster than β-Lg (Hines & Foegeding, 1993). Denaturation of BSA begins at temperatures in excess of 62 C (Ruegg et al., 1977). Baier, Decker, and McClements (2004) suggested the first stage of denaturation was a reversible transformation from a native to a moltenglobule state. Upon further heating, the molten globule undergoes an irreversible transformation into a more hydrophobic conformation, which finally leads to intermolecular hydrophobic aggregation. It is also likely that BSA’s free
SH group plays a complementary role to hydrophobic forces. Clark, Kavanagh, and Ross-Murphy (2001) proposed that during gelation of BSA, covalent
S
S
linkages, along with hydrophobic interactions, are involved in aggregate formation.

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Immunoglobulins (Ig) present in mammalian secretions are important proteins providing immune protection for newborns. Igs exist in many different isotypes, namely IgA, IgE, IgD, IgG1, IgG2, and IgM; however, in bovine milk the major isotype is IgG1 which accounts for approximately 66% of total Ig (Hurley & Theil, 2013). Lindström, Paulsson, Nylander, Elofsson, and Linkmark-Mansoon (1994) reported the Td of IgG1, as measured by DSC, to be 81 C at pH 6.6. Vermeer, Bremer, and Norde (1998) found a slightly lower Td of 74 C at pH 6.0 but also observed an exothermic aggregation peak at approximately 80 C. Irreversibility of the structural changes observed was attributed to formation of aggregates and suggests that heating of IgG1 at temperatures above 80 C may lead to extensive denaturation and aggregation. This is in keeping with the findings of Bogahawaththa, Chandrapala, and Vasiljevic (2017) who found that when solutions of IgG were heated to 100 C for 30 seconds, 55% of total IgG formed covalently bonded aggregates; when heated at 72 C for 15 seconds, no covalently bonded aggregates were formed. Whey protein streams derived from cheese manufacture can contain significant amounts (B20% of the proteins) of glycomacropeptide (GMP) (O’Loughlin, Murray, Kelly, Fitzgerald, & Brodkorb, 2012). GMP consists of the hydrophilic region of κ-casein, which is cleaved from the intact protein by chymosin during cheese manufacture. As a disorganized peptide with no free
SH groups, GMP is highly heat-stable in comparison to other whey protein constituents. Martinez, Farías, and Pilosof (2010) studied the gelation of GMP 1 β-Lg mixtures and reported that at neutral pH, heat-induced gelation did not occur in GMP solutions at concentrations up to 40% (w/w). In support of this observation, they reported that DSC of GMP solutions did not exhibit a denaturation event below 100 C at pH 3.5 and 7.0. O’Loughlin et al. (2012) found GMP to be the most heat-stable component in commercially available whey protein isolate (WPI); heating of 10% WPI solutions for 10 minutes at 80 C resulted in approximately 3% loss in native GMP compared to approximately 76% loss of β-Lg A.

6.3.2

Whey Protein 2 Whey Protein Interactions

Heat-induced interaction of β-Lg with other whey proteins has been studied both in complex systems such as whey protein concentrates (WPCs) as well as in model solutions containing mixtures of β-Lg with one or more protein (Gezimati, Singh, & Creamer, 1996; Havea, Singh & Creamer, 2001; Havea, Singh, Creamer, & Campanella, 1998; Livney, Verespej, & Dalgleish, 2003; Schokker, Singh, & Creamer, 2000). Schokker et al. (2000) studied the gelling properties of β-Lg/α-La mixtures and found that while α-La did not form aggregates when heated alone, it was incorporated into both covalently

6.3 Behavior of Whey Proteins During Thermal Processing

bonded and hydrophobic aggregates when heated in the presence of β-Lg. The presence of α-La resulted in a larger proportion of covalently bonded aggregates compared to hydrophobic aggregates, but did not alter the early stages of β-Lg unfolding. This indicates the mechanism for covalent interaction between α-La and β-Lg requires initial unfolding of β-Lg to expose the reactive free
SH group, which reacts with a sterically accessible
S
S
bond in α-La to initiate intermolecular
SH/
S
S
interchanges and hence aggregation (Livney et al., 2003; Schokker et al., 2000). In contrast, Havea et al. (2001) found heating (75 C for 15 minutes) mixtures of β-Lg and BSA dissolved in permeate resulted in aggregates which were homopolymers of each protein due in part to the lower thermal transition temperature of BSA compared to β-Lg (Havea et al., 2001; Ruegg et al., 1977). The majority of the BSA was aggregated during heating to 75 C, so that when β-Lg underwent conformational changes at higher temperatures BSA was inert. It follows the view that the heating temperature and hold-up time will have an influence on the interaction between β-Lg and BSA. This may explain why
S
S
linked aggregates of β-Lg and BSA were observed by Matsudomi, Oshita, and Kobayashi (1994) when heating at the higher temperature of 80 C for 30 minutes. Thermal interaction of α-La and BSA has been found to proceed by a similar mechanism to β-Lg/α-La interaction (Havea, Singh, & Creamer, 2000). Calvo, Leaver, and Banks (1993) found that in heated α-La/β-Lg/BSA mixtures, the rate of inclusion of α-La in aggregates was dependent on the concentration of free
SH groups in the other whey proteins. This suggests that the formation of intermolecular
S
S
bridges is central to the inclusion of α-La in whey protein aggregates. In comparison to β-Lg, α-La, or BSA, thermal association of IgG with other whey proteins has not been widely reported. Recently, Bogahawaththa et al. (2017) studied the effect of two heat treatments (72 C/15 seconds and 100 C/30 seconds) on IgG in the presence of β-Lg, α-La, and BSA. Heating at 72 C did not result in the formation of covalently bonded complexes of whey proteins; however, at 100 C, IgG was incorporated into covalently bonded aggregates along with the other whey proteins. Interestingly, heating of IgG/BSA mixtures did not result in covalently bonded aggregates. More research is required to fully understand IgG
whey proteins interactions.

6.3.3

Whey Protein 2 Casein Interactions

It is well known that industrial heat treatments of milk lead to denaturation of whey proteins and subsequent formation of complexes of whey protein aggregates and whey protein 2 casein (mostly κ-casein) complexes (Anema & Klostermeyer, 1997; Sutariya, Huppertz, & Patel, 2017; Vasbinder, Alting, & de Kruif, 2003; Vasbinder & de Kruif, 2003). Interactions of whey proteins

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with casein micelles occur specifically between β-Lg and κ-casein and/or αs2 casein via
SH/
S
S
interchange reactions. These caseins have cystine residues within their structure, which enables them to associate with whey proteins (Farrell, Malin, Brown, & Mora-Gutierrez, 2009; Jang & Swaisgood, 1990). The type of bonds responsible for formation of whey protein 2 casein micelle complexes are noncovalent (e.g., ionic and/or hydrophobic) and/or covalent, depending on the heat treatment (Anema & Li, 2003; Jang & Swaisgood, 1990; Mulvihill & Donovan, 1987; Vasbinder et al., 2003; Vasbinder & de Kruif, 2003). It is believed that noncovalent interactions are involved during the early stages of heating, while covalent interactions (mainly
SH/
S
S
interchange reactions) are involved in the later stages of heating (Mulvihill & Donovan, 1987). The association of whey proteins with casein micelles is influenced by several factors, such as pH, whey protein-to-casein ratio, ionic strength, and temperature of heat treatment (Donato & Guyomarc’h, 2009; Gaspard, Auty, Kelly, O’Mahony, & Brodkorb, 2017; Nguyen, Chassenieux, Nicolai, & Schmitt, 2017; Vasbinder et al., 2003; Vasbinder & de Kruif, 2003; Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth, 2014). Upon heating milk at pH B6.6
6.9, only part of the whey protein aggregates become associated with casein micelles and are mostly dissolved in the serum phase, while at lower pH (,6.6) almost all whey proteins are associated with casein micelles (Corredig & Dalgleish, 1996; Vasbinder et al., 2003; Vasbinder & de Kruif, 2003; Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth, 2014). According to Vasbinder and de Kruif (2003), a uniform distribution of whey protein aggregates on casein micelles was observed at pH 6.55, but the distribution was inhomogeneous as soon as the pH was reduced to pH 6.35. The uniformity of whey protein distribution on casein micelles influences the renneting behavior of milk, where a decrease in flocculation of milk is observed when whey proteins are uniformly coated onto the casein micelles. Moreover, it has been reported that the amount of denatured whey proteins associated with casein micelles considerably influences the casein micelle size changes, as the size of soluble and micelle-bound complexes decreases with increasing pH from 6.3 to 7.3 (Fig. 6.4) (Anema & Li, 2003; Donato & Dalgleish, 2006; Guyomarc’h, Violleau, Surel, & Famelart, 2010; Pesic, Barac, Stanojevic, & Vrvic, 2014; Vasbinder & de Kruif, 2003). Sutariya et al. (2017) studied the behavior of whey protein 2 casein complexes in heated concentrated milk (80 C or 90 C for 5 minutes at 50%
55% total solids) and found a similar result as previously reported by Vasbinder and de Kruif (2003). Heating the concentrated milk at pH 6.7 resulted in a higher proportion of whey protein aggregates (self-aggregates or whey proteins 2 κ-casein aggregates) in the serum phase than at pH 6.5. The denatured whey proteins were mostly associated with casein micelles when

6.3 Behavior of Whey Proteins During Thermal Processing

FIGURE 6.4 An illustration of casein micelle changes as influenced by pH. The illustrations are derived from the results of Anema and Li (2003) on skim milk heated at 90 C for 30 min.

heated at pH 6.5. Nevertheless, at such high solids content (.50% total solids), the viscosity of skim milk is mostly influenced by the extent of heatinduced whey protein denaturation rather than whey protein distributions on casein micelles or in the serum phase (Sutariya et al., 2017). This is because at a higher concentration, volume fractions and interactions of particles primarily control the viscosity. These results suggested new insight into controlling viscosity prior to spray drying for improving spray drying efficiency.

6.3.4 Aggregate Size and Structure of Thermally Denatured Whey Proteins The first stage of aggregation is generally defined as formation of oligomers, mostly dimers, trimers, and tetramers, formed by
S
S
bonds (Nicolai et al., 2011; Schokker et al., 2000; Schokker, Singh, Pinder, Norris, & Creamer, 1999). Further aggregation involves both covalent and noncovalent association of oligomers to form larger aggregates, generally due to the aforementioned charge screening and promotion of hydrophobic interactions (Schmitt, Bovay, Rouvet, Shojaei-Rami, & Kolodziejczyk, 2007). Solutions of native β-Lg tend to be stable over a wide pH range except close to its isoelectric point (5.1), where the dimer 2 monomer equilibrium is shifted toward the monomer. When the pH of the solution is very close to the isoelectric point of the protein, the structure of the aggregates changes considerably. Jung, Savin, Pouzot, Schmitt, and Mezzenga (2008) used small-angle neutron scattering to show that β-Lg aggregates prepared at pH 5.8 and with no salt had a spherical shape with a swollen gel-like, low-density internal structure with flexible chains holding the aggregates together. At pH on either side of whey proteins’ pIs (4.5
4.7 and 5.7
5.9), and at low ionic strength, stable suspensions of monodisperse spherical particles of about 200 nm were obtained on heating (Schmitt et al., 2009). Between these pH ranges,

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spherical whey protein particles associate into larger aggregates leading to macroscopic phase separation or precipitation on aging (Guyomarc’h et al., 2015). Phan-Xuan et al. (2014) reported that similar spherical particles of β-Lg are formed at pH values up to 7.5 by careful control of the Ca21/β-Lg molar ratio, and that the net charge of native proteins (and hence structure) can be modified by pH and ionic strength. Schmitt et al. (2007) found that surface hydrophobicity at pH 7.0 was twice that at pH 6.0 and that a decrease in surface hydrophobicity was associated with spherical, dense aggregates in which hydrophobic patches may have been buried in the particle interior. The relationship between structure of whey protein aggregates and pH was further demonstrated by Jung et al. (2008) using transmission electron microscopy (TEM); long and rigid strands were formed at pH 2.0, but changed to spheres at pH 5.8 or to small curved strands at pH 7.0. Zuniga et al. (2010) also used TEM to show curved protein strands at pH 6.8 and 6.4, but spherical aggregates were observed at pH 6.0. Mehalebi et al. (2008) showed that at pH from 6.0 to 8.0, the hydrodynamic radius (Rh) of steady-state aggregate structures was independent of protein concentration until close to the critical gel point. At low concentrations, Rh showed an inverse relationship with pH with aggregates of B20 nm at pH 6.0 to B12 nm at pH 8.0. Schmitt et al. (2009) and Moitzi et al. (2011) confirmed that these aggregates are covalently bonded and were formed due to unfolding of β-Lg in a narrow pH range (pH 5.8
6.2). Another contributor to changes in the size and structure of whey protein aggregates is salts, which affect the colloidal stability of proteins by salting in or salting out of the proteins (depending on the protein:salt ratio). Salts also screen repulsive protein charge density thereby promoting aggregation. Adding salt at a given pH is equivalent to altering the pH toward the pI at a given ionic strength as electrostatic interactions are reduced either by increasing or decreasing the charge density. At neutral pH, the maximum overall aggregation rate with heat is a function of sodium chloride (NaCl) concentration due to its conformation-stabilizing, salting out-like effect at concentrations from 50 to 100 mM (Verheul & Roefs, 1998). The turbidity of whey protein solutions increases with increasing NaCl concentrations due to the formation of large aggregates (Schmitt et al., 2007). At concentrations below a critical value, heating causes whey proteins to aggregate into noninteracting isolated clusters, which are well dispersed in the solution and the overall solution maintains the properties of a fluid (Mezzenga & Fischer, 2013). Above this critical concentration gels are formed. These gels have been reported to be highly turbid close to the pI and transparent at low and high pH values (Ako et al., 2009). At very low ionic strength, these gels can be transparent; in most cases heat-set gels range from

6.3 Behavior of Whey Proteins During Thermal Processing

hazy to opaque (Mezzenga & Fischer, 2013). The microstructure of gels at pH values between pH 4.3 and 5.6 indicated a network of spherical particles with radii of several hundred nanometers (Ako et al., 2009; Bromley et al., 2006). The latter authors also showed that at a concentration of 30 g/L β-Lg gels, formed at pH 5.3, were made up of spherical particles, the size of which was inversely dependent on heating rate. The critical gelation concentration also decreases with increasing ionic strength (Baussay et al., 2004) or decreasing charge density, which again is indicative of the interactive nature of factors affecting whey protein aggregation phenomena. McSwiney, Singh, and Campanella (1994) suggested that so-called soluble aggregates might be formed by heating prior to development of macroscopic gels, at concentrations below the critical gelation point. Such soluble aggregates, which may be defined as protein intermediates between the monomeric form and an insoluble gel network, appeared as monomers on SDS-PAGE gels when a reducing agent was used (McSwiney et al., 1994). Ryan (2011) showed that the thermal stability of β-Lg could be improved in the presence of salts by first heating below the critical gelation concentration at neutral pH and in the absence of salt. Heating under these conditions forms a solution with low turbidity and viscosity in which protein aggregates exist in the size range from 36 to 59 nm. Soluble aggregates were formed that were resistant to salt concentrations exceeding those typically used in nutritional beverages up to 100 mM NaCl or 15 mM calcium dichloride (CaCl2). Whey proteins are reported to form a wide range of assemblies—fibrils, multistranded ribbons, spherulites, spherical particles, flexible strands, or fractal aggregates depending on the heating conditions (Guyomarc’h et al., 2015). The ability of whey proteins to form remarkably long rigid strands at pH 2.0 without added salt has attracted much attention, in part because they resemble so-called amyloid fibrils that are responsible for a number of neurodegenerative diseases (Adamcik et al., 2010). At a concentration of 80 g/L, larger branched aggregates were formed that appeared to be agglomerates of these strands. Strands can be several microns long, with an average diameter of 4
5 nm and a persistence length of about a micron. These strands consist of multiple protein filaments that form twisted ribbons with a helical structure (Adamcik et al., 2010). Fibril formation in β-Lg upon heating at pH 2.0 is strongly influenced by ionic strength (Arnaudov & de Vries, 2006). Addition of NaCl reduces the persistence length of the protein strands. Mudgal, Daubert, and Foegeding (2009) reported that small flexible strands with length of 50
150 nm and diameter of 5
10 nm are formed at low protein concentration and at pH of 2.5
4.0 and .6.3. Randomly branched aggregates resulting from the secondary aggregation of these small flexible strands were also observed at higher protein concentration (Nicolai & Durand, 2013). Under specific pH conditions, whey proteins with opposite net charge, β-Lg or α-La

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and lactoferrin are shown to coassemble in the absence of external energy input and form well-defined microspheres (Bouhallab & Croguennec, 2013). Dry-heating is another means used to denature and form whey protein aggregates, albeit at a slower rate. Gulzar, Lechevalier, Bouhallab, and Croguennec (2012) showed that the size of aggregates in dry-heated WPI is dependent on heating time, pH of the concentrate, and the water activity of the powders. Manipulation of dry-heating conditions affects the gelation properties of reconstituted dry-heated whey proteins.

6.4 EFFECTS OF COMMERCIAL THERMAL PROCESSING ON WHEY PROTEIN FUNCTIONALITY 6.4.1

Rheological Properties

Heat-induced gels are produced by classical denaturation/aggregation processes, in which unfolded whey proteins interact with each other through electrostatic, hydrophobic interactions, and
SH/
S
S
interchange reactions. The ability of whey proteins to form gels capable of holding large amounts of water is of great importance for the food industry (Spotti, Tarhan, Schaffter, Corvalan, & Campanella, 2017). The rheology of whey protein structures is highly dependent on the interplay between temperature, concentration, pH, ionic strength, and shear. Tang, Munro, and McCarthy (1993) reported that WPC solutions at 22 C and pH 7.0 exhibit Newtonian behavior up to a concentration of about 10% total solids. Generally, the globular proteins behave as inert particles in dilute solutions or dispersions and lack the natural ability to interact with each other to form more complex structures. Increasing the whey protein concentration results in pseudoplastic behavior between 10% and 30% before showing time-dependent shear thinning behavior at solids contents of 35% and higher. The effect of temperature on apparent viscosity from 5 C to 60 C was shown to follow Arrhenius behavior. Apparent viscosity was significantly increased by addition of CaCl2 whereas NaCl had little or no effect on viscosity. Following heating above 60 C, WPC solutions changed from time-independent to time-dependent shear behavior at high concentrations, high and low pH values, and high CaCl2 concentrations. Solutions exhibited thixotropic behavior at 40% solids (at 22 C and pH 6.75). Even with these deviations from Newtonian behavior at certain concentrations, globular proteins do not tend to confer structural heterogeneity. Further work by Tang, McCarthy, and Munro (1995) showed that the structure of WPC gels was affected by pH, salt concentration, and salt type (CaCl2 or NaCl). Cooney, Rosenberg, and Shoemaker (1993) studied the rheological properties of heated WPC gels and found that the complex modulus |G*| and tan δ

6.4 Effects of Commercial Thermal Processing on Whey Protein Functionality

increased with decreasing temperature from 90 C to 30 C and 90 C to 60 C, respectively. The dependence of |G*| and tan δ on temperature remained constant during heating and cooling between 30 C and 70 C, indicating that rheological changes were reversible within this temperature range. Guo, Ye, Lad, Dalgleish, and Singh (2013) examined the large deformation properties (relevant to breakdown properties in the human mouth) of heat-set whey protein emulsions measured by uniaxial compression testing. Heating at different concentrations of NaCl formed whey protein emulsion gels with different structures. Gel hardness increased with increasing NaCl concentration and this was correlated with greater fragmentation in the human mouth. Fragmentation in the mouth correlated highly with mechanical properties of the gels. Vardhanabhuti, Foegeding, McGuffey, Daubert, and Swaisgood (2001) added various proportions of preheated whey protein (11% WPI heated at pH 7.4, 80 C for 1 hour) to native whey protein dispersions and prepared heat-set gels in the presence of NaCl (200 mM) or CaCl2 (10 mM). Addition of whey protein polymers increased gel fracture stress, storage modulus, water holding capacity and transparency. It was suggested that addition of whey protein aggregates induced a transition from particulate to a more fine-stranded gel structure. Previous work also demonstrated a transparent, fine-stranded gel network when heated according to a two-step heating method (Doi, 1993; Tani et al., 1993). These gels were significantly harder than the gels produced by a single-step heating method. McClements and Keogh (1995) observed that the gelation temperature of aggregated whey protein dispersions was significantly lower than for native proteins (48 C and 77 C, respectively). The presence of heat-aggregated whey proteins is desirable in yogurt manufacture. The influence of mechanical shearing on the small deformation properties and microstructure of heat-induced whey protein gels has been studied in some detail. The response of proteins to applied shear stresses is an inevitable factor in food processing and physiology, and offers ways in which the material properties of whey protein gels can be modified. Heavily sheared WPC formed weak gels with lower storage modulus than equivalent unsheared gels (Walkenström, Windhab, & Hermansson, 1998). Moakes, Sullo, and Norton (2015) prepared thermally stable fluid gels via heatinduced gelation of 10 wt% WPI solutions under controlled conditions of temperature and shear. The size of aggregates was inversely proportional to shear rate with particles .120 and ,40 nm, for low and high shear, respectively. All suspensions showed marked shear thinning behavior associated with particle break-up and was more apparent for larger, low-shear aggregates. Particulate systems, at high volume fraction were reported to resemble a pseudo-solid material. In addition, it was shown that at a given volume fraction, the elasticity of the suspension varied depending on processing

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conditions to the degree of particle 2 particle interactions. Badii, Atri, and Dunstan (2016) examined the viscoelastic properties of whey protein gels as a function of concentration (10 and 20%, w/w) and different shear rates (0, 50, 100, 200, and 500 second21) during gelation. The storage modulus of gels was increased by increasing the shear rate exposure during gelation, while the shear-treated gels were more elastic and showed frequencyindependent behavior. The increase in the elastic modulus was attributed to an overall increase in density of cross-links in the system. Zhang, Arrighi, Campbell, Lonchamp, and Euston (2016) examined the properties of partially denatured whey protein concentrates (PDWPCs) using a heating process that allowed control of whey protein denaturation by following changes in the free
SH content. Soluble whey protein aggregates were produced with a diverse range of particle size and morphology (compact particle gel to open fibrillar structure) with whey protein denaturation in the range from 45% to 93% and particle size of 3
17 μm. Viscosity of solutions was dependent on aggregate structure and was lower in more compact particles. Viscosity of same-size particles was also dependent on their surface morphology with higher viscosity in particles with rougher surfaces. The shear viscosity behavior of PDWPCs was shown to differ markedly from that of both WPC and microparticulated whey proteins. This study suggests that partial denaturation technology provides a means of controlling the structure of soluble WPC aggregates with specific rheological characteristics.

6.4.2

Surface Properties

Protein adsorption is a spontaneous thermodynamic process driven by (1) an entropic gain due to the higher conformational flexibility of the unfolded amino acid chain and removal of the water cage surrounding the native protein, and (2) an enthalpic gain due to the dehydration of hydrophobic surface patches of the native protein (Mezzenga & Fischer, 2013). Protein adsorption at interfaces is influenced by those factors that also drive protein denaturation and aggregation, i.e., temperature, pH, and ionic strength. It is generally accepted that during adsorption proteins undergo structural changes. During the course of emulsion formation (homogenization) whey proteins adsorb at the surface of a newly formed oil droplet and reduce the interfacial tension between the oil and water phases. Adsorbed protein layers are able to stabilize oil droplets against flocculation and coagulation. Adsorption at the interface is thermodynamically favorable because hydrophobic residues are removed from the bulk aqueous phase and are reoriented toward the oil phase. As a result, the adsorbed globular protein

6.4 Effects of Commercial Thermal Processing on Whey Protein Functionality

structure assumes an intermediate structural state also referred to as the molten globule state. Globular proteins such as β-Lg, α-La, and BSA undergo relatively minor structural changes during this process. In the case of β-Lg, partial unfolding at the interface can expose
SH groups and lead to polymerization reactions and the development of a viscoelastic layer (Dickinson & Matsumura, 1991). Globular proteins, such as β-Lg, tend to form surface layers of deformable but nonoverlapping particles (Mezzenga & Fischer, 2013). This structural rearrangement is considered to be nonreversible and once adsorbed at the interface, proteins do not tend to desorb and exchange with proteins in the continuous phase (Dickinson et al., 1990). Reducing the protein concentration in the continuous phase does not cause denatured proteins to desorb from the interface (Mezzenga & Fischer, 2013). It has been documented that when β-Lg is adsorbed at an interface, displacement by other proteins, including β-casein or αs1-casein, is unlikely to occur (Dickinson & Matsumura, 1994). The stability of an emulsion decreases over time, due to changes in the protein conformation along with the thermodynamic drive for phase separation (Tcholakova, Denkov, Ivanov, & Campbell, 2006). The improvement of the emulsifying and foaming properties of β-Lg and WPI after heat treatment has been reported in several studies (Dissanayake & Vasiljevic, 2009; Kim, Cornec, & Narsimhan, 2005; Moro, Gatti, & Delorenzi, 2001; Phillips, Schulman, & Kinsella, 1990; Zhu & Damodaran, 1994). Heat treatment affects the rheology and structure of milk protein emulsions and affects emulsion particle size. McSweeney, Mulvihill, and O’Callaghan (2004) attributed increases in emulsion particle size to aggregation of fat droplets due to interactions between adsorbed and nonadsorbed serum phase proteins. Euston, Finnigan, and Hirst (2000) suggested that such protein 2 protein interactions were the cause of enhanced creaming rates. Keowmaneechai and McClements (2006) reported emulsion viscosity, creaming instability, and attributed droplet flocculation to increasing surface hydrophobicity following heat-induced denaturation of whey proteins at the interface. Demetriades, Coupland, and McClements (1997) reported that the effects of thermal processing on emulsion behavior is dependent on the extent of protein denaturation at the oil 2 water interface. Flocculation of emulsion droplets at pH 7.0 caused an increase in emulsion particle size following heating from 65 C to 80 C in 30 minutes. Further heat treatment ( . 80 C) resulted in a decrease in particle size. Dickinson and Parkinson (2004) also showed that emulsions made with β-Lg had significantly larger particle size distribution when heated at temperatures higher than 85 C for time periods from 30 minutes to 48 hours. It was also shown that when sodium caseinate was added to the system, whey

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protein emulsion was much more stable against heat-induced flocculation. It was proposed that this increased stability was the result of a steric hindrance effect due to the long (predominantly polar) tails, which prevented flocculation. Stabilization of whey protein emulsions by aggregated proteins has also been examined by Knudsen at al. (2008) and Bernard, Regnault, Gendreau, Charbonneau, and Relkin (2011). They have shown that the apparent viscosity of emulsions could be significantly increased and that entrapment and adsorption of protein aggregates contributed to a more cohesive interfacial film. The majority of the studies using proteins as emulsifiers have reported on intact proteins rather than hydrolysates. Partial hydrolysis has been used to improve the emulsifying properties of proteins by exposure of hydrophobic amino acids and by changing surface hydrophobicity. Excessive hydrolysis, however, can lead to saturation of the continuous phase rather than adherence to the interface (Conde & Patino, 2007). Murphy et al. (2015) demonstrated that emulsions of infant milk formula containing whey proteins in which β-Lg was selectively hydrolyzed had lower viscosities than formulations containing nonhydrolyzed ingredients and therefore offer greater potential for the production of infant formula powders at high solids levels (it is advantageous to minimize the amount of water to be removed during spray-drying). Schröder, Berton-Carabin, Venema, and Cornacchia (2017) reported that surface hydrophobicity of whey protein decreased with the extent of hydrolysis. Whey proteins formed droplets with stronger interfacial layers and had higher surface coverage compared to whey protein hydrolysates (WPHs). Whey protein peptides smaller than 5 kDa formed weak interfacial films and unstable emulsions. Larger peptides (.5 kDa) were preferentially adsorbed. Small whey protein peptides (,5 kDa) formed a weak oil 2 water interfacial film, which led to unstable emulsions. Lam and Nickerson (2015) showed that pH and temperature altered the emulsifying properties of holo- (with bound calcium) and apo- (without bound calcium) α-La through changes to surface hydrophobicity and surface charge. Emulsions with holo α-La were more stable at pH 7.0 compared to pH 5.0 due to electrostatic repulsion and increased hydrophobicity. It is known that Ca21 stabilizes α-La against thermal unfolding. Buggy, McManus, Brodkorb, Mc Carthy, and Fenelon (2017) demonstrated that increasing the concentration of α-La in heat-treated infant milk formula emulsions resulted in a significant reduction in large soluble aggregates. Increasing α-La content reduced viscosity, especially in emulsions formed prior to heat treatment. Lowering the proportion of β-Lg resulted in fewer covalently bonded aggregates formed by heat treatment and led to a more stable emulsion. Schröder et al. (2017) showed that β-Lg preferentially adsorbs over α-La in whey-protein-stabilized emulsions.

6.4 Effects of Commercial Thermal Processing on Whey Protein Functionality

A significant body of work has also been done on the interfacial behavior of whey proteins with respect to foams and the effects of heating on foam formation and stability. Zhu and Damodaran (1994) showed that β-Lg monomers had pronounced interfacial activity, whereas aggregates improved foam film stability. Similarly, Davis and Foegeding (2004) determined the surface tension at various protein concentrations and confirmed that native proteins were the most surface active but that aggregates induced a bulk stabilization effect. Kim et al. (2005) reported that heating of β-Lg solutions at a range of pH values induced secondary structural changes and increased protein hydrophobicity especially at pH 4.0. Such conformational changes led to the development of highly surface active particles and a strong viscoelastic network at the interface. Rullier, Axelos, Langevin, and Novales (2010) showed that the presence of native β-Lg was required to improve the foamability but that aggregates, although poor with respect to surface activity, helped to reduce foam drainage. Ulaganathan et al. (2017) demonstrated that β-Lg is most surface active at pH 5.0 where net charge close to the pI is negligible and that adsorption kinetics of β-Lg at the water 2 air surface depend strongly on the protein effective charge, which is dictated by the solution pH and ionic strength.

6.4.3

Digestability of Whey Proteins

The quality or value of protein for human nutrition is commonly assessed by the protein digestibility-corrected amino acid score procedure (Schaafsma, 2000), which is a method recognized by the FAO/WHO. However, other methods such as the apparent or ileal (rather than fecal) digestibility, and measurement of plasma amino acids or nitrogen show strong evidence that the above methods may overestimate the nutritional value of some proteins. Furthermore, it became apparent that the kinetics of protein digestion profoundly affect the bioaccessibility, absorption, and subsequent assimilation of amino acid/nitrogens in the plasma and its metabolic response (Dangin et al., 2001). Some of the early work showed that not all proteins are equal as regards their digestion kinetics and that the molecular structure of the proteins can affect the mechanism and rate of digestion; the terms slow and fast proteins were coined (Mahe et al., 1996; Boirie et al., 1997). While the physiological response toward specific peptides may contribute to differing kinetics (Fruhbeck, 1998), it was later shown that the protein denaturation and/or aggregation behavior under gastric conditions can accelerate or delay gastric emptying, which, in turn, affects the kinetics of the overall postprandial protein metabolism. Whey proteins are associated with a number of beneficial physiological effects such as muscle synthesis, satiety, glycemic control to name but a few

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(see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins, Chapter 16, and Chapter 17). Recently, the fast digestion of whey proteins, compared to that of other commonly used food proteins, has given a new impetus to research into the digestive behavior of whey protein ingredients for products designed for sports nutrition, where fast delivery of proteins is seen as beneficial under certain circumstances. In their native state, the main whey proteins β-Lg and α-La remain soluble at the gastric pH range of 1
4. Gastric emptying of partly digested food into the small intestine via the pyloric valve is related to the caloric value of the food and also the physical structure of the stomach chyme, namely its viscosity and particle size. Hence, liquid foods such as whey protein solutions have a shorter gastric residence time than solid foods or foods that restructure under gastric conditions into solids or gels. Several studies in vitro (Lambers, van den Bosch, & Jong, 2013; Mulet-Cabero, Rigby, Brodkorb, & Mackie, 2017) and in vivo (Mackie, Rafiee, Malcolm, Salt, & van Aken, 2013) have demonstrated that gastric restructuring in dairy systems, especially the coagulation behavior of caseins under acidic conditions, seem to delay gastric emptying and the subsequent metabolic response. In contrast to this, the main whey proteins, β-Lg and α-La, are both globular proteins that exhibit a relatively high resistance to proteolysis due to the inaccessibility of the protein chain to enzymes. The resistance to pepsin hydrolysis during the gastric digestion was demonstrated by using in vitro digestion models (Dupont et al., 2010) and in vivo extraction using nasogastric tubes (Sullivan et al., 2014) or double-lumen nasogastric tube that migrated to the proximal jejunum (Boutrou et al., 2013) in healthy adults. However, most milk products undergo some type of industrial processing, most commonly heat-treatment. Temperatures above the denaturation temperature of whey proteins (B70 C) cause unfolding and subsequent aggregation, as described in this chapter. This generally facilitates the exposure of the protein chain to enzymes, hence protein hydrolysis can be is accelerated significantly by heat treatments as shown in vitro (O’Loughlin et al., 2012, 2013) and in vivo (Lacroix et al., 2008). Over the last decade, significant progress has been made in understanding the mechanism of adult digestion of whey proteins, mainly by combining static or dynamic in vitro models (Bellmann, Lelieveld, Gorissen, Minekus, & Havenaar, 2016; Dupont et al., 2010; Minekus et al., 2014) with in vivo models. However, other population groups such as infants and elderly, where an increased consumption of whey proteins is considered beneficial, lack appropriate models at this time (Levi et al., 2017). However, considerable research is ongoing due to the increasing interest in understanding the digestion of food.

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

6.5 FUTURE CHALLENGES: PREVENTION OF DENATURATION/AGGREGATION OF WHEY PROTEINS 6.5.1

Physicochemical Modifications

Whey protein intake has been linked to many health benefits, which promotes its utilization within the food industry. Despite their excellent functional properties, the stability of whey proteins has been reported to deteriorate during commercial processing, which can be problematic for their application in food industry. Processing used in the food industry promotes interactions of whey proteins with other food components, and changes to their environment conditions (temperature, pH, ionic strength) ultimately alter their functional properties. Therefore, there has been a great deal of interest in reducing/preventing denaturation and aggregation of whey proteins through physical, chemical, and structural modifications.

6.5.1.1 Physical Modification Many studies have revealed the potential application of novel processing technologies to control the heat stability of whey proteins (Ashokkumar et al., 2008, 2009; Dissanayake & Vasiljevic, 2009; Jambrak, Mason, Lelas, Paniwnyk, & Herceg, 2014; Koh et al., 2014; Martini, Potter, & Walsh, 2010; Oboroceanu et al., 2011; Spiegel, 1999; Spiegel & Huss, 2002; Zhang & Zhong, 2009; Zisu, Bhaskaracharya, Kentish, & Ashokkumar, 2010; Zisu et al., 2011). Research in such an area is continuously growing and is not only limited to thermal processing technology, but also to high-pressure processing, high-shear technology, and nanotechnologies (see also Chapter 8: Novel Processing Technologies: Effects on Whey Protein Structure and Functionality). Preheating of milk before evaporation is well known to improve the heat stability of whey proteins, especially to heating at high temperatures (.100 C) and for long times (B30 minutes) (Deysher, Webb, & Holm, 1929). Although early studies found that a combination of preheating and downstream homogenization was ineffective in improving heat stability (Deysher et al., 1929; Webb & Bell, 1942), more recent research has suggested the opposite. In this regard, the combination of preheat treatments with several processing technologies has been found to work synergistically when used to reduce denaturation/aggregation of whey proteins. For example, preheat treatment prior to sonication considerably improves the heat stability of whey proteins by deactivating free
SH groups and breaking the polymer aggregate chains (Ashokkumar et al., 2009; Martini et al., 2010; Zisu et al., 2010). The increase in heat stability was indicated by a dramatic reduction in viscosity and aggregate size. It was suggested that preheating treatments (80 C, 1 minute) produced large soluble aggregates via hydrophobic

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and
SH/
S
S
interchange reactions, which break into smaller particles upon sonication due to the physical shear forces generated during acoustic cavitation. Additionally, it was observed that ultrasonication is mostly effective at a protein concentration of 35.6% (w/w, dry basis) and when ultrasound was applied using a 20 Hz generator for 1 minute with 15 W electrical power at 60 C (Martini et al., 2010). A similar modification in particle size and viscosity was observed during microfluidization of preheated whey proteins (140 MPa) (Dissanayake & Vasiljevic, 2009). The combination of heat treatment (90 C, 20 minutes) and high-pressure shear considerably increased the heat coagulation time of whey protein samples. High-pressure shearing during microfluidization disrupts the microstructural aggregates formed during preheat treatment, and alters intra- and intermolecular interactions within or between protein molecules (Bouaouina, Desrumaux, Loisel, & Legrand, 2006). As a result, interactions between protein molecules are enhanced and the amount of available reactive sites is greatly reduced, which consequently increases the overall heat stability of whey proteins. Other research has shown the effectiveness of combining preheat treatment and Ultra-Turax high-shear mixing or homogenization to improve the heat stability of whey proteins (Koh et al., 2014). It was shown that homogenization (80 bar, 8 MPa) of preheated (80 C, 1 minute) whey protein solutions (WPC80, 5% w/w) gave similar particle size distribution and viscosity results to that of preheated and sonicated whey samples. This finding suggested the important unique role of high-shear forces alone in improving the heat stability of whey proteins, and eliminates the role of cavitation as previously postulated by Ashokkumar et al. (2009) and Zisu et al. (2010). Furthermore, the combination of preheat treatment and Ultra-Turax high-shear mixing (17,500 minute21) was less effective in reducing the average aggregate size in comparison to homogenization or sonication treatment, but heat stability of the whey proteins was still significantly improved. Another physical approach to produce heat-stable whey proteins is through the application of microencapsulation. Whey proteins are pretreated by utilizing microemulsions to modify their ability to form intermolecular bonds (Zhang & Zhong, 2009). These pretreated whey proteins are called whey protein nanoparticles; their average size is dependent on the pH of the native whey protein (5% w/w) solutions. Thermal pretreatment (90 C, 20 minutes) of a WPI solution adjusted to pH 6.8 will produce whey protein nanoparticles ,100 nm. Upon further heating to 80 C for 20 minutes and in the presence of 100 mM NaCl, the whey protein nanoparticles are very stable with no gel formation and have a translucent appearance. These studies have demonstrated the potential application of microencapsulation to produce whey protein nanoparticles with size less than 100 nm for clear

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

beverage applications. The authors, however, suggested the combination of enzymatic cross-linking and microencapsulation to reduce the turbidity of whey protein nanoparticles, thus improving their heat stability.

6.5.1.2 Chemical Modification 6.5.1.2.1 Derivatization of Amino, Carboxyl, and Sulfhydryl groups Numerous attempts have been made to improve the functional properties and heat stability of whey proteins through chemical modification, both using food and nonfood grade products. Depending on the type of reagent used for modification, the functional side chain groups in whey proteins can be modified to create protein species with substantially altered chemical and structural characteristics. Chemical reactions that take place on the amino groups, like succinylation or acetylation, decrease the positively charged amino groups due to their replacement by negatively charged succinyl or neutral acetyl groups. The derivatization of the amino groups leads to a decrease in the isoelectric point of the protein and promotes electrostatic repulsion (Alting, de Jongh, Visschers, & Simons, 2002; Vidal et al., 1998). Succinylation of β-Lg decreases the pH (pH 2.5) at which cold-set acid whey protein gels are formed in comparison with nonmodified native β-Lg (pH 5.1). Improved solubility and emulsification properties at acidic pHs was reported upon esterification of free carboxylate groups of β-Lg, which is an important finding for the application of whey proteins in acid foods or soft drinks (Chobert, 2012). Esterification of β-Lg increases the net positive charge by blocking the negative charges of carboxylates, and raises the protein’s isoelectric point to 6.2 (esterified with butanol), 8.7 (esterified with ethanol), or 9.8 (esterified with methanol). As a result, the solubility of β-Lg is significantly improved in the acidic pH range. Addition of allyl isothiocyanate (AITC) to whey protein has been found to alter the functionality of proteins by forming covalent adducts with free
SH and free amino groups (Keppler et al., 2014; Keppler, Koudelka, Palani, Tholey, & Schwarz, 2014; Keppler et al., 2017; Rade-Kukic, Schmitt, & Rawel, 2011). The AITC conjugation reaction is promoted at elevated pH upon deprotonation (Nakamura, Kawai, Kitamoto, Osawa, & Kato, 2009) and controllable to a certain degree by changing the protein:AITC ratio, indicating that the N terminal part of the protein is the most reactive to the electrophilic C atoms of AITC (Keppler, Koudelka, Palani, Tholey, et al., 2014). It was shown that binding of AITC (0.05
20 mM) to β-Lg (0.5 mM) at pH 7.1 caused dissociation of the native dimers and partial unfolding of the protein structure, whereas at pH 4.0 the native β-Lg would be transformed into a molten globule conformation (Rade-Kukic et al., 2011). Interestingly, conjugation reduced aggregation of β-Lg when heated to 85 C for 15 minutes at neutral pH (pH 7.1), but promoted formation of large aggregates at pH 4.0.

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Current findings revealed that the covalent modification of WPI (5 or 10 g/L) with AITC significantly influenced the physicochemical properties of the WPI at acidic pH (pH 2 and 4), but less significantly at pH 6.0 and pH 7.0. The results showed that the modified WPI had increased hydrophobicity and altered secondary and tertiary structures of the proteins; this resulted in a loosening of the protein folding and a consequent change in the emulsifying capacity of the protein (Keppler et al., 2017). Chemical modification of β-Lg via reactive
SH or
S
S
groups using protein-structure perturbing agents such as N-ethylmaleimide (NEM), dithio (bis)-p-nitrobenzoate (DTNB), urea, sodium deodecyl sulfate (SDS), dithiothreitol (DTT), cysteine, mercaptoethanol, glutathione, or dihydrolipoic acid (DHLA) to improve its heat stability has been reported (Boye et al., 2004; Considine, Patel, Singh, & Creamer, 2005; Sakai, Sakurai, Sakai, Hoshino, & Goto, 2000; Sawyer, 1969; Smyth, Konigsberg, & Blumenfeld, 1964; Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth 2014; Wijayanti, Oh, Sharma, & Deeth, 2013; Zhu & Labuza, 2010). These studies have enhanced our understanding of the fundamental aspects of whey proteins during heat-induced denaturation and aggregation reactions. The exposure of the
SH group is known to induce the formation of inter- and intramolecular protein complexes or aggregates via
SH/
S
S
interchange reactions, hence blocking the participation of the
SH group in the aggregation reactions can be an effective means of improving the overall heat stability of whey proteins. Addition of DTNB, a specific
SH reagent, to a denatured β-Lg solution induces formation of free thiol-p-nitrobenzoate (TNB) that can be detected by UV-VIS at 412 nm, and a covalently-linked TNB-protein derivative that can be detected by UV-VIS at 352 nm (Sakai et al., 2000). A rapid technique to identify the binding of heat-induced β-Lg with DTNB was developed using electrospray ionization-mass spectrometry technique (Wijayanti, Waanders, Bansal, & Deeth, 2015). It has been postulated that modification of β-Lg with DTNB disturbs the hydrophobic core of denatured β-Lg, which enhances dissociation of native dimers to monomers that are less reactive than the nonmodified monomers (Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth 2014). Although formation of smaller aggregates is still observed, formation of higher-molecular-weight aggregates is prevented. These authors further studied the reaction of NEM or DHLA with β-Lg (at molar ratio of 1:1) and found a significant decrease in the formation of covalently linked aggregates (Wijayanti, Bansal, & Deeth, 2014; Wijayanti, Bansal, Sharma, & Deeth, 2014). Reaction of NEM with proteins occurs via nucleophilic attack on the
SH group of cysteine (Smyth et al., 1964; Smyth, Nagamatsu, & Fruton, 1960). Wijayanti, Bansal, and Deeth (2014), Wijayanti, Bansal, Sharma, & Deeth (2014) demonstrated that the presence of NEM during heat-induced aggregation of β-Lg solution, in both pure β-Lg and a mixed whey protein system, considerably reduced the

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

formation of intermediate (trimer or tetramer) and larger molecular weight aggregates. Other authors have found that addition of blocking reagents, such as mercaptoethanol, during heating inhibits the primary aggregation of β-Lg, and thus prevents formation of protein complexes (Sawyer, 1969). This is due to dissociation of β-Lg aggregates or complexes as a result of mercaptoethanol’s specific reactivity toward the intermolecular
S
S
bonds. Addition of SDS, urea, or cysteine has also been reported to increasing the thermal stability of β-Lg (Boye, Ismail, & Alli, 1996; Boye et al., 2004; Considine et al., 2005; Sawyer, 1969). SDS and urea are known to disrupt hydrogen/hydrophobic interactions, while cysteine disrupts the
SH/
S
S
interchange reactions (Boye et al., 2004). Extensive unfolding of β-Lg and increased formation of aggregates was observed upon addition of 50 mM SDS. On the contrary, an increase in thermal stability of β-Lg was noted in the presence of 10 mM SDS (Boye et al., 2004). Addition of SDS to β-Lg at a molar protein:ligand ratio of 1:1 was found to stabilize the native structure of β-Lg against heat-induced structure flexibility, subsequent unfolding, and denaturation (Considine et al., 2005). These authors also noted the significant formation of a hydrophobically associated nonnative dimer in the presence of SDS. Zhu and Labuza (2010) reported the ability of cysteine (cys) to delay protein aggregation and hardening of WPI protein bars by reducing the
SH/S
S interchange reactions (cys/WPI 5 0.05). However, a higher relative amount of added cysteine (cys/WPI 5 0.25) accelerated the bar hardening, thus it is necessary to avoid excess addition of cysteine and determine the critical concentration of cysteine when using it as a
SH blocking reagents. More recently, Sutariya and Patel (2017) have found the potential application of hydrogen peroxide (H2O2) to improve the heat stability of WPI solutions (12.8% w/w protein, pH 6.5). In comparison to the non-H2O2 treated samples, the WPI solution treated with H2O2 remained in a liquid state and did not form a gel after heat treatment at 120 C for 150 seconds. It was proposed that treatment of whey proteins with a sufficient level of H2O2 (0.072 and 0.144 hydrogen peroxide to protein ratios) would lead to interaction of H2O2 with the reactive free
SH group of whey protein and convert the free
SH of Csy121 to a stable form (R-SO3H, cysteine sulfonic acid), thus preventing intermolecular
S
S
/
SH interchange reactions. H2O2 is permitted for use by Codex for Federal Regulation in colored whey as a bleaching agent, as an antimicrobial agent in starch (CFR, 2014), and during the preparation of modified whey by electrodialysis at a maximum concentration of 500 ppm (Sutariya and Patel, 2017). Therefore, a positive outcome of the study conducted by Sutariya and Patel (2017) would open the possibility for incorporation of H2O2 into various whey protein ingredients and enable manufacturers to benefit from an improved heat stability of whey proteins during commercial thermal processing.

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6.5.1.2.2 Enzymatic Cross-Linking Enzymatic cross-linking of whey proteins is one possible means of improving the heat stability of whey proteins through formation of covalent cross-links between the reactive proteins, thus limiting the accessible active sites for aggregation reactions with other proteins. A number of enzymes can catalyze cross-linking reactions of proteins, however, only transglutaminase (TGase) is classified as food grade and commercially available. TGase catalyzes the cross-linking reactions by acyl-transfer between a γ-carboxyamide group of glutamine residues and a ε-amino group of lysine residues forming a ε-(γ-glutamyl)lysine isopeptide bond and one molecule of ammonia (Folk & Finlayson, 1977). TGase-catalyzed cross-linking of whey proteins has been extensively studied, but the manner in which the cross-linking improves the heat stability of whey proteins remains a challenging area. This is because the native globular structure of whey proteins hinders most of the target amino acid side chains of the cross-linking enzyme (Ercili-Cura et al., 2012). Nevertheless, the reactivity of whey proteins toward TGase can be improved by complete or partial denaturation of the protein structure. Several attempts have been made to increase the extent of cross-linking, such as addition of reducing agents, increasing pH, and pretreatment by heat or high pressure (Damodaran & Agyare, 2013; Ercili-Cura et al., 2012; Tanimoto & Kinsella, 1988; Tang & Ma, 2007; Zhong, Wang, Hu, & Ikeda, 2013) (Fig. 6.5). The optimum conditions for TGase activity to cross-link β-Lg were reported to be pH 8.0 and 4 mM calcium (Tanimoto & Kinsella, 1988). Cross-linked β-Lg formed polymers with molecular masses ranging from 22
99 kDa

FIGURE 6.5 Schematic transglutaminase (TGase)-catalyzed cross-linking of β-Lg. The folded native β-Lg image was generated from PDB file using RasMol (version 2.7.4.2), where glutamine is displayed as ball and stick, and lysine as wireframe. The scheme is an illustration of the results of Tang & Ma (2007), Tanimoto & Kinsella, (1988) and Zhong et al. (2013).

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

(B40%) to .100 kDa (B35%) (Fig. 6.5). These polymers were readily dispersed and remained stable at protein concentrations up to 5% (w/v) upon heating to 100 C for 30 minutes. This is in contrast to native β-Lg, which rapidly forms gels or precipitates under similar conditions. It was not known, however, whether the increased heat stability of polymerized β-Lg was due to an increased number of intramolecular cross-links between glutamyl and lysine residues, to intermolecular cross-linking, or to a combination of both. To date, there is no clear theory on how TGase improves the heat stability of β-Lg. Additionally, extensive polymerization of β-Lg (up to β-Lg concentration of 10% w/v) has been found to reduce the gel strength, suggesting the potential application of TGase for producing soft custard-like structures useful in food product formulation. Other researchers have found the importance of dithiothreitol (DTT) in TGase-induced whey proteins cross-link reactions to cleave the protein
S
S
bonds and make lysyl and glutamyl residues accessible to the active site of TGase (Aboumahmoud & Savello, 1990). Tang and Ma (2007) found a remarkable increase in the heat stability of β-Lg upon extensive TGase pretreatment, regardless of the presence of DTT. Excess incubation treatment up to 23 hours prevented formation of oligomers and aggregates, which suggests conformational changes of β-Lg induced by TGase and the rearrangement to a more compact conformation post extensive treatment. This finding indicates the possibility of using TGase to improve the heat stability of whey proteins by increasing the extent of cross-linking, and without the use of reducing conditions. The TGase enzymatic activity was reported to have a linear correlation with thermal denaturation of whey proteins (Gauche, Barreto, & Bordignon-Luiz, 2010). A remarkable increase in enzymatic action occurred when whey proteins were heated to .85 C prior to enzymatic reaction with TGase. The denaturation of whey proteins results in the exposure of internal amino acids (e.g., glutamine and lysine) to the enzymatic reaction, which promotes polymerization. Other research has shown enhanced thermal stability of WPI (5% protein, w/w) following heat shocks (70 C for 5 or 10 minutes) and TGase treatments at neutral pH (Damodaran & Agyare, 2013). Nevertheless, significant precipitation at pH 4.0
5.0 via decreasing the hydrophilic/hydrophobic ratio of the water-accessible protein surface was observed. On the other hand, sequential preheating (80 C for 15 minutes) and TGase pretreatments (15 hours incubation time) were able to improve stability of 5% (w/v) WPI at pH 7.0 during thermal sterilization (138 C for 30 minutes) (Zhong et al., 2013). The sequentially pretreated WPI dispersions remained transparent and heat-stable even in the presence of 50 mM NaCl. Whey proteins are partially denatured during preheat treatment at 80 C for 15 minutes and the presence of NaCl (50 mM) promotes aggregation between the

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protein particles due to the increased ionic strength. Enzymatic covalent cross-linking is promoted in such conditions. The increase in heat stability resulting from reaction with TGase is an important finding for the manufacture of shelf-stable transparent beverages with a high content of whey protein (5%). The application of TGase is not only limited to improving the heat stability of whey proteins, but also to modifying the immunogenicity of β-Lg (Villas-Boas, Vieira, Trevizan, de Lima Zollner, & Netto, 2010). Cross-linked polymerization of β-Lg was promoted by either preheating (80 C for 60 minutes) or addition of cysteine (0.25 M). However, the antigenic responses were only altered when β-Lg was polymerized in the presence of cysteine, suggesting molecular rearrangement or formation of hidden antigenic sites leads to decreased antigenic properties. Although the antigenicity was not completely removed, the results have shown a new possible approach of reducing allergenicity of β-Lg through the application of TGase.

6.5.1.2.3 Interaction With Carbohydrates In addition to the numerous attempts to further improve the heat stability of whey proteins via physical, chemical, or enzymatic cross-linking treatments, nonenzymatic modification via the Maillard reaction is another potential beneficial treatment (Chobert, 2012). The reaction involves condensation of the glycosidic hydroxyl group of a reducing sugar with an amino group of proteins, resulting in a sequence of reactions that terminates with the formation of brown polymers, known as melanoidins (Chuyen, 1998; Friedman, 1996; Ledl & Schleicher, 1990). The reaction occurs spontaneously and is accelerated by heating; most importantly it does not require a chemical catalyst (Chuyen, 1998). Therefore, such a reaction is a simple way of modifying the heat stability of whey proteins since it occurs naturally during heat treatment and is possible to control. β-Lg exhibits better thermal stability and improved emulsifying properties after glycation with several sugars (arabinose, galactose, glucose, lactose, rhamnose, or ribose) (Chevalier, Chobert, Popineau, Nicolas, & Haertlé, 2001). Earlier studies revealed the possibility of improving heat stability of whey proteins using glucose, mannose, and galactose (Nacka, Chobert, Burova, Léonil, & Haertlé, 1998) or disaccharides (Jou & Harper, 1996). The modification degree is directly affected by the sugar size; sugars with a shorter carbonic chain have more open-chain forms and are more reactive with the amino group of proteins (Chevalier et al., 2001). Additionally, the covalent sugar-protein bonds are relatively heat-stable and are not cleaved upon heating up to 90 C for 1 hours. At pH values near the isoelectric point of β-Lg, the glycated β-Lg was more heat-stable than the native β-Lg. This is an interesting finding as protein aggregation is accelerated at pH values close to the

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

isoelectric point. The most heat-stable β-Lg (at 2 mg/mL) was found after glycation with ribose or arabinose (0.217 M), indicating that these sugars are more reactive toward β-Lg than other sugars studied. Chevalier, Chobert, Dalgalarrondo, Choiset, and Haertle (2002) revealed important conformational changes of β-Lg in the presence of ribose or arabinose, while β-Lg glycated with lactose or rhamnose had similar three-dimensional structures to native β-Lg. Chen, Chen, Guo, and Zhou (2015) studied the effects of sugar structure (size, position of carbonyl group, and charge state) on the heat stability of β-Lg (10 mg/mL) and found that all β-Lg 2 sugar conjugates exhibited a higher denaturation temperature than native β-Lg when the glycation extent of reducing sugars was controlled to the same level (an average number of saccharides attached per protein molecule of β-Lg is 5). It was reported from this study that β-Lg 2 sugar conjugates could maintain their solubility at pH 3.0 and 7.0 as the temperature was increased from 60 C to 90 C, while a better heat stability was observed at pH 5.0. Furthermore, the sugar structure had a significant effect on increasing the denaturation temperature of β-Lg; a sugar with larger molecular size and negative charge would be more effective in improving the heat stability of β-Lg. Sugars with larger molecular size will promote stearic hindrance and prevent formation of large aggregates during heating at high temperature, while negatively charged sugars will dramatically alter the surface charge and shift the isoelectric point in a more acid direction, hence providing more electrostatic repulsion at pH 5.0 and reducing protein 2 protein interactions. Improving heat stability of whey proteins via glycation with various sugars (mono-, oligo-, and polysaccharide) has been claimed in a patent (Smulders & Somers, 2009). According to the patent, the effectiveness of the sugars in improving heat stability of whey proteins is strongly dependent on the concentration of whey proteins, pH, type of reducing sugar, and concentration of the sugar used for glycation. Nevertheless, it seems that glycation of whey proteins with sugars works best for improving heat stability of whey proteins if performed under mild conditions. The functional properties (solubility, heat stability, emulsifying, and foaming properties) of whey proteins after glycation with several polysaccharides (dextran, carrageenan, or maltodextrin (MD) have been studied (JiménezCastaño, López-Fandiño, Olano, & Villamiel, 2005; Spotti et al., 2014; Stone & Nickerson, 2012; Wang & Zhong, 2014; Zhu, Damodaran, & Lucey, 2008, 2010). Compared with mono- or disaccharides, conjugation of polysaccharides with proteins led to significant improvement in the thermal stability, and emulsification or antioxidant properties (Dunlap & Côté, 2005; Hattori, 2002; Nakamura, Kato, & Kobayashi, 1992). According to JiménezCastaño et al. (2005), glycation of β-Lg with dextran led to formation of

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high-molecular-weight complexes and induced disulfide-linked polymerization of the protein. The conjugate formed at 60 C, 0.44 water activity (aw), and 2:1 weight ratio of dextran to β-Lg, underwent less conformational change than a conjugate formed at 55 C, 0.65 aw, and 6:1 weight ratio of dextran to β-Lg (as measured by fluorescence intensity). The initial conjugate exhibited a higher solubility than the dry-heated β-Lg around the isoelectric point of the protein, but lower than the native protein at pH 4.0. Although glycated β-Lg had lower heat stability than native β-Lg upon heating at pH 7.0, its thermal stability was higher at pH 5.0 upon heating to 85 C. Other authors have found the ability of dextran (30% by weight) to act not only as a reactant but also as a protective agent (via a macromolecular crowding effect) in preventing excessive WPI (10% w/w) denaturation and aggregation during conjugation (pH 6.5, 60 C for 24 hours) (Zhu et al., 2008). These authors postulated that the extent of conjugate formation would increase with increasing proportion of polysaccharides, because each polysaccharide molecule has only one reducing group capable of reacting with amine groups in the proteins. They also confirmed that with dextran bound to WPI via covalent interactions. Further research conducted by this research group found that the purified WPI 2 dextran conjugate, produced in aqueous solution under mild conditions (60 C for 48 hours), exhibited good solubility over the pH range from 3.2 to 7.5 and at high ionic strengths (0.05
0.2 M), as well as good heat stability, regardless of pH and ionic strength, compared to native WPI. Additionally, WPI 2 dextran conjugates had greater emulsifying ability and the emulsions had greater stability compared with native WPI or gum arabic (a natural commercial glycoprotein emulsifier). It was assumed that the improved emulsifying properties of WPI 2 dextran emulsions were due to the following reasons: formation of a thick steric barrier during conjugation; increased oil droplet surface hydrophilicity; increased adsorptive ability of conjugates on the surface of oil droplets; and decreased oil droplet aggregation. Spray-dried powder made from WPI 2 maltodextrin solutions (2.5% w/v each) adjusted to pH 4.0
7.0 and glycated at 80 C (65% relative humidity) for 1
4 hours was investigated for its physicochemical properties (Wang & Zhong, 2014). The results highlight the improvement of heat stability upon pH adjustment of WPI 2 maltodextrin mixtures to pH 6.0 and color reduction of protein ingredients, making them suitable for transparent beverage applications. At pH 6.0 the reconstituted conjugate powder (5% w/v protein) had the highest degree of glycation and denaturation temperature, with lowest surface hydrophobicity upon heating at 138 C for 1 minute (0
150 mM NaCl, pH 4
7). This finding resulted in a patent on producing heat-stable protein ingredients via cross-linking proteins with carbohydrates (Zhong, 2011). Stone and Nickerson (2012) found that the structure of

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

conjugated WPI 2 carrageenan (κ, ι and λ type) strongly depended on the pH, salt, mixing ratio, and polysaccharide type. WPI 2 κ-carrageenan and WPI 2 ι-carrageenan formed soluble and insoluble complexes at pH 5.5 and 5.3, respectively, whereas WPI 2 λ-carrageenan formed soluble and insoluble complexes at pH 5.7 and 5.5, respectively. In all types of WPI-carrageenan mixtures, maximum turbidity was found at pH 4.5, but decreased at lower pHs. The addition of NaCl disturbed interactions within WPI 2 κ-carrageenan as levels were increased, but improved interactions within WPI 2 λ/ι-carrageenan up to a critical sodium concentration. Furthermore, emulsion stability was enhanced in the mixed systems (12:1 WPI:carrageenan), regardless of the type or carrageenan. Nonreducing sugars, which do not take part in Maillard reactions, can also affect the thermal stability of whey proteins. For example, some studies have shown the potential of sucrose (Baier & McClements, 2001, 2003b; Kulmyrzaev, Bryant, & McClements, 2000) and sorbitol (Baier & McClements, 2003a; Chanasattru, Decker, & McClements, 2007) to improve the heat stability of whey proteins. Excessive addition of sucrose must be avoided as it can increase protein 2 protein interaction. It has been found that addition of sucrose to a 10% (w/w) WPI solution is limited to a maximum 10% (w/w). Addition of sucrose (10
40%, w/w) to a 1% (w/w) BSA solution (pH 6.9) increased the thermal denaturation temperature of BSA (Baier & McClements, 2001). Furthermore, the addition of sucrose (20%, w/w) to 4% (w/w) BSA (in the presence of 100 mM NaCl) improved the heat stability of BSA as indicated by an increase in the gelation temperature and a decreased final gel turbidity (Baier & McClements, 2003b). Sorbitol is more effective than glycerol in improving the heat stability of whey proteins (Baier & McClements, 2003a; Chanasattru et al., 2007). The different effects were attributed to differences in the preferential interactions of the sugars with the protein surface as well as the rate of protein 2 protein collisions (Chanasattru et al., 2007).

6.5.1.2.4 Enzymatic Hydrolysis WPHs have improved solubility, decreased viscosity and gelling, as well as considerably changing physicofunctional (solubility, foaming, emulsifying) properties in comparison to native whey proteins (Konrad, 1996; Singh & Dalgleish, 1998; Van den Berg, 1979). The common enzymes used for hydrolysis of whey proteins are trypsin, chymotrypsin, pepsin and microbial proteinases. Nevertheless, native whey proteins are not easily hydrolyzed by pepsin and trypsin, due to their aforementioned compact tertiary structure that hides most of the enzyme-susceptible peptide bonds. Therefore, physical and/or chemical denaturation of whey proteins is necessary prior to enzymatic hydrolysis. For example, thermal treatments (60
75 C) increased the

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extent of hydrolysis of WPC93 by Proteinase K in neutral and alkaline pH environments (Stanciuc, Hintoiu, Stanciu, & Rapeanu, 2010). This is because at that pH and temperature, the native dimer of β-Lg, the major protein in whey, dissociates into monomers and releases the initially hidden hydrophobic residues to the outer surface, thus increasing the accessibility of the specific peptide bonds for the enzymatic cleavage. However, the extent of hydrolysis was significantly reduced upon further heating to 80 C, probably due to formation of covalent and/or noncovalent aggregates that hide the specific peptide bonds from enzymatic cleavage. Heating proteins prior to enzymatic hydrolysis partly determines the nature of peptides released during hydrolysis, and hence their resultant functionalities (Adjonu, Doran, Torley, & Agboola, 2013). Modification of whey protein conformation by sulfitolysis has been found to increase the hydrolysis rate by pepsin and trypsin in comparison to that of the intact proteins (Kananen et al., 2000). Sulfitolysis is a reversible reaction of sulfite with
S
S
bonds and the reaction is specific toward
S
S
bonds of cystine residues, hence, side-reactions with peptides and proteins can be avoided (Kananen et al., 2000). Thus sulfitolysis offers an attractive alternative to methods based on heat and pressure for modifying the conformation of whey proteins for subsequent proteolysis. The impacts of carbohydrate conjugation on thermal stability of WPH have been well documented (Drapala, Auty, Mulvihill, & O’Mahony, 2016a; Mulcahy, Park, Drake, Mulvihill, & O’Mahony, 2016). Conjugation of 5% WPI or WPH with 5% MD (degree of hydrolysis 9.3%) enhanced the stability of whey protein/peptide solutions when subsequently thermally treated in the presence of 40 mM NaCl (Mulcahy et al., 2016). Conjugation was achieved by heating solutions at 90 C for 8 hours at an initial pH of 8.2. The improved thermal stability of whey proteins conjugated with MD is possibly due to enhanced stearic hindrance as a result of the attachment of carbohydrate side chains that helps to reduce heat-induced aggregation. The authors further confirmed that the rate and extent of whey protein 2 carbohydrate conjugation was influenced by the physical state of the whey proteins (either intact or hydrolyzed). Heat stability of model infant formula emulsions based on hydrolyzed whey protein ingredients was markedly improved by modification of the protein ingredients through conjugation with carbohydrates (Drapala et al., 2016a; Drapala, Auty, Mulvihill, & O’Mahony, 2016b). Preheating of hydrolyzed whey protein prior to its use in infant formula emulsions resulted in enhanced heat stability of the final emulsions due to reduction of the level of reactive
SH groups through protein 2 protein interactions (Drapala et al., 2016a). In comparison with WPH 2 lecithin or WPH 2 CITREM (citric acid esters of mono- and diglycerides), WPH 2 MD has superior heat stability after infant formula emulsions were heated at

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

95 C for 15 minutes, and stored at 40 C for 10 days (Drapala et al., 2016b). The authors proposed that the covalent attachment of the MD to WPH reduces the interaction at the interface between the proteins/peptides 2 MD layer and proteins/peptides in the serum due to steric interference.

6.5.1.2.5 Addition of Hydrophobic Compounds Other modifications that have been made to prevent denaturation/aggregation of whey proteins include addition of low-mass surfactants (e.g., lecithins or free fatty acids) or chaperone proteins, and formation of soluble whey protein aggregates. Improving heat stability of whey proteins through the use of low-mass surfactants has received a great deal of attention (Considine et al., 2005; Giroux & Britten, 2004; Ikeda, Foegeding, & Hardin, 2000; Loch et al., 2012; Van der Meeren, El-Bakry, Neirynck, & Noppe, 2005). Ikeda et al. (2000) studied the effect of fatty acids (butyrate, oleate, and palmitate) and phosphatidylcholine (PC) on secondary structural changes in β-Lg during heat-induced gelation. In the absence of added NaCl, addition of both fatty acids and PC induced α-helix formation in β-Lg on heating to 80 C and increased the storage moduli (G0 ) of heat-induced gels. In the presence of NaCl (500 mM), PC did not interact with β-Lg, but interfered with gel network formation. On the other hand, butyrate caused significant unfolding of β-Lg upon heating resulting in the formation of elastic gels with increased G0 values. β-Lg unfolded substantially in the presence of palmitate and oleate, with 500 mM NaCl, upon heating and formed a gel even at room temperature (25 C). The heat stability of whey protein at 75 C (0
30 minutes, pH 7.5) in the presence of SDS, sodium stearoyl-2 lactylate (SSL), and diacetyl tartaric acid esters of mono- and diglycerides (DATEM) was greatly improved (Giroux & Britten, 2004). The protein solubility at pH 4.8 and denaturation temperature increased with increasing surfactant to protein ratio (from 10 to 80 μmol/g) when heated to 75 C. Complete saturation of β-Lg sites by the surfactants was reached at a concentration greater than 80 μmol surfactant per gram of protein, which leads to the unfolding of the tertiary structure (noncooperative binding). These anionic surfactants were able to bind to the surface of whey proteins via specific binding, and the interactions were related to the nature and concentration of the surfactant used. In this regard, SDS and SSL have the strongest interactions with whey proteins and thus are the most effective anionic surfactants for improving heat stability of whey proteins. A similar finding was on the effect of SDS (1:1.1 protein/ligand ratio) on heat-induced denaturation/aggregation of β-Lg B (1.5 mg/mL), in which SDS stabilized the native structure of β-Lg when heated and prevented unfolding and denaturation (Considine et al., 2005). In comparison to SDS, palmitate bound less tightly to β-Lg due to its larger hydrocarbon chain, thus providing slightly less protection to the native β-Lg against thermal denaturation (at 40
93 C for 12 minutes). These authors also

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found that retinol was less effective in stabilizing the native structure of β-Lg due to its low affinity toward the calyx-binding site, and 8-anilino-1naphthalenesulfonate (ANS) did not stabilize β-Lg since it has weak binding toward the hydrophobic cavity of β-Lg. The binding sites of β-Lg to fatty acids were studied by Loch et al. (2012) who postulated two different sites of binding. The first site was the central calyx located on the β-sheet strands, which mostly consists of hydrophobic groups, and the second site was on the surface of the protein. A fundamental study conducted by Tran Le et al. (2011) demonstrated that protein 2 surfactant (2.75 and 1% w/v, respectively) interaction takes place at room temperature and not upon heating although the effect of the interactions toward the heat stability of whey proteins became pronounced during thermal treatment. The interactions were studied comprehensively using high resolution as well as diffusion NMR techniques. The addition of hydrolyzed soybean lecithin (which contains lysophospholipids) (1% w/v) decreased G0 of whey protein gels, which indicates that the bound surfactants reduced heat-induced protein interactions and subsequent heat-induced protein aggregation. The stabilizing effect of hydrolyzed lecithin on the whey proteins has been attributed to its ability to reduce attractive protein 2 protein interactions during heating (Van der Meeren et al., 2005). Addition of Tween 20, Tween 80, and Brij 78 had smaller effects on whey protein gelation to lysolecithin. Furthermore, addition of 1% (w/v) SDS or sodium laurate to 2.75% (w/v) WPI resulted in no gel formation, whereas addition of sucrose palmitate, sucrose oleate, and sucrose laurate increased G0 of the whey protein gels.

6.5.1.2.6 Soluble Whey Protein Aggregates and Microparticulated Whey Proteins A possible approach to improve the heat stability of whey proteins is by formation of soluble whey protein aggregates (Nicolai et al., 2011; Purwanti et al., 2011; Ryan et al., 2012; Ryan, Zhong, & Foegeding, 2013). Formation of whey protein soluble aggregates by heating whey proteins at concentrations below their critical gelation concentration can improve the thermal stability of whey proteins via physical, chemical, and structural modifications. When a solution of WPI or β-Lg (7% w/w, pH 6.8) was heated for 10 minutes at 90 C, soluble aggregates were formed (Ryan et al., 2012). These soluble aggregates had improved thermal stability compared with the native proteins when further heated (second heating) to 90 C for 5 minutes in the presence of salt (0
108 mM NaCl). Another approach to improving the heat stability of various high-protein dairy products containing whey proteins is microparticulation (Çakır-Fuller, 2015; Ipsen, 2017; Mounsey, O’Kennedy, Corrigan, Kelly, & O’Callaghan, 2009; Saglam, 2011). The microparticulates are whey protein aggregates with

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

particle sizes ranging from 1 to 10 μm. The improved heat stability of dairy products containing microparticulated whey protein is due to the limited active sites available for aggregation during secondary heat treatment; this indicates the importance of the amount of native whey proteins on heat stability of dairy products (see also Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated).

6.5.1.2.7 Addition of “Chaperone” Proteins Another method of prevention of denaturation/aggregation is addition of chaperone proteins, which are proteins that assist in stabilization of other proteins by ensuring a proper protein folding conformation (Treweek, Meehan, Ecroyd, & Carver, 2015). Caseins have been reported to act as chaperone proteins during heat-induced denaturation/aggregation of whey proteins and ultimately improve the heat stability of whey proteins (Kehoe & Foegeding, 2011; Koudelka, Hoffmann, & Carver, 2009; Morgan, Treweek, Lindner, Price, & Carver, 2005; Treweek, Thorn, Price, & Carver, 2011; Yong & Foegeding, 2008). It has been proposed that the caseins interact with the exposed hydrophobic regions of unfolded target proteins by forming soluble high-molecular-weight complexes, and subsequently the phosphate groups on the surface of the complex hold the interactions within the complex via electrostatic repulsion, which ultimately stabilize the target protein against aggregation and precipitation (Matsudomi, Kanda, Yoshika, & Moriwaki, 2004; Morgan et al., 2005). The role of phosphate groups was demonstrated in dephosphorylation trials. Dephosphorylation of αs- and β-caseins markedly reduced their chaperone activity in preventing aggregation of DTT-reduced α-La and also in preventing heat-induced amorphous aggregation of some nonmilk proteins (Koudelka et al., 2009). It was suggested that the flexible, dynamic, and amphipathic nature of αs- and β-caseins are important features contributing to their chaperone activity. Kehoe and Foegeding (2011) showed that β-casein (10 g/L) altered heat-induced aggregation of whey proteins (50 g/L) as shown by a reduction in turbidity of the whey proteins upon heating at 80 C for 20 minutes or 145 C for 60 seconds. They also found that pH and ionic strength greatly affected the chaperone-like activity of β-casein. The effectiveness of β-casein as a chaperone protein was greatly increased at pH 6.0, but diminished as the pH was decreased to 5.8 or 5.5 or as the concentration of salts was increased (5
20 mM NaCl/CaCl2). These results provided new insight into the mechanism of the chaperone ability of β-casein which alters the size or shape of the aggregates formed rather than being involved in the kinetics and thermodynamics of whey protein denaturation/aggregation. The possibility of forming nanosized whey protein particles by increasing pH and casein content in concentrated commercial dairy protein mixtures with

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high whey protein contents (casein:whey protein ratios of 5:95
30:70, total solids content of 10% w/w) was investigated by Liyanaarachchi, Ramchandran, and Vasiljevic (2015). At pH values away from their isoelectric point (pH 6.7), the aggregate size of heat-induced whey proteins was reduced to a nanolevel (10
1000 nm) with a further size reduction to 50
250 nm upon increasing the pH to 7.5 when heated from 80 C to 120 C for 60 seconds. The chaperone-like activity of caseins at higher pH is due to increasing electrostatic repulsion of individual caseins, which causes loosening of hydrophobic domains of individual caseins which subsequently leads to a loosely packed micellar structure. Such a micellar structure would be greatly favorable for hydrophobic associations with unfolded whey proteins and ultimately lead to aggregation of whey proteins into smaller particles. This theory was confirmed from FT-IR results that indicated the refolding of heatinduced whey proteins into their original secondary structure. At lower pH (5.7) solubilization of calcium phosphate occurred, resulting in dissociation of caseins from the micelles thus allowing them to engage in interactions with whey proteins. However, the caseins tended to associate with each other forming micelles during their dissociation, thereby diminishing their chaperone-like activity at this pH. More recently, the heat stability and physicochemical characteristics of whey protein/casein aggregates were studied in a high-protein milk ingredients system (milk protein concentrate, MPC80) (Gaspard et al., 2017). The MPC80 was heated to 90 C for 25 minutes after adding calcium chloride (0
5 mM) (pH adjusted to 6.7 or 7.2), and subsequently fractions of whey proteins/ κ-casein aggregates were isolated after casein micelles and micelle-bound aggregates were separated by high-speed centrifugation. The chaperone activity of κ-casein was greatest at native whey protein to κ-casein ratio of 1:0.7 and stronger in the presence of αs- and β-casein. The mechanism of chaperon-like activity of κ-casein against the heat-induced aggregation of whey proteins was postulated to be similar to that of heat-shock proteins. In this regard, κ-casein would bind to whey protein aggregates via hydrophobic interactions forming a high-molecular-weight complex, and this complex would be solubilized by the hydrophilic part of κ-caseins, hence stabilizing the denatured whey proteins. Furthermore, addition of calcium chloride did not significantly modify the morphology (hydrophobicity) of κ-casein 2 whey protein aggregates within the fractions.

6.5.1.3 Structural Modification Structural modification of whey proteins as a result of nonthermal technologies, such as pulsed electric field (PEF) treatment or supercritical carbon dioxide (ScCO2), offers new insight on the application of nonthermal

6.5 Future Challenges: Prevention of Denaturation/Aggregation of Whey Proteins

technology to reduce aggregation of whey proteins (Perez & Pilosof, 2004; Sui, Roginski, Williams, Versteeg, & Wan, 2011; Sun, Yu, Zeng, Yang, & Jia, 2011; Xiang, Ngadi, Ochoa-Martinez, & Simpson, 2011; Xu et al., 2011; Zhong & Jin, 2008). PEF processing presents unique advantages over conventional heat treatment, for example, enhancement of drying efficiency (Taiwo, Angersbach, & Knorr, 2002), modification of enzymatic activity (Giner et al., 2002; Yeom, Streaker, Zhang, & Min, 2000), preservation of certain food ingredients (Jia, Howard Zhang, & Min, 1999), or providing consumers with microbiologically safe and nutritious food (Qin et al., 2016). Perez and Pilosof (2004) reported that PEF treatment (at 12.5 kV/cm using 10 exponentially decaying pulses of 2 μs) modified 40% of the native structure of β-Lg and found formation of covalently linked aggregates using electrophoresis. Similarly, Xiang et al. (2011) found that whey protein was denatured by approximately 43.7% after treatment with PEF (at 20 kV/cm and 30 pulses). As monitored by ANS fluorescence, PEF treatments on WPI (3 and 5% w/w) increased the WPI surface hydrophobicity. Furthermore, these authors have also demonstrated the importance of electric field intensity and pulse on structural modification of WPI as denaturation is enhanced with increasing pulses. In contrast, Barsotti, Dumay, Mu, Diaz, and Cheftel (2001) observed that PEF treatments (31.5 kV/cm using 200 exponentially decaying pulses at 1 Hz) on β-Lg solutions did not induce noticeable unfolding of the native β-Lg molecule. Similar findings by Li, Bomser, and Zhang (2005) showed no changes in the secondary structure of IgG after PEF treatment at 41 kV/cm for 54 μs (,44 C). More recently, PEF has been successfully used to improve the solubility and emulsifying properties of a WPI-dextran conjugate, as well as promote formation of glycosylation products in the WPI-dextran model system (Sun et al., 2011). Compared to the nontreated WPI (1% w/v), formation of glycosylation products in a mixed WPI-dextran system treated by PEF (at 15 or 30 kV/cm) successfully prevented heat-induced aggregation of WPI in aqueous solution. Glycosylation has been known to improve the thermal stability of β-Lg (Chobert, 2012; Jiménez-Castaño et al., 2005), which is discussed in detail in Section 6.5.1.2.3. Another study by Sui et al. (2011) reported the effect of PEF treatment (30
35 kV/cm 19.2
211 μs, 30
75 C) on the physicochemical and functional properties of 1% (w/w) WPI. Under the experimental conditions tested, PEF treatment did not affect the physicochemical (protein aggregation, surface hydrophobicity, or amount of total
SH groups) and emulsion stability properties of WPI, but reduced gel strength (from 461 to 139 or 67 Pa) and increased gelation time (from 40.8 to 43.2 or 47.9 minutes) of heat-induced gels. These findings may be beneficial for concentrated whey protein preparations and prior to spray drying, where whey protein gelation or precipitation is undesirable.

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Another structural modification approach is the application of ScCO2. ScCO2 has liquid-like density and gas-like diffusivity and viscosity, which lead to rapid wetting and penetration of complex structures (Rizvi, Mulvaney, & Sokhey, 1995). The ScCO2 conditions can be achieved with a critical temperature of 31.1 C and critical pressure of 7.38 MPa (Zhang et al., 2006). Zhong and Jin (2008) reported improved functional properties of WPI and WPC dispersions and powders after treatment with ScCO2. WPI and WPC solutions (10% w/v) were treated with ScCO2 at 40 C and 10 MPa for 1 hour, whereas WPI and WPC powders were treated with ScCO2 at 65 C and 10 or 20 MPa for 1 hour. Interestingly, improved gel strength was noticed for both WPI dispersions and powders post-ScCO2 treatment, but the improvement was most significant for the ScCO2-treated WPC powders. Additionally, the powders post-ScCO2 treatment were fine and free-flowing. The enhanced functional properties of the whey proteins were postulated to be due to compositional and structural changes as measured by surface hydrophobicity and rheological techniques, which enabled faster gelation and formation of a stronger gel. It has been shown that ScCO2 changes the secondary structure of proteins, in terms of the relative amount of α-helix, β-sheet, and random coil (Liu, Hsieh, & Liu, 2004; Striolo, Favaro, Elvassore, Bertucco, & Di Noto, 2003). The secondary changes were confirmed by Xu et al. (2011) who revealed a decrease in the α-helix content and hydrogen bonds, and an increase in the amount of β-sheet as a result of ScCO2 treatment at 20 MPa and 60 C for 1 hour. The disruption of hydrogen bonds was associated with a significant increase in turbidity and particle size, indicating the unfolding of the compact structure, denaturation, and aggregation. Interestingly, the ScCO2 treated-WPI exhibited a large amount of denatured fraction and was less sensitive to heat denaturation with a higher peak temperature (Td 5 98.76 C vs Td 5 73.06 C for unprocessed WPI). Recently, a method for producing whey protein aggregates though the use of carbon dioxide has been patented (Callanan, Schmitt, Michel, Pas, & Donato-Capel, 2017). The invention relates to production of whey protein aggregates by heating a native whey protein solution (,80 C) in order to denature the proteins, and dissolving carbon dioxide in the solution under pressure (.7.39 MPa for 1 minute). Processing conditions, such as protein concentration, pressure, and time were altered in order to obtain aggregates with a wide range of size and size distributions. The solutions of whey protein aggregates were found to act as surfactants and to be able to create stable foams or emulsions. Furthermore, WPI aggregates produced at the isoelectric point (pH 5.2) with addition of ScCO2 formed smaller aggregates (D50 of 0.27 μm) in comparison to non-ScCO2 treated-WPI (D50 of 2.63 μm). The larger aggregates in nontreated ScCO2-WPI solutions could lead to spontaneous precipitation in solutions.

References

6.6

CONCLUSIONS: FUTURE PERSPECTIVES

Whey proteins have been widely used in food applications due to their high nutritional and functional properties and their versatility in influencing the structural and textural qualities on food products. Despite their excellent properties, whey proteins are subjected to heat treatments in the food industry which can lead to their aggregation or gelation, depending on the physicochemical conditions. The continuously growing research on prevention/ reduction of whey protein aggregation, especially during thermal processing, has been reviewed in this chapter. Numerous interesting findings have suggested new ways of modulating the behavior of whey proteins during thermal processing and provided strategies for optimizing industrial food/ dairy processes involving whey proteins. However, strategies on how to prevent/reduce aggregations of whey proteins applicable to industrial-scale processes are currently limited. Better knowledge of the properties of the complexes formed postmodification, physical or chemical, and of the means of controlling/modulating these properties is necessary to better understand the prevention mechanisms and to develop applications of these modifications in food processing. This includes, for example, the effect of modifications on the physicochemical properties of concentrated ( . 40% solids) and high-protein systems, on the final flavor of products, and on interactions of the modified complexes with other food components that could be beneficial for products such as infant formula.

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12. Vidal, V., Gastaldi, E., Lefebvre-Cases, E., Lagaude, A., Marchesseau, S., de la Fuente, B. T., & Cuq, J.-L. (1998). Effect of succinylation on the rennet coagulation of milk. Journal of Dairy Science, 81, 69
75. Villas-Boas, M. B., Vieira, K. P., Trevizan, G., de Lima Zollner, R., & Netto, F. M. (2010). The effect of transglutaminase-induced polymerization in the presence of cysteine on β-lactoglobulin antigenicity. International Dairy Journal, 20, 386
392. Walkenström, P., Windhab, E., & Hermansson, A.-M. (1998). Shear-induced structuring of particulate whey protein gels. Food Hydrocolloids, 12, 459
468. Wang, W., & Zhong, Q. (2014). Properties of whey protein
maltodextrin conjugates as impacted by powder acidity during the Maillard reaction. Food Hydrocolloids, 38, 85
94. Webb, B., & Bell, R. (1942). The effect of high-temperature short-time forewarming of milk upon the heat stability of its evaporated product. Journal of Dairy Science, 25, 301
311. Wijayanti, H. B., Bansal, N., & Deeth, H. C. (2014). Stability of whey proteins during thermal processing: A review. Comprehensive Reviews in Food Science & Food Safety, 13. Wijayanti, H. B., Bansal, N., Sharma, R., & Deeth, H. C. (2014). Effect of sulphydryl reagents on the heat stability of whey protein isolate. Food Chemistry, 163, 129
135.

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Wijayanti, H. B., Oh, H. E., Sharma, R., & Deeth, H. C. (2013). Reduction of aggregation of β-lactoglobulin during heating by dihydrolipoic acid. Journal of Dairy Research, 80, 383
389. Wijayanti, H. B., Waanders, J., Bansal, N., & Deeth, H. C. (2015). Identification of the binding of β-lactoglobulin (β-Lg) with sulfhydryl (
SH) blocking reagents by polyacrylamide gel electrophoresis (PAGE) and electrospray ionisation/time of flight-mass spectrometry (ESI/TOFMS). LWT-Food Science & Technology, 63, 934
938. de Wit, J. N. (2009). Thermal behaviour of bovine beta-lactoglobulin at temperatures up to 150 degrees C. a review. Trends in Food Science & Technology, 20, 27
34. Wolz, M., & Kulozik, U. (2015). Thermal denaturation kinetics of whey proteins at high protein concentrations. International Dairy Journal, 49, 95
101. Wu, S. Y., Perez, M. D., Puyol, P., & Sawyer, L. (1999). beta-lactoglobulin binds palmitate within its central cavity. Journal of Biological Chemistry, 274, 170
174. Xiang, B. Y., Ngadi, M. O., Ochoa-Martinez, L. A., & Simpson, M. V. (2011). Pulsed electric fieldinduced structural modification of whey protein isolate. Food & Bioprocess Technology, 4, 1341
1348. Xiong, Y. L., Dawson, K. A., & Wan, L. P. (1993). Thermal aggregation of beta-lactoglobulin
Effect of pH, ionic environment, and thiol reagent. Journal of Dairy Science, 76, 70
77. Xu, D., Yuan, F., Jiang, J., Wang, X., Hou, Z., & Gao, Y. (2011). Structural and conformational modification of whey proteins induced by supercritical carbon dioxide. Innovative Food Science & Emerging Technologies, 12, 32
37. Yeom, H. W., Streaker, C. B., Zhang, Q. H., & Min, D. B. (2000). Effects of pulsed electric fields on the activities of microorganisms and pectin methyl esterase in orange juice. Journal of Food Science, 65, 1359
1363. Yong, Y. H., & Foegeding, E. A. (2008). Effects of caseins on thermal stability of bovine β-lactoglobulin. Journal of Agricultural & Food Chemistry, 56, 10352
10358. Zabbia, A., Buys, E. M., & De Kock, H. L. (2012). Undesirable sulphur and carbonyl flavor compounds in UHT milk: A review. Critical Reviews in Food Science & Nutrition, 52, 21
30. Zhang, J., Burrows, S., Gleason, C., Matthews, M. A., Drews, M. J., LaBerge, M., & An, Y. H. (2006). Sterilizing Bacillus pumilus spores using supercritical carbon dioxide. Journal of Microbiological Methods, 66, 479
485. Zhang, W., & Zhong, Q. (2009). Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by sequential enzymatic cross-linking and thermal pretreatments. Journal of Agricultural & Food Chemistry, 57, 9181
9189. Zhang, Z., Arrighi, V., Campbell, L., Lonchamp, J., & Euston, S. R. (2016). Properties of partially denatured whey protein products 2: Solution flow properties. Food Hydrocolloids, 56, 218
226. Zhong, Q. (2011). Heat stable protein ingredients. Patent number US2011268680. Zhong, Q., & Jin, M. (2008). Enhanced functionalities of whey proteins treated with supercritical carbon dioxide. Journal of Dairy Science, 91, 490
499. Zhong, Q., Wang, W., Hu, Z. X., & Ikeda, S. (2013). Sequential preheating and transglutaminase pretreatments improve stability of whey protein isolate at pH 7.0 during thermal sterilization. Food Hydrocolloids, 31, 306
316. Zhu, D., Damodaran, S., & Lucey, J. A. (2008). Formation of whey protein isolate (WPI) 2 dextran conjugates in aqueous solutions. Journal of Agricultural & Food Chemistry, 56, 7113
7118. Zhu, D., Damodaran, S., & Lucey, J. A. (2010). Physicochemical and emulsifying properties of whey protein isolate (WPI) 2 dextran conjugates produced in aqueous solution. Journal of Agricultural & Food Chemistry, 58, 2988
2994.

Further Reading

Zhu, D., & Labuza, T. P. (2010). Effect of cysteine on lowering protein aggregation and subsequent hardening of whey protein isolate (WPI) protein bars in WPI/buffer model systems. Journal of Agricultural & Food Chemistry, 58, 7970
7979. Zhu, H., & Damodaran, S. (1994). Heat-induced conformational changes in whey protein isolate and its relation to foaming properties. Journal of Agricultural & Food Chemistry, 42, 846
855. Zisu, B., Bhaskaracharya, R., Kentish, S., & Ashokkumar, M. (2010). Ultrasonic processing of dairy systems in large scale reactors. Ultrasonics Sonochemistry, 17, 1075
1081. Zisu, B., Lee, J., Chandrapala, J., Bhaskaracharya, R., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. Journal of Dairy Research, 78, 226
232. Zuniga, R. N., Tolkach, A., Kulozik, U., & Aguilera, J. M. (2010). Kinetics of formation and physicochemical characterization of thermally-induced beta-lactoglobulin aggregates. Journal of Food Science, 75, E261
E268.

Further Reading Adjonu, R., Doran, G., Torley, P., & Agboola, S. (2014). Formation of whey protein isolate hydrolysate stabilised nanoemulsion. Food Hydrocolloids, 41, 169
177. Gauthier, S. F., Paquin, P., Pouliot, Y., & Turgeon, S. (1993). Surface activity and related functional properties of peptides obtained from whey proteins. Journal of Dairy Science, 76, 321
328. Gauthier, S. F., & Pouliot, Y. (2003). Functional and biological properties of peptides obtained by enzymatic hydrolysis of whey proteins. Journal of Dairy Science, 86, E78
E87. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16, 945
960. Lajoie, N., Gauthier, S. F., & Pouliot, Y. (2001). Improved storage stability of model infant formula by whey peptides fractions. Journal of Agricultural & Food Chemistry, 49, 1999
2007. Rich, L. M., & Foegeding, E. A. (2000). Effects of sugars on whey protein isolate gelation. Journal of Agricultural & Food Chemistry, 48, 5046
5052. Sağlam, D., Venema, P., van der Linden, E., & de Vries, R. (2014). Design, properties, and applications of protein micro- and nanoparticles. Current Opinion in Colloid & Interface Science, 19, 428
437.

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Whey Protein 2 Carbohydrate Conjugates James A. O’Mahony, Kamil P. Drapala, Eve M. Mulcahy and Daniel M. Mulvihill University College Cork, Cork, Ireland

7.1

INTRODUCTION

A conjugated protein is defined as a protein to which another chemical group (e.g., carbohydrate) is attached by either covalent bonding or other interactions (Wong, 1991). Whey proteins and peptides, in the presence of reducing carbohydrates, can undergo a series of complex chemical changes during heating, known as the Maillard reaction. Conjugation occurs naturally during the early stages of the Maillard reaction when a covalent bond forms between the protein and carbohydrate components. Conjugation of food proteins with carbohydrates via the Maillard reaction (i.e., glycation) is a growing area of interest, with many studies completed, particularly over the last 15 20 years, on the use of conjugation to modify physicochemical, techno-functional, and nutritional properties of proteins and peptides. Whey protein ingredients are utilized in the formulation of a wide range of food, and clinical and pharmaceutical products, due to their unique functional and nutritional attributes. In the food industry, the principal technological hurdles limiting the use of whey protein ingredients in the formulation of value-added beverages and powders are: (1) poor solubility of intact proteins in high-acid, ready-to-drink beverages, resulting in the development of turbidity and phase separation (Akhtar & Dickinson, 2007); (2) poor emulsification properties of hydrolyzed proteins (Singh & Dalgleish, 1998), causing challenges with emulsion formation, stabilization, and spray drying (e.g., powder stickiness and high free fat) during the manufacture of powdered nutritional products; and (3) physical instability such as aggregation, sedimentation, and creaming during processing and on storage in high ionic strength environments and during thermal processing (Yadav, Parris, Johnston, Onwulata, & Hicks, 2010). Conjugation has been shown to be successful in modifying the functional properties of a range of milk Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00008-4 © 2019 Elsevier Inc. All rights reserved.

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protein/peptide-based ingredients, including whey protein-based substrates. This chapter provides an overview of how the key compositional, structural, and physicochemical properties of whey protein/peptide and carbohydrate substrates influence conjugation of whey proteins/peptides. A comparison of the differences between the two main modes of achieving conjugation (i.e., dry and wet heating) is provided, in addition to a detailed description of the effects of conjugation on selected functional properties (e.g., solubility, heat stability, emulsification, foaming, and gelation) of whey protein/peptide ingredients used in the food industry. A brief overview of the impact of conjugation on the nutritional properties of whey proteins, and of approaches being developed for enriching and purifying conjugates is provided toward the latter part of the chapter. While the focus of this chapter is predominantly on the intentional production of whey protein 2 carbohydrate conjugates, mainly for enhancement of technological and/or nutritional functionality, it is important to consider that the Maillard reaction also occurs naturally under the conditions of processing and storage of several food ingredients and products containing mixtures of whey proteins and reducing carbohydrates. Examples of such ingredients and products in which Maillard-induced conjugation of whey proteins with carbohydrates is of importance include whey protein isolate (WPI), dairy-based ultra-high-temperature (UHT) treated beverages and infant nutritional products. Taking one of these systems as an example, WPI is a premium nutritional whey protein powder containing .90% protein and is used extensively in sports nutritional applications. During transport from site of production to market (often between continents by sea freight), the powders can be exposed to temperatures in excess of 45 C, often reaching 55 60 C within shipping containers. Under such conditions of high temperature during transport, coupled with high humidity in the target market, the whey proteins become conjugated with the low level of residual lactose (i.e., lactosylated) which can result in considerable changes in powder surface composition (Burgain et al., 2016) and technological functionality (Norwood et al., 2016). These changes are also generally accompanied, or rapidly followed by, deleterious changes in product sensory (e.g., browning) and nutritional (e.g., protein cross-linking) quality. In UHT-treated milk products the susceptibility of individual milk proteins to plasmin-mediated proteolysis can be influenced by lactosylation of the proteins during thermal processing (i.e., rendering lysine residues unavailable for protein cross-linking), which has important implications for gelation and shelf life of such products (Bhatt et al., 2014). The generation of advanced Maillard reaction products (AMPs), some of which confer color and flavor, have important implications for storage condition requirements, shelf life, and quality of infant nutritional products. Levels of Maillard reaction products (MRPs) produced

7.2 Maillard Reaction Chemistry

under accelerated storage conditions have been investigated as suitable predictors of storage stability at ambient conditions (Cheng et al., 2017).

7.2

MAILLARD REACTION CHEMISTRY

The Maillard reaction (Maillard, 1912) encompasses a complex series of reaction pathways, many of which proceed concurrently during heating and/or storage of protein/carbohydrate mixtures. The first simplified, integrated scheme for the Maillard reaction was developed over 60 years ago by Hodge (1953), and this has been advanced further and refined by researchers from different fields over the years (e.g., Henle, Walter, & Klostermeyer, 1991). In essence, Hodge (1953) divided the chemistry of the Maillard reaction into three stages: the early, intermediate and advanced stages (Fig. 7.1). The early stage of the Maillard reaction involves a series of individual reactions that are

FIGURE 7.1 Schematic overview of the Maillard reaction in dairy-based products. Based on O’Mahony, J. A., Drapala, K. P., Mulcahy, E. M., & Mulvihill, D. M. (2017). Controlled glycation of milk proteins and peptides: Functional properties. International Dairy Journal, 67, 16 34.

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initiated when the ε-amino group of lysine, or to a lesser extent, the imidazole and indole groups of histidine and tryptophan, respectively, and the α-amino groups of terminal amino acids in proteins/peptides condense with the carbonyl groups of reducing carbohydrates, to form a Schiff base, with the release of a molecule of water (Ames, 1996). The Schiff base is thermodynamically unstable and undergoes spontaneous rearrangement to form either an Amadori (in the case of aldoses) or Heyn’s (in the case of ketoses) product (Wrodnigg & Eder, 2001). The intermediate stage of the Maillard reaction involves the degradation of the Amadori and/or Heyn’s rearrangement products by a number of different reactions, including cyclizations, dehydrations, retro-aldolizations, isomerizations, and further condensations, which causes degradation of amino acids and carbohydrates (Ames, 1998). The advanced stages are complex and variable, depend on the reaction conditions (i.e., the environmental conditions prevailing during heat treatment and storage of protein 2 carbohydrate mixtures), and involve dehydration and decomposition of the early reaction products, resulting in the production of many AMPs and colored nitrogenous polymers and copolymers, known collectively as melanoidins. While, from a functionality perspective, it may be desirable to achieve conjugation in the early stages of the Maillard reaction, it is normally desirable to limit the progression of the Maillard reaction to advanced stages, as AMPs are largely responsible for some of the less desirable consequences of the Maillard reaction, e.g., generation of off-flavors, loss of nutritional value, protein crosslinking, and generation of potentially toxic compounds (Uribarri et al., 2005). Given the complexity of the Maillard reaction, it should not be surprising that a wide range of analytical platforms and approaches have been developed and applied for monitoring the progress of the Maillard reaction and for the identification and quantification of specific MRPs in food systems. One approach commonly used to assess progress of the Maillard reaction through the early stages is to quantify, using spectrophotometry, the reduction in chemically reactive amino groups using reagents such as trinitrobenzenesulfonic acid (TNBS) and o-phthaldialdehyde. The production of the early-stage Schiff base may also be monitored using spectrophotometry (Zhu, Damodaran, & Lucey, 2008). Various Amadori compounds (e.g., lactulosyl-lysine), produced during the early stages of the reaction, may be measured using high performance-liquid chromatography (HPLC), while the accumulation of 5-hydroxymethylfurfural (HMF) may also be determined using HPLC and is a good indicator of the intermediate stages of the reaction. Various advanced glycation end-products may be measured using fluorescence (Birlouez-Aragon et al., 1998) or enzyme-linked immunosorbent assays (Cheng et al., 2017), while color changes (i.e., accumulation of

7.3 Factors Affecting Maillard-Induced Conjugation of Whey Proteins

yellow/brown colored melanoidin-based compounds) can be measured directly using a colorimeter and volatile Strecker degradation products may be identified and quantified using gas chromatography-mass spectrometry (Jansson et al., 2014). Development and application of new analytical methods for monitoring the Maillard reaction in dairy-based systems is a very active and rapidly advancing area of research, therefore, it is not possible to give a more complete overview here, and the reader is referred to the following recent articles for further information (Arena, Renzone, D’Ambrosio, Salzano, & Scaloni, 2017; Cheng et al., 2017; Nursten, 2011; Roux et al., 2016).

7.3 FACTORS AFFECTING MAILLARD-INDUCED CONJUGATION OF WHEY PROTEINS Several factors, including temperature, time, the nature of the reactants, pH, and water activity (aw) influence the production of whey protein 2 carbohydrate conjugates (Fig. 7.2). Understanding and manipulation of these factors is critical in being able to control the yield, quality, and functionality of conjugated whey proteins. For whey protein hydrolysates (WPHs), the degree of hydrolysis (DH), molecular weight (Mw) profile, and charge of the peptides are important in determining their reactivity during Maillard-induced conjugation (Drapala, Auty, Mulvihill, & O’Mahony, 2016a, 2016b; Mulcahy,

FIGURE 7.2 Schematic representation of the influence of different environmental factors on the progression of the Maillard reaction (MR).

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Park, Drake, Mulvihill, & O’Mahony, 2016; Van Lancker, Adams, & De Kimpe, 2011). Reactivity of compounds tends to decrease with increasing Mw, due to the greater contribution of steric hindrance with increasing Mw; as an example, monosaccharides are more reactive with whey proteins than di- or oligosaccharides under conditions which favor conjugation. While the rate of the Maillard reaction generally increases with increasing heating temperature and time, temperature also influences the nature (e.g., conformation and accessibility to reactive protein functional groups) of the reactants. The proportion of reducing sugar molecules present in the open-chain form (i.e., the more reactive form) increases with increasing temperature (Van Boekel, 2001). Heat-induced conformational changes (e.g., denaturation and aggregation) of whey proteins can result in amino groups becoming less available for participation in the Maillard reaction (Chevalier, Chobert, Popineau, Nicolas, & Haertlé, 2001; Mulcahy, Park, Drake, Mulvihill, & O’Mahony, 2018). Reactivity of both the whey protein and carbohydrate components of mixtures during the Maillard reaction is strongly influenced by pH; e.g., a basic environment can catalyze the initial stages of the Maillard reaction by deprotonating amino groups on whey protein molecules, which in turn increases reactivity with carbonyl groups of reducing carbohydrates. The open-chain form of the carbohydrate and the unprotonated form of the amino group, which are considered to be the most reactive forms, are usually favored at higher pH, up to a maximum of B9 10 (Martins, Jongen, & Van Boekel, 2000). The pH of whey protein 2 carbohydrate mixtures can decrease as the Maillard reaction progresses due to the formation of acids (e.g., formic and acetic acids), the consumption of acidic amino groups (e.g., lysine) or the loss of carboxyl groups during Strecker degradation, resulting in the production of carbon dioxide. Therefore, the use of buffers to help minimize changes in pH on heating has been reported to accelerate the Maillard reaction (Van Boekel, 2001). The pH of the system also influences the reaction pathway and the profile of MRPs generated. For example, degradation of the Amadori products at pH # 7 takes place via the 1,2-enolization pathway that favors the formation of furfural or HMF, whereas, at pH .7, the degradation of Amadori products proceeds through the 2,3-enolization pathway, favoring the production of reductones and fragmentation products such as hydroxyacetone and 2,3-butanedione, minimizing the formation of HMF (Liu, Ru, & Ding, 2012). Increasing aw increases diffusion and molecular mobility and thereby generally increases the rate and extent of conjugation; however, high water concentrations/aw can negatively influence progression of the Maillard reaction (Morgan, Léonil, Mollé, & Bouhallab, 1999).

7.4 Whey Protein and Carbohydrate Substrates Used in Conjugation

7.4 WHEY PROTEIN AND CARBOHYDRATE SUBSTRATES USED IN CONJUGATION The conjugation of whey proteins has been studied using many different types of whey protein-based ingredients as substrates, including, but not limited to, whey protein concentrates (WPCs) and WPIs, individual whey protein fractions (e.g., β-lactoglobulin, β-Lg; α-lactalbumin, α-La; bovine serum albumin, BSA; and lactoferrin, LF) and WPHs. Whey proteins are more susceptible than caseins to heat-induced denaturation and aggregation under the conditions used for conjugation (particularly under wet heating conditions). While denaturation (i.e., unfolding) would be expected to increase the accessibility of carbonyl groups to amino groups on the whey protein/ peptide molecules, aggregation would be expected to restrict accessibility of carbonyl groups to amino groups on the whey protein/peptide molecules. Therefore, the balance between native, denatured, and aggregated whey proteins in the chosen substrate strongly influences conjugation. β-Lg typically represents B50% 60% of total protein in WPC, WPI, and WPH ingredients and has two disulfide bonds and one free thiol group, which are deemed responsible for the irreversible thermal aggregation and gelling properties of this protein (Brodkorb, Croguennec, Bouhallab, & Kehoe, 2016). In contrast, α-La has a single polypeptide chain, containing four disulfide bonds, and no free sulfhydryl group, making it less sensitive to heat-induced denaturation/ aggregation under the conditions used in conjugation of whey protein. It is desirable to have only low levels of nonprotein components (e.g., lactose, minerals, and lipid) in the whey protein-containing ingredients used as substrates for conjugation, as lactose can compete with other carbohydrates for conjugation to the protein substrate and contributes strongly to brown color and flavor compound formation (Lillard, Clare, & Daubert, 2009), minerals promote aggregation of whey proteins, and lipid material can contribute to off-flavor formation (Lloyd, Hess, & Drake, 2009). Based on this knowledge, high protein content WPC and WPI, or pure protein fraction ingredients are most commonly used for conjugation purposes. Hydrolysis of whey protein molecules increases the number of free amino groups available to react with carbonyl groups during conjugation and can lead to increased exposure and accessibility of previously buried lysine residues. Protein hydrolysates are generally characterized by their DH, which expresses the number of peptide bonds cleaved as a percentage of the total number of peptide bonds available (Foegeding, Davis, Doucet, & McGuffey, 2002). Hydrolysis of whey proteins, due to reduction of average Mw and levels of secondary structure, enhances their stability to heat-induced aggregation, which can facilitate enhanced retention of amino groups in a form

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accessible for conjugation during heating. For example, Mulcahy, Park, et al. (2016) reported that WPH with a low degree of hydrolysis (DH 9.3%) had 55.4% higher levels of available amino groups compared with an intact WPI counterpart, which contributed to more rapid and extensive conjugation of maltodextrin (MD) with the WPH than with the WPI. The conjugation of whey proteins has been studied using several different types of carbohydrate ingredients, including, but not limited to, lactose, MD, corn syrup solids (CSSs), dextrans, glucose, maltose, ribose, oligosaccharides, gum arabic, pectin, and corn fiber gum. From the point of view of their ability to participate in Maillard-induced conjugation of whey proteins, and the functionality of the resultant conjugates, the key differences between these carbohydrates are chain length and structure (i.e., linear vs branched and ketoses vs aldoses). In general, the shorter the chain length of the carbohydrate component, the faster the rate, and the greater the extent of conjugation. On conjugation of whey proteins with MD or CSS, having dextrose equivalent (DE) values in the range 6 38, at an initial pH 8.2, at 90 C for up to 24 hours, the extent of conjugation increased with increasing DE value of the MD and CSS ingredients (Mulcahy, Mulvihill, & O’Mahony, 2016).

7.5

APPROACHES USED TO ACHIEVE CONJUGATION

The main variables that can be controlled during conjugation of whey proteins are temperature, time, pH, moisture content, relative humidity (RH), and/or aw. These variables can be grouped to give two distinct approaches for achieving conjugation: (1) wet heating, and (2) dry heating. The wet heating approach normally involves incubation of an aqueous solution of protein and carbohydrate reactants, commonly preadjusted to a target pH (normally pH 6.0 11.0), for a predetermined time (minutes-days) at a set temperature (typically in the range 60 95 C). The conjugation reaction is normally stopped by cooling and further processing (e.g., freeze or spray drying) of the conjugated protein/peptide solution. The dry heating approach normally involves incubation of a codried mixture (commonly preadjusted to a target pH) of the protein and carbohydrate ingredients for a predetermined time (minutes to days) at a set temperature (typically in the range 60 130 C) at a set RH (typically 60 80%). Both approaches have been used extensively for conjugation of whey proteins and both have their advantages and limitations. While more detailed information on the comparisons of wet and dry heating for the production of milk protein/peptide carbohydrate conjugates can be found in O’Mahony, Drapala, Mulcahy, and Mulvihill (2017), a brief overview is provided here. The mobility of reactants is higher with the wet heating than the

7.6 Techno-Functional Properties of Conjugated Whey Proteins

dry heating approach and higher temperatures (for shorter times) are generally used with the former than with the latter. With the use of dry heating, it is necessary to prepare a solution of the two components, which is dried before being conjugated, to achieve maximum reactivity between the protein and carbohydrate components. The conjugated powder typically also requires downstream drying due to release of water during the early stages of the Maillard reaction and this can lead to localized browning of the powdered reaction mixture during conjugation due to sugar crystallization (Lievonen, Laaksonen, & Roos, 1998). In addition to the differences in energy costs and efficiency between wet and dry heating approaches, the use of dry heating (at least at temperatures ,70 C) has been shown to result in greater preservation of the native three-dimensional structure of whey proteins, compared with wet heating approaches, which has important implications for selected functional properties, such as solubility and interfacial properties. Interestingly, the use of macromolecular crowding to restrict denaturation and, in particular, aggregation of whey proteins has also shown promise on conjugation of WPI with dextran (Ellis, 2001; Perusko, Al-Hanish, Velickovic, & Stanic-Vucinic, 2015; Zhu et al., 2008). In addition to conventional wet and dry heating, alternative approaches to achieving conjugation have also shown promise in modifying whey protein functionality; e.g., Sun, Yu, Zeng, Yang, and Jia (2011) reported that WPI 2 dextran conjugates, prepared by application of a pulsed electric field (15 and 30 kV/cm, flow rate B30 mL/min) at an initial pH of 10 and at 30 C for 7.35 ms, had higher solubility (10 30% increase) at pH 4.0 6.0 than the control WPI solution treated with pulsed electric field. Sonication (20 kHz frequency for 60 minutes at pH 8.0 and B5 10 C) has also been used to achieve conjugation of WPI with arabinose and polyethylene glycol (PEG) (Perusko et al., 2015). The authors reported that the conjugated WPI 2 arabinose 2 PEG solution had a greater extent of conjugation (10% increase), due to the presence of PEG facilitating macromolecular crowding, compared to the sonicated solution of WPI 2 arabinose without PEG.

7.6 TECHNO-FUNCTIONAL PROPERTIES OF CONJUGATED WHEY PROTEINS Much of the research conducted on conjugation of whey proteins has been focused on modification and enhancement of selected techno-functional properties. This section of the chapter provides an overview of the effects of conjugation on solubility, heat stability, emulsification, foaming, and gelation properties of whey proteins.

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7.6.1

Solubility

Whey proteins used in food products are generally required to have high levels of solubility in order to facilitate expression of the desired functional properties such as gelation, aeration, water-binding, foaming, and emulsification (de Wit, 1989). The solubility of whey proteins is influenced by many physicochemical properties of the protein molecules themselves, i.e., amino acid composition, Mw, conformation (e.g., as affected by denaturation/aggregation), exposure of selected functional groups, surface hydrophobicity, and environmental factors, such as pH, temperature, ionic strength, and nature of the solvent. Several studies have been conducted involving conjugation of whey protein mixtures and their individual fractions with different carbohydrates with a view to increasing solubility and minimizing the effects of different food processing operations (e.g., thermal processing) on solubility of whey proteins. The dry heating approach has been used extensively to conjugate whey proteins with carbohydrates as it is claimed to result in less heat-induced conformational changes to the whey protein molecules than with wet heating (Zhu et al., 2008). Wang and Ismail (2012) demonstrated that WPI conjugated with dextran by dry heating at 60 C and 49% RH, for 96 hours, had enhanced protein solubility (85.7% and 89.0% increase) at pH 4.5 and 5.5, respectively, when they were subsequently heated to 80 C for 30 minutes, compared to the respective WPI control. The authors reported that suppressed intermolecular protein 2 protein interactions, along with structural/physicochemical changes to the protein, including a shift in the isoelectric point of the protein to a more acidic pH, reduction in the surface hydrophobicity of the whey protein molecules, and increased resistance to thermal denaturation, resulting in reduced exposure of free sulfhydryl groups after conjugation of the protein with dextran, were responsible for the enhanced solubility of the WPI. A further study by Wang, He, Labuza, and Ismail (2013) characterized the structural changes in whey protein molecules conjugated with dextran (at 60 C and 49% RH for 96 hours) using surface-enhanced Raman spectroscopy. The authors reported that conjugation-induced conformational changes in the whey protein molecules imparted structural rigidity to the conjugated WPI 2 dextran system, which in turn increased protein solubility on thermal treatment (75 C for 30 minutes) over a wide pH range (3.4 7.0), compared to previously unheated WPI. Jimenez-Castano, Villamiel, and Lopez-Fandino (2007) conjugated individual whey protein fractions (β-Lg, α-La, BSA) with dextran (Mw of 10 or 20 kDa) by dry heating at an initial pH of 7.0, at 60 C and 44% RH for 24 72 hours and reported that the extent of conjugation decreased in the

7.6 Techno-Functional Properties of Conjugated Whey Proteins

following order, BSA . β-Lg . α-La, also demonstrating that conjugation of β-Lg with dextran (20 kDa), for either 36 or 60 hours, improved its solubility by B40% at pH 5.0. However, the solubility of the β-Lg 2 dextran conjugate was B20% 30% lower at pH 4.0, compared to the unheated or heated β-Lg control samples, which may be attributed to the consumption of positively charged amino groups (i.e., lysine) causing a shift in the isoelectric point to a more acidic pH. In contrast, Chevalier et al. (2001) reported that β-Lg conjugated with galactose, glucose, lactose, or rhamnose, by wet heating at an initial pH of 6.5, at 60 C for 72 hours, had increased solubility of B25% at pH 4.5, compared to the respective heated β-Lg control due to changes in the conformation and hydrophobicity of the protein molecules. Jimenez-Castano et al. (2007) also reported that α-La 2 dextran conjugates exhibited a higher solubility (B5% 50% increase), compared to the unheated control, in the pH range 3.0 5.0, with the greatest increase in solubility occurring at pH 4.0; similar trends were reported for BSA dextran conjugates which had higher solubility around the isoelectric point (pH 4.7 4.9) than the unheated control. A limited number of studies have reported modification of functional properties of whey proteins conjugated with carbohydrates using wet heating conditions. The likely reason for the limited number of such studies is that heating of whey protein in an aqueous environment at $ 70 C can result in denaturation and aggregation, which is known to reduce solubility of whey proteins (Liu et al., 2012; Pelegrine & Gasparetto, 2005; Zhu, Damodaran, & Lucey, 2010). However, Jiang and Brodkorb (2012), Lillard et al. (2009), and Liu and Zhong (2015) have investigated the use of high temperatures (95 130 C) to induce conjugation of whey proteins or isolated whey protein fractions with carbohydrates, and have reported improvements in the antioxidant activity, emulsification properties, and heat stability, respectively, of whey protein 2 carbohydrate conjugates. Mulcahy, Park, et al. (2016) reported that at pH 3.5, a conjugated WPI 2 MD solution, prepared using a wet heating approach involving heating at an initial pH of 8.2, at 90 C for 8 hours, had higher protein solubility (50.7%) than the WPI solution heated without MD (26.7% solubility) (Fig. 7.3). The authors also reported that the protein solubility of a conjugated WPH 2 MD solution, prepared by heating at 90 C for 8 hours, increased to 80.9% at pH 4.0 4.5, compared with that of the heated WPH solution (75.3%). The increase in protein solubility of the conjugated WPI 2 MD and WPH 2 MD solutions, compared with solutions of heated whey protein (90 C for 8 hours) without MD, was attributed to enhanced hydration of the protein and increased steric hindrance between the protein molecules provided by the attachment of the bulky dextran molecules.

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FIGURE 7.3 Protein solubility as a function of pH for whey protein isolate (WPI) and WPI-maltodextrin (MD17) solutions unheated (- - -) or heated (—) at an initial pH of 8.2 at 90 C for 8 h: WPI (x or K) and WPI-MD17 (& or ’). Values are presented as mean 6 standard deviation of data from three independent trials. Protein solubility was measured by centrifugation of solutions at a range of pH values at 10,000 3 g for 20 min followed by filtration with the protein content of the supernatant and untreated solution being analyzed using a BCA protein assay kit. Results are reported as the protein content of each supernatant expressed as a percentage of the total protein content of the respective initial solution. Extracted from Mulcahy, E. M., Park, C. W., Drake, M., Mulvihill, D. M., & O’Mahony, J. A. (2016). Improvement of the functional properties of whey protein hydrolysate by conjugation with maltodextrin. International Dairy Journal, 60, 47 54.

Heat treatment of milk proteins (i.e., including whey proteins) can lead to the formation of reactive intermediates (e.g., methylglyoxal or dehydroalanine), which can subsequently react with the ε-amino group of lysine, resulting in the formation of protein cross-links, leading to modification of functional properties and loss of nutritional value (Pellegrino, Van Boekel, Gruppen, Resmini, & Pagani, 1999). In particular, the development of lysinoalanine (LAL) in proteins during heat treatment is associated with development of protein cross-links (Gerrard, 2002; Mulcahy, Park, et al., 2016).

7.6.2

Heat Stability

Improvement of the heat stability of whey proteins by conjugation is of considerable academic and industrial relevance as globular whey proteins are susceptible to heat-induced (.70 C) denaturation and aggregation (Wijayanti, Bansal, & Deeth, 2014), contributing to thermal processing

7.6 Techno-Functional Properties of Conjugated Whey Proteins

challenges such as viscosity increase, fouling of heat exchanger surfaces, and product quality challenges such as turbidity and sedimentation. Zhu et al. (2010) conjugated WPI with dextran (Mw 440 kDa) by heating a solution of 10% WPI and 30% dextran, at an initial pH of 6.5 at 60 C for 48 hours and measured thermal stability of the conjugated whey proteins by heating at 80 C for 30 minutes. The absorbance at 500 nm (Abs500) of the conjugated WPI 2 dextran solution did not change on heating; however, there was a B10-fold increase in Abs500 of the WPI solution that was heated at 80 C for 30 minutes in the pH range 4.5 5.5, which was attributed to the formation of large protein aggregates with the ability to scatter light. The unheated WPI had a typical differential scanning calorimetry (DSC) denaturation profile, with an endothermic peak at B74 C attributed to the denaturation of β-Lg; however, the conjugated WPI 2 dextran solution had a flat line profile suggesting that whey protein in the WPI had less secondary structure, due to the covalent attachment of the dextran which contributed to a higher denaturation temperature and improvements in thermal stability. Similar DSC profiles were reported by Hattori, Nagasawa, Ametani, Kaminogawa, and Takahashi (1994), Liu and Zhong (2013), and Wang and Ismail (2012) who showed that the denaturation temperature of whey protein 2 carbohydrate conjugates was higher than that of the corresponding unconjugated whey proteins. Chevalier et al. (2001) reported that β-Lg conjugated with either ribose, arabinose, glucose, galactose, lactose, or rhamnose, at pH 6.5 and 60 C for 72 hours in an aqueous environment (0.4% protein, 0.4% carbohydrate), exhibited greater thermal stability at pH 5.0, when heated at 70 90 C for up to 1 hour, than unheated and heated β-Lg controls (i.e., without added carbohydrate). The improvement in thermal stability of the protein solution was dependent on the choice of carbohydrate as follows, ribose . arabinose . rhamnose . glucose 5 galactose . lactose. Liu and Zhong (2013) conjugated WPI with either glucose, lactose or MD (Mw 1 kDa) by dry heating at an initial pH of 7.0 at 80 C and 80% RH for 2 hours, in a mass ratio of 1:1, and evaluated heat stability using an approach designed to simulate a hot-fill beverage process (Etzel, 2004). The solutions prepared from the conjugated WPI 2 MD and WPI 2 lactose remained transparent under all conditions tested and the increased thermal stability of the conjugated WPI was attributed to a higher denaturation temperature and a more negative net charge across the pH range 2.0 7.0 than the unheated WPI control. Several authors reported that improvements in heat stability of whey protein 2 carbohydrate conjugates can be related to the number and chain length of the carbohydrates attached to the whey protein molecules, along

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with the location at which they are attached on the protein molecule backbone; the attachment of higher Mw carbohydrates has been shown to have a greater impact on improving the thermostability of whey proteins, due to increased steric repulsion, compared to conjugation with monosaccharides (Mulcahy, Mulvihill, et al., 2016; Wooster & Augustin, 2006). WPHs generally have impaired functional properties compared to their intact counterparts. Mulcahy, Park, et al. (2016) reported that WPH (DH 9.3%) conjugated with MD (DE 17) under wet heating conditions at an initial pH of 8.2 and 90 C for 8 hours, had superior thermal stability to further heating at 85 C for 10 minutes with 40 mM NaCl added, compared to that of the unheated or heated WPH control solutions. The unheated or heated WPH control solutions with added NaCl precipitated and phase separated on heating at 85 C for 10 minutes, due to the formation of large protein aggregates (B10 50 μm), whereas, the conjugated WPH 2 MD solution heated in the presence of 40 mM NaCl remained stable and the protein aggregates present remained small (,B1 μm). The environmental conditions used during conjugation impact the thermal stability of the resulting conjugates; Wang and Zhong (2014) dry heated WPI 2 MD in the mass ratio 1:1, at 80 C and 65% RH for 4 hours, at different pHs (i.e., pH 4.0, 5.0, 6.0 and 7.0). The solutions prepared from the conjugated WPI 2 MD at pH 6.0 (5% protein, and 0 150 mM added NaCl) that was subsequently heated at 138 C for 1 minute (to simulate UHT treatment), had improved thermal stability compared to the solution prepared from the WPI 2 MD conjugated at pH 4.0. While most of the work on conjugation of whey proteins has been performed using WPI, conjugation has been shown to enhance the functional properties of WPC also as evidenced by the study of Liu and Zhong (2014) whereby a defatted WPC (34% protein) was used in conjugation by dry heating at 130 C for either 20 or 30 minutes, or 60 C for either 24 or 48 hours, at 79% RH, and considerably higher heat stability (on heating at 138 C for 1 minute with 150 mM added NaCl) was observed for the conjugated WPC compared with the unheated system. Conjugation has also been shown to be beneficial in producing heat stable whey protein nanofibrils; Liu and Zhong (2013) produced protein nanofibrils (pH 2.0, heated at 85 C for 24 hours) from solutions of WPI and lactose which had previously been conjugated under dry heating conditions (80 C and 70% RH for 2 hours). The nanofibrils prepared from the conjugated WPI 2 lactose were highly dispersible and remained transparent after heating (88 C for 2 minutes or 138 C for 1 minute) in the pH range 4.0 7.0, even with up to 150 mM NaCl added, compared to the nanofibrils formed from a WPI solution, which became turbid under all heating conditions tested.

7.6 Techno-Functional Properties of Conjugated Whey Proteins

7.6.3

Emulsification

There is considerable interest in modifying the emulsification properties of whey proteins by their conjugation with carbohydrates to allow formation of novel emulsion-based food products. Conjugation of protein with carbohydrates results in formation of a surface active ingredient consisting of two composite blocks, where, in an emulsion system, the surface-active component (i.e., protein) adsorbs at the oil/water (O/W) interface, while the hydrophilic component (i.e., carbohydrate) extends into the continuous phase of the emulsion, resulting in stabilization of the encapsulated oil via a physical barrier. Conjugation of whey proteins with carbohydrates can improve their emulsion formation properties indirectly by enhancing protein solubility, increasing their effective concentration and mobility in aqueous solution. Changes in conformation of proteins arising from conjugation (i.e., unfolding of the protein structure and exposure of hydrophobic and hydrophilic groups) result in a more flexible protein structure, enabling it to move faster toward and adsorb at the O/W interface, compared to unconjugated protein (Báez, Busti, Verdini, & Delorenzi, 2013). The initial structure of a protein influences the effect of conjugation on its emulsion formation abilities; the emulsification properties of native globular whey proteins can benefit more from conjugation than those of less-structured proteins (e.g., caseins), due to the unfolding of the compact globular structure, increasing molecular flexibility and surface hydrophobicity (Einhorn-Stoll, Ulbrich, Sever, & Kunzek, 2005; Evans, Ratcliffe, & Williams, 2013). In an analogous manner, it is reasonable to assume that the effect of conjugation on emulsification properties of hydrolyzed whey proteins/peptides would largely depend on the degree of protein hydrolysis/ conformation change and the Mw of the protein/peptide and carbohydrate components of the conjugates. Carbohydrate moieties covalently attached to protein on conjugation act like a tail, and are effectively towed by the protein as it migrates through the bulk aqueous phase toward the O/W interface. The carbohydrate component of whey protein 2 carbohydrate conjugates generally does not impede the movement of the conjugated protein through the bulk phase, except when the size ratio between the protein and carbohydrate is disproportional. The larger hydrodynamic radius of protein 2 carbohydrate conjugates, compared to the protein alone, can potentially result in a decreased rate of diffusion in the bulk phase and reduce the rate of adsorption of conjugates at the interface (Ganzevles, van Vliet, Stuart, & de Jongh, 2007). As an example, lower emulsion formation ability was reported for WPI conjugated with high Mw MD (DE 2; Mw 280 kDa), an effect which was not observed for medium Mw MD (DE 19; Mw 8.7 kDa), compared to nonconjugated WPI (Akhtar & Dickinson, 2007).

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Conjugation of milk proteins with carbohydrates generally enhances their emulsion formation and stabilization properties at high salt concentrations and under acidic conditions, due to improved protein solubility under such environmental conditions. Covalent attachment of MD or corn fiber gum to globular whey proteins (i.e., β-Lg and proteins in WPI) by conjugation has been shown to enhance the emulsifying properties of the proteins across a broad pH range (3.2 5.5), by significantly increasing protein solubility (Yadav et al., 2010). Conjugation of whey proteins can greatly improve their solubility at acidic pH and high ionic strength conditions due to the additional steric barrier provided by the conjugated carbohydrate component preventing protein aggregation and precipitation. Additionally, conjugation can shift the isoelectric point of proteins to lower pH as reported for individual whey proteins (β-Lg, α-La, and BSA) conjugated with MD (JimenezCastano et al., 2007). Such enhanced protein functionality under challenging environmental conditions offers significant potential for the development of novel emulsion-based food formulations. Emulsion stability can be further enhanced by conjugation of whey protein with charged carbohydrates; Neirynck, Van Der Meeren, Gorbe, Dierckx, and Dewettinck (2004) reported improved stability of O/W emulsions due to strong electrosteric stabilization functionality of WPI 2 pectin conjugates. In addition to electrostatic repulsion, emulsions formulated with conjugated whey proteins are also stabilized by the additional steric hindrance provided to the adsorbed conjugated protein molecules by the carbohydrate component. The carbohydrate component of the conjugate is anchored at the O/W interface by surface-active protein and, due to its hydrophilicity, it extends into the aqueous phase and acts to physically hinder interactions between oil globules. Drapala et al. (2016a, 2016b) showed that model infant formula emulsions stabilized by WPH 2 MD conjugates, were resistant to heatinduced bridging flocculation, compared to those stabilized by nonconjugated WPH. The authors reported that the conjugate-stabilized systems showed no changes in viscosity or particle size distribution after a high temperature-short time treatment of between 75 C and 100 C for 15 minutes, in contrast to emulsions stabilized by intact, hydrolyzed, or preheated hydrolyzed whey protein (Figs. 7.4 7.5). In addition, significant improvements in heat stability of O/W emulsions stabilized by WPI conjugated with low methoxyl-pectin under dry heating conditions (60 C at 74% RH for 16 days) have been reported by Setiowati, Vermeir, Martins, De Meulenaer, and Van der Meeren (2016). Low zeta (ζ) potential of oil globules near the isoelectric point of milk proteins, and screening of the electrostatic charge by excess ions, can promote flocculation of protein-coated oil globules, leading to breakage of the emulsion and phase separation (Sarkar & Singh, 2016), whereas the presence of a

7.6 Techno-Functional Properties of Conjugated Whey Proteins

FIGURE 7.4 Photographs of model infant formula emulsions (containing 1.55%, 3.50%, and 7.00% whey protein, soybean oil, and maltodextrin, respectively) stabilized with whey protein isolate (WPI), whey protein hydrolysate (WPH), WPH-maltodextrin (MD12) conjugate (WPHMD12), and preheated WPH (WPH-H) after heat treatments at 75 C for 15 min ((A): WPH) and at 95 C for 15 min in an AR-G2 controlled stress rheometer equipped with a starch pasting cell geometry ((B): WPI; (C): WPH-H; (D): WPH-MD12). Emulsions WPH-H and WPH-MD12 were also heated at 100 C for 15 min in an oil bath ((E): left 5 WPH-H; right 5 WPH-MD12). Stock WPH-MD12 conjugate and preheated WPH-H solutions were prepared prior to emulsion formulation by heating WPH and MD12 (5%, w/w, WPH and 5%, w/v, MD12) and WPH (5%, w/v) solutions (pH 8.2) at 90 C for 8 h with continuous agitation. Reproduced from Drapala, K. P., Auty, M. A. E., Mulvihill, D. M., & O’Mahony, J. A. (2016a). Improving thermal stability of hydrolysed whey protein-based infant formula emulsions by protein carbohydrate conjugation. Food Research International, 88, 42 51.

strong steric barrier provided by protein 2 carbohydrate conjugates can oppose emulsion destabilization under these environmental conditions. Lesmes and McClements (2012) demonstrated that conjugation of β-Lg with dextran, under dry heating conditions (60 C and 76% RH for 24 hours), enhanced the formation and stability of O/W emulsions prepared at pH 7 using the conjugated protein on subsequent acidification to pH 5. The authors reported that the thick interfacial layer formed with the high Mw dextran (i.e., Mw $ 40 kDa) was responsible for the greater stability of the conjugate-based emulsions, compared to emulsions made using unconjugated protein. Good stability to storage at high salt concentration (0.2 M citrate buffer) and under acidic conditions (pH 3.2) was reported for emulsions stabilized by conjugates of β-Lg or WPI with corn fiber gum prepared using dry heating conditions at 75 C and 79% RH for time periods ranging from 2 hours to 7 days (Yadav et al., 2010); in these systems, the branched nature of the corn fiber gum resulted in good emulsion stability, even at low levels of conjugation. Considerably improved resistance to flocculation for emulsions stabilized

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FIGURE 7.5 Confocal laser scanning micrographs of model infant formula emulsions (containing 1.55%, 3.50%, and 7.00% whey protein, soybean oil, and maltodextrin, respectively) stabilized by preheated whey protein hydrolysate (WPH-H) and WPH conjugated with maltodextrin (WPH-MD12) before and after heat treatment in an oil bath at 100 C for 15 min. Protein 5 red (i.e., continuous phase); oil 5 green (i.e., discontinuous phase). Scale bar (bottom right) is 25 μm (first and second image in each row) and 5 μm (third image in each row). Note: the third figure in the first row is a combination of two micrographs (i.e., left and right) to give a more comprehensive representation of the heterogeneous structure observed in the WPH-H emulsion after heat treatment. Modified from Drapala, K. P., Auty, M. A. E., Mulvihill, D. M., & O’Mahony, J. A. (2016a). Improving thermal stability of hydrolysed whey protein-based infant formula emulsions by protein carbohydrate conjugation. Food Research International, 88, 42 51.

by conjugates of β-Lg and dextran (Mw 27 200 kDa) at high mineral addition levels was also reported by Wooster and Augustin (2006). Similar findings were reported for lactoferrin 2 dextran conjugates, where, strong steric stabilization of oil globules resulted in emulsion stability at high ionic strength (Liu, Ma, McClements, & Gao, 2016). Likewise, Akhtar and Dickinson (2007) reported that emulsions stabilized by WPI 2 MD conjugates (DE 19) and containing high levels of sodium lactate (5% w/w) did not show any changes in particle size distribution after 21 days of storage at 22 C, in contrast to an approximately twofold increase in mean volume diameter for emulsions stabilized by unconjugated protein or by gum arabic (a naturally-occurring protein 2 carbohydrate conjugate). The greater thickness of the interfacial layer in conjugate-stabilized emulsions can provide additional stability of oil globules to mechanical stress and high

7.6 Techno-Functional Properties of Conjugated Whey Proteins

shear forces, commonly experienced during unit operations such as mixing, pumping, flow, or atomization (Sagis & Scholten, 2014). Wooster and Augustin (2006) reported that the thickness of the interfacial layer in O/W emulsions stabilized by β-Lg 2 dextran conjugates can be modified by using carbohydrates with different Mw. Emulsions stabilized by conjugated milk proteins display greater oxidative stability than those stabilized by protein alone, possibly due to the increased thickness of the interfacial layer and the physical barrier that restricts the access of pro-oxidant species to oxidation-sensitive components such as lipids and lipid-soluble compounds. A significant improvement in the oxidative stability of emulsions containing β-carotene, stabilized by lactoferrin 2 dextran conjugates, compared to emulsions stabilized by the protein alone, was reported recently by Liu et al. (2016), where the antioxidative effect was attributed to physical restriction of contact between prooxidants and lipids by the thick interfacial layer of the conjugate-stabilized emulsion. Whey protein 2 carbohydrate conjugate-based emulsifiers offer significant potential for applications in emulsion-based delivery systems, where their interfacial functionality can facilitate controlled release of sensitive bioactives (e.g., vitamins) in the small intestine, avoiding acid-mediated emulsion destabilization and loss of the encapsulated material in the stomach. In the study of Lesmes and McClements (2012), β-Lg 2 dextran conjugate-stabilized emulsions displayed good stability to stomach-like environmental conditions, due to strong steric stabilization and subsequent release of encapsulated fatty acids during the intestinal stage of digestion, due to emulsifier displacement by bile salts.

7.6.4

Foaming

Changes to the structure/conformation of whey proteins, resulting from their conjugation with carbohydrates, generally contribute to increased protein solubility, higher protein mobility, and faster adsorption at air/water (A/W) interfaces. Improvement in foam capacity of BSA conjugated with glucose in a wet heating process (45 C for 2 hours with continuous stirring), compared to BSA conjugated with mannose or unconjugated BSA, was reported by Jian, He, Sun, and Pang (2016). In this study, conjugation resulted in changes in protein conformation, yielding a more flexible structure, which facilitated an increased rate of protein adsorption at the A/W interface. Similar findings were reported for foams stabilized by β-Lg 2 glucose conjugates (dry heating; 50 C at 65% RH for 96 hours) (Báez et al., 2013), where improved foam capacity, compared to using unconjugated β-Lg, was explained by heatinduced conformational changes in the structure of the whey protein molecules. A combination of increased hydrophobicity, and changes in protein

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conformation, can contribute to increased foam overrun as reported for supramolecular α-La 2 glycomacropeptide complexes (i.e., stabilized by noncovalent interactions) by Diniz et al. (2014). Conjugation of whey protein with carbohydrates helps to prevent extensive aggregation of protein when the electrostatic repulsion forces are disabled (i.e., at acidic pH or high ionic strength). In effect, more dense packing of protein, without extensive aggregation, can be achieved using conjugated proteins (Rade-Kukic, Schmitt, & Rawel, 2011). Controlled aggregation under these conditions, combined with flexible, unfolded protein structures, and low electrostatic repulsion, offer significant potential for stabilization of foam systems. Another approach to improving foam stability involves increasing the thickness, and thereby elasticity, of the interfacial film by increasing the size of its building blocks (i.e., controlled protein aggregation) (Dombrowski, Johler, Warncke, & Kulozik, 2016; Tamm, Sauer, Scampicchio, & Drusch, 2012) or by conformational changes to the protein structure (i.e., partial unfolding of globular protein) (Morales, Martínez, Pizones RuizHenestrosa, & Pilosof, 2015). These approaches closely match the changes to protein structure/conformation and functionality offered by protein conjugation; increased size of interfacial building blocks, controlled protein aggregation on conjugation and opening up of the protein structure have been shown to improve stability of foams formed with conjugated milk proteins (Corzo-Martínez, Carrera-Sánchez, Villamiel, Rodríguez-Patino, & Moreno, 2012; Hiller & Lorenzen, 2010). In using whey protein 2 carbohydrate conjugates to stabilize foams, the thickness of the interfacial layer can be controlled using carbohydrates with different Mw (Wooster & Augustin, 2006). Hiller and Lorenzen (2010) reported increased stability of foams prepared with a range of milk protein (WPI, sodium caseinate, and lactose-hydrolyzed skim milk) and carbohydrate (glucose, lactose, pectin, and dextran) conjugates (produced by dry heating at 70 C and 65% RH for up to 240 hours) due to formation of thick and viscoelastic interfacial films that prevented disproportionation of gas bubbles. Increasing the thickness of the interfacial film can improve its rheological properties, in addition to providing an effective steric barrier with good dilatational properties (Dombrowski et al., 2016); Kim, Cornec, and Narsimhan (2005) reported that denaturation and unfolding of β-Lg resulted in increased shear elasticity and viscosity of the interfacial layer due to increased flexibility of the partially denatured globular protein. Facilitating dense packing and interactions between protein-based building blocks is effective in improving viscoelastic properties of the interfacial layer of a foam (Mackie & Wilde, 2005). Cai and Ikeda (2016) reported increased resistance against surfactant-induced displacement of protein from the A/W

7.6 Techno-Functional Properties of Conjugated Whey Proteins

interface in foams stabilized with WPI 2 gellan conjugates prepared by dry heating at 80 C and 79% RH for 2 hours, compared to systems containing unconjugated WPI and the surfactant Tween 20. The authors attributed the greater resistance to displacement of protein in the conjugate-based foam system to the ability of the gellan moiety, covalently attached to the whey protein molecules, to form a carbohydrate network at the interface, effectively immobilizing the conjugate-covered interface. The increase in hydrophilicity of whey proteins on conjugation can contribute to enhanced foam stability due to improved water holding capacity by the conjugate located at the interfacial layer, serving to restrict liquid drainage in the foam (Báez et al., 2013). The hydrophilic nature of the carbohydrate anchored at the A/W interface by the protein, viscoelastic properties of the interface, and higher viscosity of conjugated milk protein systems (WPI, skim milk powder, sodium caseinate), compared to native proteins, have been shown by Hiller and Lorenzen (2010) to be the main factors responsible for increased foam stability. In contrast, other authors have claimed that the increased hydrophilicity of BSA resulting from its conjugation with glucose or mannose decreased foam stability (Jian et al., 2016). Both of these, apparently contradictory, findings can hold true, with the precise impact of conjugation being very dependent on differences in protein structure (e.g., globular, ordered, unordered), nature of the carbohydrate (e.g., chain length, charge) and conditions employed for conjugation and foam formation.

7.6.5

Gelation and Textural Properties

Whey protein gels are three-dimensional, self-supporting networks, within which the aqueous solution and any dispersed elements (e.g., fat) are entrapped. Gelation of whey proteins involves a controlled increase in protein 2 protein interactions, while carefully maintaining a balance with protein 2 solvent interactions (Brodkorb et al., 2016). During gelation, the number and combined strength of protein 2 protein interactions (e.g., disulfide, hydrophobic, and electrostatic interactions) determine the mechanical and rheological properties of the resultant gel network. High protein content whey ingredients (e.g., WPC and WPI) are commonly used in food applications that require gelation of the protein for the expression of functionality (e.g., recombined meat products, desserts, puddings, mousses). It has been known for over 20 years that heating solutions of whey proteins (e.g., lysozyme and BSA) and reducing sugars (e.g., lactose, ribose, and xylose), at temperatures of 90 121 C, results in the formation of gels with higher firmness and elasticity than gels made using the proteins alone in solution (Armstrong, Hill, Schrooyen, & Mitchell, 1994). The increased strength of these protein 2 carbohydrate gels is due to Maillard reaction-mediated

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reduction in pH and by cross-linking of the protein molecules (e.g., via methylglyoxal and lysinoalanine). The gel strength (but also, undesirably, color development) increases with decreasing Mw of the sugars, while the pH required to achieve gelation decreases with increasing sugar concentration and reactivity. In combination, these effects of sugar incorporation on gelation properties of globular protein on heating, means that it is possible to reduce the amount of protein required for gel formation (Azhar, 1996; Oliver, Melton, & Stanley, 2006). More recent work has focused on the gelation properties of whey proteins conjugated with higher Mw carbohydrates under dry heating conditions, due to the challenges associated with denaturation and aggregation of whey proteins under wet heating conditions. Conjugation of whey proteins in WPI with dextran has been shown to influence the rheological properties of heatinduced gels made therefrom. Conjugation of WPI with dextran of Mw 6, 40, and 70 kDa, under dry heating conditions at 60 C for 2 9 days at 63% RH resulted in whey protein-conjugate gels with lower fracture stress and Young’s Modulus (Spotti et al., 2013) and lower gel firmness (i.e., storage modulus) (Spotti et al., 2014), compared with WPI alone or unconjugated WPI 2 dextran mixtures. Similar results were reported by Sun, Yu, Yang, et al. (2011) for WPI conjugated with dextran (average Mw 150 kDa) at 60 C for 7 days at 79% RH. The lower strength of heat-set WPI-based gels made from whey protein conjugated with dextran, compared with unconjugated whey protein or mixtures of whey protein and carbohydrates is attributed to several factors, with the relative contribution of the individual factors dependent on the system composition and conditions under which conjugation was achieved. Under the heating conditions typically required to achieve conjugation, denaturation and aggregation of whey proteins can occur, serving to alter exposure and reactivity of functional groups (e.g., free sulfhydryl and hydrophobic groups) and the surface charge of protein molecules, all of which influence protein 2 protein and protein 2 water interactions (Brodkorb et al., 2016). Covalent attachment of the carbohydrate molecules also increases the hydrophilicity and steric barrier properties of the conjugated proteins, both of which result in decreased protein 2 protein interactions and increased protein 2 water interactions.

7.7 NUTRITIONAL PROPERTIES OF CONJUGATED WHEY PROTEINS One of the most common areas of focus in considering the nutritional properties of conjugated whey proteins is the decrease in availability of the

7.7 Nutritional Properties of Conjugated Whey Proteins

essential amino acid lysine, due to both its abundance in dairy proteins and the high reactivity of its ε-amino group toward reducing sugars (Pellegrino, Masotti, Cattaneo, Hogenboom, & De Noni, 2013). LAL is formed by the addition of the ε-amino group of lysine to the dehydroalanine residues, which are promoted during high temperature treatments at alkaline condition, typical of the Maillard reaction (Cattaneo, Masotti, & Pellegrino, 2009). However, formation of LAL is more of a challenge in casein- than whey protein-based systems due to the absence of phosphoseryl residues in the whey proteins (Cattaneo, Masotti, & Pellegrino, 2012). In an extensive animal study on digestibility it has been shown that with progression of the Maillard reaction in peas, the digestibility, availability, and utilization of lysine decreased (Van Barneveld, Batterham, Skingle, & Norton, 1995). Generally, lysine can be regenerated from early-stage MRPs (i.e., Schiff bases); however, this is not the case for advanced MRPs (De Almeida, 2013). In mixed whey protein systems (e.g., whey protein concentrate), β-Lg preferentially undergoes conjugation, and as the primary function of β-Lg is believed to be nutrition, conjugation directly affects the nutritional value of β-Lg, making the lysine in β-Lg, at least partially, unavailable for protein metabolism (Dyer et al., 2016). Maillard-induced conjugation can also potentially impact protein digestibility by altering the accessibility of proteolytic enzymes to cleavage sites due to changes in the structure and surface reactivity of whey protein molecules (Moscovici et al., 2014). On the other hand, structural rearrangement of globular whey proteins, by their glycation with carbohydrates, can also expose new cleavage sites and result in release of unique peptides with potential beneficial effects (Hiller & Lorenzen, 2010). Recent studies, utilizing an in vitro gastric model, showed that structural changes to LF, induced by Maillard reaction (i.e., conjugation with glucose, fructose, and oligofructose), resulted in increased proteolytic susceptibility of the protein (Moscovici et al., 2014). The authors also reported different peptide profiles for native and glycated LF on digestion, suggesting altered enzymatic cleavage patterns as a consequence of protein conjugation, while no adverse effects on the bioactivity of LF were reported as a result of conjugation. Several studies have reported reduced allergenicity of whey proteins, in particular β-Lg, on conjugation with carbohydrates. For example, Taheri-Kafrani et al. (2009) assessed the effect of moderate glycation of β-Lg with a range of sugars (lactose, galactose, glucose, ribose, rhamnose, and arabinose) on its recognition by immunoglobulin (Ig) E from patients suffering with cows’ milk allergy and determined that low levels of glycation had a limited effect on masking the allergenicity of β-Lg, with the magnitude of the effect increasing with increasing extent of Maillard-induced conjugation of the whey

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protein molecules. The authors concluded that glycation of β-Lg results in blockage of lysyl ε-amino groups and restricted access to epitopes that bind IgE; overall, glycated β-Lg displayed reduced allergenicity compared to nonglycated β-Lg. Lucey, Zhu, and Damodaran (2009) developed a process for the production of protein 2 polysaccharide conjugates by dry heating to yield novel, functional ingredients with potential health benefits in high value food products, including reduced allergenicity infant formulae. Böttger, Etzel, and Lucey (2013) demonstrated that glycated β-Lg remained digestible using an in vitro infant digestion model. Findings from their work helped to alleviate some concerns in regards to the risks that large amounts of glycated β-Lg could potentially be fermented to toxic compounds (e.g., ammonia, amines, and phenolic compounds) in the colon. The work of Böttger et al. (2013) also suggested that a high degree of glycation of β-Lg, or the use of a high molecular mass carbohydrate for glycation, might be more effective in masking immunogenic epitopes on the protein. In addition, the conditions used for glycation of β-Lg (e.g., time, temperature, wet vs dry heating), and the site of glycation on the protein, would be expected to be key determinants of digestibility and immunogenicity of conjugated whey proteins. Bu, Lu, Zheng, and Luo (2009) studied the effect of conjugation of bovine α-La with glucose on the antigenicity of the protein and concluded that the antigenicity of α-La was significantly decreased by conjugation with glucose and that the effect was influenced by the WPI:glucose ratio and by the reaction conditions (i.e., time and temperature). MRPs are a large heterogeneous group of compounds and their digestion depends largely on their structure. There is broad general agreement that MRPs can undergo three different metabolic fates upon digestion: (1) absorption upon protein release (e.g., by digestive enzymes); (2) excretion in the urine or in the feces; or (3) fermentation by microflora of the gastrointestinal tract (GIT) (De Almeida, 2013; Faist & Erbersdobler, 2001). The lysine residues in early-stage MRPs, such as Schiff bases, have been shown, using in vivo animal digestion studies, to have similar digestibility to that of free lysine, as these early-stage MRP products are reversible in the acidic environment of the stomach (De Almeida, 2013). Conversely, with the Amadori products, which are later stage MRPs, recovery of lysine is no longer possible and these compounds are generally excreted (in feces) or decomposed by the microbiota of the GIT (De Almeida, 2013), while Amadori compounds have also been shown to accumulate in the tissue of the kidneys, liver, or pancreas (Younus & Anwar, 2016). MRPs which are precursors of melanoidins can be metabolized to some extent by reductase enzymes (Faist & Erbersdobler, 2001), while melanoidin-based advanced MRPs have been shown to escape digestion in the upper GIT, while being broken down by Gram-positive cocci in the hindgut (Nursten, 2005).

7.8 Conclusions

In a study by Luz Sanz, Corzo-Martínez, Rastall, Olano, and Moreno (2007), β-Lg was reported to be resistant to hydrolysis by pepsin under in vitro gastrointestinal digestion conditions (pH 2.5), suggesting that it would remain intact after passage through the stomach to reach the upper portion of the small intestine. This resistance of β-Lg to hydrolysis was not affected by its glycation with galactooligosaccharides (GOS), where similarly, no digestion was observed in the simulated stomach environment. However, glycation of β-Lg with GOS resulted in reduced proteolysis in the duodenal environment (using trypsin and chymotrypsin). The authors suggested a possible implication of this finding would be that conjugation of prebiotic carbohydrates to whey protein molecules could potentially allow the carbohydrate to reach the colon in a more intact manner where it could then be fermented. Some MRPs display metal chelating abilities, which can provide beneficial health effects in limiting lipid oxidation or by binding of potentially toxic metals (Nursten, 2005). The ability of MRPs to interact with metals can also improve absorption and retention of metals with a significant nutritional value, which has been demonstrated for copper, for example (DelgadoAndrade, Seiquer, & Navarro, 2002). Oh et al. (2016) demonstrated that β-Lg conjugated with lactose and glucose displayed increased reducing power and higher radical-scavenging activity compared to native β-Lg.

7.8

CONCLUSIONS

Modification of whey protein functionality through Maillard-induced glycation offers considerable potential in the development of a new generation of functional whey protein-based ingredients. To be able to realize this potential, some challenges exist and need to be overcome, such as developing strategies for limiting the progression of the Maillard reaction to advanced stages, as these later stages are largely responsible for the less desirable aspects of the Maillard reaction. Little information exists in the peer-reviewed literature on the sensory properties of whey protein 2 carbohydrate conjugate ingredients; e.g., the covalent attachment of hydrophilic carbohydrate moieties to peptides during conjugation may present opportunities for reducing bitterness in conjugated WPHs. It is desirable to enrich whey protein 2 carbohydrate conjugates from the reaction mixtures in which they are produced in order to remove unreacted carbohydrate, unreacted protein, and possibly soluble MRPs, while increasing the concentration of conjugated protein. In spite of this, there is very little work published on the enrichment/purification of whey protein 2 carbohydrate conjugates (Bund, Allelein, Arunkumar, Lucey, & Etzel, 2012; Etzel & Bund, 2011) and this is likely to be an area of focus for the future. Further research on the nutritional and toxicological aspects of whey protein/

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peptide 2 carbohydrate conjugates and how conjugation impacts protein digestion and allergenicity would help in the development of conjugatebased ingredients for hypoallergenic food applications. In using whey protein/peptide 2 carbohydrate conjugates as emulsifiers in powdered emulsion-based products, deeper fundamental understanding is needed of the interfacial behavior of such systems and how inclusion of these ingredients in formulated food systems may affect spray drying properties and stability of the resultant powders. In addition to the conventional dry and wet heating approaches, some alternative/nonthermal technologies and combinations thereof, such as sonication, microfluidization, high hydrostatic pressure, and microwave treatment, are increasingly being studied in the pretreatment of protein substrates prior to conjugation or directly in the production of protein 2 carbohydrate conjugates; these offer potential for the production of whey protein/peptide conjugates with modified functionality.

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Setiowati, A. D., Vermeir, L., Martins, J., De Meulenaer, B., & Van der Meeren, P. (2016). Improved heat stability of protein solutions and O/W emulsions upon dry heat treatment of whey protein isolate in the presence of low-methoxyl pectin. Colloids & Surfaces A: Physicochemical & Engineering Aspects, 510, 93 103. Singh, A. M., & Dalgleish, D. G. (1998). The emulsifying properties of hydrolyzates of whey proteins. Journal of Dairy Science, 81, 918 924. Spotti, M. J., Martinez, M. J., Pilosof, A. M., Candioti, M., Rubiolo, A. C., & Carrara, C. R. (2014). Influence of Maillard conjugation on structural characteristics and rheological properties of whey protein/dextran systems. Food Hydrocolloids, 39, 223 230. Spotti, M. J., Perduca, M. J., Piagentini, A., Santiago, L. G., Rubiolo, A. C., & Carrara, C. R. (2013). Does dextran molecular weight affect the mechanical properties of whey protein/dextran conjugate gels. Food Hydrocolloids, 32, 204 210. Sun, W. W., Yu, S. J., Yang, X. Q., Wang, J. M., Zhang, J. B., Zhang, Y., & Zheng, E. L. (2011). Study on the rheological properties of heat-induced whey protein isolate dextran conjugate gel. Food Research International, 44, 3259 3263. Sun, W. W., Yu, S. J., Zeng, X. A., Yang, X. Q., & Jia, X. (2011). Properties of whey protein isolate dextran conjugate prepared using pulsed electric field. Food Research International, 44, 1052 1058. Taheri-Kafrani, A., Gaudin, J. C., Rabesona, H., Nioi, C., Agarwal, D., Drouet, M., . . . Haertle, T. (2009). Effects of heating and glycation of β-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients. Journal of Agricultural & Food Chemistry, 57, 4974 4982. Tamm, F., Sauer, G., Scampicchio, M., & Drusch, S. (2012). Pendant drop tensiometry for the evaluation of the foaming properties of milk derived proteins. Food Hydrocolloids, 27, 371 377. Uribarri, J., Cai, W., Sandu, O., Peppa, M., Goldberg, T., & Vlassara, H. (2005). Diet-derived advanced glycation end products are major contributors to the body’s AGE pool and induce inflammation in healthy subjects. Annals of the New York Academy of Sciences, 1043, 461 466. Van Barneveld, R. J., Batterham, E. S., Skingle, D. C., & Norton, B. W. (1995). The effect of heat on amino acids for growing pigs: 4. Nitrogen balance and urine, serum and plasma composition of growing pigs fed on raw or heat-treated field peas (Pisum sativum). British Journal of Nutrition, 73, 259 273. Van Boekel, M. A. J. S. (2001). Kinetic aspects of the Maillard reaction: A critical review. Food/ Nahrung, 45, 150 159. Van Lancker, F., Adams, A., & De Kimpe, N. (2011). Chemical modifications of peptides and their impact on food properties. Chemical Reviews, 111, 7876 7903. Wang, Q., & Ismail, B. (2012). Effect of Maillard-induced glycosylation on the nutritional quality, solubility, thermal stability and molecular configuration of whey protein. International Dairy Journal, 25, 112 122. Wang, Q., He, L., Labuza, T. P., & Ismail, B. (2013). Structural characterisation of partially glycosylated whey protein as influenced by pH and heat using surface-enhanced Raman spectroscopy. Food Chemistry, 139, 313 319. Wang, W., & Zhong, Q. (2014). Properties of whey protein maltodextrin conjugates as impacted by powder acidity during the Maillard reaction. Food Hydrocolloids, 38, 85 94. Wijayanti, H. B., Bansal, N., & Deeth, H. C. (2014). Stability of whey proteins during thermal processing: A review. Comprehensive Reviews in Food Science & Food Safety, 13, 1235 1251. de Wit, J. N. (1989). Functional properties of whey proteins. In P. F. Fox (Ed.), Developments in dairy chemistry (Vol. 4, pp. 285 321). New York: Elsevier Applied Science.

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Wong, S. S. (1991). Application of chemical cross-linking to soluble proteins. Chemistry of protein conjugation & cross-linking (p. 226) Florida: CRC Press. Wooster, T. J., & Augustin, M. A. (2006). β-Lactoglobulin-dextran Maillard conjugates: Their effect on interfacial thickness and emulsion stability. Journal of Colloid & Interface Science, 303, 564 572. Wrodnigg, T. M., & Eder, B. (2001). The Amadori and Heyns rearrangements: Landmarks in the history of carbohydrate chemistry or unrecognized synthetic opportunities. In A. E. Stutz (Ed.), Glycoscience Epimerization, isomerization, rearrangement reactions of carbohydrates; topics in current chemistry (Vol. 215, pp. 115 152). New York: Springer. Yadav, M. P., Parris, N., Johnston, D. B., Onwulata, C. I., & Hicks, K. B. (2010). Corn fiber gum and milk protein conjugates with improved emulsion stability. Carbohydrate Polymers, 81, 476 483. Younus, H., & Anwar, S. (2016). Prevention of non-enzymatic glycosylation (glycation): Implication in the treatment of diabetic complication. International Journal of Health Sciences, 10, 261 277. Zhu, D., Damodaran, S., & Lucey, J. A. (2008). Formation of whey protein isolate (WPI) 2 dextran conjugates in aqueous solutions. Journal of Agricultural & Food Chemistry, 56, 7113 7118. Zhu, D., Damodaran, S., & Lucey, J. A. (2010). Physicochemical and emulsifying properties of whey protein isolate (WPI) 2 dextran conjugates produced in aqueous solution. Journal of Agricultural & Food Chemistry, 58, 2988 2994.

CHAPTER 8

Novel Processing Technologies: Effects on Whey Protein Structure and Functionality 1

Thom Huppertz1, Todor Vasiljevic2, Bogdan Zisu3 and Hilton Deeth4

FrieslandCampina, Amersfoort, The Netherlands, 2Victoria University, Melbourne, VIC, Australia, 3RMIT University, Bundoora, VIC, Australia, 4University of Queensland, Brisbane, QLD, Australia

8.1

INTRODUCTION

Whey proteins (WP) are characterized by exemplary digestibility, availability, amino acid pattern and biological value, sensory characteristics, and multiple functionalities for their use in food product applications. Based on the amino acid composition and the rate of peptide and amino acid release in the small intestine, they present an exclusive nutritional and physiologically functional supplement, better than any other dietary protein (Sah, McAinch, & Vasiljevic, 2016) (See also Chapters 14 & 17). WP are widely used in the food industry with some common applications including sport beverages (see also Chapter 16) and liquid meat replacements, baked products and processed meats, salad dressings, artificial coffee creams, soups, and various other dairy products (Fitzsimons, Mulvihill, & Morris, 2007). High solubility of WP over a wide range of pH is an added advantage for the exploitation of their other functionalities, such as foaming, emulsifying, gelling, heat coagulation, and water binding, over various types of food products. In addition, WP, being excellent foaming and emulsifying agents, have an ability to form cohesive and viscoelastic films by polymerization mainly via disulfide bonds and hydrophobic interactions (Bouaouina, Desrumaux, Loisel, & Legrand, 2006) (see also Chapter 11). Also, ability of WP to form heat- and cold-set stable gels to perform as a matrix for holding other components of a food is very useful in food formulations and product development (Resch, Daubert, & Foegeding, 2004). This is in addition to the superior nutritional benefits obtained from WP compared to pregelatinized starch and hydrocolloids, which also allow water holding and increasing viscosity (Resch, Daubert, & Foegeding, 2005). 281 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00009-6 © 2019 Elsevier Inc. All rights reserved.

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In the last few decades, understanding of the nutritional and economic value of WP and their myriad of functionalities has grown exponentially. With this understanding, growing interest in using WP beyond traditional applications has evolved and thus also put increasing demands on the functional properties of these proteins, and how they can be attained or retained. In addition, a number of novel processing technologies have emerged over the past few decades, providing alternatives to the traditional thermal processing technologies. In this chapter, the effects of a number of novel processing technologies on WP are described, i.e., high pressure processing, shear processing, low-frequency power ultrasound processing, pulsed electric field (PEF), and UV processing. In all cases, emphasis is made to link specific effects of these novel technologies on protein structure to functional properties of the WP which are valued and sought-after in application.

8.2 8.2.1

HIGH-PRESSURE PROCESSING Introduction

In the late 19th Century, the pioneering work by Hite (1899) showed the potential of high-pressure (HP) processing as a nonthermal preservation process. Today, several HP-treated products, such as fresh fruit jams, jellies, juices, salad dressings, rice, cakes, and guacamole are commercially available. In most cases, these applications have been based around the ability of HP processing to extend the shelf life of products without negative side reactions such as loss of nutritional value, discoloration, or loss of sensory properties. However, another interesting aspect of HP processing is its ability to modulate protein structure and hence affect protein functionality. In this section, HP-induced changes in WP are described and discussed.

8.2.2

High Pressure Processing and Its Effect on Proteins

HP processing involves the application of hydrostatic pressures in the range 100
800 MPa to samples for a defined time. Treatments are typically carried out on prepackaged samples in flexible packaging which are placed in vessels filled with a pressure-transmitting fluid, e.g., water. Once the vessel is sealed, hydrostatic pressure is increased by pumping additional fluid into the vessel until the desired pressure is reached. The pressure is typically maintained for several times ranging from 0 to 30 minutes and is subsequently released. In some cases, a heating jacket around the vessel can be used to achieve a desired temperature. Application of such high pressures results in considerable compression of the sample. At a pressure of 600 MPa, water volume is reduced by B15%. This volume reduction is the driving force for biochemical changes in the system

8.2 High-Pressure Processing

as a result of HP pressure processing. According to Le Chatelier’s principle, when a stress is applied to a system, the system will aim to counteract this stress. In the case of HP processing, this means that reactions involving a reduction in volume will be promoted. For aqueous solutions, the main changes occur in the structuring of the main component, i.e., water. Application of high pressure causes proteins to lose their native threedimensional structure and leads to denaturation and aggregation of WP and/ or interactions with each other (whey protein 2 whey protein interactions) or with the caseins (casein 2 whey protein interactions). The native three-dimensional structure of proteins is maintained by a variety of noncovalent interactions (such as hydrogen bonding, electrostatic, van der Waals’, and hydrophobic interactions) between amino acid residues within the polypeptide chain and between residues and solvent molecules. Threedimensional structure has a very important role to play in the stability and functional properties of a protein. HP processing at room temperature disrupts only relatively weak bonding, such as hydrophobic and electrostatic interactions (Balny & Masson, 1993; Silva & Weber, 1993), whereas hydrogen bonds are relatively insensitive to pressure, as are covalent bonds (Hayakawa, Linko, & Linko, 1996). This suggests that high pressure affects the tertiary and quaternary structure of proteins, i.e., the three-dimensional configuration, held together mainly by hydrophobic and ionic interactions, and the noncovalent association into dimers and higher-order structures, respectively. The effects of HP on denaturation, aggregation, and interactions of WP have been studied extensively under various conditions and in different systems such as milk (e.g., Anema, Stockmann, & Lowe, 2005; Arias, López-Fandiño, & Olano, 2000; Felipe, Capellas, & Law, 1997; Huppertz, Fox, & Kelly, 2004a, 2004b; Huppertz, Kelly, & Fox, 2002; Law et al., 1998; Patel, Singh, Havea, Considine, & Creamer, 2005; Scollard, Beresford, Needs, Murphy, & Kelly, 2000), whey protein concentrate (WPC; e.g., Van Camp, Messens, Clément, & Huyghebaert, 1997a, 1997b), whey protein isolate (WPI; Hinrichs, Rademacher, & Kessler, 1996a, 1996b; Michel et al., 2001), and pure WP (Dumay, Kalichevsky, & Cheftel, 1994, 1998; Funtenberger, Dumay, & Cheftel, 1995; Galazka, Dickinson, & Ledward, 1996; Jegouic, Grinberg, Guingant, & Haertlé, 1996; Olsen, Ipsen, Otte, & Skibsted, 1999). Reports suggest that pressure-induced reactions of WP lead to unfolding of monomeric proteins, aggregation and gelation (Fertsch, Müller, & Hinrichs, 2003; Huppertz et al., 2002; Tedford, Kelly, Price, & Schaschke, 1999; Tedford, Smith, & Schaschke, 1999; Van Camp & Huyghebaert, 1995a, 1995b; Van Camp et al., 1997a, 1997b), by formation of intra- and intermolecular bonds through hydrophobic interactions and disulfide bridges (e.g., Cheftel, 1992; Galazka et al., 1996; Hoover, 1993; Masson, 1992; Messens, Van Camp, & Huyghebaert, 1997; Trujillo, Capellas, Saldo, Gervilla, & Guamis, 2002),

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depending on the type of protein, protein concentration, pH, ionic strength, applied pressure, pressurizing temperature, and duration of the pressure treatment (Fertsch et al., 2003; Huppertz et al., 2004a; Messens et al., 1997). Temperature during HP treatment also affects the denaturation and aggregation of proteins (e.g., Huppertz et al., 2004a; Patel, 2007; Tedford et al., 1999).

8.2.3 Denaturation and Aggregation of Pure Whey Protein Fractions in Model Systems 8.2.3.1 β-Lactoglobulin (β-Lg)

Many reports have suggested that β-lactoglobulin (β-Lg) is the most sensitive of the major WP to HP and that it dominates the HP-induced denaturation, aggregation, and gelation of the whey protein system (Belloque, LópezFandiño, & Smith, 2000; Considine, Patel, Singh, & Creamer, 2005a; Considine, Patel, Anema, Singh, & Creamer, 2007; López-Fandiño, 2006a, 2006b; Patel, Singh, Anema, & Creamer, 2004; Patel et al., 2005; Stapelfeldt, Petersen, Kristiansen, Qvist, & Skibsted, 1996; Van Camp, Feys, & Huyghebaert, 1996; Van Camp et al., 1997a, 1997b). Therefore, a considerably body of work has focused on the mechanisms of unfolding and aggregation of β-Lg during pressurization or after pressure release. The majority of these reports deal with the properties of the proteins after pressure release, but some have involved measurement while at elevated pressure. At relatively low pressures (50 MPa), thiol reactivity analysis (Møller, Stapelfeldt, & Skibsted, 1998; Stapelfeldt, Olsen, & Skibsted, 1999) and NMR (Tanaka & Kunugi, 1996) studies suggest the existence of a “predenatured” state of β-Lg, i.e., a notcompletely-unfolded structure which precedes reversible denaturation. The “core” of the β-Lg molecule remains unaffected by this “predenaturation” as shown by the limited deuterium exchange at 100 MPa by 1H NMR (Belloque et al., 2000). Pressures up to 140 MPa also did not affect β-sheets in β-Lg (Subirade, Loupil, Allain, & Paquin, 1998), whereas the reactivity of the free sulfhydryl group of β-Lg increased with pressure up to 150 MPa (Tanaka, Koyasu, Kobayashi, & Kunugi, 1996; Tanaka, Nakajima, & Kunugi, 1996). A three-step denaturation process has been suggested for the HP-induced denaturation of β-Lg (Considine, Singh, Patel, & Creamer, 2005b; Stapelfeldt & Skibsted, 1999); i.e., monomerization of β-Lg dimers (Iametti et al., 1997), followed by a decrease in α-helix and β-sheet content (Hayakawa et al., 1996; Panick, Malessa, & Winter, 1999) and irreversible aggregation through intermolecular disulfide bonds (Funtenberger, Dumay, & Cheftel, 1997; Iametti et al., 1997; López-Fandiño, Ramos, & Olano, 1997; Møller et al., 1998). Protein concentration (Dumay et al., 1994), pH, ionic strength and composition (Cheftel & Dumay, 1996; Funtenberger et al., 1995), conditions

8.2 High-Pressure Processing

of HP treatment (pressure, time, temperature) (Patel, 2007; Yang, Dunker, Powers, Clark, & Swanson, 2001), and solutes such as sucrose (Dumay et al., 1994) affect the pressure-induced denaturation and aggregation of β-Lg. Different aspects of the pressure-induced unfolding and aggregation of β-Lg have been reviewed in detail (see Considine et al., 2007; López-Fandiño, 2006b), including the effects of high pressure on the functional properties of β-Lg (López-Fandiño, 2006b).

8.2.3.2 α-Lactalbumin (α-La)

Compared to β-Lg, α-La is more resistant to HP-induced denaturation (Huppertz et al., 2004a; López-Fandiño, Carrascosa, & Olano, 1996; Patel et al., 2004, 2006; Scollard et al., 2000; Tanaka & Kunugi, 1996). At neutral pH, reversible unfolding of α-La to a molten globule state commences at 200 MPa and loss of native structure becomes irreversible only beyond 400 MPa, as compared with 50 and 150 MPa, respectively, for β-Lg (Stapelfeldt & Skibsted, 1999; Tanaka & Kunugi, 1996; Tanaka, Tsurui, Kobayashi, & Kunugi, 1996; McGuffey et al., 2005). Differences in their secondary structures (which lead to a higher hydrophobicity for β-Lg) and/or in the number of disulfide bonds (four in α-La and two in β-Lg) and also the Ca-binding sites (Tanaka & Kunugi, 1996) have been suggested as reasons for the higher barostability of α-La compared to β-Lg. Considerably higher barostability has been observed for the holo- than the apo-form of α-La (Dzwolak, Kato, Shimizu, & Taniguchi, 1999; Hosseini-nia, Ismail, & Kubow, 2002). α-La does not form aggregates when treated with pressure alone at 800 MPa (Patel, 2007), but forms high-molecular-weight disulfide-bonded oligomers at high pressure in the presence of thiol reducers (Jegouic et al., 1996).

8.2.3.3 Bovine Serum Albumin BSA has been found to be quite resistant to pressure treatment up to 400 MPa (Hayakawa, Kajihara, Morikawa, Oda, & Fujio, 1992; López-Fandiño et al., 1996; López-Fandiño, 2006b; Patel et al., 2004; Patel et al., 2005; Patel et al., 2006). HP-induced changes in the secondary structure of BSA are largely reversible (Hosseini-nia et al., 2002) and the high stability of BSA is probably related to the rigid molecular structure due to the presence of 17 intramolecular disulfide bonds (Hayakawa et al., 1992; López-Fandiño et al., 1996; LópezFandiño, 2006b). At B150 MPa, BSA undergoes structural modifications, yielding an increase in the molecular volume (Ceolín, 2000), but these changes in the secondary structure of BSA are largely reversible (LópezFandiño, 2006b). Treatment at 800 MPa has a substantial effect on the secondary structure of BSA and results in polymerization of BSA through disulfide bonding involving the free sulfhydryl residue (Galazka, Ledward, Sumner, & Dickinson, 1997; Galazka, Sumner, & Ledward, 1996; Patel, 2007).

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8.2.3.4 Mixed Whey Protein Solutions In addition to the aforementioned studies using single protein systems, studies have also been conducted on the denaturation, aggregation, and gelation of WP on HP treatment of solutions of whey protein ingredients, such as WPC or WPI. Characterization of HP-treated WPC solutions using 2D-polyacrylamide gel electrophoresis (PAGE) (Patel et al., 2004; Patel et al., 2005) showed that HP generated both noncovalently bonded and disulfidebonded aggregates containing all the WP. Disulfide-linked aggregates in HP-treated WPC solutions showed disulfide-bonded dimers, trimers, tetramers, 1:1 complexes of β-Lg and α-La, as well as higher-molecular-weight disulfide-linked aggregates consisting of BSA, β-Lg and α-La. β-Lg, α-La, and BSA participate in HP-induced aggregation and gelation through intermolecular disulfide bonding, with the number of stabilizing disulfide bonds strongly influencing the textural properties of the gels. Intermolecular interactions and irreversible aggregation are favored at high protein concentration (Dumay et al., 1994; Wong & Heremans, 1988). Patel (2007) reported that the rate of aggregation of the WP in WPC solutions increased with an increase in the concentration of protein and the pressurizing temperature. The combination of low protein concentration, mild treatment pressure (200 MPa) and ambient temperature led to minimal loss of nativelike and SDS-PAGE-monomeric β-Lg, whereas the combination of high protein concentration, severe treatment pressure (600 MPa) and elevated pressurizing temperature (40 C and higher) led to significant loss of both native-like and SDS 2 PAGE-monomeric β-Lg. The sensitivity of the pressure-resistant WP, α-La and BSA, to aggregation was strongly increased at pressurizing temperatures of 40 C and higher. Self-supporting gels were formed when WPC solutions (8%
10%) were HP treated at 600
800 MPa and 20 C. At sufficiently high protein concentration, HP-induced whey protein gels formed at 200
400 MPa (Van Camp & Huyghebaert, 1995a, 1995b; Van Camp et al., 1996). HP-induced whey protein gels are typically more porous, less firm and elastic, and weaker than their counterparts from heat treatment (Cheftel & Dumay, 1996; Dumay et al., 1998; Van Camp & Huyghebaert, 1995b; Zasypkin, Dumay, & Cheftel, 1996). Microstructural analysis combined with protein interaction studies showed HP-induced whey protein gels to contain protein aggregates cross-linked by intermolecular disulfide bonds and by noncovalent interactions (Patel et al., 2006). The size and distribution of these aggregates determines the rheological properties and opacity of the samples.

8.2.4

HP-Induced Changes in Milk

In addition to changes in whey protein solutions, HP-induced changes in WP have also been studied in milk as reviewed by Huppertz et al. (2002), Huppertz, Fox, de Kruif, and Kelly (2006), Huppertz, Smiddy, Upadhyay,

8.2 High-Pressure Processing

and Kelly (2006), López-Fandiño (2006a, 2006b), and Considine et al. (2007). In milk, β-Lg is the most pressure-sensitive among the WP and B . 75% denaturation of β-Lg can occur after treatment at 400 MPa (Arias et al., 2000; García-Risco, Olano, Ramos, & López-Fandiño, 2000; LópezFandiño & Olano, 1998; López-Fandiño et al., 1996; Scollard et al., 2000). The reaction order of pressure-induced denaturation of β-Lg is 2.5 (Hinrichs, Rademacher, & Kessler, 1996b), indicating that the denaturation process is concentration-dependent and that a lower initial concentration of native β-Lg should reduce the extent of denaturation of β-Lg under pressure. In contrast, α-La in milk is stable to pressures of at least 400 MPa (Arias et al., 2000; Felipe et al., 1997; García-Risco et al., 2000; Gaucheron et al., 1997; Hinrichs et al., 1996b; Hinrichs et al., 1996a; Huppertz et al., 2004b; Huppertz et al., 2002; López-Fandiño & Olano, 1998; López-Fandiño et al., 1996; Needs et al., 2000; Scollard et al., 2000). BSA has also been found to be resistant to pressures up to 600 MPa in milk (Hayakawa et al., 1992; Hinrichs et al., 1996b; López-Fandiño et al., 1996). The pressure intensity and the holding time have also been reported to affect the level of denaturation of WP in milk (López-Fandiño & Olano, 1998; Anema et al., 2005; Hinrichs & Rademacher, 2005; Huppertz et al., 2004a). Also, β-Lg and α-La are reported to be comparatively more pressure-resistant in whey than in milk, which may be attributed to the absence of casein micelles and colloidal calcium phosphate in whey (Huppertz et al., 2004b). On HP treatment of milk at 300
600 MPa, β-Lg may form small aggregates (Felipe et al., 1997) or may interact with the casein micelles (Huppertz, Fox, & Kelly, 2004c; Needs et al., 2000; Scollard et al., 2000). It was reported that, when mixtures of κ-casein (κ-CN) and β-Lg were pressure treated at 400 MPa, the presence of β-Lg reduced the susceptibility of κ-CN to subsequent hydrolysis by chymosin, indicating interactions between the proteins (López-Fandiño et al., 1997). SDS-PAGE analysis of pressure-treated and untreated milks or solutions containing κ-CN or β-Lg or both in the presence or absence of denaturing agents showed evidence of the formation of aggregates linked by intermolecular disulfide bonds (López-Fandiño et al., 1997). Interestingly, αs2-CN occurs at the same concentration as κ-CN, and has one disulfide bond, but has not normally been reported to interact with β-Lg in milk systems heated at 85
90 C. In contrast, Patel et al. (2006) concluded that the effects of heat treatment and HP treatment on the interactions of the caseins and WP in milk were significantly different by demonstrating the formation of disulfide-linked complexes involving αs2-CN, κ-CN, and WP in heat- and pressure-treated milks. The results have been explained using modified 2D SDS- and then reduced SDS-PAGE and by proposing possible reactions of the caseins and WP in heat- and pressure-treated milk. The virtual absence of αs2-CN from the heat-induced aggregates formed at 85
90 C in milk, as reported in previous studies, might be because αs2-CN is not a

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surface component of the micelle and therefore its disulfide bond(s) are inaccessible to the denatured β-Lg. On the other hand, κ-CN is on the surface of the micelles, and its disulfide bond(s) could be readily accessible to a thiol group of β-Lg. Moreover, it has been reported that large quantities of very large aggregates that cannot enter the gel are present to a greater extent in heat-treated milk than in pressure-treated milk (Patel et al., 2006), indicating that the sizes of the aggregates are comparatively smaller in pressure-treated milks than in heat-treated milks. Such differences can be attributed to different effects of heat treatment and pressure treatment on the structure of the proteins, which may ultimately lead to different textures of the final products. Upon HP treatment of milk serum depleted of casein micelles, no sedimentable WP were observed despite high levels of whey protein denaturation, indicating that sedimentable WP in HPP-treated milk are mostly associated with the casein micelles (Huppertz et al, 2004a). The level of denatured β-Lg associated with the casein micelles increased with increase in pressure intensity, treatment time, and pressurization temperature (Anema, 2008, 2010; Huppertz et al., 2004a; Zobrist, Huppertz, Uniacke, Fox, & Kelly, 2005). No effects of β-Lg or the solids content of milk were observed (Anema, 2008). However, the association of β-Lg with casein micelles increased with increase in pH of the milk before HP-treatment (Anema, 2010; Huppertz et al., 2004a), whereas the addition of KIO3 to milk prior to HPP treatment resulted in a lower level of denatured β-Lg associated with casein micelles in HPP-treated milk, probably because the formation of disulfide bridges through thiol 2 thiol interactions rather than thiol 2 disulfide interchange reactions is favored in the oxidizing environment (Zobrist et al., 2005). In contrast to β-Lg, most denatured α-La was found in the serum phase of HPP-treated milk. The 2D-PAGE results of Patel et al. (2006) also showed that pressure treatment of milk at 200 MPa caused β-Lg to form disulfide-bonded dimers and incorporated β-Lg into aggregates, probably disulfide-bonded to κ-CN, suggesting that preferential reaction occurred at this pressure. The other WP appeared to be less affected at 200 MPa. In contrast, pressure treatment at 800 MPa incorporated β-Lg and most of the minor WP (including immunoglobulin, Ig, and lactoferrin, Lf), as well as κ-CN and much of the αs2-CN, into large aggregates. However, only a proportion of the α-La was denatured or incorporated into the large aggregates. The relatively lower degree of α-La reactivity at high pressures is probably related to the relative stability of this protein compared with β-Lg, as discussed earlier, and is based on the unusual pressure-dependent behavior of α-La (Kobashigawa, Sakurai, & Nitta, 1999; Kuwajima, Ikeguchi, Sugawara, Hiraoka, & Sugai, 1990; Lasalle et al., 2003). At higher pressures (.400 MPa), the polymerization of β-Lg becomes the

8.3 Shear-Based Processing

norm and pressure-induced β-Lg aggregation becomes similar to heatinduced β-Lg aggregation. The β-Lg in WPC or in milk is not significantly modified by the other components, i.e., β-Lg dominates the denaturation and aggregation pathway during pressure (.400 MPa) treatment, as it has been shown to dominate the reaction at high-temperature heat treatments. All these results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that result in differences among the compositions of resultant aggregation or interaction products (including their sizes) that ultimately may affect product functionalities.

8.3 8.3.1

SHEAR-BASED PROCESSING Introduction

Although WP are a unique nutritional and functional protein source, the major challenge in their applications is their heat-induced destabilization (Onwulata, Konstance, & Tomasula, 2004) or conformational changes during storage leading to, e.g., hardening of protein-rich solid foods (Purwanti, 2012) (see also Chapter 13). Native WP have a high solubility due to a high proportion of hydrophilic residues on the surface in their native state. However, in the presence of denaturants such as heat, pressure, or urea, these globular proteins unfold and subsequently aggregate with the rates and pathways of these physicochemical reactions determined by factors intrinsic to protein characteristics and extrinsic environmental factors such as protein concentration, pH, temperature, ionic strength, and solvent condition (Marangoni, Barbut, McGauley, Marcone, & Narine, 2000). On the other hand, functionality of WP may be manipulated by application of different processing methods, such as application of high pressure shearing, agitation or, in brief, microparticulation, that would either stabilize conformational properties and thus prevent complete protein denaturation, or control the rate of aggregation during or after primary denaturation that would create particles of a colloidal size (see also Chapter 3).

8.3.2

General Principles

During processing, dairy systems are subjected to various phenomena such as flow, heat convection and conduction, and diffusion. Dairy materials, whether liquid or solid, tend to flow if a force is applied onto them over a certain period of time. This force, usually defined as a stress vector (σ), which can be resolved into tangential and normal components, may cause a deformation on the system, which is defined as a change in the distance between two points in the material (Walstra, 2003) and is usually expressed as a

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_ a strain (ϒ). The time derivative of the strain is defined as the strain rate (γ), parameter which is commonly applied in food engineering. When a stress (force) is applied to a fluid, as in case of pumping or homogenization, it will follow either laminar or turbulent flow. Laminar flow exists as several types, depending on the geometrical constraints; some examples include simple shear, rotational, and elongational flows. During a simple shear flow, the following equation is applied to Newtonian fluids, such as milk or a protein dispersion at a low concentration: σ 5 ηU

dγ 5 ηUγ_ dt

ð8:1Þ

The term η is defined as viscosity and presents a measure of the extent to which a fluid resists flow. Viscosity depends on the type of flow and is always higher for the elongational flow than that for a simple shear flow. When solid spherical particles are subjected to flow, they will rotate at a constant rate; however, anisometric particles do not rotate at a completely constant rate. A deformable sphere or a polymer coil, which would be representative of proteins, can also elongate either by obtaining an orientation of about 45 degrees to the direction of flow, with the liquid rotating inside the particle (Fig. 8.1A), or by coil elongation through coil compression and extension (Fig. 8.1B). When the velocity of the fluid exceeds a certain level, laminar flow converts into turbulent flow, in which the streamlines are described as chaotic with fluid constituents subjected to rapid fluctuations both in velocity and in direction of flow. Created vortices provide a strong mixing effect and dissipation of kinetic energy. Folded conformations of proteins are only marginally stable under the most optimal conditions and can be easily disrupted with an environmental change such as increased temperature and solvent quality. Balance between the stability and instability of native proteins is governed by a great number of rather small

FIGURE 8.1 Motion of simple proteins in a simple shear flow. Protein A, α-Lactalbumin (α-La)—as an example of a deformable sphere; Protein B, β-lactoglobulin (β-Lg) dimer—as an example of a polymer coil.

8.3 Shear-Based Processing

but cooperative contributions and compensations of attraction and repulsion. These interactions include conformational degrees of freedom of the polypeptide chain and those of internal water and external hydration waters in addition to interplay between protein and solvent (Fenimore, Frauenfelder, McMahon, & Parak, 2002). In addition to enthalpic contributions, entropic effects exerted via flexibility of side chains or via presence of more or less ordered water play a substantial role, therefore depicting the native state of a protein as a well-balanced compromise between stability and flexibility (Scharnagl, Reif, & Friedrich, 2005). The change of the native state of proteins can be described as a two-state approach considering the folding
denaturing transition as a phase transition (Fersht, 1999). The free energy of change (ΔG) is associated with the transition and expressed as a difference between the free energy of the denatured state (GD) to that of the native state (GN): ΔG 5 GD 2 GN

ð8:2Þ

Since multiple factors may induce the transition, ΔG is a multidimensional function of all the independent variables such as temperature (T), pressure (P), cosolvent (x), concentration of cosolvent (cx), pH, and so on: ΔG 5 ΔGo 1 f ðT; P; x; cx ; pH; . . .Þ

ð8:3Þ

The free energy gap which governs the native from the unfolded state can be sufficiently small to induce spontaneous unfolding that may be followed by irreversible association or dissociation reactions. Such a small free energy difference of proteins between their native and denatured state is influenced by the negating effects of the entropic and the enthalpic contributions to the folding process (Walstra, 2001). The total entropy of the protein along with the associated water shell decreases as the protein folds, increasing the free energy. On the other hand, the enthalphy change becomes negative due to hydrogen bond formation. The maximum value of the free energies of unfolding is not large and ranges between 20 and 65 kJ/mol (Fersht, 1999). In addition, protein stability is greatly impacted by the solvent, which is in many cases water mixed with cosolvents impacting ionic strength, pH, and chemical affinity, all of which can induce unfolding or folding, and may also lead to significant changes of the critical physical parameters such as critical temperature or critical pressure which govern the phase boundaries between the native and the denatured states.

8.3.3 Conformational and Colloidal Modifications of Proteins Due to Shear Most of the work on structural modification of proteins due to shear was performed using enzymes as a model as they would frequently lose their activity under this condition. Shear-induced changes of proteins were originally

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studied by Charm and Wong (1970) who subjected catalase and carboxypeptidase to shearing in a narrow-gap coaxial cylinder and observed a loss of tertiary structure due to shear forces. Charm and Lai (1971) also found that catalase could be inactivated during circulation through ultrafiltration devices while rennet was not affected. They suggested that shear rate and exposure time have great importance in terms of loss of activity. Tirrell and Middleman (1975) studied urea hydrolysis by urease under shear and observed both temporary and permanent inactivation. Furthermore, Charm and Wong (1981) claimed that comparatively turbulent flow leads to greater loss in enzymatic activity than laminar flow. Later, some other studies showed that loss of enzyme activity could also be related to interfacial effects such as air bubble entrainment or adsorption to solid surfaces than to shear alone (Lee & Choo; 1989; Thomas & Geer, 2011). As indicated, all these observations of shear-induced unfolding of proteins was probed using enzyme activity assays that lack the sensitivity and time resolution of optical spectroscopic probes of protein conformation. Removing the protein from shearing flow to allow for measuring residual enzyme activity may also provide sufficent time for protein refolding before measurement. For this reason, Jaspe and Hagen (2006) subjected a well-characterized protein to high shear rates under controlled conditions using a sensitive fluorescence spectroscopy probe to detect even small conformational changes. They demonstrated that even shear rates of 200,000 s21 did not result in any detectable protein unfolding. Their model suggested that the thermodynamic stability of the protein required an extraordinarily high shear rate (B107 s21) to destabilize a typical small protein of B100 amino acids in water. For unfolding to take place, the molecular weight of a protein or viscosity of the solution needs to be sufficiently high. Therefore, protein denaturation could still occur in a highly turbulent flow. This observation was made in another study, in which Bee et al. (2009) exposed immunoglobulin-G1 (IgG1) monoclonal antibody to shear rates between 20,000 and 250,000 s21 for up to 5 minutes. They calculated that the forces applied to a protein by shear (,0.06 pN) were small in comparison to 140 pN force expected at the air
water interface, which would be close to a maximum force (20
150 pN) required to mechanically unfold proteins. Consequently their conclusion was that adsorption to solid surfaces (with a possible shear synergy) or pump cavitation stresses would be much more important causes of unfolding and aggregation than a simple shear. Most research involving WP has focused on conditions relevant to their processing, mainly heat-, pH-, and ionic strength-induced denaturation. In general, protein behavior in solution follows the general kinetics by which their stability does not result exclusively from the colloidal stability, which includes the energy barrier that prevents a collision between two protein

8.3 Shear-Based Processing

FIGURE 8.2 A simplified pathway illustrating unfolding and aggregation of a multidomain protein such as whey proteins. Unfolding unearths proteins sites, primarily hidden, prone to interactions and consequently formation of strong and ultimately irreversible interprotein bonds that stabilize aggregates. Double and single arrows denote reversible and irreversible steps, respectively. Nuclei are defined as the smallest net-irreversible aggregates. Adapted from Roberts, C.J. (2014). Therapeutic protein aggregation: Mechanisms, design, and control. Trends in Biotechnology, 32, 372
380.

molecules leading to aggregation, but also from their conformational stability, such as an energy barrier that prevents unfolding (Nicoud, Owczarz, Arosio, & Morbidelli, 2015). Graphically it can be presented as in Fig. 8.2, which shows a folded monomeric protein that is unfolded or partially unfolded and refolded dynamically while in solution. The unfolded monomers expose more hydrophobic sites that would govern initially reversible dimerization or oligomerization. Eventually, in the presence of activators (interfacial surfaces, impurities such as other proteins), these activated oligomers would establish strong hydrophobic interactions and satisfy hydrogen bonding requirements such as with interprotein β-sheets and overcome an energy barrier and create irreversible, yet soluble aggregates that would grow through various mechanisms (Roberts, 2014). Reversible and irreversible aggregation of proteins depends on a collision frequency of unfolded monomers (Taboada-Serrano, Chin, Yiacoumi, & Tsouris, 2005) and can be described by several but basically three mechanisms of aggregation: Brownian motion (perikinetic aggregation), orthokinetic aggregation, and differential settling (Meyer & Deglon, 2011) (Table 8.1). Smoluchowski (1917) proposed a model describing two-stage aggregation kinetics of particles in a flow based on the assumption that particles and aggregates move along straight trajectories up to the moment of their contact. The probability of a particle irreversibly interacting with another particle is given by the following two equations, with J1 describing the perikinetic aggregation and J2 depicting the orthokinetic aggregation: J1 5

4  kB  T  N 2 3η

ð8:4Þ

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Table 8.1 Types of Particle Collisions Leading to Aggregation (Meyer & Deglon, 2011) Mechanism

Description

Brownian motion (perikinetic) Shear (orthokinetic) Differential sedimentation

Particle collision due to random motion of particles Particles follow streamlines and collide within shear flow Particles exhibit different settling velocities due to their size resulting in collisions Particles deviate from streamlines and collide. Particle velocity is correlated to the fluid velocity Particles randomly transient among eddies and collide

Accelerative
correlated Accelerative
independent

J2 5

Continuous Phase Flow Regime Laminar Laminar and turbulent Laminar Turbulent Highly turbulent

32 UαUNUd3 Uγ_ 3

ð8:5Þ

According to the equations, a number of collisions among particles is highly dependent on the number of particles (N) and their size and, when it comes _ number to orthokinetic aggregation, is directly related to the shear rate (γÞ, of particles in a particular volume (N), and the capture efficiency (α), which is, on the other hand, related to medium viscosity (Potanin, 1991). Intermolecular bonds are created if the frequency and the energy of the collision are high enough. At higher shear rates the contact time between particles is short, which does not allow enough time for appropriate bond creation, thus formed particles are likely to remain relatively small (Zumaeta, Byrne, & Fitzpatrick, 2006). While flow motion apparently plays an important role in aggregation kinetics, it invariably creates disruptive stresses that can cause fracture of the agglomerates (Kim & Kramer, 2006). In addition, large aggregates are more easily disrupted than smaller ones as they are more likely to have weak spots. Furthermore, the net growth rate and size of any protein aggregate depends upon the equilibrium between growth and shearcontrolled breakage. Rate of aggregation is governed by mass transfer and reaction of components in a solution. At hydrodynamic shear conditions, mass-transfer-controlled aggregation proceeds as a two-stage process: diffusion-controlled (perikinetic growth) followed by hydrodynamic-shearcontrolled (orthokinetic) growth (Taylor & Fryer, 1994).

8.3.4 Impact of Shear on Properties of Whey Proteins in the Absence of Heat Walstra (2001) concluded that denaturation of globular proteins cannot occur during agitation or traditional homogenization, and for this to take place the power density of a turbulent flow needs to be sufficiently high. Recent progress in dynamic HP technology with the design of new homogenization valves that

8.3 Shear-Based Processing

can withstand extremely high pressures of up to 350 MPa have created new opportunities (Grácia-Juliá et al., 2008). These pressures are ten times those traditionally used in the dairy industry. In dynamic high pressure systems, particles are subjected to forces created by cavitation, shear, and turbulence, all of which work simultaneously (Panick et al., 1999). Another parameter that usually accompanies dynamic HP treatments is the temperature rise in the impact chamber, the effect of which is not clear as this stress lasts a very short period of time and can be controlled by efficient cooling. Most of the work in this area has focused on either improving the homogenization effect in regards to size reduction and emulsification efficiency or modifying functionality of a dairy system. The general conclusion in many instances was that the observed effects were due to subtle conformational changes of WP. For example, Subirade et al. (1998) studied the effects of dynamic HP on the conformation of β-Lg using Fourier-transform infrared spectroscopy. They concluded that the dynamic pressure used (range 0
140 MPa) had no apparent effect on the secondary structure of β-Lg. However, the treatment created three crucial differences: (1) a different pH sensitivity; (2) an improved thermostability upon treatment; and (3) different gelation behavior upon cooling. The authors attributed these effects to subtle but not obvious differences in conformation due to slightly different interactions. More recently, Ashton, Dusting, Imomoh, Balabani, and Blanch (2010) confirmed this conclusion assessing susceptibility of β-Lg to flowinduced conformational changes monitored with Raman spectroscopy and recording changes that were subtle and reversible without evidence of complete unfolding. The degree of conformational change in flow appeared to be related to structure as there appeared to be a correlation between the amount of β-structure and the extent of unfolding. Similarly, Zhong et al. (2012) investigated aggregation and conformational changes of β-Lg subjected to dynamic high pressure microfluidization. At lower pressures, ,120 MPa, behavior of β-Lg was accompanied by partial unfolding, reflected in the decrease in particle size, increase of free sulfhydryl groups, β-strand contents, and exposure of aromatic amino acid residues. At higher pressures, $ 120 MPa, β-Lg aggregated via thiol-catalyzed disulfide bond interchange resulting in an increase of particle size, formation of aggregates, decrease of free sulfhydryl groups, and β-strand contents, and slight changes of conformation around aromatic amino acid residues. The properties and behavior of β-Lg under various conditions are of particular importance as most of the time they dictate the properties of a bulk system due to its abundance in a whey protein preparation. It has a high affinity for various hydrophobic molecules and surfaces and may interact with them during treatment of milk or whey. For this reason, observations on the impact of dynamic HP processing on WP in more complex systems

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appear fairly different from those obtained in simpler systems such as a pure protein preparation in a buffer. In a more complex protein preparation such as a commercial WPI, very strong elongational flow at the entrance of the homogenizing valve and accompanying frictional forces resulted in irreversible disruption of large protein aggregates without affecting protein solubility (Bouaouina et al., 2006). The treatment appeared ineffective in changing the conformation of the proteins although very sensitive spectral techniques were not used in the assessment. The treated WP had greater stabilizing properties due to an increase in surface hydrophobicity induced by greater exposure of hydrophobic side chains enabled by aggregate fragmentation. The treatment also improved foaming ability and stability. Such conformational changes may also affect digestibility (Blayo, Vidcoq, Lazennec, & Dumay, 2016) despite the fact that aggregation of WP was avoided by efficient cooling upon processing at 300 MPa. While high pressure processing may inflict reversible changes, even such subtle conformational unfolding could be amplified in the presence of other components in the system or by downstream processing, such as in the case of milk. Homogenization combined with UHT processing of milk induced significant secondary structural loss to WP, particularly in the amounts of apparent antiparallel β-sheet and α-helix segments, as well as diminished tertiary structural content (Qi, Ren, Xiao, & Tomasula, 2015). Homogenization alone did not cause appreciable changes in the secondary structure although a disruption was evident in the tertiary structure of the WP. This indicated that even though processing of milk imposed little impairment of the stability of the secondary structure, stability of the tertiary structure was substantially compromised. Recently Mediwaththe (2017) showed that even at room temperature, applied shear had an effect on certain properties of milk proteins. The object of the work was to assess the impact of the combined effect of shearing and heating on properties of milk proteins in raw milk subjected to temperatures relevant to traditional milk processing. The control, untreated raw milk sample had an average particle size of B150 nm at 20 C, which was shifted towards higher values under applied shear to B157 nm at 500 s21 and increased further up to B172 nm at 1000 s21 (Fig. 8.3A). Shearinduced denaturation and aggregation predominated at 500 s21, indicated by a slight fading of bands associated with major WP in native PAGE (Fig. 8.3C). On the contrary, increasing shear to 1000 s21 inflicted shearinduced fragmentation of created aggregates indicating that these were held together by rather weak interparticle forces. FTIR analysis (Fig. 8.3B) showed that, in the absence of shear at 20 C, peaks were prominent at 1656 cm21 denoting native α-helical structure, and at 1637 and 1633 cm21 due to the presence of β sheets. At both shear rates (500 and 1000 s21), the intensity of β sheet at 1637 cm21 diminished indicating shear-dependent aggregation.

8.3 Shear-Based Processing

(A)

(B)

(C)

25 1637 0/s 500/s 1000/s

Absorbance (–)

Volume (%)

20 15 10

1633 1618

1656

1683 0/s 500/s 1000/s

5 0 1

10

100

1000

Particle size (nm)

10000 1700

1680

1660

1640

1620

1600

Wavenumber (cm–1)

FIGURE 8.3 Particle size distribution (A), FTIR spectra of the Amide I region (1600
1700 cm21) (B) and native polyacrylamide gel (C) of raw milk processed at 20 C at different shear rates (0, 500 and 1000 s21).

This was followed by appearance of prominent peaks at 1683 and 1618 cm21 depicting aggregation of β-sheets and intermolecular crosslinking, respectively (Kong & Yu, 2007). Increasing shear to 1000 s21, a distinguishable peak was observed at 1697 cm21 due to appearance of antiparallel β-sheet aggregated strands denoting aggregation (Qi et al., 2015); the peak observed at 1683 cm21 under 500 s21 disappeared, likely due to breakup of β-sheet aggregates, which confirmed shear-induced fragmentation of created particles. These weakly held aggregates may be created via either hydrophobic or electrostatic attractions that would enhance ever-present van der Waals attractive forces. While surface potential appeared unaffected by applied shear, the measured surface hydrophobicity declined significantly as the shear was enhanced. In their native state, WP adopt a more thermodynamically stable arrangement by having polar amino acids facing the exterior with nonpolar amino acids buried in the interior minimizing surface tensions. Due to shear-induced protein unfolding, these nonpolar residues would be exposed inducing hydrophobically driven aggregation and consequently lowering of overall surface hydrophobicity. Both shear rates induced changes on the particle surface, especially involving hydrophobic sites on the secondary structure.

8.3.5 Impact of Shear Accompanying Preprocess or Simultaneous Application of Heating on the Properties of Whey Proteins
Microparticulation Based on current knowledge, it appears that shear-induced conformational changes propagate various weak interactions among constitutive proteins that may be enhanced or lessened depending on the environment. The major

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weak (noncovalent) interactions involved are van der Waals forces, molecular and steric repulsions, depletion interactions, hydrophobic interactions, and hydrogen bonds; however, for newly shear-formed structures to be fully stabilized it would require strong, covalent bonding (i.e., S
S bonds) which is most frequently achieved by application of heat (Walstra, Wouters, & Geurts, 2006). When the WPs denature during heating, β-Lg unfolds reversibly up to around 65 C from where aggregation starts to take place. Around 65 C, a minor shifting of the α helix from the β-sheet takes place allowing for S-S interactions within the hydrophobic zone, which would induce aggregation (Creamer et al., 2004). The free -SH group on either Cys119 or Cys121 initializes SH
SS interchange reactions, which can affect exposed SS bonds on other protein molecules, making them reactive (Kinsella, 1984) and involving them in covalent interactions with other protein molecules (see also Chapter 6). Therefore, by carefully selecting processing conditions including whey protein concentration, pH, ionic strength, and quality of the mineral environment, temperature, and shear rate (Erabit, Flick, & Alvarez, 2014), it is possible to design protein-based micro- and nanoparticles with controlled size, morphology, and physicochemical properties that can be used in various applications in the food industry through a process commonly termed microparticulation (Ipsen, 2017). The size of newly created aggregates is also dependent on the protein concentration (Wolz & Kulozik, 2015). At low concentrations enlargement of aggregate size appears directly proportional to changes in concentration without a major impact of shear, while above 10%, shear influences the size of aggregates. Protein particle formation via heat-induced aggregation can be feasibly accomplished at higher whey protein concentrations; however, this depends on other environmental factors such as pH and ionic strength. Above a critical protein concentration, heating of WP typically results in bulk gelation. Therefore, traditional approaches applied in microparticulation of WP could be loosely grouped into approaches falling under A or B in Fig. 8.4. Approach A refers to heating a more concentrated protein solution under appropriate conditions with simultaneous application of shear under a particular flow regime that would limit the growth of aggregates. Approach B refers to intentional bulk gelation, during which large aggregates are formed in which primary, nuclei particles are held mainly by weak interactions that are readily disrupted by shear (Dissanayake, Liyanaarachchi, & Vasiljevic, 2012). Microparticulation has a long history of industrial applications. One of the first patents in this area was made public in, 1988, when Latella et al. described production of whey protein microparticles by thermal aggregation at high shear and low pH. The patent served as a basis for manufacturing of

8.3 Shear-Based Processing

FIGURE 8.4 Two main routes to creation of nano- and microparticles involving (A) heating with simultaneous shearing of protein dispersion or (B) heating of a protein dispersion followed by shearing of created gel. T, temperature; Cp, protein concentration; IS, ionic strength; CCa, calcium concentration; OC, other _ shear rate. components (polysaccharides, proteins, minerals); Re, Reynolds number—flow character; γ,

a commercial fat replacer (Simplesse) (Singer & Dunn, 1990). In this process, a 40% WPC solution was subjected to simultaneous application of heat (80
120 C) and shear flow (7500
10,000 s21). Queguiner, Dumay, SalouCavalier, and Cheftel (1992) applied extrusion, which was recommended previously as a novel way to texturize proteins (Kinsella, 1978), subjecting a 20% WPI dispersion to operating temperatures between 90 C and 100 C at various pH values. The authors noted that pH and temperature played an important role in aggregation processes, as the smallest particles were formed in the pH range 3.5
3.9. The morphology of created microparticles depended strongly on the temperature and lactose concentration in the mixture. Similar observations were reported later by Onwulata, Phillips, Tunick, Qi, and Cooke (2010), who modified nonfat dried milk, WPC, and WPI using a twin-screw extruder. Change of the protein state, depicted by the extent of texturization, was correlated to solubility, which along with viscoelasticity, was highly dependent on the extrusion temperature. Spiegel (1999) applied microparticulation using a single-stage scraped-surface heat exchanger treating a 10% WPC dispersion at varying concentrations of lactose and processing temperature. The author noted that low lactose concentration at 80 C induced formation of particles pH 7 with an average

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diameter of 2 μm through a fast aggregation process due to control of the denaturation step of WPC. Raising the temperature to 100 C, the rate of denaturation increased again which resulted in a great rise in the average size and compactness of the particles. A follow-up study (Spiegel & Huss, 2002) showed that process pH and concentration of Ca21 ions also influenced the properties of microparticles. Lowering the pH down to pH 4.5 substantially decreased the denaturation rate of WP, probably due to minimized reactivity of the reactive thiol groups and overall net charge of the proteins in this pH range. The concentration of Ca21 ions needed to be lowered in order to improve the denaturation stability of PC. Several more recent studies examining aggregation of WP during simultaneous heating and shearing showed that the size of aggregates decreased with increasing shear stress (Simmons, Jayaraman, & Fryer, 2007; Wolz, Mersch, & Kulozik, 2016). Since only conditions of extremely high shears accompanied with turbulence could induce protein unfolding and denaturation (Thomas and Geer, 2011), this occurrence was previously attributed to basically two mechanisms: (1) deformation and fragmentation, or (2) erosion of primary particles from the surface of large particles (Heffernan, Zumaeta, Cartland-Glover, Byrne, & Fitzpatrick, 2005, Taylor & Fryer, 1994). However, Simmons et al. (2007) and Wolz et al. (2016) demonstrated complexity of whey protein aggregation during simultaneous heating and shearing. Simmons et al. (2007) investigated the properties of bulk aggregates formed due to the denaturation and aggregation of WP in WPC dispersions under controlled conditions of shear and temperature with varying concentrations of Ca and P. Experiments were conducted below and above the temperature of β-Lg denaturation. Below the denaturation temperature, newly formed aggregates were small and weakly bonded, while above the denaturation temperature, large aggregates were created linked with strong bonds. The heating temperature governed the nature of interactions as weak van der Waals interactions prevailed at low temperature, while covalent (disulfide) bonds were formed at higher temperatures. Protein denaturation appeared strongly influenced by temperature due to the reaction kinetics. On the other hand, particle aggregation was governed by the applied shear field and magnitude of interactions among particles. Furthermore, the extent of protein denaturation and particle growth rate of aggregates was directly related to shear rate, which was attributed to frequency of particle collisions, but the final particle size showed a complex behavior with a further increase in shear rate (Simmons et al., 2007). Wolz et al. (2016) assessed the impact of heating time, shear rate, and protein concentration on the denaturation and aggregation behavior of WP. By increasing the protein concentration, smaller

8.3 Shear-Based Processing

and more compact and stable aggregates were created. The impact of shear rate appeared independent of the protein concentration. At a low concentration (5% protein), the aggregate size initially increased with concomitant increase in shear rate, likely due to greater probability and frequency of particle collisions. However, the size of aggregates declined when shear rate reached 1000 s21 and beyond due to apparent increase in shear stress. At high protein concentrations (30%), the size of the aggregates decreased continuously with increasing shear rate again due to increasing shear stress. The authors noted that the shear rate was the critical factor limiting particle size (Wolz et al., 2016). An alternative microparticulation approach to simultaneous heating and shearing is induction of bulk gelation under defined conditions to control the rate and extent of aggregation followed by application of shear (Fig. 8.4B). Dissanayake and Vasiljevic (2009) used this method to produce microparticles with an average size of approximately 10 μm. The authors used microfluidization at 140 MPa to treat a 10% WPC dispersion at pH 7. The dispersion was first heated at 90 C for 20 minutes and then microfluidized by passing the denatured proteins through the impact chamber once or five times. The microparticulation efficiency was improved when the initial pH was adjusted to pH 3, since the average size of the particles decreased by about two orders of magnitude as compared to sizes of particles prepared at neutral pH (Dissanayake et al., 2012). The authors suggested that by controlling covalent interactions at low pH, fragile protein aggregates were formed during heating that were disrupted to smaller particles during microfluidization. In summary, shear alone at levels normally used in the food processing is insufficient to induce considerable conformational changes in the secondary structure of WP, although some functionalities on a macroscale appear modified. For this to take place, extremely high shear rates and/or turbulence are required to induce irreversible protein unfolding and aggregation (Havea, Grant, Hii & Wiles, 2012). Controlling the rate and extent of whey protein denaturation, especially of β-Lg, appears to be one of the prerequisites for initiation of nucleation and the particle growth that would further be governed by applied shear. Solvent state, especially pH and ionic strength, along with the presence of other solutes including Ca, different mono- and polysaccharides, and even proteins such as caseins that may exert a chaperon like activity (Liyanaarachchi, Ramchandran, & Vasiljevic, 2015), and choice of processing temperature are some of the factors impacting denaturation of WP, while selection of shear rate is needed to ensure that the aggregate size, morphology, and surface properties of created particles meet the requirements of a specific application.

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8.4 LOW-FREQUENCY POWER ULTRASOUND TREATMENT 8.4.1

Ultrasound and Whey Processing

Industry has utilized the power of ultrasound for decades but the technology is seldom used in food processing. The ability to create material changes through physical and chemical reactions has been documented since the late 1920s (Wood & Loomis, 1927), but the earliest reported studies relevant to the food industry emerged in the 1960s where the homogenization-like effects of the technology were demonstrated. There was little interest in the technique in the decades to follow and it was not until the 1980s that research in this field became more frequent. In the two decades ahead, the technology was used widely but mainly focused on the destruction of microorganisms. From the beginning of the 21st century, however, we witnessed the emergence of a technology with novel widespread applications in food manufacturing. Research interests were broad and included work on lipids, crystallization, and separation to name a few (Dincer et al., 2014; Juliano et al., 2017; Muthukumaran, Kentish, Ashokkumar, & Stevens, 2005). In this section, the application of ultrasound to proteins, and in particular, the WP is reviewed. Although there are no documented examples of ultrasonic assisted commercial whey protein manufacturing, published research demonstrates the diverse potential of this technology. The effects of ultrasound on WP has received recent attention due to improved and efficient equipment designs, the ability to “tailor” protein functionality and increased general awareness of its potential. Examples of ultrasonicinduced effects on WP include enhancement of whey ultrafiltration (Muthukumaran, Kentish, Ashokkumar, et al., 2005; Muthukumaran, Kentish, Lalchandani, Ashokkumar, et al., 2005; Muthukumaran, Kentish, Stevens, Ashokkumar, & Mawson, 2007; Muthukumaran et al., 2004), viscosity reduction of whey solutions (Ashokkumar et al., 2009; Bates & Bagnall, 2011; Zisu, Bhaskaracharya, Kentish, & Ashokkumar, 2010), and yogurts manufactured with superior rheological properties (Vercet, Oria, Marquina, Crelier, & LopezBuesa, 2002; Riener et al., 2009c, 2010; Chandrapala, Martin, Zisu, Kentish, & Ashokkumar, 2012; Chandrapala, Zisu, Kentish, & Ashokkumar, 2012).

8.4.2

Power Ultrasound: Frequency is Critical

Power ultrasound is defined as sound waves at frequencies emitted above the human hearing range of approximately 16 Hz to 16 kHz and like all sound waves, these are longitudinal pressure waves transmitted through a medium. When it comes to whey processing, the medium is usually liquid but may also be air in de-foaming applications. Power ultrasound when used

8.4 Low-Frequency Power Ultrasound Treatment

as a processing tool is generally regarded as the sound waves emitted in the frequency range of 20 kHz to 1 MHz and diagnostic ultrasound occurs at frequencies above 1 MHz. In an acoustic field, microbubbles in solution undergo growth by rectified diffusion and by bubble 2 bubble coalescence (Ciawi, Rae, Ashokkumar, & Grieser, 2006) until they reach a maximum size and violently collapse generating mechanical, physical, and chemical effects including shockwave formation and turbulent motion (Ashokkumar, Lee, Kentish, & Grieser, 2004). These physical effects are powerful enough to break large aggregates apart (Ashokkumar et al., 2009) and in the majority of food applications, frequencies between 20 and 40 kHz (Mason, 1998) are used predominantly due to powerful cavitation effects (Earnshaw, 1998; Hem, 1967). The physical power of acoustic cavitation generated by high-intensity, low-frequency power ultrasound at 20 kHz is also demonstrated by the pitting it causes in the titanium sonotrode from which the sound waves are emitted (Fig. 8.5). At the point of cavitation, high localized temperatures are emitted and these may cause chemical changes by generating radicals (Ashokkumar & Mason, 2007). The concentration of radicals produced depends on contact time and frequency with the intermediate frequency range typically 200
500 kHz generating the greatest chemical reactions (Ashokkumar et al., 2008). Ultrasound treatment at low frequencies, below 40 kHz, typical of most whey

FIGURE 8.5 Pitting of a 20 kHz titanium sonotrode as a result of acoustic cavitation.

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applications results in high pressures (70
100 MPa) generating shockwaves, turbulence, and physical effects through transient cavitation; these dominate over chemical effects (Laborde, Bouyer, Caltagirone, & Gerard, 1998). Many of the early low-frequency whey protein studies neglected to consider radical production and often applied “excessive” treatment times; however, there is growing and gradual acknowledgement of the risk of radical production in food applications. Recent studies on milk proteins, WP, and lipids have shown that production of volatiles is proportional to energy density at both high and low frequencies (Juliano et al., 2014; Martini & Walsh, 2012; Riener, Noci, Cronin, Morgan, & Lyng, 2009b). In the study by Martini and Walsh (2012), liquid whey solutions containing 10% total protein at pH 3.5, 4.5, and 7.5 were sonicated at a frequency of 20 kHz for 15 minutes. Calculating the applied energy density based on a 50 mL sample batch size that was sonicated at 15 W for 15 minutes, this treatment delivered 270 J/mL. In the study, a trained sensory panel examined 21 sensory attributes of sonicated whey and compared these against those of untreated whey solutions. The study concluded that, overall, ultrasound did not change the sensory attributes of the whey, although two detrimental attributes described as “cardboard” and “malty” were identified as different when compared with untreated samples. This is likely a result of high localized temperatures associated with acoustic cavitation. Taking into consideration that the sensory attributes described were based on high intensity batch sonication with significantly a long contact time, this observation may not be representative of commercial processing conditions. In unrelated WPC and isolate studies, there were no unusual odors detected following 20 kHz sonication at significantly lower energy densities, below 30 J/mL, with treatment times less than 60 seconds (unpublished data; Lo, 2017). Depending on the protein source (either WPC or isolate), protein concentration and pH, these unnatural odors were only identified at energy densities greater than 150 J/mL and contact times exceeding 4 minutes. It is therefore necessary to control and characterize the applied energy density based on contact time, power drawn, and volume of treated protein solution. The use of applied energy density was demonstrated in a batch system on reconstituted WPC in the work by Koh et al. (2014) and in a separate study, this principle was also demonstrated in a continuous operation where reconstituted WPC and evaporated whey protein retentate were treated at 20 kHz (Zisu et al., 2010). In the latter study, the applied energy density was calculated based on flow rate rather than batch volume size.
Energy Density J=mL 5 Power DrawnðW Þ
3 Time ðsecÞ=Volume ðmLÞ or Flow Rate mL=min

ð8:6Þ

8.4 Low-Frequency Power Ultrasound Treatment

8.4.3 Temperature and Sonication of Whey Protein Solutions WP, and β-Lg in particular, undergo irreversible heat denaturation at temperatures above 65 C. Heat is a by-product of acoustic cavitation and although reactions are localized to imploding cavitation bubbles, these reactions are so prevalent that if left unchecked, the temperature of the sonicated solution rises. Given sufficient contact time (as in the case of many reported whey protein studies), the solution temperature may reach the whey protein denaturation point. Temperature control of sonicated whey protein solutions is therefore critical. The rate at which the temperature will rise is not only dependent on the contact time but also frequency, power, and volume. The power delivered as ultrasonic energy can be measured based on temperature rise as shown in Equation 8.7 and reported in the work by Koh et al. (2014). P 5 Cpw MðΔT=tÞ

ð8:7Þ

Where Cpw is the heat capacity of water (54.18 3 103 J/kg K), M is the mass of water (kg), ΔT is the change in temperature during sonication (K), and t is sonication time (s). Temperature is commonly controlled with a cooling jacket surrounding the reaction vessel (in both batch and continuous sonication) through which an ice slurry or other cooling medium is circulated. Without a cooling jacket, a separate container may be used in which the reaction vessel is immersed and the cooling medium regularly replaced. The heat retaining capacity of the titanium sonotrode through which soundwaves propagate is high and during continuous treatment exceeding 2 minutes (depending on the frequency, volume or flow rate, and power), it is unlikely that the cooling vessel will be sufficient to control temperature and this will slowly rise. This effect is often negligible in continuous operations with high flow rates but is often a concern in small batch operations. The surface temperature of the titanium sonotrode increases with use and this may cause partial whey protein denaturation when in direct contact. In a laboratory or experimental environment, it is best practice to cool the sonotrode between uses. When ultrasonic treatment exceeds 2 minutes of continuous operation, a break in sonication followed by cooling of the sonotrode should be considered. Using this approach, it was possible to increase the treatment time of reconstituted WPC and isolate solutions containing up to 10% protein before any noticeable sensory changes were detected (personal communication; Lo, 2017). Nguyen and Anema (2010) showed that skim milk sonicated at 22 kHz for up to 30 minutes without temperature control reached 95 C after just 15 minutes of treatment. Sonication of milk continued at this temperature

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for up to 30 minutes before acid gelation was induced. Firmness of resulting gels (G0 ) was altered but the effect was attributed largely to denaturation of WP at temperatures .65 C rather than to ultrasound-induced effects.

8.4.4 Effects of Power Ultrasound on the Solubility of Reconstituted Whey Protein Powders With all whey protein powders, it is desirable to achieve rapid dissolution across a temperature range to improve productivity through reducing processing times, minimizing loss, and producing high quality products (Fang, Selomulya, Ainsworth, Palmer, & Chen, 2011; Gaini et al., 2011). Low powder solubility causes commercial manufacturing delays when making secondary dairy products such as cheese, yogurts, and beverages and if powders are poorly dissolved there is the added risk that product defects may occur. Low-frequency power ultrasound has been shown to improve the solubility of reconstituted whey protein powders and reduce hydration times. There is strong correlation between the particle size of reconstituted powders and solubility and energy density; a gradual particle size reduction occurs in response to longer treatment times. During powder rehydration, the powder particles are disrupted into primary particles together with the simultaneous release of material into the surrounding aqueous phase (Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). Degradation of the powder particle in response to acoustic cavitation explains the size reduction initiating the release of insoluble material to the aqueous phase at a faster rate. Low-frequency (20 kHz) power ultrasound has been used to disrupt insoluble powder aggregates suspended in reconstituted WPC and isolate solutions (Ashokkumar et al., 2010; Onwulata, Konstance, & Tomasula, 2002, Zisu et al., 2011). In other studies, the solubility of reconstituted WPC (Kresic, Lelas, Jambrak, Herceg, & Brncic, 2008) and WPI (Jambrak, Mason, Lelas, Herceg, & Herceg, 2008) also improved following ultrasonication at low frequency. Solution clarity also improved when reconstituted WPC containing 5% (w/w) solids was treated at a frequency of 20 kHz. This was strongly linked to particle size reduction as larger insoluble particles were solubilized. What is lacking in many whey powder solubility studies is information regarding energy efficiency. Several of the reviewed studies show significant improvement in solubility after only a few minutes of ultrasonic treatment. Based on the reported treatment times, power, and sample volume, it appears that substantial improvements in powder solubility occur at energy densities as low as 30 J/mL. In recent controlled energy density studies it has been proven that the solubility of WPC reconstituted to 4% (w/w) protein

8.4 Low-Frequency Power Ultrasound Treatment

improved at energy densities as low as 15 J/mL (20 kHz) corresponding to a contact time of less than 30 seconds (unpublished data). At higher frequencies (.200 kHz), neither solution clarity nor particle size reduction was reported in whey protein solutions. At these higher frequencies, the physical forces of acoustic cavitation are weaker compared to those at 20 kHz (Zisu et al., 2011).

8.4.5 Effect of Power Ultrasound on the Viscosity of Whey Protein Solutions The effects of high- and low-frequency ultrasound on the viscosity of whey protein solutions have been studied. As was the case for powder solubility, high-frequency sonication had no effect on the viscosity of whey protein solutions (Ashokkumar et al., 2009). Reconstituted WPC sonicated at frequencies ranging from 20 kHz to 1 MHz (Ashokkumar et al., 2008) showed significant viscosity reduction at 20 kHz, but the viscosity of those sonicated at frequencies greater than 200 kHz was not affected. Since higher frequencies generate more radicals compared to low frequencies, the authors attributed the viscosity reduction to the physical forces generated during acoustic cavitation (Ashokkumar et al., 2009). Sonication at low frequency has been reported to both lower and increase the viscosity of whey protein solutions depending on the study. In the majority of studies, sonication at 20 kHz reduced viscosity. Many of these studies were on a laboratory scale, but larger pilot-scale data have also been published. In one such example, a 1 kW continuous flow 20 kHz sonicator delivering applied energy densities of 60
240 J/mL lowered the viscosity of whey protein retentate concentrated to 33% solids (81.5% protein) by membrane filtration. Viscosity was reduced by up to 10% in a continuous flow operation and this was correlated with particle size reduction (Zisu et al., 2010). A larger 4 kW continuous flow 20 kHz ultrasonic unit was also used to deliver energy densities of 34 to 258 J/mL and this lowered the viscosity of whey retentate by up to 33%. The total solids content of the whey protein retentate was shown to have an effect on viscosity when sonicated, with sonication efficiency improving at higher solids. The viscosity of evaporated whey protein retentate containing 54% solids was reduced by up to 40% when sonicated at 20 kHz. A small number of laboratory studies have, however, reported viscosity increases after low-frequency (20 kHz) sonication of reconstituted WPC and WPI (Jambrak et al., 2008; Kresic et al., 2008). In both studies, the whey solutions were batch sonicated for 15 minutes. The length of treatment was significantly greater than that used in other studies demonstrating a viscosity

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Table 8.2 Effect of 20 kHz Sonication on Viscosity and Surface Hydrophobicity of 5% (w/w) Reconstituted Whey Protein Concentrate Applied Energy Density (J/mL)

Ultrasonic Treatment Time (min)

Viscosity (cP at 100 s21)

Surface Hydrophobicity

0 31 155 310 620 930

0 1 5 10 20 60

38 33 31 33 36 41

232 255 270 245 232 218

Modified from Ashokkumar, M., Lee, J., Zisu, B., Bhaskharacharya, R., Palmer, M., & Kentish, S. (2009). Hot topic: Sonication increases the heat stability of whey proteins. Journal of Dairy Science, 92, 5353
5356.

reduction. It has been shown that long treatment times, exceeding 10 minutes, increase the viscosity of whey solutions at 20 kHz (Ashokkumar et al., 2009). Table 8.2 demonstrates this result and makes a clear link between treatment time and applied energy density. The viscosity of 5% (w/w) reconstituted WPC was reduced after batch sonication at 20 kHz for less than 10 minutes. This corresponds to applied energy densities up to 155 J/mL. Exceeding 10 minutes sonication times and 310 J/mL has the opposite effect and viscosity increases. Furthermore, whey protein surface hydrophobicity changes proportional to applied energy density also occur. Surface hydrophobicity either increases or decreases at the equivalent energy densities where the viscosity behavior changes. These results show that the effects of lowfrequency sonication extend beyond physical shear, i.e., particle size reduction and chemical changes may occur. It is therefore paramount that research based on applied ultrasound clearly defines all treatment conditions and associates results with applied energy density. Although low-frequency power ultrasound generates predominantly physical effects through the process of acoustic cavitation, the role of highly reactive radicals generated even at low frequency (c. 20
100 kHz) cannot be ruled out completely. This is particularly important for long treatment times and high applied energy densities. Under controlled sonication conditions where the contact time of low frequency sonication is short, the chemical effects associated with acoustic cavitation are not expected to play a major role. In the study by Kresic et al. (2008) where low frequency ultrasound (15 minutes treatment at 20 kHz) increased viscosity of reconstituted whey solutions, the authors attributed the observations to protein structural changes where the hydrophilic segments of the amino acids opened towards the surrounding aqueous phase leading to increased binding with the water molecules. In a more detailed study, whey protein structure was explored in reconstituted

8.4 Low-Frequency Power Ultrasound Treatment

whey solutions (Chandrapala, Zisu, Palmer, Kentish, & Ashokkumar, 2011). This study showed that low frequency sonication (20 kHz) had no significant effect on the protein structure after a short treatment (1 minutes). Minor changes to the secondary protein structure and protein hydrophobicity occurred only when sonicating for significantly longer times from 5 to 60 minutes. Enthalpy of denaturation decreased when sonicated for up to 5 minutes but increased thereafter due to protein aggregation. Gulseren, Guzey, Bruce, and Weiss (2007) studied the effects of sonication on a purified bovine serum albumin (BSA) whey protein fraction. The functional properties of the pure whey protein fraction were modified in what was described as the formation of an ultrasonically induced state that differed from a thermal-, mechanical- or solvent-induced state. Similar studies on pure whey protein fractions also showed minor changes following ultrasonic treatment but the extent of change depended on the pure protein fraction type and degree of purity. Applied energy density was also a likely contributing factor. However, when the same purified proteins were sonicated as a mixture resembling the composition of a typical complex product (3:1 β-Lg and α-La), the changes observed in individual protein fractions were not observed (Chandrapala, Zisu, et al., 2012). Minor changes to reactive thiol groups and surface hydrophobicity were only identified in pure protein fractions sonicated individually with the α-La being affected to a greater extent and this was proportional to applied energy density.

8.4.6

Effect of Power Ultrasound on Whey Protein Gelation

Low-frequency sonication has been used to tailor the functional properties of WP with some studies manipulating the foaming and solubility properties of reconstituted whey protein powders (Jambrak et al., 2008; Kresic et al., 2008) and others using the technique to increase the firmness of whey protein gels and reduce syneresis (Zisu et al., 2011). Not only has the gelling effect been demonstrated in the laboratory but it has also been proven at pilot scale. In pilot-scale studies, continuous-flow, low-frequency (20 kHz) sonication was used to treat whey protein retentate containing 33% solids (Zisu et al., 2010). When the sonicated whey solution was heated to $ 80 C to form gels, the strength of the gels increased by more than 25%. Whey protein retentate from the same study was further processed to manufacture whey protein powder containing 80% protein. Protein powder was reconstituted to make a 4% protein solution and heated to $ 80 C. This resulted in 25% firmer gels. This study showed that the functional properties associated with ultrasonic treatment were maintained even after drying and reconstitution. This was linked to ultrasound-induced particle size reduction which resulted in smaller particles with increased surface area. The larger

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surface area improved whey protein association creating a compact interconnected protein network during gelling (Zisu et al., 2010). In other applications, the thermal instability of WP is a major processing problem. In a manufacturing environment, WP are subjected to temperatures often exceeding their denaturation point. This creates unwanted protein aggregation through hydrophobic interactions and the formation of intermolecular disulfide bonds (Wang, Tolkach, & Kulozik, 2006). Whey protein aggregation often results in excessive thickening, fouling, or gelling during processing and later during storage of the dairy product (Morr & Richter, 1999). To address this issue, Ashokkumar et al. (2009) used a combination of heat and low-frequency (20 kHz) ultrasound. Whey protein solutions containing 4%
15% (w/w) protein were partially denatured at $ 80 C. The resulting highly viscous aggregated whey protein solutions were then physically sheared by sonication for at least 5 seconds. Acoustic cavitation was used to break whey protein aggregates apart and the resulting product was largely unaffected by further heating above the denaturation temperature of native WP (Fig. 8.6). Gordon and Pilosof (2010) used a similar approach in a subsequent WPI study.

FIGURE 8.6 Appearance of 4% whey protein solution heated at 130 C. Control sample (without preheat and sonication—left image) and sample preheated at 90 C and sonicated (right image).

8.4 Low-Frequency Power Ultrasound Treatment

Table 8.3 Composition of Formulation Closely Resembling Infant Formula Ingredient

% Contribution

SMP HH WPC80 WPC35 Maltodextrin Raftilose Lactose

23 5 12 17 0.5 16

Temperature (°C)

Control pH 7

Treated pH 7 Blocking

135 134 133 132 131 130 129 128 127 126 125

Out of sample -

Temp Temp

0

10

20

Temp

Temp

30

40

50

60

70

80

Time (min)

FIGURE 8.7 High-temperature processing of infant formula containing native (control) and sonicated heat-stable whey powders (treated).

The heat stability effect was maintained after spray drying and reconstitution (Ashokkumar et al., 2009; Zisu et al., 2010). Partially denatured whey protein solutions were made by continuous heating at 85 C and sonication at an applied energy density of 210 J/mL, then spray dried. The resulting whey powders were inert to secondary heating when reconstituted and heated at 80 C for 30 minutes (Zisu et al., 2010). These heat-stable whey protein powders were used in high-temperature processing of infant formula. Heat-stable whey powder (WPC35) was used to replace an equivalent amount of commercial whey powder in the formulations described in Table 8.3 and adjusted to pH 7. Bench-top heating was maintained at 131 C (Fig. 8.7). Infant formula containing native whey protein ingredients was unstable during heating and the temperature was constantly regulated until the unit finally blocked after 60 minutes of processing. Replacing native WPC35 with the sonicated heat-stable powder allowed

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processing to occur for 80 minutes without regulating temperature or blocking of the system. It is probable that the effects of acoustic cavitation lower the surface hydrophobicity of WP after partial heat denaturation (at temperatures $ 80 C) and low-frequency sonication which prevents re-aggregation of proteins through hydrophobic interactions. In summary, ultrasound has numerous applications in whey processing including solubility improvement, viscosity control, modification of gelation properties, heat stability, and various other aspects of tailored ingredient functionality. Although the potential application of this technology is widespread, much of the documented work has only been proven in the laboratory and at pilot scale. Without improvement in ultrasonic equipment design specifically focused on food applications, it is probable that some of the applications covered in this chapter may never reach commercialization due to high energy requirements or other constraints. Applications that deliver the greatest benefits with the lowest energy requirement stand the greatest chance of commercial implementation. From the applications reviewed in this chapter, the possibility of commercial success may come from the simplest, yet one of the most common applications, powder rehydration. Viscosity control in concentrated protein solutions and heat stability of WP are also feasible applications.

8.5 8.5.1

PULSED ELECTRIC FIELD TECHNOLOGY Introduction

PEF technology involves treatment of a pumpable food with very short pulses (microseconds) at very high electric field strengths. In general the treatments are performed at around room temperatures but higher temperatures up to B60 C can be used. However, as the treatment temperature is increased, any changes in the product are increasingly due to thermal effects rather than the PEF. Research on milk has largely focused on inactivation of bacteria and shelf life extension although its effect on other milk constituents such as WP, including enzymes, has received some attention. PEF treatments are typically carried out at electric field strengths of 10
50 kV/cm in multiple short pulses (typically 1
5 μs) at frequencies of 200
400 Hz. The total treatment time, which is the product of the pulse width and the number of pulses, is usually much less than 1 seconds. The field strength can be determined by the voltage produced by the power supply and the distance between the electrodes in the treatment chamber; a voltage of 10 kV across a distance of 0.25 cm yields a field strength of 40 kV/cm. Several authors cite, in addition to the electric field strength and treatment time, the overall energy input from a treatment in kJ/kg or kJ/L; this facilitates comparison of treatments. However, the shape of the pulse

8.5 Pulsed Electric Field Technology

wave also affects the outcome of the treatment. Common shapes are the exponential decay and square wave, both of which can be administered as mono- or bipolar forms. Higher field intensities are reached, momentarily, in exponential decay pulses than in square wave pulses where a moderate field intensity is maintained for a longer time than in exponential decay pulses. Several factors affect the outcome of PEF treatments. These are both process and product factors. Process factors include electric field strength, pulse width, pulse frequency, number of pulses, shape of the pulse wave, treatment chamber format, flow rate, and flow conditions (laminar or turbulent), while product factors include electrical conductivity, temperature, viscosity, pH, and composition. Because of the large number of factors which can influence the results of PEF trials, valid comparisons between the data from different researchers is often difficult. This is either because all salient parameters are not specified or the processes are fundamentally different, e.g., different pulse wave forms or different treatment chamber configurations. As suggested by Alvarez, Pagan, Condon, and Raso (2003), total specific energy input (in kJ/k or kJ/L), electric field strength, and treatment times are essential process factors which should be reported to enable reasonable comparisons of treatments in different laboratories. Of the product factors, temperature and conductivity are very important. The temperature increases during PEF treatment so both the inlet and outlet temperatures should be reported. The following data from Craven et al. (2008) are an indication of the increases in temperature which occur during PEF treatment. Milk processed at a field intensity of 28 kV/cm and specific energy input of 111.6 kJ/L reached final temperatures of 15 C, 40 C, 50 C, or 55 C from inlet temperatures of 10.5 C, 30.5 C, 40.5 C, and 45.5 C. PEF is more effective in liquids with low conductivity. Milk has a conductivity of around 4.5 mS/cm (Grahl & Märkl, 1996) and is suitable for PEF, although some researchers have diluted milk before PEF treatments to enhance the effectiveness (de Luis et al., 2009). PEF equipment consists of: a high-voltage power supply (up to 50 kV); an energy storage capacitor; a pulse generator and switching system; a treatment chamber containing two electrodes between which the liquid food is continuously pumped; a temperature measurement and control system; a unit for controlling and monitoring voltage, current, and electric field strength; and a product handling and packaging system. The energy from the high-voltage power supply is stored in the capacitor, discharged at high levels of power at an extremely fast rate (1
5 μs). The interval between discharges varies from 1 ms to seconds. When the electrical energy is applied as pulses, the disadvantages of continuous high voltage current treatments, electrolysis, and ohmic heating, are minimized.

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The design of the treatment chamber is significant for the efficiency of the PEF treatment. Importantly, it should not contain “dead spots” where the product receives little treatment. In some systems, the dead spot problem is alleviated by using two or more treatment chambers in series. Common configurations for continuous chambers are parallel plate, coaxial, and co-field. The chambers may be slightly pressurized to prevent bubble formation which can cause electrical arcing.

8.5.2

Effect on Whey Proteins

In general, PEF has minimal effect on WP. Table 8.4 summarizes the relevant published reports. As indicated above, reporting the results of research on PEF treatment is not straightforward as the conditions used can vary considerably as many different factors can be altered. Table 8.4 lists the conditions used in the various studies to facilitate comparisons between the studies. In the main, the PEF conditions and other details of the reports summarized in Table 8.4 are not repeated in the discussion below. Most authors have reported no effect on the physicochemical, immunochemical, and structural properties by PEF on the major WP, β-Lg, α-La, BSA, and immunoglobulins (Barsotti, Dumay, Mu, Diaz, & Cheftel, 2002; de Luis et al., 2009; Li, Zhang, Lee, & Pham, 2003, 2005). These proteins were studied as individual proteins dissolved in a variety of solvents (water, buffers, soymilk). By contrast, Odriozola-Serrano, Bendicho-Porta, and Martin-Belloso (2006) reported denaturation levels of BSA, β-Lg, and α-La of 24.5%, 20.1%, and 40%, respectively, based on densitometry of PAGE gels. The different results from various researchers may relate to the different PEF conditions used, including different wave forms (Li et al., 2003, 2005; Odriozola-Serrano et al., 2006 used bipolar pulses, while Barsotti et al. (2002) used exponential decay pulses) and/or different analytical methods, e.g., ELISA, circular dichroism (CD) (Li et al., 2003, 2005), electrophoresis (Odriozola-Serrano et al., 2006), UV spectroscopy (Barsotti et al., 2002), and radial immunodiffusion (de Luis et al., 2009), for determining change in proteins. Guan et al. (2010) reported the effect of PEF on a solution of BSA, with and without dextran, on the secondary structure of BSA (by CD) and on the progress of the Maillard reaction. They found that PEF caused no change in the secondary structure of BSA in the absence of dextran but marked changes when treated in the presence of dextran. PEF accelerated the Maillard reaction as indicated by increases in Maillard reaction products, browning, and antioxidative activity. Sui, Roginski, Williams, Versteeg, and Wan (2011) treated a WPI solution with PEF and found no effect on a range of physicochemical and

Table 8.4 Summary of Reports on the Effects of Pulsed Electric Field Technology on Whey Proteins (Arranged in Chronological Order) Protein

Treated Product

Treatment

Analytical Method

Effect

Reference

Lactoperoxidase

Whole milk

Assay by Sigma method

25% decrease in activity

Grahl & Märkl (1996)

β-Lactoglobulin (β-Lg)

2% solution in phosphate buffer pH 7. Whole milk

21.5 kV/cm, total energy input 400 kJ/kg, ,40
50 C Exponential decay pulses, 260 μs, 30 kV/cm, ,30 C 500 μs, 19 kV/cm

4th derivative UV

No significant unfolding or aggregation

Barsotti et al. (2002)

Assay with 2,2-azinobis(3-ethyl-benzothiazoline6-sulphonic acid) (ABTS) as substrate 1 H2O2; spectophotometric at 412 nm ELISA; immunoactivity against Salmonella enteridids Circular dichroism, immunoactivity by ELISA

No change in activity even after energy input of 500 kJ/kg.

Van Loey et al. (2002)

No effect

Li et al. (2003)

No detectable change to secondary structure or immunoactivity

Li et al. (2005)

Densitometry of PAGE gels

Denatured by 20%, 40%, and 24% respectively

OdriozolaSerrano et al. (2006)

400 μs; 37.6 kV/cm, total energy input 920 kJ/kg, 20
, 35 C

Radial immunodiffusion

No effect

de Luis et al. (2009)

735 μs; 10 or 20 kV/ cm, square wave pulse

Circular dichroism (CD), free amino acid analysis by OPA, SDS-PAGE.

Accelerated the Maillard reaction; marked changes to chemical structure of BSA only in presence of dextran

Guan et al. (2010)

Lactoperoxidase

Immunoglobulin IgG

IgG in soymilk

Immunoglobulin IgG

IgG in borate buffer, pH 6.83

β-Lg, α-lactalbumin (α-La), bovine serum albumin (BSA) β-Lg, α-La, immunoglobulins, lactoferrin, lactoperoxidase BSA

Whole milk

Milk, whey, diluted with water to electrical conductivity of B 2 mS/cm BSA-dextran -salt solution electrical conductivity of B 4.05 mS/cm

54 μs; 41.1 kV/cm, bipolar square-wave form, 15
46.4 C 54
91.4 μs; 41.1 kV/cm, bipolar square-wave form, 15
46.4 C 1000 μs; 35.5 kV/cm , monopolar squarewave pulse form, ,40 C

Continued

Table 8.4 Summary of Reports on the Effects of Pulsed Electric Field Technology on Whey Proteins (Arranged in Chronological Order) Continued Protein

Treated Product

Treatment

Analytical Method

Effect

Reference

Lactoperoxidase

Raw whole milk

12.5
75 μs; 15
35 kV/cm

LPO assay with ABTS 1 H2O2 as above

Lactoferrin (Lf)

Lf in SMUF

19.2 μs; 35 kV/cm, mono- or bipolar square wave pulse; 30, 50, 60, 65, 70 C (outlet temperature); energy input 40.6 kJ/L

Spectrophotometric (A280); ELISA

Riener, Noci, Cronin, Morgan, and Lyng (2009a) Sui et al. (2010)

Lactoferrin (Lf)

Lf in SMUF at 0.2
2 3 normal concentration

Spectrophotometric (A280); ELISA and CD; iron by ICP-OES; hydrophobicity

All whey proteins

WPI, 1 & 10% in SMUF

19.2 μs; 35 kV/cm, bipolar square wave pulse; 30, 50 60, 65, 70 C (outlet temperature); energy input 40.6 kJ/L 19.2 μs; 30
35 kV/cm, energy input, 989.5131.9 kJ/L or 211 μs; 30 kV/cm, energy input, 1083 kJ/L; bipolar square wave pulse; 30, 60, 65, 70, 75 C (outlet temperature);

No change to LPO activity even under severe conditions: 75 μs at 35 kV/cm No effect on the physicochemical properties of Apo-LF, N-LF, or Holo-LF at # 50, # 60, # 65 C; hydrophobicity changes at higher temperatures due to thermal effect, not PEF No effect on the physicochemical and structural properties; iron released at high SMUF concentrations

Sui et al. (2011)

Whey proteins

Raw whole milk

No effect on physiochemical, including themal stability, and emulsification properties; increased gelation time & decreased strength of heat-induced gel No clear evidence of effect on whey proteins; no change in hydrophobicity at # 20 kV/cm

34 μs; 20
26 kV/cm; bipolar square wave pulse; 21.4
23.1 C

PAGE, HPSEC, hydrophobicity, -SH content, DSC, emulsification & gelation (rheology) properties

Hydrophobicity, Td

Sui et al. (2010)

Sharma et al. (2016)

8.5 Pulsed Electric Field Technology

emulsification properties. However, they did find that when the PEF-treated samples were heated, gel formation was delayed and the formed gels had lower strength than untreated controls. This suggests that PEF had some effects on the WP which were not detected by the analytical tests performed (PAGE, size exclusion chromatography, sulfhydryl content, emulsification capacity). Sharma, Oey, and Everett (2016) reported the effects of PEF of whole milk on the thermal properties of some milk components including WP. A clear effect on the WP was not evident from the results but the authors found that PEF treatment at # 20 kV/cm did not significantly increase hydrophobicity (as did heat treatments) and concluded that PEF treatments may cause less unfolding and therefore less adsorption onto the milk fat globule membrane surface than heat treatments. There has been considerable interest in the effect of PEF on the bioactive WP because of the potential of using PEF rather than heat to inactivate bacteria in functional foods containing these proteins. de Luis et al. (2009) reported no change in Lf after treatments of skim or whey (diluted to onethird to adjust conductivity to 2 mS/cm at 20 C) with PEF treatments in which the temperature did not exceed 35 C. Similarly, Sui, Roginski, Williams, Versteeg, and Wan (2010) treated Lf (native-Lf, iron-depleted (apo-Lf), and iron-saturated (holo-Lf)) in simulated milk ultrafiltrate (SMUF) at temperatures ranging from 30 C to 70 C and found no effect on the physicochemical properties provided the temperature did not exceed a critical value for each Lf type, namely, 50 C, 60 C, and 65 C for apo-Lf, native-Lf, and holo Lf, respectively. Changes in hydrophobicity occurred at higher temperatures but these were attributed to thermal effects not to the PEF treatment. Sui et al. (2010) investigated the effect of the ionic strength of the PEF treatment medium on the iron-binding capacity of Lf. PEF caused a decrease in iron binding with increasing concentrations of the medium (SMUF). In one-fifth strength SMUF, no iron was released by PEF; however, after PEF of Lf in double-strength SMUF only half the iron remained bound. This could have beneficial effects as iron-depleted Lf (apo-Lf) is more effective against microorganisms than native or ironsaturated Lf (holo-Lf). The authors suggested that PEF had potential as a method for preparing apo-Lf. Lactoperoxidase activity has been shown by some authors to be not affected by PEF under a range of conditions, namely, 75 μs at 35 kV/cm (Riener et al., 2009a) and 19
36.6 kV/cm field strength and 500
920 kJ/kg total energy input (de Luis et al., 2009; Van Loey et al., 2002). However, Grahl and Märkl (1996) found a 25% decrease in activity with a PEF treatment of field strength 21.5 kV/cm and total energy input 400 kJ/L.

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In summary, PEF has minimal effect on WP. This could be beneficial for the use of the technology for inactivating bacteria in liquid functional food products incorporating WP, particularly heat-sensitive bioactive proteins such as immunoglobulins, lactoferrin, and lactoperoxidase.

8.6 8.6.1

ULTRAVIOLET IRRADIATION Introduction

Irradiation of foods with UV-C light (200
280 nm wavelength) is effective in killing most microorganisms. It has been used for many years for decontaminating air, water, and surfaces of packaging materials and equipment. It was recently approved by the European Food Safety Association for production of pasteurized milk (EFSA, 2016). For liquids, its effectiveness is reduced by opacity, turbidity, and presence of particles. Hence it is most suitable for water and clear solutions such as clarified whey; milk presents a challenge because of its opacity. UV treatment equipment typically involves a UV-penetrable tube through which the liquid product is pumped. Ideally the flow in the tube is turbulent. Turbulent flow continuously renews the surface and ensures all parts of the liquid come into contact with the UV light. This is essential for an opaque product such as milk. This can be achieved with the use of static mixers (Altic, Rowe, & Grant, 2007; Kristo, Hazizaj, & Corredig, 2012) or a complex swirling flow such as achieved through a corrugated spiral tube in the commercial SurePure Turbulator system (Cilliers et al., 2014). An alternative arrangement to turbulent flow is laminar flow in a very thin film. Research on UV irradiation of milk has been conducted over many decades (Burton, 1951). UV-C has been shown to reduce bacterial numbers but the dose required to achieve a sufficient reduction for pasteurization (a 5-log reduction) causes unpleasant, light-induced flavors in milk. Of seven milkborne pathogens tested by Crook, Rossitto, Parko, Koutchma, and Cullor (2015), Listeria monocytogenes, the most UV resistant, required 2 kJ/L of UV-C exposure to reach a 5-log reduction while Staphylococcus aureus, the most sensitive, required 1.45 kJ/L for a 5-log reduction. Reinemann, Gouws, Cilliers, Houck, and Bishop (2006) described the UV-induced flavors as cooked, barny, rancid, and unclean off-flavors in milk treated with UV at 1.5 kJ/L which achieved a 3-log reduction in standard plate counts of the mixed microflora. They concluded that the treatment limit to avoid off-flavor development in milk was 1 kJ/L. However, others have observed sensory defects in milk treated with 1.045 kJ/L (Cilliers et al., 2014) and 0.88 kJ/L (Rossitto et al., 2012).

8.6 Ultraviolet Irradiation

A technology related to continuous UV-C irradiation is pulsed-light treatment which involves the use of a broad spectrum of light in the wavelength range of 180
1100 nm. Therefore, the light used includes UV wavelengths but also visible and infrared. It is often referred to as “pulsed-UV” light (Krishnamurthy, Demirci, & Irudayaraj, 2007). The treatment is delivered in short pulses (flashes) of durations of B0.3 ms. Pulsed-light treatment is reported to be more effective than continuous UV treatment for inactivating bacteria (McDonald et al., 2000). The relative effectiveness can be gained from the report by McDonald et al. (2000) that pulsed light treatment of 40 J/m achieved the same inactivation of Bacillus subtilus spores as continuous UV of 80 J/m.

8.6.2

Effect on Whey Proteins

An important potential application of UV irradiation in the dairy industry is treatment of cheese whey. This is significant as whey often has to be stored for some time before being processed into WPC or WPI and cannot be thermally pasteurized. Reduction of the bacterial load by a nonthermal process such as UV irradiation to improve its keeping quality is therefore attractive. Whey produced during cheese-making is not clear as it contains a relatively high level of suspended solids and has poor transmittance to UV light (Mahmoud & Ghaly, 2004). Therefore it has to be clarified before the UV treatment to ensure the treatment achieves an acceptable level of destruction of bacteria. It is therefore important to know the effect of such a UV treatment on the WP. Unfortunately, there has been a limited amount of research on this topic. The effects of UV-C irradiation on proteins are mostly attributable to absorption of the light by the aromatic amino acids, tryptophan, tyrosine, and phenylalanine. At the wavelength commonly used, 254 nm, these compounds have absorption coefficients of 1, 0.11, and 0.05, respectively, indicating that tryprophan has the highest probability of undergoing changes during UV-C irradiation (Kristo et al., 2012). However, it should be remembered that WP have a low concentration of aromatic amino acids (Ustunol & Mert, 2004) and hence could be expected to be less affected by UV-C than other proteins. Research on the effects of UV irradation on WP falls into two categories: whey protein mixtures, as occur in whey, and individual WP. In the first category, Kristo et al. (2012) studied the structural changes in WP caused by UV treatment at 254 nm in a continuous Taylor-Couette reactor type UV system equipped with a Teflon FEP (Fluorinated Ethylene Propylene) fluid conduit and a static mixing element. The UV dosage was varied by changing the rate at which the WPI solutions were pumped through the reactor. The flow rate was varied from 30 to 800 mL/min, which resulted in UV dose levels of

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6.6
0.285 kJ/L. The authors treated both 1% and 5% WPI solutions. On the basis of fluorescence spectroscopy and hydrophobicity determinations (using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, ANS), the UV treatment was shown to cause changes in the tertiary structure of the proteins. The authors also observed denaturation and aggregation of the proteins, formation of oxidation products (N-formylkynurenine from tryptophan and dityrosine from tyrosine), increased susceptibility of the proteins to hydrolysis by pepsin, and an increase in sulfhydryl groups. In all aspects, the effects on the proteins in the 1% solution were greater than those in the 5% solution. This was attributed to the higher turbidity and lower UV penetration of the 5% solutions. Ustunol and Mert (2004) investigated UV-C treatment (0.324 kJ/cm for 180 minutes) as a protein cross-linking procedure in the preparation of whey protein films (see also Chapter 11). Cross-linking by UV radiation is due to formation of free radicals of aromatic amino acids, such as tyrosine and phenylalanine, which become involved in formation of intermolecular covalent bonds (Rhim, Gennadios, Fu, Weller, & Hanna, 1999). The UV treatment was compared with the use of chemical cross-linking agents (glutaraldehyde, formaldehyde, polymeric (starch) dialdehyde, and carbonyldiimidazole). Use of UV irradiation and chemical cross-linking agents led to an increase in film tensile strength but, in contrast to the chemical treatments, UV treatment had no effect on solubility, water vapor permeability. and oxygen permeability of the film. Schmid, Held, Hammann, Schlemmer, and Noller (2015) irradiated WPI films with UV-C at doses of up to 0.0314 kJ/cm for up to 200 minutes. This increased the tensile strength of the films but had no effect on their mechanical properties (Young’s modulus, elongation at break) or barrier properties. The result is similar to that of Ustunol and Mert (2004) who irradiated the whey protein solution before formation of the film. Schmid et al. (2015) considered the lack of effect on barrier properties to be beneficial as food packaging in which such films could used are exposed to UV irradiation during storage (see also Chapter 11). With regard to effects of UV on individual WP, Vanhooren, Devreese, Vanhee, Van Beeumen, and Hanssens (2002) irradiated solutions of goat milk at 280 or 295 nm. They observed cleavage of disulfide bonds, partial unfolding (from fluorescence spectroscopy at 3 C), and some dimerization and oligomerization, although most α-La molecules remained monomeric. The disulfide bond cleavage occurred in the Cys6 2 Cys120 and Cys73 2 Cys91 bonds. Each cleavage resulted in the formation of one free thiol, Cys120 and Cys91, respectively. A thioether linkage between Cys73 and Trp60 was also formed. Tammineedi, Choudhary, Perez-Alvarado, and Watson (2013) studied the effects of UV-C irradiation on the stability and allergenicity of WP. Treatment

8.7 Conclusions

for 15 minutes (calculated energy available B6 J/cm) resulted in reduced intensities of the bands of β-Lg and α-La and complete removal of the bands for BSA and immunoglobulins on SDS-PAGE gels. It also caused a significant reduction in IgE binding values compared to control samples. This indicated a 27.7% reduction in allergenicity of the WP. Interestingly, Tammineedi et al. (2013) cited a Masters thesis (Anugu, 2009) in which a 7.4-fold reduction of the allergenicity of whey was obtained by a pulsed UV treatment. Cilliers et al. (2014) carried out an extensive study of the microbiological, chemical, and sensory aspects of milk irradiated in a SurePure Turbulator, without recirculation, at a dosage of 1,045 kJ/L. They found, inter alia, that this UV treatment had no effect on the activities of the whey enzyme, lactoperoxidase. Elmnasser et al. (2008) used the pulsed-light (“pulsed UV”) process to treat solutions of β-Lg and α-La. The treatment was performed at a distance of 4 cm from the xenon lamp at an energy of 2.2 J/m, and with up to 10 pulses. They reported some similar effects to those reported for continuous UV-C. The main results were an increase in A280 of β-Lg and α-La, which was interpreted as due to protein aggregation, a red shift (to longer maximum wavelength) of 3
7 nm of the intrinsic tryptophan fluorescence, indicating a change in the microenvironment of tryptophan to a more polar environment, and the formation of dimers of β-Lg after 5
10 pulses due to formation of disulfide bonds. The effects on α-La were less than those on β-Lg; no dimers were observed after treatment of α-La. Overall these authors concluded that pulsed-light treatment did not cause very significant changes in the proteins. In summary, UV irradiation causes some changes in WP, mostly due to absorption of the light by the aromatic amino acids, particularly tryptophan. They include changes to the tertiary structure of the proteins, low levels of denaturation and aggregation, formation of oxidation products, increased susceptibility to proteolysis, decrease in allergenicity, cleavage of disulfide bonds, and increase in sulfhydryl groups.

8.7

CONCLUSIONS

In this chapter, the effects of several novel processing technologies on WP are described. It is clear that all described technologies, i.e., HP processing, shear-based treatments, ultrasound, PEF, and UV treatment have extensive and often unique effects on WP, either in model systems or, more complex systems such as milk, WPC, or WPI. However, compared to the wealth of scientific information available on the effect of these technologies on the WP, and the many exciting opportunities that appear to exist, industrial

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uptake has been rather limited to date. For this next step to be taken, several other aspects become important, such as the scale and economics of operation and the uniqueness of the treatments in relation to what is already achievable with the traditionally established technologies. Combining such assessments with the technological advantages will allow these novel technologies to establish themselves as advances rather than alternatives to traditional technologies, and thus allow sustainable implementation.

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254). Cambridge: Royal Society of Chemistry. Walstra, P. (2003). Physical chemistry of foods. New York: Marcel Dekker. Walstra, P., Wouters, J. T. M., & Geurts, T. J. (2006). Dairy science & technology (2nd ed.). Boca Raton: CRC Press. Wang, Q., Tolkach, A., & Kulozik, U. (2006). Quantitative assessment of thermal denaturation of bovine α-Lactalbumin via low-Intensity ultrasound, HPLC and DSC. Journal of Agricultural & Food Chemistry, 54, 6501
6506. Wolz, M., & Kulozik, U. (2015). Thermal denaturation kinetics of whey proteins at high protein concentrations. International Dairy Journal, 49, 95
101. Wolz, M., Mersch, E., & Kulozik, U. (2016). Thermal aggregation of whey proteins under shear stress. Food Hydrocolloids, 56, 396
404. Wong, P. T. T., & Heremans, K. (1988). Pressure effects on protein secondary structure and hydrogen deuterium exchange in chymotrypsinogen: A Fourier transform infrared spectroscopic study. Biochimica et Biophysica Acta, 956, 1
9. Wood, R. W., & Loomis, A. L. (1927). The physical and biological effects of high-frequency sound-waves of great intensity. Philosophical Magazine, 4, 417
436. Yang, J., Dunker, A. K., Powers, J., Clark, S., & Swanson, B. G. (2001). β-Lactoglobulin molten globule induced by high pressure. Journal of Agricultural & Food Chemistry, 49, 3236
3243. Zasypkin, D. V., Dumay, E., & Cheftel, J. C. (1996). Pressure- and heat-induced gelation of mixed β-lactoglobulin/xanthan solutions. Food Hydrocolloids, 10, 203
211. Zhong, J. Z., Liu, W., Liu, C. M., Wang, Q. H., Li, T., Tu, Z. C., . . . Xu, Y. J. (2012). Aggregation and conformational changes of bovine β-lactoglobulin subjected to dynamic high-pressure microfluidization in relation to antigenicity. Journal of Dairy Science, 95, 4237
4245. Zisu, B., Bhaskaracharya, R., Kentish, S., & Ashokkumar, M. (2010). Ultrasonic processing of dairy systems in large scale reactors. Ultrasonics Sonochemistry, 17, 1075
1081. Zisu, B., Lee, J., Chandrapala, J., Bhaskaracharya, R., Palmer, M., Kentish, S. E., & Ashokkumar, M. (2011). Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. Journal of Dairy Research, 78, 226
232.

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Zobrist, M. R., Huppertz, T., Uniacke, T., Fox, P. F., & Kelly, A. L. (2005). High pressure-induced changes in rennet-coagulation properties of bovine milk. International Dairy Journal, 15, 655
662. Zumaeta, N., Byrne, E. P., & Fitzpatrick, J. J. (2006). Predicting precipitate particle breakage in a pipeline: Effect of agitation intensity during precipitate formation. Chemical Engineering Science, 61, 7991
8003.

CHAPTER 9

Whey Protein Ingredient Applications Phil Kelly Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

9.1

WHEY PROTEIN-FORTIFIED BEVERAGES

The dairy industry has had a long and rich history of producing both fermented and nonfermented milk-based beverages. Until man mastered the art of fermentation, natural souring of milk with indigenous lactic acid-fermenting wild cultures provided acidified beverages that also marginally extended the shelf life of milk, particularly in circumstances where modern methods of preservation were unavailable. A turning point for milk fermentation was the discovery by Metchnikoff in 1907 that lactic acid bacteria could normalize bowel health and prolong life by displacing putrefactive bacteria. Today, probiotics are defined as live microorganisms which confer health benefits to the host when consumed in adequate amounts, hence a new emphasis on producing fermented milks containing specific probiotic bacteria that enhance the vitality of the human microbiota. Thus, the potential formats for formulating ready-to-drink (RTD) dairy-based beverages include a range of substrates, e.g., milk, buttermilk, whey, and whey protein, with the option in most cases of producing products at neutral pH (nonfermented) or acidified (fermented). Milk-based beverages may typically include emulsified fat and micellar casein, while it is also possible to formulate them with the nonmicellar caseinate form using ingredient recombination technology. Fresh sweet whey in its own right has been explored as a base for beverage preparations over the years and a number of examples have gained some prominence internationally, e.g., Swiss-based “Rivello,” Austrian-based “Latella,” and an American cultured whey product “Suero Viv.” Whey has a liquid consistency not unlike that of fruit juices, hence formulation and blending of ingredients may appear to be readily compatible. Whey protein concentrates (WPCs) prepared by ultrafiltration (UF) and gel permeation of acidic Cottage cheese whey to remove low molecular weight materials possessed the solubility, stability, and flavor necessary for incorporation at up to Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00010-2 © 2019 Elsevier Inc. All rights reserved.

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1% by weight into carbonated beverages (Holsinger, 1978). One percent whey protein solutions at pH 2.0
3.5 remained clear on heating at 80 C for 6 hours and were further stabilized against heat denaturation under acidic conditions when formulated with other soft drink ingredients such as sugar (Holsinger, 1978). However, sensory adjustment is required to mask the prevalence of whey-like flavors and their carry-over effect should not be underestimated. Acidification of UF-prepared whey protein retentates (13% total solids; 80% protein in dry matter) prepared from Cheddar cheese whey to pH 3.5 before spray drying decreased off-flavors in spray-dried WPC; this suggests that the mechanism for off-flavor reduction is a result of decreased protein interactions with volatile compounds at low pH in liquid WPC or increased interactions between protein and volatile compounds in the resulting powder (Park, Bastian, Farkas, & Drake, 2014). In a competitive market, product differentiation between whey-based and regular fruit-based beverages is key to ensuring commercial viability by using appropriate nutritional messaging and labeling to help consumers appreciate and commit to the proposition. The recent market success of whey proteins for human performance and recovery applications (Burrington, 2009) has demanded retail spray-dried and RTD beverage formats. Formulating functional beverages with whey proteins brings opportunities and challenges. Delipidated whey protein in the form of WPI and the β-lactoglobulin (β-Lg)-enriched fraction may be appropriate substrates for the formulation of clear acid-stable beverages. However, whey proteins are particularly heat labile and care is required during formulation to “manage” denaturation and aggregation particularly in light of the thermal process used to confer long shelf life. An additional challenge is to increase protein loading in beverage formulations in light of consumer desire to achieve higher protein intake per unit volume consumed. WPI is a concentrated source of high quality protein for supplementation purposes with a Protein Digestibility Corrected Amino Acid Score (PDCAAS) of 1.14, Biological Value (BV) of 104, and a high concentration of Branched Chain Amino Acids (BCAA) that are metabolized more rapidly during exercise, particularly glutamine and leucine. In a study using eight resistancetrained athletes, McMaster’s Exercise Metabolism Research Group found that participants who ingested a carbohydrate drink containing 10 g of whey protein with 21 g of fructose following resistance exercise saw a rise in muscle protein synthesis (Tang et al., 2007). Specifications will vary depending on the origin of the whey and manufacturing process, e.g., glycomacropeptide released during cheesemaking will be entrapped by microfiltration-processed WPI, but not by its ion-exchange produced variant where a higher concentration of β-Lg is emphasized. WPI’s capacity to provide clarity and heat stability is a key functional attribute for transparent beverage applications such as

9.1 Whey Protein-Fortified Beverages

protein waters, isotonics, and protein shots. WPI is soluble or forms a stable colloidal dispersion at ambient temperature and under all pH conditions. WPI’s wide stability across the entire food pH range enables clear hot-fill beverages with good stability and solubility to be formulated at ca. pH 3. However, specialized processes and stabilization systems are necessary when processing neutral pH protein-based beverages in order to counter whey protein instability. Compared to casein-based ingredients, WPI generally provides lower viscosity on a protein basis when used in liquid product formulations subject to thermal processing history and the stabilizing system employed. WheyUP, a sports drink that boasts 20 g of whey protein isolate (WPI) in an energy formula, is targeted at health-conscious individuals and marketed as “ideal to drink before or during your work-out to fuel your body.” Accelerade Advanced Sports Drink promotes a 15 g of WPI per serving. According to the sports drink company’s website, the protein in the RTD beverage to be consumed during exercise “facilitates rehydration, minimizes the breakdown of muscle that occurs during endurance exercise and speeds up the recovery process.” Following formulation, whey- and whey-protein-based beverages are subjected to thermal processing in order to achieve the desired shelf life stability commensurate with the degree of heat treatment applied and conditions under which packaging is undertaken. Shorter shelf life is usually associated with pasteurization using either in-container systems, e.g., tunnel pasteurization or high-temperature, short-time plate or tubular heating processes in combination with a hot- or cold-filled packaging system. In the latter case, product shelf life may be compromised if sterility shortcomings prevail at the point of beverage filling and/or if presterilization of the packaging material is not undertaken. When aiming for longer-shelf life beverages, aseptic processing based either on a combination of ultra-high temperature (UHT) technology with aseptic packaging or in-container retort sterilization is required. These processes usually place extra demands on the thermal stability of the product and, in particular, the whey protein ingredients used during formulation.

9.1.1

Neutral pH Beverages

Neutral pH beverages are required to be pasteurized, and either refrigerated until consumed or else thermally sterilized (rendered “commercially sterile”) via aseptic or retort processing. A broad pH range (pH 4.6
7.5) encompasses neutral pH beverages in order to accommodate the sensory effects of different flavors used. Caseins in the form of milk protein concentrates (MPCs) are predominantly used in neutral pH beverages and provide some

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protection and heat stability to the whey proteins present through complexation to prevent whey protein aggregation and precipitation. Whey proteins are sometimes added in these beverage formulations, but are generally not the predominant proteins used. Whey proteins that are not modified to have more heat stability will not be stable as the sole protein ingredient at levels above 3% protein (Cho, Shin, Sung-Moon Hong, & Cheol-Hyun Kim, 2015). Unmodified whey proteins will gel or precipitate under these conditions unless a stabilizing system is used involving one or more of the processes and component interactions described by Wijayanti, Bansal, and Deeth (2014). Further details are elaborated in the text that follows.

9.1.2

Acidified Whey Beverages

Acidified whey protein beverages are generally formulated to a pH range of 2.8 to 4.0. Their acidic nature means that a mild thermal pasteurization is sufficient to achieve shelf-stable status at room temperature. In the case of acidic beverages, milder pasteurization temperatures combined with low pH (2.8
4.0) provide the necessary microbial lethality and shelf life on condition that the product is subsequently hot-filled into containers which have been microbially decontaminated by prerinsing or other appropriate sanitizing technique. The delipidated nature of WPI with its low level of minerals enables beverage formulations with high clarity and minimal turbidity to be achieved within the pH range 2.8
3.5 even at high levels of protein.

9.1.3

Heat and Emulsion Stability

It is widely accepted that casein’s lack of tertiary structure and unique colloidal status confers considerable heat stability characteristics to milk—an important attribute that enables an extensive range of thermal processing options to be applied during dairy product manufacture, up to and including ultra-high-temperature and retort sterilization conditions. The highly folded structures of native whey proteins in milk, on the other hand, are heat-labile so that they progressively denature, aggregate and precipitate in the serum phase as temperature increases .60 C (see also Chapter 6). The classical heat stability profiles of milk reveal its pH sensitivity, concentration dependency, and susceptibility to the potential destabilizing effect of native whey proteins present. The latter problem is usually remedied by means of a high temperature milk preheat treatment before evaporation and drying during the course of heat-stable skim milk powder manufacture. Permitted processing aids that may be employed to augment the heat stability of milk such as the addition of simple phosphate salts (mono- and diphosphates) are unlikely to be of support when working with whey-based formulations. However, polyphosphates are claimed to improve the heat

9.1 Whey Protein-Fortified Beverages

stability of whey protein beverages, but require a degree of polymerization of approximately four units to be effective when stabilizing clear beverages containing more than 5% protein during retort sterilization (Rittmanic & Burrington, 2006). Increasing turbidity of beverages was accompanied by loss of soluble protein as whey proteins aggregated during storage. A slow protein aggregation process controlled the rate of formation of sediment and turbidity according to LaClair and Etzel (2009) based on a 6-week storage study undertaken with a heat-treated model beverage containing 1.25% whey protein at pH of 4. Burrington (2012) reviewed various approaches to improve whey protein heat stability including: controlling the size of protein aggregates through sugar addition, enzymatic cross-linking, mineral chelation, and ultrasonication, or modifying whey protein to prevent aggregation through molecular chaperones, enzyme hydrolysis, electrostatic repulsion, conjugation with carbohydrates, and protein encapsulation (see also Chapter 6). Windows of opportunity are afforded during the management of whey protein aggregation during which it is possible to control and stabilize particle size under optimized ionic conditions. The challenge for product developers is to identify such conditions, given the origin and diversity of the original whey plus the extensive range of WPC protein concentrations subsequently manufactured. Some commercial examples include heat-stable WPC 80 manufactured by Davisco Inc. from sweet whey which is claimed to be heat stable due to the controlled modification of the mineral profile as outlined in its technical specification: sodium 950 mg, phosphorus (P) 600 mg, calcium (Ca) 390 mg/100 g powder (Anon, 2015). A lower protein heat-stable WPC 34 with a mineral content of 7% is claimed by Foremost Farms (USA) (Anon, 2014) to be stable across a pH range 2
12. The specific mineral contents outlined in its specification include Mg 100 mg, P 517 mg and Ca 527 mg/100 g p powder. The molecular mechanisms and controlling factors for moisture-induced whey protein aggregation in a model system were investigated by Zhou, Xiaoming, and Labuza (2008). All major whey proteins were involved in the formation of insoluble aggregates. These aggregates rapidly formed during the first 3 days of storage at 35  C with a slower rate thereafter. Intermolecular disulfide bonds were the main mechanism for protein aggregation. The heat stability of a 5% WPI solution that had been preheated at 80 C for 15 min prior to cross-linking with transglutaminase (TGase) was enhanced to such an extent that it was capable of remaining transparent after heating at 138 C for 30 min in the presence of 0 and 50 mM NaCl at pH 7 (Wang, 2013). AFM images indicated that the cross-linked whey proteins were resistant to aggregation and disassociated during heating at 138 C to smaller particles that do not contribute to turbidity. Wang (2013) also attached

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seven molecules of maltodextrin to whey proteins by means of glycation and succeeded in making a 7% protein solution transparent within a pH range of 3
7 that, furthermore, remained clear after heating at 88 C for 2 min. An approx. 90% decrease in turbidity occurred when ultrasound (20 Hz, 60 C, 15 W for 15 min) was applied to a whey suspension of 28.2% solids containing 35.6% of protein on a dry basis (Martini, Potter, & Walsh, 2010). However, at higher protein (88%) concentration, ultrasound had the opposite effect by increasing turbidity. Interacting dextran sulfate with β-Lg decreased the protein’s denaturation temperature and improved its thermal stability at near neutral pH by altering its aggregation and not by stabilizing its native state (Vardhanabhuti, Yucel, Coupland, & Foegeding, 2009). Nanofibrils formed by glycating lactose with WPI were highly dispersible and remained transparent after heating at pH 3.0
7.0 and 0
150 mM NaCl (Liu & Zhong, 2013). The presence of the glycated lactose on nanofibril surfaces provided steric hindrance enabling dispersibility, thermal stability, and enhanced viscosity in various clear beverage products. High-temperature heating (85  C for 15 min and 90  C for 2 min) allowed the stabilization of WPI 2 pectin (LMP) complexes formed at pH 4.5 to a further pH adjustment to 7 (Gentès, St-Gelais, & Turgeon, 2010). The more severely heated complexes (90 C) could be preserved for up to 28 days of storage at 4 C without affecting their stability. Thus, protein 2 polysaccharide complexes formed under electrostatic associative conditions may have enhanced stability over a wide pH range by exposing the complex to high temperatures. When fats and oils are included in beverage formulations, then emulsion capacity and stability of the proteins present are of critical importance. Whey proteins are satisfactory emulsifiers, subject to optimum formulation and processing conditions being used. The protective effect of lecithins (phospholipids) against heat aggregation is only evident in fat-containing beverages. The mechanism by which lecithin improves heat stability is associated with protein 2 lecithin interactions mainly at the interface of the fat droplet. Regular, hydrolyzed, and acetylated lecithins improve the heat stability of emulsions containing up to 5% whey proteins. Modified lecithins with higher hydrophilic
lipophilic balance (HLB) values provide more protection against heat denaturation than regular lecithin. Emulsions containing hydroxylated lecithin were less sensitive to the addition of NaCl, which suggests that the binding of hydroxylated lecithin to unfolded monomers or intermediate products of β-Lg denaturation reduces the extent of their heat-induced aggregation and consequently decreases the interactions between unadsorbed β-Lg and adsorbed protein (JiménezFlores, Ye, & Singh, 2005).

9.1 Whey Protein-Fortified Beverages

9.1.3.1 Storage Stability Optimization of the parameters showed that emulsions containing 5% protein and 3% fat formulated with 0.3% lecithin and homogenized at 90 MPa had better stability when tested over 28 days of storage (Perez-Hernandez, 2005). However, creaming of the emulsions was still evident. The use of additives capable of increasing viscosity without affecting the heat stability of the emulsion is needed to improve the creaming stability of whey protein retort beverages. Another approach could be the incorporation of casein proteins such as found in MPC. Caseins have a disordered molecular structure and can protrude longer distances from the fat droplet interface, thus increasing steric repulsion and improving heat and emulsion stability. Formation of protein aggregates create undesirable turbidity and sedimentation during storage of heat-treated acidic (pH # 4.6) whey protein beverages (containing 12.5 g/L protein). LaClair and Etzel (2009) confirmed that aggregates formed during heat treatment acted as nuclei to mediate deposition of soluble protein during subsequent 6-week storage—a process that the authors characterized as fitting first- and second-order kinetic models.

9.1.3.2 The Formation and Role of Soluble Whey Protein Aggregates Soluble whey protein aggregates are formed by heating whey proteins at concentrations below their critical gelation concentration (approx. 12%). Schmitt, Bovay, Rouvet, Shojaei-Rami, and Kolodziejczyk (2007) characterized the nature of whey protein soluble aggregates following heat treatment (85 C for 15 min) in the presence of increasing amounts of added NaCl (5 to 120 mM)—the associated pH rise from 6.0 to 7.0 with increasing concentration of NaCl resulted in a higher yield of soluble aggregates from 75 to 95%. The soluble aggregates involved the major whey protein fractions and exhibited a maximum of activated thiol group content at pH .6.6. The hydrodynamic radius and the surface hydrophobicity index of the soluble aggregates increased from pH 6.0 to 7.0, but the molecular weight and zetapotential decreased. Schmitt et al. (2007) also showed that the transformation to a more fibrillar/elongated structure at pH 7.0 from the spherical/ compact structure of pH 6.0 soluble aggregates explained the loss of apparent density and decrease in zeta-potential. In order to achieve maximum heat stability, the soluble aggregates should be small and roughly spherical, have high surface charge, and low surface hydrophobicity. Ryan et al. (2012, 2013) suggested that they would be ideal for producing protein drinks with low viscosity and turbidity. Soluble aggregates can be produced with specific physicochemical properties for improving the heat stability of food products. Soluble aggregates formed from 7% w/w WPI (diluted to 3% w/w) were more heat stable in solutions containing 108 mM NaCl than were native whey proteins of the same concentration (Ryan et al., 2012). The improved

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heat stability was reflected by lower turbidity and viscosity, and also by greater solubility. The presence of salt in whey protein solutions enhances aggregation and increases the particle size of whey proteins when heated. Ryan et al. (2012) proposed that the enhanced heat stability of soluble aggregates may be due to their higher overall negative charge, more compact structure with less branching, and small size which make them resistant to secondary interactions when heated in salt solutions. Lu (2014) sought to spray dry soluble whey protein aggregates in order to produce ingredients suitable for use in food application, but discovered that they tend to lose their dispersibility upon dehydration. A follow-up study (Lu, 2014) revealed critical determinants for the solubility loss of spray-dried aggregates such as initial protein concentration, influences of storage, content of co-solutes, size of aggregates, and length of the heating process used to form the aggregates. Optimum conditions for production of soluble whey protein aggregates were identified, such as heating low solids (0.78% w/v) WPI solutions to produce powders that remained fully dispersible over 15-month storage, and by aiming to achieve aggregate suspensions that contained 30% w/w small aggregates and denatured monomers and oligomers (SMO) (,150 kDa) or more. In addition, secondary aggregation during spray drying may be prevented by increasing the proportion of SMOs to 49% w/w or more. Heatstable whey protein beverages may also be enabled by manipulating the pH-sensitive interaction between chitosan and whey protein, e.g., at pH 4.0, a small amount of chitosan prevented the heat-induced denaturation and aggregation of whey proteins, while the formation of chitosan 2 whey protein complexes at pH 5.5 improved the heat stability of dispersions without evidence of precipitation during 20 days of storage (Zhao & Xiao, 2016). The dispersion with a medium amount of chitosan (chitosan:whey protein, 1:5) produced the more stable particles, which had an average radius of 135 6 14 nm and a zeta potential value of 36 6 1 mV. In contrast, at pH 6.0, only the dispersion with a high amount of chitosan (chitosan:whey protein, 1:2) showed good shelf stability of up to 20 days.

9.1.3.3 Chaperone-Protein Effects The traditional technological practice of invoking heat-induced denaturation and associated molecular unfolding of whey proteins during the course of simultaneous interaction with casein in milk is of significance to shaping the textural and functional characteristics of manufactured dairy products. In recent years, there has been an increasing realization that natural mechanisms involving proteins themselves are at play as chaperones to assist covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. Thus, a new perspective on heat-induced casein/whey interactions was opened up when O’Kennedy and Mounsey (2006) found that whole micellar casein and some of its fractions (αs1- and β-casein) exhibited

9.1 Whey Protein-Fortified Beverages

protective behavior by restricting aggregation during heat treatment. The authors attributed the chaperone-like function to the hydrophobicity and flexibility of the casein structure which they were able to demonstrate by reconstructing model milk systems based on micellar casein/WPI mixtures dispersed in simulated milk ultrafiltrate (SMUF) heated at 85 C for 10 min. While denaturation was not prevented, aggregation could be controlled even at acidic pH values (pH 5.4). FTIR spectroscopic analysis indicated that αand β-caseins exhibited a protective effect on β-Lg during heating and changed the order of events in β-Lg’s unfolding, again confirming that hydrophobic bonding is involved in casein
β-Lg interactions (Grygorczyk, 2009). Complementary analysis using surface plasmon resonance (SPR) spectroscopy by the same author revealed that that there was a specific interaction between these proteins in the order β-casein . α-casein . κ-casein . calcium caseinate (pretreated using UHT). When exploring the chaperone effect at the higher protein concentrations and under the thermal processing temperatures applicable to beverage applications, Yong and Foegeding (2008) confirmed that β-casein (0.01 2 2% w/v) was a feasible component to stabilize higher concentrations (6% w/v) of whey protein in beverages. Mounsey and O’Kennedy (2010) also succeeded in demonstrating that a nonmicellar form of casein, sodium caseinate, had a heat-stabilizing effect by reducing uncontrolled β-Lg aggregation when present during heating (85  C for 10 min, pH 6.0) of the two proteins in different concentrations of SMUF. β-casein was shown to be more effective as a chaperone at higher, near-neutral pH 6 and with increasing ionic strength (CaCl2 . NaCl) without altering the denaturing temperature of β-Lg (Kehoe & Foegeding, 2011). The chaperone effect prevailed at temperatures up to 145 C. The authors concluded that β-casein competes with whey protein during the aggregation process such that the aggregates formed in the presence of β-casein are smaller in size than those formed during whey protein self-aggregation. By increasing the casein fraction of concentrated protein mixtures (10% total solids) within casein:whey protein ratios (5:95
30:70) heated to 80
120 C at pH 5.7
7.5 under constant shear (500 s21), Liyanaarachchi, Ramchandran, and Vasiljevic (2015) reduced the average size of WP aggregates and surface hydrophobicity with simultaneous reduction in apparent viscosity at pH 6.7. Thus, by increasing pH and the casein content in the system it was possible to form nano-sized whey protein particles. Interestingly, Grygorczyk (2009) noted that proteins alone were not entirely responsible for age gelation of high protein beverages as the condition manifested only when other beverage components were present along with the protein ingredients. Improved heat stability of WPI-inulin conjugates was achieved by dry-heating WPI-inulin at 2:1, 4:1, and 6:1 weight ratios at 80 C for 12 to 72 h without relative humidity control (He, 2015). A decrease in turbidity and particle size was noted after heating a 6% w/w protein solution

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at pH 6.0 without significant change in flow behavior. The functional improvement resulting from this limited degree of glycation was attributed to an increase in negative charge as well as increased stabilization of the protein. Other carbohydrates tested for reaction with β-Lg at aw of 0.53 (45 C) for different periods to reach an average degree of saccharide attached per protein molecule β-Lg (average DSP) of 5 included ribose, glucose, maltose, maltotriose, fructose, and galacturonic acid (Chena, Chena, Guob, & Zhoua, 2015). The results showed that all conjugates exhibited higher denaturation temperature than β-Lg. Larger molecular size, ketose, and negative charge had significant effects on increasing the denaturation temperature. β-Lg and conjugates at pH 3 and 7 could maintain their solubility when temperature was increased from 60 to 90  C. The conjugates showed better thermostability than β-Lg at pH 5 and sugar structure contributed to the improvement. WPI was optimally glycated with maltodextrin in a 2:1 ratio during 96 h incubation at 0.49 aw to produce a Partially Glycated Whey Protein (PGWP) constituting B94% protein and B4% carbohydrate based on reaching a plateau in Amadori compound formation, limited browning, and minimal loss of free amino groups (11%) and lysine (0.45%) (Savre, 2016). The PGWP had enhanced solubility (B8% loss) and thermal stability near the pI of WPI (pH 4.5) and under neutral conditions (pH 7) where WPI lost B60% of solubility. Under neutral conditions, the decrease in solubility of PGWP (B15% loss) upon heating was half that of WPI (B32% loss). Savre (2016) attributed the enhanced solubility and thermal stability of PGWP to resistance to denaturation and reduced protein 2 protein interactions upon glycation. The emulsification capacity of WPI, on the other hand, was improved upon glycation by B12%, while its stability was reduced. The improvement in emulsification capacity was attributed to the conformational changes induced by glycation. Savre (2016) was confident that this conjugated PGWP with a high protein content ( . 90%) possesses sufficient enhanced solubility and thermal stability, even at the pI of whey protein, to qualify it for application in both acidic and neutral beverages with an anticipated longer shelf life at protein concentrations .4.2%. The heat-stabilizing effect induced during microparticulation of whey proteins at low pH is of particular relevance for acid-stable beverage applications. At low pH, Dissanayake, Liyanaarachchi, and Vasiljevic (2012) utilized a combination of heat and high-pressure shearing to produce WP microaggregates with colloidal stability superior to those produced by microparticulation of denatured WP at neutral pH. As a result, the solubility of denatured microparticulated whey protein (MWP) powders was significantly higher. Controlled microaggregate size is also at the core of the case example cited by FoodNavigator-USA.com (www.foodnavigator-usa.com/ Promotional-Features/Way-forward-with-whey-protein) where it is illustrated

9.1 Whey Protein-Fortified Beverages

in a commercial example that the protein-loading of a neutral emulsion-based beverage could be increased from 4% (for standard MWP) to 11% without compromising texture or triggering gelation defects during subsequent storage (see Chapter 8 for further discussion of the effect of shear on whey proteins). Nanoscale structures of 5% WPI dispersions pretreated with microbial transglutaminase (mTGase) at 2.0 to 10.2 U/g WPI for 1 to 15 h before adjustment of pH to 7.0 and NaCl within the range 0
100 mM followed by subsequent heating (80 C for 15 min) were correlated to zeta-potential, surface hydrophobicity, thermal denaturation properties, macroscopic turbidity, and viscosity (Wang, Zhong, & Hu, 2013). The zeta potential and surface hydrophobicity of WPI increased after all pretreatment steps. Preheating increased cross-linking reactivity of WPI by mTGase, corresponding to significantly increased denaturation temperature. Particle size analysis and atomic force microscopy revealed that nanoscale structures of sequentially pretreated WPI remained stable after heating at 100 mM NaCl, corresponding to transparent dispersions (Wan Wang et al., 2013). A 9% protein-based fermented whey beverage (FWB) was formulated with WPC 80, skim milk powder, and sucrose, and fermented with Lactobacillus plantarum DK211 isolated from vegetable sources (kimchi) or mixed with commercially sourced Lactococcus lactis R704 starter culture (Cho et al., 2015). With a viable cell count of 109 CFU/mL following a 10 h fermentation that remained stable throughout storage at 15  C for 28 days, the FWB was characterized by high antioxidant and antimicrobial activity. State diagrams were developed for evaluation of the colloidal stability (protein solubility, turbidity, and macroscopic appearance) of whey protein beverages after UHT thermal treatment (Wagoner, Ward, & Foegeding, 2015). Individual whey proteins were highly soluble at pH 3, but beverages at pH 5 were characterized by poor solubility, high turbidity, and aggregation/gelation of whey proteins (Wagoner et al., 2015). Stability increased at pH 6, due to increased solubility of α-lactalbumin. FTIR-ATR spectra analyzed by multivariate regression analysis were used to build calibration models for the sensory astringency scores of an acidic whey protein beverage (Wang, Tan, Mutilangi, Plans, & Rodriguez-Saona, 2016). Major absorption bands explaining astringency scores were associated with carboxylic groups and amide regions of proteins. WPC and WPH (whey protein hydrolysate) beverages showed an increase in astringency as solution pH was lowered, but without evidence of any particular interrelationship. However, beverage astringency scores assessed by this portable FTIR-ATR spectrometer could be explained by pH, protein type (WPC, WPI, or WPH), and source (manufacturer) (see Chapter 10 for a detailed discussion of the flavor aspects of whey protein ingredients).

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9.1.3.4 Whey Protein Hydrolysates for Improved Heat and Storage Stability Whey proteins are typically digested by gastric enzymes in vivo, hence it is common to find enzymes of gastrointestinal (GI) origin, e.g., trypsin, chymotrypsin, and pepsin, in use for the preparation of protein hydrolysates under both laboratory and commercial conditions. Non-GI tract enzymes produced from bacterial and fungal sources are also commonly used. Proteolytic enzymes vary extensively in terms of their hydrolysis kinetics based on enzyme:substrate ratios, incubation conditions, specificity, and profiles of released peptides. Generally whey protein hydrolysates intended for heat stability application would have undergone 5
10% degree of hydrolysis (%DH) during processing. The resulting effects in terms of released peptides, exposed hydrophobic and ionic groups contribute to improved solubility and decreased viscosity compared to the original protein. While the desired WPH functionality may be brought about with moderate levels of %DH, excessive hydrolysis could lead to a loss of heat stability and functionality. This is also relevant to sensory characteristics—bitter flavors are associated with the release of peptides during hydrolysis and bitter tasting WPHs can result from extensive DH. Peptide self-aggregation may potentially occur particularly during heatinactivation of the enzyme-containing hydrolysate mixture following processing. Gauthier and Pouliot’s (2003) review of their research highlights identified that a bacterial enzyme-led hydrolysis of whey proteins was successful in improving heat stability of whey proteins in an acidic beverage, while some tryptic peptides demonstrated improvements in heat stability and modification of the thermal aggregation of whey proteins. Evaluation by Kankanamge et al. (2015) of five commercial enzymes (flavourzyme, protease A, protease M, protease S, and trypsin) during hydrolysis of WPCs containing 50 and 60% protein revealed that extensively hydrolyzed WPHs by protease A, and mild hydrolysates of proteases S and M were preferred for beverage fortification because of their relevant functional efficiencies for such applications. While emulsion capacity decreased with increasing degree of WPC hydrolysis, a beverage containing hydrolyzed WPC, skim milk powder, cocoa, liquid glucose, sugar, and vegetable fat was successfully formulated, according to analyses of physicochemical properties, sensory attributes, and keeping quality (Sinha, Radha, Prakash, & Kaul, 2007).

9.1.3.5 Novel Fermented Whey-Based Drinks Cheese whey was ultrafiltered by a factor of 15 3 in order to generate liquid WPC retentates with protein contents of 6
7% (Pereira, Henriques, Gomes, Gomez-Zavaglia, & de Antoni, 2015). The retentates were subjected to thermal denaturation and homogenization before addition of either

9.2 Yogurt

probiotic cultures, kefir grains, or a combination of both culture sources followed by fermentation at 25 C for 24 h. The authors also fermented the UF permeate with the same cultures following 2 3 volumetric concentration by reverse osmosis (RO) to generate a concentrate containing 10.8
11.5% total solids. All the experimentally produced fermented drinks showed acceptable physicochemical and sensorial properties, and contained above 7 log CFU/mL of Lactococci and Lactobacilli and 6 log CFU/mL of yeasts after 14 days of refrigerated storage, which the authors claimed to be in agreement with the standards required by international organizations like the European Food Safety Authority (EFSA) and Food and Drug Administration (FDA) for products containing probiotics. Fermentation of both retentate and permeate streams emphasized the authors desire for an almost total utilization of whey apart from the water permeated during RO.

9.2

YOGURT

A particular feature of yogurt manufacture is the extensive degree of heat treatment applied to the yogurt milk in order to denature whey proteins and induce whey protein interactions with the casein present prior to fermentation. The increased water binding capacity resulting from whey protein denaturation is instrumental in contributing to texture development during subsequent fermentation. Additional issues to watch for include the incidence of whey syneresis during product storage. Traditionally, skim milk powder addition has been used as a means of milk solids fortification of yogurt milk preparations. However, over time additional ingredients such as added proteins and stabilizing agents have been incorporated into yogurt because of functionality benefits in terms of texture enhancement and syneresis control. Additional technological challenges have also arisen for yogurt manufactures because of consumer preferences now dominating the market in quantitative terms for low-fat and zero-fat yogurts. Hence, the loss of solids’ contribution in yogurt milk formulations arising from fat depletion requires compensatory measures to not only maintain texture, but also ensure that yogurt sensory attributes are not unduly compromised. The yogurt market has also been enjoying a renaissance following burgeoning consumer interest in Greek-style yogurts that began in the USA led by the “Chobanis” brand. In addition, consumers’ search for higher protein versions of regular dairy products in light of positive outcomes to clinical trials has also triggered the development of high-protein versions of yogurts. Two percent fat yogurt milks fortified to 5% protein content with either SMP, BMP (buttermilk powder), or WPC75 yielded yogurts with K- and

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n-values and viscosities similar and intermediate between those obtained for sodium caseinate or WPC35—the least susceptible formulation to shear thinning, while also having the lowest viscosities at all shear rates (Guinee, Mullins, Reville, & Cotter, 1995). Interestingly, the yogurts fortified with the WPC ingredients were less susceptible to shear thinning than gelatinestabilized yogurt. However, the susceptibility to syneresis of dairy-ingredient based yogurts decreased in the order WPC35 .. sodium caseinate . WPC45 D WPC60 D WPC75 . SMP . BMP (Guinee et al., 1995). Sodini, Montella, and Tong (2005) compared five commercially-sourced WPC34 (i.e., with a total protein content of 34% each) during fortification of yogurt milk protein to 4.5% alongside skim milk powder as a control. Three of the WPCs generated yogurts with similar behavior to the control, while the other two produced yogurt with lower values for firmness, Brookfield viscosity, yield stress, complex viscosity, and apparent viscosity than the control. It was noted that the yogurts with the lowest firmness and viscosity were produced with WPC34 containing the highest amount of nonprotein nitrogen fraction (160 g/kg versus 126 g/kg of the total nitrogen), and the highest amount of denaturation of the whey protein (262 g/kg versus 200 g/kg of the total nitrogen)—the latter observation being consistent with the view that whey protein denaturation in the presence of casein is preferable for the formation of a more homogeneous and firmer acid gel. The Karam, Gaiani, Hosri, Burgain, and Scher (2013) review on the use of dairy proteins in yogurt suggests that the performance of added WPC is highly dependent on the specifications of the powders employed while the authors appeared to have more certainty on the applicability of WPC as an ingredient for drinking yogurt production (Guzmán-González, Morais, Ramos, & Amigo, 1999). However, they did concede that by using blends of different dairy ingredients (e.g., including sodium caseinate with WPC) in a proper ratio appears to be a useful and interesting perspective for yogurt mix fortification. Zhao, Wang, Tian, and Mao (2016) showed that the rheological properties (storage modulus, G0 , yield stress, and yield strain) of low-fat yogurt were greatly enhanced, the fermentation period shortened, and microstructure became more compact with smaller pores as the casein (CN) to whey protein (WP) ratio decreased from 4:1, 3:1, 2:1, and 1:1 by replacing reconstituted skim milk with WPC. In physicochemical terms, more disulfide bonds are formed and a greater degree of hydrophobic interactions occur during heat treatment, which can improve the rheological properties and microstructure of low-fat yogurt. Model yogurt studies undertaken by O’Kennedy and Kelly (2000) using combinations of micellar casein and WPI showed clearly the effects of protein synergies on texture over the course of acid gel acidification. It was also demonstrated that these synergies could be further elaborated by preconditioning of the individual ingredients before

9.2 Yogurt

blending together as a simulated nonfat yogurt milk. Combining WPI with a hydrocolloid blend of xanthan (X) and locust bean gum (L) (WPI-X/L) produced similar effects on yogurt consistency, pseudoplasticity, and apparent viscosity as gelatin, and higher sensory scores for thickness and stickiness than gelatin (Pang, Deeth, Prakash, & Bansal, 2016). However, a lower score for smoothness was observed with WPI-X/L than with gelatin. In addition to the use of added ingredients, new technologies have also been employed by researchers alongside regular thermal-based pretreatments prior to fermentation in order to determine the effects on milk proteins and gel structure. Needs, Capellas, Bland, Manoj, and MacDougal (2000) obtained similar levels of β-Lg denaturation (.90%) when comparing high pressure treatment (600 MPa for 15 min) and heating at 85 C for 20 min of skim milk fortified with WPC. However, there were considerable differences in the changes that took place at the molecular level, e.g., casein micelles were in a highly dissociated state as a result of the applied pressure in the case of the former while intact caseins were maintained in the latter. The microstructure of the resulting gels and their rheological properties were very different. Pressuretreated milk yogurt had a much higher storage modulus and yielded more readily to large deformation than the heated milk yogurt. Four novel technologies were reviewed by Loveday, Sarker, and Singh (2013)—high pressure processing (HPP), high pressure homogenization (HPH), ultrasonic processing (USP), and protein cross-linking using transglutaminase (TGase) with a view to reducing dependency on the use of hydrocolloid stabilizers in yogurt formulations. The benefits of HPH and USP depended on the response of the fat content present, while HPP and TGase work best in combination with other processes and demonstrate a strong potential for improving protein ingredients. Both stirred and low-fat yogurts benefit from the use of new technologies (Loveday et al., 2013). US and UHP improve the texture of full-fat and low-fat yogurts, but have little or no benefit for nonfat formulations. On the other hand, microfluidization of low-fat milk resulted in yogurt with modified microstructure, giving more interconnectivity in the protein networks with embedded fat globules, but with similar texture profiles and water retention compared to yogurt prepared from conventionally homogenized milk (Ciron, Gee, Kelly, & Auty, 2010). Another perspective on the use of novel technologies is how they may be used to modify and adapt the functionality of ingredients intended for yogurt application in liquid form before drying. The approach works for TGase and may be applicable for HPP. As USP pretreatments work largely on milk droplet size, it is unlikely to be suitable for manufacturing dry ingredients because of the challenges in controlling droplet size during drying and subsequent rehydration (see Chapter 8 for detailed discussion of the effect of novel technologies on whey proteins).

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In 1984, inventors Norman S. Singer, Shoji Yamamoto, and Joseph Latella of Labatt Canada, filed for a U.S. patent for Simplesset—an egg white and whey mixture that formed small particles resembling the texture of fat when subjected to combined heat and shear (microparticulation process), and licensed the invention to Nutrasweet. In 1993, Labatt subsidiary Ault Foods entered into an agreement with Pfizer to market the processed whey protein-based version of the fat substitute internationally under the trade name Dairy-Lot. With Simplesse accounting for less than one-third of the calories of real fat, the dairy industry now had the prospect of generating a compatible fat substitute for inclusion in milk products. This coincided with a time of significant market change reflecting increasing demand by health-conscious consumers for reduced-fat varieties of most dairy products. As always, the challenge was (and still is) to identify an ingredient and/or process adaptation that was capable of making up for the loss of milk solids due to fat depletion while at the same time maintaining sensory attributes resembling as close as possible those of its full-fat parent variety. Tamime, Kalab, Muir, and Barrantes (1995) compared yogurt prepared using reconstituted skim milk powder to which anhydrous milk fat (AMF) was added/homogenized as fat source (control) and Simplesse 100 incorporated as fat substitute on an equal percentage basis. Both the AMF and Simplesse interacted with the protein matrix during yogurt acidification and gelation. However, the softer texture and higher levels of serum separation of yogurts prepared with Simplesse were attributed to the likelihood that the larger-sized integrated fat substitute/protein particles may have affected the structural integrity of the yogurt differently (Tamime et al., 1995). Scanning electron micrographs showed that the protein matrix of the reduced-fat yogurts made with and without fat replacers showed differing structures, which in general terms were more open and less dense than that of their full-fat equivalents (Sandoval-Castilla, Lobato-Callerosa, & Aguirre-Mandujano, 2004). The same authors also confirmed that MWP formed part of the yogurt protein matrix and its casein micelles had similar spatial distribution as reduced fat yogurt. Yogurt made with blends of WPC and MWP had textural characteristics resembling those of full-fat yogurt (Sandoval-Castilla et al., 2004). A contrary view was expressed by Janhøj and Ipsen (2006) who inferred that MWP was not integrated in the protein network of acid gels prepared milks preheated to 80 C for 30 min. Water holding capacity was practically identical irrespective of whether MWP was added before or after preheating, while yogurt firmness was slightly higher in the case of MWP added after heating. Ten types of microparticulated whey proteins (MWP) with different particle sizes and denaturation degree, generated at an industrial source by undisclosed processing conditions, were added to low-fat stirred yogurts to obtain

9.2 Yogurt

two protein levels (4.25 and 5.0%, w/w) (Torres, Janhøj, Mikkelsen, & Ipsen, 2011). Samples were compared to reference yogurts manufactured using reconstituted skim milk powder: a full-fat (3.5%, w/w, fat; 3.5%, w/w, protein) and two low-fat (0.5%, w/w, fat; 4.25 or 5.0%, w/w, protein) yogurts. A high native-to-denatured whey protein ratio (0.94
1.33) in microparticulated whey protein (MWP) powders provided yogurts with high creaminess and viscosity (high yield stress values and elastic modulus), a slow meltdown in the mouth, as well as creamy flavor and low syneresis. These sensory characteristics were related to those of the reference full-fat yogurt. Correlations between rheological and sensory variables showed that viscometry and oscillatory frequency sweep data are useful in predicting texture attributes such as creaminess. The sensory characteristics of low-fat yogurts (0.5% w/w fat; 4.25 or 5.0% w/w protein, the latter adjusted with added MWP) manufactured with microparticulated whey protein were distinguishable according to the characteristics of the microparticles added (Torres et al., 2011). These results emphasize that the degree of denaturation of the whey proteins included in the MWP powder used as fat replacer is important for the sensory and rheological properties of the final yogurts. As a result, the authors emphasized the importance of having full control of the parameters influencing the degree of denaturation of the whey proteins during the manufacture of the MWP powder in order to maintain the sensory and rheological properties of lowfat yogurts incorporating MWP powder as a fat replacer. The surface reactivity of the microparticles also appears to play a more important role in the final perception of graininess in the yogurts than the initial particle size of the MWP aggregates. Thus, a large particle size among the initial aggregates may in fact yield yogurts with low graininess perception if the microparticles become an integral part of the yogurt matrix during fermentation of the milk. Some sensory attributes of yogurts may be effectively predicted by rheological tests such as an oscillatory frequency sweep, Posthumus funnel, or viscometry analysis. This can ease the task of adapting the functionality of MWP or other ingredients to the sensory perception of yogurt. Using fractal analysis to characterize the microstructure of low-fat stirred yogurt manufacture with MWP, Torres, Amigo Rubio, and Ipsen (2012) showed that the amount of native and soluble whey proteins present in the microparticles had a positive influence on the structure of the formed gel. The resulting yoghurts were dominated by dense aggregates and lower amounts of serum pores, and had an increased degree of self similarity or fractality with the full-fat variants. Microparticles, with both higher proportions of soluble particles and initial amount of native whey protein, yielded yoghurts with a structure dominated by dense aggregates and low amount of serum pores. Some ingredient companies aim to provide “packaged ingredient solutions,” e.g., a combined hydrocolloid blend of WPC and pectin (proprietary name

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Vitex AYS 08) is promoted by Cargill for viscosity/body control in yogurt applications but with the additional goal of being able to replace modified food starch and improve ingredient label declaration. There is also increasing interest in the contribution of in situ yogurt culture-generated exopolysaccharides (EPS) that may take place simultaneously during fermentation depending on the specific chosen culture and its growth characteristics. Cognisance needs to be taken of the potential interaction of added protein ingredients with EPS present as demonstrated by Purwandari (2009) who found that when heat-untreated WPI or WPC was incorporated into calciumfortified fermented milk produced using a capsular ropy EPS culture, the combined effect of both supplements was to reduce consistency to that of a drinking yogurt. On the other hand, the addition of heated WPI or WPC succeeded in maintaining a similar consistency to that of the control yogurt, but with the option of creating a harder yogurt texture by increasing the addition of whey protein. Connolly (2013) recommended the use of dairy protein blends containing in the range 50 to 70% casein and 30% to 40% whey proteins to achieve firm yogurt gels with smooth texture in Greek style yogurt applications. Such blends would typically comprise of the casein-to-whey protein ratios based on combining 60 to 85% MPC 80 and 15 to 40% WPC 80, with the final ratio of casein to whey protein being dictated by the desired total yogurt protein content. Should a higher target protein (.10%) Greek style yogurt be desired, then it is recommended to increase the addition of whey proteins in order to maintain a smooth gel texture commensurate with the increased protein content. Whey protein powder was used to fortify yogurt mixes at levels ranging between 0.6 to 4% w/w (considered as an upper limit) (Lee & Lucey, 2010; Tamime & Robinson, 2007). However, the typical recommendation for whey protein addition to dairy products is around 1 to 2% w/w since higher levels may impart an undesirable whey flavor as well as a grainy texture under some conditions (González-Martínez et al., 2002; Lucey & Singh, 1998). Addition of WPC to milk bases seems to be a more popular practice than WPI addition even though WPI powders contain higher whey protein content and branched-amino acids (.90% vs 60%
85%) and relatively lower concentration of lactose and minerals (Considine et al., 2011). Sfakianakis and Tzia‘s (2014) review of innovative developments indicates that WPC fortification during fermentations involving probiotic cultures improves coagulum formation and contributes to increased product firmness and adhesiveness. Protein fortification of yogurts is also being explored with the aim of developing biofunctional products that promote particular physiological events. Yogurts with whey protein added as WPC alone or in combination with

9.2 Yogurt

calcium caseinate maintained higher consistency index values throughout in vitro digestion which the authors used a marker for increased gastric distension and, ultimately, may contribute to an extended feeling of fullness (Morell, Fiszman, Llorca, & Hernando, 2017). The expectation is that rapid gastric emptying of whey protein in a more unaltered form than casein may result in a stronger increase in postprandial plasma amino acid concentration and associated satiety signaling. (See Chapter 15 for further discussion of the nutritive aspects of whey proteins.) Protein cross-linking of skim milk fortified with skim milk powder, sweet whey protein powder, or mixtures of both to obtain different total casein-towhey protein ratios in yogurt milk ranging from 4.0 to 1.6 by a microbial transglutaminase (mTGase) preparation was significantly enhanced by the presence of added glutathione (GSH) without recourse to application of milk preheat treatment beforehand (Bönisch, Huss, Weitl, & Kulozik, 2007). A positive relationship was established based on degree of polymerization in the range of 10%
30% with casein-to-whey protein ratio and rheological properties of the yogurt gels. It was claimed that this novel process enables stirred yogurt gels to be created at reduced protein content without syneresis and without the need for an additional heat treatment step prior to cross-linking. The authors believe that in the case of cross-linked casein micelles, denatured whey proteins may act as a protective colloid to support integration into the three-dimensional gel network and inhibit the risk of coarse and lumpy gel formation that may result from use of higher casein-to-whey protein ratio milks. TGase cross-linking of preheated (95 C for 5 min) milk blends where between 20% and 30% of the milk was substituted by natural fresh whey contributed to higher cohesiveness of the resulting fermented yogurts (Gauche, Tomazi, Barreto, Ogliari, & Bordignon-Luiz, 2009). However, in the absence of added whey, i.e., 100% milk, TGase increased fracturability, hardness, and gumminess texture profile characteristics of the yogurts. The effects of TGase and a commercial fat replacer (Dairy-Lo) were compared for the manufacture of nonfat set yogurt (Ë Sanli, 2015). Physical properties of the nonfat yogurt were improved by TGase over the course of 20-day storage. The Dairy-Locontaining nonfat yogurt had the lower serum separation, but its gel strength was weaker than those made with mTGase. Overall, the mTGase-prepared yogurt had a better sensory profile with taste and aroma compared to that of the control yogurt. Dairy-Lo was also compared with inulin, both singly and in combination for the preparation of low-fat set-style yogurts (Seydim, Sarikus, & Okur, 2005). All of the fat substitutes resulted in smooth textured and satisfactory flavored yogurts with the Dairy-Lo sample contributing slightly higher gel strength over a 14-day storage period. Henriques, Gomes, Pereira, and Gill (2013b) prepared medium-fat (1.5%) and full-fat (9%) set-type yogurts using liquid WPC (LWPC) prepared by UF concentration (20 3 ) of

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cheese whey to B14.5% total solids (B5.0% protein), heat denatured at 90 C for 1 min followed by homogenization at 100 bar to maintain aggregate particle size ,10 μm. Medium-fat (1.5%) yogurts with a target dry matter (DM) content of 15% were formulated with 100% skim milk powder (SMP) (control) or its partial replacement with 15% LWPC (test 1) or 30% LWPC (test 2). The full-fat (9.0%) experimental variant with a target DM of 19% was conducted in a similar manner to test 2, i.e., partial replacement of SMP with 30% LWPC. Viscosity was improved with the addition of LWPCs, particularly in the case of full-fat yogurts. LWPC also decreased the syneresis of all yogurts during storage (Henriques et al., 2013a). Sodini, Mattas, and Tong (2006) prepared a range of WPC34 powders from wheys collected at (a) the draining pH 6.4 and (b) following further manual acidification of (a) using an acidulant to pH 5.8. After pasteurization of all wheys at 72 C for 15 s, two lots of the wheys received further heat treatments of 82 C and 88 C for 78 s before UF and preparation of WPC. Yogurt milk was subsequently fortified from 3.4% to 4.5% protein using the experimentally produced WPC34 powders. Higher water holding capacities were obtained when yogurt was fortified with WPC from whey that had received lower heat treatment but which originated as UF feedstock from the higher (unadjusted) pH whey (pH 6.4). The results suggest that a high pH and a mild heat treatment during whey processing favors the production of functional WPCs for yogurt manufacture. The authors conclude that a more acidic pH of the yogurt mix is likely to contribute to a more inhomogeneous coverage of the casein micelles by the denatured whey protein during heating, thus resulting in a leaky gel network later.

9.3

PROTEIN BARS

Phenomenal growth in the market for protein snack bars in recent years (e.g., sales growth of 26% occurred in the USA during 2010
15) has presented dairy companies with excellent opportunities for the supply of protein-based ingredients to the processors of such snack bar products. The global market for whey protein is projected to reach US$13.5 billion in 2020 from US$9.2 billion in 2015, reflecting a 5-year annual compound growth (CAGR) of 6.5% (Bizzozero, 2016). The food and beverage segment is expected to grow from US$4.3 billion in 2015 to US$5.5 billion in 2020, and the sports nutrition segment was projected to reach US$1.7 and 2.7 billion in 2015 and 2020, respectively. While there is some suggestion that market growth is steadying in the meantime due to competition from other product formats such as healthy beverages, consumers high purchase rate of snack bars at 57% signifies the importance of the sector which is becoming increasingly more differentiated according to lifestyle and functionality

9.3 Protein Bars

preferences such as (a) snack bars (mainly protein and fiber-containing granola-types), (b) nutrition bars (nutrient-fortified; nonathletic), (c) performance bars (fueling during exercise and postexercise recovery for enhanced fitness), and (d) weight loss bars. Protein bars offering the convenience of meal replacement such as breakfast “on the go” started a process of influencing lifestyle changes around mealtimes. As a result, continuing sophistication in the formulations and formats of bars marketed to different consumer sets in categories (b), (c), and (d) is extending the market and building customer loyalty around other varied daily meal intake occasions. Manufacturers are addressing more specific concerns such as creating products to supplement perceived nutritional deficits that might be incurred in a weight-loss program. High-protein, low-carbohydrate, or balancedgastrointestinal formulations have seen good market uptake. Scientific evidence to substantiate claims is becoming increasingly available. Clinical trials undertaken with a whey protein 2 polydextrose (PPX) formulated bar proved that daily energy intake was lower irrespective of the manner of consumption (free-living vs habitual daily consumption over a 14-day period) (Astbury, Taylor, French, & Macdonald, 2014). In addition, the PPX bar was associated with lower glucose and ghrelin and higher glucagon-like peptide 1 and peptide tyrosine-tyrosine responses. Incorporating high resistant starch into protein bar formulation reduced in vitro digestibility but not in vivo glucose or insulin responses (Wolever et al., 2016). The whey protein present, on the other hand, reduced glucose but disproportionately increased insulin. The authors cautioned that the inclusion of whey protein in cereal bar formulations to reduce glycemic response may be associated with a disproportionate increase in insulin as judged by an increased insulin-to-glucose iAUC ratio. From the outset, the formulation of protein bars has generally followed a simple rule of thumb based on the 30/40/30 rule where ingredients are selected to contribute 30 calories from protein, 40 calories from carbohydrates, and 30 calories from fat. Meanwhile, there is an increasing tendency to load more protein in bar formulations in line with consumer expectations of acquiring greater nutritional and functional benefits following their ingestion. Optional protein bar production formats include baked or nonbaked varieties—the latter based on simple mixing and molding protocols. The resulting protein bars are shelf-stable since they fulfil the requirements of intermediate-moisture foods. Consequently, all ingredients are in a concentrated state within the relatively low moisture environment of snack bars, and the proteins present have a history of being reactive. These include aggregation following formation of intermolecular disulfide bonds and noncovalent interactions (Zhou et al., 2008), Maillard reactions, which may result in protein polymerization (Tran, 2009), moisture migration (Labuza &

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Hyman, 1998; Liu, Langer, & Klibanov, 1991; Loveday, Hindmarsh, Creamer, & Singh, 2009), and phase separation phenomena (Loveday, Hindmarsh, Creamer, & Singh, 2010; McMahon, Adams, & McManus, 2009). Hence, the combined effects of swelling, molecular reorganization and aggregation of proteins add up to sufficient material science changes over time that manifests in bar hardening. However, the relatively high content of protein causes texture problems due to increased hardening over the course of the shelf life of the product, and much formulation adaptation is given towards countering the advance of this defect. Protein ingredients are prone to jamming (solidification) depending on their critical volume fractions which gives rise to subsequent hardening. Diagnosis using FT-IR spectra suggests that there may be a molecular explanation for hardening due to solvent-induced plasticization and reconformation of protein secondary structures (Hogan, Chaurin, O’Kennedy, & Kelly, 2012). In high-protein bars, hardness development appears to result from nonequilibrium changes due to the hydration behavior of individual components and the competition for available moisture (Hogan et al., 2012). Hence, minimization of water activity differences between ingredients provides a means of controlling hardness. Bar formulations containing whey protein hydrolysates (WPH) did not harden to the same extent as those containing intact WPI (Hogan, O’Loughlin, & Kelly, 2015). For WPI, the onset of solidity (ψ) occurred at a volume fraction (φ) of 0.73, compared with approximately 0.55 for two of the WPHs evaluated. Bars containing the most extensively hydrolyzed proteins did not exhibit an equivalent liquid
solid transition. Hardening was lower in systems in which ψ occurred at low φ. Hogan et al. (2015) concluded that rheological characterization of the liquid 2 solid boundary provides a better means of understanding structural development in concentrated food systems. Imtiaz, Kuhn-Sherlock, and Campbell (2012) identified instrumental texture parameters that correlated with sensory texture measurements of protein bar preparations, e.g., peak force (Force 1) and maximum negative force (Force 2) representing firmness and cohesiveness (being opposite of crumbliness) parameters, respectively. The texture measurement technique was applied to mixture experiments involving two modified MPCs and one WPC where the authors (Imtiaz et al., 2012) were able to achieve certain desired textural synergies along with stable hardness over 12-month storage at 20 C in their bar formulations. (See Chapter 13 for further information on whey-protein nutrition bars.)

9.4 Desserts and Ice Creams

9.4 9.4.1

DESSERTS AND ICE CREAMS Fresh Dairy Desserts

Whole milk and reduced-fat milks usually provide the base ingredient for the formulation of dairy-based desserts. Other ingredients such as starches and hydrocolloids are added in order to provide the desired texturization during subsequent cooking and chilling. Whey proteins are versatile ingredients due to their wide range of functionalities, e.g., emulsification, water-binding, and gelation, which makes them suitable for consideration in dessert formulations. In general, whey proteins tend to contribute lower viscosity compared to caseinand caseinate-based ingredients. However, modified WPCs in the form of enhanced gelation, e.g., hi-gelling WPC, and cold-set gelling whey proteins are now available to enable formulators explore the potential of these enhanced functional ingredients. Because of the complexities of interactions with other ingredients present, adaptations of formulation protocols may be necessary in order to ensure that the modified WPCs deliver their specific functional effect. κ-Carrageenan is a hydrocolloid that contributes to the functionality of dairy desserts in a similar manner to other hydrocolloids, but also because of its potential to interact with casein present. Mleko, Chan, and Pikus (1997) investigated the influence of κ-carrageenan on textural properties of WPI gels formed by heating at 80  C for 30 min in the pH range from 1
12. WPI at a lower concentration of 3% significantly enhanced the shear stress values of 0.5% κ-carrageenan, particularly at pH 6
7. Nongelling polysaccharide guar gum at 0.5% addition had a disruptive effect during thermal gelation of WPI due to excessive protein aggregation in response to segregative interactions (Fitzsimons, Mulvihill, & Morris, 2008). However, at a low optimum concentration of B0.1% guar gum a large (B12-fold) enhancement in gel strength (G0 ) was evident in comparison to WPI alone. The rheology of starch granule suspensions may be modified by cold-set whey protein gelation in order to create specific gel-like or paste-like characteristics (Chung, Degner, & McClements, 2013). The different effects were leveraged by increasing WPI (up to 10%) and calcium concentration (20 mM). It appears that the presence of swollen starch granules in the WPI 2 starch mixtures causes discontinuous protein network formation and leads to lower gelation kinetics in comparison to protein-only WPI gels. Designing healthier pannacotta-like desserts aimed at weight management involved the addition of extra whey protein to a low-cream formulation in order to create a denser, firmer matrix in the expectation of increasing satiety (Borreani, Llorca, Quiles, & Hernando, 2017). As a second objective to their study, the authors monitored the in vitro gastric digestion behavior of whey proteins and casein in the heat-treated desserts and noted that the whey

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proteins present were more resistant to pepsin digestion than caseins, thus reinforcing the anticipated increased satiety effect. Better texture profile characteristics such as firmness, elasticity, chewiness, and gumminess were obtained when higher WPC concentrations (0, 1.5, 3.0, and 4.5%) were added to a fat-free dairy dessert base formulation consisting of sugar (12.0%), skim milk powder (10.0%), corn starch (5.0%), vanilla flavor (2.5%), and κ-carrageenan (0.02%) (Vidigal et al., 2012). The simulated processing protocol consisted of mixing all ingredients with water and maintaining the mix under agitation in a water bath at 40 C for 2 min. The mix was then pasteurized at 90 6 2 C for 15 min before reducing the temperature to 40 C and mixing for a further 2 min. It is believed that the presence of WPC promotes the formation of a stronger gel structure as a result of protein 2 protein interactions. WPC (4%) was equally versatile when formulated with 3% potato starch, 0.1% i-carrageenan, 10% sucrose, 3% milk fat, and 3% chocolate to produce products with acceptable rheological properties and good shelf life (El-Garawany & Abd El Salam, 2005). The effects of the heating time (30 min) and temperature (100
130 C) were found to be small relative to the multiple interactions between proteins, polysaccharides, fat, and minerals which impair the formation of three-dimensional networks of whey protein, amylose-amylopectin, and carrageenan.

9.4.2

Frozen Desserts

Acceptable frozen yogurts were produced using a formulation in which 100% of the SNF was replaced by WPC (Opdahl & Baer, 1991). The mix contained 6% milk fat, 10.5% WPC, 11% sucrose, 3% corn syrup solids, 0.3% stabilizer and emulsifier blend, and 30.8% total solids, and acidification was facilitated by the addition of 3.1% yogurt culture fermented WPC (sourced from the same batch). Frozen yogurt manufacture, unlike ice cream, is affected by physicochemical changes occurring during acidification that impacts on physical and textural characteristics. Indirect acidification (blending of plain acidified milk with ice cream mix) had a superior textural effect as it leveraged the functionality of added hydrocolloids (guar gum, xanthan and carboxymethylcellulose) more effectively than when used with direct lactic culture-based fermentation (Soukoulis & Tzia, 2008). Addition of 0.2% xanthan gum and the partial substitution (at a ratio of 3:1) of skim milk powder by whey powder increased overall acceptance and creaminess by controlling coarseness and wheying off during melting without imparting sandiness.

9.4.3

Ice Cream

Ice cream may be described as a functional multistranded emulsified, aerated, frozen dessert—the characteristics of which are dominated by key

9.4 Desserts and Ice Creams

physical transformations such as homogenization, whipping, freezing, and crystallization during its preparation. It is the interaction of these physical processes with the components of the ice cream mix that will ultimately determine final product texture and sensory attributes. Traditionally, nonfat dry milk solids (skim milk) have provided the base to which milk fat or vegetable oils are added along with sugar and other processing aids such as emulsifiers and stabilizers. Milk proteins function as emulsifiers by adsorbing at the fat interface during homogenization to render stability to the fat globule. However, additional low-molecular-weight emulsifiers are used to displace some of this protein and render the fat globule susceptible to partial coalescence. The most commonly used low-molecular-weight emulsifiers originate as cleavage by-products of fats and oils, e.g., mono- and diglycerides (E 471), while the commercial emulsifier polysorbate 80 (“Tween 80”) is derived from sorbitol. Stabilizers, likewise, improve structure, but also the texture by slowing the growth-rate of the ice crystals in ice cream, and reducing its melt-down rate. Their sponge-like behavior enables them to both absorb and immobilize liquid in ice cream. Today, most commercial stabilizers are gums of vegetable and microbial origins, e.g., agar-agar, guar, locust bean, xanthan, gellan, and carrageenan. Ingredient suppliers usually prepare proprietary ready-made stabilizer mixtures composed of one or more of the aforementioned hydrocolloids which have been optimized for particular ice cream mix formulations. Over time, the milk solids-not-fat portion of ice cream has been increasingly substituted with milk-based dry ingredients comprised of proprietary blends that include partially denatured/aggregated WPCs, MPCs, sodium caseinate, modified milk proteins, and whey powder or lactose blended to give protein contents of 18
25% (Goff, 2008). Casein micelles are more easily displaced from the fat interface than are either whey proteins or caseinates. Milk protein adsorption at air 2 bubble interfaces along with adsorbed fat globules during whipping/aeration is an equally important function which must be balanced with the competing demands of their fat interfacial role to ensure sufficient coverage of both microstructures (Zhang & Goff, 2004). Unadsorbed proteins will influence liquid phase viscosity during processing and contribute to ice cream structure also. The incompatibility between milk proteins and polysaccharide stabilizers that manifests in the form of depletion flocculation leads to phase separation between the two, and requires optimization through adjustment of casein:whey protein ratios in milk protein blends (Syrbe, Bauer, & Klostermeyer, 1998). Varied viscosity and pH changes were observed depending on the degree of substitution of milk solids nonfat with either fresh UF retentate or WPC in ice cream mixes (Lee & White, 1991). Interestingly, UF retentate at a 25% level of replacement had a higher flavor, body, and texture scores than that made with WPC.

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Double homogenization of ice cream mix and partial replacement of its skim milk powder on an equal basis with 1% WPC36 had no effect on sensory properties although mean ice crystal size was lowered when stabilizer was added. The authors (Ruger, Baer, & Kasperson, 2002) felt that their partial substitution of SMP by WPC may have been insufficient to support their objective of achieving greater control over the formation of smaller ice crystals. Improved functional effects were achieved when native whey proteins were replaced by predenatured whey proteins in terms of (i) more homogeneity in air bubble size, (ii) more attachment of fat globules to the air
serum interface, and (iii) closer contact between the fat globules in the continuous matrix. These differences in the microstructures of whipped frozen emulsions were attributed to the differing surface heterogeneity of adsorbed protein particles to fat globule interfaces (Relkin, Sourdet, Smith, Goff, & Cuvelier, 2006). Whey protein (WPI) addition with transglutaminase treatment improved the physical and sensory properties of reduced-fat ice cream more favorably than either whey protein addition or transglutaminase treatment alone (Danesh, Goudarzi, & Jooyandeh, 2017). The added whey protein with or without transglutaminase treatment caused an increase in apparent viscosity and a decrease in flow index of the reduced-fat ice cream. Commercially-sourced whey protein phospholipid concentrates (WPPC) with B15% fat content and 50
70% protein were evaluated at 1 and 2% of ice cream formulation to replace synthetic emulsifiers, polysorbate 80, and mono- and di-glycerides (Levin, Burrington, & Hartel, 2016). The WPPCbased ice creams showed slight differences in viscosity, flow index, and yield stress and had a lower overrun than the control ice creams. It was concluded that WPPC is unable to reproduce the identical attributes contributed by synthetic emulsifiers in ice cream, but did show a small increase in partially coalesced fat comparable to a number of commercial products. Making ice creams with higher protein content seems possible following the work of Patel, Baer, and Acharya (2006) in which the mix protein content was increased by either 30, 60, or 90% using either WPC82 or milk protein concentrate (MPC70) in formulations where emulsifiers and stabilizers were omitted. Satisfactory quality ice creams were made with a 30% higher protein (B5.0%) supplement from WPC82, while it was possible to go to 60% higher protein (B6.0%) when using MPC70.

9.5

CHEESE

The opportunity to increase cheese yield is a regular motivation for greater incorporation of whey proteins during cheesemaking. The benefits are immediate in the case of large-scale natural cheese manufacturing plants where

9.5 Cheese

incremental increases in cheese yields are quickly reflected in higher financial returns per unit of milk solids processed and improved utilization of manufacturing plant. Incorporating more whey proteins in cheese may be summarized according to cheese category (1) fresh cheeses, (2) natural cheeses, and (3) processed cheeses. Several modes of incorporation are possible, e.g., (1) in situ incorporation of whey proteins during the course of cheese milk UF and preconcentration, (2) addition of whey proteins recovered from whey in either native or denatured forms to cheesemilk during processing, and (3) addition of MWP as fat replacer to cheesemilk (Hinrichs, 2001). In general, fresh and high moisture natural cheeses (e.g., Feta and other cast cheeses) have been suitable candidates for added whey protein incorporation without unduly compromising their unique characteristics. In the case of fresh cheese, Thermoquarg relies on heat-induced complexation of whey proteins with casein prior to fermentation and centrifugal separation, while whey protein retention during UF of fermented milk offers an alternative processing approach. The higher levels of UF concentration of milk required when adapting the manufacturing protocols of semihard and hard cheeses results in high retentate viscosity and resulting challenges during subsequent cheese curd formation and handling. In addition, the retained whey proteins are likely to affect cheese structure and flavor in these varieties.

9.5.1 The Contribution of Commercial Whey Protein-Based Fat Replacers The initial quest for incorporating native whey proteins in cheese owed much to the technologically driven developments in UF membranes and membrane processes during the last quarter of the 20th century. However, it soon became apparent that these developments could not be readily adapted to semihard and hard cheese varieties without compromising quality. The effects of incorporating MWP into reduced fat cheeses have been documented for a quite a number of varieties ever since Simplesse- and Dairy-Lobased MWP became commercially available, e.g., Mozzarella (McMahon, Alleyne, Fife, & Oberg, 1996), Kashar (Koca & Metin, 2004; Sahan, Yasar, Hayaloglu, Karaca, & Kaya, 2008), Gouda-type (Schenkel, Samudrala, & Hinrichs, 2013), and Cheddar (Aryana & Haque, 2001). The addition of Simplesse to cheese milk improves the texture of reduced-fat hard to semihard cheeses such as Cheddar, Gouda, Mozzarella, and Kashar (as reflected by the reduced fracture stress, fracture strain or hardness) and has little influence on flavor (Fenelon & Guinee, 1997; Koca & Metin, 2004; Lucey & Gorry, 1994; Sahan et al., 2008). Simplesse significantly reduced the hardness, gumminess, cohesiveness, and springiness of reduced- and low-fat

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Gouda cheeses where the moisture contents of the corresponding pairs of control- and Simplesse-containing cheeses were equal (Schenkel et al., 2013). Dairy-Los has also been found to increase moisture content and reduce hardness (Fenelon & Guinee, 1997), but the extent of softening is less than that reported for Simplesses (Koca & Metin, 2004). The sensory properties of low-fat white pickled cheeses were adversely affected by the use of fat replacers during cheesemaking, while rheologically all the low-fat cheeses containing fat replacers were considered different from the full-fat cheese (Kavas, Oysun, Kinik, & Uysal, 2004). However, no cheese off-flavor or bitterness was noted. Furthermore, since natural cheeses represent live biological processes involving microbiological and biochemical changes taking place during the course or ripening, much attention is given to the microstructural changes underpinning evolving texture developments. Transmission electron microscopy microstructure images of reduced-fat Cheddar containing Simplesses 100 indicate that the microparticulated whey protein acts as a noninteracting filler in the cheese matrix during the formation of a filled gel, thus mimicking the structural properties of fat (Mackey & Desai, 1995). It appears that Simplesse retained its spherical particles within a relatively narrow size distribution (0.5
1.0 μm diameter) in the serum channels between the protein fibers of reduced-fat Mozzarella (McMahon et al., 1996). The resulting improvement in cheese texture coincided with an increase in moisture content of Simplessecontaining cheese, and was attributed to the combined effects of increased water binding by the whey proteins per se and the impedance to shrinkage of the para-casein network by the occluded whey protein particles. Natural cheeses intended for cooking and prepared consumer food applications demand satisfactory melt characteristics as represented by flowability indices. In the case of low-fat (4
5% w/w) Mozzarella cheese, Simplesses D100 (53% w/w, protein) and Dairy-Los (35% protein), added at levels of 0.06 and 0.23% w/w resulted in higher moisture contents than the control low-fat cheese (e.g., 55.3% w/w vs 53.0% w/w) but did not significantly affect the flowability or apparent viscosity of the melted cheese over a 28-day ripening period (McMahon et al., 1996). The flowability of all reduced-fat (B7% w/w) Kashar (Turkish semihard cheese), including those variants that contained 1% w/w Simplesses D100 or Dairy-Los added to the original cheesemilk, was markedly inferior to that of the full-fat cheese equivalents (Koca & Metin, 2004). Interestingly, comparable heat-induced flowability between the reduced-fat Kashar containing MWP and the control reduced-fat cheese was achieved over a 90-day ripening period even though the MWPcontaining cheeses had 2%
4% higher moisture. A subsequent study on Kashar (Sahan et al., 2008) found that reduced-fat (13.5% fat-in-dry matter (FDM)) Kasher cheese containing added Simplesses D100 (1% w/w addition to the cheesemilk) had significantly increased heat-induced flowability

9.5 Cheese

compared to the control low-fat version (17.5% FDM) where the moisture contents of both the Simplesse-containing and control cheeses were similar (B55.5%). However, the flow of both reduced-fat cheeses was significantly lower than that of the control full-fat cheese (43% FDM). Overall, Guinee’s (2016) review of the relevant literature concluded that addition of WPCs as a potential means of improving the texture and cooking properties of reduced-fat cheeses is marked by inconsistencies in results; they relate, in particular, to the characterization of cooking properties, e.g., flowability as measured by modifications of the Schreiber and Price-Olson methods, apparent viscosity, temperature related changes in maximum loss tangent (LTmax), and sol-to-gel transition temperature. Guinee (2016) draws attention to underlying interstudy discrepancies such as the level of whey protein added, the type of whey protein treatment (e.g., microparticulated or not), point in the process at which the whey proteins are added (before or after pasteurization), degree of heat treatment of the milk, differences between control and treatment cheeses (in moisture, fat, protein, and pH), method of evaluation of cheese properties, and other factors. Few of the studies provide data on calcium content of the cheeses, despite extending the set-to-cut times and curd-treatment times prior to whey separation and variation in the calcium-to-casein ratio, which has been shown to have a major impact on cooking properties. Similar gross compositions and levels of primary proteolysis in full-fat-, reduced-fat-, and low-fat- (LFC) Gouda-type cheeses made with 1% added Simplesse 100 and without (controls) were obtained (Schenkel et al., 2013). The Simplesses D100-containing cheeses did not become any more fluid than the corresponding control cheeses on heating to 80 C, but acquired their fluidity at lower temperatures. The authors concluded that whey protein microparticles act as inert fillers in the cheese matrix and behave as spherical barriers between the casein strands. Consequently, they reduce the number of interactions between the caseins and, thereby, facilitate easier thermal-induced displacement of the para-casein network. Six-month old ripened low-fat Cheddar cheeses made with or without the addition of Simplesse and Dairy Los had a rippled surface microstructure, in contrast to the undulated and rough surface microstructure caused by the inclusion of carbohydrate-based fat replacers (Aryana & Haque, 2001). Void areas observed around the Simplesse particles are believed to contribute to Cheddar softening and reduce the number of layers that could conceivably resist crushing. Hence, Punidadas et al. (1999) found that high pressure homogenization (and microparticulation) of a denatured whey protein dispersion (obtained by pH adjustment of whey to 4.6, high heat treatment, and sedimentation) prior to addition to cheese milk (at a level of B0.01%, w/w protein) significantly improved the meltability of reduced-fat Mozzarella-style cheese.

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9.5.2

Denatured Whey Protein Concentrates

Whey protein preparations with varying degrees of denaturation may be in formats other than microparticulates such as Simplesse or Dairy-Lo. Traditional precipitation-based processes such as Centriwhey and Lactal typically generate partially denatured WPCs (PDWPCs). Varying results have been obtained with the addition of PDWPC to milk for the manufacture of hard or semihard cheese, such as Cheddar and Gouda. There is general agreement that the addition of PDWPC increases moisture content, actual yield, and moisture-adjusted yield, with the extent of the increase being correlated positively with the degree of denaturation of the added PDWPC (Baldwin et al., 1986; Banks & Muir, 1985; Brown & Ernstrom, 1982; van den Berg, 1979). However, the addition of PDWPC has, generally, been found to cause defective body (greasy, soft) and flavor (unclean, astringent) characteristics in Gouda and Cheddar cheese (van den Berg, 1979), with the intensity of the defects becoming more pronounced with increasing level of the PDWPC added. It has been suggested that these defects may be a consequence of large sized whey protein particles (aggregates) not fitting compactly within pores so that shrinkage of the para-casein matrix is impeded (van den Berg, 1979) and relatively large increases in moisture content result. The question of whether denatured whey protein particles act as interacting or noninteracting fillers is important in the context of whether such aggregates interact with renneted casein micelles and impair gel properties. Thus, Perreault, Turcotte, Morin, Pouliot, and Britten (2016) heat-denatured WPC according to a proprietary process (Agropur, Canada) followed by spray drying to create a denatured WPC (DWPC) containing 56% protein with 77% denaturation (based on protein insolubility at pH 4.6). In the follow-on study, the experimental DWPC ingredient was added to cheese milk to examine the combined effect of two fillers, fat globules (varying cheese milk fat levels from 0.1% to 3.4%) and whey protein particles (fortification with DWPC from 0.0 to 0.75% in the nonfat fraction), on the properties of rennet gels (Perreault et al., 2016). The reconstituted DWPC was firstly homogenized during hydration in order to restore the particle size characteristics of the dispersion as closely as possible to those of its original fresh ingredient, i.e., [d4,3] 56.38 μm and d (v, 0.9) # 12.10 μm. In contrast, a milk protein isolate powder dispersion used for comparison in the same study had the following particle size profile: [d4,3] # 1.2 μm and d (v, 0.9) # 0.3 μm. The voluminosity of denatured whey proteins (estimated at 5.2 mL/g) was comparable with that for casein micelles (4
4.5 mL/g). However, the authors found that increasing DWPC in milk negatively affected the coagulation properties and contraction capacity of rennet gels leading to the conclusion that the behavior of DWPC is different to globular milk fat’s noninteracting filler role. One interesting outcome from the study was that a

9.5 Cheese

high protein cheese milk generated a para-casein network capable of supporting a higher rate of substitution of denatured whey proteins for milk proteins, thus contributing to higher cheese yield at the higher levels of DWPC and milk fat inclusion. In further studies (Perreault et al., 2017), a pressing-by-centrifugation method was developed to produce cheese samples with the same solids composition but variable moisture contents. Thus, with the aid of this technique the authors were able to separate the direct contribution of the DWPC to cheese rheological properties from the contribution of moisture. Hence, when similar cheese moisture contents were obtained by modulating the pressing conditions, the contribution of the DWPC proteins to cheese rheological properties proved to be similar to the contribution of caseins. The same authors explored the effects of different fractions of their DWPC to show that MWP appear to act as active fillers that confer some rigidity in pressed cheeses so that MWP as an ingredient is partially capable of replacing casein as well as functioning as a fat replacer.

9.5.3

Protein Cross-Linking and Whey Protein Incorporation

At low levels of addition, unlike with casein fractions, transglutaminase generally does not affect whey proteins in unheated cheese milk. However, it has been shown that the effects of transglutaminase-induced cross-linking in milk prior to cheesemaking can decrease the amount of protein released to whey (Cozzolino et al., 2003). Moreover, when further amounts of whey were added to the milk during the manufacturing process in the presence of TGase, whey protein-enriched dairy products could also be obtained. Thus, it is felt that such technology provides an opportunity for developing cheeses with elevated whey protein concentration and novel textures. TGase-catalyzed (40 U/g whey proteins) cross-linking of whey protein in whey at 40 C for 60 minutes at pH 5.0 also benefited subsequent recovery during UF (Wen-qiong, Lan-wei, Xue, & Yi, 2017). Among the observations were a recovery rate increase of 15
20%, lactose rejection rate decrease by 10%, and relative permeate flux increase of 30
40% compared to the control whey. The improved performance was attributed to improvements in the membrane fouling dynamics, e.g., decreased total resistance and cake resistance following enzyme catalysis.

9.5.4 Incorporation of Whey Proteins Into Pasteurized Processed Cheese Products and Analogue Cheese Products Processed cheese as a product evolved from the transformation of natural cheeses by melting and pasteurization with the aid of emulsifying salts (ES) before filling into molds and packaging. Processed cheeses have a distinct

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milder flavor profile to that of natural cheeses, which frequently appeals to consumers encountering the taste of cheese for the first time. Logistically, processed cheeses have a longer shelf life by virtue of tighter quality assurance protocols surrounding their pasteurization, hot-filling and packaging. In addition, processed cheeses are produced in a variety of textures such as blocks, slices, spreads, and dips that add taste and convenience to food preparations. In general, processed cheese or processed cheese products (PCPs) are categorized as incorporating the highest (up to 80% possible) level of natural cheese in their formulations. Accordingly, as the amount of natural cheese is reduced in PCP formulations due to replacement with other ingredients, the resulting products may need to be redefined in accordance with relevant national legislation as summarized by Guinee (2016). Examples according to the Food and Drugs Administration
FDA (2012) include pasteurized processed cheese, pasteurized processed cheese spread, and pasteurized processed cheese food. The type and level of optional ingredients permitted are determined by the product type, and include dairy ingredients, vegetables, meats, stabilizers, ES, flavors, colors, preservatives, and water. A minimum cheese content of 51% (w/w) of the final product is required in pasteurized processed cheese foods and spreads, in which non-cheese ingredients (e.g., dairy ingredients) may be used at levels up to B15% depending on the composition of the PCP (Guinee, 2016). In contrast, analogue cheese products (ACPs) generally contain no added cheese, except where small amounts are added to impart a cheese flavor or as required by customer specifications. Similar to PCPs, ACPs contain added stabilizers, ES, flavors, colors, preservatives, and water. ACPs may be categorized arbitrarily as dairy, partial dairy, or nondairy, depending on whether the fat and/or protein components are from dairy or vegetable sources (Shaw, 1984). Partial dairy analogues employing vegetable oil as fat source (e.g., soy oil, palm oil, rapeseed, and their hydrogenated equivalents) and dairy-based proteins (usually rennet casein and caseinate) are commonly formulated with ES and water in a manner similar to PCPs. As the proportion of natural cheese is reduced in formulations, the opportunity is presented to incorporate more milk protein ingredients (e.g., rennet casein, acid casein, caseinates, whey proteins) in ACPs and PCPs in accordance with customer specification and conformance with prevailing legislation. Product innovation in these cheese categories relies substantially on the availability of emerging novel ingredients to create new textures or just simply facilitate least-cost formulation objectives by virtue of enhanced functionality of added proteins; these can have a marked influence on the physicochemical and rheological properties, stability, and usage appeal characteristics of pasteurized PCPs and ACPs (Abou El-Nour, Schurer, Omar, & Buchheim, 1996;

9.6 Conclusions

Guinee, 2009; O’Riordan, Duggan, O’Sullivan, & Noronha, 2011; Savello, Ernstrom, & Kaláb, 1989). Regular sweet whey powder with B12
15% protein is used widely in PCPs as a cost-effective filler to impart a mild sweet taste and a smooth consistency, especially desirable in highly processed cheese spreads and dips (Guinee, 2016). WPCs and WPIs are less used due to their relatively high cost, hence their inclusion may be to address specific functions such as (1) controlled melting of PCPs distributed in meat-based products, and (2) enhancing stiffness and viscosity of high-moisture spreadable PCPs. The effects of added whey proteins on the texture and cooking characteristics of PCPs is associated with a concentration-dependent loss of flowability irrespective of degree of denaturation (French, Lee, DeCastro, & Harper, 2002; Gupta & Reuter, 1993; Hill & Smith, 1992; Kaminarides & Stachtiaris, 2002; Mleko & Foegeding, 2000, 2001; Mounsey, O’Kennedy, & Kelly, 2007; Savello et al., 1989). The functional behavior of processed cheese with added whey proteins during heating is likened to a two-component system (Mleko & Foegeding, 2001) involving a melting casein network and nonmelting whey proteins. Hence, there is a focus on the capacity of whey protein particles/ aggregates to interact and form larger particles or a network in the processed cheese environment at typical cooking temperatures (80
100 C) during manufacture or later at the point of food service. A high calcium content and relatively low pH (B # 6.0) of the processed cheese environment is conducive to a high degree of interaction of heat-denatured whey proteins by covalent (disulfide), hydrophobic, and electrostatic interactions. Coprecipitated casein 2 whey protein ingredients provide an adventitious approach to the incorporation of whey protein when such protein complexes are used in place of regular casein/caseinates. Once again, the control of heat-induced denaturation and aggregation of whey protein with respect to the size/gelation capacity of the resultant reaction products is pertinent to the functionality of casein 2 whey protein coprecipitates (CWPCPs) (Donato & Guyomarc’h, 2009; Mleko & Foegeding, 1999). Mounsey et al. (2007) varied the coprecipitation conditions, e.g., pH of skim milk (9.5, 7.5, 3.5), before heating (90 C 3 20 min) and prior to reacidification to pH 4.6 on the performance of the resultant liquid CWPCPs during cooking of model PCPs. Meltability and fluidity of PCPs improved significantly as the pH of the skim milk at heating was increased. Conversely, reducing the pH at heating to 3.5 had the opposite effect. Substitution of acid-casein powder with CWPCPs in a PCP formulation at a level of B8% contributed 1.0
1.4% whey protein.

9.6

CONCLUSIONS

Whey proteins are versatile food ingredients which are utilized for both nutritional and functional reasons. Both attributes are frequently demanded

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at the same time in several applications, e.g., in the case of performance nutrition beverages, whey protein-enriched ingredients such as WPI may be required to satisfy solubility demands not only at low pH, but also during subsequent formulation and thermal processing. For many years, the heat-labile nature of whey proteins led to judgements of their functionality based on the status of their native and nonnative (unfolded/denatured) states. Considerable progress has been made in recent years into understanding and exploiting the microstructural changes that are possible with whey proteins. Heat-induced aggregation occurring during whey protein unfolding/denaturation may be adapted to create soluble aggregates which are versatile when formulating heat-stable protein-based beverages. A related physical structure in the form of microparticles generated by microparticulation enables whey proteins to simulate the textural attributes of fat and, thus, act as a fat replacer in dairy and other food applications. Modification of the conditions under which whey microparticulation takes places further influences functionality such as enhanced emulsification capacity for application in high-protein beverages, microparticle surface reactivity in relation to yogurt graininess, and the interrelationship between native and denatured proteins. Related studies when reducing the fat content of cheese are shedding some light on the extent to which whey protein microparticles act as inert fillers like fat in the cheese matrix.

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whey protein concentrate, Code 349. http://www. [accessed foremostfarms.com/Commercial/pdfs/Specifications/TDS_Daritek_HS_349.pdf 06.08.2017]. Anon. (2015) Whey protein concentrate 80%
heat stable https://daviscofoods.com/docs/2015/ Whey%20Protein%20Concentrate%2080%20Heat%20Stable.pdf [accessed 06.08.2017]. Astbury, N. M., Taylor, M. A., French, S. J., & Macdonald, I. A. (2014). Snacks containing whey protein and polydextrose induce a sustained reduction in daily energy intake over 2 weeks under free-living conditions. American Journal of Clinical Nutrition, 99, 1131
1140. Aryana, K. J., & Haque, Z. U. (2001). Effect of commercial fat replacers on the microstructure of low-fat Cheddar cheese. International Journal of Food Science & Technology, 36, 169
177. Baldwin, K. A., Baer, R. J., Parsons, J. G., Seas, S. W., Spurgeon, K. R., & Torrey, J. S. (1986). Evaluation of yield and quality of cheddar cheese manufactured from milk with added whey protein concentrate. Journal of Dairy Science, 69, 2543
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Flavor Aspects of Whey Protein Ingredients Mark Stout and MaryAnne Drake North Carolina State University, Raleigh, NC, United States

10.1

INTRODUCTION

Whey protein products are primarily added to foods as an ingredient for their functional and nutritional properties (Kosikowski & Mistry, 1997). However, flavor remains a primary driver of whey protein ingredient sales (Drake, Miracle, & Wright, 2009; Morr & Ha, 1991; Russell, Drake, & Gerard, 2006). Common off-flavors in whey protein ingredients may be noticeable and affect the flavor of final products (Carunchia Whetstine, Croissant, & Drake, 2005; Drake, 2006; Evans, Zulewska, Newbold, Drake, & Barbano, 2010; Oltman, Lopetcharat, Bastian, & Drake, 2015; Wright, Zevchak, Wright, & Drake, 2009). Whey protein ingredients should ideally have a bland flavor (Childs, Yates, & Drake, 2007; Croissant, Kang, Campbell, Bastian, & Drake, 2009). Pure undegraded proteins should theoretically be flavorless, but no dried ingredients are pure proteins. Fat, carbohydrate, ash, and other trace compounds as well as unit operations in the process of manufacturing can dramatically influence the flavor of whey protein ingredients through lipid oxidation and degradation of protein amino acid side groups (Drake et al., 2009). Lipid oxidation can cause volatile off-flavors through aldehyde and ketone formation and oxidation of sulfur-containing amino acid side groups (Carunchia Whetstine et al., 2005; Wright, Whetstine, Miracle, & Drake, 2006). Carbohydrates in the presence of protein and heat can cause Maillard reaction products (Whitfield, 1992). Protein proteolysis or side group reactions can cause bitter and metallic tastes, as well as flavor-active volatile compounds which further impact flavor (Campbell, Miracle, Gerard, & Drake, 2011a; Leksrisompong, Gerard, Lopetcharat, & Drake, 2012; Wright et al., 2006). Understanding the parameters that influence flavor generation in whey protein ingredients can help mitigate off-flavors. Many of the reactions related to off-flavors are encouraged by heat, oxygen exposure, and presence of chemical oxidizing agents (Carunchia Whetstine, Parker, Drake, & Larick, Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00011-4 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 10.1 Flow diagram of general whey processing from raw milk to powdered whey ingredients. Adapted from Bylund, G. (2003). Dairy processing handbook (2nd ed.). Lund, Sweden: Tetra Pak Processing Systems AB.

2003; Jervis et al., 2012). Many off-flavors in whey protein ingredients largely originate from processing and handling of milk and whey (Park, Stout, & Drake, 2016a; Singh, Cadwallader, & Drake, 2006). Processing variables such as pasteurization, separation, bleaching, spray drying, and storage all influence flavor development in dried whey protein ingredients (Fig. 10.1) (Jervis et al., 2012; Liaw, Eshpari, Tong, & Drake, 2010; Park & Drake, 2014; Smith, Gerard, & Drake, 2015). The objective of this chapter is to address the current research on whey protein ingredient flavors and the influence of processing and handling of whey on flavor generation.

10.2

SENSORY ANALYSIS

Sensory science analyzes all aspects of food or other materials which are perceived by human senses (Drake, 2007). A full understanding of the flavor

10.3 Origin of Flavors in Whey

profile, as well as consumer perception of a product is critical for maintaining a competitive edge in food-related markets (Drake, 2007). Many objective and subjective sensory tests exist which can be applied to dairy products (Drake et al., 2009; Lawless & Heymann, 1999; Meilgaard, Civille, & Carr, 2007). These tests fall into two major categories: analytical tests and affective tests (Lawless & Heymann, 1999). Analytical sensory tests provide objective results, utilizing trained or screened panelists. This category of tests includes descriptive analysis and discrimination testing. Affective tests use untrained consumers to provide subjective data (Lawless & Heymann, 1999). In these tests, participants often indicate preference or degree of liking (Meilgaard et al., 2007). Off-flavors are determined by consumers and are therefore determined by affective testing. The appropriate sensory test must be determined prior to data collection or meaningful conclusions cannot be made (Drake, 2007). Sensory attributes of fluid whey and dried whey proteins may be described using standardized terms and can be documented by trained panelists (Table 10.1). These attributes and their intensities are typically applied to rehydrated powders (10% w/v) or directly to fluid whey by a trained descriptive panel. More concentrated liquids are generally diluted to 10% solids (w/v). The application of a standardized and clearly defined sensory language provides the platform for understanding whey protein flavor, identifying flavor sources, and relating to consumer perception.

10.3

ORIGIN OF FLAVORS IN WHEY

Flavors in fluid and dried whey ingredients can be grouped into two categories, dairy and nondairy flavors (Carunchia Whetstine et al., 2005). Whey products are often described as having dairy-like flavors such as sweet aromatic, buttery, and cooked/milky notes (Carunchia Whetstine et al., 2003; Singh et al., 2006; Smith, Smith, & Drake, 2016b). These dairy flavors develop during heat processing of milk (Contarini, Povolo, Leardi, & Toppino, 1997; Singh et al., 2006). Buttery flavors can originate from starter cultures, as many cheese cultures naturally produce diacetyl (Campbell et al., 2011a; Carunchia Whetstine et al., 2005). However, other flavors are present in whey and whey ingredients which are not typically associated with dairy products, such as cardboard, wet dog, grass, and cabbage (Carunchia Whetstine et al., 2005; Evans et al., 2010; Gallardo-Escamilla, Kelly, & Delahunty, 2005a; KaragülYüceer, Drake, & Cadwallader, 2003; Whitson, Miracle, & Drake, 2010). These nondairy flavors are generally considered off-flavors (Carunchia Whetstine et al., 2003; Drake, 2006; Marsili, 2003). Although several chemical processes can be involved in the development of these off-flavors, lipid oxidation is the primary cause of off-flavors in fluid whey and dried milk and whey ingredients (Carunchia Whetstine et al., 2003, 2005; McClements & Decker, 2008;

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Table 10.1 Sensory Language for Descriptive Analysis of Fluid Whey and Whey Proteins Term

Definition

Overall aroma intensity Sweet aromatic

The total orthonasal aroma impact

Buttery/diacetyl

Sweet aromatics associated with dairy products Sour aromatics associated with dairy fermentation Aromatic associated with diacetyl

Cooked/milky

Aromatics associated with cooked milk

Cardboard/wet paper Cabbage brothy

Aromatics associated with wet cardboard or paper Aromatics associated with boiled cabbage

Potato brothy

Aromatics associated with either broth or boiled potatoes Aromatics associated with freshly sliced cucumber Aromatics associated with dried grasses

Sour aromatic

Cucumber Grassy/hay Doughy

Aromatics associated with canned biscuit dough

Fried fatty/ painty Pasta water/ cereal Metallic/serumy

Aromatics associated with old frying oil and lipid oxidation products Aromatics associated with water after pasta has been boiled in it or oatmeal Aromatic associated with metals or with juices of raw or rare beef Aromatics associated with wet dog hair

Animal/wet dog Cowy/barny Soapy Bitter Astringency

Aromatics associated with cow feces and urine Aromatics associated with soap Basic taste associated with bitterness Chemical feeling factor characterized by a drying or puckering of the oral tissues

Reference

Vanillin in milk Cultured sour cream Diacetyl Cooked skim milk Cardboard paper Boiled cabbage

Methional (E)-2-nonenal Alfalfa or grass hay (Z)-4-heptenal

2,4-decadienal Boiled pasta or plain boiled oats Juices from seared beef Knox brand gelatin p-cresol Lauric acid Caffeine Alum

Example/preparation Evaluated as the lid is removed from the cupped sample Vanilla cake mix or 20 mg/kg vanillin in milk

1 mg/kg diacetyl onto filter paper strips in 125 mL sniff jar Heating skim milk to 85 C for 30 min Brown paper bag cut into strips and soaked in water Cabbage leaf boiled in 500 mL water for 5 min, 1 ppb dimethyl trisulfide 1 mg/kg methional in water or canned potatoes 1 mg/kg (E)-2-nonenal or freshly sliced cucumbers

1 mg/kg (Z)-4-heptenal, canned biscuit dough, or cooked pasta water Old (stored) vegetable oil Pasta boiled in water for 30 min Aroma of fresh raw beef steak or juices from seared beef steak One bag of gelatin (28 g) dissolved in two cups of distilled water 20 mg/kg p-cresol in skim milk 1 mg/kg lauric acid or shaved bar soap Caffeine, 0.5% in water Alum, 1% in water

Adapted from Carunchia Whetstine, M.E., Parker, J.D., Drake, M.A., & Larick, D.K. (2003). Determining flavor and flavor variability in commercially produced liquid Cheddar whey. Journal of Dairy Science, 86, 439 448; Drake, M.A., Karagul-Yuceer, Y., Cadwallader, K.R., Civille, G.V., & Tong, P.S. (2003). Determination of the sensory attributes of dried milk powders and dairy ingredients. Journal of Sensory Studies, 18, 199 208; Karagul-Yuceer Y.K., Vlahovich, K., Drake, M.A., & Cadwallader, K.R. (2003). Characteristic Aroma Components of Rennet Casein. J. Agric. Food Chem. 51:6797 6801; Drake, M.A., Miracle, R.E., & Wright, J.M. (2009). Sensory properties of dairy proteins. In A. Thompson, M. Boland, & H. Singh (Eds.) Milk proteins: From expression to food (pp. 429 448). San Diego, CA: Academic Press; Kussy, D., & Aylward, E. (2009). Pasteurized process cheese. In S. Clark, M. Costello, M.A. Drake, & F. Bodyfelt (Eds.), The sensory evaluation of dairy products (pp. 387 401). New York, NY: Springer; Smith, T.J., Campbell, R.E., & Drake, M.A. (2016a). Sensory properties of milk protein ingredients. In P.L.H. McSweeney & J.A. O’Mahony (Eds.) Advanced dairy chemistry Vol. 1B: Proteins: Applied aspects (4th ed.) (pp. 197 223). London: Springer.

10.3 Origin of Flavors in Whey

Smith, Campbell, Jo, & Drake, 2016c). Lipid oxidation produces volatile compounds such as aldehydes, ketones, and short-chain fatty acids which remain in the whey during processing and are present in spray-dried powders. Free radicals from lipid oxidation can also attack protein amino acid side groups and create other flavor-active degradation products (Gallardo-Escamilla, Kelly, & Delahunty, 2005b; Wright et al., 2006). Thus lipid oxidation of the milk, liquid whey, or concentrated and fractionated whey during processing may cause off-flavors in the final ingredient and negatively influence consumer liking (Carunchia Whetstine et al., 2005; Drake et al., 2009; Whitson, Miracle, Bastian, & Drake, 2011; Wright et al., 2009). Volatile lipid oxidation compounds are present in fresh fluid milk and whey, but their concentrations increase due to heat, storage, and other processing steps which increase oxidation (Gallardo-Escamilla et al., 2005b; Huffman, 1996; Jervis et al., 2012; Kelly, Kelly, & Harrington, 2002; Park, Parker, & Drake, 2016b). Understanding oxidation is essential in understanding off-flavor formation in whey products (McClements & Decker, 2008). Peroxidation is a process by which molecular oxygen reacts directly with organic compounds, affecting conformation and structure (Frankel, 1998). Free radicals, which are the most common reactive species in autoxidation, are unpaired electrons which can cause hydrogen abstraction in lipids. As a biradical, oxygen contains two unbound electrons, which facilitates the transfer of free radicals onto an organic system causing oxidation through hydrogen abstraction (Frankel, 1998). The lipid oxidation process is divided into three sections, initiation, propagation, and termination. During initiation, lipids lose a hydrogen radical, forming a lipid containing an alkyl radical. This free radical is stabilized across the double bond structure (McClements & Decker, 2008). During propagation, the fatty acid containing a free radical reacts with molecular oxygen forming peroxyl radicals (LOO ). Because molecular oxygen contains two radicals, this excess electron density attracts a hydrogen atom forming a hydroperoxide (LOOH) and passes a free radical on to another molecule (McClements & Decker, 2008). During termination, two hydroperoxides interact with each other forming an unstable tetroxide which quickly degrades into a nonradical product (Frankel, 1998). In fluid whey, dried proteins, and dried whey protein ingredients, compounds associated with off-flavors are likely to develop from the oxidation of unsaturated oleic, linoleic, and linolenic acids (Frankel, 1998). Oleic, linoleic, and linolenic acids represent 32.5%, 2.5%, and 0.6% of the total fatty acids of the fat in liquid Cheddar whey, respectively (Tomaino, Turner, & Larick, 2004). This oxidation process begins in fluid milk and can be encouraged through the cheesemaking process. Campbell et al. (2011a) observed that cheeses which used a starter culture encouraged further lipid oxidation in the whey

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stream. By the time fresh fluid whey is formed and drained from the cheese vat, lipid oxidation products are present (Carunchia Whetstine et al., 2003).

10.4

FLUID MILK

The first factor that affects the flavor of whey protein ingredients is the flavor of milk (Fig. 10.2; Drake et al., 2009). As milk is stored, autoxidation, lipolysis, and proteolysis occur, resulting in the formation of volatile compounds that can be flavor active (Singh et al., 2006). Autoxidation of unsaturated fatty acids can cause the formation of many of the same volatile compounds which cause off-flavors in whey, such as aldehydes, alcohols, and esters (Bendall, 2001; Croissant, Washburn, Dean, & Drake, 2007). Lipolysis and proteolysis from microbial growth can result in free fatty acid release and production of flavor precursors from proteolysis, which may cause off-flavors in whey (Brennand, Ha, & Lindsay, 1989; Singh et al., 2006). As such, microbial quality of raw milk influences milk flavor which impacts whey flavor. Although many of these off-flavor compounds are present in fresh milk, they increase with storage time and temperature (Bassette, Fung, Mantha, & Marth., 1986; Lee, Barbano, & Drake, 2016). Storage and microbial quality are not the only factors that influence flavor formation in fluid milk. The composition of a cow’s diet also influences the flavor of the milk (Bendall, 2001; Park, Armitt, & Star, 1969). There is a distinct

FIGURE 10.2 Trained sensory panel profiles of 1.5% fat pasteurized milk from pasture-based and total mixed ration (TMR) feeding. Attributes were scored using a universal 0 15-point intensity scale. Adapted from Croissant, A.E., Washburn, S.P., Dean, L.L., & Drake, M.A. (2007). Chemical properties and consumer perception of fluid milk from conventional and pasture-based production systems. Journal of Dairy Science, 90, 4942 4953.

10.5 Flavor Aspects of Liquid Whey

difference in flavor between milk from pasture fed cows and from traditional total mixed ration (TMR) fed cows (Bendall, 2001; Croissant et al., 2007). Grassy and mothball flavors have been documented in milk, cheeses, dried milk, and whey protein ingredients from milk from cows fed a pasture-based diet (Bugaud, Suchin, Coulon, Hauway, & Dupont, 2001; Drake et al., 2005; Drake, 2004; Kelly, Kolver, Bauman, van Amburgh, & Muller, 1998; Khanal et al., 2005). Bendall (2001) demonstrated that most aroma active compounds present in milk were found in milk from both pasture-fed cows and TMR-fed cows, but the concentration of specific volatile compounds accounted for the observed differences in the flavor of pasture-fed milk and TMR milk. One of the most dramatic differences between volatiles of pasture-fed milk and TMR fed milk was increased concentrations of skatole and indole, which are likely components of grassy/mothball flavor (Bendall, 2001; Croissant et al., 2007; Drake et al., 2005). Pasture-based diets are less energy-dense and contain more L-tryptophan than TMR diets, which encourages gluconeogenesis of L-tryptophan. This process in turn increases the concentration of skatole in milk from pasture-fed cows (Bendall, 2001; Croissant et al., 2007). These compounds and subsequent grassy and mothball flavors are present in pasture-fed milk and will also be present in the resulting whey stream. Heat treatments promote autoxidation of unsaturated fatty acids in milk (Contarini et al., 1997). Concentrations of many volatile aldehydes, ketones, and alcohols increase due to pasteurization time and temperature (Jousse, Jongen, Agterof, Russell, & Braat, 2002; Vazquez-Landezverde, Torres, & Qian, 2006). Heating can also encourage the release of sulfur compounds from protein, encouraging formation of volatile sulfhydryl compounds such as methanethiol and dimethyl trisulfide, causing intense cooked, sulfurous, and eggy flavors (Contarini et al., 1997; Friedman, 1996; Lee, Barbano, & Drake, 2017; Lee, Lo, & Warthesen, 1996). Caramelized flavors develop in fluid milk subjected to heat treatments greater than traditional HTST pasteurization, due to sugar degradation through Maillard reactions (Calvo & de la Hoz, 1992; Lee et al., 1996). This process leads to the formation of pyrazines, furans, and other Maillard products which impart cooked, malty, and brown flavors (Singh et al., 2006). These volatile compounds will also be present in fluid whey and dried whey ingredients from heat-treated milk.

10.5 10.5.1

FLAVOR ASPECTS OF LIQUID WHEY Liquid Whey

During cheese production, some carbohydrates, fat globules, proteins, minerals, and organic acids are not incorporated into the curd. These unincorporated compounds stay in the aqueous phase with the fluid whey where they can

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impact flavor and be flavor precursors (Carunchia Whetstine et al., 2003; Smith, Campbell, & Drake, 2016a). Depending on the cheese, fluid whey is either classified as sweet whey or acid whey (Drake et al., 2009; Smith et al., 2016b). Sweet whey is the by-product of cheese produced from rennet coagulation and generally has a pH of 6.0 6.4, while acid whey is the byproduct of acid-coagulated cheese and thus has a much lower pH of 4.3 4.6 (Morr & Ha, 1991. Cheese type will also impact the flavor of sweet liquid whey, as will be discussed later in this chapter (Liaw, Miracle, Jervis, Listiyani, & Drake, 2011; Smith et al., 2016b). Generally, sweet fluid whey is described as having milky and sweet aromatic flavors as well as low cardboard and metallic flavors (Carunchia Whetstine et al., 2003; Gallardo-Escamilla et al., 2005a; Lubran, Lawless, Lavin, & Acree, 2005). Acid whey generally contains higher concentrations of organic acids, and increased calcium, potassium, and iron compared to other fluid wheys (Durham, Hourigan, Sleigh, & Johnson, 1997; GallardoEscamilla et al., 2005a). The higher organic acids and minerals cause acid whey to have increased lipid oxidation and sour aromatic and cardboard flavors, as well as increased sour taste compared to sweet wheys (GallardoEscamilla et al., 2005a; Smith et al., 2016b) (Table 10.2). Fluid whey contains # 0.5% fat by weight; however this is enough for lipid oxidation to be a primary source of off-flavors (Carunchia Whetstine et al., 2003; Drake et al., 2009; Smith et al., 2016a). Many of the same aldehydes, methyl ketones, and free fatty acids that contribute to off-flavors in stored Table 10.2 Trained Sensory Panel Profiles of Fluid Cheddar Wheys Mean Intensities Descriptor

W1

W2

W3

W4

Aroma intensity Cooked/ milky Sweet aromatic Buttery Cardboard Cooked/ milky Sweet Astringent

2.0 3.0 1.5 1.0 1.4 3.0 2.0 1.5

2.2 3.3 ND ND 1.0 3.3 2.2 1.5

2.1 3.0 1.0 ND 0.8 3.0 1.7 1.9

2.5 3.0 2.5 1.4 ND 3.0 2.0 1.5

W1 W4, fluid wheys. Scores based on a universal 0 to 15-point intensity scale. ND, Not detected. Adapted from Campbell, R.E., Miracle, R.E., Gerard, P.D., & Drake, M.A. (2011a). Effects of starter culture and storage on the flavor of liquid whey. Journal of Food Science, 76, S3254 S3261; Carunchia Whetstine, M.E., Parker, J.D., Drake, M.A., & Larick, D.K. (2003). Determining flavor and flavor variability in commercially produced liquid Cheddar whey. Journal of Dairy Science, 86, 439 448; Liaw, I.W., Miracle, R.E., Jervis, S.M., Listiyani, M.A.D., & Drake, M.A. (2011). Comparison of the flavor chemistry and flavor stability of Mozzarella and Cheddar wheys. Journal of Food Science, 76, 1188 1194.

10.5 Flavor Aspects of Liquid Whey

milk are also found in fluid whey (Cadwallader & Singh, 2009; Carunchia Whetstine et al., 2003). As fluid whey is stored, more lipid oxidation occurs than in powdered whey due to increased water activity (Liaw et al., 2010, 2011; McDonough, Hargrove, & Tittsler, 1968; Park et al., 2016b). These reactions are sensitive to heat and oxygen exposure, thus autoxidation of fat must be considered at every stage of fluid whey processing and storage.

10.5.2

Chymosin and Proteolysis

The critical factor in proteolysis-related off-flavor in fluid whey is the presence of chymosin and other proteases (Carunchia Whetstine et al., 2003). Chymosin is used to coagulate the curd during cheese production and can be present at residual levels in cheese whey along with any proteases produced by the starter culture that carry over into the whey (Campbell & Drake, 2013b; Holmes, Duersch, & Ernstrom, 1977). These enzymes encourage proteolytic degradation which in turn can cause increased astringency as well as bitter and metallic tastes (Harwalker, Cholette, McKellar, & Emmons, 1993). Stevenson and Chen (1996) stated that proteins may also hydrophobically bind to volatile flavor compounds in fluid whey which may then be released when spray dried. Thus proteolysis during fluid whey processing may influence flavor of powdered products through volatile compound 2 peptide binding.

10.5.3 Cheese Type and Influence of Culture on Whey Flavor The type of starter culture used to make the cheese influences fluid whey flavor. Whey from cheese produced without culture using rennet (rennet whey) is characterized by sweet aromatic and cooked milky flavors as well as sweet taste (Campbell, Miracle, & Drake, 2011b; Smith et al., 2016c). Rennet wheys also have distinct cooked notes and will develop cardboard flavors when stored (Campbell et al., 2011a; Smith et al., 2016c). The simple flavor profile of whey can be complicated by cheese cultures which encourage oxidation, proteolysis, and lipolysis (Campbell et al., 2011a, b; El Soda, Law, Tsakalidou, & Kalantzopoulos, 1995; Liaw et al., 2010). Fatty acids found in milk are released by lipolytic enzymes from starter cultures, are oxidized and contribute to off-flavors (Cadwallader & Singh, 2009). Although free fatty acid degradation occurs naturally, starter cultures can increase the rate of free fatty acid release and degradation in milk and whey (Drake et al., 2009; Gallardo-Escamilla et al., 2005a, b). Fluid wheys produced from cultured cheeses are characterized by higher rates of lipid oxidation and related offflavors compared to rennet wheys (Campbell et al., 2011a; Drake et al., 2009; Smith, Foegeding, & Drake, 2016d).

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Gallardo-Escamilla et al. (2005b) compared the composition of the volatiles of fluid whey produced with Cheddar cheese cultures and rennet whey. The whey produced with Cheddar cheese culture had elevated volatile lipid oxidation compounds as well as increased proteolysis and lipolysis compared to the rennet whey (Gallardo-Escamilla et al., 2005b). Campbell et al. (2011a) demonstrated that after 48 hours storage at 4 C, fluid whey produced with Cheddar cheese cultures had increased cardboard and sour aromatic flavors compared to fresh fluid Cheddar wheys and fluid rennet wheys. Not all flavors related to cheese cultures are off-flavors. Many cheese cultures produce diacetyl, which contributes a buttery flavor to fluid whey (Campbell et al., 2011a; Carunchia Whetstine et al., 2003). Campbell et al. (2011a) demonstrated that different starter cultures also influenced fluid whey flavor in a different manner. Mesophilic starter cultures used for Cheddar whey caused greater lipid oxidation than thermophilic starter cultures used in Mozzarella whey (Campbell et al., 2011b; Liaw et al., 2011). As such, fresh Cheddar whey had increased cardboard flavor and decreased cooked/milky flavor compared to fresh Mozzarella whey (Campbell et al., 2011a). Although Mozzarella whey contained less lipid oxidation products than Cheddar whey, lipid oxidation still occurred. Mozzarella whey also had more oxidation compared to rennet whey, and storage of fluid Mozzarella whey reduced flavor quality (Campbell et al., 2011a; Whitson et al., 2011). Liaw et al. (2010) demonstrated that optimal fat separation and possibly the addition of an antioxidant reduced lipid oxidation in both Cheddar and Mozzarella wheys during storage. Cottage cheese curds are formed through acidification, meaning the product ferments until the pH of the milk is below the isoelectric point of casein (around pH 4.6). Fluid Cottage cheese whey is, therefore, an acid whey. Increased organic acid concentration and prolonged heat exposure cause fluid Cottage cheese whey to be characterized by acidic, sweaty, potato, oxidized, and stale flavors (Gallardo-Escamilla et al., 2005a; Smith et al., 2016b). Smith et al. (2016b) characterized fluid Cottage cheese whey by a high level of sour aromatic flavor and distinct potato/brothy flavor compared to Cheddar or rennet fluid wheys. Cottage cheese fluid whey also had much higher concentrations of diacetyl, which was likely the result of extended fermentation (Durham et al., 1997; Gallardo-Escamilla et al., 2005a; Smith et al., 2016d). The higher diacetyl concentration contributes to intense buttery flavors associated with Cottage cheese liquid whey (Smith et al., 2016d). Many of the flavor characteristics of fluid Cottage cheese whey carry over into dried protein ingredients from Cottage cheese whey. Smith et al. (2016d) reported that Cottage cheese WPI was higher in sour aromatic and potato/brothy flavors and lower in sweet aromatic flavor than Cheddar, Mozzarella, or rennet whey WPI. Cottage cheese whey is often acidified at least in part by microbial growth; acid whey can also be

10.5 Flavor Aspects of Liquid Whey

produced by direct acid addition, known as acid casein whey. Acid casein whey does not have the distinct buttery notes of Cottage cheese whey, but contains similar sour aromatic and stale flavors as well as rancid and chemical flavors and bitter taste (Gallardo-Escamilla et al., 2005a) (Figs. 10.3 and 10.4).

FIGURE 10.3 Trained panel flavor profiles of fluid whey from Mozzarella cheese, Cheddar cheese, or acid casein manufacture. Adapted from GallardoEscamilla, F.J., Kelly, A.L., & Delahunty, C.M. (2005a). Sensory characteristics and related volatile flavor compound profiles of different types of whey. Journal of Dairy Science, 88, 2689 2699.

FIGURE 10.4 Trained panel cardboard flavor intensities of fluid wheys before and after storage at 4 C. Scores based on a universal 15-point intensity scale a,b,c Means within rows with different letters are statistically different (P , .05) Adapted from Liaw, I.W., Eshpari, H., Tong, P.S., & Drake, M.A. (2010). The impact of antioxidant addition on flavor stability of Cheddar and Mozzarella whey and Cheddar whey protein concentrate. Journal of Food Science, 75, C559-C569; Smith, S., Smith, T.J., & Drake, M.A. (2016b). Flavor and flavor stability of cheese, rennet, and acid wheys. Journal of Dairy Science, 99, 3434 3444.

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10.6 10.6.1

FLUID WHEY PROCESSING Pasteurization

When fluid whey is drained from the cheese vat, cheese cultures continue to ferment and reduce the pH. Pasteurization is conducted to inactivate the starter culture, but the lactose content of fluid whey can encourage Maillard reaction products to form due to the heat (Mahajan, Goddick, & Qian, 2004; Whitfield, 1992). Although some of these Maillard reaction products contribute to cooked/milky flavors, many contribute to Strecker degradation volatile compounds (Whitfield, 1992). Strecker degradation is the process of heat-induced oxidative deamination, which forms odorous volatile compounds as well as potent intermediates in lipid oxidation (Whitfield, 1992). Lipid oxidation compounds such as methyl ketones and aldehydes also react with Maillard reaction products encouraging further development of oxidation-related flavors in fluid whey (Newton, Fairbanks, Golding, Andrewes, & Gerrard, 2012). These factors are temperature- and time-dependent, thus control over pasteurization parameters is critical in minimizing off-flavor production.

10.6.2

Separation

As might be expected, the concentration of lipids in the whey dramatically impact lipid oxidation in both fluid and powdered products. To mitigate these effects, fluid whey is subject to centrifugal fat separation, where fat concentrations reach should be # 0.5% of the total weight. Liaw et al. (2011) demonstrated that lipid oxidation in both Cheddar and Mozzarella wheys was reduced by fat separation. After 3 days of storage at 4 C, fat-separated wheys were compared to nonseparated wheys. Aldehydes and cardboard flavor were reduced in fluid fat-separated Cheddar and Mozzarella wheys compared to those which were not fat separated (Liaw et al., 2011) (Fig. 10.5).

10.7 10.7.1

COMPOSITION OF WHEY INGREDIENTS Whey Protein Concentrates and Isolates

The composition of a dried whey ingredient has a direct impact on its flavor. Products with high protein content are distinct in flavor compared to products with lower protein content. Sweet fluid whey is approximately 93.7% water, 4.8% lactose, and 0.8% protein (Kosikowski & Mistry, 1997). Water can be removed to concentrate the total solids in fluid whey. By removing water through reverse osmosis or evaporation, fluid whey can be concentrated to a product that when dried is sweet whey powder (SWP), which contains 75% lactose and 13% protein (Mahajan et al., 2004) (Fig. 10.6). The flavor of SWP

10.7 Composition of Whey Ingredients

FIGURE 10.5 Trained panel cardboard flavor intensities of Cheddar and Mozzarella liquid wheys after 72 h of storage at 3 C. Scores based on a universal 15-point intensity scalea,b,cMeans within rows with different letters are statistically different (P , .05). Adapted from Liaw, I.W., Miracle, R.E., Jervis, S.M., Listiyani, M.A.D., & Drake, M.A. (2011). Comparison of the flavor chemistry and flavor stability of Mozzarella and Cheddar wheys. Journal of Food Science, 76, 1188 1194.

is characterized by cooked/milky and oxidized flavors and sweet taste (Sithole, McDaniel, & Goddik, 2006). The high concentration of lactose in SWP increases sugar caramelization and Maillard reaction products in SWP. These reactions encourage formation of Strecker degradation compounds, furans, and pyrroles, which contribute to the predominant caramelized and cooked flavors in SWP as well as many oxidized flavors (Sithole et al., 2006). Often an ingredient is needed with a higher ratio of protein to lactose than SWP. By utilizing ultrafiltration (UF), water and lactose can be separated from the protein through size exclusion (Fig. 10.6). By this method, lactose can be separated out until 35% of the total solids is protein, creating 34% whey protein concentrate, or WPC34 (Onwulata, 2008). Additional concentration is performed by diafiltration (or the addition of deionized water to facilitate further filtration), which allows protein concentration to increase further, allowing for the creation of WPC80 (Onwulata, 2008; Rosenberg, 1995). Powder with a protein concentration above 90% of the total solids is referred to as whey protein isolate (WPI). For WPI, fat must be removed through either microfiltration or anion exchange (Huffman, 1996; Smith et al., 2016a) (see also Chapter 4). The lactose and fat concentration play a critical role in the sensory attributes of dried whey products. WPC is generally characterized by sweet aromatic, milky, cardboard, and fatty flavors although these flavors vary based on

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FIGURE 10.6 Flow diagram of whey process from cheese vat to whey ingredients. Adapted from Bylund, G. (2003). Dairy processing handbook (2nd ed.). Lund, Sweden: Tetra Pak Processing Systems AB. SWP 5 Sweet whey protein, WPC 5 Whey protein concentrate, WPI 5 Whey protein isolate.

protein concentration. The lower protein content and higher lactose content of WPC34 result in a product that has distinct cooked/milky and sweet aromatic flavors and sweet taste compared to higher protein whey ingredients (Evans, Zulewska, Newbold, Drake, & Barbano, 2009; Listiyani, Campbell,

10.8 Serum Protein

Table 10.3 Trained Sensory Panel Profiles of Rehydrated WPC34, WPC80, and WPI Mean Scores Attributes

WPC34

WPC80

WPI

Aroma intensity Sweet aromatic Cardboard Cooked/milky Sweet taste

2.3a 2.6a 1.5c 3.6 2.3

2.5a 1.4b 2.0b ND ND

2.0b ND 2.3a ND ND

Scores based on a universal 0 to 15-point intensity scale. ND 5 Not detected. a,b,c Means in a row followed by different letters are significantly different (p , 0.05). Adapted from Carunchia Whetstine, M.E., Croissant, A.E., & Drake, M.A. (2005). Characterization of dried whey protein concentrate and isolate flavor. Journal of Dairy Science, 88, 3826 3839; Evans, J., Zulewska, J., Newbold, M., Drake, M.A., & Barbano, D.M. (2009). Comparison of composition, sensory, and volatile components of thirty-four percent whey protein and milk serum protein concentrates. Journal of Dairy Science, 92, 4773 4791; Evans, J., Zulewska, J., Newbold, M., Drake, M.A., & Barbano, D.M. (2010). Comparison of composition and sensory properties of 80% whey protein and milk serum protein concentrates. Journal of Dairy Science, 93, 1824 1843.

Miracle, Dean, & Drake, 2011). As protein content increases, cooked/milky and sweet aromatic flavors decrease. During diafiltration for WPC80 production, lactose is removed and protein and fat are both concentrated. WPC80 contains between 4.6% 6.5% fat (on a dry weight basis). Lipid oxidation and off-flavor development occur in this product due to bleaching, storage, and drying (Campbell et al., 2011b; Carunchia Whetstine et al., 2005; Evans et al., 2010). WPI has much lower concentrations of fat (between 0.15% and 0.60% on a dry weight basis) as fat removal is a necessary step in concentrating protein above 90% of the total solids (Carunchia Whetstine et al., 2005; Smith et al., 2016d; Whitson et al., 2011). The reduced fat content in WPI can reduce lipid oxidation and off-flavors. Carunchia Whetstine et al. (2005) demonstrated that WPI powders contained fewer aroma-active lipid oxidation products than WPC80 powders. Although lipid content influences offflavor formation, the binding capacity of volatile compounds to protein in WPI may also decrease the perception of off-flavor in WPI compared to WPC80 (Stevenson & Chen, 1996) (Table 10.3).

10.8

SERUM PROTEIN

Serum proteins refer to whey proteins that are removed from milk before cheese manufacture (Drake et al., 2009; Evans et al., 2009; 2010; Nelson & Barbano, 2005). Serum proteins are not exposed to the cheesemaking process which reduces lipid oxidation and proteolysis in these products compared to traditionally produced whey protein (Campbell et al., 2011b; Nelson & Barbano, 2005; Smith et al., 2016a). As a result, serum proteins tend to have

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Table 10.4 Trained Sensory Panel Profiles of Rehydrated SPC34, SPC80, and SPI Mean Score Descriptor

SPC34

SPC80

SPI

Aroma intensity Sweet aromatic Cardboard Cooked milky Sweet taste Astringent

1.7 1.8 ND 2.0 2.0 1.5

1.2 1.2 1.0 1.1 ND 1.8

1.3 1.5 1.6 ND ND 1.9

Scores based on a universal 0 to15-point intensity scale. ND 5 Not detected. Adapted from Evans, J., Zulewska, J., Newbold, M., Drake, M.A., & Barbano, D.M. (2009). Comparison of composition, sensory, and volatile components of thirty-four percent whey protein and milk serum protein concentrates. Journal of Dairy Science, 92, 4773 4791; Evans, J., Zulewska, J., Newbold, M., Drake, M.A., & Barbano, D.M. (2010). Comparison of composition and sensory properties of 80% whey protein and milk serum protein concentrates. Journal of Dairy Science, 93, 1824 1843.

a mild flavor compared to traditional whey protein. To create serum protein, skim milk is first subject to microfiltration, where the serum protein and lactose are separated from the casein and fat of the milk (Nelson & Barbano, 2005; Saboya & Maubois, 2000). At this point, lactose is removed through UF, the retentate produced is serum protein concentrate (SPC) with concentrations ranging from 34% protein (SPC34) to 80% protein (SPC80) or serum protein isolate (SPI) with protein concentration above 90% (Evans et al., 2009; 2010; Nelson & Barbano, 2005; Saboya & Maubois, 2000). The microfiltration process used to remove serum protein from skim milk also removes fat (Evans et al., 2009; Nelson & Barbano, 2005). As such, SPCs contain less fat than WPC at the same protein concentration (0.53% vs 4.67% on a dry weight basis), which reduces lipid oxidation and oxidation related off-flavors (Evans et al., 2009, 2010). Evans et al. (2009) reported that SPC34 had lower diacetyl and cardboard flavors compared to WPC34. Evans et al. (2010) compared the flavor profile of SPC80 to WPC80, and demonstrated that SPC80 had lower oxidation and culture-related flavors than WPC80, including cardboard and diacetyl flavors. SPI, which contains greater than 90% protein on a dry weight basis, exhibited lower flavor intensities compared to WPI and, like other serum protein products, lower oxidation (Table 10.4).

10.9

HYDROLYSATES

Whey protein hydrolysates (WPH) are whey protein products (WPC or WPI) which have undergone enzymatic hydrolysis of peptide bonds and an aggressive heat step to inactivate the added enzymes (Drake et al., 2009; Leksrisompong, Miracle, & Drake, 2010; Smith et al., 2016a) (see also

10.10 Bleaching

Chapter 4). Hydrolysis improves digestibility of proteins and alters functional properties including solubility and heat stability (Nnanna & Wu, 2006; Nongonierma & FitzGerald, 2015; Pedrosa, Pascual, Larco, & Esteban, 2006; Tang, Moore, Kujibida, Tamopolsky, & Phillips, 2009). Despite the health benefits, their use in food applications is limited by strong negative flavor attributes. WPH differ in flavor from WPC and WPI due to their enzymatic treatment and heating step (Leksrisompong et al., 2010). The degree of hydrolysis, enzyme used, and hydrolysis conditions all influence WPH flavor (Leksrisompong et al., 2010; Ziajka, Dzwolak, & Zubel, 1994). WPH generally have distinct bitter taste due to proteolysis and the bitter taste intensity generally is associated with the degree of hydrolysis (Harwalker et al., 1993; Leksrisompong et al., 2010, 2012). Leksrisompong et al. (2010) demonstrated that bitterness in WPH was correlated with smaller peptides (,600-4142 Da) which contained hydrophobic residues at the C-terminal end. Larger peptides (3000 6000 Da) had much less bitter taste, likely due to the ability of these peptides to bind their own C-terminal hydrophobic sites, blocking hydrophobic interactions (Leksrisompong et al., 2012; Pedrosa et al., 2006). Peptides between three and six amino acids in length contributed to bitterness, as did many amino acids with L-conformation and hydrophobic side chains (Leksrisompong et al., 2010). Thus the degree to which a product is hydrolyzed is less important to the bitterness of WPH than the concentration of peptides (Newman et al., 2014a). WPH have many other sensory challenges than just bitter taste. Leksrisompong et al. (2010) demonstrated that WPH contained high levels of cooked/sulfur, potato/brothy, tortilla, and animal flavors due to the extended heating and proteolysis needed to produce WPH. Newman, O’Riordan, Jacquier, & O’Sullivan (2014b) confirmed these findings, and developed a lexicon for WPH and casein based hydrolysates, which contained metallic, cabbage, cereal, and burnt flavors. This sensory language was similar to the one independently developed and used by Leksrisompong et al. (2010). Unsurprisingly, protein degradation compounds play a pivotal role in the composition of the volatiles of WPH. These compounds include sulfur compounds such as dimethyl trisulfide which imparts cabbage flavor and methional which imparts potato flavor in WPH and other whey protein ingredients (Leksrisompong et al., 2010; Wright et al., 2006) (Fig. 10.7).

10.10 10.10.1

BLEACHING Impact of Bleaching on Whey

Annatto, the colorant used to color Cheddar cheese in some countries, contributes no flavor directly to whey or whey ingredients but provides unique

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FIGURE 10.7 Principal component analysis biplot of trained panel sensory profiles of selected rehydrated whey protein hydrolysates (WPH). H1H10 5 WPH samples. Adapted from Leksrisompong, P.P., Miracle, R.E., & Drake, M.A. (2010). Characterization of flavor of whey protein hydrolysates. Journal of Agricultural & Food Chemistry, 58, 6318 6327.

flavor challenges for whey ingredients (Campbell et al., 2011a; Jervis & Drake, 2013; Kang, Campbell, Bastian, & Drake, 2010). Once separated from the cheese curd, fluid whey retains approximately 10% of the total added annatto (Kang et al., 2010; Smith, Li, & Drake, 2014). Annatto is composed of two carotenoids, water-soluble norbixin and fat-soluble bixin, but norbixin is the primary colorant used in cheese production due to its solubility (Giuliano, Rosati, & Bramley, 2003; Smith et al., 2014). The residual annatto causes an unfavorable color in dried whey products, which necessitates bleaching (Croissant et al., 2009; Kang et al., 2010; McDonough et al., 1968) (Fig. 10.1).

10.10.2

General Bleaching

Due to its long carbon chain and double bond configuration, norbixin is susceptible to oxidation (Jervis et al., 2012; Kang et al., 2010; Scotter, 2009).

10.10 Bleaching

Oxidative cleavage along the carbon backbone of norbixin causes disruption of the chromophore and renders the compound colorless (Giuliano et al., 2003). Oxidative bleaching is the industry standard to remove color from fluid whey (Kang et al., 2010). Bleaching is accomplished chemically or enzymatically (Campbell, Kang, Bastian, & Drake, 2012; Kang et al., 2010; Kang, Smith, & Drake, 2012). Both of these approaches are general oxidative bleaching methods and have adverse effects on the flavor of whey ingredients since the process is not specific to annatto (Campbell & Drake, 2014; Jervis et al., 2012; Jervis, Smith, & Drake, 2015; Listiyani et al., 2012; Smith et al., 2015). However, different bleaching agents impact whey ingredient flavor differently and can be optimized to mitigate off-flavor (Campbell & Drake, 2014; Campbell et al., 2012; Jervis et al., 2012).

10.10.3

Chemical Bleaching

The two prominent chemical bleaching agents used for fluid whey are hydrogen peroxide and benzoyl peroxide. Both have been in use for more than 20 years. Hydrogen peroxide is a water soluble bleaching agent that causes a 37% 44% reduction in norbixin when applied at concentrations up to 500 mg/kg followed by deactivation by the addition of catalase (Croissant et al., 2009; Gilliland, 1969; Jervis et al., 2012; Kang et al., 2010; Teply, Derse, & Price, 1958; Listiyani et al., 2012; Fox, Smith, Gerard, & Drake, 2013). Benzoyl peroxide is a fat-soluble bleaching agent that can result in .95% norbixin removal at concentrations between 20 and 50 mg/kg, but leaves a benzoic acid residue when used, which is prohibited in some countries (Jervis et al., 2012; Listiyani et al., 2011; Smith et al., 2015). Croissant et al. (2009) first documented that flavor differences existed between wheys bleached with hydrogen peroxide or benzoyl peroxide. Bleaching with hydrogen peroxide (500 mg/kg) resulted in more lipid oxidation compared to benzoyl peroxide (20 mg/kg). Consequently, increased cardboard flavor was observed. Listiyani et al. (2012) demonstrated that higher bleaching temperature increased lipid oxidation in fluid whey when bleached by hydrogen peroxide. The effect of temperature on benzoyl peroxide bleaching of fluid whey, in contrast, was less pronounced than hydrogen peroxide (Fox et al., 2013; Listiyani et al., 2012; Smith et al., 2015). Off-flavor production is a barrier to use of hydrogen peroxide, as hydrogen peroxide bleaching efficiency in fluid whey is reduced at lower temperatures. The balance between efficient norbixin bleaching and reduction of off-flavor must be considered when using hydrogen peroxide (Fox et al., 2013; Listiyani et al., 2012). As bleached fluid whey is concentrated and dried, these differences in flavor profile due to bleaching agents remain noticeable. WPC80 from whey bleached with hydrogen peroxide contained distinct potato, cardboard, and

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FIGURE 10.8 Comparison of trained panel cardboard flavor intensity and percent increase in total aldehydes compared to control in rehydrated WPC80 bleached with 500 mg/kg hydrogen peroxide (HP250) or 50 mg/kg benzoyl peroxide (BP50). Adapted from Jervis, S., Campbell, R., Wojciechowski, K.L., Foegeding, E.A., Drake, M.A., & Barbano, D.M. (2012). Effect of bleaching whey on sensory and functional properties of 80% whey protein concentrate. Journal of Dairy Science, 95, 2848 2862.

fatty flavors compared to WPC80 from whey bleached with benzoyl peroxide (Jervis et al., 2012). In SWP and WPC34, hydrogen peroxide bleaching also encouraged lipid and protein oxidation as well as cardboard and fatty flavors when compared to benzoyl peroxide bleaching (Jervis et al., 2015; Listiyani et al., 2011). Bleaching can either be applied before or after filtration. However, bleaching whey retentate (whey protein after concentration) with hydrogen peroxide or benzoyl peroxide increased cardboard and fatty flavors compared to samples bleached before filtration (Fox et al., 2013) (Fig. 10.8).

10.10.4

Enzymatic Bleaching

Enzymatic bleaching of whey involves the use of either the native lactoperoxidase system or an exogenous peroxidase (Campbell & Drake, 2013b, 2014; Campbell et al., 2012; Kang et al., 2010). Lactoperoxidase is a native enzyme to milk which, in the presence of low levels of hydrogen peroxide, converts thiocyanate to the active hypothiocyanate (see also Chapter 1). The resulting radical causes oxidation and can be used to bleach whey (Campbell & Drake, 2013b, 2014; Campbell et al., 2012; Reiter & Harnulv, 1982). The lactoperoxidase system has a strong oxidizing capacity and is self-perpetuating until terminated by catalase, thus this system is extremely efficient at bleaching norbixin, causing .90% destruction compared to 37% 44% norbixin destruction by hydrogen peroxide bleaching (Campbell & Drake, 2014; Reiter & Harnulv, 1982). Unfortunately, this enzymatic system is also nonspecific, and also encourages lipid and protein oxidation (Kang et al., 2010). Campbell et al. (2012) demonstrated that bleaching fluid whey with

10.11 Storage

lactoperoxidase had higher norbixin destruction but also increased total aldehydes and cabbage flavors in WPC80 compared to WPC80 from fluid whey bleached with hydrogen peroxide. The lactoperoxidase system can also be variable, as enzyme concentration varies depending on the lactation cycle of the cow, season, feeding regime, and breed (Campbell & Drake, 2014; Kussendrager & van Hooijdonk, 2000). A solution to lactoperoxidase variability is the addition of an exogenous peroxidase. Exogenous peroxidases are peroxidases isolated from a nondairy source, and do not require thiocyanate in milk to activate them (Campbell & Drake, 2014; Kang et al., 2010). WPC80 bleached with exogenous peroxidase had similar flavor to lactoperoxidase-bleached WPC80 (Campbell & Drake, 2013a). Some offflavor can be mitigated by enzymatic bleaching at 4 C, particularly with exogenous peroxide, which resulted in faster bleaching than lactoperoxidase at lower temperatures (Campbell & Drake, 2013a). The process of bleaching, regardless of the agent, generally has a negative impact on whey ingredient flavor (Croissant et al., 2009; Jervis et al., 2012; Campbell & Drake, 2013a). A decrease in sweet aromatic flavor and increased cardboard flavor are ubiquitous among bleached whey ingredients compared to their unbleached counterparts (Jervis et al., 2012). Both hydrogen peroxide and enzymatic bleaching may cause fatty, cabbage, and potato flavors (Campbell & Drake, 2013b; Campbell et al., 2012). Benzoyl peroxide has the bleaching efficiency of enzymatic bleaching with lower negative impacts on flavor, but the issues of benzoic acid residues remain (Campbell et al., 2012; Jervis et al., 2012; Smith et al., 2015).

10.11

STORAGE

Storage of fluid whey prior to spray drying increases lipid oxidation and has a negative impact on the sensory attributes of liquid whey and dried ingredients manufactured from stored fluid whey. Commercially, fluid or concentrated whey may be stored before drying (Fig. 10.2). Lipid oxidation still occurs during storage at 3 C (Liaw et al., 2011; Park et al., 2016b; Whitson et al., 2011). Storage of liquid WPC80 or WPI retentate for 48 hours at 3 C prior to drying increased lipid oxidation and resulted in increased cardboard and decreased sweet aromatic flavors in spray-dried protein powders (Whitson et al., 2011). Fluid storage for more than 6 hours was detrimental to the flavor of spray-dried protein powders. However, steps can be taken to minimize storage effects. Park et al. (2016b) compared storage of liquid whey to storage of liquid WPC80 and determined that lipid oxidation was mitigated in unbleached whey by storing the whey as liquid WPC80 instead of as fluid whey. However, in bleached whey, storage as fluid WPC80 increased lipid oxidation compared to storage of fluid whey with lower solids.

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10.12

SPRAY DRYING AND DRY STORAGE

Drying fluid dairy products can be an efficient method to facilitate transport and storage, and slow lipid oxidation (Fig. 10.1). As one of the most common drying methods in the dairy industry, spray drying removes moisture by atomizing liquid and exposing the product to brief but intense heat. Due to rapid heating and evaporative cooling, the interior temperature of the whey particles generally does not exceed 60 C (Park, Bastian, Farkas, & Drake, 2014a). This system is minimally invasive; however the heating step introduces other opportunities for heat-related off-flavors to develop. As whey products are exposed to the intense inlet temperatures (often above 180 C), Maillard reactions, oxidation, and sugar caramelization can encourage flavor development (Park & Drake, 2014). Products high in lactose such as SWP, are particularly susceptible to Maillard reactions during the drying stage, which encourages the formation of sweet aromatic and milky flavors, as well as oxidized flavors (Sithole et al., 2006). This process can be avoided through freeze drying, which does not cause Maillard reaction products (Evans et al., 2009). However, freeze drying increased lipid oxidation compared to spray drying, possibly due to the longer processing time (Evans et al., 2009). Spray-dried WPC34 had sweet aromatic and cooked/milky flavors, indicative of Maillard reactions, and lower cardboard flavor compared to freeze-dried product. In dried high-protein whey products (WPC and WPI), lipid oxidation occurs during storage at ambient temperatures and is responsible for off-flavors at the end of shelf-life (Wright et al., 2009). Wright et al. (2009) demonstrated that dried WPC and WPI stored at 21 C for 6 24 months had higher levels of cardboard, cucumber, and fatty flavors than fresh dried WPC and WPI. They concluded that shelf-life of regular nonagglomerated WPC80 and WPI was 12 15 months. Javidipour & Qian (2008) found that powdered WPC80 stored at temperatures between 35 45 C had elevated volatile lipid oxidation compounds after 15 weeks. Shelf life of dried WPC and WPI can be influenced by processing parameters such as agglomeration and instantization with lecithin (Javidipour & Qian, 2008; Wright et al., 2009). Agglomeration of powdered whey protein ingredients is a process of creating small clumps of particles which allow for increased dispersability and reduced dispersion time in fluids (Turchiuli, Eloualia, Mansouri, & Dumoulin, 2005). Instantization of whey protein is the process of agglomerating product with lecithin to aid in dispersion (Wright et al., 2009). These processes both encourage formation of volatile lipid oxidation compounds and development of detectable off-flavor in WPC and WPI more quickly than nonagglomerated WPC and WPI stored under the same conditions (Wright et al., 2009).

10.12 Spray Drying and Dry Storage

Park et al. (2014a) demonstrated that spray drying parameters influenced lipid oxidation and subsequently flavor, by exposure of surface free fat (Park & Drake, 2014; Park et al., 2014a). During spray drying, the sample is atomized and sprayed into small droplets, which are evaporated under hot air, leaving dry particles. During drying, fat can migrate out of the globular membrane becoming surface free fat, where it is more susceptible to lipid oxidation (Park & Drake, 2014). The inlet temperature of the spray dryer and the feed solids concentration determines the extent to which fluid migration can occur and thus influence lipid oxidation rates (Kelly et al., 2002; Park et al., 2014a). Increasing the inlet temperature during spray drying increased particle drying rates and decreased surface free fat (Park et al., 2014a). Park et al. (2014a) demonstrated that WPC80 spray dried with an inlet temperature of 220 C had decreased cardboard flavor intensity compared to WPC80 spray dried with an inlet temperature of 180 C. The rate of fat migration from within the globular membrane was also influenced by solids concentration, with higher solids decreasing lipid oxidation by decreasing surface free fat. Park et al. (2014a) demonstrated that WPC80 spray dried at 10% solids had higher surface free fat, smaller particle size, and increased cardboard and cabbage flavors compared to WPC80 spray dried at 18% or 25% solids (Fig. 10.9). The pH of fluid product also influences lipid oxidation and off-flavors in spray-dried whey proteins. WPC and WPI used for acidic beverages are often

FIGURE 10.9 Trained sensory panel profiles of rehydrated WPC80 spray dried at different solids concentrations. All samples were spray dried at an inlet temperature of 200 C. Adapted from Park, C.W., Bastian, E., Farkas, B., Drake, M.A. (2014a). The effect of feed solids concentration and inlet temperature on the flavor of spray dried whey protein concentrate. Journal of Food Science, 79, C19 C24.

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acidified prior to spray drying, as this can reduce turbidity in the beverage as well as improve flavor (White, Fox, Jervis, & Drake, 2013). Park, Bastian, Farkas, & Drake (2014b) demonstrated that liquid WPC80 acidified to pH 3.5 prior to spray drying had reduced lipid oxidation compounds and decreased off-flavors compared to WPC80 adjusted to pH 5.5 or 6.5. At a pH of 3.5, the whey protein β-lactoglobulin has a conformation that encourages more lipid oxidation compounds to bind to its hydrophobic regions (Park et al., 2014b). Park et al. (2014b) hypothesized that lipid oxidation compounds were bound to protein at pH 3.5 and they were less able to volatilize and impact flavor.

10.13

CONCLUSIONS

Whey proteins are valuable as ingredients for their functional and nutritional aspects, but flavor is a major driver in the acceptance of whey protein ingredients. Understanding the flavor attributes of whey products is essential for the continued success of the whey industry. Ideally whey protein ingredients should be flavorless, as this allows for the greatest versatility in ingredient applications. However, off-flavors easily develop in whey protein products due to lipid oxidation, proteolysis, and Maillard reactions. These flavors begin to develop in fluid milk, being affected by the animal’s diet and milk handling. They also develop in whey during unit operations in whey processing, ingredient manufacture, and storage. There has been a significant amount of research regarding the flavor of whey protein ingredients; however the full potential of whey protein ingredients will only be realized through continued research on flavor reduction.

Acknowledgments Funding was provided in part by the National Dairy Council (DRI; formerly Dairy Management Inc. (DMI) Rosemont, IL). The use of trade names does not imply endorsement or lack of endorsement of any product used.

References Bassette, R., Fung, D. Y. C., Mantha, V. R., & Marth, E. H. (1986). Off-flavors in milk. CRC Critical Reviews in Food Science & Nutrition, 24, 1 52. Bendall, J. G. (2001). Aroma compounds of fresh milk from New Zealand cows fed different diets. Journal of Agricultural & Food Chemistry, 49, 4825 4832. Brennand, C. P., Ha, J. K., & Lindsay, R. C. (1989). Aroma properties and thresholds of some branched-chain and other minor volatile fatty acids occurring in milkfat and meat lipids. Journal of Sensory Studies, 4, 105 120.

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Cheddar cheese flavor in Ireland, New Zealand, and the United States of America. International Dairy Journal, 15, 473 483. Drake, M. A., Miracle, R. E., & Wright, J. M. (2009). Sensory properties of dairy proteins. In A. Thompson, M. Boland, & H. Singh (Eds.), Milk proteins: From expression to food (pp. 429 448). San Diego, CA: Academic Press. Durham, R. J., Hourigan, J. A., Sleigh, R. W., & Johnson, R. L. (1997). Whey fractionation: Wheying up the consequences. Food Australia, 49, 460 465. El Soda, M., Law, J., Tsakalidou, E., & Kalantzopoulos, G. (1995). Lipolytic activity of cheese related microorganisms and its impact on cheese flavor. In G. Charalambous (Ed.), Food Flavors, Generation, Analysis and Process Influence (pp. 1823 1847). Amsterdam: Elsevier Science. Evans, J., Zulewska, J., Newbold, M., Drake, M. A., & Barbano, D. M. (2009). Comparison of composition, sensory, and volatile components of thirty-four percent whey protein and milk serum protein concentrates. Journal of Dairy Science, 92, 4773 4791. Evans, J., Zulewska, J., Newbold, M., Drake, M. A., & Barbano, D. M. (2010). Comparison of composition and sensory properties of 80% whey protein and milk serum protein concentrates. Journal of Dairy Science, 93, 1824 1843. Fox, A. J., Smith, T. J., Gerard, P. D., & Drake, M. A. (2013). The influence of bleaching agent and temperature on bleaching efficacy and volatile components of fluid whey and whey retentate. Journal of Food Science, 78, C1535 C1542. Frankel, E. N. (1998). Oxidation in multiphase systems. Lipid oxidation (pp. 259 297). Dundee, Scotland: The Oily Press. Friedman, M. (1996). Food browning and its prevention. Journal of Agricultural & Food Chemistry, 44, 631 653. Gallardo-Escamilla, F. J., Kelly, A. L., & Delahunty, C. M. (2005a). Sensory characteristics and related volatile flavor compound profiles of different types of whey. Journal of Dairy Science, 88, 2689 2699. Gallardo-Escamilla, F. J., Kelly, A. L., & Delahunty, C. M. (2005b). Influence of starter culture on flavor and headspace volatile profiles of fermented whey and whey produced from fermented milk. Journal of Dairy Science, 88, 3745 3753. Gilliland, S. E. (1969). Enzymatic determination of residual hydrogen peroxide in milk. Journal of Dairy Science, 52, 321 324. Giuliano, G., Rosati, C., & Bramley, P. M. (2003). To dye or not to dye: Biochemistry of annatto unveiled. Trends in Biotechnology, 21, 513 516. Harwalker, V. R., Cholette, H., McKellar, R. C., & Emmons, D. B. (1993). Relation between proteolysis and astringent off-flavor in milk. Journal of Dairy Science, 76, 2521 2527. Holmes, D. G., Duersch, J. W., & Ernstrom, C. A. (1977). Distribution of milk clotting enzymes between curd and whey and their survival during Cheddar cheese making. Journal of Dairy Science, 60, 862 869. Huffman, L. M. (1996). Processing whey protein for use as a food ingredient. Food Technology, 50 (2), 49 52. Javidipour, I., & Qian, M. C. (2008). Volatile component change in whey protein concentrate during storage investigated by headspace solid-phase microextraction gas chromatography. Dairy Science & Technology, 88, 95 104. Jervis, M., Smith, T. J., & Drake, M. A. (2015). The influence of solids concentration and bleaching agent on bleaching efficacy and flavor of sweet whey powder. Journal of Dairy Science, 98, 2294 2302.

References

Jervis, S., Campbell, R., Wojciechowski, K. L., Foegeding, E. A., Drake, M. A., & Barbano, D. M. (2012). Effect of bleaching whey on sensory and functional properties of 80% whey protein concentrate. Journal of Dairy Science, 95, 2848 2862. Jervis, S. M., & Drake, M. A. (2013). The impact of iron on the bleaching efficacy of hydrogen peroxide in liquid whey systems. Journal of Food Science, 78, R129 R137. Jousse, F., Jongen, W., Agterof, W., Russell, S., & Braat, P. (2002). Simplified kinetic scheme of flavour formation by the Maillard reaction. Journal of Food Science, 67, 2534 2542. Kang, E. J., Campbell, R. E., Bastian, E., & Drake, M. A. (2010). Annatto usage and bleaching in dairy foods. Journal of Dairy Science, 93, 3891 3901. Kang, E. J., Smith, T. J., & Drake, M. A. (2012). Alternative bleaching methods for Cheddar cheese whey. Journal of Food Science, 77, C818 C823. Karagül-Yüceer, Y., Drake, M. A., & Cadwallader, K. R. (2003). Aroma-active components of liquid cheddar whey. Journal of Food Science, 68, 1215 1219. Karagul-Yuceer, Y. K., Vlahovich, K., Drake, M. A., & Cadwallader, K. R. (2003). Characteristic aroma components of rennet casein. J. Agric. Food Chem., 51, 6797 6801. Kelly, J., Kelly, P. M., & Harrington, D. (2002). Influence of processing variables on the physiochemical properties of spray dried fat-based milk powders. Lait, 82, 401 412. Kelly, M. L., Kolver, E. S., Bauman, D. E., van Amburgh, M. E., & Muller, L. D. (1998). Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows. Journal of Dairy Science, 81, 1630 1636. Khanal, R. C., Dhiman, T. R., Ure, A. L., Brennand, C. P., Boman, R. L., & McMahon, D. J. (2005). Consumer acceptability of conjugated linoleic acid-enriched milk and Cheddar cheese from cows grazing on pasture. Journal of Dairy Science, 88, 1837 1847. Kosikowski, F. V., & Mistry, V. V. (1997). Whey and whey foods. Cheese and fermented milk foods: Vol. 1: Origins and principles (pp. 422 453). Wesport, CT: F. V. Kosikowski. Kussendrager, K. D., & van Hooijdonk, A. C. M. (2000). Lactoperoxidase: Physio-chemical properties, occurrence, mechanism of action, and applications. British Journal of Nutrition, 84, S19 S25. Kussy, D., & Aylward, E. (2009). Pasteurized process cheese. In S. Clark, M. Costello, M. A. Drake, & F. Bodyfelt (Eds.), The sensory evaluation of dairy products (pp. 387 401). New York, NY: Springer. Lawless, H. T., & Heymann, H. (1999). Sensory evaluation of food: Principles and practices. New York, NY: Chapman & Hall. Lee, A. P., Barbano, D. M., & Drake, M. A. (2016). The effect of raw milk cooling on sensory perception and shelf life of high-temperature, short-time (HTST)-pasteurized skim milk. Journal of Dairy Science, 99, 9659 9667. Lee, A. P., Barbano, D. M., & Drake, M. A. (2017). The influence of ultra-pasteurization by indirect heating versus direct steam injection on skim and 2% fat milks. Journal of Dairy Science, 100, 1688 1701. Lee, K. D., Lo, C. G., & Warthesen, J. J. (1996). Removal of bitterness from the bitter peptides extracted from Cheddar cheese with peptidases from Lactococcus lactis ssp. cermoris SK11. Journal of Dairy Science, 29, 1521 1528. Leksrisompong, P., Gerard, P., Lopetcharat, K., & Drake, M. A. (2012). Bitter taste inhibiting agents for whey protein hydrolysate and whey protein hydrolysate beverages. Journal of Food Science, 77, S282 S287. Leksrisompong, P. P., Miracle, R. E., & Drake, M. A. (2010). Characterization of flavor of whey protein hydrolysates. Journal of Agricultural & Food Chemistry, 58, 6318 6327. Liaw, I. W., Eshpari, H., Tong, P. S., & Drake, M. A. (2010). The impact of antioxidant addition on flavor stability of Cheddar and Mozzarella whey and Cheddar whey protein concentrate. Journal of Food Science, 75, C559 C569.

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Liaw, I. W., Miracle, R. E., Jervis, S. M., Listiyani, M. A. D., & Drake, M. A. (2011). Comparison of the flavor chemistry and flavor stability of Mozzarella and Cheddar wheys. Journal of Food Science, 76, 1188 1194. Listiyani, M. A. D., Campbell, R. E., Miracle, R. E., Dean, L. O., & Drake, M. A. (2011). Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey proteins. Journal of Dairy Science, 94, 4347 4359. Listiyani, M. A. D., Campbell, R. E., Miracle, R. E., Barbano, D. M., Gerard, P. D., & Drake, M. A. (2012). Effect of temperature and bleaching agents on bleaching of liquid Cheddar whey. Journal of Dairy Science, 95, 36 39. Lubran, M. B., Lawless, H. T., Lavin, E., & Acree, T. E. (2005). Identification of metallic-smelling 1-octen-3-one and 1-nonen-3-one from solutions of ferrous sulfate. Journal of Agricultural & Food Chemistry, 53, 8325 8327. Mahajan, S. S., Goddick, L., & Qian, M. C. (2004). Aroma compounds in sweet whey powder. Journal of Dairy Science, 87, 4057 4063. Marsili, R. T. (2003). Flavours and off-flavours in dairy foods. In H. Roginski, J. W. Fuquay, & P. F. Fox (Eds.), Encyclopedia of dairy science (pp. 1069 1081). London: Academic Press. McClements, D. J., & Decker, E. A. (2008). Lipids. In S. Damodaran, K. L. Parkin, & O. R. Fennema (Eds.), Fennema’s food chemistry (4th ed, pp. 155 216). Boca Raton, FL: CRC Press. McDonough, F. E., Hargrove, R. E., & Tittsler, R. P. (1968). Decolorization of annatto in Cheddar cheese whey. Journal of Dairy Science, 51, 471 472. Meilgaard, M. C., Civille, G. V., & Carr, B. T. (2007). The Spectrumt descriptive analysis method. In M. C. Meilgaard, B. T. Carr, & G. V. Civille (Eds.), Sensory evaluation techniques (3rd ed, pp. 187 199). Boca Raton, FL: CRC Press. Morr, C. V., & Ha, E. Y. W. (1991). Off-flavors of whey protein concentrates: A literature review. International Dairy Journal, 1, 1 11. Nelson, B. K., & Barbano, D. M. (2005). A microfiltration process to maximize removal of serum proteins from skim milk before cheese making. Journal of Dairy Science, 88, 1891 1900. Newman, J., Egan, T., Harbourne, N., O’Riordan, D., Jacquier, J. C., & O’Sullivan, M. (2014a). Correlation of sensory bitterness in dairy protein hydrolysates: Comparison of prediction models build using sensory, chromatographic and electronic tongue data. Talanta, 126, 46 53. Newman, J., O’Riordan, D., Jacquier, J. C., & O’Sullivan, M. (2014b). Development of a sensory lexicon for dairy protein hydrolysates. Journal of Sensory Studies, 29, 413 424. Newton, A. E., Fairbanks, A. J., Golding, M., Andrewes, P., & Gerrard, J. A. (2012). The role of the Maillard reaction in the formation of flavor compounds in dairy products not only a deleterious reaction but also a rich source of flavor compounds. Food & Function, 3, 1231 1241. Nnanna, I. A., & Wu, C. (2006). Dairy protein hydrolysates. In Y. H. Hui (Ed.), Handbook of Food Products Manufacturing (Vol 2, pp. 537 556). Hoboken, NJ: John Wiley & Sons Inc. Nongonierma, A. B., & FitzGerald, R. J. (2015). The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A review. Journal of Functional Foods, 17, 640 656. Oltman, A. E., Lopetcharat, K., Bastian, E., & Drake, M. A. (2015). Identifying key attributes for protein beverages. Journal of Food Science, 80, S1383 S1390. Onwulata, C. I. (2008). Milk whey processes: Current and future trends. In C. I. Onwulata, & P. J. Huth (Eds.), Whey processing, functionality, & health benefits (pp. 369 391). Ames, IA: Wiley-Blackwell.

References

Park, C. W., & Drake, M. A. (2014). The distribution of fat in dried dairy particles determines flavor release and flavor stability. Journal of Food Science, 79, R452 R459. Park, C. W., Bastian, E., Farkas, B., & Drake, M. A. (2014a). The effect of feed solids concentration and inlet temperature on the flavor of spray dried whey protein concentrate. Journal of Food Science, 79, C19 C24. Park, C. W., Bastian, E., Farkas, B., & Drake, M. A. (2014b). The effect of acidification of liquid whey protein concentrate on the flavor of spray-dried powder. Journal of Dairy Science, 97, 4043 4051. Park, C. W., Stout, M. A., & Drake, M. A. (2016a). The effect of spray-drying parameters on the flavor of nonfat dry milk and milk protein concentrate 70%. Journal of Dairy Science, 99, 9598 9610. Park, C. W., Parker, M., & Drake, M. A. (2016b). The effect of liquid storage on the flavor of whey protein concentrate. Journal of Dairy Science, 99, 4303 4308. Park, R. J., Armitt, J. D., & Star, D. P. (1969). Weed taints in dairy produce. II. Coronopus or land cress taint in milk. Journal of Dairy Research, 36, 37 46. Pedrosa, M., Pascual, C. Y., Larco, J. I., & Esteban, M. M. (2006). Palatability of hydrolysates and other substitution formulas for cow’s milk-allergic children: A comparative study of taste, smell, and texture evaluated by healthy volunteers. Journal of Investigational Allergology & Clinical Immunology, 16, 351 356. Reiter, B., & Harnulv, B. G. (1982). The preservation of refrigerated and uncooled milk by its natural lactoperoxidase system. Dairy Industries International, 47, 13 19. Rosenberg, M. (1995). Current and future applications for membrane processes in the dairy industry. Trends in Food Science & Technology, 6, 12 15. Russell, T. A., Drake, M. A., & Gerard, P. D. (2006). Sensory properties of whey and soy proteins. Journal of Food Science, 71, 447 455. Saboya, L. V., & Maubois, J. L. (2000). Current developments in microfiltration technology in the dairy industry. Lait, 80, 541 553. Scotter, M. (2009). The chemistry and analysis of annatto food coloring: A review. Food Additives & Contaminants, 26, 1123 1145. Singh, T. K., Cadwallader, K. R., & Drake, M. A. (2006). Biochemical processes in the production of flavor in milk and milk products. In Y. H. Hui (Ed.), Handbook of food products manufacturing (pp. 715 748). Hoboken, NJ: John Wiley & Sons, Inc. Sithole, R., McDaniel, M. R., & Goddik, L. M. (2006). Physiochemical, microbiological, aroma, and flavor profile of selected commercial sweet whey powders. Journal of Food Science, 71, 157 163. Smith, S., Smith, T. J., & Drake, M. A. (2016b). Flavor and flavor stability of cheese, rennet, and acid wheys. Journal of Dairy Science, 99, 3434 3444. Smith, T. J., Li, X. E., & Drake, M. A. (2014). Norbixin and bixin partitioning in Cheddar cheese and whey. Journal of Dairy Science, 97, 3321 3327. Smith, T. J., Gerard, P. D., & Drake, M. A. (2015). Effect of temperature and concentration on benzoyl peroxide bleaching efficacy and benzoic acid levels in whey protein concentrate. Journal of Dairy Science, 98, 7614 7627. Smith, T. J., Campbell, R. E., & Drake, M. A. (2016a). Sensory properties of milk protein ingredients. In P. L. H. McSweeney, & J. A. O’Mahony (Eds.), Advanced dairy chemistry Vol. 1B: Proteins: Applied aspects (4th ed, pp. 197 223). London: Springer. Smith, T. J., Campbell, R. E., Jo, Y., & Drake, M. A. (2016c). Flavor and stability of milk proteins. Journal of Dairy Science, 99, 4325 4346.

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Smith, T. J., Foegeding, E. A., & Drake, M. A. (2016d). Flavor and functional characteristics of whey protein isolates from different whey sources. Journal of Food Science, 81, C849 C857. Stevenson, R. J., & Chen, X. D. (1996). A study of the volatile “trapping” in spray-dried protein concentrate by “crushing” and/or vacuuming, and detection by solid-phase microextraction/ gas chromatography/mass spectrometry. Food Research International, 29, 495 504. Tang, J. E., Moore, D. R., Kujibida, G. W., Tamopolsky, M. A., & Phillips, S. M. (2009). Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men. Journal of Applied Physiology, 107, 987 992. Teply, L. J., Derse, P. H., & Price, W. V. (1958). Composition and nutritive value of cheese produced from milk treated with hydrogen peroxide and catalase. Journal of Dairy Science, 41, 593 605. Tomaino, R. M., Turner, L. G., & Larick, D. K. (2004). The effect of Lactococcus lactis starter cultures on the oxidative stability of liquid whey. Journal of Dairy Science, 87, 300 307. Turchiuli, C., Eloualia, Z., Mansouri, N. E., & Dumoulin, E. (2005). Fluidised bed agglomeration: Agglomerates shape and end-use properties. Powder Technology, 157, 168 175. Vazquez-Landezverde, P. A., Torres, J. A., & Qian, M. C. (2006). Effect of high-pressure 2 moderate-temperature processing on the volatile profile of milk. Journal of Agricultural & Food Chemistry, 54, 9184 9192. White, S. S., Fox, K. M., Jervis, S. M., & Drake, M. A. (2013). Influence of heating and acidification on the flavor of whey protein isolate. Journal of Dairy Science, 96, 1366 1379. Whitfield, F. B. (1992). Volatiles from interactions of Maillard reactions and lipids. CRC Critical Review in Food Science & Nutrition, 31, 1 58. Whitson, M. E., Miracle, R. E., & Drake, M. A. (2010). Sensory characterization of chemical components responsible for cardboard flavor in whey protein. Journal of Sensory Science, 25, 616 636. Whitson, M. E., Miracle, R. E., Bastian, E., & Drake, M. A. (2011). Effect of liquid retentate storage on flavor of spray-dried whey protein concentrate and isolate. Journal of Dairy Science, 94, 3747 3760. Wright, B. J., Zevchak, S. E., Wright, J. M., & Drake, M. A. (2009). The impact of agglomeration and storage on flavor and flavor stability of whey protein concentrate 80% and whey protein isolate. Journal of Food Science, 74, S17 S29. Wright, J. M., Whetstine, M. E. C., Miracle, R. E., & Drake, M. A. (2006). Characterization of cabbage off-flavor in whey protein isolate. Journal of Food Science, 71, C86 C90. Ziajka, S., Dzwolak, W., & Zubel, J. (1994). The effect of processing variables on some properties of whey protein hydrolysates. Milchwissenschaft, 49, 382 384.

CHAPTER 11

Whey Protein-Based Packaging Films and Coatings Markus Schmid1 and Kerstin Müller2

1

Albstadt-Sigmaringen University, Sigmaringen, Germany, 2Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany

11.1

INTRODUCTION

Due to rising environmental awareness, the development of bio-based plastics has been gaining more and more attention in recent years. Renewable raw materials used for biopolymers such as proteins, polysaccharides or lipids derive from a variety of crops and even more promisingly from waste streams accruing from processing by the agro-food industry. Proteins have been successfully used for the formation of films and coatings. Well-studied proteins such as whey protein, casein, wheat gluten, soy protein, or zein have been used to develop films that are gained from renewable resources with faster degradability than other polymeric materials (Cinelli et al., 2014; Ramos, Fernandes, Silva, Pintado, & Malcata, 2012). Moreover, films derived from agricultural proteins develop new market opportunities for agricultural products, by-products, and waste streams within the food process chain (Embuscado & Huber, 2009). The ability of globular proteins to unfold and cross-link to new polymeric structures under certain conditions makes them excellent raw materials for films and coatings. Compared to their polysaccharide-based counterparts, cross-linked proteins films are more stable and often depict longer durability (Barone & Schmidt, 2006). In terms of packaging, promising oxygen, nitrogen, and carbon dioxide barriers can result from densely packed network structures developed from a variety of interactions and bonds between the protein chains and general protein hydrophilicity (Schmid, Zillinger, Müller, & Sängerlaub, 2015c). Properties of such protein films and coatings initiated extensive research within the movement towards more sustainable biopolymers for industrial applications. Thus, this chapter includes relevant discussion of the application of protein films and coatings as well as an outlook on their current and future industrial potential. Whey proteins with suitable functionalities as Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00012-6 © 2019 Elsevier Inc. All rights reserved.

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food packaging materials are described together with the different technologies for processing and current state of the art about film-forming formulations for tailored barrier, mechanical, surface, and end-of-life properties. Furthermore, the regulative background about packaging use, end-of-life, and perspectives is covered, especially with focus on the environmental impact of packaging materials. Existing drawbacks with regard to mechanical and barrier properties of whey protein-based packaging films and coatings are discussed, as well as strategies to overcome those limitations in order to match existing solutions. Physical, chemical, and biochemical methods can be applied for this purpose. Therefore, an overview of the effects of various treatments on whey protein-based films and coatings is given, focusing on food packaging applications. However, most of those modifications are still not fully grasped at a fundamental level. Understanding these modifications is an important step towards the industrial implementation of protein-based films and coatings (Zink, Wyrobnik, Prinz, & Schmid, 2016).

11.2

WHEY PROTEIN FILM FORMATION

11.2.1 Protein Structure Related Properties and Preconditions for Film Forming Proteins are macromolecules that naturally display different sequences of amino acids that are combined by peptide bonds. For protein film formation, chain alignment and structure highly depend on the present arrangement of amino acids. Geometrical structures based upon the primary amino acid sequences depict α-helices, β-sheet, or other steric turns and loops stabilized by intramolecular interactions varying in strength. Besides, strong disulfide bridges, hydrogen bonds, van der Waals-, electrostatic-, and hydrophobic interactions take place and are responsible for the final, three-dimensional protein structure. With regard to protein-based films, the initial amino acid sequence is the determining factor for interactions between the protein chains themselves as well as other film components (Belitz, Grosch, & Schieberle, 2008; Cheftel, Cuq, & Lorient, 1985). In terms of whey proteins, especially, the contained thiol groups of cysteine residues are able to form disulfide bridges, both intra- and intermolecular (Barone, Dangaran, & Schmidt, 2006). Whey proteins include different globular proteins; β-lactoglobulin (β-Lg) (B57%), α-lactalbumin (α-La)(B19%), bovine serum albumin (B7%), several immunoglobulins (B13%), and the polypeptides proteose-peptone (B4%) (Lent, Vanasupa, & Tong, 1998) (see Chapter 1 for more details on whey proteins). Being the major whey protein, β-Lg dominates gelation and aggregation behavior in whey protein formulations (Hammann & Schmid, 2014).

11.2 Whey Protein Film Formation

Native β-Lg is a small globular protein with a molecular weight of about 36.6 kDa with defined secondary and tertiary structure. At room temperature in aqueous solution (pH 5 7) it is present mainly as a dimer with a molecular weight of 18.3 kDa of each subunit (Jovanovi´c, Miroljub, & Ognjen, 2005). The molecule is made up of α-helical, β-sheet, and random coil structures. It consists of 162 amino acids, of which the nonpolar amino acids can be found on the inside and the polar residues on the outside of the globular structure. This explains the good water solubility of native β-Lg molecules (De Wit, 2001b). It contains five cysteine residues of which Cys 121 is freely available and decisive for film formation, although buried inside the protein structure under normal conditions (De Wit, 2001a; Morr & Ha, 1993). α-La is the second largest fraction in whey proteins and is a small acidic protein with a molecular weight of 14.2 kDa. The single polypeptide chain is made of 123 amino acids, including eight cysteine residues. Four disulfide bonds and Ca21 ions participate in the tertiary structure stabilization (Belitz, Grosch, & Schieberle, 2007; Jovanovi´c et al., 2005). It consists of a large α-helical domain and a small β-sheet domain, connected by a calcium-binding loop. Due to its disulfide bonds and the absence of free sulfhydryl (-SH) groups, α-La has the greatest thermal stability besides the proteose-peptone fraction (Jovanovi´c et al., 2005). The capabilities of whey proteins to change chain conformations and interact with each other to form modified three-dimensional networks are excellent properties to be used for films and coatings.

11.2.2

General Steps for Film Formation

The formation of whey protein-based films can be generally separated into different steps. Since whey proteins are globular proteins, the first step is unfolding of the protein’s native state by relieving low-energy intermolecular bonds. Protein unfolding and dissociation can be caused by several treatments such as change in temperature, change in pH, shear forces, or addition of organic solvents or salts. The generated, entangled protein chains expose reactive, mainly hydrophobic, functional groups and can now be newly orientated and arranged. The exposed groups are now able to form a threedimensional chemical network stabilized by new bonds such as disulfide bridges or physical linkages including van der Waals interactions, hydrogen bonding, and electrostatic and hydrophobic interactions (Onwulata & Huth, 2008). Although several processes are possible for film forming, the mainly applied technique to form coherent films is thermal denaturation. The denaturation temperature for whey proteins normally lies at about 78 C, although it depends on the formulation composition (Plackett, 2011). The degree of

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denaturation in whey proteins is determined by β-Lg. Not only is it the main fraction of whey proteins, the rate of aggregation also depends on the concentration of free sulfhydryl groups which only occur in β-Lg. Additionally, the free sulfhydryl groups appear to increase denaturation reactions by inducing cleavage of intramolecular disulfide bonds in α-La and other whey proteins (Calvo, Leaver, & Banks, 1993). At pH 6, thermal denaturation takes place in two steps. In the first step, above 40 C, dimers dissociate into monomers. At temperatures around 70 C the globular molecules start unfolding and can interact via sulfhydryl groups, forming small aggregates with either β-Lg molecules or other thiol-containing proteins. Secondly, small aggregates interact forming high-molecular-weight aggregates. At higher temperatures, irreversible degradation of the protein starts. However, the influence of pH, salt, sugar, and protein concentration must be considered concerning thermal behavior of β-Lg. (Dewit & Klarenbeek, 1984; Jovanovi´c et al., 2005). Because the pH directly affects protein conformation, it also influences solubility and structure and thereby denaturation behavior. Dewit and Klarenbeek (1984) showed that β-Lg has an increased thermostability at pH 3 and a decreased thermostability at pH 7.5, which is explained by increased reactivity of thiol groups due to dissociation at alkaline pH. Initial products to form whey protein-based films are whey protein isolate, whey protein concentrate, or β-Lg in aqueous solution with different plasticizers. Final film properties vary with the used proteins and formulation additives such as plasticizer, chemical agents, or the addition of lipids or salts (Coltelli et al., 2016), as well as the applied denaturation process. However, native whey protein formulations are also able to form coherent films. Here, film cohesion is mainly based on low-energy bonding like van der Waals forces, electrostatic interactions, or hydrogen bonding (Pérez-Gago & Krochta, 1999). For further information on whey protein denaturation, also see Chapter 6 in this book.

11.3 TECHNOLOGIES FOR PROCESSING FILMS AND COATINGS 11.3.1

Wet Coating

Wet processing techniques are widely used for the formation of thin layer films. Stand-alone films can be obtained by casting solutions or suspensions while coatings are always associated with different substrates. The common method used for academic research regarding formulation evaluation is casting since it represents a quick and simple method to determine film-forming properties. Especially solvent casting is a cost-efficient method for the

11.3 Technologies for Processing Films and Coatings

development of protein polymer films with equipment available from laboratory to pilot scale (Embuscado & Huber, 2009). Among others, coating techniques can mainly be divided into deposition from solution such as spray-, dip-, roll- or spin-coatings, and physical vapor deposition (Coltelli et al., 2016). For all the coating techniques, of which the mainly applied ones are lacquering or spraying, rheological behavior of the coating formulations is decisive (Tracton, 2005). Furthermore, final film properties are influenced by the applied drying techniques which range from ambient condition drying to conventional hot-air drying, infrared or microwave drying (Embuscado & Huber, 2009). Due to solvent evaporation, the applicable coat weight can be much lower compared to extrusion coatings (Rastogi & Samyn, 2015). When compared to multilayer films, lower material usage is demanded (Mihindukulasuriya & Lim, 2014), bringing both economic and ecological advantages. When packaging applications are concerned, coatings on paper or polymeric substrates are used to attain or improve material properties such as barrier, mechanical, surface, or antimicrobial properties. Protein solutions used for casting or coating are usually denatured by heat to initiate cross-linking for film formation. The step of heat treatment can be performed either before, after, or during the coating process (Schmid, Noller, Wild, & Bugnicourt, 2013b), although the formation of native protein films is possible, too (Perez-Gago & Krochta, 2001a). To prevent protein films from brittleness caused by excessive cross-linking in the protein network, the use of plasticizers is necessary (Plackett, 2011), while compatibility of all film components must be considered since they directly influence final film properties (Lent et al., 1998; Mahmoud & Savello, 1993). When stand-alone films are processed via solvent casting, the film-forming solution is spread out evenly on a chosen surface such as a petri dish or Teflon plate before it can be removed therefrom after drying. Protein-based coatings are either applied on substrates such as paper or polymer films or directly applied on food products as edible coatings. In all cases, solvent removal is required by drying (Gennadios, 2002), limiting the coating thickness. Solvents used to prepare protein-based films are usually based on water, alcohol, or a blend of other solvents. At each film preparation step, the final film properties are influenced by the formulation and method applied. Therefore, materials obtained via wet processing display different functional properties according to the protein concentration used in solution, the pH of the solution, the solvents and additives used, and the drying conditions applied, such as drying rate and temperature (Gennadios, Weller, & Testin, 1993). Another important fact needs to be taken into account, namely the post cross-linking of whey protein-based films and coatings during storage.

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Schmid, Reichert, Hammann, and Stäbler (2015b) observed that after film forming and drying further alterations of molecular interactions in whey protein-based films occur. A significant decrease in oxygen permeability was observed during storage caused by changes in covalent and noncovalent bonds between the polypeptide chains. It was concluded that the crosslinking by disulfide bonds as well as hydrogen bonds are the predominant interactions formed during the initial days of storage, accompanied by a fast decrease in oxygen permeability. The contribution of hydrogen bonds in relation to disulfide bonds increases during storage leading to an additional, but slower, decrease in oxygen permeability until 2 3 weeks after initial protein-film production (Schmid et al., 2015b).

11.3.2

Extrusion

Extrusion processing is the conventional method for preparing plastic granules and films and has therefore also been used and adapted for processing protein-based plastics (Verbeek & van den Berg, 2010). Globular proteins such as whey proteins tend to unfold and cross-link under the influence of heat, leading to a closely networked material with thermoset behavior. During continuous extrusion, the raw material mass melts under the supply of heat and energy input caused by friction between the screws, becomes formable, and is pressed through the extruder die into a desired shape (Domininghaus, Eyerer, Elsner, & Hirth, 2008). During the whole process the mass can be compressed, mixed, plasticized, homogenized, chemically transformed, degasified, or gasified (Braun, 2003; Kaiser, 2007). The extrusion process, however, requires thermoplastic properties of the raw material. Since proteins naturally do not have a thermoplastic behavior, formulation modifications such as the use of additives are essential for protein extrusion and haves been widely studied for whey proteins (HernandezIzquierdo & Krochta, 2008, 2009; Hernandez-Izquierdo, Reid, McHugh, Berrios, & Krochta, 2008; Schmid, Müller et al., 2014; Schmid, Sängerlaub, Wege, & Stäbler, 2014, 2016; Verbeek & van den Berg, 2010). Final extrudate properties are influenced by applied process and formulation parameters. Referring to the extruder plant itself, mixing of raw materials is more efficient when using corotating twin-screw extruders. The configuration of the screws can include conveying as well as reconveying elements, dividing the process into different zones to fulfill process requirements such as conveying, compression, plasticization, mixing, or homogenization of the mass. The main physical characteristic of an extruder is, however, the length-to-diameter ratio, or L/D ratio (Giles, Wagner, & Mount, 2013). The specific mechanical energy input (SME) caused by friction of the screws is characteristic for each product and applies regardless of the extruder size. High SME inputs promote extensive cross-linking and should be kept low during extrusion of

11.4 Protein Modification for Optimized Film Formation and Performance

protein-based plastics, which can be realized with low screw speeds and high mass flow rates. Furthermore, the SME highly depends on the rheological properties of the melt, macromolecular transformations, and interactions between all formulation components (Coltelli et al., 2016). Formulation adjustments are usually made by the use of plasticizers and chemical additives. Modifiers that reduce intermolecular interactions are plasticizers and reducing agents; both generally reduce thermal decomposition temperature and increase the flexibility of thermoplastic protein plastics. The main efficient plasticizers used for whey protein processing are water, glycerol, sorbitol, and sucrose. Conversely, cross-linking agents or other additives that increase intermolecular interactions lead to stronger materials with high strength and stiffness and slightly increased decomposition temperatures. In both cases the melt viscosity is affected, being decreased or increased, respectively. The temperature setting itself is another critical parameter. While slight increases result in increased chain mobility, higher temperatures enable hydrophobic chain interactions due to unfolding and cross-linking of exposed reactive functional groups. A combination of high temperature and low moisture can even lead to protein degradation. Therefore, a suitable balance between melt viscosity and profile temperature is important for thermoplastic protein processing.

11.4 PROTEIN MODIFICATION FOR OPTIMIZED FILM FORMATION AND PERFORMANCE 11.4.1

Physical Modifications

The main applied physical modification for the production of proteinbased films and coatings is heat treatment. Proteins tend to aggregate and form three-dimensional networks when exposed to temperatures above their denaturation temperature (see Chapter 6 of this book for further details on denaturation and aggregation of whey proteins). Thermal denaturation of whey proteins begins at approximately 70 C (Parris, Purcell, & Ptashkin, 1991); however, denaturation conditions in protein formulations are strongly connected to the protein concentration and the additives used as well as the solvents (Renkema, Lakemond, Jongh, Gruppen, & van Vliet, 2000). Besides the extent of heat treatment, the resulting film properties are, furthermore, a function of ionic strength and the presence of other molecules, since they directly influence the developing protein network (Nicolai, Britten, & Schmitt, 2011; Nicorescu et al., 2008). Films processed under increased temperature, which also involves a reduction of the water content, exhibit higher brittleness and strength due to extensive cross-linking and less plasticizing.

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Processes such as mixing, coating, and extrusion all transfer shear stress. Under the influence of shear, proteins can aggregate, de-aggregate, and denature (Zink et al., 2016), although intense shear rates are needed for protein denaturation (Thomas & Geer, 2011). Pressure changes occurring due to shearing of a liquid formulation can be followed by deformation and fragmentation of contained protein particles (Taylor & Fryer, 1994). Therewith, eventual aggregates can be redispersed. Conversely, high particle collision rates can also facilitate aggregate formation. Fluid viscosity, also being a function of the protein ratio, directly influences shear-induced aggregation or deaggregation. Higher whey protein ratios, up to 30% w/w, result in smaller, stable aggregates due to higher viscosity and shear stress compared to formulation with lower protein ratios (Wolz, Mersch, & Kulozik, 2016). Since film-forming protein formulations are mainly aqueous, hydrostatic pressure (HP) is another possible treatment for physical modification of whey proteins (see also Chapter 8). However, only a few studies have used HP to form protein-based films until now (Condés, Añón, & Mauri, 2015; Fetzer & Ramachandran, 1979; Molinaro et al., 2015). The influence of high pressure can also lead to disaggregation and unfolding of globular proteins by affecting their conformation. Therewith, hydrophobic groups as well as disulfide groups are exposed and are able to form new inter- and intramolecular bonds (Bouaouina, Desrumaux, Loisel, & Legrand, 2006; Dumay, Kalichevsky, & Cheftel, 1994; Dumay, Kalichevsky, & Cheftel; 1998; Olsen, Ipsen, Otte, & Skibsted, 1999; Tedford & Schaschke, 2000). In comparison to heat-induced protein gels, high-pressure processed β-Lg gels show weaker intermolecular interactions resulting in more porous and less rigid structures with strong water exudation and solubility, and are prone to aggregation during storage (Tedford & Schaschke, 2000). When increased pressure is applied, however, stronger gels with lower solubility can be generated (Famelart, Chapron, Piot, Brulé, & Durier, 1998; Kanno, Mu, Hagiwara, Ametani, & Azuma, 1998). A combination of a suitable heat pretreatment (B55 C) and high pressure also showed improved film-forming properties (Phillips et al., 1990). Another tool for physical protein modification is ultrasound treatment. Ultrasonic cavitation caused by compression and decompression cycles of the sonic waves transfers high amounts of energy, being able to disrupt physical and chemical interactions (See also Chapter 8). Therefore it has been widely used for dispersing, emulsifying, crushing, and activating particles (Xia & Wang, 2003). With regards to proteins, shear forces and energy input arising from cavitation are able to split covalent bonds in aqueous formulations (Brennan, 2006; Gülseren, Güzey, Bruce, & Weiss, 2007). Therefore, high-intensity ultrasound can induce protein denaturation with a combination of shear stress and free radicals arising from water sonolysis

11.4 Protein Modification for Optimized Film Formation and Performance

(Riezs & Kondo, 1992; Suslick, Casadonte, Green, & Thompson, 1987). Especially for the production of whey protein-based nanocomposites, sonication is used to improve final film properties such as mechanical strength and barrier properties by reducing filler aggregation and favoring a homogenous distribution of the nanofiller. However, ultrasound has not been used for the protein-film formation itself so far. Whey protein films, particularly the needed cross-linking for film formation, can also be generated via irradiation. Ionizing irradiation, such as γ-irradiation, is able to induce irreversible conformation changes in the proteins by oxidation of amino acids, formation of protein free radicals, breakage of covalent bonds, and recombination and polymerization reactions (Hammann & Schmid, 2014). Basically, γ-irradiation generates hydroxyl radicals from water that are prone to react with amino acid residues, of which aromatic amino acids are preferred rather than aliphatic amino acids (Sabato et al., 2001). Regarding whey proteins, γ-irradiation forms dityrosine bridges between the protein chains and is able to form insoluble and sterilizable films (Brault, D’Aprano, & Lacroix, 1997). However, dityrosine bridges in whey protein films are naturally limited due to the low number of tyrosine residues in β-Lg (Etzel, 2004; Wong, Camirand, & Pavlath, 1996). Ultraviolet (UV) radiation can be used for film formation by inducing covalent cross-linking in whey proteins. UV exposure of double bonds and aromatic rings leads to free radical formation of amino acid residues which are able to form new cross-links to generate a protein-film network (Gennadios, Rhim, Handa, Weller, & Hanna, 1998; Rhim, Gennadios, Fu, Weller, & Hanna, 1999). Since increased radiation doses lead to increased interactions, UV-irradiated whey protein-based films show increased strength; however, barrier properties are not significantly influenced (Schmid, Held, Hammann, Schlemmer, & Noller, 2015a; Ustunol & Mert, 2004).

11.4.2

Chemical Modifications

Chemical modification of proteins generally involves reactions with chemical agents or pH alteration (Zink et al., 2016). Chemical reactions include alkylation, acylation, acetylation, and succinylation. However, they are not suitable for significant functionality improvements of whey protein-based films and coatings, due to the used chemical agents lacking food-safety approval. Yet, chemical grafting, meaning the incorporation of fatty acid chlorides by an acylation reaction, could gain importance for whey protein modification. Here, long alkyl chains are integrated to the protein chains and can act as internal plasticizers. Just like external plasticizers, intermolecular interactions between the protein side chains are reduced, resulting in changed thermal properties and folding of the proteins

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(Winkler, Vorwerg, & Schmid, 2015; Zink et al., 2016). Beside the number of available functional groups, the solvent also influences the number of bonded fatty acid chlorides. Incorporation rates for β-Lg, e.g., were higher when organic solvents were used instead of aqueous solutions (Creuzenet et al., 1992). This way of chemical grafting results in higher protein hydrophobicity, giving improved water resistance and lower water absorption and, with regards to films and coatings, improved moisture barriers (Wihodo & Moraru, 2013). Additionally, modified proteins show melting behavior at temperatures between 150 and 200 C and therefore further process and application possibilities (Bräuer, Meister, Gottlöber, & Nechwatal, 2007). Nevertheless, just like other reactions with chemical reagents, alkylated films used for chemical grafting often require toxic agents leading to nonedible films (Ghorpade, Li, Gennadios, & Hanna, 1995). Other chemical modifications can be obtained by pH alteration. Nonenzymatic protein hydrolysis can be acidic as well as alkaline. Protein hydrolysis generally reduces chain length and molecular weight by splitting the initial protein into smaller peptide structures. Another important factor influenced by pH is the protein conformation depending on the net charge when pH differs from the isoelectric point. When pH is lower than the isoelectric point, a positive net charge is present and vice versa (Wihodo & Moraru, 2013). While at pH lower than 3.5 and higher than 8 a monomer is present, octamers are formed between pH 3.5 and 5.2 and dimers occur in the pH range between 5.2 and 8 (Onwulata & Huth, 2008; Verheul, Roefs, & de Kruif, 1998). Consequently, whey protein films derived from formulations with different pH also have different functional properties. While Young’s modulus and stress at break depicted a maximum at pH 7 and 8, the elongation at break increased with increasing pH from 7 to 9 (Anker, Stading, & Hermansson, 1999). Barrier properties are only slightly affected by pH. However, maximum values for both oxygen and water vapor barrier properties depict their maximum at the isoelectric point of around pH 5 (Pérez-Gago & Krochta, 1999; Zink et al., 2016).

11.4.3

Biochemical Modifications

Enzymatic hydrolysis cleaves the peptide bonds in proteins, generating smaller peptide chains or even single amino acids. Peptidases are generally divided into exopeptidases and endopeptidases, depending on their cleavage site within the protein chain. A consequent reduction of the molecular weight results in reduced intermolecular forces along the protein chains, giving increased free volume and higher flexibility of the protein chains (Sothornvit & Krochta, 2000b; Verbeek & van den Berg, 2009). The key parameter to determine protein hydrolysis is the degree of hydrolysis

11.4 Protein Modification for Optimized Film Formation and Performance

(DH), which is defined as the percentage of cleaved peptide bonds in relation to the total number of peptide bonds per protein (Nielsen, Petersen, & Dambmann, 2001). Studies show increased film flexibility with the use of whey protein hydrolysates at constant oxygen permeability (Schmid, Hinz, Wild, & Noller, 2013a; Sothornvit & Krochta, 2000b). As a consequence, the utilization of hydrolyzed whey protein may reduce the amount of technologically necessary plasticizer (Sothornvit & Krochta, 2000b). Other enzymatic modifications for whey protein-based films are made with the use of transglutaminases. Transglutaminases are able to form a ε-(γ-glutamyl) lysine bond by catalyzing the acyl transfer between the γ-carboxyl group of a glutamine side chain and the ε-amino group of a lysine side chain (Ichinose, Bottenus, & Davie, 1990). In this way, peptide chains of the same or of different proteins can be cross-linked, giving more mechanical stability with increased strength to protein-based films. Most transglutaminase cross-linked films also show improved barrier performance against water vapor (DeJong & Koppelman, 2002; Eissa, Puhl, Kadla, & Khan, 2006; Schmid, Müller et al., 2014; Schmid, Sängerlaub et al., 2014; Truong, Clare, Catignani, & Swaisgood, 2004) due to the dense protein network. However, some studies also showed diverse behavior, which could be explained by different formations of the cross-linked network. Further information about protein hydrolysates is given in Chapter 14 of this book.

11.4.4

Film Modifications: Blends and Composites

A common technique used to improve limited properties of polymers such as protein-based films is blending with other polymers, either natural or synthetic. The resulting combination shows mechanical as well as barrier enhancements (Gupta & Nayak, 2015; Plackett, 2011; Schmid, Müller et al., 2014; Schmid, Sängerlaub et al., 2014). Additionally, polymer blending can also offer improved processability, plant flexibility, and quick formulation changes (Utracki, 2003). In addition to conventional methods such as blending with other polymers, the incorporation of nanoparticles or clays into biopolymeric matrices such as whey protein films has become a common modification method to overcome drawbacks (Hassannia-Kolaee, Khodaiyan, Pourahmad, & ShahabiGhahfarrokhi, 2016; Oymaci & Altinkaya, 2016; Zolfi, Khodaiyan, Mousavi, & Hashemi, 2014). Many studies, summarized by Müller et al. (2017), reveal an improvement in film properties by addition of inorganic nanoparticles to whey protein isolate (WPI) based matrices. Just like conventional fillers, nanoparticles give improved mechanical strength to the final films. Additionally, barrier improvements for gases are possible by creating a so-called “tortuous path”

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for the permeating molecules. However, the improvement of film properties highly depends on the particle ratio and geometry, distribution, and orientation within the matrix. Another possibility to enhance the naturally poor water vapor barrier of whey protein-based films is the addition of hydrophobic materials such as lipids and waxes. However, some lipids negatively affect mechanical and optical properties of the films by decreasing film strength and increasing opacity. Studies showed that low lipid or wax contents and smaller particle sizes give the best overall improvements in the protein films (Janjarasskul, Rauch, McCarthy, & Krochta, 2014; Pérez-Gago & Krochta, 2001b; Talens & Krochta, 2005). Apart from approaches for further mechanical and barrier property enhancements, the incorporation of bioactive compounds such as antioxidants or antimicrobial agents for active and intelligent packaging is gaining attention in recent years. Active packaging systems use materials that interact with the packaged goods leading to extended shelf life, improved security, and preserved quality (Koshy, Mary, Thomas, & Pothan, 2015). For example, the use of essential oils, tocopherols, or ascorbic acid showed antioxidant effects in packaged foods, extending their shelf life (Lee & Krochta, 2002; Oussalah, Caillet, Salmiéri, Saucier, & Lacroix, 2004; Pérez-Gago, Serra, & Del Rio, 2006). Edible films with encapsulated antimicrobial agents showed effective growth reduction of inoculated bacteria (Joerger, 2007) and could therefore be able to control growth of pathogenic bacteria in foods.

11.5 11.5.1

PACKAGING RELEVANT PROPERTIES Barrier Properties

The ability of packaging films and coatings to protect packaged goods from negative environmental factors can be determined by barrier property characterization. Water vapor, oxygen, or other gases as well as fluids or flavorings can have a significant impact on the product shelf life when coming into contact with the packaged goods. Depending on the product, high or low barrier properties for certain substances are required. Barrier performance of packaging materials can be determined by permeation measurements. Permeation describes the mass transport of fluids or gases through the packaging material. Permeation is based on the physical process of sorption and/or adsorption of the transported substances in/on the packaging material and the contents, and diffusion through interfaces between packaging and content, or packaging and atmosphere (Piringer, 1993) and can be mathematically described by Henry’s law and Fick’s law.

11.5 Packaging Relevant Properties

Barrier properties of whey protein films or coatings mainly depend on the coating composition, thickness, and the different layers used, such as the substrate. Various studies revealed relatively low oxygen permeability of whey protein-based films, making them potentially useful for coatings or other film material used for oxygen sensitive products (Bugnicourt, Schmid, McNerney, & Wild, 2010; Mate & Krochta, 1996; McHugh & Krochta, 1994; Schmid et al., 2012; Sothornvit & Krochta, 2000a, 2000b). However, poor humidity barriers due to the general intrinsic protein hydrophilicity limit their application. Additionally, induced interaction with water is followed by swelling (Barrer, 1941) and apparent thickness effects (McHugh, Avenabustillos, & Krochta, 1993) leading to a deviation from Henry’s law and Fick’s law (Avena-Bustillos & Krochta, 1993; Barrer, 1941; Schmid, Müller et al., 2014; Schmid, Sängerlaub et al., 2014).

11.5.1.1 Water Vapor Permeability Sensory quality and food shelf life are critically affected by water activity. Not only the extent of microbial growth and chemical and enzymatic reactions that can take place during storage depend on present water activity, but also other sensory factors such as textural appearance. Thus, water vapor permeability is an important property for packaging characteristics. Films and coatings derived from whey proteins display relatively high permeability for water vapor owing to the hydrophilic character of the proteins. Generally, denser film networks, depending on the degree of cross-linking or film modification, result in lower water permeability. Relative humidity of films and coatings as well as the chosen plasticizer loosen the network by generating free volume and reducing protein chain interactions and therefore significantly influence the moisture permeation properties (Khwaldia, Perez, Banon, Desobry, & Hardy, 2004; Pérez-Gago & Krochta, 2002). To reduce water vapor permeability, hydrophobic compounds can be included in the film formulation. This can take place either by the addition of lipids or waxes to the film-forming solution itself, or by lamination of the film with a lipid layer (Ramos et al., 2012). However, for products such as fresh fruit and vegetables, where respiration takes place during storage, water vapor condensation, being the main factor for microbial spoilage, can be prevented with nonmodified whey proteinbased packaging.

11.5.1.2 Oxygen Permeability Many decomposition reactions in food and other products are oxygenrelated. Fat rancidity, microbial growth, enzymatic browning, and vitamin loss are just some examples. Hence, many products require oxygen-protective packaging. For fresh fruit and vegetable products, however, oxygen and

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carbon dioxide are essential for respiration during storage and demand moderate barrier packaging. Protein-based films usually display lower oxygen permeability than conventional synthetic polymers such as polyethylene under similar conditions (Pérez-Gago & Krochta, 2002). Compared to other proteins such as soy or wheat gluten, whey protein-based films and coatings present higher oxygen-barrier performance (McHugh & Krochta, 1994). Further enhancements can be realized by protein modifications and formulation-related optimizations such as plasticizer type and content, since the affected polymer free volume directly influences oxygen permeability (Ramos et al., 2012).

11.5.2

Mechanical Properties

Packaging materials have to withstand mechanical stress due to transportation, storage, and consumers themselves. This can lead to diminished containment protection. Since protection is the first and most important purpose of the packaging material, determination of its mechanical properties is indispensable. To prevent breaks or cracks, a material with high tensile properties, such as tensile strength and elongation at break, is demanded. Mechanical properties of whey protein-based films and coatings mainly depend on film-forming conditions, meaning the formulation itself and applied processes. Furthermore, film thicknesses as well as the testing conditions directly influence measured values. As for conventional packaging materials, tensile tests are mainly used to determine characteristic properties such as tensile strength, elongation, or Young’s modulus (Ramos et al., 2012). Tensile strength is defined as the first stress maximum during a tensile test, while maximum sample elongation is specified as the percentage of original length before breaking. The modulus gives information about the stiffness of a material and is measured at low strains in a linear part of the stress strain curve. Two main factors highly influence mechanical properties of whey proteinbased films. First of all, the three-dimensional protein network is formed during film processing. An intensive cross-linked network leads to stronger and stiffer materials, resulting in higher modulus and strength. However, this is often accompanied by lower elongations since the material also becomes less flexible. Using cross-linkers, such formaldehyde or glutaraldehyde, mechanical strength of glycerol-plasticized WPI films could be significantly enhanced (Galietta, Di Gioia, Guilbert, & Cuq, 1998; Ustunol & Mert, 2004). However, those agents are often not suitable for food packaging due to lacking food safety approval.

11.5 Packaging Relevant Properties

A second aspect is the plasticizers present, including water. Therefore, relative humidity of the film as well as storage conditions are factors that cannot be discounted and should be controlled. Relative humidity considerably affects mechanical properties by causing reduced modulus and strength and increased elongation values of the films and coatings when the water content is increased (Guilbert, Cuq, & Gontard, 1997; Ramos et al., 2012; Wu, Weller, Hamouz, Cuppett, & Schnepf, 2002). The same effect can be seen with other plasticizers. With increasing content, materials become less rigid resulting in lower tensile strength and hence more flexibility giving extended elongation values (Wihodo & Moraru, 2013). This effect can be related to fewer intermolecular interactions between the protein chains due to plasticizer incorporation. Not only the ratio, but also the type of plasticizer has a major impact on the mechanical performance. Using different plasticizers for β-Lg films, it was shown that glycerol gave the highest elongation increase and tensile strength decrease, followed by polyethylene glycol (200), sucrose and polyethylene glycol (400) (Sothornvit & Krochta, 2001). Films with other plasticizers like sorbitol and xylitol also showed significantly lower tensile strength in comparison to glycerol-plasticized WPI-based films (Shaw, Monahan, O’Riordan, & O’Sullivan, 2002). Schmid et al. (2013a) showed that hydrolyzed whey protein isolate (HWPI) can act as an internal plasticizer. Increasing the HWPI concentration in whey protein-based films at constant glycerol concentrations increased film flexibility significantly while barrier properties were maintained. Schmid (2013) considered these facts and developed formulations with increased barrier performance while maintaining film flexibility. Glycerol was partially replaced by HWPI as the internal plasticizer in WPI-based cast films resulting in improved oxygen and water vapor barrier properties, while the maintaining the mechanical properties. With this approach film flexibility was maintained, even though the external plasticizer concentration was decreased (Schmid et al., 2013a; Schmid, 2013).

11.5.3

Surface Properties

Especially for coatings but also for multilayer systems, cohesion and adhesion on surfaces are decisive. Cohesion of the polymer itself results from strong interactions and formation of bonds between the polymer chains, and the associated prevention of separation. This mainly depends on the polymeric structure itself including its geometry, molecular weight distribution, type and position of lateral functional groups, and general molecular strength (Guilbert, Gontard, & Gorris, 1996). Like most hydrophilic edible coatings, the adhesion of whey protein films is relatively low per se. Therefore, surfactants that reduce surface tension can be used to increase

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compatibility between substrate and coating (Lin & Krochta, 2005; Ramos et al., 2012). Additionally, hydrophobicity of whey protein films was also increased by using microbial transglutaminase, indicating an exposure of hydrophobic groups due to moderate cross-linking (Tang & Jiang, 2007). Whey protein-based coatings adhere very well to polyesters such as PET, PBAT, or PLA but also to corona pretreated polyolefins such as PP or PE (Cinelli et al., 2014; Schmid et al., 2012; Schmid et al., 2015b; 2015c).

11.5.4

Optical Properties

Optical properties of a material such as transparency, color, and even ultraviolet- or light-barrier properties are important features, especially when packaging applications are concerned. Especially polymers based on natural raw materials, such as whey proteins, often contain organic colored molecules. Since human color perception is not a physical quantity, it is not measurable by normal engineering methods. However, colors can be described in an objective manner by quantifying them with three distinctive color values in a three-dimensional color space (Klein, 2010) such as the CIE L*a*b* system, Hunter Lab, CIE LCH, or CIE XYZ. Colorimeters using such systems can calculate color differences ΔE* between two color points in the color space. Compared to conventionally used synthetic polymers, whey protein-based films and coatings exhibit comparable transparency (Ramos et al., 2012; Schmid et al., 2012) and slight color differences due to contained yellow metabolites (Schmid et al., 2013a). However, the final properties are also a function of film thickness.

11.6 POTENTIAL APPLICATIONS OF WHEY PROTEINBASED FILMS AND COATINGS IN FOOD PACKAGING 11.6.1

Packaging Requirements of Food Products

For maintaining quality and shelf life of products, especially in the food industry, packaging materials have to meet high standards. The primary responsibility of food packaging is to protect the packaged food from environmental impact along its whole product life cycle. Therefore, packaging does not only have to withstand mechanical stress during transport and consumer handling but also environmental effects such as product contamination and factors leading to deteriorative reactions. Besides sufficient mechanical stability, materials have to display specific barrier properties with respect to light, moisture, and gases such as water vapor or oxygen, depending on the type of packaged food. Packaging atmosphere including reasonable oxygen and carbon dioxide levels as well as respiration rates have to be considered for optimal food preservation and prevention of fat oxidation,

11.6 Potential Applications of Whey Protein-Based Films and Coatings in Food Packaging

microbial deterioration, and deviation of taste or color (Petersen et al., 1999; Schmid et al., 2012). Fig. 11.1 shows gas barrier requirements of different pharmaceutical and food products. Generally, dry products have a long shelf life due to their low water activity. Therefore, dry conditions can be maintained with good moisture barrier packaging. Oxygen-sensitive products such as fat- and oil-containing foods require light- and oxygen-excluding packaging to prevent oxidative rancidity. Fresh fruits and vegetables continue respiration during storage demanding varying packaging systems to control atmosphere. Fresh meat products require either high or low oxygen levels to assure red color, which can be obtained with modified atmosphere packaging (MAP) or vacuum packaging, both demanding high oxygen-barrier materials. Gas compositions vary depending on the type of meat. Seafood, egg, and dairy products also require high oxygen-barrier materials to prevent harmful oxidation processes and microbial growth (Petersen et al., 1999).

Ketchup, sauces Nuts, snacks

Tablets (blister)

100

(at 23°C, 50% r.h.)

Oxygen permeability / (cm3d–1m–2bar–1)

1000

Cooking oil

10

Vakuum-Kaffee Instant coffee Meat/MAP

1

UHT milk Vacuum coffee 0.1

Special, infusion, baby food

Beer

0.01 0.01

0.1

1

10

100

Water vapor transmission rate / (gd–1m–2) at 23°C, 85% r.h.

FIGURE 11.1 Gas barrier requirements of selected pharmaceutical and food products. Adapted from Schmid, M., Dallmann, K., Bugnicourt, E., Cordoni, D., Wild, F., Lazzeri, A., & Noller, K. (2012). Properties of whey protein coated films and laminates as novel recyclable food packaging materials with excellent barrier properties. International Journal of Polymer Science, 2012, Article 562381.

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11.6.2 Merging Food Packaging Requirements and Properties of Whey Protein-Based Films and Coatings To match those requirements, a combination of different materials is necessary to optimize techno-functional properties, usually performed by multilayer films. However, multilayers obtained from coextrusion or lamination processes are often expensive and use fossil-fuel-based raw materials. Especially regarding oxygen-sensitive products, ethylene vinyl alcohol copolymers (EVOH) are widely used. Furthermore, the combination of many different layers complicates recycling processes that mainly rely on monomaterials of high purity (Endres & Siebert-Raths, 2011). Thus, sustainable packaging materials suitable to replace present conventionally used plastics are part and parcel of today’s research and development. Among others, whey proteinbased films and coatings displaying excellent oxygen barriers can provide a bio-based and biodegradable solution for such composite films (Schmid et al., 2012). One example to name is the project WHEYLAYER (http://www. wheylayer.eu/) which developed whey protein-coated films and laminates with high oxygen-barrier performance and enhanced recyclability due to the biodegradable whey protein-based layer (Schmid, Bischur, Wild, & Noller, 2009). Fig. 11.2 shows the barrier performance of commonly used plastic materials for packaging as well as the WPI-based Wheylayers material. Bugnicourt et al. (2013) presented a study which demonstrated the scalability of the production of a whey protein-based coating. This was a preliminary requirement for its commercialization. The new bio-based coating solution was formulated using WPI and plasticizers to prevent brittleness. The most promising formulations among those evaluated by Schmid et al. (2012) at pilot scale were selected for scale-up. The coating was performed on PET at semi-industrial rates with optimized processing parameters for minimized energy consumption in order to reduce the environmental impact of this manufacturing stage as much as possible. This patented process also allows the correct denaturation of the protein-based coating on PET substrate films for improved barrier properties (Schmid et al., 2013b). After lamination with PE as a sealing and potential food contact layer, a full laminate structure suitable for packing sensitive food products was ready to be characterized. Whey protein-coated films and laminates achieved much better barrier properties compared to other bioplastics. The results also indicate that the oxygen barrier values of whey protein-based coatings approached those of EVOH with high ethylene content and were higher than the oxygen barrier properties of polyamide (PA). The produced laminates were used for food packaging and storage validation with very positive results. Several studies (Bugnicourt et al., 2013; Schmid et al., 2012) have proved the suitability of the whey protein-based coatings for packaging applications and show the potential of substituting other synthetic barrier layers used for food

11.7 Food Safety and Regulatory Aspect

10000

PS

1000

PE-HD

PP

PVC-P

PC

BOPP COC

(at 23°C, 50% r.h.)

3 –1 –2 –1 Oxygen permeability/ (cm d m bar )

PE-LD

PLA

100 PVC-U PET

10

PA 6

PAN PEN

1

Wheylayer PVDC

EVOH, 38%

0.1

Cellulose

EVOH, 44% EVOH, 32% EVOH, 27%

(LCP)

0.01 0.01

0.1

1

10 –1 –2 Water vapor transmission rate / (gd m )

100

1000

at 23°C, 85% r.h.

FIGURE 11.2 Barrier performance of selected packaging materials as well as the Wheylayers material. Adapted from Schmid, M., Dallmann, K., Bugnicourt, E., Cordoni, D., Wild, F., Lazzeri, A., & Noller, K. (2012). Properties of whey protein coated films and laminates as novel recyclable food packaging materials with excellent barrier properties. International Journal of Polymer Science, 2012, Article 562381.

packaging. In terms of validation of the obtained material for food packaging, laminates derived from whey protein-coated films were tested for storing different sensitive food products. The storage tests showed similar results regarding shelf life and sensory quality when compared to conventional reference packaging (Bugnicourt et al., 2013). Additional tests also showed those laminates fulfilled food contact compliance regulations according to the EU regulation 10/2011 in terms of global migration.

11.7

FOOD SAFETY AND REGULATORY ASPECT

Using whey protein films and coatings, applications include direct, edible coatings, coatings on substrates, and stand-alone films. Therefore, they match different categories such as food contact materials, ingredients, food additives, or even food products, resulting in different regulation aspects

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(Debeaufort, Quezada-Gallo, & Voilley, 1998). Therefore, different legislation must be applied depending on the respective case. When used as edible coating, the components of the coating formulation are usually food grade and are regulated as additives by legislation such as Regulation (EC) No. 1333/2008. In terms of other regulatory requirements, it must be considered that milk proteins used for films and coatings are possible food allergens. Therefore, especially regarding edible coatings, allergens must be clearly labeled (Gennadios, Hanna, & Kurth, 1997). In the case of coatings on other substrates or when used for flexible films, protein-based packaging is regarded as food-contact material. Within the European Union (EU) those materials are primarily covered by Framework Regulation 1935/2004 (EC). Following good manufacturing practice, components that could endanger human health, cause intolerable food composition changes or cause a decline in organoleptic properties should not exceed respective quantities when transferred to the food. Regarding plastic packaging, materials and additives approved for food contact and eventual migration limits are given for the EU in EC 10/2011. However, many substances used for protein-based films and coatings are not listed. (Coltelli et al., 2016) In summary, respective regulatory aspects always depend on formulations and application and need to be considered separately.

11.8 END-OF-LIFE OPTIONS FOR WHEY PROTEINBASED FILMS AND MULTILAYER LAMINATES According to Badia, Gil-Castell, and Ribes-Greus (2017) long-term properties and end-of-life of polymers are not antagonist issues. They are linked by durability and degradation. The combination of appropriate valorization techniques, i.e., material, energetic, and/or biological at the most suitable stage, should be targeted. The consideration of the end-of-life of a material for a specific application should be the fundamental focus (Badia et al., 2017). This chapter covers two key end-of-life options for whey protein-based films and multilayer laminates, namely biodegradation and recycling.

11.8.1 Biodegradation of Whey Protein-Based Films and Laminates This section deals with the valorization of whey protein-based coatings on biodegradable plastics from the point of view of the reincorporation of polymers into the carbon cycle under biotic conditions. According to Badia et al. (2017), the design of plastic materials for consumer applications such as

11.8 End-of-Life Options for Whey Protein-Based Films and Multilayer Laminates

packaging is moving towards the design of polymers with controlled degradability and enhanced bioreintegration. Biodegradation of plastics can occur through different stages, which erode and disintegrate the polymeric segments by depolymerization followed by reintegration into the carbon cycle by assimilation and mineralization in the media. Three main stages can be distinguished: 1. Deterioration 2. Fragmentation 3. Assimilation These stages are described in detail by Badia et al. (2017). Biodegradation and environmental degradation aspects of proteins in general were summarized by Coltelli et al. (2016) and Cinelli et al. (2014) specifically studied how a whey protein-based coating can improve the oxygen-barrier properties of commercial compostable plastic film while not hindering the biodegradation nor harming the quality of the compost. They applied a whey protein-based coating on to a biodegradable commercial film which was certified to meet the requirements of the composting standard DIN 13432. They found that the oxygen barrier was significantly improved by the WPI-based coating. This is of specific interest since biodegradable packaging films generally do not retain their barrier properties and the application of nondegradable materials to improve gas barriers compromises the composting of the final packaging concepts. They also assessed the biodegradability of the whey protein layer itself as natural polymers may become nonbiodegradable when cross-linked or bonded with nondegradable additives. They found that the material based on denatured WPI and plasticizer presented very fast biodegradability, also in combination with a commercial film. It can be concluded that these results show the huge potential of whey protein-based films and laminates as part of new ecological food packaging concepts (Cinelli et al., 2014). Li and Chen (2000) investigated the biodegradability of WPI and whey protein concentrate (WPC) films from glycerol-plasticized aqueous solutions. They used Pseudomonas aeruginosa under commercial composting conditions. WPC-based films degraded faster than those obtained from WPI, which might show dependence on film composition and extent of cross-linking. Under commercial composting conditions, WPI-based films lost more than 80% of total solids within 7 days (Coltelli et al., 2016; Li & Chen, 2000).

11.8.2 Recyclability of Whey Protein-Based Multilayer Laminates Polymer-based multilayer packaging materials are commonly used in order to combine the respective properties of different polymers. With this

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approach, tailored functionality of packaging concepts is created to sufficiently protect sensitive food products and to obtain extended shelf life. Due to their poor recyclability, most multilayers are incinerated or landfilled, counteracting efforts towards a circular economy. In the waste hierarchy, mechanical recycling is number three, after waste avoidance and reuse but should be favored to energy recovery and disposal. According to Lazarevic, Aoustin, Buclet, and Brandt (2010) recycling generally is the environmentally preferred treatment option when compared to municipal solid waste incineration. Even though multilayer plastic films provide a range of properties, their recyclability is still an open issue and should be improved. As already reported in detail in this chapter, the possibility exists of using whey protein-based coatings as an excellent barrier against oxygen. Whey protein-based coating is capable of replacing petrochemical nonrecyclable materials such as EVOH in multilayer laminates. Cinelli, Schmid, Bugnicourt, Coltelli, and Lazzeri (2016) followed an innovative approach in their study in order to achieve recyclability of the substrate films in multilayer laminates by separating them. They used a simple process compatible with industrial procedures, in order to promote recycling processes leading to high-value products. This could potentially beneficially impact the packaging and food industries. They prepared polyethylene terephthalate (PET)/polyethylene (PE) multilayer laminate films based on PET coated with a whey protein layer. This structure was then laminated with PE. As whey proteins constituting the coatings can be degraded by enzymes, the coatings can be washed off the plastic substrate layer. The enzyme types, dosage, time, and temperature optima used, are compatible with procedures in industrial waste recycling. The separation of the respective films of the samples based on PET/whey and PET/whey/PE were efficient when performed with an enzymatic detergent containing proteases. Different types of enzymatic detergents presented positive results in removing the whey protein-based layer from the PET substrate and from the PET/whey/PE multilayer films. These results showed the possibility of separating the whey-based multilayer film by washing with different available commercial enzymatic detergents. This allows a better recycling of the two different polymers. Mechanical properties of the plastic substrates (e.g., stress at yield, stress, and elongation at break), did not significantly change by the washing and separation process with enzymatic detergents (Cinelli et al., 2016). This enables an efficient multilayer recycling process in the near future. Certainly, it will take time until enough whey protein-based plastic laminate materials are available in the market at quantities sufficiently high to be worthwhile to sort and separate from waste streams. However, if protein- and in particular whey protein-based, multilayer laminates are available they potentially could be recycled according to the process mentioned above.

11.9 Conclusions and Industrial Perspectives

11.9

CONCLUSIONS AND INDUSTRIAL PERSPECTIVES

In contrast to standard plastics derived from fossil-fuel sources, bio-based polymers are from renewable biomass sources. The use of this class of materials is not new. In fact, e.g., back in the 1940s, Ford introduced a motor car body fully made of cellulose fibre and resin extended with by-products of the soybean oil extraction process. However, later the interest in this class of materials was reduced with the development of more durable and resistant fossil-fuel based plastics. Now, research has been intensified in the field of biopolymers revealing a large range of possible resources and extending the spectra of applications. Thanks to the improvement in their properties, biopolymers are more competitive with their synthetic counterparts. The sources for naturally occurring biopolymers range from proteins (animal- or plant-sourced) to lipids and polysaccharides (e.g., starch- and cellulose-based biopolymers). Some biobased polyesters such as polyhydroxyalkanoates (PHAs) are naturally produced in microbial cultures. Other biopolymers, such as polylactic acid (PLA), are produced from bio-derived monomers (e.g., lactic acid from corn starch) which are then polymerized. The same principle applies to the newly commercially available bio-based polyethylene terephthalate (Bio-PET) or polyethylene (Bio-PE). The so-called “drop-in solutions” are now the biggest segment in terms of production volume among biopolymers. Packaging is the biggest market for the plastics industry with a share of approximately 40% of all plastic produced in Europe. New developments in that sector are driven by legislation and market requirements such as weight reduction, recyclability, waste reduction, and utilization of sustainable/ renewable raw materials. When providing similar properties as their synthetic counterparts, biopolymers could provide a solution for conserving depleting fossil-fuel resources, and reducing CO2 emissions and environmental pollution in the case of biodegradability as an end-of-life option. However, research into the development of tailor-made solutions for this sector is a key because most biopolymers do not meet the requirements of packaging for sensitive food products due to their low barrier properties and thus do not guarantee product quality throughout its shelf life. Requirements in terms of barriers against light and gases are specific to the type of food packed. To obtain the optimal combination of final properties, multilayer laminates are commonly used. However, due to the impossibility of separating the materials during recycling operations, the recyclability of multilayer packaging is often economically not possible. As described in this chapter, the development of a biopolymer-based coating for plastic films based on whey protein is able to replace current synthetic

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oxygen-barrier layers used in food packaging such as EVOH. These developments are not far from reaching the market. Since most whey is a by-product of cheese manufacturing, it is not in direct food competition. In summary, it seems to be obvious that, especially in the field of packaging, the ecological advantages of biopolymers, such as whey protein-based films and coatings, are significant over traditional plastics. Bioplastics are produced from an increasing range of renewable resources including wastes and/or by-products which are not in competition with food. The research on bioplastics is very dynamic and there is still a lot of progress to be made. For example, the whey protein-based films and coatings described in this chapter exhibited excellent optical and barrier properties outperforming existing biopolymers. Whey protein-based films and coatings seem to be very promising as a replacement for synthetic barrier polymers used in food packaging concepts. Whey protein-based multilayer laminates were positively validated for storing various food products. This novel whey protein-based coating can be removed to allow multilayer films to become recyclable. Whey protein-based packaging concepts could provide a valuable contribution to sustainability due to the possibility of recycling materials as opposed to incinerating, as is done for laminates containing EVOH or PA, but also due to the utilization of bio-based raw materials which are a by-product from the agro-food industry.

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Molinaro, S., Cruz-Romero, M., Sensidoni, A., Morris, M., Lagazio, C., & Kerry, J. P. (2015). Combination of high-pressure treatment, mild heating and holding time effects as a means of improving the barrier properties of gelatin-based packaging films using response surface modeling. Innovative Food Science & Emerging Technologies, 30, 15 23. Morr, C., & Ha, E. (1993). Whey protein concentrates and isolates: Processing and functional properties. Critical Reviews in Food Science & Nutrition, 33, 431 476. Müller, K., Bugnicourt, E., Latorre, M., Jorda, M., Echegoyen Sanz, Y., Lagaron, J., . . . Schmid, M. (2017). Review on the processing and properties of polymer nanocomposites and nanocoatings and their applications in the packaging, automotive and solar energy fields. Nanomaterials, 7, Article74. Nicolai, T., Britten, M., & Schmitt, C. (2011). β-Lactoglobulin and WPI aggregates: Formation, structure and applications. Food Hydrocolloids, 25, 1945 1962. Nicorescu, I., Loisel, C., Vial, C., Riaublanc, A., Djelveh, G., Cuvelier, G., et al. (2008). Combined effect of dynamic heat treatment and ionic strength on the properties of whey protein foams Part II. Food Research International, 41, 980 988. Nielsen, P. M., Petersen, D., & Dambmann, C. (2001). Improved method for determining food protein degree of hydrolysis. Journal of Food Science, 66, 642 646. Olsen, K., Ipsen, R., Otte, J., & Skibsted, L. H. (1999). Effect of high pressure on aggregation and thermal gelation of β-lactoglobulin. Milchwissenschaft, 54, 543 546. Onwulata, C., & Huth, P. (2008). Whey processing, functionality and health benefits. Ames, IA: John Wiley & Sons. Oussalah, M., Caillet, S., Salmiéri, S., Saucier, L., & Lacroix, M. (2004). Antimicrobial and antioxidant effects of milk protein-based film containing essential oils for the preservation of whole beef muscle. Journal of Agricultural & Food Chemistry, 52, 5598 5605. Oymaci, P., & Altinkaya, S. A. (2016). Improvement of barrier and mechanical properties of whey protein isolate based food packaging films by incorporation of zein nanoparticles as a novel bionanocomposite. Food Hydrocolloids, 54, 1 9. Parris, N., Purcell, J. M., & Ptashkin, S. M. (1991). Thermal denaturation of whey proteins in skim milk. Journal of Agricultural & Food Chemistry, 39, 2167 2170. Pérez-Gago, M., Serra, M., & Del Rio, M. (2006). Color change of fresh-cut apples coated with whey protein concentrate-based edible coatings. Postharvest Biology & Technology, 39, 84 92. Pérez-Gago, M. B., & Krochta, J. M. (1999). Water Vapor Permeability of Whey Protein Emulsion Films as Affected by pH. Journal of Food Science, 64, 695 698. Pérez-Gago, M. B., & Krochta, J. M. (2001a). Denaturation time and temperature effects on solubility, tensile properties, and oxygen permeability of whey protein edible films. Journal of Food Science, 66, 705 710. Pérez-Gago, M. B., & Krochta, J. M. (2001b). Lipid particle size effect on water vapor permeability and mechanical properties of whey protein/beeswax emulsion films. Journal of Agricultural & Food Chemistry, 49, 996 1002. Pérez-Gago, M. B., & Krochta, J. M. (2002). Formation and properties of whey protein films and coatings. In A. Gennadios (Ed.), Protein-based films and coatings (pp. 159 180). Boca Raton: CRC Press. Petersen, K., Nielsen, P. V., Bertelsen, G., Lawther, M., Olsen, M. B., Nilsson, N. H., & Mortensen, G. (1999). Potential of biobased materials for food packaging. Trends in Food Science & Technology, 10, 52 68. Phillips, L. G., German, J. B., O'Neill, T. E., Foegeding, E. A., Harwalkar, V. R., Kilara, A., et al. (1990). standardized procedure for measuring foaming properties of three proteins, a collaborative study. Journal of Food Science, 55, 1441 1444.

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Piringer, O. G. (1993). Verpackungen für Lebensmittel: Eignung, Wechselwirkungen, Sicherheit. Weinheim: Wiley-VCH. Plackett, D. (2011). Biopolymers: New materials for sustainable films and coatings. Chichester: John Wiley and Sons. Ramos, Ó. L., Fernandes, J. C., Silva, S. I., Pintado, M. E., & Malcata, F. X. (2012). Edible films and coatings from whey proteins: A review on formulation, and on mechanical and bioactive properties. Critical Reviews in Food Science & Nutrition, 52, 533 552. Rastogi, V., & Samyn, P. (2015). Bio-based coatings for paper applications. Coatings, 5, 887. Renkema, J. M., Lakemond, C. M., Jongh, H. H. d, Gruppen, H., & van Vliet, T. (2000). The effect of pH on heat denaturation and gel forming properties of soy proteins. Journal of Biotechnology, 79, 223 230. Rhim, J.-W., Gennadios, A., Fu, D., Weller, C. L., & Hanna, M. A. (1999). Properties of Ultraviolet Irradiated Protein Films. LWT - Food Science & Technology, 32, 129 133. Riezs, P., & Kondo, T. (1992). Free radical formation induced by ultrasound and its biological implications. Free Radical Biology & Medicine, 13, 247 270. Sabato, S. F., Ouattara, B., Yu, H., D’Aprano, G., Le Tien, C., Mateescu, M. A., et al. (2001). Mechanical and barrier properties of cross-linked soy and whey protein based films. Journal of Agricultural & Food Chemistry, 49, 1397 1403. Schmid, M. (2013). Properties of cast films made from different ratios of whey protein isolate, hydrolysed whey protein isolate and glycerol. Materials, 6, 3254 3269. Schmid, M., Bischur, G., Wild, F., & Noller, K. (2009). Whey coated plastic films to replace expensive polymers and increase recyclability. Paper presented at the 12th Tappi European Place Conference, 18-20 May 2009, Budapest, Hungary, Budapest. Schmid, M., Dallmann, K., Bugnicourt, E., Cordoni, D., Wild, F., Lazzeri, A., & Noller, K. (2012). Properties of whey protein coated films and laminates as novel recyclable food packaging materials with excellent barrier properties. International Journal of Polymer Science, 2012, Article 562381. Schmid, M., Hinz, L.-V., Wild, F., & Noller, K. (2013a). Effects of hydrolysed whey proteins on the techno-functional characteristics of whey protein-based films. Materials, 6, 927. Schmid, M., Noller, K., Wild, F., & Bugnicourt, E. (2013b). Whey protein coated films: WO/ 2013/014493. Schmid, M., Müller, K., Sängerlaub, S., Stäbler, A., Starck, V., Ecker, F., & Noller, K. (2014). Mechanical and barrier properties of thermoplastic whey protein isolate/ethylene vinyl acetate blends. Journal of Applied Polymer Science, 131, Article41172. Schmid, M., Sängerlaub, S., Wege, L., & Stäbler, A. (2014). Properties of transglutaminase crosslinked whey protein isolate coatings and cast films. Packaging Technology & Science, 27, 799 817. Schmid, M., Held, J., Hammann, F., Schlemmer, D., & Noller, K. (2015a). Effect of UV-radiation on the packaging-related properties of whey protein isolate based films and coatings. Packaging Technology & Science, 28, 883 899. Schmid, M., Reichert, K., Hammann, F., & Stäbler, A. (2015b). Storage time-dependent alteration of molecular interaction property relationships of whey protein isolate-based films and coatings. Journal of Materials Science, 50, 4396 4404. Schmid, M., Zillinger, W., Müller, K., & Sängerlaub, S. (2015c). Permeation of water vapour, nitrogen, oxygen and carbon dioxide through whey protein isolate based films and coatings 2 Permselectivity and activation energy. Food Packaging & Shelf Life, 6, 21 29.

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Schmid, M., Herbst, C., Müller, K., Stäbler, A., Schlemmer, D., Coltelli, M.-B., et al. (2016). Effect of potato pulp filler on the mechanical properties and water vapor transmission rate of thermoplastic WPI/PBS blends. Polymer-Plastics Technology and Engineering, 55, 510 517. Shaw, N. B., Monahan, F. J., O’Riordan, E. D., & O’Sullivan, M. (2002). Physical properties of WPI films plasticized with glycerol, xylitol, or sorbitol. Journal of Food Science, 67, 164 167. Sothornvit, R., & Krochta, J. M. (2000a). Oxygen permeability and mechanical properties of films from hydrolyzed whey protein. Journal of Agricultural & Food Chemistry, 48, 3913 3916. Sothornvit, R., & Krochta, J. M. (2000b). Water vapor permeability and solubility of films from hydrolyzed whey protein. Journal of Food Science, 65, 700 703. Sothornvit, R., & Krochta, J. M. (2001). Plasticizer effect on mechanical properties of β-lactoglobulin films. Journal of Food Engineering, 50, 149 155. Suslick, K. S., Casadonte, D. J., Green, M. L. H., & Thompson, M. E. (1987). Effects of high intensity ultrasound on inorganic solids. Ultrasonics, 25, 56 59. Talens, P., & Krochta, J. M. (2005). Plasticizing effects of beeswax and carnauba wax on tensile and water vapor permeability properties of whey protein films. Journal of Food Science, 70, E239 E243. Tang, C.-H., & Jiang, Y. (2007). Modulation of mechanical and surface hydrophobic properties of food protein films by transglutaminase treatment. Food Research International, 40, 504 509. Taylor, S. M., & Fryer, P. J. (1994). The effect of temperature/shear history on the thermal gelation of whey protein concentrates. Food Hydrocolloids, 8, 45 61. Tedford, L.-A., & Schaschke, C. J. (2000). Induced structural change to β-lactoglobulin by combined pressure and temperature. Biochemical Engineering Journal, 5, 73 76. Thomas, C. R., & Geer, D. (2011). Effects of shear on proteins in solution. Biotechnology Letters, 33, 443 456. Tracton, A. A. (2005). Coatings technology handbook (3rd ed). Boca Raton: CRC Press. Truong, V. D., Clare, D. A., Catignani, G. L., & Swaisgood, H. E. (2004). Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase. Journal of Agricultural & Food Chemistry, 52, 1170 1176. Ustunol, Z., & Mert, B. (2004). Water solubility, mechanical, barrier, and thermal properties of cross-linked whey protein isolate-based films. Journal of Food Science, 69, FEP129-FEP133. Utracki, L. A. (2003). Polymer blends handbook. Dordrecht: Kluwer Academic Publishers. Verbeek, C. J., & van den Berg, L. E. (2009). Recent developments in thermo-mechanical processing of proteinous bioplastics. Recent Patents on Materials Science, 2, 171 189. Verbeek, C. J. R., & van den Berg, L. E. (2010). Extrusion processing and properties of proteinbased thermoplastics. Macromolecular Materials & Engineering, 295, 10 21. Verheul, M., Roefs, S., & de Kruif, K. G. (1998). Kinetics of heat-induced aggregation of betalactoglobulin. Journal of Agricultural & Food Chemistry, 46, 896 903. Wihodo, M., & Moraru, C. I. (2013). Physical and chemical methods used to enhance the structure and mechanical properties of protein films: A review. Journal of Food Engineering, 114, 292 302. Winkler, H., Vorwerg, W., & Schmid, M. (2015). Synthesis of hydrophobic whey protein isolate by acylation with fatty acids. European Polymer Journal, 62, 10 18. Wolz, M., Mersch, E., & Kulozik, U. (2016). Thermal aggregation of whey proteins under shear stress. Food Hydrocolloids, 56, 396 404. Wong, D. W. S., Camirand, W. M., & Pavlath, A. E. (1996). Structures and functionalities of milk proteins. Critical Reviews in Food Science & Nutrition, 36, 807 844.

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Whey Proteins in Infant Formula Mark A. Fenelon, Rita M. Hickey, Aoife Buggy, Noel McCarthy and Eoin G. Murphy Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

12.1

INTRODUCTION

The global nature of the infant formula (IF) industry is underpinned by strongly branded nutritional products of multinational companies and provides whey ingredient producers with a vital route to market across large geographical regions. This is recognized within individual countries as an important channel for utilization of whey from cheese and other products. Technological advancements in concentration and separation (covered in Chapters 2 and 3 of this book) have led to valorization of the cheese process through the development of technologies such as demineralization in the early 1960s. This process can combine ion-exchange and electrodialysis to remove minerals from whey and has been instrumental in the development of 1st stage (0
6 months) IF, often referred to as whey-dominant formulas. IF can be categorized into 1st stage (0
6 months), 2nd stage (6
24 months), and follow-on formulas (.24 months), and can use different levels of whey protein based on nutritional requirements. Typically, 1st stage infant formulations are based on bovine skim milk solids mixed with whey protein, lactose, vegetable oils, and mineral/vitamin premixes in ratios designed to mimic the nutritional profile of human milk. Bovine milk differs from human milk in its total protein concentration, whey protein to casein ratio, and amino acid profile, and hence the need to fortify with additional whey protein. Similarly, developments in ingredient technology have enabled the use of high-protein (.80% protein) whey protein concentrates (WPC) and whey protein isolates (WPI) in IF. The calculations used to assess the minimum levels of protein required for IF are based around simulating the amino acid composition of human milk. The whey ingredients used in IF can have different forms; e.g., the source of whey protein, can be either as a WPC, 439 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00013-8 © 2019 Elsevier Inc. All rights reserved.

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WPI, and/or whey protein hydrolysate (WPH), in either liquid of powdered form. Recent technological advances in microfiltration have led to the development of a whey type referred to as “native whey” or “micellar casein whey,” produced by membrane separation (microfiltration and ultrafiltration) of skim milk and this can also be incorporated into infant formulations. Bovine milk has a different composition compared to human milk, i.e., in both type and content of fat, protein, carbohydrate, minerals, and vitamins (Table 12.1). Particular interest has surrounded the production of whey protein-dominant IF, the nutritional content of which must closely mimic that of human milk. Bovine milk ingredients are the most common source of nonfat constituents in IF (Nasripour, Scher, & Desorby, 2006) and target the same available quantity of each individual, and some conditionally indispensable, amino acids as is in human milk to attain the protein requirements needed for sufficient growth and development of an infant (European Commission, 2006). When bovine skim milk, for example is used as an ingredient in 1st stage IF manufacture, protein and mineral contents must be reduced. This can typically be achieved by addition of lactose and/or demineralized whey powder (DWP; .80% w/w lactose). Addition of DWP and/or WPC changes the casein to whey protein ratio from that of bovine milk (approximately 80:20) to that of human milk which is most commonly cited as 40:60, but subject to change depending on stage of lactation (Lönnerdal, 2003). While casein to whey protein ratio is not specified in Europe, IF are commonly manufactured to include more whey protein; however, Chinese regulations do require that the protein in IF contains a minimum of 60% whey protein (People’s Republic of China, Ministry of Health, 2010). The lower casein content in

Table 12.1 Composition of IF, Human, and Bovine Milk IF

Human

Bovine

71 0.9 0.3 0.6 3.8 7

69 3.3 2.6 0.7 3.7 4.8

Per 100 mL Energy (kcal) Total protein (g) Casein Whey protein Fat (g) Carbohydrate (g)

60
70 1.2
2.0a N.S N.S 2.9
3.9a 5.9
9.1a

Data: Gurr (1981), Nasripour et al. (2006), Thompkinson and Kharb (2007). NS, not specified. a Calculated from European Commission (2006) for energy content 5 65 kcal/100 mL.

12.1 Introduction

human milk helps to produce a softer and more flocculent curd, which is more easily digested and leads to a faster rate of gastric emptying, compared to the curd from bovine milk, which is coarse, flaky and firm, and more difficult to digest (Nakai & Li-Chan, 1989; Thompkinson & Kharb, 2007). Human milk proteins also have a lower buffering capacity than bovine milk proteins and, therefore, reduce gastric acidity to a greater extent (Davis, Harris, Lien, Pramuk, & Trabulsi, 2008). While altering the casein:whey protein ratio is beneficial for simulating the amino acid profile of human milk, it has also been shown to affect infants’ microbiota in a similar manner to human milk (Hascoet et al., 2011). IF must contain at least the same quantity of essential and semiessential amino acids (see Table 12.2) as human milk (Koletzko et al., 2005; Thompkinson & Kharb, 2007). The amino acid composition of human milk provides all 11 essential amino acids necessary for infant growth and development (Dewey et al., 1996). However, while bovine milk contains many amino acids in excess, it is deficient in methionine, cysteine, and tryptophan (Lien, 2003); differences in phenylalanine:tyrosine and cysteine:methionine ratios between bovine and human milk are considered particularly important (Thompkinson & Kharb, 2007). Therefore, to ensure an adequate supply of all amino acids, the protein content of IF (1.3
1.5 g/100 g) is higher than that of human milk (B0.8
1.0 g/100 g). The levels of indispensable and conditionally indispensable amino acids in human milk are shown in Table 12.2. Table 12.2 The Minimum Levels of Indispensable and Conditionally Indispensable Amino Acids in Infant Formulas, Based on Human Milk, and Expressed in mg per 100 kJ and 100 kcal Amino Acid

mg/100 kJ

mg/100 kcal

Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine

6 11 17 37 29 7 15 19 7 14 19

24 45 72 156 122 29 62 80 30 59 80

Commission Directive 2006/141/EC (European Commission, 2006); amended by Commission Regulation (EC) No 1243/2008 amending Annexes III and VI.

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The minimum crude protein content of IF manufactured with bovine milk proteins is 1.8 g/100 kcal (Codex Alimentarius, 2007; European Commission, 2006; People’s Republic of China, Ministry of Health, 2010). IF manufactured with hydrolyzed bovine proteins and soy proteins should contain a minimum protein content of 2.25 g/100 kcal (Codex Alimentarius, 2007; European Commission, 2006; People’s Republic of China, Ministry of Health, 2010). Many different whey-based ingredients are suitable for use in IF and selecting the correct ingredient is dependent on nutritional, technological, marketing, compositional specification, and price
functionality interrelationships. The diversity in functionality of whey proteins, interaction with processing parameters and their important nutritional and physiological roles are covered in this chapter.

12.2 PHYSIOLOGICAL FUNCTION OF COMPONENTS FOUND IN WHEY PROTEIN INGREDIENTS Evidence that whey proteins and peptides have health benefits beyond basic infant nutrition has increased dramatically in recent years. Emerging findings from in vitro, animal, and a limited number of human studies suggest a range of associated beneficial bioactivities can be linked to whey protein in its various derivative forms, i.e., concentrate, isolate, hydrolysate, and individual proteins and peptides (see Chapters 15
17 for further information on health and nutrition aspects of whey proteins). The sections below summarize the key studies and their findings, focusing where possible on infant studies. In particular, the health benefits attributed to the presence of the highly valuable individual proteins in whey are reviewed. Table 12.3 provides a summary of whey components and their known benefits to infant health.

12.2.1

Whey-Based Infant Formula

Whey derived from cows’ milk contains many of the same components found in human milk and for this reason, is a key ingredient in a wide variety of IF, including those for premature infants. While, breastfeeding is preferred, IF containing whey proteins are currently the best alternative when breastfeeding is not an option. Whey-predominant and whey hydrolysate formulas, e.g., have been shown to support the infant immune system. Whey hydrolysate formula is less allergenic when compared to standard IF and possibly decreases the risk that the infant will later develop allergies (Szajewska, Mrukowicz, & Stoinska, 2004). A meta-analysis review revealed that the incidence of atopic dermatitis was noticeably lower among infants fed partially

Table 12.3 Major Constituents of Whey and Their Functions Constituent

Conc. (g/L)

MW

Potential Benefits to Infant Health

References

α-Lactalbumin

1.2
1.5

14,200

β-Lactoglobulin

3.0
4.0

18,300

Brȕck et al. (2003), Kamau, Cheison, Chen, Liu, and Lu (2010), Lien (2003), Lönnerdal (2014), Nestlé Nutrition Institute (2014), Oevermann, Engels, Thomas, and Pellegrini (2003) Kontopidis, Holt, and Sawyer (2004), Tai, Chen, and Chen (2016)

Immunoglobulins

0.6
0.9

150,000
900,000

Glycomacropeptide

B1.2

6,700

Serum albumin

0.3
0.6

66,000

Lactoferrin

0.05

78,000

Osteopontin

0.015
0.020*

B60,000

Antimicrobial, antiviral, immunostimulatory, source of essential and branched chain amino acids, e.g., tryptophan, which helps regulate sleep and response to stress Binds vitamins A and D, palmitic acid, fatty acids, phospholipids, and aromatics compounds, source of essential and branched chain amino acids Antimicrobial, antiviral, promotes growth of beneficial bacteria, immunomodulatory Does not contain amino acid phenylalanine, used in IF for infants with phenylketonuria, antimicrobial, antiviral, prebiotic that promotes the growth of beneficial bacteria, source of branched chain amino acids, immunomodulatory, may have an inhibitory effect on atopic dermatitis Source of essential amino acids, binds fatty acids Bactericidal, bacteriostatic, antifungal, antiviral, antiprotozoan, modulates inflammation, promotes growth of beneficial bacteria, improves neural functions in piglets, antioxidant, regulates iron absorption and bioavailability Inhibits ectopic calcification, immunomodulatory, possible role in cognitive and intestinal development, influences cell proliferation and repair

Korhonen and Marnila (2009), Korhonen, Marnila, and Gill (2000), Kvistgaard et al. (2004) Brück, Kelleher, Gibson, Graverholt, and Lönnerdal (2006), Krissansen (2007), Muñoz et al. (2017), Neelima, Sharma, Rajput, and Mann (2013), Oh, Worobo, Kim, Rheem, and Kim (2000), Sawin et al. (2015)

Gupta, Prakash, Garg, and Gupta (2012), Kassem (2015) Actor, Hwang, and Kruzel (2009), Bruni et al. (2016), Chen et al. (2015), Liu and Newburg (2013), Lönnerdal, Erdmann, Thakkar, Sauser, and Destaillats (2017), Nguyen et al. (2016), Rai et al. (2014), Sherman, Bennett, Hwang, and Yu (2004) Anborgh, Mutrie, Tuck, and Chambers (2011), Ashkar et al. (2000), Jiang, Prell, and Lönnerdal (2015), Lönnerdal, Kvistgaard, Peerson, Donovan, and Peng (2016), Schack et al. (2009a)

Continued

Table 12.3 Major Constituents of Whey and Their Functions Continued Constituent

Conc. (g/L)

MW

Potential Benefits to Infant Health

References

Lactoperoxidase

(0.06)

(78,000)

Lysozyme Folate binding protein PP3

(0.0004) 0.005
0.008*

(14,000) B30,000
35,000

Lactoperoxidase catalyzes the oxidation of molecules in the presence of hydrogen peroxide, antibacterial, antiviral Lysozyme is antibacterial, antiviral Sequester folate from blood plasma

0.3

28,000

Antibacterial, antiviral, immunostimulatory, inhibits lipase activity, proteolysis results in bioactive peptides

Growth factors

Various

Various

Stimulate cell proliferation, suppress apoptosis, MRG4 possible role in protection against NEC

Cytokines

Various

Various

Lactose

48

340

Oligosaccharides

0.06

B300
1,500

Involved in gut development and maturation, reduce allergy, protect against infection, stabilize epithelial barrier function Encourages growth of beneficial bacteria Prebiotic activity, antiadhesive activity, antiinflammatory properties, modification of cell surface sugars, a role in brain development, influences growth-related characteristics of intestinal cells

Gurtler and Beuchat (2007), Lönnerdal (2003), Golinelli, Del Aguila, Flosi Paschoalin, Silva, and Conte-Junior (2014) Lee-Huang et al. (1999) Rosenberg and Selhub (2006), Yu, Gilar, Kaska, and Gebler (2005) Barzyk, Campagna, Wieclaw, Korchowiec, and Rogalska (2009), Girardet and Linden (1996), Girardet, Linden, Loye, Courthaudon, and Lorient (1993), Inagaki et al. (2010), Mikkelsen, Bakman, Sørensen, Barkholt, and Frokiaer (2005), Sugahara et al. (2005) Davis-Fleischer and Besner (1998), McElroy et al. (2014), Nair, Warner, and Warner (2008), Pavelic, Matijevic, and Knezevic (2007), Pouliot and Gauthier (2006) Oddy et al. (2003), Hering et al. (2011), Penttila (2010), Ustundag et al. (2005)

Conc., concentration; MW, molecular weight.

Paterson (2009), Venema (2012) Kunz and Rudloff (2008), Bode (2015), Goehring et al. (2016)

12.2 Physiological Function of Components Found in Whey Protein Ingredients

hydrolyzed whey protein-based formula compared to those fed the intact protein cows’ milk formula (Alexander, Schmitt, Tran, Barraj, & Cushing, 2010). The findings suggest that whey-based formula might protect infants from atopic dermatitis. Furthermore, a randomized, double-blind, placebocontrolled study of 43 infants with diagnosed colic was performed to examine whether a hypoallergenic, hydrolyzed whey protein formula was superior to a standard cows’ milk formula (Lucassen, Assendelft, Gubbels, van Eijk, & Douwes, 2000). After a 1-week qualification period, infants in the study were randomized to receive either whey or cows’ milk formula for 1 week. A clinically significant result was observed in the whey formula group, with crying time reduced to less than 1 hour per day which was a one-hour reduction when compared to infants fed cows’ milk formula. Whey protein may also play a role in influencing the infant gut microbiota. Species such as bifidobacteria are abundant in the breast-fed infant gut and are particularly important for inhibiting the growth of pathogenic organisms, modulating mucosal barrier function, and promoting immunological and inflammatory responses (Westermann, Gleinser, Corr, & Riedel, 2016). Balmer, Scott, and Wharton (1989) analyzed the fecal flora of 33 infants who received a whey-protein formula, 29 babies who received a casein formula, and 38 breast-fed babies over a 15-week period. Infants who received the whey-protein formula had higher counts of bifidobacteria than those who received the casein-predominant formula at 2 weeks of age. In a double-blind study conducted in Germany, 102 healthy infants of less than 2 weeks of age were randomized to receive either a standard cows’ milk formula or an IF containing partially hydrolyzed whey protein (Schmelzle et al., 2003). The results indicated that the whey protein-fed infants had greater numbers of bifidobacteria in their stools. More recently, Hascoet et al. (2011) demonstrated that infants who received a formula low in phosphate and protein, comprising mainly of whey protein, developed a stool microbiota profile similar in bifidobacteria composition to that for breast-fed infants. These studies suggests that even in infants not exclusively breast-fed, a breast-fed gut microbiota could be achieved, in part at least, by supplementing with a type of formula having a composition similar to human milk. Apart from supporting immunity and gut health, whey protein may provide several other benefits to infants (reviewed by Nestlé Nutrition Institute, 2014). For example, there is clinical evidence to suggest that whey protein has the potential to increase muscle mass (Hoppe et al., 2008), which may be beneficial for a healthy body composition in infants. Whey protein contains amino acids with a very similar pattern to muscle proteins and a particularly high amount of branched-chain amino acids, which may promote protein synthesis in muscle (Mølgaard, Larnkjær, Arnberg, & Michaelsen, 2011).

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Whey protein also results in higher whole body protein synthesis when compared to casein since leucine, a key amino acid in protein metabolism, is present in higher concentrations in whey protein than in casein (Mølgaard et al., 2011). Other studies suggest that whey protein can aid gastric emptying. For example, Billeaud, Guillet, and Sandler (1990) conducted a study to determine the influence of gastroestophageal reflux and the effect of milk composition on gastric emptying in 201 infants (age: 0
1 year). Gastric emptying was measured by cineoesogastroscintigraphy. Infants were fed different types of milk (e.g., human milk, whey-predominant formulas, casein-predominant formulas, follow-up formulas, whey-hydrolysate formulas, or cows’ milk). Gastric emptying was more rapid with the consumption of whey-predominant formulas when compared with casein-predominant formulas suggesting that the greater the casein concentration in milk, the slower the gastric emptying. The above studies highlight the known benefits which can be associated with whey-based infant formulations. To match the performance of breast-fed infants, the addition to formula of individual bioactive components similar to those found in human milk is an attractive option for formula-fed infants. However the composition of IF is strictly regulated in many countries (WHO/FAO, 2011) and this therefore can be a challenging task. The addition of individual bioactive components requires not only that these are safe but that they also result in clear benefits to the recipient infants (Lönnerdal, 2014). The section below reviews the possible benefits associated with individual whey proteins and their applications in infant formulations where examined.

12.2.2

Major Whey Proteins and Peptides

12.2.2.1 α-Lactalbumin

α-La is a relatively small whey protein with a molecular mass corresponding to 14,070 Da in human milk and 14,178 Da in bovine milk. Its content in bovine whey is approximately 1.2 g/L, making up 20%
25% of the total whey proteins while in human milk at 2
3 g/L, α-La makes up 41% of the whey proteins (Kamau et al., 2010). The amino acid sequences of human and bovine α-La are very similar, sharing 72% homology. Hence, bovine α-La may be an excellent substitute for human α-La in infant nutrition. In IF, the extent of protein digestibility becomes a critical issue for both essential amino acid supply and the potential bioactivities of bovine-milk proteins. When lowering the protein concentration in IF, the first limiting amino acid becomes tryptophan (Lönnerdal & Lien, 2003). Tryptophan is a precursor of serotonin, a neurotransmitter that regulates the response to stress, the sleep
wake rhythm and other physiologic processes (Lien, 2003; Nestlé Nutrition Institute, 2014). α-La has a relatively high proportion of

12.2 Physiological Function of Components Found in Whey Protein Ingredients

tryptophan and is commercially available as an enriched whey fraction. Clinical studies have shown that lowering the protein amount in IF coupled with an increased proportion of α-La results in plasma tryptophan concentrations similar to those in breast-fed infants (Davis et al., 2008; Sandström, Lönnerdal, Graverholt, & Hernell, 2008). α-La is also relatively high in other essential amino acids, namely, lysine, and cysteine, which amount to 11%, and 6% moles of total amino acids in α-La, respectively (Appel, Bairoch, & Hochstrasser, 1994). Cysteine is a constituent of the tripeptide glutathione, a vital element of the neonatal antioxidant system. It is also the precursor to taurine, an amino acid that may play a role in the brain development (Lien, 2003). α-La also serves as a regulatory unit of the enzyme galactosyltransferase which is responsible for lactose synthesis from galactose and glucose in milk. More precisely, after α-La has attached to galactosyltransferase, the conversion of galactose to N-acetylglucosamine is enabled and synthesis of lactose from the uridyldiphosphate-galactose and glucose follows (Kamau et al., 2010; Kassem, 2015; Lisak Jakopovi´c, Barukˇci´c, & Boˇzani´c, 2016). Therefore, α-La plays an important role in milk formation overall through lactose production and secretion. It is likely that several peptides are released from α-La during digestion which are also likely to exert bioactivities. These peptides are possibly formed in the upper part of the gastrointestinal tract but may exert functions during their passage through the more distal part of the small intestine as well as in the colon (Lönnerdal, 2014). α-La-derived peptides have been shown to have antibacterial and immunostimulatory properties (Jaziri et al., 1992; MiglioreSamour et al., 1992; Lönnerdal, 2003, 2014; Lönnerdal & Lien, 2003). In one study, three polypeptide fragments from α-La exerted antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermis, Streptococci, and Candida albicans (Pellegrini, Thomas, Bramaz, Hunziker, & von Fellenberg, 1999). Other in vitro studies have demonstrated that α-La-derived peptides were antiviral against herpes simplex virus type 1 and human immunodeficiency virus type 1 (HIV- 1) (Berkhout et al., 1997; Oevermann et al., 2003). Peptides from α-La have also been shown to possess prebiotic activity through stimulating the growth of bifidobacteria (Kee et al., 1998). Although these observations have been made in vitro, it is possible that these types of peptides are formed in vivo and exert their antibacterial activities in the colon. This may explain the inhibitory effect of α-La-supplemented IF on enteropathogenic E. coliinduced diarrhea in infant rhesus monkeys (Brück., 2003). The potential combined effect of prebiotic and antimicrobial peptides released from α-La during digestion may aid in preventing dysbiosis in the formula-fed infant gut. Clinical studies involving formulas with added α-La have been conducted, but they have mainly focused on growth, plasma amino acids, and

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acceptability. Future studies are required to reveal the full extent of the biological effects associated with α-La as suggested from the in vitro studies described above.

12.2.2.2 β-Lactoglobulin

β-Lg is a major whey protein in bovine milk accounting for approximately 10%
15% of total milk proteins and 58% of whey protein. It exists as a dimer with a molecular weight of 36 kDa at the normal pH of bovine milk (Kontopidis et al., 2004). The complete amino acid sequence (162 aa) of β-Lg has been elucidated and eight genetic variants are known to exist (A, B, C, D, E, F, G, and Dr) (Creamer, Parry, & Malcolm, 1983). β-Lg is not present in human milk and the exact biological function of bovine β-Lg is unclear. It is of nutritional value given its amino acid composition, but it has other properties which may suggest further biological roles. β-Lg could play a role in metabolism of phosphate in the mammary gland (Farrell, Bede, & Enyeart, 1987) or in the transfer of passive immunity to the new born (Warme, Momany, Rumball, Tuttle, & Scheraga, 1974). Based on the structure of β-Lg, it is thought to bind ligands and transport small hydrophobic ligands (Perez & Calvo, 1995). Given that β-Lg has multiple ligand binding sites, it is capable of binding to vitamins A and D, palmitic acid, and other hydrophobic compounds. In addition, β-Lg exhibits strong binding affinities for fatty acids, phospholipids, and aromatic compounds (Tai et al., 2016). β-Lg is a retinol-binding protein and may play a role in the transport of retinol (Puyol, Perez, Ena, & Calvo, 1991). Papiz et al. (1986) demonstrated that β-Lg increases the uptake of retinol in the gut of neonate calves. Native β-Lg is resistant to gastric digestion in vivo but is digested by intestinal enzymes. However, some intact β-Lg can be detected as far as the ileum (Sanchón et al., 2018). As β-Lg is not a component of human milk, the intact protein is reportedly an allergen to a minority of infants (Chatterton, Nguyen, Bering, & Sangild, 2013) and its removal could provide the basis of a hypoallergenic IF. Intake of intact β-Lg stimulates the production of Treg cells specific for β-Lg in murine Peyer’s patch cells (Adel-Patient et al., 2011). β-Lg is cleaved in the intestine, and compared to intact β-Lg, hydrolyzed β-Lg induces only local stimulation of Treg and no stimulation of an allergic response in mice (Adel-Patient et al., 2011). Interestingly, β-Lg has a high (83%) amino acid homology with human glycodelin A, a protein involved in the maintenance of the feto-maternal immune system (Van Cong et al., 1991). Glycodelin A suppresses all main immune cells, including both Th1 and Th2 responses (Ogge et al., 2011; Scholz et al., 2008). Moreover, monoclonal antibodies raised against β-Lg cross-react with glycodelin A (Dutta, Mukhopadhyay, Roy, Das, & Karande, 1998). It is not known whether natural antibodies to β-Lg cross-react with

12.2 Physiological Function of Components Found in Whey Protein Ingredients

glycodelin A, or whether β-Lg is associated with similar activities to those of glycodelin A (Chatterton et al., 2013).

12.2.2.3 Immunoglobulins Immunoglobulins (Igs) represent about 10%
15% of whey proteins in both human and bovine milk (Golinelli et al., 2014; Kulczycki & MacDermott, 1985; Marshall, 2004). The major Ig classes in both human and bovine milk are IgA, IgG, and IgM (Korhonen & Marnila, 2009). In human milk and colostrum, the major Ig is IgA. IgA is also present in bovine milk where IgG predominates (Butler, Seawright, McGivern, & Gilsdorf, 1986). The main role of immunoglobulins is to agglutinate bacteria, neutralize toxins, inactivate viruses, and stimulate the growth of beneficial bacteria (Bojsen et al., 2007; Kvistgaard et al., 2004; McWilliams, 1986). In other words, milk Igs give the offspring an immunological protection against infections in the gastrointestinal tract and respiratory system. Numerous studies (reviewed by Jasion & Burnett, 2015) have been performed demonstrating that Ig preparations derived from human sera as well as bovine colostrum and sera survive digestion and are present in fecal matter. IgG was the predominant Ig in these preparations, but often IgA and/or IgM were present in smaller amounts. The glycan chains of milk Igs may protect the Ig protein from digestion by proteolytic enzymes, allowing the intact or only partially digested Ig to reach the intestine for absorption into the blood (O’Riordan, Kane, Joshi, & Hickey, 2014). Cross-species activity between human and bovine immune-related milk proteins has been reported (den Hartog et al., 2011; van Neerven, Knol, Heck, & Savelkoul, 2012), and cows’ milk IgG binds to gastrointestinal pathogens that also infect humans, such as Shigella flexneri, Clostridium difficile, E. coli, Cryptosporidium parvum, Helicobacter pylori, Streptococcus mutans, rotavirus (Korhonen et al., 2000), and human respiratory syncytial virus (den Hartog et al., 2011). Not taking into account colostrum, the total amounts of Igs in bovine milk are lower than in human milk, and levels are significantly lower or absent in IF. This could partly account for the increased incidence of infections in formula-fed infants, compared with breast-fed infants (Dewey, Heinig, & Nommsen-Rivers, 1995), supporting the supplementation of formulas with Igs. Indeed, studies have been performed where bovine colostrum-derived Igs have been added to IF and resulted in a significant reduction in diarrhea and related morbidities among a large population of Iraqi infants (Tawfeek, Najim, & Al-Mashikhi, 2003). High concentrations of antibodies against a particular pathogen may be achieved by immunizing cows with the pathogen or its antigens. Through a strategy of vaccination and established purification procedures in the dairy industry, large-scale manufacture of various IgGs is possible. On average, 500 g/L of IgG can be acquired from each immunized cow immediately after

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calving (Kramski et al., 2012). Such hyperimmune milk products have been tested in prophylaxis and treatment of viral infections (reviewed by Hurley & Theil, 2011).

12.2.2.4 Glycomacropeptide Glycomacropeptide (GMP) is a casein-derived glycoprotein released into whey due to the action of the enzyme, chymosin, on κ-casein during cheesemaking. GMP is also released in the adult human gastrointestinal (GI) tract by pepsin-mediated hydrolysis after milk ingestion (Chabance et al., 1998). The protein comprises 20%
25% of the proteins in WPI and WPC that are produced from cheese whey. GMP has unique characteristics due to the absence of phenylalanine, tryptophan, tyrosine, histidine, arginine, or cysteine residues (Neelima et al., 2013). The absence of phenylalanine makes this protein a valuable dietary ingredient for patients who are suffering from phenylketonuria. Thus, whey can be used as a potential source of GMP to fulfill the nutritional profile of such patients. Moreover, GMP is rich in branchedchain amino acids (Marshall, 2004; Krissansen, 2007). GMP can neutralize enterotoxin (Oh et al., 2000), inhibit virus or bacterial adhesion to cells (Brück et al., 2006; Feeney, Ryan, Kilcoyne, Joshi, & Hickey, 2016), inhibit gastrointestinal secretions (Brody, 2000), promote proliferation of beneficial bacteria (Janer, Pelaez, & Requena, 2004), and exert an immunoregulatory function (Otani & Hata, 1995). Moreover, GMP supplementation in the diet protected weaning piglets against E. coli K88 infection (Rong et al., 2015). The most important functional group of GMP is sialic acid (SA) (N-acetylneuraminic acid). It plays an important role in defence mechanisms in vivo. Human milk contains a high concentration of SA, which is closely related to infants’ development and immune system maturation under infection conditions (Wang, Brand-Miller, McVeagh, & Petocz, 2001). SA is also involved in development of the nervous systems and has been linked to improvement in learning and memory (Wang & Brand-Miller, 2003). Recently, SA isolated from GMP was found to have antibacterial activity against Helicobacter in rats (Kim et al., 2016). Indeed, more than 75% of the SA in milk is attached to GMP, indicating that GMP’s physiological function is very important. The addition of GMP to IF has been questioned due to its high threonine content (12
13 threonine residues) which results in higher plasma threonine concentrations (hyperthreoninemia) in infants fed GMP-containing formula when compared with breast-fed infants (Rigo et al., 2001). However, later work (Sandström et al., 2008) did not find any such disparity and suggested that the difference in plasma threonine concentrations between formula-fed and breast-fed infants could be due to a difference in threonine metabolism.

12.2 Physiological Function of Components Found in Whey Protein Ingredients

In mice, GMP was found to act as a prebiotic, based on reducing Desulfovibrio numbers, increasing short-chain fatty acid production and lowering indexes of inflammation compared with casein and amino acid-based diets (Sawin et al., 2015). A study in rats demonstrated that the prebiotic action of GMP led to an allergy-protective microbiota, resulting in an increase in transforming growth factor beta (TGF-β) production and a reduction in mast cell response to allergens (Jiménez et al., 2016). Results from a separate rat trial have recently indicated that GMP has an inhibitory effect on atopic dermatitis through downregulating the Th2-dominant immune response (Muñoz et al., 2017). Taken together, a unifying feature and potential mechanism for the reported health-promoting properties associated with GMP may be its role as a prebiotic that promotes a beneficial gut microbiota and modulates immune function.

12.2.2.5 Serum Albumin Serum albumin is a large protein present in human and bovine milk and makes up approximately 10%
15% of total whey protein. It is a source of essential amino acids but there is very little available information regarding its potential therapeutic activity (Gupta et al., 2012; Kassem, 2015). Given that its properties in milk are similar to those in blood, it is thought that it may not be synthesized by the mammary gland (Lönnerdal, 2003). Instead, it is believed to be transferred from maternal circulation. While serum albumin does serve as a source of amino acids for the breast-fed infant, the question of whether it has other physiologic functions in human milk is unclear. In blood, serum albumin binds many ligands, including fatty acids, trace elements, calcium, and other molecules. Similarly, in milk, serum albumin has been associated with zinc, copper and thyroxine (Etling & Gehin-Fougue, 1984; Lönnerdal, Hoffman, & Hurley, 1982). However, it is unlikely that serum albumin plays a major role as a nutrient binder or a source of nutrients for infants because its associations with these ligands are weak and binding to these ligands would not persist in the infant gut (Lönnerdal, 1985). Bovine serum albumin (BSA) is also known to bind to fatty acids, as well as other small molecules (Fletcher, Spector, & Ashbrook, 1971; van der Vusse, 2009).

12.2.3

Minor Whey Proteins and Peptides

12.2.3.1 Lactoferrin Lf is the second most abundant protein in human milk, with concentrations ranging from 6 g/L in early milk to 2 g/L in mature milk (Rai et al., 2014). In bovine milk concentrations are much lower and range from 0.8 g/L in early milk to 0.1 g/L in mature milk (Sanchez, Aranda, Perez, & Calvo, 1988). The high homology in protein sequence (77%) between human lactoferrin (hLf) and bovine lactoferrin (bLf) suggests that supplementation of bLf to IF may

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exert beneficial effects in a similar manner to that of hLf in human milk (Manzoni et al., 2010; Nguyen, et al., 2016). Structurally, bLf is an ironbinding glycoprotein and consists of a single polypeptide chain of approximately 700 amino acids (B78 kDa MW). Although the exact function of the glycans is not fully understood, they may protect Lf from proteolysis and are involved in receptor recognition (Barboza et al., 2012; Zimecki, Artym, Kocie ba, Duk, & Kruzel, 2014.). The bioactivity of Lf is well documented and many recent comprehensive reviews exist examining its role in detail (Aly, Ros, & Frontela, 2013; Donovan, 2016; Mayeur, Spahis, Pouliot, & Levy, 2016). Lf is both bactericidal and bacteriostatic in that it limits the growth of several pathogens and kills others. The iron-free form of Lf, its most common form in human milk, has been shown to kill Pseudomonas aeruginosa, Streptococcus pneumonia, Str. mutans, Vibrio cholera, E. coli, and C. albicans (Arnold, Brewer, & Gauthier, 1980; Lönnerdal et al., 2017). The bacteriostatic effects of Lf result, in part, from its ability to withhold iron from bacteria that require it for growth. It also exhibits antiviral, antifungal, and antiprotozoan activities that are likely distinct from its ability to chelate iron (Lönnerdal et al., 2017). The peptides resulting from Lf proteolysis also have antibacterial activities (Bruni et al., 2016). For instance, lactoferricin, a peptide derivative of Lf, is reported to exhibit numerous biological activities in common with those of Lf (Walzem, Dillard, & German, 2002). Lf also promotes the proliferation of Lactobacillus and Bifidobacterium, thereby contributing to a beneficial intestinal microbiota (Petschow, Talbott, & Batema, 1999; Sherman et al., 2004). Lf is also involved in modulating inflammatory and immune responses with evidence suggesting that it increases the number and activity of B lymphocytes, T lymphocytes, and natural killer cells. It can accelerate B and T cell maturation, and increase the expression of cellular receptors (Actor et al., 2009; Liu & Newburg, 2013). These preclinical findings are supported by the discovery that administration of bLf to very low-birth-weight infants protects against late-onset sepsis and necrotizing enterocolitis (NEC) resulting from a variety of infections (Manzoni et al., 2012, 2014). Recently, Donovan (2016) reviewed the role of LF in gastrointestinal and immune development and function from a preclinical perspective. Many of the trials performed have used the piglet as a model for the human infant and have shown that bLF is well tolerated and retains bioactivity within the gut. One such study involving preterm pigs (Nguyen et al., 2016) demonstrated that bLf dose-dependently affects intestinal epithelial cells via metabolic, apoptotic and inflammatory pathways. It is, therefore, important to select an appropriate dose when feeding bLf to neonates to avoid detrimental effects which could result from high doses. LF can also cross the blood
brain barrier via receptor-mediated transcytosis and has suppressive effects on psychological distress (Kamemori, Takeuchi,

12.2 Physiological Function of Components Found in Whey Protein Ingredients

Hayashida, & Harada, 2004; Kamemori et al., 2008). These studies suggested a potential involvement of Lf in neural functions and indeed Chen et al. (2015) found that Lf can improve neural development and cognition in postnatal piglets. Currently, available studies on the role of Lf in iron absorption by the gastrointestinal tract have resulted in varied findings which require further clarification (reviewed by Mayeur et al., 2016). EFSA (2012) accepted and approved bLf as a food ingredient. Currently, bLf is added as a supplement to IF in many countries including Indonesia, South Korea, Spain, Japan (Conesa, Calvo, & Sánchez, 2010; Wakabayashi, Yamauchi, & Takase, 2006). and the United States (Johnston et al., 2015).

12.2.3.2 Osteopontin Osteopontin (OPN) is a multifunctional protein present in most tissues and body fluids, with the highest concentrations found in milk (Christensen & Sørensen, 2016). Human milk contains B138 mg/L while bovine milk contains B18 mg/L. Standard IF contains on average B9 mg/L OPN (Schack et al., 2009a). Bovine and human milk OPN are structurally very similar with regard to amino acid sequence and phosphorylation pattern, and both proteins are O-glycosylated in the same region of the protein. OPN is involved in a variety of physiological processes such as inhibition of ectopic calcification, cancer metastasis, bone remodeling, and immune modulatory functions (Anborgh et al., 2011; Sodek, Ganss, & McKee, 2000; Wang & Denhardt, 2008). OPN can act as an opsonin, as it interacts directly with several bacterial strains leading to enhanced phagocytosis by macrophages (Schack et al., 2009b). OPN is relatively resistant to proteolysis by neonatal gastric juice (Chatterton, Rasmussen, Heegaard, Sørensen, & Petersen, 2004) and can induce Th1 type immunity by inducing interleukin-12 expression in macrophages (Ashkar et al., 2000). These examples suggest that milk OPN plays an important role in the development of the infant immune system. In fact, milk OPN is also thought to play essential roles in cognitive and intestinal development in infants (Christensen & Sørensen, 2016; Jiang & Lönnerdal, 2016). For instance, new-born wild-type mice nursing knock-out dams lacking milk OPN showed impaired cognitive development, reduced brain OPN and myelin-related proteins compared with pups nursing wildtype dams suggesting a role for OPN in promoting brain development (Jiang et al., 2015). Transcriptomic analysis of intestinal biopsies from infant rhesus monkeys fed formula with added bovine milk OPN demonstrated that OPN positively regulated intestinal proliferation, cell migration, and cellular chemotaxis via binding to integrin receptors and generally resulted in a shift to gene expression profiles similar to that of breastfed monkeys (Donovan et al., 2014a).

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Given the similarity between human and bovine milk OPN, it has been suggested to use bovine OPN in IFs to close the concentration gap to match that in human milk (Boskey, Christensen, Taleb, & Sørensen, 2012; Christensen, Klaning, Nielsen, Andersen, & Sørensen, 2012; Christensen, Nielsen, Haselmann, Petersen, & Sørensen, 2005; Christensen & Sørensen, 2016; Sørensen, Højrup, & Petersen, 1995). Processes for isolation of OPN from bovine milk for use in IF have been developed and studies have investigated the effects of oral administration of milk OPN (reviewed by Christensen & Sørensen, 2016). A proprietary whey protein-based product supplied by Arla Food Ingredients P/S Group (Lacprodan OPN-10) that contains approximately 95% bovine milk OPN has been tested and found in in vitro and in vivo trials not to be genotoxic (Kvistgaard, Matulka, Dolan, & Ramanujam, 2014). Formula containing Lacprodan OPN-10 has also recently been investigated in human infants (Lönnerdal et al., 2016). In a randomized controlled trial, mothers either breast- or formula-fed their infants from 1 to 6 months of age. The formula-fed infants received either standard formula or formula containing OPN representing B50% and 100% of the OPN concentration in human milk (double-blinded). The study showed that addition of OPN resulted in the plasma levels of several amino acids and cytokines in formula-fed infants being more similar to those observed in breastfed infants, though not all changes were statistically significant. Interestingly, addition of OPN to formula significantly lowered the levels of the proinflammatory cytokine TNF-α but significantly increased levels of interleukin-2 that plays key roles in oral tolerance (Lönnerdal et al., 2016). Importantly, there were no differences in appetite, growth, weight, or height among the different infant groups (Lönnerdal et al., 2016). Furthermore, the groups that received OPN-containing formulas had significantly fewer fever days than the group that received standard IF (Lönnerdal et al., 2016). Supplementing IF with 50% OPN concentration was also indicated to support improved immune development in infants, as it significantly shifted the gene expression of peripheral blood mononuclear cells to be more similar to breast-fed infants (Donovan et al., 2014b). West et al. (2017) examined frequencies and composition of peripheral blood immune cells by four-color immune-flow cytometry of formula-fed infants at ages 1, 4, and 6 months, and compared the results with a breast-fed reference group. Feeding infants formula with levels of OPN at the same concentration as found in human milk increased the proportion of circulating T cells compared with both standard formula and formula with added OPN at B50% of the concentration in human milk. This suggests that OPN may favorably influence immune ontogeny in infancy and that the effects appear to be dose-dependent (West et al., 2017). Overall, addition of bovine milk OPN to IF may improve the performance of formula-fed infants through bridging the gap between formula and human milk.

12.2 Physiological Function of Components Found in Whey Protein Ingredients

12.2.3.3 Enzymes Whey contains several classes of enzymes such as oxidoreductases, hydrolases, transferases, lyases, isomerases, and ligases. A major whey enzyme is lactoperoxidase (LP), which in the presence of hydrogen peroxide (formed in small amounts in different cell reactions) catalyzes the peroxidation of thiocyanate (which is present in biological fluids such as saliva and milk) forming hypothiocyanate, which is effective against both Gram-positive and Gram-negative bacteria (Golinelli et al., 2014; Steele & Morrison, 1969). Thus, LP in human milk may prevent infections in the mouth and upper gastrointestinal tract of the new born (Shin, Hayasawa, & Lönnerdal, 2001). This action is so promising that LP has been promoted in cows’ milk for decades by the dairy industry in developing countries to delay microbial spoilage of milk where poor processing logistics prevail. It accounts for 0.25%
0.50% of total protein found in whey and levels are greater in bovine milk than in human milk (Chatterton et al., 2013). Gurtler and Beuchat (2007) demonstrated that LP can be used to control the growth of Cronobacter sakazakii in reconstituted IF. Lysozyme is another component of the whey fraction in human milk and is capable of degrading the outer cell wall of Gram-positive bacteria (Lönnerdal, 2003; Lönnerdal et al., 2017). This enzyme may work synergistically with Lf and Igs in killing bacteria (Ellison & Giehl, 1991; Lonnerdal, 1985). Lysozyme, along with other factors including Igs, Lf, and LP, may limit the migration of neutrophils into damaged tissue by acting as an antiinflammatory agent (Leon-Sicairos et al., 2006). A clinical trial in which recombinant human lysozyme and human Lf were given in a rice-based oral rehydration solution to children hospitalized with acute diarrhea may validate these in vitro observations (Zavaleta et al., 2007). The incidence and duration of diarrhea as well as relapse rate were significantly reduced by the milk proteins when compared with the control. However, whether human lysozyme or human Lf alone may have caused the effect was not determined. Lysozyme may also inhibit the growth of HIV in vitro (LeeHuang et al., 1999), although the mechanism for its antiviral activity is unknown.

12.2.3.4 Folate Binding Protein Folate binding proteins (FBP) have been separated and identified in both bovine and human milk (Ford, Salter, & Scott, 1969). In contrast to bovine milk, human milk contains two forms of FBP; one particulate and one soluble (Antony, Utley, Marcell, & Kolhouse, 1982; Svendsen, Hansen, Holm, & Lyngbye, 1982), the latter being glycosylated which may help it survive gastric digestion. FBP in bovine milk is made up of 220
237 amino acids, with a molecular weight (deglycosylated) of approximately 26.5 kDa (Yu et al., 2005).

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Many different methods with varying results (reviewed by Nygren-Babol and Jägerstad, 2012) exist for measuring FBP in milk and dairy products. For instance, Indyk, Filonzi, and Gapper (2006) using surface plasmon resonance technology, detected 10
60 mg/kg of FBP in commercial milk powder. The suggested role of FBP as a folate-trapping protein in milk has been well documented in a number of studies. Experiments using rat intestinal cells have shown that folate uptake was higher as an FBP complex compared to the free form, suggesting that FBP may facilitate folate uptake (Mason & Selhub, 1988). Similarly evidence from studies on goat kids suggest that FBP-bound folate plays a physiological role during lactation through absorption (Ford, Knaggs, Salter, & Scott, 1972; Salter & Blakeborough, 1988). This is most likely also an important mechanism for newborn infants. Rosenberg & Selhub (2006) found that human milk folate when bound to protein is similarly absorbed, largely intact, through the permeable infant intestine after birth with concentrations ranging between 109
204 nmol/L, which is fiveto 10-fold higher than that of maternal plasma (Tamura & Picciano, 2006). A positive relationship exists between human milk folate and folate-binding protein concentrations, while human milk folate-binding capacity exceeds the available folate by B68 nmol/L (Tamura & Picciano, 2006). The folatebinding capacity of human milk is reported to range between 180 and 270 nmol/L (Selhub, Arnold, Smith, & Picciano, 1984). The excess folate-binding capacity may act to concentrate human milk folate for secretion against a concentration gradient. Plasma folate concentrations in breast-fed infants are generally high (45
68 nmol/L) in the first 6 months of life and decline to 23
45 nmol/L by 12 months, when foods other than human milk are consumed (Tamura & Picciano, 2006). This shift indicates the important role of FBP in enhancing the bioavailability of folate from human milk. Although FBP definitely plays an essential role in folate bioavailability in neonates, intake of cows’ milk containing up to equimolar amounts of folate and FBP actually decreases folate uptake significantly in human gastrointestinal-simulating models. The decreasing effect of FBP is dosedependent and also dependent on the folate form (Nygren-Babol, Sternesjo, Jagerstad, & Bjorck, 2005). For infants, a much higher survival of FBP may be expected due to a less-mature, more-permeable gastrointestinal system, where the gastric pH is higher compared to that of adults. Milk formula prepared from spray-dried milk powder could contain active FBP. As milk formula is fortified with folic acid, the effect of FBP on folic acid bioavailability could be significant, especially when milk formula is the sole dietary source of folate before weaning. Infant food formulators should consider this and, as suggested by Nygren-Babol and Jägerstad (2012), either denature the FBP or replace the folic acid with a form to which FBP has less affinity, such as 5-methyltetrahydrofolic acid.

12.2 Physiological Function of Components Found in Whey Protein Ingredients

12.2.3.5 Proteose Peptone Component 3 (PP3) The proteose peptone fraction of bovine milk accounts for approximately 10% of total whey protein and consists of a complex mix of low-molecularweight heat-stable, acid-soluble proteins. Proteose peptone component 3 (PP3), a 28-kDa phosphorylated glycoprotein also known as lactophorin (Girardet & Linden, 1996), is the principal component of the fraction (25%) and is the bovine homolog of murine glycosylation-dependent cell adhesion molecule-1 (Girardet et al., 1995). Sørensen, Rasmussen, Møller, and Petersen (1997) confirmed the presence of PP3 on the milk fat globule membrane (MFGM) but the group also detected PP3 in the whey fraction suggesting PP3 is weakly associated with the MFGM and is easily lost into the whey fraction of milk. PP3 is not present in human milk which makes its occurrence in bovine milk at 300 mg/L appear relatively high (Sørensen & Petersen, 1993). The function of PP3 in vivo is not clear. PP3 has previously been shown to inhibit lipase activity in milk by competitive adsorption (Girardet et al., 1993). PP3 and derived fragments also show immune-stimulating properties (Mikkelsen et al., 2005; Sugahara et al., 2005). A larger fragment of PP3 was observed to act as a potent inhibitor of human rotavirus infections in embryonic monkey kidney cells and suckling mice (Inagaki et al., 2010). A peptide (called lactophoricin), encompassing residues 113
135 of the C-terminal amphipathic helix of PP3, has been found to form pores in planar lipid bilayers as well as to display antibacterial activity against both Gram-positive and Gram-negative strains of bacteria (Barzyk et al., 2009; Campagna, Cosette, Molle, & Gaillard, 2001; Campagna, Mathot, Fleury, Girardet, & Gaillard, 2004). Moreover, a 26-residue C-terminal peptide of PP3 displayed antibacterial activity against Str. thermophilus. The PP3 full-length protein did not display the same properties, which could indicate that PP3 functions as a precursor protein that upon proteolysis, releases a bioactive antibacterial peptide (Pedersen et al., 2012).

12.2.3.6 Growth Factors Whey contains several growth factors including insulin-like growth factor (IGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) (Pouliot & Gauthier, 2006; Ryan & Walsh, 2016). IGFs are an important part of the putative growth factors in milk. Human and bovine IGF-I share 100% homology suggesting that the two forms may have comparable functionality. Levels of both IGF-I and IGF-II are greater in bovine milk when compared to human milk. Several studies have shown that IGFs stimulate cell proliferation and suppress apoptosis (Chatterton et al., 2013; Pavelic et al., 2007). FGF21 in human milk contributes to neonatal intestinal function and appropriate signaling of FGF21 to the infant is necessary to ensure optimal

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digestive and endocrine function in the developing intestine (GavaldàNavarro et al., 2015). Delivery of EGF via human milk induces proliferation in the mouse gut lumen, enhances growth and maturation and intestinal cell restitution (Nair et al., 2008). Heparin-binding EGF (HB-EGF) stimulates proliferation and migration of enterocytes (Davis-Fleischer & Besner, 1998). Neuregulin 4 (NRG4) is a member of the neuregulin family that shares a common structure with epidermal growth factor (EGF)-like domains. NRG4 is present in human breast milk but has not been detected in formula. Importantly, NRG4 promotes epithelial cell survival and was recently found to protect against experimental necrotizing enterocolitis (NEC) (McElroy et al., 2014). Supplementation of IF with biologically active growth factors similar to those found in human milk seems a next logical step in the prevention or treatment of NEC. It may be that bovine whey may provide a source from which to isolate such growth factors.

12.2.3.7 Cytokines Human milk contains a variety of cytokines (Interleukin [IL]-1β, IL-2 IL-6, IL-8, tumor necrosis factor-alpha [TNF-α]) (Ustundag et al., 2005). Cytokines are among the agents with potential immunological properties in milk and they play vital roles in the regulation and behavior of immune system cells. Cytokines are small soluble glycoproteins that act in autocrine
paracrine fashions by binding to specific cellular receptors, operating in networks, and orchestrating immune system development and functions. These cytokines are present in picogram quantities. However, early-lactation milk has an abundance of cytokines at a time when neonatal organ systems are immature, suggesting that these bioactive components of milk might be important in neonatal development (Oddy et al., 2003; Wallace et al., 1997). Numerous cytokines (e.g., TNF-α, IFN-γ, GM-CSF, IL-8, IL-12) have also been detected in bovine milk (Alluwaimi & Cullor, 2002; Leutenegger, Alluwaimi, Smith, Perani, & Cullor, 2000). TGFβ is the main cytokine in human milk where it exists as five isoforms with 66%
80% amino acid homology and similar, but not identical, activities. In addition to its importance for neonatal gut development and maturation, it stimulates protection against infection, reduces the incidence of allergic manifestations, and induces oral tolerance (Oddy et al., 2003; Penttila, 2010; Verhasselt et al., 2008). TGFβ-2 predominates in bovine milk and 100% amino acid homology exists between active human and bovine TGFβ-2 forms. Milk-derived WPC rich in TGFβ has been developed by the food industry for infant and children’s nutrition products and has been proposed to stabilize epithelial barrier function and protect against inflammatory barrier impairment, as, e.g., in dextran sulfate sodiuminduced colitis (Sprong, Schonewille, & van der Meer, 2010). This was

12.2 Physiological Function of Components Found in Whey Protein Ingredients

further elucidated when a WPC enriched in TGFβ was found to strengthen the intestinal barrier by upregulating Claudin-4 in human colonic HT-29/ B6 cells (Hering et al., 2011).

12.2.4

Other Bioactive Components in Whey

12.2.4.1 Lactose Lactose (4-O-β-D-galactopyranosyl-D-glucose) can be recovered from cheese whey or, more likely, from whey permeate by crystallization (Paterson, 2009). Due to its lower concentration in cows’ milk (4.4%
5.2% in bovine milk compared to 7% in human milk), lactose is added indirectly via other dairy ingredients (skim milk powder (SMP); DWP; WPC) or directly using edible-grade crystalline lactose (99.5% purity) during preparation of IF (Ryan & Walsh, 2016). Lactose can also be used for the direct production of various other adapted ingredients which are used in formula such as prebiotics. In particular, galactooligosaccharides (GOS) are frequently produced from lactose and are known to have a positive effect on human health by encouraging the growth of probiotic bacteria in the gut (Golowczyc et al., 2013; Jovanovic-Malinovska, Fernandes, Winkelhausen, & Fonseca, 2012).

12.2.4.2 Oligosaccharides Oligosaccharides are the third largest solid component of human milk following lactose and lipids, with concentrations reaching up to 50 g/L or more in colostrum to an average of 10
15 g/L in mature milk (Kunz & Rudloff, 2008). Given their abundance, it became obvious that human milk oligosaccharides (HMO) have specific biological functions. Such functions may include prebiotic activity, antiadhesive activity, antiinflammatory properties, modification of the entire complement of cell surface sugars, a role in brain development, influencing growth-related characteristics of intestinal cells and other uncharacterized effects (for reviews see Bode, 2012, 2015; Donovan & Comstock, 2016). However, until recently, there were very few commercial products on the market that capitalize on these functions. This is mainly due to the fact that the large quantities of human milk oligosaccharides required for clinical trials have been unavailable. Recently, Abbott Laboratories have developed a formula containing a major HMO, 20 -fucosylactose (20 -FL). Moreover, a study in healthy infants has shown that infant fed formula supplemented with 20 -FL exhibit lower plasma and ex vivo inflammatory cytokine profiles, similar to those of a breast-fed reference group (Goehring et al., 2016). However, one of the most striking features of human milk is the diversity of oligosaccharides found with over 150 identified to date (Wu, Tao, German, Grimm, & Lebrilla, 2010; Wu, Grimm, German, & Lebrilla, 2011). It may be that a mixture of oligosaccharides is even more beneficial to the infant. Recent research has demonstrated that bovine milk and dairy

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streams (whey streams) contain several oligosaccharides, with a number of them structurally similar to those found in human milk and with some structures common to both milks (Barile et al., 2009; Mehra et al., 2014; Mehra & Kelly, 2006). As the chemical structure of oligosaccharides influences their biological activities, it may be expected that oligosaccharides from both human and bovine milks would have similar physiological effects. As a result, there is also interest in adding mixtures of these valuable molecules to IFs to better match the nutritional value of breast milk. However it should be noted that concentrations of oligosaccharides in bovine milk are much lower when compared to human milk.

12.3 INCORPORATION OF WHEY PROTEIN INGREDIENTS INTO INFANT FORMULAS 12.3.1 Development of Whey Protein Ingredients for Infant Formula Selecting whey protein ingredients for use in IF is dependent on many factors including nutrition, cost, label claim specification, compositional calculations (based on need to fit other ingredients), and capacity to enrich with bioactive nutrients. A manufacturer must ensure that nutritional needs are met while delivering high-quality liquid or powder IF. To achieve these targets, a range of different whey protein ingredients are used in keeping with nutrition and also manufacturing requirements. Liquid sweet whey, the most common liquid whey used for whey ingredient production, is a pale green liquid with a protein concentration of B0.7% (w/w), and is a by-product produced during hard and semihard cheese manufacture. When concentrated and dried post clarification (removal of curd fines), a whey powder with a protein content of approx. 10%
14% (w/w) is produced depending on the composition of the original liquid whey. Generally, this type of product is not used in IF production as the mineral content of this powder is too high to meet nutritional specifications. The major whey proteins in bovine milk are, in order of concentration, β-Lg (50% of total whey protein), α-La (B20%), and BSA (5
10%) (Fox & McSweeney, 1998b; Thompkinson & Kharb, 2007). α-La is the most abundant whey protein in human milk (Thompkinson & Kharb, 2007; Shi et al., 2011) and through advances in separation and ion exchange technology, α-La-enriched bovine WPCs and WPIs (Holt et al., 1999) are now available for addition to IFs. Demineralized whey is manufactured using electrodialysis and/or ionexchange technology; a more detailed explanation of these processes is given by Greiter, Novalin, Wendland, Kulbe, and Fischer (2002). Whey streams can be demineralized to a varying extents, typically to levels of 70 or 90%,

12.3 Incorporation of Whey Protein Ingredients Into Infant Formulas

providing an ingredient suitable for use in 1st stage IF formulations (Okawa et al., 2015); demineralized whey is not normally used in 2nd stage or follow-on formulas where a 60:40 whey casein ratio is not required. WPCs used in infant formulations can have a protein content ranging from 35% to 80%. Production of these ingredients include the removal of fat, lactose, and minerals to varying extents (dependent on targeted protein) through centrifugation, ultrafiltration, evaporation (in some instances), and spray-drying (Fig. 12.1; see also Chapter 3). High-protein WPCs (typically greater than 65% protein) are suitable for use in 1st stage IF and can replace demineralized whey; however, the higher the protein content of the WPC, the more fortification with lactose that is required. Less-expensive lower-protein whey concentrates, e.g., 35% and 65%, can only be partially used in 1st stage formulas due to their higher mineral content; however such restrictions do not apply to 2nd stage and follow-on formulas. WPIs are manufactured using microfiltration for maximal removal of fat, followed by ultrafiltration and diafiltration (Wang & Lucey, 2003) to concentrate the protein to greater

FIGURE 12.1 Typical manufacturing process, with heat map (excluding spray drying temperatures), for demineralized and nondemineralized whey, whey protein concentrates and whey protein isolate.

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90%; these ingredients provide a useful platform for development of specialized whey ingredients enriched in certain bioactives and are commonly used for the development of new IF formulations. Further purification of WPI can be achieved using ion-exchange chromatography to remove the GMP generated during the renneting process of cheesemaking, increasing the concentration of individual whey proteins compared to WPI made using membranes and evaporation alone. The manufacturing parameters used to produce WPC and WPI ingredients, determine the nutrient composition and protein denaturation levels, both key characteristics in ingredient selection for nutritional products including beverages (Holt et al., 1999; Wang & Lucey, 2003). The development of processes for α-La and Lf-enriched WPC and WPI, in particular, has generated interest (Lien, 2003; Satue-Gracia, Frankel, Rangavajhyala, & German, 2000). α-La-enriched whey protein ingredients are rich in tryptophan, cysteine, and histidine, thus making the production of lower protein IFs possible (O’Callaghan & Wallingford, 2002). The manufacturing processes for different whey ingredients is illustrated in Fig. 12.1; the heat map depicts, as a guide, the thermal load contributing to denaturation and aggregation of the proteins which in-turn can impact ingredient functionality during subsequent use in IF. In some instances, bovine milk proteins in an infant’s diet have been associated with development of allergic response. These allergies have been reported to be associated with β- Lg as this whey protein fraction is absent in human milk (Asselin, Amiot, Gauthier, Mourad, & Hebert, 1988); however, evidence suggests that infants can sometimes suffer from allergic responses to both major and minor whey proteins (α-La, BSA, and Igs) and casein components (κ-casein, β-casein, and αs-casein), all of which are found in IF (Gjesing, Schwartz, Wahn, & Løwenstein, 1986). Hydrolyzed whey protein (HWP) may be used as an alternative protein ingredient for infants who show allergic response to products containing intact dairy proteins and are currently used in the manufacture of specialized comfort formulas. Whey protein ingredients are commercially available in extensively hydrolyzed form, and are clinically proven to be effective against milk protein allergy. Protein allergenicity can be reduced by altering a protein’s secondary structure via enzymatic hydrolysis, thus lowering its molecular weight (Chobert, Bertrand-Harb, & Nicolas, 1988; Kelly, O’Mahony, Kelly, & O’Callaghan, 2016). Generally, HWPs are easier to digest compared to intact protein, and are more readily broken down into small peptides and amino acids, improving absorption and providing nutritional benefits. The degree of hydrolysis of a HPW is expressed as the percentage of protein which has been altered through enzymatic hydrolysis and is determined by the percentage of peptide bonds which have been cleaved (Adler-Nissen, 1979). However, the ability of HWP to act as a surfactant to stabilize an emulsion against coalescence

12.3 Incorporation of Whey Protein Ingredients Into Infant Formulas

and flocculation is altered. Thus, hydrolysis of whey proteins can affect emulsification capacity (Agboola, Singh, Munro, Dalgleish, & Singh, 1998; Okawa et al., 2015), impacting in-process stability and finished product quality of IF; in this instance additional emulsifiers, such as lecithin, monoglycerides, or esterified acids, are sometimes used to aid emulsification. Although sweet whey is traditionally used in the production of IF, interest in the use of acid whey protein ingredients in IF has increased in recent years. Acid whey is a by-product of acid casein and other fermented consumer products such as strained yogurt or cottage cheese. Acid whey has a number of differences compared to cheese (sweet) whey, such as lower protein and lactose, lower pH (4.6
5) and lactic acid content, higher mineral content (e.g., calcium, phosphorus, and lactate), no GMP, color additives, residual rennet, starter bacteria and associated proteinases and peptidases. GMP is a C-terminal part (f 106
169) of κ-casein which is released in whey during cheesemaking by the action of chymosin in rennet (Neelima et al., 2013). This glycosylated protein can comprise 20%
25% of the total protein (Farías et al., 2010) in whey concentrate powders derived from sweet whey. Commercial ingredients enriched in GMP with 78%
83.7% protein content containing 7%
9% of sialic acid are available (Neelima et al., 2013). Although not a protein present in human milk, during infant digestion it is formed and arguably has some benefits to an infant’s health, i.e., antimicrobial, mineral enhancing, and prebiotic effects (Brück, 2003). As a nutrient, the role of GMP in IF is commonly debated; theoretically the protein content of an IF can be lowered if a whey ingredient without GMP is used. For example, with acid whey, it may be possible to meet the specific amino acid profile required for an infant through a lower overall protein content. Nonprotein nitrogen (NPN) components, i.e., those soluble in 12% trichloroacetic acid (TCA), are also present in whey; in bovine milk, the percentage of TCA-soluble nitrogenous compounds is 5%
6%. NPN components are as follows: urea, peptides, uric acid, amino sugars and alcohols, ammonia, creatine, creatinine, nucleic acids, low-molecular-weight peptide hormones, free amino acids, growth factors, carnitine, and choline (Atkinson & Lönnerdal, 1995). Up to 25% of the total nitrogen in human milk can be in the form of NPN. This NPN contains free amino acids such as taurine that can be converted to cysteine which is important in infant development. In some instances IF are fortified with taurine to emulate levels in human milk (O’Mahony & Fox, 2013). Urea is the largest component of NPN in bovine milk comprising of 50%, and differences in its quantity vary with stage of lactation and season causing variability in heat stability (Muir & Sweetsur, 1976). The degradation of urea during heating produces subproducts, including ammonia and isothiocyanate, which buffer milk protein systems against changes in pH. For this reason, the urea content of whey is a contributing factor to in-process thermal stability.

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12.3.2

Formulation Dynamics

Depending on nutritional requirements, process efficiency, and ingredient availability, producers adopt different protocols for manufacture of IF. In addition to protein, WPC contains bioactive compounds, carbohydrate in the form of lactose, fat, minerals, and vitamins. WPI being typically greater than 90% protein by weight, is virtually depleted of fat and lactose. Both WPC and WPI have a mild taste profile and can be incorporated into IF with little impact on sensory properties. WPH are partially hydrolyzed and as a result have higher levels of NPN while also containing lactose, fat minerals, and vitamins. The nutrient content of a whey ingredient is determined by the separation process used, which in turn governs the calculations to formulate a finished beverage to a required nutritional specification. Ultimately, the protein content determines the level at which a whey ingredient is added to an IF formulation and therefore influences the quantity of all other innate nutrients, i.e., minerals, vitamins, bio-actives etc., contributed; this is a key consideration during development of a new whey ingredient for use in IF and determines that level at which a bioactive compound in a whey ingredient can be added. For formulation of 1st stage IF, use of a demineralized whey ingredient is desirable because it contains a high level of lactose (B80%) relative to, for instance, a WPC with a protein content of .70% and lower lactose; the lower protein content (B12.5%) of demineralized whey means that a greater quantity of this ingredient is required to meet the protein specification compared to the higher protein WPC. For a target of 1.4% protein (per 100 mL) on reconstitution (1st stage formulation; 60:40 whey protein to casein ratio), taking into account the whey protein in the added skim, addition of 5.6 g of a demineralized whey powder (12.5% protein) is required, of which 4.48 g is lactose. In contrast, if WPC80 powder (80% protein) is used instead of demineralized whey powder, 0.88 g w/w of ingredient is required per 100 mL reconstituted formula, which contributes 0.07 g lactose per 100 mL to the final formulation. The typical lactose content in a 1st stage formulation is 7.3 g per 100 mL. Taking into account the lactose contributed by the skim powder (1.04g) and the innate lactose present from either the added demineralised whey powder (4.48g) or WPC80 powder (0.07g), the approximate additional lactose required for a 1st stage formulation will be 1.8 g versus 6.2 g of lactose per 100 mL, respectively. These calculations, while not complex, are an important consideration for developers of new whey ingredients, as the quantity of powder added determines the amount of other nutrients incorporated into the finished formulation. Another key criterion for ingredient selection is the level of ionic 3 species present, e.g., Ca21, Mg21, Na1, K1, PO32 4 , and citrate
in the soluble phase which strongly affects the thermal stability of an IF during heating. If for instance a WPC35 is replaced with a WPC80, the levels of soluble ions

12.3 Incorporation of Whey Protein Ingredients Into Infant Formulas

are reduced due to more extensive membrane separation/diafiltration used during manufacture of the latter. This increases the level of fortification required to attain targeted individual mineral levels which, in many instances, is done using more insoluble salts. In this scenario, the reduction in soluble ions, e.g., Ca21, can result in a reduction in viscosity during heat treatment, thus facilitating longer manufacturing times before cleaning (CIP) is required and less downtime.

12.3.3 Whey Protein 2 Casein Interactions in Infant Formula Heat-induced interactions between whey proteins and other ingredients in IF are inevitable and can have a positive or negative impact on the quality of the product. These interactions can have a significant effect on both the physical and chemical characteristics of IF during processing, e.g., in-process control of heat stability, viscosity, and gelation, or in the finished powder, e.g., wettability, dispersibility, or formation of “white flecks” on rehydration. The physical phenomena of precipitation, coagulation, and gelation are the resulting effect of whey protein denaturation/aggregation which is dependent on environmental conditions such as temperature, pH, protein, and mineral concentration. Caseins are, for the most part, present in many commercial infant formulations, including 1st stage and follow-on, and their physicochemical interaction with whey proteins is an essential mechanism contributing to heat stability during thermal treatment (Murphy et al., 2015). Caseins have relatively little tertiary structure and are more heat-stable than whey proteins (Donella-Deana et al., 1985; Swaisgood, 2003); as an example, sodium caseinate can be held at 140 C for more than 1 h without visible changes in physicochemical properties (Fox & McSweeney, 1998b). However, caseins do interact with whey proteins during heat treatment and these reactions are central to producing a stable IF. The heat stability of skim milk, e.g., is linked to cysteine residues in κ-casein located at the surface of the casein micelle and their interaction with the whey protein β-Lg. When mixtures of β-Lg and κ-casein are heated, they aggregate through the formation of disulfide bonds and hydrophobic interactions (Guyomarc’h, Nono, Nicolai, & Durand, 2009). When skim milk is heated, depending on pH, the formation of complexes of β-Lg and κ-casein can either have a favorable or detrimental effect on heat stability (Tessier & Rose, 1964). Jeurink and De Kruif (1993) found that unfolding of β-Lg and subsequent association with casein micelles was responsible for increasing the viscosity of skim milk after heating at 85 C. Heat treatment at pH ,6.9 allows for the formation of β-Lg covalent bonds with casein micelles via κ-casein, and in turn helps stabilize the micelle against precipitation (Wijayanti, Bansal, & Deeth, 2014). However, when milk is heated at pH . 6.9, κ-casein-β-Lg complexes dissociate from casein micelles, enhancing release of κ-casein which

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results in micelle destabilization (Singh & Fox, 1987). Similarly, interactions between casein and whey proteins in IF have been shown to be pHdependent. At pH 6.5
6.8, β-Lg in a dispersion of SMP and electrodialyzed whey (whey protein 60%, casein 40%) was found to co-sediment with caseins after heating (140 C for 80 s); at pH 6.9
7.1 β-Lg was found in the supernatant after sedimentation (McSweeney, Mulvihill, & O’Callaghan, 2004). The effect of pH is important as IF manufacturers target a pH typically between 6.7 and 6.8 during batch make-up by careful selection of mineral salts and their order of addition to the liquid mix. Understanding casein 2 whey protein interactions and pH is further complicated by the drop in pH during concentration on evaporation (Murphy et al., 2015). It is important to monitor the pH of IF during processing as a significant drop in pH (e.g., pH ,6.5) can lead to increased viscosity and protein precipitation during evaporation as the pH drops further, thus causing fouling and contributing to manufacturing downtime. It is thought that pH affects the availability of κ-casein which subsequently has an effect on the type of aggregates that can be formed (Creamer, Berry, & Matheson, 1978). Donato, Guyomarc’h, Amiot, and Dalgleish (2007) reported that excess κ-casein or sodium caseinate in skim milk of neutral pH and heated to 90 C, had a minor influence on the size and number of protein aggregates formed, with excess whey protein having a much more prolific effect, where both the quantity and size of protein aggregates were altered. κ-Casein, when added in excess, is structurally different to that of κ-casein found on the surface of the casein micelle as it resides in the serum phase and has insignificant interactions with whey protein and does not affect the casein micelle. κ-Casein residing on the surface of the micelle is more accessible than when in the serum phase, as it is widely distributed across the surface giving greater access for whey proteins to interact, compared to that of the aggregated κ-casein in the serum fraction. Goodison (2017) demonstrated that heating WPI and caseins together in a model IF solution, compared to heating individually and then mixing, resulted in increased heat stability. The greater stability was attributed in-part to the significantly lower level of exposed free thiol groups compared to those formulations where the skim milk and whey protein were heated separately. Murphy et al. (2015) showed that casein at a concentration similar to that used in IF increases the temperatures at which β-Lg denatures and also the onset of viscosity increase during heating.

12.4 IMPACT OF WHEY PROTEIN FUNCTIONALITY ON STABILITY OF INFANT FORMULAS DURING PROCESSING 12.4.1

Whey Proteins and In-Process Emulsion Stability

IF is a typical oil-in-water emulsion formed though homogenization of aqueous and oil phases in the presence of proteins and/or other emulsifiers.

12.4 Impact of Whey Protein Functionality on Stability of Infant Formulas During Processing

Coarse emulsions are produced during the batch make-up process, i.e., the shearing of protein ingredients, carbohydrate, minerals and vitamins with a fat blend usually by recirculation through a high-shear powder entrainment system. This mix is unstable owing to its nonuniform emulsion droplet size characterized by a bimodal fat globule size distribution ranging from small to large droplets which are likely to cream or flocculate. A two-stage valvetype homogenization step is typically used to form a homogenous and stable IF emulsion. The initial stage during homogenization involves the disruption of existing fat droplets and particles creating a narrower fat globule size distribution (Lam & Nickerson, 2015; McCarthy, Gee, O’Mahony, Kelly, & Fenelon, 2015). For IF manufacture, the oil-in-water mixture is forced through a valve, typically at a pressure between 150 and 180 bar (15
18 MPa), inducing, depending on certain factors, a uniform and stable emulsion. A second homogenization step, generally carried out at B20% of the first-stage pressure, is used to break up flocculates that may have formed immediately after the first homogenization step ensuring that all large fat globules have been reduced to the smallest droplet size. McCarthy et al. (2015) used a factorial statistical design to determine the optimal total solids (TS), preheat treatment temperature, and first- and second-stage homogenization pressures for maximum emulsion stability in a model IF during processing. The dispersion of fat in the aqueous phase forms an interface where proteins can interact and adsorb, reducing interfacial tension, increasing stability of emulsion droplets through the formation of hydrodynamically and electrically charged protein layers, providing resistance to creaming and coalescence of fat globules (McClements, 2004; Ye, 2008). Formation of a stable emulsion with mono-modal fat globule size distribution is a critical step in the manufacture of IF and determines subsequent stability throughout evaporation, drying, and powder rehydration. Insufficient emulsification can cause an unstable emulsion to flocculate and/or coalesce, resulting in free fat and poor reconstitution of the IF powder. The affinity of a protein to adsorb to the interface relates to the flexibility, linearity, and hydrophobicity of its structure (Dalgleish, Goff, Brun, & Luan, 2002). In mixed protein systems containing both the same ratio of casein to whey proteins, preferential adsorption of caseins occurs as they contain little secondary structure and are more open flexible molecules. In particular, β-casein and αs1-casein are highly surface active, containing hydrophobic residues which anchor to the interface (Dickinson, 2001). In comparison, the globular whey proteins are less susceptible to adsorption, especially in their native globular form (Dickinson, 1998). However, upon unfolding during thermal treatment, these proteins become more flexible and are more likely to adsorb to an emulsion interface in conjunction with caseins. Lam & Nickerson (2015) found that subjecting α-La to a preheat treatment of 65 C while increasing pH to 7, improved its emulsifying properties, attributed to an

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increase in surface electrostatic forces. In IF processing, emulsions are primarily stabilized by dairy proteins, however, emulsifiers such as lecithins and monoacylglycerols/diacylglycerols can be added to help increase emulsion stability in certain formulations, e.g., those made using WPH ingredients. As protein or an emulsifier is needed to stabilize the interfacial surface of each fat droplet formed during homogenization, the protein to fat ratio plays an important role in the stability of an emulsion both during and post processing (Creamer & Parry, 1981; McDermott, 1987; McCarthy et al., 2013; Ye, 2008). McCarthy et al. (2012) described how a reduction in the protein (60%, w/w, whey protein) to fat ratio of a model 1st stage IF from 0.43 to 0.21, significantly increased fat globule size from 0.74 to 0.81 μm (Sauter mean diameter; D[3,2]) post homogenization. Furthermore, the fat globule size for all emulsions significantly increased on evaporation, with the greatest increase being observed when the protein to fat ratio was lowered to 0.21. The study suggests that the critical protein to fat ratio needed to achieve a stable IF is 0.26, and when the protein to fat ratio is decreased below this level, emulsion stability is significantly reduced, particularly on evaporation. Removal of water from the continuous phase can compress an emulsion, reducing the quantity of water between the interface of neighboring droplets causing distortion, disruption, and short-range interactions leading to coalescence (Aranberri, Binks, Clint, & Fletcher, 2004). While IF emulsions are primarily stabilized by the protein found at the fat droplet interface, pH and ionic strength are important; fat globule stability is governed by van der Waals interactions, electrostatic repulsion and short-range interactions all of which are sensitive to changes in pH and ionic strength (Demetriades, Coupland, & McClements, 1997a, 1997b). As the isoelectric point of the majority of dairy proteins is in the range of 4 to 6, the pH of the continuous phase can affect the strength of electrostatic repulsions provided by proteins embedded on the surface of fat droplets. Lowering the pH within this range reduces the net charge of the protein on the surface of droplets causing flocculation and destabilization of the emulsion. Depending on the composition of protein on the surface of the emulsion droplet, emulsions may deflocculate when the pH is either further reduced or increased. Both caseinand whey protein-based emulsions are destabilized at pH B5. However, at this pH, casein undergoes irreversible protein precipitation while emulsions stabilized by whey proteins can be deflocculated when the pH is decreased to pH ,3 or increased to pH .7 (Dickinson, 2010). For example, higher levels of α-La are found at the interface at pH 3 due to preferential adsorption of its monomeric structures favored by the lower pH (Hunt & Dalgleish, 1994). Emulsions containing globular whey proteins are thermodynamically unstable during heat treatment due to molecular rearrangement of protein molecules on the surface of the oil droplets (Tcholakova, Denkov, Ivanov, & Campbell, 2006). Without sufficient preheating, β-Lg, exposed on emulsion

12.4 Impact of Whey Protein Functionality on Stability of Infant Formulas During Processing

droplets may destabilize due to partial molecular rearrangement induced by heat-denaturation and homogenization (Dickinson, 1998), an important consideration for manufacturers of whey-dominant IF. This partial rearrangement can result in protein 2 protein bridging and flocculation of protein molecules on the surface of oil droplets, or cause depletion flocculation where detached surface proteins interact with unfolded protein in the aqueous phase, thus causing coalescence of oil droplets and a subsequent increase in creaming rates. This effect is confirmed by recent research which shows that increasing α-La within a whey protein fraction (with concomitant reduction in β-Lg) of a model IF, significantly increased stability of the emulsion and reduced viscosity during subsequent evaporation (Buggy, McManus, Brodkorb, Mc Carthy, & Fenelon, 2017; Crowley, Dowling, Caldeo, Kelly, & O’Mahony, 2016). Replacing β-Lg with α-La reduced the number of free thiol groups within the protein system and less aggregation was observed. As a consequence both the surface of emulsion droplets and the continuous phase contain fewer molecules with exposed free thiol groups, thus reducing the occurrence of depletion flocculation, bridging between emulsion droplets and increase in viscosity on concentration. WPH ingredients present some challenges regarding in-process emulsion stability depending on the degree and extent of hydrolysis. Low levels of hydrolysis result in the formation of peptides which have greater flexibility and a greater number of available hydrophobic residues producing a polypeptide with better emulsification properties compared to the original native globular protein (Singh, 2005). However, if the degree of hydrolysis is considerable (B20%), there is the propensity for the formation of shorter peptides which lack flexibility, have reduced steric hindrance and electrostatic repulsion, and are less effective at stabilizing an emulsion leading to coalescence of oil droplets (Kelly et al., 2016; Singh & Dalgleish, 1998). Schröder, Berton-Carabin, Venema, and Cornacchia (2017) showed that extensive hydrolysis of whey protein decreases surface hydrophobicity and emulsification capacity. Introducing a greater amount of higher-molecular-weight peptides (B5000 Da) through (i) specific proteolysis of whey protein; (ii) fractionation of WPH by ultrafiltration; and (iii) addition of higher peptide concentrations (protein/oil ratio B1:1), can help to alleviate this complication and produce an emulsion capable of being stabilized by peptides (Agboola et al., 1998; Gauthier, Paquin, Pouliot, & Turgeon, 1993). Murphy et al. (2015) made a 1st stage IF using a whey ingredient in which β-Lg was selectively hydrolyzed and α-La remained mostly intact. The resulting formulation had similar in-process stability and finished powder properties to an IF made with nonhydrolyzed protein (60:40; whey protein:casein), and improved stability compared to IF made with partially hydrolyzed whey and casein (60:40). Targeted hydrolysis of β-Lg reduces the role of free thiol groups and

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susceptibility to formation of covalently linked whey protein aggregates, thus lowering viscosity. An increased number of reactive thiol groups has been linked to a faster first-order rate constant and more spherical aggregates with higher molecular weight (McGuffey, Otter, van Zanten, & Foegeding, 2007). Murphy et al. (2015) found that IF emulsions made with WPC in which β-Lg was selectively hydrolyzed, had lower volume mean diameter (D[4,3]) and viscosity in the feed to the spray dryer than IF emulsions prepared with intact protein or more typical hydrolyzed whey protein systems. The lower viscosity resulted in a finer spray on atomization, smaller powder particle size, and lower final powder moisture content due to an increased rate of evaporation in the dryer, highlighting the importance of whey protein functionality in IF manufacture.

12.4.2 Thermal Stability of Infant Formula Containing Whey Proteins Thermal treatment of IF or IF ingredients is needed to reduce bacterial load and ensure the product is safe for consumption. However, whey proteins are heat-labile, and can decrease the stability of IF during heat treatment, especially when concentrated, ultimately influencing finished powder functionality. In particular, α-La, β-Lg, and BSA undergo conformational changes including unfolding, denaturation, and aggregation which are irreversible under particular conditions (Tolkach & Kulozik, 2007). The presence of free thiol groups in both β-Lg and BSA monomers can induce, when heated above B65 C, the disruption of inter- and intramolecular bonds resulting in disulfide bonding and bridging (Havea, Singh, & Creamer, 2001; Morr, 1975; Verheul, Roefs, & de Kruif, 1998). β-Lg, as the main whey protein in bovine milk, plays an important role in determining the physical characteristics of IF during heating. During heat treatment, denatured and unfolded β-Lg participate in intermolecular reactions via thiol 2 disulfide interchanges leading to aggregation with adjacent β-Lg molecules (Galani & Apenten, 1999; Manderson, Hardman, & Creamer, 1998), other whey proteins (Schokker, Singh, & Creamer, 2000), and casein micelles (Fox & Morrissey, 1977). α-La is stabilized by four disulfide bonds and association with Ca21, but does not contain a free thiol group to initiate covalent aggregation (Brew, 2003). Consequently, denaturation of pure α-la has been reported to be reversible, and gel formation upon heating is substantially reduced in comparison to β-Lg (Boye, Alli, & Ismail, 1997). However, most systems contain both β-Lg and α-La, e.g., IF, and α-La can be incorporated into aggregates as a result of thiol 2 disulfide interchanges and hydrophobic interactions (Dalgleish, Senaratne, & Francois, 1997; Schokker et al., 2000). The GMP fraction in whey is the hydrophilic region of the κ-casein which is separated from the intact protein during manufacture of cheese or rennet

12.4 Impact of Whey Protein Functionality on Stability of Infant Formulas During Processing

casein. It contains no sulfydryl groups (Fox & McSweeney, 1998a) and as a result does not undergo physical changes due to heat treatment. O’Loughlin, Murray, Kelly, Fitzgerald, and Brodkorb (2012) found the GMP fraction of WPI to be resistant to heat treatments up to 80 C for 10 min, especially when compared to the β-Lg A, β-Lg B, and α-La fractions which were reduced by 77%, 65%, and 64%, respectively. At neutral pH, gelation does not occur in GMP solutions at concentrations of up to 40% (w/w) (Martinez, Farías, & Pilosof, 2010). Uncontrolled aggregation of whey proteins is undesirable, as it can lead to fouling or burn-on in heat exchangers which is time-consuming and costly to clean and can also cause defects in finished products, e.g., burnt particles (Zuniga, Tolkach, Kulozik, & Aguilera, 2010). Thermal exposure, ionic environment, and protein concentration used during heat treatment of whey ingredients determine the level of denaturation and aggregation which, in turn, governs the reactivity of whey proteins during sequential heating steps required during IF manufacture. In most IF processes, a preheat step is used to denature/aggregate whey proteins to control functionality during subsequent processing. The effect of preheating a WPI solution on the subsequent heat stability of a model IF containing a 40:60 casein to whey protein ratio was investigated by Joyce, Brodkorb, Kelly, and O’Mahony (2017). The study, used preheating temperatures of 72 or 85 C to denature whey protein with or without added Ca21 ions (to promote aggregation) prior to HTST pasteurization. Protein particles with different surface reactivity (differences in -SH groups) and hydrophobicity were produced and model IF formulations, in which whey proteins were preheated prior to pasteurization, had increased stability during subsequent heating. In another study, Buggy, McManus, Brodkorb, Hogan, and Fenelon (2018) showed that both concentration and pH affect the formation of heat-induced whey protein aggregates; heating (85 C, 30 s, pH 6.7) 8% w/w whey protein solutions resulted in a greater amount of soluble aggregates and smaller particle size compared to 1% or 4% whey protein solutions. The smaller particle size contributed to a more heat-stable system when heated subsequently. Buggy et al. (2017) demonstrated, by partially replacing β-Lg with α-La, that the destabilizing effect of upstream homogenization (when compared to downstream) was due to insufficient β-Lg denaturation/aggregation prior to emulsification. In agreement, Crowley et al. (2016) showed that increasing the α-La to β-Lg ratio significantly increased heat stability in model IF systems. The studies confirm that in-process stability of IF can be attributed to the structural changes and interactions of its individual whey proteins, in particular, β-Lg which is the most abundant whey protein in bovine milk and highly reactive in its denatured state (Murphy et al., 2015). Loss of α-helical secondary structure occurs when β-Lg is heated to temperatures greater than 60 C, exposing a free thiol

471

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group and inducing disulfide linkages between β-Lg and adjacent β-Lg molecules and other proteins (Brodkorb, Croguennec, Bouhallab, & Kehoe, 2016). These chemical changes, along with hydrophobic interactions, and the ionic environment in IF due to mineral fortification, accelerate protein aggregate formation, increase viscosity, and ultimately induce gelation (Brodkorb et al., 2016; Crowley et al., 2016) during concentration prior to drying.

12.4.3 Reducing Thermal Load on Whey Proteins During Infant Formula Manufacture Generally two types of heat treatment are used in the dairy and IF industry: (i) indirect, where heat is transferred across a physical barrier which separates product from a heating medium (e.g., plate or tubular heat exchangers); and (ii) direct, where the product and heating medium are in contact (e.g., steam injection or infusion). Direct heat treatments have a lower associated heat load than indirect treatments (Lewis & Deeth, 2009) and can achieve ultrahigh temperatures in as little as 1 s, compared to in excess of 10 s for indirect treatments. As a result, the level of denaturation of β-Lg and α-La is lower in direct UHT compared to indirect UHT (Tran, Datta, Lewis, & Deeth, 2008). The application of high velocity steam injection and its impact on colloidal stability of concentrated whey protein based IF emulsions has been investigated as a possible alternative to indirect heating systems (Murphy, Tobin, Roos, & Fenelon, 2013). Adamopoulos and Petropakis (1999) described direct steam injectors where a two-phase mixture of steam and liquid is accelerated to supersonic velocity as a result of the injector’s geometry, producing cavitation and rapid changes in shear rate within the injector resulting in homogenization of fat particles in the mixture. Using this type of injection system it was possible to produce a stable emulsion with relativity low levels of denaturation of β-Lg and α-La compared to an indirect system. The process resulted in lower viscosity in 60:40 whey protein:casein IF emulsions (18.9 mPa.s at 55% solids) compared to more typical indirect heating (43 mPa.s at 55% solids), which could facilitate higher solids feed to the spray dryer and more efficient energy utilization (Murphy et al., 2013).

12.4.4 Behavior of Whey Proteins During Concentration of Infant Formula Emulsions Batches of IF are typically manufactured at relatively low dry matter (DM) contents (for example 20%
40%) prior to concentration by evaporation to 50%
58% DM and finally spray-dried. A primary concern for an IF manufacturer while producing a whey-dominant formula is heat-induced aggregation, leading to an uncontrolled increase in viscosity, e.g., to .80 mPa.s in

12.4 Impact of Whey Protein Functionality on Stability of Infant Formulas During Processing

the concentrate feed to the dryer. Heat-induced changes in the physical properties (denaturation, viscosity and gelation) of whey proteins are dependent on concentration and interactions with other macro- and micronutrients such as lactose and minerals during IF manufacture. Studies documenting the effect of increasing DM content on the denaturation of whey protein are summarized in Table 12.4. The most commonly reported finding is that whey protein, or β-Lg, denaturation decreased with increasing concentration (Anema, 2000; Hillier, Lyster, & Cheeseman, 1979; McKenna & O’Sullivan, 1971). Anema (2000), however, reported the effect of concentration reduced as heating temperature increased and was negligible at 100 C. It was proposed that the thermodynamic favorability of the reduction in interactions between whey protein and lactose could explain these observations; increased levels of lactose at high DM content may prevent transition of β-Lg from dimeric to monomeric form, which was suggested to be rate determining at ,90 C. At .90 C, aggregation is the rate determining step of β-Lg Table 12.4 Studies Showing the Effect of Increasing Dry Matter Content on Whey Protein Denaturation Study

Year

Harland et al.

1952

Guy et al.

1967

McKenna & O’ Sullivan

1971

Hillier et al.

1979

Oldfield

1996

Anema

2000

Anema

2001

Protein/ System

Conc.

Heating Method and Temperatures

Findings

Whey/in skim milk Whey/in cottage cheese whey Whey/in skim milk

9%
36% (w/w) 5%
40% (w/w)

Not available. Data from Anema (2000) Heated at 87 C, 84.5 C, and 74 C in water batch for up to 30 min

Concentration had little effect on whey protein denaturation Whey protein denaturation was at a minimum at 20% (w/w)

9%
44% (w/w)

Whey protein denaturation decreased with concentration

α-La and β-Lg/in cheese whey α-La and β-Lg/in skim milk β-Lg/in skim milk

1.9
11.4 mg protein /mL

Heated to 75 C and 80 C in test tubes for from 5 to 20 min Small volumes sealed in capillaries and heated from 70 C to 130 C. No heating times mentioned Heated to 110 C in a direct steam injection plant 75
100 C in sealed plastic tubes in water bath for up to 15 min

α-La/in skim milk

9.6%
38.4% (w/w)

6%
13% (w/w) 9.6%
38.4% (w/w)

75
100 C in sealed plastic tubes in water bath for up to 15 min

Denaturation of α-La increased with concentration; Denaturation of β-Lg decreased with concentration Whey protein denaturation increased with concentration β-Lg denaturation decreased with increasing concentration; effect of concentration decreased with increased heating temperatures—at 100 C there was no affect α-La denaturation was not affected by concentration

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denaturation in milk; lactose is less effective in stabilizing against these interactions, and hence, the effect of increasing dry matter content was not effective in preventing denaturation at elevated temperatures. The above explanation was consistent with the observation that increased lactose and DM contents did not affect α-La denaturation (Anema, 2001); α-La is a monomeric species, therefore no rate-determining shift in molecular association occurs. Oldfield (1996) found that increased DM content resulted in increased denaturation of whey proteins at 110 C, suggesting that at temperatures approaching UHT, increasing dry matter content has the opposite effect compared to ,90 C. This is in contrast with Hillier et al. (1979) who found that increasing dry matter content reduced β-Lg denaturation and increased α-La denaturation at temperatures .100 C. The studies used different methods of heat treatment, the former employing a dynamic direct contact heat treatment of skim milk, while the latter employed a static treatment of unknown duration on cheese whey. These differences likely account for the contrasting results; the same may be said for earlier studies mentioned in Table 12.4, i.e., Harland, Coulter, and Jenness (1952) and Guy, Vettel, and Pallansch (1967). Increasing DM content also affects the thermal properties of an IF concentrate, which can affect overall energy requirements during heat treatment. The specific heat capacity (Cp; kJ kg-1 K-1) will decrease with increasing concentration due to relatively larger amount of energy required to break hydrogen bonds in water (Edsall & Wyman, 1962; Fernandez-Martin, 1971). Thermal conductivity (k; W m-1 K-1) of milk, e.g., also decreases with increasing dry matter content (More & Prasad, 1988). Prior to spray drying IF formulations are generally concentrated to, depending on constituents, between 50% and 60% w/w. Alternatively, high solids processing with no evaporation can be achieved if powder and/or preconcentrated liquid ingredients are used. Water is generally removed by falling film evaporators which have lower associated energy costs compared to spray drying (Fox, Akkerman, Straatsma, & de Jong, 2010). In falling film evaporators the wet-mix flows by gravity through a number of tubes, forming a film on the inside of each tube as it flows downwards. The extent of concentration achievable is determined by viscosity and, therefore, control of aggregation of whey proteins is key to preventing any unwanted fouling or blockages. For example, to ensure efficient drying, postevaporation viscosity of whole milk should not exceed 60
100 mPa s (Westergaard, 2004). In addition to changes in viscosity, evaporation can affect the physical state of wet-mix constituents. Liu, Dunstan, and Martin (2012) reported that concentration of skim milk affected micellar hydration, aggregation, and the amount of calcium associated with the micelle. Transfer of calcium phosphate from the soluble to colloidal micellar state reduces pH during evaporation (Singh, 2007) causing increased viscosity. In whey-dominant IF concentrate; it is a

12.4 Impact of Whey Protein Functionality on Stability of Infant Formulas During Processing

requisite that whey proteins receive the correct heat treatment prior to evaporation to ensure thermal stability as a consequence of the concentration of ions and associated pH drop. McCarthy et al. (2012) observed an increase in fat globule size in IF emulsions during concentration on evaporation, likely due to shearing (particularity where any cavitation occurs due to incorporation of air), concentration of ionic species, and reduction in pH. Murphy et al. (2015) attempted to decouple macronutrient (casein, whey protein, lactose, and fat) interactions during heating of model systems, by measuring physical changes in the behavior of macronutrients, in isolation and combination, over the range of concentrations used during the manufacture of 1st stage IF. Addition of both casein (as phosphocasein—PCN) and lactose to whey protein (as WPI) solutions elevated the denaturation temperature (Td) of β-Lg and the temperature at which viscosity started to increase upon heating from 40 to 95 C at a rate of 22 C min21 (Tv). After holding at 95 C for 5 min, mixtures of WPI and PCN exhibited similar viscosity increase to that of WPI alone, confirming the important role of whey protein in viscosity development in IF as emphasized throughout this chapter. Under the same heating conditions, dispersions containing PCN and lactose, but not WPI, did not exhibit the same extent of viscosity increase. The extent of viscosity increase was linearly correlated with whey protein content (Fig. 12.2). The effect of lactose, at the level used in IF, delays secondary structural changes and increases the temperature and which both denaturation and viscosity occurs, reducing postheat-treatment viscosity in protein solutions. The work demonstrates the central role that whey proteins have on in-process stability of whey-dominant IF concentrates and highlights that viscosity is not only a function of concentration but is dependent on interactions between whey proteins and other macronutrients like lactose and casein. IF powders are primarily produced using a spray dryer and the effect of whey proteins on the viscosity of an IF feed to the dryer determines the atomization efficiency. Hot air is used to remove water from wet-mixes which have been atomized into fine droplets (10
400 μm) to increase the area of contact with the hot air (Westergaard, 2004). Changes in viscosity through molecular interactions of whey proteins and other ingredients, affect the size of the atomized IF concentrate, evaporative capacity and final powder moisture content. IF powders are generally produced using two- or three-stage spray dryers consisting of a large drying chamber in which the bulk of water is removed, followed by supplementary drying using an internal fluidized bed (stage 2, located at the bottom of the drying chamber) and/or external fluidized bed (stage 3). Murphy et al. (2013, 2015) showed that the IF final powder particle size, produced using a lower pressure two-fluid nozzle in a pilot scale multistage dryer, was dependent on the extent of heat-induced viscosity generated prior to spray drying; this further highlights the effect that

475

Whey Proteins in Infant Formula

50 40

µpostHT/µpreHT(-)

CHAPTER 12:

30 20 10 0

0

2

4 6 8 % Whey protein (w/w)

10

0

2

4 6 8 % Whey protein (w/w)

10

50 40

µpostHT/µpreHT (-)

476

30 20 10 0

FIGURE 12.2 Ratio of apparent viscosity (40 C; 16.8 s21) post (μpostHT) and pre (μpreHT) heat treatment (95 C, 5 min) in Whey protein isolate (WPI) containing systems. Upper graph: WPI (▲), WPI 1 lactose (Δ). Lower graph: WPI 1 phosphocasein (V), WPI 1 phosphocasein 1 lactose (e) From Murphy, E.G., Fenelon, Mark A., Roos, Y.H., and Hogan, S.A. (2014) Decoupling Macronutrient Interactions during Heating of Model Infant Milk Formulas. Journal of Agriculture & Food Chemistry, 62(43), 10585
10593) Murphy, Fenelon, Mark, Roos, & Hogan, 2014).

protein conformational change and associated viscosity increase can have on finished product quality.

12.5

CONCLUSION

IF manufacturers have access to an increasingly wide range of whey proteinbased ingredients, which, depending on location, can be availed of as liquid

References

concentrates as well as powdered equivalents. At the macro level, in the case of 1st stage formulas, IF composition mimics that of human milk, with differences in protein fractions being addressed by incorporation of, for instance, α-La-enriched WPC. Meanwhile, research on minor and trace-level constituents in both human and bovine milks continues as scientists explore and attempt to validate their biological functionalities. In recent years, increased research activities have addressed the application of dairy chemistry and colloid science principles to the understanding of IF systems at both in-process and postproduction levels. Interaction between process and composition of dairy systems has a large effect in determining the physical characteristics of concentrates and resulting powders. The chapter describes the physical properties of globular whey proteins, their interaction during processing, and their influence on the physical state of IF wet mixes. Clearly, whey proteins, and in particular β-Lg, have a major role in determining the stability of 1st stage IF. The relationship between whey protein denaturation, aggregation, and in-process viscosity is determined by the concentration, temperature, and pH at which the IF formulation is heat-treated. Formulations containing whey proteins which are heated at low total solids content behave differently than those heated at high solids due to a variety of factors. Understanding the level of denaturation and aggregation of whey proteins is essential to producing an ingredient which is stable in IF formulations where emulsification and sequential heating steps are used. Understanding the mechanisms associated with aggregation can be complex, as whey proteins generally undergo heating steps prior to use in IF formulations. The IF industry’s manufacturing operations historically adhere to proven production practices; however new challenges are emerging in the area of sustainability and climate change, encouraging continued exploration of advanced technologies and novel ingredients.

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268.

CHAPTER 13

Whey Protein-Based Nutrition Bars Naiyan Lu and Peng Zhou Jiangnan University, Wuxi, People’s Republic of China

13.1

HIGH-PROTEIN NUTRITION BARS

High-protein nutrition bars are typically a high-protein intermediatemoisture food, and usually have a water activity (aw) in the range of 0.5 0.8. The growth of most microorganisms is inhibited at this low water activity. However, the shelf life of the product could still be limited by various chemical or physical factors, such as Maillard reactions and protein aggregation during storage. The protein content in high-protein nutrition bars is usually 15% 45% (Zhu & Labuza, 2010). Such products are used, e.g., as space food, military food, and emergency food. Dairy and soy proteins are the most commonly used proteins due to their favorable balance between health benefits and cost-effectiveness. Other components like chocolate, sugars, nuts, vitamins are also added to improve the taste, flavor, texture, or nutritional properties.

13.1.1

Health Benefits of High-Protein Diets

The nutritional functionality of high-protein diets has been extensively investigated. Compared to carbohydrate-based foods, protein-rich foods with the same caloric value can deliver more satisfaction. Intake of a protein-rich drink before lunch leads to less energy consumption at lunch than after a carbohydrate-rich drink (Bertenshaw, Lluch, & Yeomans, 2008). With respect to the specific protein type, whey proteins seemed to induce a satiety signal, which would influence the food intake both short and long term (Luhovyy, Akhavan, & Anderson, 2007) (see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins). High-protein diets led to lower subsequent food intake (Halton & Hu, 2004; Luhovyy et al., 2007; Westerterp-Plantenga & Lejeune, 2005; Westerterp-Plantenga et al., 2006) and dramatically decreased total body weight. The decrease was more obvious in people undertaking long-term diets (6 months or longer) (Westerterp-Plantenga & Lejeune, Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00014-X © 2019 Elsevier Inc. All rights reserved.

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2005). In comparison with the control group, people with a higher protein intake gained less weight and had a lower waist circumference (Lejeune, Kovacs, & Westerterp-Plantenga, 2005). In addition to the effect of the protein source, other factors, including dose, the form of the product, the treatment and formulation, the duration between two meals, and the presence of other macronutrients, also contributed to the satiety influence (Luhovyy et al., 2007). The concentration of satiety hormones, energy expenditure, amino acid concentration, and gluconeogenesis also positively influenced the satiety (Veldhorst et al., 2008). Moreover, a high-protein diet can reduce the progression of sarcopenia, especially for the elderly (Campbell & Leidy, 2007). The recommended dietary protein amount (0.8 g/kg per day) is generally lower than the needs of the elderly to maintain their fat-free mass and their muscle mass (Campbell & Leidy, 2007; Gersovitz, Motil, Munro, Scrimshaw, & Young, 1982; Kurpad & Vaz, 2000).

13.1.2

Formulation of High-Protein Nutrition Bars

Protein is the most important component in high-protein nutrition bars, and although there is no official standard, such foods usually contain 15% 45% protein by weight (Zhu & Labuza, 2010). The most commonly used protein sources are dairy and soy proteins. Egg proteins are also used in some products (Rao, Fisher, Guo, & Labuza, 2013; Rao, Rocca-Smith, & Labuza, 2013). Carbohydrates usually make up 10% 50% of the protein bar by weight (Zhu & Labuza, 2010). They are supplied as a mixture of crystalline sugars such as sucrose, and sugar syrups such as high-fructose corn syrup (HFCS), corn syrup, brown rice syrup, and glucose syrup. In addition, sugar alcohols are often used as low calorie sweeteners and for their humectant properties. Sorbitol and maltitol are two common sugar alcohols used in high-protein nutrition bars. Glycerol, a polyol with a water activity-lowering effect, may also be incorporated into high-protein nutrition bars (Liu, Zhou, Tran, & Labuza, 2009; Loveday, Hindmarsh, Creamer, & Singh, 2009; Loveday, Hindmarsh, Creamer, & Singh, 2010). Lipids make up about 10% 15% of the bar (Zhu & Labuza, 2010), although higher percentages in commercially produced bars, especially lowcarbohydrate varieties, are available. Lipids are often incorporated into the high-protein nutrition bars as vegetable shortening (McMahon, Adams, & McManus, 2009) or cocoa butter (Loveday et al., 2009, 2010). Other oils, such as canola, peanut, or soy, can also be added (Gautam & Simon, 2006). In addition, the bars contain water that acts as a plasticizer in maintaining stability (Purwanti, van der Goot, Boom, & Vereijken, 2009). High-protein nutrition bars are intermediate moisture foods with relatively lower moisture

13.2 Quality and Stability of High-Protein Nutrition Bars

content and aw (Loveday et al., 2009). Moisture content may be so low that added water may be almost excluded from a bar formulation (McMahon et al., 2009) or added at less than 15% of the formulation (Loveday et al., 2009, 2010). Low aw is needed to prevent microbial growth and ensure consumer safety since many high-protein nutrition bars are not subjected to a heat treatment (Liu et al., 2009). Besides the high-protein matrix, other components in high-protein nutrition bars add value, consumer appeal, and increase eating quality. Components include flavor layers (e.g., chocolate, peanut butter, and strawberry paste), textural components (e.g., crisps, nuts, and wafers), and nutritional supplements (e.g., fiber, vitamins, and minerals) (Loveday et al., 2009).

13.2 QUALITY AND STABILITY OF HIGH-PROTEIN NUTRITION BARS 13.2.1

Quality of High-Protein Nutrition Bars

13.2.1.1 Sensory Properties of High-Protein Nutrition Bars Flavor is one of the challenges faced by high-protein food manufacturers. Proteins can generate different flavors when incorporated into food products, sometimes off-flavors and after tastes (see also Chapter 10: Flavor Aspects of Whey Protein Ingredients). Drake, Karagul-Yuceer, Cadwallader, Civille, and Tong (2003) developed a profile for descriptive characterization of the sensory properties of dry dairy ingredients. The language included flavors of whey protein isolate (WPI) and whey protein concentrate (WPC), and was used along with instrumental analysis by Carunchia Whetstine, Croissant, and Drake (2005) to determine flavor and flavor compounds generated using WPC80 and WPI. Flavor variations of WPC80 and WPI can be demonstrated using this system. The profile was expanded by Russell, Drake, and Gerard (2006) to include sensory flavors from soy protein concentrate (SPC). The comparison study of flavor from rehydrated SPC70, WPC80, soy protein isolate (SPI), and WPI and the differences in flavor were documented. In products such as high-protein nutrition bars or high-protein beverages, dairy proteins and soy proteins are the most commonly used protein sources. Knowledge of differences in flavor between dairy and soy proteins is thus of importance for formula development for high-protein foods. Besides, the flavor quality differences with the same kind of protein also affect the final product. According to the report of Caudle, Yoon, and Drake (2005), flavor quality and consumer acceptance of the final product was closely related to the off-flavors generated from proteins during processing, which varied widely when different skim milk powders were used. They suggested that a practical evaluation system of sensory flavor would be helpful to the high-protein food industry.

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Formulation has a significant impact on the sensory properties of commercial meal replacement bars and beverages. This has been extensively studied to determine the effect of using different flavoring and stabilizing additives to obtain favorable sensory properties. However, the current information on how different protein sources impact the sensory properties of high-protein foods and beverages is insufficient. When it comes to the effect of protein sources on sensory properties, bars containing whey proteins have been shown to generate higher intensities of sweet aromatic or cardboard flavor notes, and bitter taste. WPI bars showed more surface gloss, together with higher adhesiveness and cohesiveness. Soy protein bars tend to have higher intensities of nutty, cereal, and hay flavors. Bars made with soy proteins were characterized as having higher hardness, denseness, and particulates in comparison with whey protein formulations. A combined use of whey and soy proteins in the bar leads to lower sweet aromatic flavor and higher hay flavor compared to bars made with only whey proteins. Compared to bars made with whey proteins or soy protein, bars made with the combined proteins showed the highest level of cohesiveness, hardness, and denseness (Childs, Yates, & Drake, 2007). By comparing model systems of dairy proteins with soy proteins, the typical beany flavor compounds, such as 2-ketone and 2-octanone, were observed in systems containing SPI (Heenan et al., 2012). In addition, by comparison of the GC-MS peaks of sorbitol and fructose systems at day 30, the typical volatile products of the Maillard reaction, such as furfuryl methanol, 5-methyl-2furfuryl methanol, dihydro-2-methyl-3-furanone, and acetyl-2-furan, were observed in model systems containing fructose, particularly for those containing dairy proteins. Moreover, some volatile compounds were observed after 30 days of storage in model bars with either sorbitol or fructose systems, including pyrazine derivatives such as 2,5-dimethylpyrazine and 2,6-dimethylpyrazine, which gave a barbecue flavor. Thus, different flavor profiles were generated in model systems made with dairy and soy proteins, and protein bars formulated with reducing sugars, such as fructose, generated more caramel and barbecue flavors due to the Maillard reaction (Zhang, 2014).

13.2.1.2 Texture of High-Protein Nutrition Bars The protein source will impact bar texture, flavor, consumer acceptance, and stability (Childs et al., 2007). Protein blends and hydrolysates can be used to improve flavor, texture, and stability. WPC can be used in milk protein concentrate (MPC) bars to reduce firmness (Imtiaz, Kuhn-Sherlock, & Campbell, 2012). Lu et al. (2016) studied the textural properties of high-protein nutrition bars made with WPI, sodium caseinate (NaCas), and SPI. They found these

13.2 Quality and Stability of High-Protein Nutrition Bars

proteins to be very different using a strain recovery test (Fig. 13.1). For WPI bars, the overall textural features of the samples before and after storage (25 C, sealed) were quite similar to each other. The samples stayed soft, although the maximum force in the compression test increased nonsignificantly. They could not maintain the original shape and samples had to be reshaped before testing. The samples were also sticky and easily adhered to

FIGURE 13.1 Extrinsic appearance changes pre- and post-test of high-protein nutrition bar model systems made with sodium caseinate (NaCas), soy protein isolate (SPI), and whey protein isolate (WPI) during storage (Lu et al., 2016).

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the probe. While for SPI and NaCas bars, the overall texture features were quite different before and after storage. For SPI bars, the newly prepared samples were relative hard, solid, and grainy, and collapsed in the compression test. After storage, the texture of the samples became harder and more integrated as the fracture force increased over time. Newly prepared NaCas bars also collapsed like the SPI bars in the compression test. After a 7-day storage, the bars became a typical elastomer. The samples remained intact after 50% compression and the postcompression recovery rate increased significantly. Other studies also found that bar texture varied according to the protein sources. Hogan, Chaurin, O’Kennedy, and Kelly (2012) found that highprotein nutrition bars made with NaCas were significantly harder than bars containing other dairy proteins. Loveday et al. (2010) reported that highprotein bars made with WPI remained soft throughout storage and were not firm enough to carry out a fracture test. Calcium caseinate (CaCas) bars had a firm texture and hardened during storage with a sharp initial hardening and hardened further with subsequent long-term storage. MPC bars hardened over time without a sharp initial increase (Loveday et al., 2009), which was quite different from the CaCas bars.

13.2.1.3 Stability of High-Protein Nutrition Bars High-protein nutrition bars are formulated to maintain palatable texture, and only limited texture changes are desired during storage. Typically, high-protein nutrition bars should maintain shelf stability for a minimum of 6 months if stored at room temperature (McMahon et al., 2009). However, many properties related to the quality of such foods like color (Banach, Clark, & Lamsal, 2014), flavor (Massaro & Labuza, 1990), and texture (Loveday et al., 2010) would be adversely affected by the physicochemical changes in the food matrix during storage. Among the major problems for high-protein nutrition bars, texture hardening during storage is the most severe one, which often makes the food inedible (Hogenkamp, Stafleu, Mars, Brunstrom, & de Graaf, 2011) and limits the shelf life (Gautam & Simon, 2006). The mechanism(s) for texture hardening of protein bars is complicated. On the one hand, intrinsic factors include bar ingredients, physical structure, component distribution within the matrix, and interaction among different ingredients. On the other hand, storage conditions including temperature are also relevant to hardening (Wilkinson, Dijksterhuis, & Minekus, 2000).

13.2.1.4 Texture Hardening of High-Protein Nutrition Bars Many previous studies have focused on the cause of hardening of high-protein nutritional bars and a number of mechanisms have been proposed to explain why some high-protein nutrition bar formulations are unstable and become

13.2 Quality and Stability of High-Protein Nutrition Bars

unpalatable with the development of rapidly increasing hardness. The main mechanisms proposed for high-protein nutrition bar hardening include protein aggregation (Zhou & Labuza 2007; Zhou, Liu, & Labuza, 2008a), Maillard reactions (Chen, Liang, Liu, Labuza, & Zhou, 2012; Corzo-Martinez, Moreno, Olano, & Villamiel, 2008; Gerrard et al., 2005; Imtiaz et al., 2012), sugar crystallization (Belcourt & Labuza, 2007), phase separation (Loveday et al., 2009; McMahon et al., 2009), and moisture migration (Loveday et al., 2009, 2010; Lu et al., 2016; McMahon et al., 2009). The hardening of high-protein nutrition bars can occur in two stages: the early stage and the later stage (Hogan et al., 2012; Loveday et al., 2010; McMahon et al., 2009). The early stage often refers to the initial week or month of storage, during which the redistribution of water and small molecular plasticizers is the dominating factor that induces a fast increase in hardness. During the later stage, after several months (length of this period depends on the bar’s formula and storage conditions), physical changes and chemical reactions lead to further hardening.

13.2.1.4.1 Moisture Migration Moisture content, water activity, and osmotic pressure influence the migration of moisture in high-protein nutrition bars (Loveday et al., 2010). After the high-protein nutrition bar components are mixed together, water may migrate, not necessarily from components of high moisture to low moisture, but from those with high aw to those of low aw (Li, Szlachetka, Chen, Lin, & Ruan, 2008). Water may migrate from sugar syrups into protein powders that were only partially hydrated during bar manufacture, meaning that the syrups will lose their ability to act as plasticizers and the bars will harden as a function of the moisture migration during the early stage of storage (Li et al., 2008). Lu et al. (2016) showed that, in high-protein nutrition bar systems, moisture migration in the early stage of storage decreased the mobility of small molecules, which would further change the microstructure of the product and could lead to texture hardening. The hardening might be different as the hydrophobicity of the proteins varies. For NaCas and SPI systems, the hardness of the samples increased rapidly after preparation. NMR spectra of model high-protein nutrition bars made with different protein sources showed that water may migrate from the continuous phase to partially hydrated protein, leading to changes in microstructure. For NaCas model bars, the protein particles were further hydrated, merged, and formed a network structure (Dickinson, 2006). This network structure was so strong in NaCas model bars that it resulted in unacceptable hardening of the product. For the SPI system, as soy proteins are much more hydrophobic, small

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molecules, high mobility still existed without being hydrated according to the NMR spectra. The protein particles did not form a network structure, but swelled and became larger over time. A higher effective volume fraction of the protein particles was achieved, raising the viscosity and elastic shear modulus of the system, and leading to texture hardening. For WPI model bars, the protein particles were much more hydrophilic (Jean, Catherine, & Marie, 1988) and almost fully hydrated during preparation. The samples showed a uniform structure and the texture remained soft and sticky during storage, without significant changes in the mobility of the small molecules. Moreover, decreasing protein solubility was only found in the SPI bars, which further demonstrated that moisture migration was the major factor causing bar hardening during the early stage of storage of those bars.

13.2.1.4.2 Protein Aggregation 1. Moisture and disulfide bond-induced protein aggregation. Denaturation and aggregation of proteins is a problem which often occurs during processing and storage of high-protein foods, and can lead to undesirable changes in microstructure and texture of the matrix. Whey proteins are subject to denaturation, and aggregation with exposure of active residues such as thiol groups, especially in systems with moderate or high aw. “Moisture-induced protein aggregation” often refers to the aggregation of proteins in the solid or semisolid state (Liu, Langer, & Klibanov, 1991; Zhou et al., 2008a). It can be induced by either intermolecular noncovalent bonds or disulfide bonds, and sometimes a combination of both. Two mechanisms are responsible for the formation of intermolecular disulfide bonds: One is the thiol disulfide interchange reaction between proteins, and the other involves broken disulfide bridges formed from beta-elimination, followed with disulfide interchange reaction catalyzed by free thiol groups. During processing and storage of high-protein foods, like high-protein nutrition bars, this moisture-induced protein aggregation may lead to loss of consumer acceptability of the product (Costantino, Langer, & Klibanov, 1994; Liu et al., 1991; Zhou, Liu, & Labuza, 2008b). In processing and storage of diluted systems like dairy beverages, denaturation of whey proteins can induce formation of aggregates through protein protein disulfide bonding or intermolecular noncovalent interactions, which lead to precipitation of the protein component. At higher protein concentrations, a gel network may form. The structure and texture of whey protein gels are not only dependent on the composition of the system, but also on the environmental factors including temperature, pH, ionic strength, and pressure (see also Chapter 6: Thermal Denaturation, Aggregation, and Methods of Prevention).

13.2 Quality and Stability of High-Protein Nutrition Bars

Under different conditions, whey proteins can form either a particulate gel or a filamentous network. The particulate gels are nontransparent, with large aggregates (which can be as large as several thousand nanometers) dispersed in the matrix. On the other hand, the filamentous network is transparent and contains “flexible strands or more rigid fibrils” (Zhou et al., 2008a). In a matrix with lower aw, Liu et al. (2009) reported that when whey proteins were in a solid amorphous state, moisture-induced aggregation could occur during storage at a relatively low temperature without protein denaturation. It was suggested that disulfide bonds formed through thiol disulfide interchange interactions between proteins were responsible for aggregation of whey proteins, and noncovalent intermolecular interactions also played a role (Liu, Zhou, Liu, & Labuza, 2011). Protein aggregation leads to formation of insoluble compounds. When it comes to model high-protein nutrition bars made with WPI and water, insoluble protein aggregates were formed rapidly in the first 3 days of storage, and a longer period with a lower formation rate followed. Further investigations showed that all major whey proteins (β-lactoglobulin (β-Lg), α-lactalbumin (α-La), and bovine serum albumin (BSA)) took part in the formation of aggregates (Zhou et al., 2008b). 2. Maillard reaction-induced protein aggregation. One of the major problems in dairy protein bars is Maillard reaction-induced quality loss. A blend of HFCS and a sugar alcohol (sorbitol or maltitol) syrup is usually the carbohydrate source in protein bars. However, high fructose content increases the risk of bar quality loss induced by the Maillard reaction. The Maillard reaction in dairy protein bars during long-term storage could induce variable quality losses in extrinsic appearance, nutritional value, sensory texture, and flavor of the products (Chen et al., 2012; Corzo-Martinez et al., 2008; Imtiaz et al., 2012; Lederer & Klaiber, 1999), which would result in loss of consumer acceptance. For example, there are 12 lysine residues per molecule for α-La and 16 for β-Lg that are vulnerable to participation in the Maillard reaction. In early phases of the Maillard reaction in high-protein nutrition bars, it is believed that protein glycation only has a limited impact on bar texture. As the reaction proceeds, however, the system keeps hardening during long-term storage because of the formation of protein aggregates. In model systems prepared with WPI and glucose or fructose, both soluble and insoluble protein aggregates were formed during 6 months of storage (Chen et al., 2012). β-Lg was glycated more heavily than α-La, BSA, and caseins, according to SDS-PAGE results. Therefore, more β-Lg took part in the formation of aggregates during long-term storage, which is indicated by the

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significant reduction of free β-Lg. Glucose was more active than fructose in this system due to its higher reducibility with moderate aw. The hardening process in the later storage was correlated with the formation of protein aggregates, which suggested that the Maillard reaction-induced protein aggregation was the major cause of texture hardening during storage of the model system (Chen et al., 2012). 3. Phase separation-induced protein aggregation. Preparation of protein bars involves a process of mixing different ingredients in either the solid or liquid state to form a two- or three-phase matrix. The continuous phase consists of sugar syrup and dissolved proteins, while the dispersed phases include protein particles and in some cases fat droplets. The proteins are still not completely hydrated after 24 hours of storage due to a low water/protein proportion (  0.5 w/w) in the system. Hydration of proteins is a slow and gradual process during storage, and the hydrated protein dissolves in the sugar syrup and intermixes with the continuous phase. A true protein solution cannot be formed as the water in the system is not sufficient. It has been shown using confocal laser scanning microscopy that phase separation in protein bars is more complex during long-term storage than in simple sugar protein systems. In simple sugar protein systems, phase separation is commonly related to sugar crystallization or the glass transition. In protein bars, however, it seems that interferences are found between the macromolecules in the dispersed phase and the dissolved small molecules. For protein bars, however, the rearrangement of components between the continuous phase composed of dissolved proteins and sugar syrup and the dispersed phase of protein particles and fat globules is the main cause of phase separation in this unique system. Firstly, as neither protein nor sugar is soluble in lipid, they are excluded from fat globules during storage, while only some protein molecules can be distributed on the lipid/syrup interface. Secondly, along with gradual hydration of proteins, more and more water is bound to the proteins, and water in the continuous phase is pulled into the dispersed phase (Lu et al., 2016). This water removal, however, can be reversed in some cases. If the sugar is not sufficiently dissolved during manufacture of protein bars, water absorbed by protein particles can be pulled back into the continuous phase (Tolstoguzov, 2003). Aside from rearrangement of components between phases, another explanation is the low entropy of the system due to the compressed configuration of protein and sugar molecules at high concentrations, which makes the particles-in-syrup dispersion thermodynamically unstable. Anyway, it is the partially hydrated protein particles (or dehydrated particles in some cases) that make the major contribution to the hardness of protein bars, while the syrup phase and fat globules act as plasticizers. When the syrup phase is disrupted by the protein hydration or dehydration process, the texture changes. When lipid is introduced into the

13.2 Quality and Stability of High-Protein Nutrition Bars

system, phase separation is even more complicated. During storage of model systems containing model lipids in either the solid or liquid state, there are two opposite effects, phase separation and phase integration. On one hand, there is a trend that small lipid droplets merge with each other under the drive of higher surface potential, which leads to phase separation. On the other hand, the large interface area between lipid droplets and the highprotein continuous phase increases the chance that lipid molecules could seep into the continuous phase, thus enhancing lipid protein interaction (Lopez et al., 2008). The macroscopic low fluidity of lipid in the solid state slows down the first effect, and provides enough time for the second effect. In the end, the combination of these two effects leads to the coexistence of two states of lipids: free lipids in large droplets and protein-combined lipids (Lopez et al., 2008). Coexistence of these two forms resulted in wider DSC peaks for the lipid phase transition as observed after storage, especially for the WPI/solid lipid system. In the WPI/solid lipid system, as the protein was more hydrophilic, the second effect was slower, and the system did not reach an equilibrium until the end of 14-days storage (Lopez et al., 2008).

13.2.1.4.3 Sugar Crystallization The most commonly used saccharides in high-protein nutrition bars are sucrose, glucose, fructose, high fructose syrup, and corn syrup. Among these sugars, the solubility of sucrose and glucose are relatively lower in water, and excessive addition of sugar may lead to sugar crystallization in the system during storage. Sugar crystallization can induce bar hardening in two ways. Firstly, sugar crystallization leads to the formation of sugar particles. Secondly, crystallization reduces the sugar in the continuous phase available as a plasticizer (Loveday et al., 2010). This is similar to the texture change induced by sugar crystallization in soft cookies (Belcourt & Labuza, 2007). Fortunately, sugar crystallization can be avoided by using sugar sources with better solubility, such as HFCS.

13.2.1.5 Other Quality Losses in High-Protein Nutrition Bars 13.2.1.5.1 Color Changes The major cause of color change in high-protein nutrition bars is the Maillard reaction. Maillard browning occurred in model high-protein nutrition bars prepared with partially hydrolyzed WPI (HWPI) and HFCS, but was limited in model bars made with WPI and sorbitol (McMahon et al., 2009). The bars formulated with HFCS remained softer but became darker, whereas the bars formulated with sorbitol had less color change and became firmer. Maillard browning was determined to occur during high-protein nutrition bar storage, but was not identified as a major mechanism of hardening (McMahon et al., 2009), although, the Maillard reaction leads to quality decline in high-protein nutrition bars. However, the generation of

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Maillard browning products may lead to increased covalent protein crosslinking and thus some attention should be given to these reactions (Le, Bhandari, Holland, & Deeth, 2011). The rate of the Maillard reaction increases with temperature. The progress of the Maillard reaction, determined by the browning assay, shows a dependence on the sugars involved. Studies using β-Lg model systems showed that glucose had a higher reaction rate than fructose with β-Lg, due to the higher reactivity of its α-hydroxy carbonyl (Zhang, Ames, Smith, Baynes, & Metz, 2012). The change in color is an indicator of the progress of the Maillard reaction (Fig. 13.2). The systems containing fructose became darker with L* decreasing as the storage time increased. Using sorbitol instead of fructose in

FIGURE 13.2 Color changes of high-protein nutrition WS: whey protein bar with sorbitol CS: casein bar with sorbitol SS: soy protein bar with sorbitol

bar model systems caused by Maillard reaction during storage for 48 days. WF: whey protein bar with fructose CF: casein bar with fructose SF: soy protein bar with fructose.

13.2 Quality and Stability of High-Protein Nutrition Bars

model systems effectively inhibited color change during 48 days of storage. Storage experiments with whey protein, casein, and soy protein systems showed that protein source also had a major impact on browning. After 48 days of storage, the color of model systems made with whey proteins and caseins became dark brown, while soy protein model bars had better resistance to color change. The darkness of fructose systems prepared with different proteins was as follows: whey proteins . caseins . soy proteins, probably because whey proteins contain the most active lysine residues (12 lysine residues per molecule for α-La, 16 for β-Lg) for the Maillard reaction.

13.2.1.5.2 Nutrition Loss Lysine is the first limiting amino acid in many high-protein foods. In highprotein nutrition bars made with both dairy and soy proteins, the reduction in active lysine induced by the Maillard reaction leads to a significant quality loss during storage (Ha & Zemel, 2003). As mentioned earlier, glycation is dependent on the reducing sugar involved, depending on the reactivity of the α-hydroxy carbonyl of the sugar molecule. Substitution of fructose with sorbitol can significantly slow down active lysine reduction during storage (Fig. 13.3). Overall, the Maillard reaction is responsible for the quality losses of many protein bars during long-term storage, and exclusion of reducing sugar seems to be the most efficient solution to inhibit these undesired changes.

FIGURE 13.3 Active lysine of high-protein nutrition bar model systems made of whey proteins with sorbitol (WS) or fructose (WF).

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13.3 SOLUTIONS TO AVOID HARDENING OF HIGHPROTEIN NUTRITION BARS 13.3.1

Inhibition of Disulfide Bond Formation

A possible method to avoid hardening of high-protein foods is to inhibit the formation of protein protein disulfide bonds. Theoretically, it is not difficult to prevent the thiol disulfide interchange reaction in food systems. As Dalgleish, Senaratne, and Francois (1997) reported, e.g., reducing pH to 6 decreased the thiol disulfide interchange reaction in a model system with 10% WPI. However, this may or may not be effective in a system with a lower aw. The initial pH value of B7 in systems with lower aw generally decreases by approximately 1.5, which would give a pH close to the nominal pI of whey proteins (4.8 5.5). When the pH value falls below 6, the solubility of WPI proteins decreases significantly (Anema, Hunter, & Hemar, 2006). The free thiol group blocking reagents, such as N-ethylmaleimide (NEM), 2-iodoacetamide, and iodoacetic acid, or disulfide bond splitters, such as β-mercaptoethanol (2-Me), dithiothreitol, and p-chlormercuribenzoate, could inhibit protein protein thiol disulfide interchange reaction effectively. However, these thiol group blockers and disulfide bond splitters are mostly toxic and cannot be used in food formulas. There have been some attempts to avoid disulfide formation, although generally with nonfood grade additives. Addition of the disulfide dithiothreitol (DTT) in whey protein gels as a reducing agent resulted in a more brittle texture compared to gels without DTT (Errington & Foegeding, 1998). The use of a free-thiol group blocking agent, NEM, was also effective in reducing hardness of whey proteins gels. Nevertheless, to do this type of inhibition using food grade ingredients that are consistent with consumer preferences remains a challenge (Alting, Hamer, de Kruif, Paques, & Visschers, 2003; Matsudomi, Rector, & Kinsella, 1991). A promising additive for food usage is L-cysteine (Cys) hydrochloride. Cys is a food additive allowed in the United States (21CFR, 184. 1272) as a dough strengthener, and can be used to reduce the processing time of baked foods. Cys can also generate a meat-like flavor. Also, Cys is used to reduce nonenzymatic browning reactions and thus increase the whiteness of foods. In dietary supplements, Cys can be used as a free radical scavenger and antioxidant. And in high-protein nutrition bars, Cys might be a useful free thiol group blocker to inhibit protein protein thiol disulfide interchange reaction and thus delay bar hardening. WPI model bars with and without Cys or NEM showed different shelf lives when stored at 45 C as part of an accelerated storage test, and difference in

13.3 Solutions to Avoid Hardening of High-Protein Nutrition Bars

formation of insoluble aggregates and rearrangement of microstructure were observed during storage for up to 35 days (Zhu & Labuza, 2010). Texture hardening in this model system (WPI:water 5 6:4) is induced by insoluble protein aggregates formed through thiol disulfide interchange reactions. According to the electrophoresis results, both α-La and β-Lg took part in the formation of aggregates through protein protein thiol disulfide interchange reactions in the early stage of storage and this led to rapid bar hardening, whereas aggregates formed via noncovalent interactions were responsible for later stage hardening during long-term storage. The model system with NEM (WPI/NEM 5 2) had a sixfold longer shelf life (based on an unacceptable hardness of 12 N with a texturometer) compared to the control system, because almost no thiol disulfide interchange reactions can take place during storage. Compared to the model systems without Cys, The addition of Cys in bar formulas significantly inhibited the formation of protein aggregates with appropriate concentration of Cys (WPI/Cys 5 0.05), which extended the shelf life of the model bars for B14 days. An excessive addition of Cys (WPI/Cys 5 0.25), however, accelerated the hardening process of the model bars. It was suggested that protein protein thiol disulfide interchange reactions were delayed by an appropriate amount of Cys, especially for α-La, while excessive Cys addition would accelerate the thiol disulfide interchange reactions (Zhu & Labuza, 2010).

13.3.2

Adding Protein Hydrolysates

Information on how to inhibit texture hardening of high-protein nutrition bars is limited, and the application of hydrolyzed protein is one of the most reported methods. Both the storage time and concentration of HWPI significantly affects the hardening of high-protein nutrition bars. After manufacture, bars made with different ratios of WPI and HWPI had similar hardness of B34 N using a penetration test. However, significant differences in bar hardness occurred after being stored at 32 C for 7 days. Bars with 0% and 25% HWPI showed a hardness of B17 N and were not significantly different from each other, but were both much harder than bars with higher HWPI content (Hogan, Chaurin, O’Loughlin, & Kelly, 2016; McMahon et al., 2009). Bars with 100% HWPI were the softest with a hardness of B5.2 N. These different trends in changes of hardness continued throughout storage. The observation, that all bars were similar in initial hardness and microstructure regardless of the ratio of HWPI/WPI used, can be explained by the fact that the dry protein particles had only been dispersed in the model bar without being completely hydrated, and the whole matrix had not reached equilibrium after manufacture. Gautam and Simon (2006) reported that bars

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made of more hydrophilic proteins are softer and are more stable during storage. Hydrolysis of proteins generally leads to more hydrophilicity and higher water binding capacity, which makes it harder for sugars to pull water from the protein phase and thus inhibit phase separation (Sinha, Radha, Prakash, & Kaul, 2007). Another mechanism of phase separation, the steric effect, can also be limited due to the lower molecular weight of hydrolysates compared to intact proteins. A similar attempt was carried out to enhance the storage stability of protein bars made with dried hen egg white (DEW). The addition of hydrolysates of egg white (HEW) resulted in a softer initial texture of the model bars, and the hardening rate was also decreased. Higher proportions of HEW were more effective, and the combination of 75% HEW 1 25% DEW decreased the hardness of the model bars by nearly 50% throughout storage compared to 50% HEW 1 50% DEW bars (Rao, Rocca-Smith, & Labuza, 2013). Hydrolyzed proteins can reduce the initial hardness of the bar in some cases, but they have their own drawbacks that limit their application. The relatively high cost of hydrolysates is one obstacle. Quality problems include undesired bitter flavors and sticky mouthfeel. Furthermore, the hydrolyzed proteins are commonly unstable during processing, which causes new difficulties in product development. Bars containing hydrolyzed proteins also tend to stick to equipment, making them harder to process. Overall, although addition of hydrolyzed protein might be a useful method to inhibit bar hardening, more efforts are needed before it becomes practical.

13.3.3

Creation of Heterogeneity by Pretexturization

The concept of pretexturization of proteins used in high-protein nutrition bars is based on the hypothesis that hardening of high-protein nutrition bars is not only related to the properties of the proteins, but also dependent on the component distribution and microstructure of the bar, which determines the dynamics of the physiochemical changes during storage. Therefore, the hardening process of the high-protein nutrition bars is related to the state of different components in the system, especially the dispersion of the protein phase. The pretexturization of proteins aims to form a more stable system by protein modification to a more stable microstructure, thus enhancing the stability of different phases and making the system more resistant to texture changes during storage (Sun et al., 2015).

13.3.3.1 Extrude and Toast Banach et al. (2014) reported that extrusion and toasting used to modify MPC80 before preparation of high-protein nutrition bars could modify the proteins used in model high-protein nutrition bars to limit texture hardening.

13.3 Solutions to Avoid Hardening of High-Protein Nutrition Bars

MPC80 toasted at 75 (T75) or 110 C (T110) for 4 hours were used. However, the two types of bars hardened at a similar rate and extent to the control MPC80 bars, suggesting toasting is not effective. However, MPC80 extruded at die temperatures of 65 (E65) and 120 C (E120) decreased bar hardness after storage when compared with high-protein nutrition bars made with unmodified MPC80, and the E65 were significantly more stable than the E120 during storage. Overall, extruding MPC80 before preparation of high-protein nutrition bars might be a promising method to inhibit texture hardening. Since high-protein nutrition bars formulated with extruded MPC80, especially at 65 C, retained a softer texture, the discussion will focus on a proposed mechanism. The whey proteins, β-Lg and α-La, were not able to contribute to internal disulfide bond formation because they were already disulfide bonded, as demonstrated by their reappearance in the presence of a reducing agent when using SDS-PAGE with a reducing agent. Extrusion enhanced disulfide bond formation prior to incorporation into the highprotein nutrition bar thus decreasing internal aggregation and decreasing peptide solubility during storage. Extrusion also has a significant impact on the aw of high-protein nutrition bars. When stored at 25 C, aw of model bars made with unmodified MPC80 increased slightly. Although aw differences among model high-protein nutrition bars made with extruded, toasted, and unmodified MPC80 were limited at 32 C, the aw was more stable during storage for model bars made with unmodified MPC80 compared to those made with E65. Increase in aw of bars made with extruded MPC80 might be related to lower water uptake of the modified proteins. Fewer water molecules are bound to protein particles, and more move to the continuous phase (Li et al., 2008). There are consistent results from other studies on the increased aw in model high-protein nutrition bars during storage, and it is suggested that high-protein nutrition bars in which the aw rises quickly also suffer from rapid texture hardening compared to those with nearly constant aw (Li et al., 2008; McMahon et al., 2009). Except for textural changes induced by redistribution of components in the matrix, protein molecules that lack bound water molecules are more likely to form disulfide bonds or other intermolecular interactions with other neighboring proteins. They may subsequently cause protein aggregation, which is also reported to be one of the major mechanisms of texture hardening during storage of high-protein nutrition bars (Zhou et al., 2008a, 2008b).

13.3.3.2 Jet-Milling Banach, Clark, and Lamsal (2017) used a jet-milled MPC in model highprotein nutrition bars to reduce hardening during storage. MPC85 was jetmilled using a Aveka CCE Technologies mill (Cottage Grove, MN, USA) with

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an Aveka 100/20 jet-mill air classifier system. JM-coarse and JM-fine powders were obtained at classifier rotor speeds of 1000 and 2500 rpm, respectively. Separately, MPC85 was rehydrated at 5% protein (w/w) at room temperature using Millipore water with continual overhead mixing for 2 hours. After holding the solution for 5 hours at 4 C, it was frozen (220 C) and freezedried. Freeze-dried material was mechanically milled into the freeze-drying sample using a L’Equip NutriMill (St. George, UT, USA). As a result, the MPC powder particle size and shape affected the functional properties and textural properties of the high-protein nutrition bars. Finely jet-milled MPC85 produced high-protein nutrition bars that were firmer and more cohesive than the control with unmodified MPC85. More importantly, high-protein nutrition bars formulated with finely jet-milled MPC85 or freeze-dried MPC85 were less prone to texture change during storage. Particle size reduction removed occluded air from the spray-dried MPC85 and allowed for denser particle packing in the high-protein nutrition bars. Reducing the particle size of MPC85 improved its ability to rehydrate during high-protein nutrition bar production, which translated into improved plasticization and high-protein nutrition bar cohesion. Particle size, shape, and physical properties should be considered when evaluating the functional properties of protein powder concentrates and their effect on high-protein nutrition bar texture and its change during storage.

13.3.3.3 Superfine Grinding Purwanti, Moerkens, van der Goot, and Boom (2012) used WPI microparticles made using superfine grinding with an ultracentrifuge mill to prepare protein gels. After superfine grinding, the average particle size of WPI powder can be decreased from 62 to 15 μm, and superfine grinding is an effective method to reduce the mean particle size of WPI powder. When added into model system at the same total protein content, systems with microparticles hardened slower during storage compared to those with only the original WPI powder. It was suggested that hardening of the model systems was related to dissociation and reconstruction of the cross-linked network in the gel. Addition of microparticles led to a different mechanism inducing moisture redistribution between the protein particles and continuous phase. Addition of 20% (w/w) microparticles into the formulation significantly limited hardness changes of the model system, and the texture of the matrix was rather stable during storage (Purwanti, van der Veen, van der Goot, & Boom, 2013). The rate of change of hardness in the model systems containing WPI microparticles was relatively low. However, a limited slow stiffening occurred throughout storage. It was suggested that the addition of microparticles prolonged the hardening process compared to the systems without microparticles. This suggests that there might be an additional mechanism related to

13.3 Solutions to Avoid Hardening of High-Protein Nutrition Bars

the microparticles which contributes to the long-term hardening. Microscopic observation provided a probable explanation. Although changing slowly, deformation of microstructure was observed during storage, which might be related to slow rearrangements of molecules within the microparticles. One of the factors responsible for this deformation effect is physical bonding between microparticles and the continuous phase. Hardening of the matrix is well correlated to migration of water from the continuous phase into the microparticles, which not only reduced free water available as a plasticizer in the continuous phase, but also induced swelling of the particles, resulting in a larger volume fraction of the solid phase, and contributing to the increase of hardness. As the stiffening process is related to the state of the particles as a dispersed phase, the use of a dispersed protein phase does not reduce the rate of hardening compared to microparticles. Dissociation and reconstruction of the cross-linked network within the microparticles, as well as redistribution of water, might be responsible for microstructural and texture changes of the model system during storage. Overall, introduction of microparticles leads to a different water distribution in the matrix compared to model systems without microparticles, which results in a different microstructure and texture of the two phase system (Sun et al., 2015).

13.3.3.4 Protein Cross-Linking by Transglutaminase Transglutaminase (TGase) is an enzyme which cross-links glutamine residues with intra- or interprotein lysine residues (see also Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated). TGase produced through biotechnology has been widely used to help many kinds of solid foods like restructured meats, fish pastes, and breads to improve their texture. Banach, Clark, Metzger, and Lamsal (2016) prepared low- and high-cross-linked MPC using TGase, and compared their texture in high-protein nutrition bars. The initial bar hardness was slightly decreased with TGase cross-linked proteins, and the development of bar hardening was slowed during storage, although their texture became more similar with those formulated with untreated proteins after long-term storage. When the amount of cross-linking increased, the hardness of highprotein bar would be reduced. The TGase cross-linking of proteins also improved the overall cohesiveness of high-protein nutrition bars and produced less crumbly bars by adding structure. Also, protein aggregation induced by internal disulfide bond formation and the Maillard reaction, which contribute to bar hardening might be slowed as protein cross-linking would decrease the initial free amine groups content, affecting both molecular mobility and chemical reactivity. It should be noted that, although protein cross-linking practically reduced the hardness of high-protein nutrition bars significantly, the small magnitude of the differences was not sufficient to make it a practical solution.

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13.4

CONCLUSION

Whey proteins are one of the most commonly used protein sources in highprotein nutrition bars due to their high nutritional value and desired flavor and functionalities. Compared to bars made from other protein sources, whey protein-based nutrition bars have a softer initial texture and better general homogeneity because of their highly hydrophilic nature. However, whey protein-based nutrition bars suffer from quality losses during storage which limit the shelf life of such products. Previous studies have revealed the mechanisms of the quality changes in high-protein nutrition bars during storage, and possible solutions have been suggested to slow down or inhibit the undesired physiochemical changes. Further efforts are needed to effectively prolong the shelf life of whey protein-based high-protein nutrition bars.

Acknowledgment We wish to thank Professor Joe Regenstein at Cornell University for extensive help in the preparation of this chapter.

References Alting, A. C., Hamer, R. J., de Kruif, C. G., Paques, M., & Visschers, R. W. (2003). Number of thiol groups rather than the size of the aggregates determines the hardness of cold set whey protein gels. Food Hydrocolloids, 17, 469 479. Anema, S. G., Hunter, R. J., & Hemar, Y. (2006). Effects of storage temperature on the solubility of milk protein concentrate (MPC85). Food Hydrocolloids, 20, 386 393. Banach, J. C., Clark, S., & Lamsal, B. P. (2014). Texture and other changes during storage in model high-protein nutrition bars formulated with modified milk protein concentrates. LWT-Food Science & Technology, 56, 77 86. Banach, J. C., Clark, S., Metzger, L. E., & Lamsal, B. P. (2016). Textural performance of crosslinked or reduced-calcium milk protein ingredients in model high-protein nutrition bars. Journal of Dairy Science, 99, 6061 6070. Banach, J. C., Clark, S., & Lamsal, B. P. (2017). Particle size of milk protein concentrate powder affects the texture of high-protein nutrition bars during storage. Journal of Food Science, 82, 913 921. Belcourt, L. A., & Labuza, T. P. (2007). Effect of raffinose on sucrose recrystallization and textural changes in soft cookies. Journal of Food Science, 72, 65 71. Bertenshaw, E. J., Lluch, A., & Yeomans, M. R. (2008). Satiating effects of protein but not carbohydrate consumed in a between-meal beverage context. Physiology & Behavior, 93, 427 436. Campbell, W. W., & Leidy, H. J. (2007). Dietary protein and resistance training effects on muscle and body composition in older persons. Journal of the American College of Nutrition, 26, 696S 703S. Carunchia Whetstine, M. E. C., Croissant, A. E., & Drake, M. A. (2005). Characterization of dried whey protein concentrate and isolate flavor. Journal of Dairy Science, 88, 3826 3839. Caudle, A. D., Yoon, Y., & Drake, M. A. (2005). Influence of flavor variability in skim milk powder on consumer acceptability of ingredient applications. Journal of Food Science, 70, S427 S431.

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Purwanti, N., Moerkens, A., van der Goot, A. J., & Boom, R. (2012). Reducing the stiffness of concentrated whey protein isolate (WPI) gels by using WPI microparticles. Food Hydrocolloids, 26, 240 248. Purwanti, N., van der Veen, E., van der Goot, A. J., & Boom, R. (2013). Stiffening in gels containing whey protein isolate. International Dairy Journal, 28, 62 69. Rao, Q. C., Fisher, M. C., Guo, M. F., & Labuza, T. P. (2013). Storage stability of a commercial hen egg yolk powder in dry and intermediate-moisture food matrices. Journal of Agricultural & Food Chemistry, 61, 8676 8686. Rao, Q. C., Rocca-Smith, J. R., & Labuza, T. P. (2013). Storage stability of hen egg white powders in three protein/water dough model systems. Food Chemistry, 138, 1087 1094. Russell, T. A., Drake, M. A., & Gerard, P. D. (2006). Sensory properties of whey and soy proteins. Journal of Food Science, 71, S447 S455. Sinha, R., Radha, C., Prakash, J., & Kaul, P. (2007). Whey protein hydrolysate: Functional properties, nutritional quality and utilization in beverage formulation. Food Chemistry, 101, 1484 1491. Sun, C. C., Liu, R., Wu, T., Liang, B., Shi, C. Y., & Zhang, M. (2015). Effect of superfine grinding on the structural and physicochemical properties of whey protein and applications for microparticulated proteins. Food Science & Biotechnology, 24, 1637 1643. Tolstoguzov, V. (2003). Some thermodynamic considerations in food formulation. Food Hydrocolloids, 17, 1 23. Veldhorst, M., Smeets, A., Soenen, S., Hochstenbach-Waelen, A., Hursel, R., Diepvens, K., . . . Westerterp-Plantenga, M. (2008). Protein-induced satiety: Effects and mechanisms of different proteins. Physiology & Behavior, 94, 300 307. Westerterp-Plantenga, M. S., & Lejeune, M. P. G. M. (2005). Protein intake and body-weight regulation. Appetite, 45(2), 187 190. Westerterp-Plantenga, M. S., Luscombe-Marsh, N., Lejeune, M. P. G. M., Diepvens, K., Nieuwenhuizen, A., Engelen, M. P. K. J., . . . Westerterp, K. R. (2006). Dietary protein, metabolism, and body-weight regulation: Dose-response effects. International Journal of Obesity, 30, S16 S23. Wilkinson, C., Dijksterhuis, G. B., & Minekus, M. (2000). From food structure to texture. Trends in Food Science & Technology, 11, 442 450. Zhang, Q. B., Ames, J. M., Smith, R. D., Baynes, J. W., & Metz, T. O. (2012). A perspective on the Maillard reaction and the analysis of protein glycation by mass spectrometry: Probing the pathogenesis of chronic disease. Journal of Proteome Research, 8, 754 769. Zhang, X. (2014). The storage stability of high protein high sugar intermediate-moisture foods. Unpublished master dissertation. Jiangnan University, Wuxi. Zhou, P., & Labuza, T. P. (2007). Effect of water content on glass transition and protein aggregation of protein powder during short-term storage. Food Biophysics, 2, 108 116. Zhou, P., Liu, X. M., & Labuza, T. P. (2008a). Effects of moisture-induced whey protein aggregation on protein conformation, the state of water molecules, and the microstructure and texture of high-protein-containing matrix. Journal of Agricultural & Food Chemistry, 56, 4534 4540. Zhou, P., Liu, X. M., & Labuza, T. P. (2008b). Moisture-induced aggregation of whey proteins in a protein/buffer system. Journal of Agricultural & Food Chemistry, 56, 2048 2054. Zhu, D., & Labuza, T. P. (2010). Effect of cysteine on lowering protein aggregation and subsequent hardening of whey protein isolate (WPI) protein bars in WPI/buffer model systems. Journal of Agricultural & Food Chemistry, 58, 7970 7979.

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CHAPTER 14

Bioactive Peptides from Whey Proteins Bimlesh Mann, Syamala Athira, Rajan Sharma, Rajesh Kumar and Prabin Sarkar ICAR-National Dairy Research Institute, Karnal, Haryana, India

14.1

INTRODUCTION

Whey is the liquid portion produced as a by-product during cheesemaking or during coagulation of milk. It contains the lactose and noncasein proteins of milk, and its elevated content of organic matter is associated with a high biological oxygen demand and high perishability. Whey used to be considered as the most important pollutant of the dairy industry, not only due to its high organic load, but also due to its high volume (Walzem, Dillard, & German, 2002). The growing concern over pollution and environmental control has renewed the pressure on dairy industries to stop dumping whey into streams and municipal sewage systems. These legislative restrictions on whey disposal encouraged a deeper exploration of the widely recognized but less well understood physical, chemical, nutritional, and biological properties of whey components. So, in the light of global food shortage, the most logical use was to return whey to the human food chain in palatable form (Smithers, 2008). The relationship between diet and health is now well known to be one of the keys to preventing disease and promoting well-being. Indeed, it is on this basis that there has been major growth in the market for functional foods. These are foods that exert a positive influence on human health over and above their nutritive value (Mills, Ross, Hill, Fitzgerald, & Stanton, 2011). Whey contains more than half of the solids of original whole milk, including whey proteins (20% of total protein) and most of the lactose, minerals, and water soluble vitamins. It represents a rich and varied mixture of secreted proteins with wide-ranging chemical, physical and functional properties (Smithers et al., 1996). These proteins not only play an important role in nutrition as an exceptionally rich and balanced source of amino acids (Regester, McIntosh, Lee, & Smithers, 1996), but in a number of instances Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00015-1 © 2019 Elsevier Inc. All rights reserved.

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also appear to have specific physiological activity in vivo. It has long been recognized that several whey proteins confer nonimmune protection to neonates against disease, and others have putative biological and physiological effects. Such proteins include α-lactalbumin (α-La), β-lactoglobulin (β-Lg), lactoferrin (Lf), lactoperoxidase, immunoglobulins, glycomacropeptide (GMP), proteose peptone, and a variety of growth factors. These proteins have been implicated in a number of biological effects observed in human and animal studies ranging from anticancer activity to influence on digestive function (see also Chapter 1, Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins, Chapter 16: Sports and Exercise Supplements, and Chapter 17: Whey Proteins in Functional Foods). From the nutritional point of view, whey proteins are superior to the other proteins, especially caseins. They are rich in essential, branched-chain amino acids (BCAAs) (Devries & Phillips, 2015) including leucine, isoleucine, and valine that play crucial roles in metabolism, blood glucose homeostasis, and neural function. Leucine is known to regulate skeletal muscle protein synthesis and cysteine is the building block of glutathione, the dietary antioxidant (Micke, Beeh, & Buhl, 2002). It is vital for fighting against oxidative stress and preventing redox imbalance-caused diseases (Trachootham, Lu, Ogasawara, Valle, & Huang, 2008). In the last couple of decades, much research has concentrated on the therapeutic potential of milk proteins (Madureira, Pereira, Gomes, Pintado, & Malcata, 2007; Zimecki & Kruzel, 2007). For example, it has been demonstrated that whey proteins are superior to other dietary proteins for suppression of tumor development, due to components such as Lf, β-Lg, α-La, and serum albumin (Parodi, 2007). Indeed, under acidic conditions in the presence of oleic acid, α-La and oleic acid form a complex, termed HAMLET (human α-La made lethal to tumor cells) which has been shown to inhibit a wide array of tumors via an apoptosis-like event (Svanborg, Agerstam, Aronson, Bjerkvig, & Duringer, 2003). The bovine counterpart of HAMLET, termed BAMLET, was recently shown to display potent cytotoxic activity against eight cancer cell lines tested via a mechanism involving lysosomal membrane permeabilization (Rammer et al., 2010). A specialized whey fraction (high in leucine, bioactive peptides, and milk calcium) with the commercial name Prolibra has been shown to promote loss of body fat mass and greater preservation of lean muscle in a randomized human clinical trial lasting 12 weeks (Frestedt, Zenk, Kuskowski, Ward, & Bastian, 2008). The proteins are well documented as an essential source of amino acids, but recently it has been recognized that dietary proteins exert many other functionalities in vivo by means of biologically active peptides. Such peptides are inactive within the sequence of the parent protein and can be released by digestive enzymes during gastrointestinal transit or by fermentation or

14.2 Bioactive Peptides Derived From Whey Proteins

Hydrolysis with commercial proteases

Functional food products enriched with whey protein hydrolysate

Antihypertensive Antioxidant Antiappetizing

Whey proteins

Fermentation with starter culture

Antidiabetic

Fermented foods with inherent bioactive peptides

Therapeutic effects Mineral binding Antimicrobial

Hydrolysis by gastrointestinal enzymes during digestion

Release of bioactive peptides in intestine

FIGURE 14.1 Generation of bioactive peptides from whey proteins.

ripening during food processing. Bioactive peptides have been defined by Kitts and Weiler (2003) as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health. At present, milk proteins are considered the most important source of bioactive peptides. Upon oral administration, bioactive peptides may affect the major body systems—namely, the cardiovascular, digestive, endocrine, immune, and nervous systems (Fig. 14.1). For this reason, the potential of distinct dietary peptide sequences to promote human health by reducing the risk of chronic diseases or boosting natural immune protection has aroused increasing scientific and commercial interest over the past decade (Hartmann & Meisel, 2007). The activity of these peptides is based on their inherent amino acid composition and sequence. The size of active sequences may vary from 2 to 20 amino acid residues, and many peptides are known to possess multifunctional properties (Table 14.1).

14.2 BIOACTIVE PEPTIDES DERIVED FROM WHEY PROTEINS Bioactive peptides from milk can be divided into different categories based on their physiological effect on the body or the protein from which they have been derived: antihypertensive, antioxidant, antithrombotic, opioid, casein phosphopeptides (CPPs), antimicrobial, cytomodulatory, immunomodulatory, and miscellaneous peptides (Hayes, Ross, Fitzgerald, & Stanton, 2007). These functions relate to general health conditions or a reduced risk of certain chronic diseases (for reviews see Korhonen, 2009; Korhonen & Pihlanto, 2006; Madadlou & Abbaspourrad, 2016; Mills et al., 2011; Nongonierma & FitzGerald, 2015a, b, c).

Opioid effect Cytomodulatory

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Table 14.1 Bioactive Peptides From Whey Proteins With Different Physiological Properties Sl. No

Peptide Sequence

Fragment of Parent Protein

Reference

β-Lg f(71
75) β-Lg f(78
83) β-Lg f(142
148) β-Lg f(106
111) β-Lg f(94
100) β-Lg f(15
19) β-Lg f(81
83) β-Lg f(22
25) β-Lg f(32
40) α-La f(99
108) α-La f(50
52)

Power, Fernández, Norris, Riera, and FitzGerald (2014)

α-La f(101
104) α-La f(115
118) α-La f(99
108) β-Lg f(92
100) β-Lg f(84
91) β-Lg f(75
82) β-Lg f(19
29) β-Lg f(42
46) β-Lg f(145
149) β-Lg f(94
100) β-Lg f(123
131) β-Lg f(58
61) β-Lg f(95
101) β-Lg f(151
155) β-Lg f(15
20) β-Lg f(19
22)

Sadat et al. (2011)

α-La f(104
117) α-La f(105
115) α-La f(110
117) α-La f(4
11) β-Lg f(46
54) β-Lg f(46
57) β-Lg f(78
86) β-Lg f(78
83)

Lacroix and Li-Chan (2014b)

1. Antihypertensive Peptides 1 2 3 4 5 6 7 8 9 10 11

IIAEK IPAVFK ALPMHIR CMENSA VLDTDYK VAGTW VFK LAMA LDAQSAPLR VGINYWLAHK YGL

Mullally et al. (1997) Pihlanto-Leppälä et al. (2000)

2. Antioxidant Peptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

INYW LDQW VGINYWLAHK VLVLDTDYK IDALNEK KTKIPAVF WYSLAMAASDI YVEEL MHIRL VLDTDYK VRTPEVDDE LQKW LDTDYKK FNPTQ VAGTWY WYSL

Mann et al. (2015)

Hernández-Ledesma et al. (2005)

Athira et al. (2015) Contreras, Hernández-Ledesma, Amigo, Martín-Álvarez, and Recio (2011) Power et al. (2014) Zhang, Wu, Ling, and Lu (2013)

3. Antidiabetic Peptides 1 2 3 4 5 6 7 8

WLAHKALCSEKLDQ LAHKALCSEKL LCSEKLDQ TKCEVFRE LKPTPEGDL LKPTPEGDLEIL IPAVFKIDA IPAVFK

Power et al. (2014)

Continued

14.2 Bioactive Peptides Derived From Whey Proteins

Table 14.1 Bioactive Peptides From Whey Proteins With Different Physiological Properties Continued Sl. No

Peptide Sequence

Fragment of Parent Protein

Reference

β-Lg f(84
91) β-Lg f(125
135) β-Lg f(15
20) β-Lg f(25
40) β-Lg f(92
100) β-Lg f(78
83) α-La f(1
5) α-La f(17
31)S-S(109
114)

Demers-Mathieu et al. (2013)

4. Antimicrobial Peptides 1 2 3 4 5 6 7 8 9

IDALNENK TPEVDDEALEK VAGTWY AASDISLLDAQSAPLR VLVLDTDYK IPAVFK EQLTK GYGGVSLPEWVCTTF/ ALCSEK CKDDQNPH/ISCDKF

Pellegrini et al. (2001)

Pellegrini et al. (1999)

α-La f(61
68)S-S(75
80)

5. Opioid Peptides 1 2

YGLF YLLF

α-La f(50
53) β-Lg f(102
105)

Pihlanto-Leppälä (2001)

Caetano-Silva et al. (2015)

6. Mineral-Binding Peptides 1 2 3 4

TPEVDDE VLDTDYK VEELKPTPEGDLEI FD

β-Lg β-Lg β-Lg β-Lg

5 6

CKDDQNPH DDDLTDDI

α-La f(61
68) α-La f(82
89)

14.2.1

f(125
131) f(94
100) f(43
56) f(136
137)

Zhao, Huang, Cai, Hong, and Wang (2014) Caetano-Silva et al. (2015)

Antihypertensive Peptides

Cardiovascular diseases (CVDs) are the major cause of death in humans globally (WHO, 2017). High blood pressure is a modifiable risk factor for CVD. The angiotensin I-converting enzyme (ACE, peptidyldipeptide hydrolase, EC 3.4.15.1) has been associated with the renin
angiotensin system, which regulates peripheral blood pressure. Inhibition of this enzyme can exert an antihypertensive effect. ACE-inhibitory peptides are the most studied group of bioactive peptides and a large number of such peptides have been isolated from milk proteins hydrolysates. Apart from ACE inhibition, milk peptides may exert antihypertensive effects also through other mechanisms, such as inhibition of the release of endothelin-1 by endothelial cells (Maes et al., 2004), stimulation of bradykinin activity (Perpetuo, Juliano, & Lebrun, 2003), enhancement of endothelium-derived nitric oxide

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production (Sipola, Finckenberg, Korpela, Vapaatalo, & Nurminen, 2002), and enhancement of the vasodilatory action of binding to opiate receptors (Nurminen et al., 2000). Milk fermentation using lactic acid bacteria (LAB) or using their proteinases has been described as a strategy to release ACE-inhibitory peptides from milk proteins, especially caseins (Hayes, Ross, Fitzgerald, Hill, & Stanton, 2006; Hayes et al., 2007). For example, the ACE-inhibitory peptides Val-Pro-Pro (VPP; Clare, Catignani, & Swaisgood, 2003; Hamel, Kielwein, & Teschemacher, 1985; Juillard et al., 1995) and Ile-Pro-Pro (IPP; Chabance et al., 1995; Drouet et al., 1990), derived from casein following fermentation with Lactobacillus helveticus and Saccharomyces cerevisiae, respectively, are found in the commercial sour milk product, Calpis (Calpis, Co. Ltd., Tokyo). The antihypertensive effect of Calpis was shown to yield a systolic blood pressure decrease of 17.7 mmHg following administration of 5 mL/kg body weight of Calpis sour milk drink over an 8-hour period in spontaneously hypertensive rats (Yamamoto, Akino, & Takano, 1994). Likewise, a significant reduction in BP was observed in mildly hypertensive patients following oral consumption of 95 mL Calpis over an 8-week period (Hata et al., 1996). Particularly, it has been shown that nonhydrolyzed β-Lg has very low ACE-inhibitory activity (Mullally, Meisel, & FitzGerald, 1997), but its hydrolysis (using pepsin, trypsin, chymotrypsin, and/or other proteases) resulted in high levels of ACE inhibition (73%
90%). Pihlanto-Leppälä et al. (1999) demonstrated that the synthetic peptides corresponding to the sequences β-Lg f(102
105) and α-La f(50
53) are bioactive peptides presenting ACE-inhibitory activity. Hydrolysates prepared with different enzymes and from different protein sources result in in vitro ACE inhibition implying that ACE inhibition is induced by a variety of peptides and peptide combinations. The ACEinhibiting activity depends on the protein source and proteolytic enzymes used for hydrolysis. It was reported that whey protein hydrolysates (WPHs) prepared with elastase were less effective ACE inhibitors than hydrolysates from other pancreatic enzymes. Hydrolysis with trypsin generally results in hydrolysates with good ACE-inhibiting activity (Mullally et al., 1997; Naik, Mann, Bajaj, Sangwan, & Sharma, 2013), but does not always yield hydrolysates with the best ACE inhibition (Hyun & Shin, 2000). Apart from enzyme specificity, hydrolysis conditions such as hydrolysis time and enzyme-tosubstrate ratio also determine the final ACE-inhibiting activity (Naik et al., 2013). For some proteases, high ACE inhibition is reached after short hydrolysis times, yet with other enzymes longer hydrolysis time is needed (Lee, Kwon, Shin, & Yang, 1999). ACE inhibition by hydrolysates seems to be mainly caused by low-molecular-weight peptides, as permeate fractions obtained after filtration by 1 kDa membranes yield relatively high inhibition

14.2 Bioactive Peptides Derived From Whey Proteins

(Pihlanto-Leppälä, Koskinen, Piilola, Tupasela, & Korhonen, 2000). Two potent ACE-inhibitory peptides, LLF and LQKW, were identified from a hydrolysate of β-Lg prepared using thermolysin (Hernández-Ledesma, Recio, Ramos, & Amigo, 2002). Subsequently, the antihypertensive effect of these two peptides was evaluated in spontaneously hypertensive rats (HernándezLedesma, Beatriz, Lourdes, Mercedes, & Isidra, 2005; Hernández-Ledesma, Quiros, Amigo, & Recio, 2007).

14.2.2

Antioxidant Peptides

Oxidative stress, the increased production of reactive oxygen species (ROS) and reactive nitrogen species, combined with overtaking endogenous antioxidant defence mechanisms, is a significant causative factor for the initiation or progression of several lifestyle-mediated diseases (Moskovitz, Yim, & Chock, 2002). Dietary consumption of antioxidants appears to benefit endogenous antioxidant defence strategies in the fight against oxidative stress (Fang, Yang, & Wu, 2002). Recent studies have shown that peptides having antioxidant properties can be released from milk proteins (Pihlanto, 2006). The use of synthetic antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, and tert-butylhydroquinone, has been restricted because of their potential toxic effects on humans. On the other hand, bioactive peptides are considered natural antioxidants and have attracted a great deal of interest because of their safety and availability from a wide range of protein sources (Brandelli, Daroit, & Correa, 2015). The mechanism of action of antioxidant peptides studied by various researchers involves inhibition of lipid peroxidation, scavenging of free radicals, and chelation of transition metal ions (Moure, Dominguez, & Parajo, 2006; Qian, Jung, & Kim, 2008; Rajapakse, Mendis, Jung, Je, & Kim, 2005), but the defined mechanism pertaining to the antioxidant activity of peptides has not been fully understood to date. In addition, it has been reported that antioxidative peptides exert their effect by intracellular conversion of cysteine to glutathione, a potent intracellular antioxidant that keeps cells safe from damage by ROS through the induction of genes (Marshall, 2004). Antioxidative properties of the peptides are more related to their composition, structure, and hydrophobicity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998). Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that have antioxidant activity (Wang & Mejia, 2005). Contributing to the antioxidant potency are amino acids with aromatic residues that can donate protons to electron-deficient radicals. This property improves the radical-scavenging properties of the amino acid residues. Rajapakse et al. (2005) proposed that antioxidative activity of His-containing peptides relates to the hydrogen-donating, lipid peroxyl radical-trapping, and/or the metal

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ion-chelating ability of the imidazole group. On the other hand, the SH group in cysteine has an independently crucial antioxidant action due to its direct interaction with radicals (Qian et al., 2008). In addition to the presence of relevant amino acids, their correct positioning in the peptide sequence plays an important role in antioxidant activity of peptides (Rajapakse et al., 2005). Saito et al. (2003) reported that any change in the amino acid sequence in tripeptides resulted in different antioxidant activities. Peptide linkage and/or specific structural features of the peptides have been claimed to influence antioxidant capacity. In contrast to these findings, other results indicated that the peptide bond or its structural conformation can reduce the antioxidant activity of the constituent amino acids. Therefore, peptide conformation appears to behave as a double-edged sword; i.e., it can show both synergistic and antagonistic effects, as far as the antioxidant activity of free amino acids is concerned (Hernández-Ledesma, Beatriz, Lourdes, Mercedes, & Isidra, 2005). Moreover, it has been stated that the configuration of peptides can also affect antioxidant activity. Chen, Muramoto, Yamauchi, and Nokihara (1996) found that substitution of L-His with D-His in an antioxidative peptide leads to reduction in the activity. They concluded that the correct positioning of the imidazole group is the key factor influencing the antioxidant activity. Finally, it has been postulated that the overall antioxidative activity must be ascribed to the integrated effects of these actions rather than to the individual actions of peptides (Chen et al., 1998). Some commonly used antioxidant capacity assays are the Trolox equivalent antioxidant capacity assay utilizing the 2,20 -azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid radical, the 2,2-diphenyl-1-picrylhydrazyl assay, which detects both electron transfer and hydrogen atom transfer, and the ferric ion reducing antioxidant power assay, which evaluates electron transfer (Brandelli et al., 2015). Peng, Kong, Xia, and Liu (2010) reported that the hydrolysis of whey protein isolate (WPI) by alcalase enhanced the antioxidant activity in liposome model systems. In another study, Hernández-Ledesma et al. (2005) reported that the hydrolysates of α-La and β-Lg possess high radical-scavenging activity and the antioxidant activities of a 3 kDa permeate obtained from α-La and β-Lg by corolase PP were 71% and 85% higher than the total activity of their corresponding hydrolysates. Athira, Mann, Sharma, and Kumar (2013), Athira et al. (2015) reported that the in vitro antioxidant activity of the WPHs prepared using alcalase was 1.18 6 0.015 μmol of Trolox/mg of protein as compared to the commercial whey protein concentrate (WPC) with an antioxidant activity of 0.19 6 0.010 μmol Trolox/mg protein and showed an ameliorative potential against paracetamol-induced oxidative stress in mice. The mice that were given WPH, either intraperitoneally or orally at the rate of 4 mg/kg of body weight, showed increased activities of antioxidant

14.2 Bioactive Peptides Derived From Whey Proteins

enzymes and a reduction in thiobarbituric acid reactive substances in liver homogenate compared with the paracetamol control group. It also successfully mitigated the increase in the concentration of oxidative biomarkers and restored the level of blood urea nitrogen to normal in sera of mice in which oxidative stress was induced with an overdose of paracetamol. Antioxidant activity of WPHs depends on the proteolytic enzymes used for hydrolysis. Mann et al. (2015) reported that WPH prepared using corolase (1.42 6 0.12 μmol Trolox/mg protein) showed maximum antioxidant activity as compared to WPHs prepared with Flavourzyme (0.81 6 0.04) and alcalase (1.16 6 0.05). Furthermore, when these WPHs were incorporated into flavored milk at 2%, the antioxidant activity of flavored milk was enhanced by up to 42% with WPH prepared using corolase. A recent study was conducted with elite Brazilian soccer players who were supplemented with a WPH (0.5 g protein/kg of body mass per day) for 12 weeks. The supplementation led to a decrease in creatine kinase (242%) and lactate dehydrogenase (238%), which are in vivo markers of oxidative stress and tissue damage (Lollo et al., 2014).

14.2.3

Antidiabetic Peptides

Diabetes is a serious chronic disease that occurs either when the pancreas does not produce enough insulin (a hormone that regulates blood sugar, or glucose), or when the body cannot effectively use the insulin it produces. Diabetes is one of four noncommunicable diseases targeted for action by global researchers. Over the years the prevalence of diabetes has been progressively increasing. Globally, an estimated 422 million adults were living with diabetes in 2014, compared to 108 million in 1980. The global prevalence (age-standardized) of diabetes has nearly doubled since 1980, rising from 4.7% to 8.5% in the adult population. In 2012 alone, diabetes caused 1.5 million deaths. Diabetes complications can lead to heart attack, stroke, blindness, kidney failure, and lower limb amputation. On World Health Day 2016, WHO issued a call for action on diabetes, drawing attention to the need to step up prevention and treatment of the disease (WHO, 2016a,b). Consumption of milk protein-derived bioactive peptides has been linked with serum glucose regulatory properties in humans. Different mechanisms may include an insulinotropic activity, incretin secretagogue action, as well as activity on different metabolic enzymes involved in the regulation of serum glucose such as dipeptidyl peptidase IV (DPP-IV), α-amylase, and α-glucosidase (Lacroix & Li-Chan, 2014a, b). The individual components which are responsible for this insulinotropic activity of milk protein hydrolysate have not been clearly identified in humans. It is thought that the active

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components may comprise a combination of BCAA and milk protein-derived peptides (Morifuji et al., 2010). Free amino acids can directly act at the β-cell level to release insulin through various mechanisms including membrane depolarization and mitochondrial signaling affecting insulin secretion (Newsholme, Gaudel, & McClenaghan, 2010). The insulinotropic properties of milk-derived components have been particularly attributed to whey-derived peptides (Oseguera-Toledo, González de Mejía, Reynoso-Camacho, Cardador-Martínez, & Amaya-Llano, 2014). However, it has been shown that both WPHs and casein hydrolysates (CNHs) are able to induce an insulinotropic response in humans. Most milk protein-derived hydrolysates which have been evaluated in humans appear to exist as proprietary products. In contrast, a human trial was recently conducted with a WPI hydrolysate generated at a laboratory-scale with flavourzyme (Goudarzi & Madadlou, 2013). The insulinotropic effects of milk protein hydrolysates have been seen both with (Geerts et al., 2011) or without carbohydrates (Akhavan, Luhovyy, Brown, Cho, & Anderson, 2010) and in the absence or presence of Leu (Geerts et al., 2011), an amino acid which has been shown to have a strong insulinotropic activity. The insulinotropic activity of WPHs has been related to intestinal amino acid absorption and the increased plasma concentration of free amino acids (Leu, Ile, Phe, Arg, Tyr, Thr, Val, Ala, and Lys), BCAA-containing dipeptides (e.g., Ile-Leu, LeuLeu, and Val-Leu), and possibly cyclic dipeptides (Morifuji et al., 2010). Contradictory results exist where no direct correlation between plasma BCAA and the consumption of an insulinotropic WPHs could be established (Power, Hallihan, & Jakeman, 2009). Consumption of 10 g of commercial WPHs by humans did not induce a reduction in post-meal serum glucose, although insulin secretion was increased (Akhavan et al., 2010). This was explained by the fact that the WPHs did not affect gut hormone secretion (i.e., CCK, GLP-1, and gastric inhibitory polypeptide) and consequently gastric emptying. In addition, the response observed in humans with various WPHs is possibly dependent on their free amino acid and peptide compositions. The extent of protein hydrolysis may dictate the in vivo plasma amino acid level following ingestion of protein hydrolysates (Morifuji et al., 2010). Two fragments of β-Lg, LKPTPEGDL and LKPTPEGDLEIL (IC50 5 45 and 57 μM, respectively) with potent DPP-IV inhibition activity, were identified from pepsin-treated whey proteins (Lacroix & Li-Chan, 2014b).

14.2.4

Mineral-Binding Peptides

Due to its prevalence worldwide, osteoporosis is considered a serious public health concern. According to International Osteoporosis Foundation (https:// www.iofbonehealth.org/facts-statistics), over 200 million women worldwide

14.2 Bioactive Peptides Derived From Whey Proteins

suffer from this disease. Worldwide, one in every three women and one in every five men over 50 years of age have suffered from osteoporotic fractures. On the other side is anemia, which is also a widespread nutritional deficiency particularly common in children and women. Food supplementation with mineral salts is a challenge for the food industry, since various metal salts may result in changes in the physical and sensory properties of foods (Guo et al., 2014). In addition, when ingested in the form of salts, they have low bioavailability, may promote the formation of ROS, and may be responsible for gastric mucosa damage (Chaud et al., 2002). Radicals can initiate peroxidation of lipids in biological membranes, and cause enzyme inactivation and damage to the DNA structure (Saiga, Tanabe, & Nishimura, 2003). Therefore, organic mineral supplementation is currently a popular research topic to overcome these problem (Narin, Benjamas, Nualpun, & Wirote, 2013). Metal-chelating peptides have been identified as potential functional ingredients to improve bivalent mineral bioavailability (Guo et al., 2014). Iron
peptide complexes have been considered as an alternative to mitigate the problems related to iron fortification, and have been considered as one of the best choices to replace iron supplements (Wang, Huang, & Jiang, 2013). Whey protein-derived peptides, obtained from proteolytic digestion have shown considerable capacity for binding cations such as calcium, iron, and zinc (Caetano-Silva, Bertoldo-Pacheco, Paes-Leme & Netto, 2015; Chaud et al., 2002; Zhao et al., 2015; Zhou et al., 2012). Regarding the mechanism of mineral 2 peptide complexation, Reddy and Mahoney (1995) suggested that the net charge, side chain length, and functional groups of the amino acids and peptides seem to be directly related to the extent of complex formation with iron. Studies with iron
peptide complexes show that the major iron binding site corresponds primarily to the carboxyl groups (Caetano-Silva et al., 2015; Chaud et al., 2002; Lee & Song, 2009; Zhao et al., 2015), although the ε-amino nitrogen of lysine, the guanidine nitrogen of arginine, and the imidazole nitrogen of histidine may also have been involved in iron
peptide bonding (Reddy & Mahoney, 1995). Glycine and proline could also be involved in iron complexation (Storcksdieck, Bonsmann, & Hurrell, 2007). A tripeptide, Tyr-Asp-Thr was identified from WPH with strong calcium-binding capacity of 79.5 μg/mg and indicated that the major binding sites included the oxygen atom of the carbonyl group and the nitrogen of amino or imino groups (Zhao et al., 2015). Kim et al. (2007) studied the effect of different enzymes like pancreatin, alcalase, flavourzyme, esperase, neutrase, papain, pepsin, and trypsin on the hydrolysis of whey proteins together with their effectiveness to bind iron. The results indicated that iron-binding by alcalase hydrolysate with degree of hydrolysis of 13.06 was the highest (97.6%) of all hydrolysates. CaetanoSilva et al. (2015) identified 28 β-Lg fractions with a size of ,1 kDa from

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iron-binding WPHs produced using pancreatin. They observed Glu and/or Asp in all the sequences, and their carboxylic groups were amongst the main iron-binding sites. Alcalase hydrolysate of whey protein with good ironbinding ability had higher Lys, Ala, and Phe contents as reported by Kim et al. (2007). A hydrolysate of neutrase-treated WPC was shown to be a promising facilitator of iron absorption in a Caco-2 cell model (Ou et al., 2010). The effect was attributed to the amino acids Asp, Glu, Gly, Cys, His, and Pro as well as peptides of #10 kDa. Udechukwu, Downey, and Udenigwe (2017) evaluated the effect of everlaseand papain-hydrolyzed whey protein on zinc-chelating capacity and assessed the stability of zinc complexes with these WPHs. They concluded that higher net negative surface charge of the peptides was associated with higher zincchelating capacity; everlase-hydrolyzed whey protein exhibited a greater capacity to bind zinc as compared to papain-hydrolyzed whey protein due to relatively higher negative surface charge of the former, despite having a lower content of metal-chelating amino acid residues. They also found that zinc complexes with these hydrolysates are gastric-stable. This study demonstrates that WPHs can be used as delivery agents to promote zinc nutrition in humans. CPPs function as carriers for different minerals, playing an important role in their bioavailability. These CPPs have phosphorous bond via monoester linkages to seryl residue in their structure. CPPs contain highly polar acidic sequence of three phosphoserines followed by two glutamic acid residues (SpSpSpEE) which are the binding sites for minerals (FitzGerald & Meisel, 2003). The presence of SpSpSpEE embedded in the bioactive peptide is a distinctive feature for all functional CPPs. β-casein-4P (1
28) is one of the main casein-derived proteose peptone fractions which is the N-terminal segment of β-casein released by the action of indigenous milk enzyme plasmin and considered a part of whey proteins as proteose peptone component 8f has such domain at position 17
21. A part of this segment is the prominent CPP, i.e., β-casein 4P (f2
25) or β-casein f(2
28) 4P identified by a number of researchers (Holt, 1996; Naito, 1990; Saini et al., 2014; Schlimme & Meisel, 1993; Schmidt, Both, Visser, Slangen, & van Rooijen, 1987). Iron bound to β-casein f(1
25) remains soluble in the digestive tract, is not altered by ionic strength because of the presence of coordination links preventing iron from insolubilization or from interactions with other minerals or trace elements, and maintains iron in a protected stable state until it reaches the enterocyte receptor, thereby favoring iron absorption (Ait-Oukhatar et al., 2002; Chaud et al., 2002). This fraction contains four of the five phosphoserine residues of the native β-casein and can bind four atoms of iron (Bouhallab, Léonil, & Maubois, 1991). In a single experiment, Zidane et al. (2012)

14.2 Bioactive Peptides Derived From Whey Proteins

determined that one mole of β-casein(1
25)4P could bind two moles of Ca21, Mg21, and Zn21 under identical experimental conditions close to those of the ileum, with relatively low binding affinity constants (K 5 4900
11,200 M21). In an earlier study, the chelation of zinc on β-casein (1
25)4P was measured by atomic absorption spectrometry and it was found that one mole of peptide is able to bind 4 moles of Zn21 at pH 5.3 (Pérès et al., 1999).

14.2.5

Antimicrobial Peptides

Milk is a rich source of antimicrobial proteins and peptides, capable of exerting antimicrobial activities comparable to antibiotics. This effect is due to the synergistic activity of naturally occurring peptides and defence proteins such as Lf, lactoperoxidase, and lysozyme and is greater than any individual contribution (Clare et al., 2003). The most studied are the lactoferricins, derived from bovine and human Lf (Kitts & Weiler, 2003). Also, a few negatively charged peptides derived from β-Lg (Pellegrini, Dettling, Thomas, & Hunziker, 2001) and α-La (Pellegrini, Thomas, Bramaz, Hunziker, & von Fellenberg, 1999) showed antibacterial activity against Gram-positive bacteria and a weak activity against Gram-negative bacteria. The disruption of normal membrane permeability is at least partly responsible for the antibacterial mechanism of lactoferricins. The difference in net charge, charge distribution, size, amino acid sequence, and secondary structure along with amphipathicity might be responsible for the differential behavior of identified antibacterial peptides against bacteria (Brandelli et al., 2015). The physiological importance of antimicrobial milk peptides is not established yet but Shimizu (2004) proposed that these peptides may modulate the intestinal microflora if generated during milk protein digestion in vivo.

14.2.6

Opioid Peptides

Opioid peptides are those peptides having pharmacological similarities to opium. Opioid peptide sequences typically contain Tyr-Gly-Gly-Phe at their N-terminal side, and in the case of atypical opioid peptides, a Tyr residue is found at the N-terminus (Teschemacher, Koch, & Brantl, 1997). These peptides have been reported to behave like morphine in the brain. These have been found within milk proteins, both in casein (αs1-, αs2-, β-, and κ-) (casomorphins) and whey proteins (lactorphins). However, the major opioid peptides are fragments of β-casein, called β-casomorphins (Clare & Swaisgood, 2000). On the other hand, all κ-casein fragments, known as casoxins behave as opioid antagonists (Severin & Wenshui, 2005). Opioid peptides have also been found encrypted within the primary sequence of whey proteins such as Lf, β-Lg, and bovine serum albumin (Belem, Gibbs, & Lee, 1999).

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Opioid peptides are thought to be biologically very potent; potentially, micromolar amounts may be sufficient to exert physiological effects (Meisel & FitzGerald, 2000). Opioid-like sequences are encrypted in the primary structure of major whey proteins: human and bovine α-La f(50
53) and bovine β-Lg f(102
15), which have been termed α- and β-lactorphins, respectively (Pihlanto-Leppälä, 2001). The whey protein-derived peptide, α-lactorphin, with proven opioid activity, although with lower affinity toward μ-opioid receptors than β-casomorphin 7, can induce mucin secretion and mucin gene expression in human colonic goblet-like cells (Martínez-Maqueda et al., 2012). A similar effect was observed for a trypsin β-Lg hydrolysate and β-lactorphin, probably operating through an opioid pathway (Martínez-Maqueda, Miralles, Ramos, & Recio, 2013). WPHs with the ability to modulate mucin production could be promising for improving gastrointestinal protection (Brandelli et al., 2015).

14.2.7

Immunomodulatory Peptides

Milk protein hydrolysates and peptides derived from caseins and major whey proteins can enhance immune cell functions, measured by lymphocyte proliferation, antibody synthesis, and cytokine regulation (Gill, Doull, Rutherfurd, & Cross, 2000). The protective effect of a casein-derived immunopeptide on resistance to microbial infection by Klebsiella pneumoniae has been demonstrated in mice (Migliore-Samour, Floch, & Jolles, 1989). Korhonen and Pihlanto (2003a) proposed that immunomodulatory milk peptides may alleviate allergic reactions in atopic humans and enhance mucosal immunity in the gastrointestinal tract. In this way immunomodulatory peptides may regulate the development of the immune system in newborn infants. Recently, it was demonstrated that commercial WPIs contain immunomodulating peptides which can be released by enzymatic digestion (Mercier, Gauthier, & Fliss, 2004). This information is of high relevance when developing infant formula with optimized immunomodulatory properties (Korhonen & Pihlanto, 2006). Peptides derived from hydrolysis of WPI with trypsin/chymotrypsin seem to modulate immune parameters in vivo using noninfected and Escherichia coli-infected mice. In particular, the basic peptide fraction showed promising results, stimulating serum TGF-β1 secretion, which coincided with a significant increase in IgA levels (Saint-Sauveur, Gauthier, Boutin, Montoni, & Fliss, 2009). Eriksen, Vegarud, Langsrud, Almaas, and Lea (2008) investigated the immunomodulatory properties of whey protein-derived peptides prepared using different enzymes. Samples of cow and goat whey were hydrolyzed with either commercial enzymes pepsin and corolase PP or human gastric and duodenal juices. Whey protein samples from both goat and cow showed dose-dependent inhibition of peripheral blood mononuclear cell proliferation in vitro. This effect is

14.2 Bioactive Peptides Derived From Whey Proteins

suggested to be associated with intact or hydrolyzed components in whey samples that affect the generation of important activating signals, thus inhibiting further lymphocyte proliferation. Eleven synthetic peptides derived from theoretical release from β-Lg and α-La by hydrolysis with trypsin or chymotrypsin were evaluated for their immunomodulatory properties. The peptides β-Lg f(15
20), f(55
60), f(84
91), f(92
105), f(139
148), f(142
148), and α-La f(10
16) stimulated proliferation to different extents, whereas β-Lg f(15
20), f(55
60), and f(139
148) also induced various inhibiting and/or stimulating effects on cytokine secretion (Jacquot, Gauthier, Drouin, & Boutin, 2010). These results confirm that hydrolysis of α-La and β-Lg by digestive enzymes may result in peptides that have the potential to influence the specific immune response through modulation of splenocyte proliferation and cytokine secretion (Brandelli et al., 2015). Recent reports indicate that the addition of whey peptides has a positive effect in the development of immune-modulating diets in both mouse and rat models (Brandelli et al., 2015).

14.2.8

Cytomodulatory Peptides

Cytomodulatory peptides, which have been shown to inhibit cell growth and stimulate the activity of immune competent and neonatal intestinal cells, respectively, have been isolated from a variety of fermented dairy products (Hayes et al., 2007). Such peptides can be classified as potential anticarcinogens and have been reported to act as specific signals that can inhibit the viability of cancer cells (Meisel, 2005). GMP and its derivatives have been shown to exhibit a range of immunomodulatory functions, such as immunosuppressive effects on the production of IgG antibodies (Manso & López-Fandiño, 2004) and immunoenhancing effects on proliferation and phagocytic activities of human macrophage-like cells U937 (Li & Mine, 2004). On the basis of results obtained in mouse model studies, Matar,
(2003) concluded that peptides released by LeBlanc, Martin, and Perdigon bacterial proteolysis might have important implications in modulation of the host’s immune response and have an impact on inhibition of tumor development. WPC has been attributed with anticancer activities, linked to its ability to donate cysteine to the glutathione antioxidant system, one of the principal cellular protection mechanisms (Bounous, 2000). Remarkably, the combination of a WPI, termed Immunocal, with the anticancer drug baicalein enhanced the cytotoxicity of baicalein by inducing more apoptosis in the human hepatoma cell line Hep G2 (Tsai, Chang, Chen, & Lu, 2000). Most recently, whey peptides from Mozzarella di Bufala Campana cheese were shown to exert a significant antiproliferative effect on a Caco-2 cell line (De Simone et al., 2009).

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14.3 PRODUCTION AND ENRICHMENT OF BIOACTIVE PEPTIDES The release of bioactive peptides from a parent protein sequence is the prerequisite for their functional role in a living system. Milk proteins are recognized as a primary source of bioactive peptides which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of LAB is an attractive approach for generation of functional foods enriched in bioactive peptides for a low-cost and positive nutritional image associated with fermented milk products. Basically, biologically active peptides can be produced from milk proteins in the following ways: (1) enzymatic hydrolysis by digestive enzymes; (2) fermentation of milk with proteolytic starter cultures; and (3) proteolysis by enzymes derived from microorganisms or plants. In many studies, a combination of (1) and (2) or (1) and (3) has proven effective in generation of short functional peptides (Korhonen & Pihlanto, 2003b).

14.3.1

Enzymatic Hydrolysis

The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. Most of the known bioactive peptides have been produced using digestive enzymes, usually pepsin and trypsin. Trypsin is the most commonly used digestive enzyme for the production of angiotensin-converting enzyme (ACE)-inhibitory peptides and calciumbinding CPPs (FitzGerald, Murray, & Walsh, 2004). Other gastrointestinal enzymes and different enzyme combinations of proteinases used in the food industry, including alcalase, chymotrypsin, pancreatin, pepsin, and thermolysin as well as enzymes from bacterial and fungal sources, have also been used to produce bioactive peptides from various proteins (Korhonen & Pihlanto, 2003a, 2003b). Nine milk protein substrates were hydrolyzed in vitro with five proteases and the highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin, and Bacillus licheniformis protease. The IC50 values for thermolysin hydrolysates of caseins and whey proteins were 45
83 and 90
400 mg/mL respectively with α-La hydrolysates giving the highest inhibitory activity (Otte, Shalaby, Zakora, Pripp, & El-Shabrawy, 2007). In vitro gastrointestinal digestion of whey protein produced high ACE-inhibitory activity with an IC50 value of 0.041 mg/mL (Vermeirssen, Van Camp, & Verstraete, 2005). The thermolysin-catalyzed hydrolysates of α-La and β-casein were fractionated by size-exclusion chromatography and reversed-phase high performance liquid chromatography and it was found that a peptide contained IPP as the C-terminal sequence and had IC50 values of 4 and 5 mM and an ACE-inhibitory peptide from β-casein, identified a LYQQP, had less activity

14.3 Production and Enrichment of Bioactive Peptides

(Otte et al., 2007). Athira et al. (2015) reported that whey protein hydrolyzed by alcalase for 8 hours at pH 9 and 55 C had maximum antioxidant activity of 1.18 6 0.015 μmol Trolox/mg protein. Mizuno et al. (2005) measured the ACE-inhibitory activity of CNHs upon treatment with nine different commercially available proteolytic enzymes and found that the hydrolysate produced by a protease isolated from Aspergillus oryzae had the highest ACE-inhibitory activity in vitro per peptide. Further, Hernández-Ledesma, Miguel, et al. (2007), Hernández-Ledesma, Quiros, et al. (2007) identified bioactive peptides with ACE-inhibitory and antioxidant activity in hydrolysates of several samples of human milk and infant formula after digestion with pepsin and pancreatin.

14.3.2

Microbial Fermentation

Many dairy starter cultures are highly proteolytic. So, during the manufacture of fermented dairy products, formation of bioactive peptides is one of the major biochemical changes. Through microbial proteolysis, the release of different bioactive peptides from milk proteins is now well documented (Matar et al., 2003). Many studies have confirmed that L. helveticus strains, in particular, are capable of releasing antihypertensive peptides, the best known of which are the ACE-inhibitory tripeptides VPP and IPP. The antihypertensive capacity of these peptides has also been established in rat models and human studies (Aihara, Kajimoto, Hirata, Takahashi, & Nakamura, 2005; Mizushima et al., 2004; Seppo, Jauhiainen, Poussa, & Korpela, 2003). Also yogurt and cheese starter bacteria and commercial probiotic bacteria have been demonstrated to produce different bioactive peptides in milk during fermentation (Donkor, Henriksson, Vasiljevic, & Shah, 2007). Virtanen, Pihlanto, Akkanen, and Korhonen (2006) demonstrated that fermentation of milk with single industrial dairy cultures generated antioxidant activity in the whey fraction. The activity increases with the degree of proteolysis suggesting that peptides generated during fermentation contribute toward the antioxidative activity. Chen, Tsai, and Sun Pan (2007) observed that fermentation of milk with a commercial starter culture mixture of five LAB strains followed by hydrolysis with a microbial protease increased ACE-inhibitory activity of the hydrolysate. Two strong ACE-inhibitory tripeptides (Gly-Thr-Trp) and (Gly-Val-Trp) were identified and an antihypertensive effect of the hydrolysate containing these peptides was demonstrated in an animal model study using spontaneously hypertensive rats. Quiros et al. (2007) identified several novel ACE-inhibitory peptides in milk fermented with Enterococcus faecalis strains isolated from raw milk. Two β-casein derived peptides f(133
138) and f(58
76) showed distinct antihypertensive activity when administered orally to spontaneously hypertensive rats. Donkor et al. (2007) studied the proteolytic activity of several dairy LAB cultures and probiotic strains

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(Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei) as a determinant of growth and in vitro ACE-inhibitory activity in milk fermented with these single cultures. All the cultures released ACE-inhibitory peptides during growth with a Bifidobacterium longum strain and the probiotic L. acidophilus strain showing the strongest ACE-inhibitory activity. Lorenzen and Meisel (2005) demonstrated that trypsin treatment of yogurt milk prior to fermentation with yogurt cultures resulted in a release of phosphopeptide-rich fractions. In particular, the release of the CPP sequences β-casein f(1
25)-4P and αs1-casein f(43
79)-7P during trypsin treatment was pronounced whereas the proteolysis caused by peptidases of the yogurt cultures was not significant. Kilpi, Kahala, Steele, Pihlanto, and Joutsjoki (2007) studied the impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of the L. helveticus CNRZ32 strain on the ACE-inhibitory activity in fermented milk by taking advantage of peptidase-negative derivatives of the same strain. Increased ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The results suggest that both peptidases are involved in the release or degradation of ACE-inhibitory peptides during the fermentation process.

14.4

GLYCOMACROPEPTIDE

GMP is a hydrophilic peptide released by the cleavage of a specific peptide bond (Phe105-Met106) of κ-casein by rennet during the manufacturing of cheese. GMP comprises of 64 amino acids of the C-terminal part of κ-casein and contains all the glycans present in κ-casein (Farias, Martinez, & Pilosof, 2010). GMP is the fourth most abundant protein/peptide, next to β-Lg, α-La, and bovine serum albumin in whey products manufactured from cheese whey; its concentration is between 1.2 and 1.5 g/L contributing between 20% and 25% of the total protein of whey (Thoma, Krause, & Kulozik, 2006). Studies have indicated biological activity of GMP such as anticariogenic activity, inhibition of cholera toxin, modulation of immune response, and hemagglutination inhibition which have been ascribed mainly to the glycan part of the peptide, particularly sialic acid-containing glycans. GMP may have a beneficial role in modulating the gut microflora, as this macropeptide is known to promote the growth of bifidobacteria, due to its carbohydrate (mainly sialic acid) content (Manso & López-Fandiño, 2004). Further, GMP lacks aromatic amino acids and the high content of BCAAs in GMP have attracted the attention of the pharmaceutical industry (Neelima, Sharma, Rajput, & Mann, 2013). There have been efforts to develop largescale GMP production processes that are able to maintain the molecule’s biological and nutritional properties for use in functional foods (see Chapter 16: Whey Proteins in Functional Foods).

14.4 Glycomacropeptide

14.4.1

Isolation of Glycomacropeptide From Whey

GMP isolation from cheese/rennet whey or WPC usually involves steps such as: (1) selective precipitation of other proteins induced by adjustment of the physical properties of the solution (heating, alcoholic precipitation, precipitation using trichloroacetic acid or acetic acid); (2) membrane filtration (dialysis, ultrafiltration, microfiltration, reverse osmosis, electrodialysis); and (3) selective adsorption (ion-exchange chromatography) (Tullio, LazzariKarkle, & Cândido, 2007). A common industrial isolation procedure involves ultrafiltration of the heated whey followed by either selective isolation of GMP or impurities (Neelima et al., 2013). Ultrafiltration of cheese whey alone has also been attempted after adjusting the pH (Li & Mine, 2004) and making aggregates of GMP. Kawasaki et al. (1993) reported that GMP aggregation can be controlled by changing the pH. At pH 3.5, GMP present in cheese/rennet whey permeates through UF membranes with molecular weight cutoffs ranging from 20 to 50 kDa and the majority of the whey proteins are retained in the retentate. The pH of the permeate is then adjusted to 7.0 where GMP forms noncovalent linked polymers and is then concentrated using the same membrane. The low recovery (less than 34%) makes the method unsuitable for commercial use. The formation of self-assembled aggregates of GMP at low pH has been reported by Martinez, Carrera Sánchez, Rodríguez Patino, and Pilosof (2009) and Farias et al. (2010). The difference in pI of GMP (B4.0) and other major whey proteins (.4.8) has been exploited for the development of an anion-exchange membrane adsorption chromatography method (Kreuß & Kulozik, 2009). The system consisted of a spiral-packed cellulose-based strong anionic membrane coiled around a solid core. More than 91% purity of GMP could be achieved. Using an altogether different approach, Tolkach and Kulozik (2005) isolated GMP by an enzymatic cross-linking technique. This approach involves pretreatment of WPC with the enzyme transglutaminase (TGase). GMP is preferentially crosslinked by TGase due to the presence of glutamic acid and lysine residues in its primary structure. Further, it has been stressed that presence of sugar residues in GMP provides hydrophilic character which facilitates the enzyme action. On the other hand, native whey proteins show much less sensitivity to cross-linking by TGase due to their globular structure with relatively little exposure of the protein chain to the exterior. The GMP aggregates are then removed by microfiltration or diafiltration. The abovementioned approaches for the isolation of GMP from cheese whey yield GMP with quite a high content of other contaminating whey proteins. Literature indicates that a combination of ion-exchange chromatography and UF provides the best result in terms of recovery as well for scale-up operations. The manufacturer of GMP would be interested in the methods by which the spent whey can be used for other food applications. Since the pI of GMP varies with the extent of

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glycosylation, more research may be required to elaborate the optimum conditions, such as pH, type of exchanger, elution buffer, and temperature, to recover the maximum amount of GMP from cheese whey (see also Chapter 1 for information on GMP).

14.5

CONCLUSION

It is well documented that whey is a potential source of bioactive peptides. Peptides can be released from WPs in sufficient quantities either by enzymatic hydrolysis or during in situ production of fermented products incorporating whey proteins. Research is needed to ascertain the optimal processing conditions and the storage stability of these bioactive peptides. In addition, clinical studies are required to assess the bioavailability, health claims, and safety of these bioactive peptides.

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Nutritive and Therapeutic Aspects of Whey Proteins 1

15.1

Veronique Lagrange1 and David C. Clark2

Consultant, Washington, DC, United States, 2Bovina Mountain Consulting LLC, Englewood, FL, United States

INTRODUCTION

For many decades, whey from cheese manufacture was considered a waste product, the disposal of which frequently was problematic to producers (Tunick, 2008). The first approach to add more value to cheese whey, other than drying it as sweet whey powder, was by demineralization. In the early days, the removal of ash from whey was achieved by either batch ion exchange or electrodialysis processes. The resulting demineralized whey, however, had limited functionality and application potential, mainly in confectionary and infant formula, due to its low protein content of approximately 11% 12% (w/w) (Bonnaillie & Tomasula, 2008). It is only since the widespread commercialization of membrane filtration processes and in particular ultrafiltration (UF) processing, that the real nutritional potential of whey, the protein fraction, has become accessible. Initially, whey protein concentrates (WPCs) were produced containing 34% protein (WPC34). These concentrates, which have an overall protein content that matches that of skim milk powder (SMP), were developed as cost-effective substitutes for SMP in a wide range of applications (Bonnaillie & Tomasula, 2008). The addition of diafiltration steps to the UF process and subsequently ion exchange led to the widespread availability of high protein concentration products such as WPC80, containing about 80% protein and whey protein isolate (WPI), containing at least 90% protein (see also Chapter 2: History of the Development and Application of Whey Protein Products and Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated). The high protein content of these preparations made them ideal for exploitation of both the functional and nutritional properties of this protein fraction. The extraordinary nutritional properties of whey proteins continue to fuel research. Perhaps the most surprising fact is that it took so long to invest in this fertile area given that nature had Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00016-3 © 2019 Elsevier Inc. All rights reserved.

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designed the whey protein complex through evolution specifically to protect and nourish young mammals. Important applications of whey proteins in infant and sports nutrition are discussed in other chapters of this book (see also Chapter 12: Whey Proteins in Infant Formula and Chapter 16: Sports and Exercise Supplements). To some extent, however, research into the effects and benefits of whey proteins in these market areas opened the door to investigation of their potential in other nutritional application fields. These fields include overnutrition (e.g., obesity); nutrition for aging populations, in particular addressing age-related muscle loss caused by sarcopenia; and undernutrition, including severe acute malnutrition (SAM) and moderate acute malnutrition (MAM) and the associated wasting and stunting caused by chronic, sustained hunger. A substantial body of research has been accrued which shows that whey protein delivers startling benefits in all these “therapeutic” and medical nutrition application fields. Recent important findings will be discussed in the sections below.

15.2

WHEY PROTEIN IS HIGH-QUALITY PROTEIN

Whey protein is considered a high-quality protein. According to the Digestible Indispensable Amino Acid Score (DIAAS) method developed by The Food and Agricultural Organization of the United Nations (FAO) (FAO, 2013), which measures the digestion of specific amino acids rather than crude protein levels, whey protein is a higher quality protein than soy protein. High-quality whey proteins have been shown to have advantageous digestive and absorptive properties that facilitate rapid, yet lasting, delivery of essential amino acids to the body (Pasiakos, 2015).

15.3 WHEY PROTEIN: A POTENTIAL ROLE IN HELPING TO REDUCE UNDERNUTRITION The FAO estimates that approximately 795 million people out of a global population of 7.3 billion, or more than 10% of the world’s population, suffered from chronic undernourishment in 2014 16 (World Hunger Education Service, 2016). Acute malnutrition, resulting from severe undernourishment due to insufficient calorie and protein intake, manifests itself in one of two ways: wasting, where an individual has low weight or is excessively thin for height; or stunting, where an individual is proportionally sized but has low height for age. The World Health Organization (WHO) has set an internationally agreed target to reduce stunting and wasting in children by 40% by 2025

15.3 Whey Protein: A Potential Role in Helping to Reduce Undernutrition

(Suarez Weise, 2014) as part of the process to meet the United Nations Sustainable Development Goals of ending all forms of malnutrition by 2030 (UN, 2014). Generally, wasting is reversible by provision of appropriate nutrition to support recovery. Stunting, however, is of major concern because it is in most cases irreversible. Aside from reducing physical stature, childhood stunting is associated with increased morbidity, underdeveloped cognitive function, increased incidence of chronic diseases in later life, and significantly reduced earning potential (Hoddinott, Alderman, Behrman, Haddad, & Horton, 2013; Hoddinott, Maluccio, Behrman, Flores, & Martorell, 2008; Martorell, Melgar, Maluccio, Stein, & Rivera, 2010). Stunting was estimated to affect 154.8 million children aged under 5 worldwide in 2016 (UNICEF, WHO, & World Bank Group, 2017). Stunting is a slow cumulative process that can begin in utero when preconception and pregnant females lack sufficient key nutrients. Studies have shown that faltering in linear growth is greatest during the first 1000 days, conception to 24 months (Leroy, Ruel, Habicht, & Frongillo, 2014), and that interventions to prevent stunting should target mothers and children during this period. Inadequate nutrition of women during pregnancy can result in intrauterine growth retardation (IUGR) of the fetus and delivery of an infant with low birth weight (,2500 g). Numerous dietary pattern studies have been conducted within the last decade and these have been reviewed recently (Clark, 2016; Grieger & Clifton, 2015). Although the studies were conducted on mainly healthy and well-nourished Western female populations, they did reveal links between consumption of dairy products and lower risk of IUGR and increased birth weight. In contrast, subjects consuming diets with a high content of processed foods, confectionery, and soft drinks showed an increased occurrence of low birth weight deliveries. Low birth weight is a risk factor for childhood stunting (Aryastami et al., 2017). Out of six dietary pattern studies reviewed by Brantsaeter, Olafsdottir, Forsum, Olsen, and Thorsdottir (2012), four showed a positive association between birth weight and moderate milk consumption by the pregnant mother. Two of the studies reviewed by Brantsaeter et al. (2012) indirectly identified a possible role of whey protein in fetal growth in utero. Heppe et al. (2011) analyzed data collected in the Dutch Generation R Study that addressed fetal life until young adulthood in Rotterdam. They found that maternal milk consumption of greater than 3 glasses per day was associated with fetal weight gain in the third trimester of pregnancy, resulting in an 88 g (95% CI: 39, 135 g) higher birth weight compared to consumption of 0 1 glasses per day. This association appeared to be limited to whey protein as intake of protein from nondairy sources or from cheese was not associated with birth weight increase. Similarly, Olsen et al. (2007) using data from the Danish National Birth Cohort, found that milk consumption was positively associated with higher birth weight for

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gestational age, lower risk of small for gestational age (SGA) births, and higher risk of large for gestational age births. Interestingly, Olsen et al. (2007) compared associations relating to nondairy proteins, milk products excluding cheese and ice cream and cheese protein. Their findings revealed that birth weight was constant across quintiles of nondairy protein intake, suggesting that the associations found with dairy protein were not a general protein attribute (Fig. 15.1). In addition, Olsen et al. (2007) observed that compared to dairy protein, cheese protein intake predicted only a slight increase in birth weight. The authors, however, did caution that the range of intake of cheese protein was much narrower than that for milk protein. Leaving that aside, by deduction,

200 150 100 Difference in birth weight (g)

552

50 0 Q1

Q2

Q3

Q4

Q5

–50 –100

Quintile of protein intake –150 –200 –250 Dairy protein intake-Olsen et al. (2007)

Dairy protein intake-Heppe et al. (2011)

Cheese protein intake-Olsen et al. (2007)

Cheese protein intake-Heppe et al. (2011)

FIGURE 15.1 Difference in birth weight observed over quintiles of consumption of total dairy protein intake compared to cheese protein intake for pregnant mothers. Dairy protein intake was associated with a positive increase in birth weight with increased consumption. Cheese protein intake showed either no association or a slight negative association with birth weight. Adapted from data reported by Olsen, S. F., Halldorsson, T. I., Willett, W. C., Knudsen, V. K., Gillman, M. W., Mikkelsen, T. B., . . . & Consortium, N. (2007). Milk consumption during pregnancy is associated with increased infant size at birth: Prospective cohort study. American Journal of Clinical Nutrition, 86, 1104 1110 and Heppe, D. H., van Dam, R. M., Willemsen, S. P., den Breeijen, H., Raat, H., Hofman, A., . . . & Jaddoe, V. W. (2011). Maternal milk consumption, fetal growth, and the risks of neonatal complications: The Generation R Study. American Journal of Clinical Nutrition, 94, 501 509.

15.3 Whey Protein: A Potential Role in Helping to Reduce Undernutrition

this finding suggests that whey protein intake during pregnancy may be associated with increased birth weight. Currently, the available evidence is insufficient to come to an informed conclusion about the possible association of maternal whey protein intake with birth weight, but the findings described above are intriguing and merit more focused investigation. Further study is stimulated by indirect evidence from other areas of research that could explain how whey proteins could be implicated in fetal growth (Clark, 2016). These findings include: 1. Leucine improves growth performance in IUGR of piglets and whey proteins are a rich source of leucine (Xu et al., 2016). 2. Breast milk contains approximately three times the whey protein content of cow’s milk, and the level of whey proteins in breast milk is significantly higher in mothers of preterm babies (Narang, Bains, Kansal, & Singh, 2006). 3. Based on breast milk intake, young infants’ requirement for indispensable amino acids is most acute immediately after birth; the infant requirement in month one is twice that of month four on a mg/ kg body weight basis, and there is reason to assume that the fetal requirement is similarly acute, a demand that could in part be met by maternal whey protein consumption (WHO, FAO, & UNU, 2007). 4. Chondral plate growth, a determinant of fetal linear growth is regulated by the mTORC1 pathway, which in turn is controlled by plasma leucine levels. Stunted children have been shown to have lower serum levels of essential amino acids (Semba et al., 2016). On the other hand, caution is required following the observation by Olsen et al. (2007) that milk intake (and by deduction, whey protein) displays an association with large-for gestational-age (LGA) births. As with low birth weight and SGA, LGA births are at an increased risk of chronic diseases in later life, including type 2 diabetes, obesity, and coronary heart diseases (Godfrey & Barker, 2000). Other studies, however, suggest that the association between high protein consumption and LGA births is the protein:carbohydrate (P:C) ratio (Maslova, Halldorsson, Astrup, & Olsen, 2015; Moore, Davies, Willson, Worsley, & Robinson, 2004). Indeed, the impact that LGA has on risk of nontransmissible disease in later life may be further confounded by phenotype as data from the Growing Up in Singapore Towards health Outcomes (GUSTO) study indicated that higher maternal animal protein intake, but not plant protein, during pregnancy was associated with lower neonate internal adipose tissue (Chen et al., 2016). Currently, there is strong evidence supporting the involvement of whey protein intake and increase in lean body mass (Miller, Alexander, & Perez, 2014). It remains to be seen if this observation extends to the pregnant female, fetal, and infant development.

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15.3.1

Managing Malnutrition During Infancy

SAM and MAM are conditions that afflict those within the first 1000 days of life, conception to 24 months, as well as other children living in vulnerable countries (WHO, 2007). The impact of SAM and MAM is most devastating in early life, associated with increased stunting, morbidity, and mortality (WHO, 2007). These conditions are managed with ready-to-use therapeutic foods (RUTFs), the established standard of care for the community-based management of SAM (WHO, 2007). Whey proteins were recently demonstrated to be an effective and affordable recovery agent for malnutrition in young children (Bahwere et al., 2014; Stobaugh et al., 2016). In a randomized clinical effectiveness trial among rural Malawian and Mozambican children, significantly more children recovered from MAM when fed a whey-based supplement compared to a soybased supplement (Stobaugh et al., 2016). In addition, children fed the whey-based supplement demonstrated better markers of growth during treatment (Stobaugh et al., 2016). Of importance is that when tested against a standard milk-based recovery supplement, a whey protein-based supplement was equally effective at treating SAM, and was a cheaper alternative to standard milk-based supplements (Bahwere et al., 2014). A requirement for the inclusion of dairy protein in RUTF and fortified blended foods (FBFs) was established by the US Department of Agriculture in 2015 (USDA, 2015). The regulation requires 50% of protein to be from dairy in RUTF, and 33% in RUSF. Whey proteins and other milk proteins can be used to meet these requirements. Earlier in 2008, the USAID food aid quality review project recommended whey proteins be included in FBF. UNICEF introduced a standard in Codex in 2016, which features a minimum content of dairy proteins, along with a substantial body of evidence that demonstrates higher and faster recovery rates from SAM when dairy ingredients are included (Stobaugh et al., 2016).

15.3.2 Why Linear Growth Is Important, and How Whey Proteins Contribute to This Effect A recent metaanalysis examined the relationship between linear growth and child development in low- and middle-income countries (Sudfeld et al., 2015). Researchers reviewed 68 published studies from 29 different countries, and found the data indicated that improvement in height-for-age over time or recovery from a stunted to a nonstunted height-for-age score was associated with improved cognition. They concluded that in low- and middle-income countries, linear growth was positively associated with cognitive and motor development, and that “without intervention, early cognitive deficits may persist throughout childhood for children experiencing restricted

15.3 Whey Protein: A Potential Role in Helping to Reduce Undernutrition

linear growth during the first 2 years of life.” One of the plausible mechanisms explaining the relationship is that chronic infection and protein malnutrition may delay the development of early motor skills (Sudfeld et al., 2015). Loss of muscle or decreased muscle fiber size may also directly lead to motor delay. Children who are more lethargic or less active may have reduced ability to engage with their environment and a reduced ability to explore and extract learning opportunities from their physical and social surroundings (Sudfeld et al., 2015). There is little research available on how children’s dietary patterns are related to growth. A recent study conducted in Peru examined the dietary intakes of children aged 6 8 months and found that their dietary pattern score specific to dairy intake was positively associated with changes in length of age scores (Arsenault, Lopez de Romaña, Penny, & Brown, 2016). The authors stated that “the introduction of animal source foods, such as dairy products, to children’s diets is important given their nutrient profile that includes highquality protein, bioavailable nutrients and possibly other bioactive components that are essential for growth.” Whereas breastfeeding throughout the first year is recommended, the study suggests it is important to introduce dairy products as part of complementary foods to optimize linear growth (Arsenault et al., 2016). Overall, nutritional interventions have been only moderately effective at preventing stunting. Recent research indicates that insufficiency in amino acids may be limiting the linear growth of children (Bhutta et al., 2013). In fact, it has been estimated that even if current evidence-based nutritional interventions were all applied at 90% coverage, stunting would be reduced only by 20% (Bhutta et al., 2013). Although some new research has been published since this assessment, and new products developed, it remains critical to continue to improve the supplementary products used for interventions, and more research is needed to understand the pathogenesis of stunting. Overall, researchers agree that there is a clear association between the quality of protein available at national level and the prevalence of stunting, and that more research is needed in infants 6 months and older to examine the relationship between protein quality, linear growth, and the prevention of stunting (Ghosh, Suri, & Uauy, 2012). According to Manary, Callaghan, Singh, and Briend (2016), the effect of using proteins of different quality on recovery is not fully understood. Researchers examined the results of six clinical trials and established correlations between rates of weight gain and protein quality. Using the DIAAS Food Aid Score, the findings indicated the greatest recovery outcome per unit protein digestibility score (Manary et al., 2016). The researchers concluded “when looking at all the protein quality scores, dairy protein is likely to be higher, and particularly for malnourished children, dairy proteins are associated with higher growth” (Manary et al., 2016).

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Furthermore, considering the specific pattern of whey proteins, it is clear they can be used to improve the pattern of complementary foods based on local ingredients and boost the overall DIASS score. Additional research is underway to document the specific benefits of dairy proteins with regards to linear growth, in particular the exact amount that will prove to be most costeffective.

15.3.3

Whey Protein, Weight Gain and Lean Body Mass

A study of weight gain and recovery rates amongst Malawian children (Ackatia-Armah et al., 2015) found that weight gain from baseline was greater with RUSF containing a whey/soy isolate blend than with the locally processed vegetable protein-containing blends, and was intermediate with CSB11, also known as Super Cereal Plus, which is an FBF containing some skimmed milk powder. Sustained recovery rates were higher with the RUSF compared to any of the other treatments. It is important to note that the protein content of the RUSF at 12.5 g was significantly lower than any of the other treatments (15.5 18.5 g). These findings indicate that quality of protein, rather than quantity of protein, is critical. Further, Manary et al. (2016) provided evidence that the current method for assessment of protein quality, the DIAAS (FAO, 2013), significantly underestimates the demand for indispensable or essential amino acids by malnourished individuals as the calculation of protein quality is based on a reference pattern from healthy children. While DIAAS is currently considered superior to the measurement standard known as PDCAAS (Protein Digestibility 2 Corrected Amino Acid Score). This claim may be supported by the results of a recent study by Batra et al. (2014) which examined two levels of dairy proteins, 15% and 33% of total protein, in RUSF fed under supervision to school children (3 5 years) in Guinea-Bissau. Researchers found that RUSF-33%, but not RUSF-15%, eliminated a decrease in mid-upper arm circumference (MUAC), a measure of wasting, that was observed in controls (20.01 cm in RUSF-33% compared with 20.34 cm in controls, P , .05). Currently, more extensive studies are needed to establish ideal protein levels. Noriega and Lindshield (2014) reviewed evidence supporting the inclusion of animal-sourced protein in FBFs. They concluded that whilst epidemiological data was consistently associated with improved growth outcomes with animal-sourced protein inclusion, there was little evidence from isocaloric or isocaloric/isonitrogenous interventions. They did find evidence indicating that whey proteins benefit muscle accretion, but not linear growth. It is quite likely that the amount of whey proteins studied was a confounding factor in their conclusion. This uncertainty surrounding effectiveness of dairy protein inclusion in FBFs needs resolving, because cost remains a common question for dairy protein

15.3 Whey Protein: A Potential Role in Helping to Reduce Undernutrition

incorporation into RUSF/FBF (Suri, Moorthy, & Rosenberg, 2016). The effectiveness of WPC as an economical alternative to SMP/nonfat dried milk has been proposed (Hoppe et al., 2008) and is the subject of several promising studies. Stobaugh et al. (2016) compared the effectiveness of a peanut-based RUSF containing whey protein and whey permeate with soy proteincontaining control in a trial conducted in rural Malawi and Mozambique. They found that a significantly higher number of children recovered from MAM in the whey protein-containing RUSF group (83.9%) compared to the group receiving the soy protein-containing RUSF (80.5%), which had a higher protein and calorie content. In addition, those consuming the whey protein RUSF demonstrated better growth markers. The researchers concluded that the whey protein RUSF resulted in higher recovery rates and improved growth than did the soy protein even though the whey protein RUSF provided less protein and energy than the soy protein control. Hoppe et al. (2008) studied the effectiveness of FBF containing whey protein or skimmed milk powder, and determined that whereas protein quality was improved, cost was increased. Thus, randomized intervention trials testing the effectiveness of milk-protein-based FBF in vulnerable populations is warranted (Hoppe et al., 2008). In the context of MAM, Bahwere et al. (2014) reported that milk powder comprised about 30% of the ingredient content of RUTF and contributed to approximately 50% of the cost. The researchers explored the economics and effectiveness of substitution of the skimmed milk powder component with WPC35 and reported that the effectiveness of the WPC-based RUTF was comparable to the SMP control with respect to recovery rate from SAM, average weight gain, and length of stay. Weber and Callaghan (2016) described a free access, linear programming tool that alongside other capabilities, supports the calculation of lowest cost formulations for supplementary foods products that to date has been used successfully in the design of over 20 products. While the dairy component of RUFs is indeed more expensive than alternative proteins, some authors have suggested that the actual difference of cost in a daily ration is minimal. Batra et al. (2014) stated that “although milk protein is more expensive than ingredients such as soy isolate, the difference is currently not large. . .it was only $0.0017 per 92 g daily ration (2015 cost).” The study demonstrated that only products containing 33% dairy resulted in increases in MUAC (Batra et al., 2014). Stobaugh et al. (2016) conducted a study where the production cost of the soy supplement was $2.78/kg, and that of the whey protein supplement was $3.13/kg. Researchers calculated that the total amount of RUSF provided until recovery for a 7 kg child was just over 3 kg, resulting in a very small

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difference per child who recovers. The authors further stated that, “in the larger context of the operational costs of a supplementary feeding program that includes staff, logistical support and facilities, this additional cost is quite minimal for the significantly higher recovery rate achieved,” and that “. . .inclusion of dairy leads to improved outcomes in children with MAM with only a marginal increase in cost.” Whereas recovery from MAM as a simple outcome is important, what is really critical is not only linear growth but also lean body mass accretion. In South Asia, and part of Southeast Asia, it has been documented that low birth weight is linked to low lean body mass, which results in a higher fat mass versus lean body mass ratio later in life (Kulkarni et al., 2014). Fetal undernutrition, and lack of lean body mass accretion early in life predisposes one to hyperlipidemia, insulin resistance, and central obesity (Kulkarni et al., 2014). Researchers now advocate for the provision of supplementary foods fortified not only in micronutrients but also in quality proteins. Ideally, mothers’ milk does provide large quantities of whey proteins, but in developing countries, where the mother is herself malnourished, or may not be able to lactate beyond a few months, the reality is that high-quality complementary foods, providing whey proteins, need to be provided (Kulkarni et al., 2014). In conclusion, there is significant evidence from numerous studies showing an association between inclusion of dairy proteins, including whey proteins, and reduction in the incidence of wasting and stunting in the prenatal diet and from 6 months and up. To a limited extent this is supported by data from randomized, controlled trials but more data are needed to define the most efficient and economical treatment. Such trials must not only establish the minimum effective level of dairy or whey protein as a function of recovery rate, such that the cost-effectiveness of inclusion of dairy/whey protein can be accurately assessed. A key strategy is to provide enrichment of complementary foods with high-quality proteins such as whey proteins. In the past, availability of these proteins was limited, but they are now widely available in international trade, and can be used for therapeutic purposes for the management of malnutrition, the cost-effective prevention of stunting, and optimization of lean body mass.

15.4 WHEY PROTEIN: A POTENTIAL ROLE IN HELPING TO REDUCE OVERNUTRITION 15.4.1

Higher Protein Diets and Weight Loss

Overweight and obesity, a global epidemic of overnutrition, are now linked to more deaths worldwide than underweight (WHO, 2016a). As of 2014,

15.4 Whey Protein: A Potential Role in Helping to Reduce Overnutrition

41 million children under the age of 5 were overweight or obese, 1.9 billion adults were overweight, and over 600 million adults were obese (about 13% of adults, worldwide) (WHO, 2016a). Once considered conditions of the developed world, overweight and obesity are now affecting low- and middleincome countries (WHO, 2016a). For example, overweight or obesity nearly doubled in Africa from 5.4 to 10.6 million between 1990 and 2014 and nearly half of the world’s children under the age of 5 who were overweight or obese in 2014 lived in Asia (WHO, 2016a). Overweight and obesity are primary risk factors for noncommunicable diseases such as cardiovascular diseases, diabetes, musculoskeletal disorders, and some cancers (WHO, 2016a). Higher protein diets may be effective for weight loss because of changes in energy metabolism and appetite signaling that promote decreased energy intake (Leidy et al., 2015). Dietary protein has been shown to create a greater postprandial thermic effect of food than an equal amount of dietary carbohydrate or fat (Halton & Hu, 2004). Whereas dietary carbohydrate requires 5% 10% of its usable energy to be expended for metabolism and/ or storage, and dietary fat requires just 0% 3%, dietary protein requires 20% 30% usable energy (Leidy et al., 2015; Westerterp-Plantenga, Nieuwenhuizen, Tome, Soenen, & Westerterp, 2009). Furthermore, during weight loss, high-protein diets can help maintain resting energy expenditure (Eisenstein, Roberts, Dallal, & Saltzman, 2002; Halton & Hu, 2004; Leidy et al., 2015). Protein also has a greater effect on satiety than carbohydrate or fat, which may lead to reduced energy intake (Bosse & Dixon, 2012; Leidy et al., 2015). Body Mass Index (BMI), a measure of adiposity based on measurements of height and weight, is a crude but useful measure for overweight and obesity, because it is simple to measure and is standardized globally (WHO, 2016a). BMI, as a sole indicator for health, however, has its limitations. It once seemed indisputable that higher BMI was associated with increased risk for all-cause death (Whitlock et al., 2009), but recently published research has indicated lower mortality rates in overweight and obese persons than in normal weight persons (Costanzo et al., 2015; Greenberg, 2013; Lavie, Alpert, et al., 2013; Lavie, Cahalin, et al., 2013; Vapattanawong, Aekplakorn, Rakchanyaban, Prasartkul, & Porapakkham, 2010). A recent cohort study demonstrated that low BMI and high body fat percentage are independently associated with increased mortality (Padwal, Leslie, Lix, & Majumdar, 2016). Emerging evidence indicates that aiming for optimal body composition, rather than simply weight loss, will be imperative to improving rates of morbidity and mortality (Prado & Heymsfield, 2014).

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Over the last two decades, dietary strategies to increase dietary protein consumption, compared with carbohydrate and fat consumption, have shown promising results in improving body composition and metabolic health (Abete, Astrup, Martinez, Thorsdottir, & Zulet, 2010; Champagne et al., 2011; Krieger, Sitren, Daniels, & Langkamp-Henken, 2006; Skov, Toubro, Ronn, Holm, & Astrup, 1999). Evidence indicates that increased dietary protein promotes improved body composition under iso-, hypo-, and hypercaloric conditions (Bosse & Dixon, 2012). A robust body of evidence indicates that the consumption of 1.2 1.6 g of protein/kg body weight per day, with at least 25 30 g of protein per meal, will reduce body weight and fat mass while preserving lean mass and can be considered a successful strategy in the prevention or treatment of obesity (Clifton, 2012; Dong, Zhang, Wang, & Qin, 2013; Leidy et al., 2015; Te Morenga & Mann, 2012; WesterterpPlantenga, Lemmens, & Westerterp, 2012). In a randomized trial of 20 young men, higher protein diets of up to 2.4 g of protein/kg body weight per day contributed to improved body composition by promoting lean body mass and loss of fat mass during energy deficiency combined with intense exercise for four weeks compared with higher protein diets of 1.2 g of protein/kg body weight per day (Longland, Oikawa, Mitchell, Devries, & Phillips, 2016). These results indicate that dietary protein consumption higher than the Recommended Dietary Allowance during an energy deficit may help to further preserve lean body mass, particularly when combined with exercise (Longland et al., 2016).

15.4.2

Whey Protein and Optimal Body Composition

It is well accepted that resistance training in combination with adequate amounts of dietary protein can increase muscle protein synthesis in healthy adults, regardless of their age or gender (Hulmi, Lockwood, & Stout, 2010). An emerging body of evidence, however, indicates that dairy protein, specifically whey proteins, may stimulate the greatest rise in muscle protein synthesis, thereby optimizing body composition compared with other nonmeat protein sources (Hulmi et al., 2010). This is likely because whey proteins are considered to be high-quality protein (Loenneke et al., 2012). In a study that determined the relationship between the amount of quality protein, carbohydrate, and fat consumed and percentage of central abdominal fat, quality protein consumption was inversely associated with central abdominal fat (Loenneke et al., 2012). A randomized trial in nonresistance-trained men and women who consumed daily isocaloric supplements containing carbohydrate, whey protein, or soy protein showed that gains in lean body mass were greater in the group consuming whey protein than in the groups consuming carbohydrate and soy (Volek et al., 2013). The results indicate that protein quality may be an important determinant of lean body mass,

15.4 Whey Protein: A Potential Role in Helping to Reduce Overnutrition

particularly following resistance exercise (Volek et al., 2013) (see also Chapter 16: Sports and Exercise Supplements). High-quality protein, such as whey protein, has also been demonstrated to be effective in improving body composition in untrained, free-living adults (Baer et al., 2011). In a randomized trial that was designed to test the effect of supplemental whey protein, soy protein, and carbohydrate on body weight and composition in adults, body weight and fat mass were lower in the groups consuming whey protein and soy protein than the group consuming carbohydrate (Baer et al., 2011). Whereas lean body mass did not differ between groups, the group consuming whey protein had a greater decrease in waist circumference than the groups consuming soy protein and carbohydrate (Baer et al., 2011). A metaanalysis of 14 randomized controlled trials found that whey protein, with or without resistance exercise, improved body composition in adults (Miller et al., 2014). The effects of whey protein supplementation were beneficial whether the whey protein was added to the diet or used as a replacement for other proteins or carbohydrates in the diet, but were most pronounced when consumed as part of an overall healthy eating plan and combined with resistance exercise (Miller et al., 2014). Maintaining a healthy body composition is particularly important for aging adults, as it is associated with gait-speed (Beavers et al., 2013), and is a predictor of functional status and mortality in older adults (Studenski et al., 2011). A randomized controlled trial conducted in 60 older adults indicated that supplementation with a milk-based protein matrix consisting mostly of whey protein at breakfast and lunch for 24 weeks improved lean tissue mass compared with an isocaloric carbohydrate control (Norton et al., 2016). The beneficial effect of whey protein on body composition in older adults has been demonstrated during caloric restriction-based weight loss as well, and was attributed to not only the preservation of lean body mass, but the preferential reduction of adipose tissue (Coker, Miller, Schutzler, Deutz, & Wolfe, 2012). As interest in nonpharmacologic approaches to maintain skeletal health in adult life increases, the safety of dietary protein intake has received attention (Shams-White et al., 2017). According to a systematic review and metaanalysis of 16 randomized controlled studies and 20 prospective cohort studies, current evidence shows no adverse effects of higher protein diets on bone health in adults (Shams-White et al., 2017). A trial that assessed whey protein supplementation, specifically, found that after 36 weeks of supplementation, whey protein, regardless of doses up to 60 g/day, did not influence bone mineral density or bone mineral content following the intervention and was not associated with reduction of total or regional bone mineral

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density or bone mineral content over time (Wright, McMorrow, WeinheimerHaus, & Campbell, 2017). Current evidence from a large body of research indicates that consuming whey protein, with or without resistance exercise, helps to optimize body composition in both young and older adults (Coker et al., 2012; Miller et al., 2014). Whey protein can easily be incorporated into the daily diet by mixing it into shakes and smoothies or by adding it as an ingredient to regular everyday recipes.

15.4.3

The Double Burden of Malnutrition

Paradoxically, whereas many countries are experiencing an increase in obesity-related noncommunicable diseases, they continue to suffer from undernutrition and the conditions associated with it (WHO, 2016a). This coexistence of over- and undernutrition within countries, and even within households and families, is referred to as “the double burden of malnutrition” (WHO, 2017). This double burden mostly affects low- and middleincome countries, but is not absent from developed and affluent countries (Tzioumis & Adair, 2014). Individuals can be both obese, but undernourished (micronutrient-deficient), contributing to a multitude of chronic conditions (Tzioumis & Adair, 2014). Further, there is an association between childhood stunting and adult obesity and its comorbidities, such as cardiovascular diseases and type 2 diabetes (Tzioumis & Adair, 2014). The current challenge for public health programs and policies is to adequately address undernutrition while remaining vigilant against obesity and the comorbidities associated with it (Tzioumis & Adair, 2014). A systematic review and metaanalysis of prospective cohort studies comprising over 46,000 children and adolescents indicated that dairy consumption was inversely associated with the risk of childhood overweight and obesity (Lu, Xun, Wan, He, & Cai, 2016). In adults, it appears dairy may aid in weight loss only when coupled with energy restriction (Abargouei, Janghorbani, Salehi-Marzijarani, & Esmaillzadeh, 2012). While more research is needed to understand the potential mechanisms behind dairy and weight maintenance, one randomized controlled trial in adults showed that in combination with a calorie-restricted diet, a specialized whey fraction high in leucine, bioactive peptides, and milk calcium led to a greater loss of fat and preservation of lean body mass than an isocaloric control (Frestedt, Zenk, Kuskowski, Ward, & Bastian, 2008). This is an important point for public health officials who are looking for strategies to manage undernutrition without contributing to overnutrition.

15.5 Whey Protein and Chronic Diseases

15.5

WHEY PROTEIN AND CHRONIC DISEASES

Chronic diseases, such as cardiovascular disease and diabetes, are major causes of morbidity and mortality in developed and developing countries globally (Halpin, Morales-Suárez-Vaerla, & Martin-Moreno, 2017). Unhealthy diet is among several shared risk factors beyond genetics that are associated with the global burden of chronic diseases (Halpin et al., 2017). While a multifaceted approach to public health is necessary to chronic disease management and prevention, understanding how certain components in the diet, such as whey proteins, may contribute to health and wellness is also of importance.

15.5.1

Cardiovascular Diseases

Cardiovascular diseases are the leading cause of death in the developed world, with high blood pressure being a leading modifiable risk factor (Mozaffarian et al., 2016). A randomized controlled trial conducted in 42 adults demonstrated that the consumption of whey proteins lowered 24-hour ambulatory blood pressure and improved vascular reactivity and biomarkers of endothelial function (Fekete, Giromini, Chatzidiakou, Givens, & Lovegrove, 2016). Another clinical study, in overweight and obese men, showed improvements in blood pressure after a whey protein preload administered 30 minutes before an ad libitum meal (Tahavorgar, Mohammadreza, Shidfar, Mahmoodreza, & Heydari, 2015). Dyslipidemia is another modifiable risk factor associated with increased risk for cardiovascular diseases (Mozaffarian et al., 2016). While a trial conducted in 52 participants demonstrated that whey protein supplementation resulted in a beneficial effect on one aspect of lipid metabolism, a decrease in apolipoprotein B-48 response (Bohl et al., 2015), current evidence from randomized controlled trials indicates that whey protein supplementation has only a modest effect on lipid metabolism, with a lowering effect on triglycerides, but no effect on total cholesterol, low-density, or high-density lipoprotein cholesterol (Zhang et al., 2016).

15.5.2

Diabetes

Diabetes, a chronic disease linked to obesity and heart disease, affects nearly 10% of adults globally (WHO, 2016b). Whey protein supplementation was recently demonstrated to be an effective contributor to the management of type 2 diabetes (Jakubowicz & Froy, 2013; Jakubowicz et al., 2014). Whey proteins may work to suppress appetite and encourage insulinotropic effects (Jakubowicz & Froy, 2013; Jakubowicz et al., 2014), but research in this area

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is ongoing. While whey proteins have shown promise for improving insulin secretion in patients with type 2 diabetes, longer-term studies focused on primary disease endpoints are warranted (Pasin & Comerford, 2015).

15.6 15.6.1

WHEY PROTEINS AND SARCOPENIA Sarcopenia and Its Economic Impact

Sarcopenia is a condition associated with loss of muscle mass and function (Santilli, Bernetti, Mangone, & Paoloni, 2014). The term sarcopenia was first used by Rosenberg (1989) and is derived from the Greek words, “sarx” meaning flesh and “penia” meaning loss. Sarcopenic muscle loss is correlated with physical disability, loss of mobility, and ultimately death. Although primarily observed in advanced stages in the elderly, individuals begin losing muscle from about age 40 years or even younger and the rate of muscle loss accelerates with age (Paddon-Jones & Leidy, 2014) (Fig. 15.2). Other risk factors associated with sarcopenia are gender and level of physical activity. The economic impact of sarcopenia is enormous due to the steadily increasing aging population of the world. The number of people aged $ 60 years is predicted to reach 1.2 billion by 2025 and will increase to over 2 billion by 2050. The prevalence of diagnosed sarcopenia in 60 70-year-olds is in the range of 5% 13%, but rises to 11% 50% amongst 80-year-olds (Palmio & Udd, 2014). Conservatively, sarcopenia will affect over 200 million people Middle-aged (52 ± 4 years)

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FIGURE 15.2 Age-dependence of rate of leg muscle loss during bed rest for young (28 days), middle-aged (14 days), and elderly (10 days). The extent of muscle loss increases with length of inactivity (bed rest) and age with the rate of loss being highest amongst the elderly. Adapted from Paddon-Jones, D., & Leidy, H. (2014). Dietary protein and muscle in older persons. Current Opinions in Clinical Nutrition & Metabolic Care, 17, 5 11.

15.6 Whey Proteins and Sarcopenia

between now and 2050. The estimated health care costs directly associated with the treatment of sarcopenia in the United States in 2000 were US$18.5 billion (Janssen, Shepard, Katzmarzyk, & Roubenoff, 2004).

15.6.2

Sarcopenia and Protein Intake

There is an important correlation between inactivity and muscle loss, but management of sarcopenia also requires adequate nutrition. Both the quantity and quality of food, in particular protein, can impact the development and course of sarcopenia. Age-related reductions in appetite can result in reduced weight and muscle mass (Ahmed & Haboubi, 2010). Food intake by elderly populations reduces by approximately 25% between 40 and 70 years of age (Nieuwenhuizen, Weenen, Rigby, & Hetherington, 2010). In addition, the effectiveness of utilization of dietary amino acids in muscle protein synthesis decreases with age. Indeed, the elderly appear more resistant to several factors that stimulate muscle protein synthesis, including plasma amino acid levels (Fry & Rasmussen, 2011). In the United States, the current Recommended Dietary Intake for protein remains at 0.8 g/kg body weight per day for adults. New research, however, shows that this may not be enough, especially in an older population (Wolfe, 2012). Given the hypothesis that older adults may have a blunted anabolic response to dietary protein and likely need more to preserve lean muscle mass and functional ability, current research now shows that protein intake of at least 1.0 g/kg body weight and as high as 1.5 g/kg body weight may be optimal. This would mean as much as 102 g of protein daily for a person weighing 150 pounds (67 kg) (Morley et al., 2010). Recommendations from a European Society for Clinical Nutrition and Metabolism (ESPEN) workshop in 2013 suggested that alongside resistance exercise for healthy older people, the diet should provide at least 1.0 1.2 g protein/kg body weight and for older people, who are malnourished or at risk of malnutrition due to acute or chronic illness, the diet should provide 1.2 1.5 g protein/kg body weight per day (Deutz et al., 2014).

15.6.3 Whey Protein Supplementation and Sarcopenia in the Elderly Over the past decade, there have been multiple trials conducted examining the benefit of substitution or supplementation of protein intake in the diet with whey proteins compared to other sources of protein, comparing young and old subjects or patients with sarcopenia in the presence or absence of resistance exercise with generally positive outcomes. For example, Chale et al. (2013) reported that supplementation of mobility-limited older adults with 40 g protein/day from WPC resulted in increased lean body mass

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(1.3% vs 0.6%) and muscle protein cross-sectional area (4.6% vs 2.9%) compared to those receiving a maltodextrin-based isocalorific control drink. The observed improvements in stair-climbing in both supplemented and control groups, however, were not statistically different. Studies have become sufficiently numerous to allow several metaanalyses to be conducted. Cermak, Res, de Groot, Saris, and van Loon (2012) conducted a metaanalysis of data that examined protein supplementation in combination with exercise in both young and older subjects. In the analysis of a subset of trials focusing on older subjects, they identified five trials that exclusively used dairy sources of protein (whey, milk, or casein) and one which examined a combination of animal-sourced proteins that included dairy. Individually, these studies showed no significant benefit compared to placebo on fat free mass. When the data from the studies were combined (215 older subjects), however, protein supplementation resulted in 38% more fat-free mass and a 33% increase in strength when compared to placebo. This outcome led the authors to conclude, “protein supplementation increases muscle mass and strength gains during prolonged resistance exercise training in both young and older subjects.” A recent retrospective analysis of data from multiple laboratories (Moore et al., 2015) revealed that whilst basal myofibrillar protein synthesis rates were comparable between older and young men, the plateau synthetic rate in older men was 0.40 6 0.19 g/kg body mass, which was only two-thirds that of younger men. Colonetti et al. (2017) published a systematic review of studies specifically addressing whey supplementation in elderly subjects who were participating in resistance exercise programs. A total of 632 studies were screened and a metaanalysis was carried out on five studies involving 391 participants. Whey protein supplementation was associated with higher total protein ingestion and a mean increase in plasma leucine concentration ranging from 406 to 490 µmol/L compared to a control group. Critically, supplementation with whey proteins was associated with increased mixed muscle protein fractional synthesis rate (1.26; 95% CI: 0.46, 2.07) compared to the control group (Colonetti et al., 2017). The metaanalyses described above reviewed studies that delivered essential amino acids solely in protein-bound form. In contrast, Rondanelli et al. (2016) studied the effect of a supplement that combined whey protein (22 g) and free essential amino acids (10.9 g, of which 4 g was leucine) along with vitamin D (100 IU) and that was administered to a mixed gender group of sarcopenic elderly people (average age 80.3 years) over 12 weeks in combination with physical activity. They found that the supplement not only

15.6 Whey Proteins and Sarcopenia

boosted fat-free mass (1.7 kg gain; P , .001) and hand grip strength, but also enhanced other aspects that contribute to well-being in sarcopenic elderly. Whey protein is associated with greater stimulation of muscle protein synthesis than other protein sources. The association between whey protein intake and lean mass in elderly subjects may be specific. For example, in the study by Farsijani et al. (2016), it was found that the observed decline in lean mass amongst a cohort of men (n 5 351) and women (n 5 361), aged 67 84 years, declined by 2.0% 2.5% over a period of 2 years and was not affected by the total quantity nor the distribution of total protein intake. Indeed, Burd et al. (2012) demonstrated that even the different classes of dairy protein—casein and whey protein—showed different levels of muscle protein synthesis-stimulating activity. They compared the effect of WPI and casein ingestion on myofibrillar protein synthesis in elderly subjects at rest and after exercise. Burd et al. (2012) found that leucine concentration peaked 60 minutes after consumption of a protein (20 g) containing drink and was greater in amplitude in the WPI-consuming subjects. Myofibrillar protein synthesis in the subjects’ rested legs was 65% higher after ingestion of whey proteins compared to ingestion of micellar casein. Similarly, resistance exercise-stimulated synthesis rates in the exercised leg were significantly higher after whey protein ingestion (0.059% per hour) compared to micellar casein (0.035% per hour). The effectiveness of whey protein supplementation in stimulation of muscle protein synthesis has been compared with supplementation with other proteins, including soy. Yang, Breen, et al. (2012), Yang, Churchward-Venne, et al. (2012) compared the fractional synthetic rate (FSR) of muscle protein accretion in elderly subjects (average age 71 years) at rest and after a leg resistance exercise for two different levels, 20 and 40 g of either whey or soy protein. The results showed a predictable trend of increased FSR after exercise compared to rest and a progressive upward trend in FSR with protein level. Only whey protein intake, however, delivered a statistically significant increase in FSR between both 20 and 40 g levels and when compared to controls at rest and after exercise. In contrast, soy protein showed a statistically significant difference at only the highest level (40 g) and only after exercise.

15.6.4 Hypotheses About the Mechanism of Action of Whey Protein Intake on Sarcopenia Previously, Dangin et al. (2003) had attributed differences between leucine balance following ingestion of an isonitrogenous dose of casein or whey protein as being linked to the higher leucine content of whey proteins and whey

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proteins being “fast” digesting proteins in comparison to “slow” digesting casein. These conclusions were supported in a later study by Pennings et al. (2011) where it was shown that whey proteins stimulated muscle protein accretion more effectively than either casein or casein hydrolysate. A further critical finding from the study by Dangin et al. (2003) was that the higher leucine balance observed following consumption of whey proteins compared to casein was significantly magnified (58% greater) in the elderly (average age 72 years) compared to younger subjects (average age 24 years). Such observations support the hypothesis that the stimulants of muscle protein synthesis are in some way blunted with age. A possible explanation of the superior effectiveness of whey proteins over soy proteins has been offered by Mitchell, Della Gatta, Petersen, CameronSmith, and Markworth (2015). Muscle protein synthesis is primarily controlled by the regulation of translation initiation. The mechanistic Target Of Rapamycin (mTOR) complex integrates signals from nutrients, growth factors, and physical exercise via a cascade of protein phosphorylation events that trigger protein synthesis. Mitchell et al. (2015) observed that ingestion of 30 g of either whey or soy protein resulted in equivalent levels of phosphorylation of a key protein in the mTOR pathway 2 hour postexercise. Unlike whey proteins, however, soy protein failed to promote prolonged phosphorylation at 4 hour postexercise. This difference was attributed to the different leucine content of whey and soy protein resulting in a greater peak concentration of leucine in the blood and a more prolonged elevation in leucine levels following whey protein ingestion. This explanation, however, may be oversimplistic. In a study conducted by Katsanos et al. (2008), the muscle protein stimulating effect of whey proteins (15 g) was compared to that of a mixture of its constituent essential amino acids (6.72 g) and a mixture of its constituent nonessential amino acids (7.57 g). The results showed that intact whey protein was significantly more effective at promoting muscle protein synthesis than its constituent essential amino acids. This would seem to suggest that there is more to whey protein and its muscle protein synthesis stimulating effect than simply its high content of leucine. In summary, multiple studies have indicated that supplementary intake of whey proteins especially in combination with resistance exercise results in increased muscle protein synthesis. This effect appears most marked in elderly subjects. Without doubt, the high quality of whey proteins and in particular its leucine content and fast digestion/absorption are implicated in whey protein-induced stimulation of muscle protein synthesis. Further research is needed to unravel full details of the mechanism of action of whey to exploit its potential in delaying the debilitating impact of sarcopenia in older subjects.

15.7 Conclusion

Currently, many of the protein supplements provided to hospitalized or elderly individuals consist of ready-to-drink, shelf-stable, and retorted products. Because caseinates exhibit good heat stability, they have been used extensively in such products. Their organoleptic qualities, however, can be improved, especially for these populations who may lack appetite, and suffer from nausea. Monotony of the supplementation is also a factor impacting compliance, and therefore overall effectiveness of the supplementation. In recent years, suppliers of such products have included milk protein concentrates in their formulations, yet whey protein usage remains limited because of the relative lack of stability (due to pH, heat-treatment, etc.) (see Chapter 6: Thermal Denaturation, Aggregation, and Methods of Prevention). Lessons can be learned from developments achieved in the field of child malnutrition. The lipid-based, peanut butter products used in ready-to-eat therapeutic foods, e.g., are an excellent matrix that can contain 50% more protein from whey proteins. They are palatable, shelf-stable, and mostly easy to consume as snacks, even by subjects who may have disabilities, and difficulties swallowing. Whey proteins form gels and as such, can be formulated into high-protein puddings and other desserts (see Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated). Single-serve whey protein sachets, e.g., would offer convenience for self-supplementation and use in every day foods such as soups, sauces, and fruit or vegetable purees. Advances in whey protein manufacture have resulted in improvement of their flavor profile (see Chapter 10: Flavor Aspects of Whey Protein Ingredients), making their widespread use in a variety of foods a great asset in home care or institutional settings. It is likely this sector will become a vibrant segment of innovation, contributing to the effective management of sarcopenia and the promotion of healthy aging.

15.7

CONCLUSION

Whey protein is a safe, high-quality protein source for medical nutrition worldwide. With nearly a quarter of the children under the age of 5 around the world stunted, and nearly 52 million wasted (UNICEF et al., 2017), whey proteins can be used in supplementary and therapeutic foods to help improve malnutrition recovery rates, promote linear growth, and most importantly deliver lean body mass accretion benefits. In fact, new evidence indicates that the provision of high-quality proteins, such as whey proteins, to pregnant mothers, does have an impact not only on birth weight, but perhaps most importantly, on the lean body mass of their baby. This, in turn, along with the provision of high-quality proteins during the first 2 years, is likely to have a lasting impact, protecting from central fat obesity, diabetes, and other chronic diseases later in life. Just like clean water,

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vaccinations, mosquito nets, and micronutrients, whey protein supplementation should be part of the health arsenal available to meet the United Nation’s Sustainable Development Goals. At the other end of the spectrum, developed countries face another challenge: an aging population. New scientific evidence indicates that whey proteins can be helpful in managing cardiovascular diseases and diabetes. Furthermore, growing evidence points to the critical importance of preserving, or restoring, lean body mass in the elderly, and helping manage sarcopenia. Whey protein is widely available as an ingredient and traded worldwide. Advances in processing have resulted in great improvements in their functionality and organoleptic properties. A wide range of products are available at various price points, allowing their use in cost-effective, therapeutic products in a wide variety of contexts, cultures, and settings.

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Palmio, J., & Udd, B. (2014). Borderlines between sarcopenia and mild late-onset muscle disease. Frontiers in Aging Neuroscience, 6, Article 267. Pasiakos, S. M. (2015). Metabolic advantages of higher protein diets and benefits of dairy foods on weight management, glycemic regulation, and bone. Journal of Food Science, 80(Suppl. 1), A2 A7. Pasin, G., & Comerford, K. B. (2015). Dairy foods and dairy proteins in the management of type 2diabetes: A systematic review of the clinical evidence. Advances in Nutrition, 6, 245 259. Pennings, B., Boirie, Y., Senden, J. M., Gijsen, A. P., Kuipers, H., & van Loon, L. J. (2011). Whey protein stimulates postprandial muscle protein accretion more effectively than do casein and casein hydrolysate in older men. American Journal of Clinical Nutrition, 93, 997 1005. Prado, C. M., & Heymsfield, S. B. (2014). Lean tissue imaging: A new era for nutritional assessment and intervention. Journal of Parenteral & Enteral Nutrition, 38, 940 953. Rondanelli, M., Klersy, C., Terracol, G., Talluri, J., Maugeri, R., Guido, D., . . . Perna, S. (2016). Whey protein, amino acids, and vitamin D supplementation with physical activity increases fat-free mass and strength, functionality, and quality of life and decreases inflammation in sarcopenic elderly. American Journal of Clinical Nutrition, 103, 830 840. Rosenberg, I. (1989). Summary comments: Epidemiological and methodological problems in determining nutritional status of older persons. American Journal of Clinical Nutrition, 50, 1231 1233. Santilli, V., Bernetti, A., Mangone, M., & Paoloni, M. (2014). Clinical definition of sarcopenia. Clinical Cases in Mineral & Bone Metabolism, 11, 177 180. Semba, R. D., Shardell, M., Sakr Ashour, F. A., Moaddel, R., Trehan, I., Maleta, K. M., . . . Manary, M. J. (2016). Child stunting is associated with low circulating essential amino acids. EBioMedicine, 6, 246 252. Shams-White, M. M., Chung, M., Du, M., Fu, Z., Insogna, K. L., Karlsen, M. C., . . . Weaver, C. M. (2017). Dietary protein and bone health: A systematic review and meta-analysis from the National Osteoporosis Foundation. American Journal of Clinical Nutrition, 105, 1528 1543. Skov, A. R., Toubro, S., Ronn, B., Holm, L., & Astrup, A. (1999). Randomized trial on protein vs carbohydrate in ad libitum fat reduced diet for the treatment of obesity. International Journal of Obesity Related Metabolism Disorders, 23, 528 536. Stobaugh, H. C., Ryan, K. N., Kennedy, J. A., Grise, J. B., Crocker, A. H., Thakwalakwa, C., . . . Trehan, I. (2016). Including whey protein and whey permeate in ready-to-use supplementary food improves recovery rates in children with moderate acute malnutrition: A randomized, double-blind clinical trial. American Journal of Clinical Nutrition, 103, 926 933. Studenski, S., Perera, S., Patel, K., Rosano, C., Faulkner, K., Inzitari, M., . . . Guralnik, J. (2011). Gait speed and survival in older adults. Journal of the American Medical Association, 305, 50 58. Suarez Weise, A. (2014). Global Nutrition Targets 2025: Stunting policy brief. ,http://apps.who.int/ iris/bitstream/10665/149019/1/WHO_NMH_NHD_14.3_eng.pdf. Accessed 05.06.17. Sudfeld, C. R., McCoy, D. C., Danaei, G., Fink, G., Ezzati, M., Andrews, K. G., & Fawzi, W. W. (2015). Linear growth and child development in low- and middle-income countries: A metaanalysis. Pediatrics, 135, e1266 e1275. Suri, D. J., Moorthy, D., & Rosenberg, I. H. (2016). The role of dairy in effectiveness and cost of treatment of children with moderate acute malnutrition: A Narrative Review. Food & Nutrition Bulletin, 37, 176 185. Tahavorgar, A., Mohammadreza, V., Shidfar, F., Mahmoodreza, G., & Heydari, I. (2015). Beneficial effects of whey protein preloads on some cardiovascular diseases risk factors of overweight and obese men are stronger than soy protein preloads A randomized clinical trial. Journal of Nutrition & Intermediary Metabolism, 2, 69 75.

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N u t r i t i v e a n d T h e r a p e u t i c As pe c t s o f W h e y P r o t e i n s

Te Morenga, L., & Mann, J. (2012). The role of high-protein diets in body weight management and health. British Journal of Nutrition, 108(Suppl. 2), S130 S138. Tunick, M. H. (2008). Whey protein production and utilization: A brief history. In C. I. Onwulata, & P. J. Huth (Eds.), Whey processing, functionality and health benefits. Ames, IA: Wiley-Blackwell. Tzioumis, E., & Adair, L. S. (2014). Childhood dual burden of under- and overnutrition in lowand middle-income countries: A critical review. Food & Nutrition Bulletin, 35, 230 243. UN. (2014). United National Open Working Group of the General Assembly on Sustainable Development Goals. Sustainable Development Goals. ,https://sustainabledevelopment.un. org/content/documents/1579SDGs%20Proposal.pdf. Accessed 05.06.17. UNICEF, WHO & World Bank Group. (2017). Levels and trends in child malnutrition. In Joint Child Malnutrition Estimates. ,https://data.unicef.org/wp-content/uploads/2017/05/JME2017-brochure-1.pdf. Accessed 05.06.17. USDA. (2015). USDA commodity requirements. Ready-to-use nutrition food for use in international food assistance programs. ,https://www.fsa.usda.gov/assets/USDA-FSA-Public/usdafiles/CommOperations/pdf/ruf.pdf. Accessed 05.06.17. Vapattanawong, P., Aekplakorn, W., Rakchanyaban, U., Prasartkul, P., & Porapakkham, Y. (2010). Obesity and mortality among older Thais: A four year follow up study. BioMed Central Public Health, 10, Article 604. Volek, J. S., Volk, B. M., Gomez, A. L., Kunces, L. J., Kupchak, B. R., Freidenreich, D. J., . . . Kraemer, W. J. (2013). Whey protein supplementation during resistance training augments lean body mass. Journal of the American College of Nutrition, 32, 122 135. WHO. (2007). World Food Programme, United Nations System Standing Committee on Nutrition, & United Nations Children’s Fund. Community-based management of severe acute malnutrition. ,http://apps.who.int/iris/bitstream/10665/44295/1/9789280641479_eng.pdf? ua 5 1. Accessed 05.06.17. WHO. (2016a). Obesity and overweight fact sheet. ,http://www.who.int/mediacentre/factsheets/ fs311/en/. Accessed 05.06.17. WHO. (2016b). Global report on diabetes. ,http://apps.who.int/iris/bitstream/10665/204871/1/ 9789241565257_eng.pdf?ua 5 1. Accessed 05.06.17. WHO. (2017). Double burden of malnutrition. ,http://www.who.int/nutrition/double-burdenmalnutrition/en/. Accessed 05.06.17. WHO, FAO & UNU. (2007). Protein and amino acid requirements in human nutrition. World Health Organization Technical Report Series, 935, 1 265. Weber, J., & Callaghan, M. (2016). Optimizing ready-to-use therapeutic foods for protein quality, cost, and acceptability. Food & Nutrition Bulletin, 37(Suppl. 1), S37 S46. Westerterp-Plantenga, M. S., Lemmens, S. G., & Westerterp, K. R. (2012). Dietary protein Its role in satiety, energetics, weight loss and health. British Journal of Nutrition, 108(Suppl. 2), S105 S112. Westerterp-Plantenga, M. S., Nieuwenhuizen, A., Tome, D., Soenen, S., & Westerterp, K. R. (2009). Dietary protein, weight loss, and weight maintenance. Annual Reviews in Nutrition, 29, 21 41. Whitlock, G., Lewington, S., Sherliker, P., Clarke, R., Emberson, J., Halsey, J., . . . Peto, R. (2009). Prospective Studies Collaboration report: Body-mass index and cause-specific mortality in 900 000 adults: Collaborative analyses of 57 prospective studies. Lancet, 373, 1083 1096. Wolfe, R. R. (2012). The role of dietary protein in optimizing muscle mass, function and health outcomes in older individuals. British Journal of Nutrition, 108(Suppl. 2), S88 S93.

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CHAPTER 16

Sports and Exercise Supplements Ajmol Ali, Sung-Je Lee and Kay J. Rutherfurd-Markwick Massey University, Auckland, New Zealand

16.1

INTRODUCTION

Protein supplements are one of the most popular dietary supplements used by athletes, recreationally active adults, and soldiers striving to increase muscle mass, improve exercise recovery, and improve performance (McLellan, Pasiakos, & Lieberman, 2014). The global sports nutrition market was worth US$20.7 billion in 2012, and is expected to rise to US$37.7 billion by 2019 (Persistence Market Research, 2017) indicating a growing, financially lucrative business sector. Resistance and endurance exercise can induce a net negative protein balance and, if the body is lacking sufficient levels of amino acids (AAs), both preand postexercise, muscle wasting and delayed exercise recovery can occur (Wilborn et al., 2013). When protein intake is too low, maximal strength and lean mass gains cannot be achieved, even with maintenance of a positive nitrogen balance (Tarnopolsky et al., 1992). Therefore, many people, and in particular athletes, consume protein supplements to meet the additional demands from training and/or competition. A higher protein intake is advantageous for muscle and strength development and, therefore, bodybuilders and other strength athletes widely use protein supplements to achieve high protein intakes (up to three times the recommended daily allowance; Cribb, Williams, Carey, & Hayes, 2006). Whey protein (WP) is a high-quality protein which contains a higher concentration of essential amino acids (EAAs) than other protein sources, is rapidly digested, absorbed, and utilized (Bawa, 2007), and increases blood AA concentrations and stimulates muscle protein synthesis (MPS; for up to 2 hours) compared to an equivalent or larger dose of casein (Cribb et al., 2006). This chapter will initially examine key attributes which make WP a “high-quality” protein within sport and exercise settings. Resistance exercise (weight training, Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00017-5 © 2019 Elsevier Inc. All rights reserved.

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body building) and cardiorespiratory endurance training are markedly different and may benefit from WP supplementation for several distinct reasons. Furthermore, consuming WP before, during, or after exercise—with or without other bioactives—may confer distinct advantages to exercisers. Therefore, the effect of WP intake for different sports and activities, as well as timing of ingestion, and issues relating to dosage and coingestion with other ingredients, will also be examined. It is not only trained athletes who may benefit from WP supplementation, and so the potential benefits in different user groups will also be explored. Mechanisms by which WP ingestion may enhance sports performance—of which there are several—will be discussed, as well as potential contraindications of supplementation. Finally, a brief discussion on limitations of current research and suggestions for future study will be given.

16.2

DIFFERENT SOURCES OF PROTEIN

Adequate protein consumption and hence provision of AA is essential for maintaining life, playing an important role in growth and repair of the body and maintenance of good health. Food proteins derived from animal sources including meat, fish, poultry, eggs, and milk contain all EAA, and are thus referred to as high-biological-value proteins. In contrast, some proteins from plants, such as cereals and legumes, are deficient in one or more EAA, thus they are incomplete sources of protein. The most common proteins widely used for consumption before, during, and/ or after exercise or training by athletes are bovine milk proteins (Davison, 2014). Milk proteins have several desirable attributes: (1) favorable AA composition including high levels of EAA and branched-chain amino acids (BCAAs); (2) high biological value (BV); (3) readily digested and absorbed by the human body; and (4) abundant and readily available natural protein.

16.2.1

Dairy (Milk) Proteins: Caseins and Whey Proteins

Bovine milk contains 3.3% 3.5% protein which consists of the two main types of protein, namely caseins (80%) and WP (20%) (Haug et al., 2007). These two types of proteins can be separated from milk based on their solubility at pH 4.6 (Huppertz, Fox, & Kelly, 2004). Caseins are defined as proteins that become coagulated and precipitated from skim milk when the pH of milk is adjusted to pH 4.6 at 20 C. WP is the protein remaining soluble at pH 4.6 after the precipitation of caseins, and includes β-lactoglobulin (β-Lg), α-lactalbumin (α-La), bovine serum albumin (BSA), immunoglobulin (Ig), and lactoferrin (Lf) (Farrell et al., 2004; Morr & Ha, 1993). WP is often considered a superior protein relative to casein and soy protein for athletes, which may relate to the AA contained within the different proteins.

16.3 Types of Whey Protein Products

16.2.2

Soy Protein

Soy protein products are also commercially available as a vegetable protein source, such as soy protein concentrates (SPCs) and soy protein isolates (SPIs). These soy protein products, containing a mixture of soy proteins (α-, β-, and γ-conglycinins and glycinin and other globulins), have some food applications, such as water- and fat absorption, foaming, gelation, and binding properties (Fukushima, 2004). However, commercial SPC and SPI products are not highly water soluble but can be dispersed in water. Therefore, the use of SPC and SPI in some aqueous food systems, such as clear beverages and drinks (e.g., sports drinks), is relatively limited. In addition, compared to milk proteins, the quality of soy protein is not ideal because it is deficient in methionine and low in lysine (Friedman & Brandon, 2001). In addition, some strong off-flavors, such as grassy, beany, bitter, and astringent, often limit its use in food applications (Fukushima, 2004).

16.3

TYPES OF WHEY PROTEIN PRODUCTS

Different types of WP products are commercially available as ingredients in the form of dry powders, including sweet whey (or whey powder), acid whey, whey protein concentrates (WPCs), whey protein isolates (WPIs), and whey protein hydrolysates (WPHs) (see also Chapter 3: Manufacture of Whey Protein Products: Concentrates, Isolate, Whey Protein Fractions and Microparticulated and Chapter 15: Bioactive Peptides). These whey products differ in their chemical composition and physicochemical properties, depending on the methods and processing conditions used to produce them. Sweet whey is obtained from cheese production after coagulation and precipitation of caseins through the enzymatic action of rennet, whereas acid whey is produced from the acid-induced coagulation of caseins in cheesemaking, such as Cottage cheese, acid casein production (Tunick, 2009), and Greek-style yogurt production. WPC, WPI, and WPH are produced using a combination of several different membrane filtration techniques, such as microfiltration, ultrafiltration, diafiltration, electrodialysis, nanofiltration, and reverse osmosis (Tunick, 2009). WPC and WPI powders are made by removing lactose and minerals using ultrafiltration and diafiltration; therefore, WPC and WPI have higher protein and AA contents, and less lactose and salts than acid and sweet whey powders (Tamime, Robinson, & Michel, 2007). The levels of protein in WPC range from 35% to 80% but the most common protein levels in WPC are 35%, 55%, and 80% (Kilara & Vaghela, 2004). WPI contains a higher protein content, $ 90%, with almost all the lactose removed and a very low fat content (Jovanovic, Barac, & Macej, 2005;

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Tunick, 2009). Commercial WPC and WPI powders contain native (undenatured) WPs; therefore, their physicochemical (functional) properties are largely retained, making them readily soluble in water over a wide pH range (Wong, Camirand, & Pavlath, 1996). Other benefits of WPC and WPI as protein ingredients include low caloric content due to their low levels of fat and carbohydrate (CHO) (e.g., lactose), their low level of salts (e.g., sodium) as well as their desirable sensory attributes (e.g., sweet aromatic and milky flavors) (Tunick, 2009). When compared to WPC, the WPI products are more desirable to use for individuals with lactose intolerance as they have a more purified high protein content with little or no lactose. WPC and WPI are commonly used in sports drinks, due to their high protein and favorable AA contents. In addition, an aqueous solution of WPI is transparent due to its high water solubility and protein purity and therefore WPI is highly suitable for use in drinks and beverages. WPH are produced by cleaving WPs into shorter chain peptides using enzymes (see also Chapter 15: Bioactive Peptides). The producer can control the degree of fractionation of a given protein, hence the fractions can range from whole proteins, to peptides, to individual AA. WPH are more easily digested and absorbed than the parent product, and are thus suitable for use in infant formulas, and sports and medical nutrition products (Boland, Golding, & Singh, 2014).

16.3.1

Amino Acid Composition of Whey Proteins

Following digestion, milk proteins perform many physiological functions (Kanekanian, 2014) such as providing bioactive peptides and EAA which are necessary for the growth and maintenance of the body. WP is a high-quality protein due to high EAA content, rapid digestibility, and high bioavailability (Pereira, 2014), which means that they are metabolized more efficiently in the human body (Pordesimo & Onwulata, 2009). Protein quality can be assessed in various ways, including BV, protein digestibility (PD), net protein utilization (NPU), protein efficiency ratio (PER), and protein digestibility corrected amino acid score (PDCAAS) (Walsh & FitzGerald, 2004). WPs have higher BV, PD, NPU, and PER values of 104, 100, 92, and 3.6, respectively, compared to whole milk (91, 95, 86, and 3.1) and caseins (77, 100, 76, and 2.9) (European Dairy Association, 1997). WP is digested more rapidly than caseins, resulting in faster absorption and release of AA into the postprandial plasma (Boirie et al., 1997). This is because WP is soluble whereas caseins are clotted in the stomach, thus slowing gastric emptying resulting in slow absorption and release of AA into the plasma (Boirie et al., 1997). This implies that following consumption WP render a high concentration of AA in the plasma in a shorter time than caseins.

16.3 Types of Whey Protein Products

Dietary proteins high in EAA, BCAA and, particularly, leucine (Leu) have been shown to stimulate MPS and increase weight loss and body fat loss while decreasing the levels of triglycerides in the plasma (Etzel, 2004; Wester, Lobley, Birnie, & Lomax, 2000). WP contain all the EAA and have a relatively high ratio of BCAA (e.g., leucine, isoleucine, and valine) (Haug, Høstmark, & Harstad, 2007; Onwulata, 2009; Paul, 2009). In addition to EAA and BCAA triggering and promoting MPS, these AA also suppress protein catabolism, serving as substrates for gluconeogenesis (Haug et al., 2007). The levels of BCAA and leucine in WPs are 26 and 14 g per 100 g protein, respectively, which are higher than in any other food proteins (e.g., muscle: 18 and 8 g; soy: 18 and 8 g; wheat: 15 and 7 g) (Layman, 2003). Compared to theoretical “average” values calculated for other proteins, β-Lg contains 17% more EAA, 33.5% more BCAA, and 74% more Leu (Mehra & O’Kennedy, 2009). Interestingly, the ratio of all the EAA in milk proteins (caseins and WP) is similar to their ratio in the skeletal muscle of the human body (Kvistgaard, Schroder, Jensen, Setarehnejad, & Kanekanian, 2014). The high level of BCAA found in WP, which is important for energy provision as well as protein synthesis, has made them a commonly utilized source of protein for sports snacks and drinks (Tawa & Goldberg, 1992). WP is also rich in sulfurcontaining AAs, such as cysteine (Cys) and methionine (Met), making it the protein of choice for athletes aiming to maintain and build body mass for enhanced sports performance (Pordesimo & Onwulata, 2009).

16.3.2

Meals Versus Supplements

If adequate micro- and macronutrients are eaten by athletes in their daily meals, then this should negate the need for supplements (Rodriguez, Di Marco, & Langley, 2009). Thus, the rationale for using protein supplements is that they will enhance net protein synthesis above and beyond eating meals (Paddon-Jones, Sheffield-Moore, Aarsland, Wolfe, & Ferrando, 2005). To test this hypothesis, Paddon-Jones et al. (2005) gave a between-meal supplement containing 30 g CHO and 15 g EAA (mix of casein and WP) to recreationally active men; the control group only consumed the three standardized meals without the supplement. Over the 16-hour study period the anabolic stimulus was greater in the supplement group than with meals alone; phenylalanine balance was also greater in the supplement group than the meal group, even though both the meal and supplement contained 15 g EAA. The authors postulated that the slower digestion of meals enhanced the efficiency of splanchnic uptake of AA relative to the supplement. In contrast, the rapid digestion of the supplement may have decreased the efficiency with which the splanchnic tissues (which are largely responsible for whole-body protein synthesis) could extract AA, leaving a greater proportion of EAA available to the peripheral muscle tissue (Paddon-Jones et al., 2005).

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16.3.3

Other Food Issues

Proteins derived from different sources are widely added into foods to not only improve the nutritional quality but also to achieve desired sensory characteristics (e.g., texture, mouthfeel, appearance, taste) and functional properties (e.g., solubility, thickening, foaming, emulsifying, gelling) (Luyten, Vereijken, & Buecking, 2004). The multiple functional properties of WP (viscosity, gelation, emulsifying, and foaming), including water solubility at high concentrations over a wide pH range, make it desirable for use in drinks and beverages. Such beverages include nutritional beverages, meal replacement beverages, sports beverages, and protein-fortified citrus beverages which require the dissolution of protein in aqueous solution (Kilara & Vaghela, 2004). In addition, WP can also be utilized in other types of products including yogurt drinks, ice cream, low-fat frozen desserts, yogurt, sour cream, coffee whiteners, creamy salad dressings, soups, gravies, and infant formula, due to their properties as an emulsifying agent to form and stabilize oil-in-water emulsions (Kilara & Vaghela, 2004). However, there are many factors that should be taken into account when WP is used as an ingredient, including environmental (e.g., pH, temperature, salts, ionic strength) and processing (e.g., temperature, pressure) conditions, as the functional behavior of WP is affected by such factors as well as when WP is mixed with other ingredients. It is expected that the demand for the use of WP in foods and beverages will continue to grow as it can provide a high nutritive value as well as many functional properties required in some food systems (Kilara & Vaghela, 2004).

16.4 EFFECT OF WHEY PROTEIN ON EXERCISE PERFORMANCE 16.4.1

Resistance Exercise

Individuals undertake resistance training to improve muscle strength, endurance, and power, and to increase muscle mass. Although resistance training may be viewed as activities for athletes and body builders, this type of training can also be beneficial for rehabilitation, recovery from injury, for attenuating age-related declines in muscle function and muscle mass, and for weight management. In a seminal study, Biolo, Maggi, Williams, Tipton, and Wolfe (1995) examined muscle protein balance and AA transport in untrained men at rest and following resistance exercise. Although resistance exercise increased muscle protein breakdown (MPB) by 50%, it increased MPS by 100%, thus resulting in improved net muscle protein balance (relative to resting trial). However,

16.4 Effect of Whey Protein on Exercise Performance

they concluded that exercise needs to interact with protein ingestion to promote muscle anabolism. Therefore, ingesting a high-quality protein like WP may be beneficial for enhancing strength performance and muscle mass.

16.4.1.1 Strength Performance and Muscle Mass Many studies show positive effects of WP intake and resistance exercise on strength performance, muscle mass, and other metabolic changes in trained and untrained humans and animals (Table 16.1). Furthermore, WP intake also leads to superior gains in muscle strength relative to other proteins. Cribb et al. (2006) found that 1.5 g/kg body weight per day hydrolyzed WPI intake, provided in smaller servings throughout the day during a 10-week resistance training programme, showed significantly greater gains in muscle strength and muscle mass than casein ingestion. However, not all studies show improvements in strength performance following WP supplementation. Erskine, Fletcher, Hanson, and Folland (2012) pair-matched untrained males into PRO (protein) 1 CHO (40 g WP 1 6.7 g lactose) or placebo (PL; 6.8 g lactose) groups and all subjects completed 12 weeks (3 times/week) upper body resistance training. In both groups maximal elbow flexor strength increased by B41% and isometric strength by B13%, with no differences between trials. Furthermore, there were no differences in neural adaptations or muscle morphology between WP and PL groups. The sensitivity of MPS to protein persists for up to 24 hours after a bout of resistance exercise, thus prolonged sensitivity to MPS following each training session may have enabled the protein content within the regular meals of the PL group to stimulate similar MPS to the WP group (Erskine et al., 2012). Since a relatively small muscle group was trained, the protein intake of both WP and PL groups may have been sufficient to maintain optimal MPS, and so protein supplementation may only be necessary for wholebody resistance training. Joy et al. (2013) reported comparable effects of ingestion of 48 g WPI or 48 g rice protein isolate on strength performance following resistance training (3 times/week for 8 weeks) in trained male athletes. Their findings indicate differences in composition of protein are not relevant when protein is consumed in high doses throughout the resistance training period. Weisgarber et al. (2012) also showed no improvement in strength performance following 0.3 g/kg WP ingestion (relative to 0.3 g/kg CHO) after 8 weeks of resistance training in untrained men. However, morphological adaptations typically commence several weeks after the commencement of training (Narici et al., 1996), and so the effects may take longer in previously untrained men. Issues relating to dose of WP and training status of individuals will be covered in more detail later in this chapter. Nevertheless, the results of several meta-analyses support significantly greater improvements in strength and muscle mass with WP ingestion compared to

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Table 16.1 Summary of Publications on Resistance Exercise/Training and Whey Protein Supplementation Training Programme

Reference

Participants

Supplementation

Type and Intensity

30 males (26 years), military personnel (includes resistance- and endurance-trained athletes)

Randomly assigned to consume 19.7 g WP 1 6.2 g leucine powder or PL (0.0 g protein) daily for 8 weeks, one packet 30 min before and another packet 30 min after exercise

Pre- and postsupplement training sessions. Max bench press, chin ups, sit-ups and push-ups in 1 min with 3 min break in between exercises. 5 min rest then 3 mile timed run; asked to sprint for last 40 yards. 10 min break before cognitive tests 8 12 reps/sets for several exercises

Frequency (Times /Week)

Duration (Weeks)

Key Performance and Metabolic Changes

Trained Athletes Walker et al. (2010)

Colker, Swain, Fabrucini, Shi, and Kaiman (2000)

16 healthy athletic males

WP (40 g/day) or WP combination (40 g/ day whey, 3 g/day BCAA and 5 g/day glutamine)

Minimum 3

8

G

G G G G

3

10

G G G G

Wilborn et al. (2013)

16 female basketball players (20 years, 1.55 m, 67 kg, 26% BF)

WP 5 24 g CP 5 24 g Supplement given 30 min before and after each training session for 8 weeks

Day 1 5 vertical jump, broad jump, agility run/shuttle Day 2 5 bench press, leg press, maximal repetitions Had periodized anaerobic resistance training programme

4

8

G

G

G

3.9% improvement in bench press with WP (55% of WP subjects showed 5% improvement) 12.8% improvement in push-ups with WP FFM and LBM increased with WP No difference in cognitive tests, chin ups, sit ups However, training programmes were not standardized

Higher body mass with WPComb compared to WP after 10 weeks In first 5 weeks, WPComb showed higher FFM Improved bench press repetitions in WPComb Trend for improved leg press repetitions in WPComb Improvements in leg press, bench press, vertical jump, and broad jump in both groups; no difference between groups Positive net AA balance with both supplements through expression of leucine and phenylalanine in blood Improvements in body fat, lean mass, and fat mass in both groups; no difference between groups

Tipton et al. (2007)

17 healthy trained young males and females (26.5 years, 73 kg, 170 cm)

300 mL solution containing 20 g intact WP either immediately before (PRE) or 1 h following (post) exercise

10 sets of 8 repetitions of leg extension exercises

G

G

G

Arterial AA concentration was elevated by 50% and net AA balance switched from negative to positive following WP ingestion at either time No difference between PRE and POST trials AA uptake from beginning of exercise or from ingestion of each drink However, no placebo group was used

Moderately Trained/Recreational Athletes Joy et al. (2013)

24 healthy males (21.3 years, 76 kg, 177 cm)

48 g WPI (5.5 g leucine) or 48 g rice protein isolate (3.8 g leucine) immediately following exercise 1 48 h postexercisefollowed diet (25% protein, 50% CHO, 25% fat)

3 sets of leg press, bench press, pushups, pull-ups, bentover rows, barbell curls

3

8

G

G G G

G

Yang et al. (2012)

Cribb et al. (2006)

37 moderatelyactive elderly men (70 years, 173 cm, 80 kg)

13 male recreational body builders

Ingested either 0, 10, 20 or 40 g WPI dissolved in 400 mL water, as beverage immediately after exercise

3 sets of unilateral knee extension with predetermined load based on 10RM shown to increase rates of MPS

Supplemented normal diet with WPH or CP (1.5 g/kg body weight per day)—divided into smaller servings with breakfast, lunch,

Supervised RT programme—high intensity workouts, mostly compound exercises with free weights. Strength

G

G

10

G

G G

Increased muscle thickness of vastus intermedius and vastus lateralis from baseline to 8 weeks Increased LBM with in both trials Bench and leg press strength increased in both trials No nonsupplemented control group, cannot conclude how beneficial protein supplementation is relative to resistance training High dose of protein providing 5.5 and 3.8 g leucine—which is higher than 2.0 and 3.5 g leucine proposed to maximally stimulate MPS MPS increased above basal, fasting values by B65 and 90% for W20 and W40, but not with lower doses of whey W20 and W40 ingestion postexercise increased MPS above W0 and W10 exercised values, and W40 was greater than W20 Greater improvements in strength in each assessment with WPH compared to CP Greater gain in lean mass in WPH than CP Reduced fat mass in WPH compared to CP

Continued

Table 16.1 Summary of Publications on Resistance Exercise/Training and Whey Protein Supplementation Continued Training Programme

Reference

Cooke et al. (2011)

Sheikholeslami and Ahmadi (2012) WP 1 CHO

Rindom et al. (2016)

Participants

Supplementation

Type and Intensity

10 healthy recreationally active males (20 years, 174 cm, 78.5 kg)

directly after training and with dinner 10 g WP (containing 5.25 g EAA) or PL (10 g maltodextrin) given 30 min before each session

assessed by 1RM in 3 exercises 2 separate bouts of RT; each session involving only 1 leg and each separated by 2 weeks; 4 sets of 8 10 repetitions at 75 80% 1RM on the angled leg press and knee extension exercises 60% 70% 1RM each session. Squat, bench press, lat pull down, standing EZcurls, cable triceps extensions and back press

30 healthy males (23 years, 25 30 BMI)

12 healthy men (24 years, 79 kg, 183 cm) recreationally trained, 2 3 times/week

35 g either WP (30 g WPI 1 5 g orange juice powder) or PL (30 g starch 1 5 g orange juice powder) 3 3 /day (immediately after exercise, lunch 1 dinner). Split into 3 groups: 1) RT 1 WP, 2) RT 1 PL, 3) control WP or CP in first trial then switched to other type in second trial. Given food for the entire week 1 1.4 g protein/ kg body weight with 25 g WP or CP added 5 2 g protein total

Frequency (Times /Week)

Duration (Weeks)

Key Performance and Metabolic Changes G

1

2

G

G G G

3

6

G

G

G

2 identical training periods, each training period 5 4 sessions HI-resist exercise during 5 days. Performance was evaluated 3, 24 1 48 h after training periods. Prior to 1 at 48 h after training periods performance time to exhaustion was evaluated

1

G

G

No change in plasma glutamine levels in either group Serum insulin higher at 30 min postingestion and 15 and 120 min postexercise; but no supplement 3 test interaction No interaction effect for Akt/mTOR signaling intermediates Higher IRS, mTOR and p70S6K at 15 min postexercise with WP Decrease in 4E-BP1 with WP 15 min postexercise Total antioxidant capacity, cholesterol and HDL varied significantly in RT 1 WP group compared to pretest. Significant changes in both groups for glutathione, Vitamin C, LDL and triglyceride levels. In the posttest, TAC, glutathione and HDL levels were higher in RT 1 WP compared to control No regain in exercise performance and attenuated muscle soreness with WP compared to CP No effect of supplement with different types of protein on muscle strength, anaerobic power or aerobic capacity

Staples et al. (2011) WP 1 CHO

9 young recreational active males (23 years, 80 kg, 24 BMI)

WP (25 g WPI) or PRO 1 CHO (25 g WPI 1 50 g maltodextrin after exercise

Unilateral leg extension on each leg. 4 sets of 8RM to 12RM failure

WPC (40 g/day) or an isocaloric control powder. Consumed 2 serves/day, 1 at breakfast and immediately after RT or after evening meal if no RT that day

Supervised high intensity RT. Leg press, seated row, leg extension, chest press, leg curl at 80% of their 1RM

15 g WPI or nonenergetic PL before and after exercise

5 3 10 leg press 2 5 sets, 6 20 reps, 40% 85% 1RM for 12 14 exercises (exercise including: leg press, bench press, knee extension)

1

2

G

G

Areas under the glucose and insulin curves were 17.5-fold and 5-fold greater for PRO 1 CHO than for PRO Phosphorylation of Akt was greater in PRO 1 CHO than PRO

Untrained Individuals Chale et al. (2012)

Hulmi et al. (2009)

8 mobility-limited adults (70 85 years)

31 healthy males (25 years, 182 cm, 75 kg, 17% BF)— untrained

3

24

G

G

G

2

21

G G G

G

Erskine et al. (2012) WP 1 CHO

33 untrained young men (23 years, 1.76 cm, 75.2 kg)

Weisgarber et al. (2012)

17 untrained young men (9) and women (8), 24 years, 86 kg, 170 cm

250 mL beverage. 20 g WP 1 6.7 g lactose or PL: (6.8 g lactose) consumed immediately before and after exercise WP (0.3 g/kg body weight containing 0.15 g/kg body weight EAA) or isocaloric PL (0.2 g/ kg body weight corn starch maltodextrin 1 0.1 g/ kg body weight sucrose) before,

4 6 sets of elbow flexion

3

12

G

G

3 sets of 6 10 repetitions of 9 whole-body exercises to fatigue with 2 min rest between sets for each exercise (90 min training sessions)

4

8

G

G

Muscle strength and stair climb performance increased 16% 50% in both groups. Lean mass increased by 1.3% in WPC and 0.6% in control, no sig difference between groups. Muscle CSA was increased 4.6% WPC vs 2.9% control, no sig difference between groups. 24.3% improvement in isometric leg extension with WP WP increased muscle CSA in vastus lateralis Increase in cell-cycle kinase cdk2 mRNA expression in the vastus lateralis muscle (molecular signals of cell growth) WP prevented 1 h post-RE decrease in myostatin and myogenin mRNA expressions No difference in maximum isometric voluntary force or 1RM strength between groups No difference in muscle CSA between groups 1RM strength in the chest press increased in both groups without any between-group difference Increase in muscle mass in both groups; no difference between groups

Continued

Table 16.1 Summary of Publications on Resistance Exercise/Training and Whey Protein Supplementation Continued Training Programme

Reference

Reidy et al. (2013)

Cooke et al. (2010)

Participants

Supplementation

19 healthy young subjects. (17 M, 2 F, 24 years, 25 BMI, 24% FFM). Active, but did not engage in regular RT

during and after exercise 300 mL protein blend (PB; 19.3 g PRO, 50% from sodium caseinate, 25% WPI, 2.5% SPI) or WP (17.7 g) 1 h posthigh intensity leg RE bout

17 untrained males (23 years, 180 cm, 80 kg)

Type and Intensity

8 sets of 10 repetitions at 55% (set 1), 60% (set 2), 65% (set 3), 70% (sets 4 8) of participants previously determined 1RM, with 3 min rest between sets

1.5 g/kg body weight per day (B30 g) of either WPH or CHO immediately after exercise, with breakfast, lunch, in the afternoon and after evening meal for 14 days

The damage session 5 unilateral eccentric contraction-based resistance exercise session consisting of 4 sets 10 repetitions at 120% max voluntary contraction of leg press, leg extension and leg flexion

WP 5 150 g/kg body weight CP 5 140 g/kg body weight Split into 4 groups: Group

Exercise trial: jumps out of water with weights attached to chest as resistancemimicking weight-

Frequency (Times /Week)

Duration (Weeks)

Key Performance and Metabolic Changes

2

1

G

G

G

G

G

G

Higher and earlier (BCAA) after WP; PB created lower initial rise in (BCAA) but sustained elevated levels of (AA) later into recovery Postexercise fractional synthetic rate increased in both groups during early period, but remained elevated only in PB group during late period mTORC1 signaling increased, but no increase in S6K1 phosphorylation was seen in WP group 5 h postexercise Isometric knee extension strength was higher following WPH 3 and 7 days into recovery from exerciseinduced muscle damage compared to CHO Greater strength after 7-day recovery following WPH compared to CHO Plasma LDH levels tended to be lower in WPH during recovery

Animals (Untrained) Haraguchi, Silva, Neves, Dos Santos, and Pedrosa (2011)

32 male rats

5

8

G

G

WP treatment increased total liver glutathione levels—no difference among WP groups WP and exercise increased muscle protein carbonyl content and

1 5 WP 1 sedentary, Group 2 5 WP 1 RE, Group 3 5 CP 1 sedentary, Group 4 5 CP 1 RE

lifting training; 4 sets of 10 jumps/day G

G

Haraguchi et al. (2014)

32 male rats

Fed standard diet modified with WP or control protein (casein) WP 5 150 g/kg body weight CP 5 140 g/kg body weight Split into 4 groups: Group 1 5 WP 1 sedentary, Group 2 5 WP 1 RE, Group 3 5 CP 1 sedentary, Group 4 5 CP 1 RE

Exercise: jumps out of water with weights attached to chest as resistance-mimicking weight-lifting training; 4 sets of 10 jumps/ day

5

8

G

G

G

maintained low levels of TBARS in exercised and sedentary rats WP and exercise generated higher body and muscle weight than exercise without WP Increased muscle glycogen content in exercise rats fed WP (relative to other trials) WP exercised rats exhibited higher body and muscle weight gain compared with control-exercise rats mTOR expression was reduced by exercise but increased when WP was consumed as a dietary protein MAFbx was reduced only by WP ingestion independent of exercise

Abbrevations: AA, amino acids; CHO, carbohydrate; CP, casein protein; CSA, muscle cross sectional area; EAA, essential amino acids; FFM, fat-free mass; LBM, lean body mass; MPS, muscle protein synthesis; PB, protein blend; PL, placebo; PRO, protein; RE, resistance exercise; RM, repetition maximum; RT, resistance training; SPI, soy protein isolate; WP, whey protein; WPC, whey protein concentrate; WPH, whey protein hydrolysate; WPI, whey protein isolate.

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control supplements when combined with prolonged ($4 weeks) resistancetraining interventions (Cermak, Res, Groot, De Saris, & Van Loon, 2012; Miller, Alexander, & Perez, 2014; Naclerio & Larumbe-Zabala, 2016). WP is considered a “fast” digesting protein whereas casein is considered a “slow” digesting protein. Ingesting a blend of proteins with different digestion rates has been suggested to enhance skeletal MPS, prolong AA delivery to muscles and promote superior gains in muscle mass compared to ingesting a single protein source or a blend with similar digestion rates (Paul, 2009). Reidy et al. (2014) had 16 young adults ingest either WP (B17 g WPI containing 2 g leucine, 0.6 g phenylalanine, 1 g valine, and 9 g EAA) or a protein blend (50% casein, 25% WPI, 25% SPI composed of B20 g total protein containing 2 g leucine, 1 g phenylalanine, 1 g valine, and 9 g EAA) 1 hour after resistance exercise. Phenylalanine transport into the muscle, myofibrillar protein synthesis and mRNA expression of AA transporters increased similarly in both groups. However, the protein blend enhanced AA transporter gene expression and also prolonged the positive phenylalanine balance compared to WP; nevertheless, WP had a greater initial anabolic stimulus. Therefore, it appears that the blend stimulates an initial anabolic stimulus from WP (with its high leucine content), but prolongs anabolism longer from the slower-releasing casein and soy proteins. The increase in blood leucine concentration triggers leucine oxidation and thus slows delivery of leucine and other AAs to muscle (Paul, 2009). Furthermore, ingestion of 10 g EAA with high (3.5 g) leucine stimulated skeletal muscle AA transporter expression more than with low (1.8 g) leucine, suggesting leucine content may be a key regulator in AA transporter protein expression (Reidy et al., 2014) (see also Section 16.9.5).

16.4.1.2 Recovery From Resistance Exercise Resistance training can lead to muscle damage, resulting in greater protein degradation than synthesis, and subsequently to increased muscle soreness and impaired muscle function (Allen, Whitehead, & Yeung, 2005). Therefore, WP intake may help to reduce MPB and promote MPS, thus helping the athlete recover for subsequent exercise sessions. Cooke, Rybalka, Stathis, Cribb, and Hayes (2010) randomized 17 untrained males into WPH (1.5 g/kg per day) or isocaloric CHO supplement groups. Participants underwent a muscle-damaging bout of resistance exercise followed by 14 days of supplementation with WPH or CHO. WPH improved isometric muscle strength on days 3 and 7 following the damaging exercise. A similar trend was also observed in isokinetic strength, with lower lactate dehydrogenase (LDH; a marker of muscle damage) levels in the WPH group. The authors speculated

16.4 Effect of Whey Protein on Exercise Performance

that an accumulation of the benefits with continual training over time could benefit the athlete, even if only providing the smallest advantage (Cooke et al., 2010). However, athletes who train frequently are less likely to be affected by muscle damage and muscle soreness as they develop protective responses to damaging exercise (“repeated bout effect”; Nosaka, Sakamoto, Newton, & Sacco, 2001). Rindom et al. (2016) also reported that strength, anaerobic, and aerobic performance were markedly reduced following high-intensity resistance training and high-quality WP supplementation did not produce noteworthy improvements in the regain of exercise performance compared with low-quality collagen protein. These results suggest additional protein supplementation is not necessary to enhance recovery when the body is in a state of positive nitrogen balance from dietary protein intake.

16.4.1.3 Body Composition and Weight Loss Weight loss regimes that only focus on body weight reduction may also reduce lean mass; the resultant decrease in basal metabolic rate may make it more difficult to lose weight and/or result in weight regain (WesterterpPlantenga, Nieuwenhuizen, Tome, Soenen, & Westerterp, 2009). However, increasing muscle mass raises resting energy expenditure and therefore may help with weight loss. WP intake, in conjunction with resistance exercise, appears to have a greater capability to augment body composition changes during exercise than casein intake (Cribb et al., 2006). Furthermore, decreases in body weight, BMI, body fat, and waist circumference have also been observed when WP was used as a dietary replacement compared to CHO (Miller et al., 2014). However, there was no effect of resistance exercise combined with WP supplementation on percent body fat and lean body mass (LBM) in an 8-week trial (Weisgarber, Candow, & Vogt, 2012), or body fat percentage in a 6-week trial (Burke et al., 2001). Therefore, resistance training combined with WP supplementation should be undertaken over a minimum of 10 12 weeks (with 3 5 sessions per week) to improve body composition (Miller et al., 2014). The addition of supplements to a balanced diet may increase energy intake, thus being counterproductive for weight management. However, participants given high supplement doses (1.5 g/kg per day) only increased their daily protein intake by a small amount, possibly due to individuals replacing a large portion of their normal daily protein intake with the supplement that contained a rich source of EAA; over a period of weeks this may have resulted in improved body composition during intense resistance training (Cribb et al., 2006).

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16.4.2

Endurance Exercise

Cardiorespiratory endurance exercise (as opposed to muscular endurance) primarily relies on the aerobic system to provide energy for performance of the activity. From a practical point of view exercising for more than a few minutes (such as an 800 m run) to several hours (such as a marathon) would constitute endurance exercise. The predominant fuels for endurance exercise are CHO and fats but the oxidation of AA (especially BCAA) can contribute between 5% (Tarnopolsky, 2004) and 20% (Rennie, Bohe, Smith, Wackerhage, & Greenhaff, 2006) toward total energy metabolism. Therefore, prolonged exercise can create changes to whole-body and skeletal muscle protein turnover and changes to the AA pool (Rennie et al., 2006; Tarnopolsky, 2004). Furthermore, endurance exercise can cause remodeling of the structure of skeletal muscle, and result in new mitochondria, increased activity of oxidative enzymes and various transport proteins (Hansen, Bangsbo, Jensen, Bibby, & Madsen, 2015). Repetitive eccentric muscle actions (where muscle is in tension but actively lengthens, e.g., running) can also damage skeletal muscle and so delay the recovery process; this is especially relevant for athletes who train regularly (up to three sessions per day). Therefore, WP supplementation may be beneficial for those wishing to improve protein oxidation during exercise and/or aid recovery from prolonged exercise through tissue repair.

16.4.2.1 Endurance Capacity and Performance Most studies exploring the efficacy of WP ingestion for endurance performance have also included CHO (Section 16.8.1.2); here we examine studies specifically investigating WP ingestion. Chen, Huang, Chiu, Chang, and Huang (2014) investigated the independent and additive effects of ingesting 4.1 g/kg WP and endurance exercise (longterm intensive aerobic swimming) in mice. After 6 weeks of training, time-toexhaustion (TTE) was significantly higher in the exercise 1 WP group relative to controls. Aoi et al. (2011) examined the effect of a WPH diet relative to an energy-matched casein diet on mice who underwent training 5 times/ week for 4 weeks; after the intervention the mice performed a run-toexhaustion test. The WPH diet improved performance by 58% relative to the casein diet. However, greater improvements in endurance performance have been observed in studies using rodents, as animals have better compliance, more variables can be controlled, and, following euthanasia more complete metabolic and anthropometric analysis can be undertaken (Cooke et al., 2011). Hansen et al. (2015) explored the effects of ingesting WPH before and WPH 1 CHO after each exercise session (compared with isocaloric CHO ingestion) on performance in elite orienteers at the end of a strenuous

16.4 Effect of Whey Protein on Exercise Performance

1-week training camp. One group ingested a WPH drink 10 minutes before and a WPH 1 CHO drink 15 minutes after exercise (over the 13 training sessions) whereas the control group consumed an electrolyte-matched CHO drink before and after exercise. The WPH group improved 4-km time trial (TT) performance by 1.9% with no change in performance in the CHO-only group. However, whether the beneficial effect on performance was a result of the pre- (WPH) and/or the postexercise (WPH 1 CHO) protein supplements alone or in synergy cannot be determined. Not all studies have shown improvements in endurance performance with WP supplementation. Schroer, Saunders, Baur, Womack, and Luden (2014) examined the effect of consuming WPH (45 g/h) relative to a noncaloric taste-matched placebo in 10 endurance-trained cyclists. The drinks were provided at regular intervals during 120 minutes of constant load cycling exercise and also during a subsequent 30-km TT. Based on magnitude-based inferential statistics, the authors suggested that WPH may have impaired performance by 2.1%. However, some of the participants in the WPH trial experienced “serious nausea symptoms” during exercise, which may have impacted on their performance. The authors suggested that WPH may need to be consumed with CHO to provide ergogenic benefits. Discrepancies in performance data between studies may relate to the way performance was measured. Some studies used tests of exercise capacity, i.e., exercise at a constant intensity until volitional fatigue (also known as TTE tests); these tests typically show much larger differences in performance between experimental and placebo groups. However, TT, completing a set distance as fast as possible or completing as much work as possible within a set time, appear to have lower variation (CV ,5%) than TTE (CV .25%) protocols and may provide a more valid representation of sporting events (Currell & Jeukendrup, 2008). Since the difference between finishing first and second in many sporting events is ,1% it is important to be able to detect small differences in performance (sensitivity of measurement).

16.4.2.2 Muscle Protein Synthesis Following Endurance Exercise Endurance training does not typically lead to changes in muscle size. Chen et al. (2014) showed that WP supplementation in mice improved strength with no concomitant muscle hypertrophy. Nevertheless, changes in MPS are relevant for endurance athletes to enable tissue repair and remodeling of muscle (Burd, Tang, Moore, & Phillips, 2009). Carraro et al. (1990) showed that low-intensity exercise (treadmill walking) alone can promote MPS, thus it is tempting to speculate that the ingestion of WP will increase MPS further. The results of a systematic review suggest the use of protein supplements creates an enhanced anabolic state within muscle after endurance exercise, which may beneficially affect the recovery of muscle function (McLellan et al., 2014).

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Morifuji et al. (2012) had eight trained men complete a 70-minute cycle test, followed by TT after a 2-hour recovery session, on three different occasions. The cyclists ingested either (1) CHO (17.5 g maltodextrin); (2) CHO 1 low WPH (17.5 1 3 g); or (3) CHO 1 high WPH (17.5 1 8 g) immediately after exercise and 30, 60, 90, and 120 minutes later. Plasma concentrations of AA during the recovery period were higher following ingestion of a high dose of WPH, whereas they only marginally increased with a lower dose of WPH and decreased in the CHO trial. These results indicate that the amount of protein ingested in the postexercise period affects the concentrations of plasma AA (Morifuji et al., 2012). However, it is worth noting that the drinks were not isocaloric and so the increased MPS could be due to increased energy supply. Nevertheless, using an isocaloric CHO control, Rustad et al. (2016) showed that ingestion of WP 1 CHO (0.8 g/kg per hour CHO 1 0.4 g/kg per hour WP) resulted in a positive nitrogen balance (from both reduced MPB and increased MPS) during recovery after exhaustive cycling exercise, an anabolic state. However, CHO (1.2 g/kg per hour) ingestion alone and PL resulted in a negative nitrogen balance, a catabolic state (Rustad et al., 2016).

16.4.3

Multiple-Sprint Sports

Multiple-sprint sports combine elements of both resistance and endurance exercise, i.e., prolonged ( . 60 minutes) exercise punctuated by short periods of power activities like sprinting, jumping, or tackling. Therefore, team sport athletes may benefit from WP supplementation in several ways including improvements in anaerobic power, increasing muscle mass, provision of fuel during exercise, muscle repair, and/or muscle glycogen recovery following exercise. Alghannam (2011) examined the effect of WP 1 CHO (2.1% WP 1 4.8% CHO) intake on various performance indictors in six soccer players during and following a soccer-specific running test (relative to isocaloric CHO or no-energy PL). The WP 1 CHO supplementation improved running capacity following exercise, compared to isocaloric CHO alone, possibly due to higher blood glucose at the point of fatigue, and an increase in extramuscular CHO oxidation. Glycogen levels were restored twice as fast in the initial 40 minutes of recovery compared to the CHO trial. Furthermore, ratings of perceived exertion (RPE) were lower in WP 1 CHO trial compared to CHO alone or PL, possibly due to the increased BCAA intake from WP (Alghannam, 2011) and thus likely responsible for the enhancement in performance in the WP 1 CHO trial. WPC and WPH intake immediately before and after high-intensity training has also been shown to increase muscle mass in elite soccer players despite endurance exercise not being the most appropriate exercise for stimulating

16.5 Timing of Whey Protein Ingestion

hypertrophy (Lollo et al., 2014). However, increasing body weight should not be a main aim for team sport players due to the downstream effects of carrying additional mass, e.g., reduced maximal oxygen uptake (VO2max) and/or reduced speed. Nevertheless, even though WP supplementation increased LBM in elite hockey players following 8 weeks of training, sprint performance was significantly faster following WP supplementation (Hofman, Smeets, Verlaan, Lugt, & Verstappen, 2002). Again, not all studies show improvements in performance with WP supplementation. Naclerio, Larumbe-Zabala, Cooper, Jimenez, and Goss-Sampson (2014) observed no effect of ingesting WP 1 CHO relative to a CHO supplement before, during, or immediately after exercise on repeated sprint ability in recreationally trained males. However, the amount of CHO ingested (53 g in CHO and 46 g in WP 1 CHO) during the 90-minute exercise may have been too low for optimal CHO delivery rates during exercise. Therefore, Naclerio et al. (2014) concluded that the most important factor in attenuating performance loss during intermittent exercise is the energy content of the supplement rather than mixing WP and CHO per se. However, too much energy (especially from protein) can lead to ergolytic effects due to nausea (Schroer et al., 2014).

16.5

TIMING OF WHEY PROTEIN INGESTION

The timing of WP intake may enable athletes to maximize anabolic and performance gains and/or speed up recovery. For instance, athletes may ingest supplements prior to exercise to supercompensate nutrients, enable exogenous substrates for fuel during exercise, and/or expedite recovery process by consuming supplements immediately postexercise.

16.5.1

Resistance Exercise and Training

WP ingestion prior to resistance exercise may increase insulin secretion and thus reduce MPB or increase AA availability and so “prime” the system (Morton, McGlory, & Phillips, 2015). Tipton et al. (2001) showed a greater anabolic response when an EAA 1 CHO solution was provided before exercise relative to immediately postexercise, due to increased AA delivery to the muscle. In a subsequent study (Tipton et al., 2007), untrained young men and women consumed 2 g WP either immediately before or immediately after heavy leg resistance exercise. The increase in AA during exercise was 100% for EAA (Tipton et al., 2001) but only 30% for WP (Tipton et al., 2007) when ingested immediately before exercise. Thus AA delivery during exercise is greater when EAA is ingested immediately before exercise than intact WP. However, the authors (Tipton et al., 2007) suggested that certain individuals may be more responsive to timing of protein ingestion than others.

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However, not all studies support beneficial effects of preexercise protein consumption. Although consumption of 25 g of whey and casein proteins 30 minutes before resistance exercise increased insulin secretion, it also decreased serum growth hormone, testosterone, and free fatty acid levels in young men (Hulmi, Volek, Selanne, & Mero, 2005). The authors suggested that preexercise protein intake may redistribute blood flow away from active muscles to the gastrointestinal tract, which may lower the muscle uptake of hormones during exercise (Hulmi et al., 2005). Furthermore, given the synergistic response of aminoacidemia following resistance exercise, preexercise aminoacidemia may blunt the subsequent postresistance exercise MPS response to AA availability due to an overlap in the aminoacidemic responses and a “muscle full effect” (Morton et al., 2015). Although there is equivocal information with regards WP ingestion, consumption of preworkout supplements containing other bioactives may lead to ergogenic effects. Fukuda, Smith, Kendall, and Stout (2010) showed 10% 12% improvements in TTE during high-intensity exercise following consumption of a preworkout supplement containing WP, caffeine, and creatine. Although the supplement may have increased BCAA supply, it was suggested that the caffeine was most likely to benefit performance due to decreased perception of pain during exercise (Fukuda et al., 2010). The delivery of protein during exercise may also increase MPS. Beelen et al. (2008) gave 10 recreationally active men either CHO (0.15 g/kg per hour) or PRO 1 CHO (0.15 g/kg per hour CHO plus 0.15 g/kg per hour casein hydrolysate) solution every 15 minutes during a 2-hour resistance training session. The PRO 1 CHO solution reduced MPB and increased MPS relative to CHO only; this led to positive net protein balance (NPB) in the coingestion trial but a negative NPB in the CHO-only trial. However, although the authors suggest that the increase in NPB was due to the protein intake, there was significantly higher energy intake in the coingestion trial. No studies have examined the intake of WP ingestion on MPS or performance during resistance exercise. Although resistance exercise alone increases MPS, providing protein immediately postexercise increases skeletal MPS rate to an even greater extent (Biolo, Tipton, Klein, & Wolfe, 1997). Therefore, there is an “anabolic window” within the first 2 3 hours after resistance exercise and protein should be consumed within this time frame for the most beneficial effects on MPS (Paul, 2009). However, the results of a metaanalysis (Schoenfeld, Aragon, & Krieger, 2013) suggests that if an anabolic window of opportunity does exist then the window for consumption is much longer, i.e., potentially 4 6 hours depending on the size and composition of the meal. The interaction of timing and quantity of protein is also an important consideration. Areta et al. (2013) concluded that the optimal delivery of WP is 4 3 20 g within the 12hour period following resistance exercise for optimal MPS.

16.5 Timing of Whey Protein Ingestion

Schoenfeld et al. (2013) noted that any positive effects associated with protein timing on muscle protein accretion disappeared after controlling for covariates, the most significant of which was the total amount of protein ingested. Hoffman et al. (2009) gave athletes a WP-casein supplement to consume in the morning and evening or immediately before and after workouts (4 times/week for 10 weeks). Supplementation also occurred at the same time of day during the 3 nontraining days and the control group did not receive any supplementation. No differences were found for changes in muscle strength or power among the groups, regardless of supplementation or its timing. Hoffman et al. (2009) suggested WP-casein supplementation did not enhance performance since the athletes were in positive nitrogen balance at the beginning of the study and consumed high daily dietary protein (.1.6 g/kg). Nevertheless, even though total protein intake is the strongest predictor of muscle hypertrophy, and that protein timing does not necessarily influence hypertrophy, athletes should still consider consuming WP in the postexercise period as this also enables rehydration, refueling (CHO), and repair of damaged tissues (Morton et al., 2015). Most research on timing of protein intake has been conducted in young, trained or untrained men. Due to issues relating to sarcopenia, elderly individuals may benefit to a greater extent from higher protein intake in the postexercise period (see also Chapter 15: Nutritive and Therapeutic Aspects of Whey Proteins). Esmarck et al. (2001) gave elderly (70 75 year-old) men a WP supplement (containing 10 g WP, 7 g CHO, 3 g fat) either immediately following or 2-hour postresistance exercise (3 times/week for 12 weeks). Muscle hypertrophy and muscle strength significantly improved over the training period in the men who were provided the supplement immediately postexercise, whereas there was no change in the participants who were provided the same supplement 2 hours later. Therefore, an increase in AA availability is crucial for elderly participants to support resistance training-induced adaptations.

16.5.2

Endurance Exercise and Training

Ingestion of CHO 1 WPH prior to exercise may activate key proteins in skeletal muscle that regulate glycogen synthesis and glucose uptake during exercise, resulting in an attenuation of glycogen depletion during exercise (Morifuji, Kanda, Koga, Kawanaka, & Higuchi, 2011). Moreover, ingestion of WP 1 CHO (within 200 mL beverage) every 20 minutes during intermittent cycling for 3 hours increased subsequent cycling TTE by 36% compared to CHO alone (Ivy, Res, Sprague, & Widzer, 2003). However, many studies showing improved benefits to endurance athletes compared WP 1 CHO with CHO alone (i.e., not isocaloric drinks), and the consensus view is that addition of WP to CHO drinks during exercise is only beneficial when the delivery of CHO is less than optimal (McLellan et al., 2014).

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Post endurance exercise protein ingestion stimulates synthesis of myofibrillar protein and related muscle proteins, which is important for tissue remodeling. Furthermore, coingestion of WP with CHO may increase muscle glycogen resynthesis (see Section 16.9.1).

16.6 16.6.1

DIFFERENT USER GROUPS Training Status of Athletes

While trained athletes respond positively to WP ingestion and resistance training (Naclerio & Larumbe-Zabala, 2016), there is equivocal data for untrained individuals. Hulmi et al. (2009) randomized 31 men into a WP group (15 g consumed before and after exercise), a PL group, and a control group; heavy resistance training was undertaken twice per week for 21 weeks in the WP and PL groups, whereas the control group continued with other types of exercise. The main finding was that WP ingestion before and after strength training further enhanced resistance-exercise-induced muscle hypertrophy in previously untrained individuals. However, not all studies report beneficial effects. Erskine et al. (2012) gave previously untrained men 20 g WP before and 20 g WP after resistance training and showed improved muscle mass and muscle strength, but there was no difference between WP and CHO-only groups. Furthermore, Weisgarber et al. (2012) reported no effect of 26 g WPI ingested during resistance exercise on muscle mass or strength in previously untrained male and female recreational exercisers. Narici et al. (1996) showed that the initial improvements in strength performance following resistance exercise was due to neurological changes (electromyography), with muscle hypertrophyrelated changes leading to strength gains after several weeks of training. Therefore, changes in LBM and muscle strength during the initial week of resistance training are not influenced when protein supplements are provided to untrained individuals (Pasiakos, McLellan, & Lieberman, 2015). Training status can also affect postexercise MPS. Well-trained athletes are likely to return to basal levels of postexercise MPS quicker than untrained individuals (Burd et al., 2009). Therefore, it is more critical for well-trained athletes to consume protein within the first few hours following resistance exercise, i.e., to improve the timing of AA delivery when the initiation of MPS is most sensitive (Burd et al., 2009).

16.6.2

Older Age Adults

The World Health Organisation (2007) states that the number of people aged 60 years and over is growing faster than any other age group, with an estimated increase from 688 million in 2006 to almost 2 billion in 2050.

16.6 Different User Groups

Maintaining mobility and function is of paramount interest to those wishing to sustain independent living as they age. However, sarcopenia, the agerelated loss of muscle mass, strength, and function, contributes toward physical frailty and increases risk of reduced mobility, falls, and fractures (Narici & Maffulli, 2010). Therefore, WP ingestion, combined with physical activity, may help older adults offset muscle and strength loss due to sarcopenia. Eliot et al. (2008) examined the independent and additive effects of WP and creatine supplementation, in conjunction with 3 times/week resistance training for 14 weeks, in 42 72 year-old men. Although there were no changes in body composition in any of the supplement trials, the authors suggested that supplementation doses that are effective in improving body composition in young adults are unlikely to produce the same effect in older adults. This reduced sensitivity of skeletal muscle tissue to dietary protein intake has been termed “anabolic resistance” and can lead to reduced muscle mass, reduced strength, and poorer activities of daily living (Moore, 2014). Therefore, older adults’ muscles may be less sensitive to anabolic stimulus (i.e., “anabolic resistance”) compared with younger adults, even in the presence of what may appear adequate total protein intake (Bauer et al., 2015). Moore et al. (2009) showed that young adults can achieve maximal MPS following resistance exercise with as little as 5 10 g protein, with intakes of 20 and 40 g having no further increase in MPS. However, older individuals require higher protein intake for increases in MPS. Pennings et al. (2012) randomly assigned 33 older men (.70 years old) to ingest either 10, 20, or 35 g WP, and used intrinsically labeled WP to assess digestion and absorption of WP and incorporation into newly synthesized muscle protein. The ingestion of 35 g WP allowed more AA to be absorbed and used to synthesize new muscle protein, relative to 10 and 20 g WP. Therefore, Pennings et al. (2012) concluded that “anabolic resistance” could be offset by consuming greater amounts of WP in older adults. In a later study, Moore et al. (2014) showed that while younger individuals needed 0.24 g/kg body weight to maximally stimulate MPS, 0.40 g/kg body weight was needed to maximally stimulate MPS in older adults. Thus, protein recommendations are 0.8 g/kg body weight per day for young adults and 1.0 1.5 g protein/kg body weight per day for older adults (Devries & Phillips, 2015). Older people also have a higher “leucine threshold” and thus require greater amounts of leucine to stimulate MPS at rest and following resistance exercise (Phillips, 2016). Yang et al. (2012) showed that 20 g WP stimulated myofibrillar MPS above rest in the elderly, with increases in whole-body leucine oxidation observed with increasing doses of WP. A blunted sensitivity of myofibrillar MPS was seen at low doses of EAA (in 10 g intact WP) whereas 40 g WP increased exercise-stimulated rates of

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MPS above 20 g. At least 2 g of leucine (contained in 20 g WP) is required to pass the “leucine threshold” and increase rates of myofibrillar MPS in the elderly, however in young adults this can be achieved at lower doses (Yang et al., 2012). As older individuals have reduced energy intake, which often coincides with appetite suppression, their increased needs may be met in lower doses through ingestion of a higher quality protein such as WP (Phillips, 2016). Furthermore, the elderly tend to consume a large portion of their protein with the evening meal (Berner, Becker, Wise, & Doi, 2013). However, older adults should distribute dietary protein intake equally across their daily meals. For example, if the optimal protein intake is 60 g/day, they should consume 20 g at each mealtime (breakfast, lunch, and dinner) to enable better MPS throughout the day (Phillips, 2016). Resistance exercise alone can increase MPS in the elderly. Yarasheski et al. (1999) provided a resistance training programme (3 times/week for 3 months) to 12 frail 76 92-year-old men and women. Compared to the control group the resistance-trained individuals significantly improved their MPS and muscle strength performance. Therefore, a combination of resistance exercise and a leucine-rich WP may be an effective strategy for older adults to offset muscle loss and muscle strength due to sarcopenia.

16.6.3

Female Exercisers

In general, men and women of a similar health status and BMI have similar protein turnover rates (Markofski & Volpi, 2011). However, there is very little research specifically investigating the effects of WP intake on female athletes. Wilborn et al. (2013) gave female basketball players either 24 g WP or 24 g casein immediately pre- and postresistance exercise (4 times/week) for 8 weeks. Although there were improvements in performance and body composition, there were no differences between the two supplementation groups. In an earlier study male body builders showed greater improvements in strength and LBM with WP relative to casein (Cribb et al., 2006). The differences in the findings of Wilborn et al. (2013) and Cribb et al. (2006) may relate to the sex of the athletes, but it is unclear why. However, other studies that used male and female subjects showed no differences in responses to protein CHO intake and resistance exercise (Rasmussen, Tipton, Miller, Wolf, & Wolfe, 2000; Tipton et al., 2001). Indeed, even though women have a 10-fold lower testosterone concentration than men, they can still undergo substantial hypertrophy of their muscles, indicating that it is local rather than systemic androgen hormones that are responsible for promoting increases in MPS (Burd et al., 2009). In older

16.6 Different User Groups

postmenopausal women, however, the differences between sexes may become more apparent. Smith et al. (2008) compared MPS in postabsorptive conditions and during feeding in 65 80 year-old men and women. They found that elderly women had 30% greater basal rate of MPS, which is consistent with the finding that although women have a smaller muscle mass the age-associated decrease is slower in women. Furthermore, Smith, Fukuda, Kendall, and Stout (2010) reported that elderly women have an inability to increase MPS in response to protein feeding, possibly due to reduced capacity and responsiveness to anabolic signaling (Burd et al., 2009). Future research should further explore effects of age and sex on performance and MPS following resistance training and WP intake (Naclerio & Larumbe-Zabala, 2016). Women also typically rely less on protein metabolism during endurance exercise than men of a similar health and training status (Burd et al., 2009). Therefore, the same recommendations with regards WP ingestion can be applied as those for male endurance exercisers.

16.6.4

Children and Adolescents

Resistance training can be beneficial for children and adolescents for numerous reasons including improved muscle strength, bone mineral density, body composition, and positive attitudes to physical activity (Benjamin & Glow, 2003). Furthermore, adequate protein intake is required for normal growth and development of children, which should be able to be met by consuming a balanced diet without requiring supplementation (Petrie, Stover, & Horswill, 2004). Nevertheless, if young athletes consume inadequate energy intake (including 12% 15% protein of total energy intake) this will cause protein to be used as a substrate rather than for synthesizing lean tissues (Petrie et al., 2004). There are few experimental studies on the effects of exercise and protein intake in children and adolescents. Laskowski and Antosiewicz (2003) reported that a group of 12 adolescent elite judo athletes receiving a soy protein supplement (2 g/kg body weight) increased peak work capacity, peak anaerobic power, and work output compared with a control group after 4 weeks of training. Volterman, Moore, Obeid, and Timmons (2016) also showed that postexercise milk consumption improved fluid retention in 10 12 year-old children who were exercising in the heat. Children are not “miniature adults” (Atan, Foskett, & Ali, 2014) and more research is needed to determine whether recommendations for adults with regard to WP intake for resistance and/or endurance exercise, can be applied to children and adolescents.

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16.7

DOSE-RESPONSE

The most recent position stands of the Academy of Nutrition and Dietetics, Dietitians of Canada, and American College of Sports Medicine is that dietary protein intake necessary to support metabolic adaptation, repair, remodeling, and for protein turnover ranges from 1.2 to 2.0 g/kg body weight per day (Thomas, Erdman, & Burke, 2016). However, individuals have often exceeded these recommendations in the thought that “if some is good, more must be better.” Nevertheless, evidence suggests that although a doseresponse may exist, exceeding recommended levels will not provide any additional benefits. In an early study (Tarnopolsky et al., 1992) a group of young males underwent 4 days/week resistance exercise for 2 months before the study; a second group were sedentary age-matched control subjects. Participants were randomly assigned one of three levels of protein for 13 days: low (0.86 g/kg body weight per day), moderate (1.4 g/kg body weight per day), and high protein (2.4 g/kg body weight per day). The low-protein diet was adequate for sedentary individuals but five of the seven strength-trained athletes were in negative nitrogen balance. Tarnopolsky et al. (1992) also showed that protein consumed in excess of requirements resulted in a plateau in whole body protein synthesis and excess dietary protein was oxidized as energy rather than stored as lean muscle tissue. Moore et al. (2009) gave six healthy men doses of 0, 5, 10, 20, or 40 g of whole egg protein postresistance exercise (five separate trials). Maximal protein synthesis and maximal albumin protein synthesis increased in a dosedependent manner from 0 to 20 g, however there was no further benefit of ingesting 40 g protein. Therefore, 20 g WP has been suggested to be the optimal amount of protein for young healthy men; any more protein is oxidized and results in urea production (Witard et al., 2001), indicating there is a limit of AA that can be used for muscle protein balance, which has been termed the “muscle full effect” (Atherton et al., 2010). A potential method of overcoming the “muscle full effect” may be to provide WP in smaller doses throughout the day. Areta et al. (2013) investigated the effect of providing different boluses of 80 g WP following resistance exercise in 24 well-trained male athletes. Following resistance exercise athletes were split into three groups and provided WP in 8 3 10 g boluses (every 1.5 hours), 4 3 20 g (every 3 hours), or 2 3 40 g (every 6 hours). All supplement regimes increased MPS during the 5-day recovery period (compared to rest) but the 4 3 20 g (every 3 hours) feeding pattern was far superior to the other two methods. These results support the “muscle full effect” where, when AA delivery is sufficient (B20 g), AA are no longer used for MPS

16.8 Coingestion With Other Bioactives

and become oxidized. Thus, it appears beneficial to ingest protein in moderate doses (B0.4 g/kg body weight per meal) throughout the day (Morton et al., 2015).

16.8 16.8.1

COINGESTION WITH OTHER BIOACTIVES Carbohydrate

The primary reason for CHO coingestion with protein is to stimulate insulin release beyond that seen with protein ingestion alone (Morton et al., 2015). As increased insulin secretion helps with enhancing MPS and reducing MPB; coingestion of WP and CHO may be beneficial for both resistance and endurance-trained athletes.

16.8.1.1 Resistance Exercise Following resistance exercise CHO ingestion alone has no effect on MPS but may decrease MPB (Børsheim et al., 2004). Coingesting CHO and protein has no further stimulatory effect on MPS and does not suppress MPB if protein intake is adequate. Staples et al. (2011) had nine recreationally active males undertake resistance exercise followed by ingestion of 25 g WP or 25 g WP plus 50 g maltodextrin (WP 1 CHO). They found no difference in MPS, intramuscular signaling, or blood flow to the quadriceps between trials and suggested that 25 g WP was sufficient in stimulating insulin-mediated MPS and inhibiting MPB during hyperaminoacidemia without the addition of CHO. However, when protein intake is below adequate levels (,0.25 g protein/kg body weight), coingestion with CHO can increase insulin levels, suppressing MPB and enhancing AA delivery to the muscle (Morton et al., 2015).

16.8.1.2 Endurance Exercise Endurance exercise can lead to depletion of muscle glycogen stores and necessitate repair of damaged skeletal muscle tissue; prolonged activity also increases energy requirements during exercise itself. Therefore, coingestion of CHO with WP alongside endurance exercise makes more intuitive sense. Refueling with CHO immediately postexercise is common practice by athletes to optimize muscle glycogen restoration, i.e., “time window” of opportunity within 2 hours of ceasing exercise (Ivy, Katz, Cutler, Sherman, & Coyle, 1988), but adding protein may enable greater glycogen resynthesis. Zawadzki, Yaspelskis, and Ivy (1992) were the first to show improvements in muscle glycogen resynthesis following endurance exercise with WPI 1 CHO coingestion relative to WPI or CHO ingestion alone. They suggested that the interaction of WPI and CHO ingestion allowed higher insulin secretion, which increased glucose transport and glycogen synthase activity, and

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resulted in greater postexercise glycogen resynthesis. However, the three supplements used by Zawadzki et al. (1992) were not isocaloric (112 g CHO; 40.7 g WPI; 112 g CHO plus 40.7 g WPI) and therefore the glycogen resynthesis rates could have been due to the higher energy intake in the WPI 1 CHO trial. Therefore, the prevailing view is that coingestion of WPI 1 CHO may only be beneficial for muscle glycogen resynthesis when insufficient CHO is consumed (Morton et al., 2015). Morifuji, Kanda, Koga, Kawanaka, and Higuchi (2010) showed coingestion of WPI 1 CHO to be more effective than other protein sources for increasing postexercise skeletal muscle glycogen stores in rats. It may be that WP 1 CHO accelerates glucose uptake in the muscle, by enhancing glycolregulatory enzyme activity, to a greater extent than casein or soy (Aoi et al., 2011). Furthermore, WP is more insulinotropic compared to caseins and other protein sources and independent of CHO coingestion; the increased plasma insulin during recovery and earlier reduction in plasma glucose concentration in the WPI 1 CHO group likely explains the enhanced recovery of muscle glycogen (Breen et al., 2011). Coingestion of WP 1 CHO during endurance exercise also shows mixed results. Saunders, Kane, and Todd (2004) had 15 trained cyclists complete a cycling TTE followed, 12 15 hours later, by a second TTE. The cyclists received beverages containing either 7.3% (26 g) CHO or 7.3% CHO plus 1.8% (6.5 g) WP during and following exercise. In the WP 1 CHO trial the cyclists performed 29% and 40% longer in the first and second TTE, respectively. However, there was a 20% lower caloric intake in the CHO-only trial, and so the improved performance in the WP 1 CHO trial could have been due to the higher energy supply. van Essen & Gibala (2006) provided welltrained cyclists optimal delivery rates of WP 1 CHO (2% WP 1 6% CHO), CHO (6%), or noncaloric PL during an 80-km TT. Although both were superior to PL, there was no difference between performance times or glucose levels for WP 1 CHO relative to CHO-only group (van Essen & Gibala, 2006). Other studies also show no difference in performance when exogenous rates of CHO delivery are optimal (Breen, Tipton, & Jeukendrup, 2010; Jeukendrup, Tipton, & Gibala, 2009; Valentine, Saunders, Todd, & St Laurent, 2008) or if the exercise test is of short duration (Toone & Betts, 2010). Therefore, it appears the addition of WP to CHO can provide an ergogenic effect on performance when delivery of CHO is less than optimal. However, when CHO supplementation is delivered at or above 60 g/h, the addition of WP seems to provide no further performance-enhancing benefits. CHO ingestion alone may not be sufficient to stimulate significant MPS and the adaptive response to endurance exercise (Hill, Stathis, Grinfeld, Hayes, & McAinch, 2013). Breen et al. (2011) had 10 trained cyclists complete

16.8 Coingestion With Other Bioactives

90 minutes of fatiguing exercise followed by ingestion of either 25 g CHO or 10 g WP plus 25 g CHO. They reported increased myofibrillar but not mitochondrial protein synthesis with WP 1 CHO relative to CHO alone. Therefore, Breen et al. (2011) suggested that WP 1 CHO ingestion post endurance exercise may enhance the synthesis of proteins associated with fatigue resistance and myofibrillar protein breakdown, thus potentially enhancing the adaptive response and better recovery of muscle function. In addition to improvements in performance in the first and second rides, Saunders et al. (2004) reported 83% lower plasma creatine phosphokinase— a measure of muscle damage—following WP 1 CHO supplementation relative to CHO-only trial. The authors suggested that the WP 1 CHO supplement regime increased protein synthesis outside the cell which may have increased protein synthesis and repair, and led to improvements in subsequent performance.

16.8.2

Caffeine

An analysis of over 20,000 urine samples from elite athletes showed that 74% consume caffeine before or during competition, and 19% coingested caffeine with other substances (Del Coso, Muñoz, & Muñoz-Guerra, 2011). The benefits of caffeine intake include improved exercise capacity, muscle endurance, cognition, repeated sprint ability, and altering perceived effort or fatigue (Sokmen et al., 2008). Caffeine is metabolized by many organs within the body and various mechanisms of action have been proposed (Rutherfurd-Markwick & Ali, 2016). However, it is caffeine’s role as an adenosine antagonist (due to caffeine having a similar molecular structure to adenosine) that seems to be the main reason for its ergogenic benefit. Caffeine may increase the secretion of β-endorphins during exercise and therefore improve exercise performance due to the analgesic properties of β-endorphins which decrease the perception of pain (Hartwig, 1991). Some evidence also suggests that caffeine may help enhance fat utilization and spare intramuscular glycogen (Rutherfurd-Markwick & Ali, 2016). Caffeine is typically added to preworkout supplements that also contain other active ingredients. Some studies have shown improvements in various performance aspects when using these multinutrition supplements in acute (Fukuda et al., 2010) and repeated (Smith et al., 2010) exercise sessions. Fukuda et al. (2010) had 10 moderately trained males and females complete two TTE on separate days and ingest a multinutrition supplement (including caffeine, WPC, BCAA) or isocaloric PL (containing maltodextrin and flavorings) 30 minutes before testing on an empty stomach. The WPC-caffeine-containing supplement improved anaerobic running capacity by 10.8% and increased TTE by 10% 12% during high intensity exercise (Fukuda et al., 2010).

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In another study, Smith et al. (2010) had 24 moderately trained men and women ingest either a mixed nutrition supplement (including 100 mg caffeine, 9 g WP, 1 g BCAAs, 1.5 g creatine) or an isocaloric PL prior to highintensity interval training sessions. After 3 weeks, the mixed nutrition supplement was shown to improve critical velocity (maximal running velocity that can be maintained for an extended period of time using only aerobic energy), VO2max, LBM, and total training volume compared to PL. WP 1 BCAAs may have helped recovery between sessions and maintaining LBM whereas caffeine ingestion helped aerobic performance and training volume (Smith et al., 2010). However, the fact that these supplements contain a wide range of ingredients means that it is difficult to pinpoint the independent and/or additive effects of caffeine, WP, and BCAA.

16.8.3

Creatine

Creatine is a naturally occurring compound in the body, 95% of which is found in muscle and predominantly in type II (fast twitch) muscle tissue (Greenhaff, 1996). Phosphocreatine (PCr) is a limiting factor in anaerobic and sprint exercise, i.e., as it runs out, work rate is reduced (fatigue). Therefore, creatine supplementation may increase muscle creatine availability, thus delaying the rate of PCr depletion and enable faster PCr resynthesis during recovery from sprints, thus improving performance. Harris, Soderlund, and Hultman (1992) reported increased muscle creatine levels and increased PCr content following 5 g creatine supplementation (6 times/ day for 1 week); however, they did not include any performance measures. In a well-controlled study, 36 young males consumed either WP (1.2 g/kg per day), WP 1 creatine monohydrate (1.2 g/kg per day WP and 0.1 g/kg per day creatine), or isocaloric PL (1.2 g/kg per day maltodextrin) in four equal servings across the day and completed resistance training 3 times/week for 6 weeks (Burke et al., 2001). WP 1 creatine significantly increased LBM and bench press strength compared to WP and PL. Furthermore, WP 1 creatine and WP significantly increased knee extension peak torque with training compared to PL. Therefore, WP and creatine provided independent and additive ergogenic benefits for resistance-trained athletes (Burke et al., 2001). Older adults may not benefit from WP 1 creatine supplementation as much as younger individuals. Eliot et al. (2008) had 42 older men (42 72 years) consume 480 mL of a commercially-available sports drink (PL) with either WP 1 creatine (35 g WP, 5 g creatine), WP (35 g WP) or creatine (5 g creatine) preexercise. Resistance training sessions were performed 3 times/week for 14 weeks. No additional benefit/changes to body composition were observed for any supplement when combined with resistance training relative to resistance training alone. Eliot et al. (2008) suggested that older men

16.8 Coingestion With Other Bioactives

may not have responded to the dose of WP and creatine since their rate of muscle synthesis is lower due to decreased testosterone and myosin heavy chain protein synthesis compared to young men. Creatine also causes weight gain which can be beneficial for those wishing to increase muscle mass; however, with excess body weight, VO2max and sprint speed may decrease because of the need to carry the increased mass. Furthermore, the extent of creatine retention is highly variable with 20% 30% of individuals not responding to creatine supplementation (Greenhaff, 1996). It appears that athletes with the lowest presupplementation levels of creatine typically show the biggest gain in muscle creatine. Therefore, individuals should carefully consider the reasons for including creatine within a WP-containing supplement. Excess creatine is excreted by the kidneys as creatinine in urine. Early studies usually provided too high a dosage and so the current guidelines recommend an initial loading of 20 g (4 3 5 g) per day for 1 week, followed by maintenance of 2 g per day for a length of time. A summary of the guidelines suggest that individuals add a small amount of creatine (0.1 g/kg) to WP 1 CHO supplements postexercise to optimize the adaptations of resistance training (Potgieter, 2013).

16.8.4

Vitamin D

The ingestion of serum 25-hydroxyvitamin D, at an optimal rate of 60 75 nmol/L (800 IU/day), has been suggested to enhance lower-body strength and reduce risk of falls and fractures (Bischoff-Ferrari, 2014). Inadequate protein intake can also reduce muscle mass and muscle strength and contribute to increased risk of falls and fractures (Narici & Maffulli, 2010). Therefore, adding vitamin D to WP makes intuitive sense, especially for elderly adults undertaking resistance exercise. Bauer et al. (2015) divided 380 sarcopenic older (.65 years) adults into two groups. The intervention group consumed a WP 1 vitamin D and leucine supplement (20 g WP, 3 g leucine, 800 IU/day vitamin D, 9 g CHO, mix of vitamins and minerals per serving) twice daily for 13 weeks; the PL group consumed an isocaloric control (CHO and fat) two times/day for 13 weeks. Serum 25-hydroxyvitamin D concentrations improved from 34 66 to 48 100 nmol/L in the supplemented group. There were also improvements in the chair-stand test and muscle mass in the WP 1 vitamin D group compared to control (Bauer et al., 2015). In another study, sarcopenic elderly (70 90 years) individuals ingested either a WP 1 vitamin D supplement (22 g WP, 10.9 g EAA including 4 g leucine and 2.5 μg vitamin D) or isocaloric PL in conjunction with regular

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physical activity, for 12 weeks (Rondanelli et al., 2016). The WP 1 vitamin D supplementation improved fat-free mass, muscle mass, hand grip strength, insulin-like growth factor (IGF-1), and overall daily living measures relative to PL. These improvements could have been due to changes in gene expression and MPS (Rondanelli et al., 2016). WP and vitamin D supplementation has also been shown to reduce the risk of sarcopenia in obese older adults (Verreijen et al., 2015) and healthy, early postmenopausal women (Holm et al., 2008). A systematic review showed that there was a positive effect of vitamin D supplementation on improving muscle strength, especially for those with serum 25-hydroxyvitamin D levels of ,30 nmol/L, and that supplementation was more effective in older (.65 years) relative to younger adults (Beaudart et al., 2014). However, there was no significant effect of vitamin D supplementation alone on improving muscle mass or muscle power (Beaudart et al., 2014). Therefore, the addition of WP to vitamin D appears to be the most successful supplementation regime in terms of improvements in LBM and muscle strength (Naclerio & LarumbeZabala, 2016).

16.9 16.9.1

MECHANISMS OF ACTION Insulin Signaling and Glycogen Synthesis

Compared to casein and vegetable proteins both WPH and WPI are more insulinotropic (Hill et al., 2013; Power, Hallihan, & Jakeman, 2009), which means they are able to stimulate insulin production, ultimately counteracting muscle catabolism. Increased insulin levels lead to increased muscle glycogen levels via stimulation of translocation of glucose transporters (GLUT-4) to the plasma membrane (Zorzano, Palacín, & Gumá, 2005), which increases glucose uptake (Klip et al., 1993), and also via activation of the enzyme glycogen synthase (Armstrong, Bonavaud, Toole, & Yeaman, 2001). This activation is brought about by increased insulin levels upregulating the PI3-kinase pathway which leads to phosphorylation of Akt which inhibits glycogen synthase kinase 3 (GSK3); this inactivation removes the normal inhibitory effect that GSK3 has on glycogen synthase activity (Armstrong et al., 2001). However, GLUT-4 translocation can also occur in an insulin-independent manner with taurine being able to independently activate the insulin pathway (Carneiro et al., 2009). Physical exercise also increases the translocation of GLUT-4 to the plasma membrane (Kennedy et al., 1999). WP is widely recognized as being able to increase glycogen stores. Normally, glycogen synthase activity will reduce as muscle glycogen levels recover

16.9 Mechanisms of Action

following exercise; however, long-term consumption of WPH appears to maintain glycogen synthase activity even when muscle glycogen levels are high. Interestingly, ingestion of WPH caused significantly greater increases in skeletal muscle glycogen levels than nonhydrolyzed WP even though their AA composition was the same (Morifuji et al., 2010). A study in rats (Morato et al., 2013) has shown that consumption of WPH significantly increased plasma membrane GLUT-4 concentrations (an effect enhanced by exercise) and glycogen levels in the heart, skeletal muscle, and liver, with no accompanying changes in GLUT-1 or insulin, indicating the mechanism of action in this case is likely to be insulin-independent. Consumption of WPH increased Akt phosphorylation in both the sedentary and exercised groups and also increased plasma taurine levels (Morato et al., 2013). In addition, BCAA from WPH have been shown to activate skeletal muscle glucose uptake via the PI3-kinase and atypical PKC pathways, a mechanism different from insulin-induced GLUT-4 translocation (Morifuji et al., 2009). Hence, multiple mechanisms may have contributed to the insulin-independent increase in plasma GLUT-4 in response to WP consumption seen in the study by Morato et al. (2013). In skeletal muscle, the control of protein and/or glycogen synthesis is the result of a number of enzymes including Akt, mammalian target of rapamycin (mTOR), p70S6 kinase, rpS6, GSK3, and glycogen synthase. In addition, both exercise and nutrient supplementation are able to impact on the activities of each of these enzymes. For example, AA activation of p70S6K kinase not only activates protein synthesis via the mTOR pathway but also transiently inhibits GSK3, resulting in glycogen synthase activation (Armstrong et al., 2001; Peyrollier, Hajduch, Blair, Russell, & Hundal, 2000). Coingestion of CHO and WP increases the rate of muscle glycogen storage after exercise, likely due to the greater insulin response upregulating phosphorylation of Akt via the PI3-kinase pathway which leads to activation of mTOR (leading to protein synthesis), phosphorylation of GSK3 with resulting dephosphorylation and activation of GS (Ivy, Ding, Hwang, CialdellaKam, & Morrison, 2008). Premeal consumption of WP has been shown to improve postmeal glucose control, potentially by slowing gastric emptying (Akhavan, Luhovyy, Brown, Cho, & Anderson, 2010). WP and other proteins have been shown to slow stomach emptying by various gut hormones such as cholecystokinin from the intestinal enteroendocrine cells (Pupovac & Anderson, 2002).

16.9.2

Increase Muscle Mass

The methods used to process WP and level of hydrolysis of the protein can impact on rate of absorption, with studies in animals and humans showing

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that peptides from WP are absorbed faster than either whole protein or individual AA (Cribb et al., 2006; Manninen, 2004) and this may impact on muscle anabolism. Results from a 21-week study carried out in previously untrained young subjects (25 30 years), showed that ingestion of WP before and after resistance exercise was able to further increase muscle hypertrophy above resistance training alone (Hulmi et al., 2009). Alongside the increase in muscle area was an increase in mRNA expression of cell-cycle kinase (cdk2) in the vastus lateralis muscle. A similar study in older men (57 72 years) also showed significantly increased cdk2 mRNA levels in the group receiving WP supplementation (Hulmi, Kovanen, Lisko, Selänne, & Mero, 2008). cdk2 activity is restricted to and essential for the G1-S transition phase of the cell cycle and therefore an important regulator of cell proliferation. In addition, the intake of WP appeared to prevent decreases in mRNA expression of myostatin and myogenin, which were evident 1-hour post resistance exercise in the control group (Hulmi et al., 2009). Myostatin, a member of the TGF-B family, is a protein produced and released by myocytes which limits muscle hypertrophy in two ways: firstly, it has an autocrine action, inhibiting muscle cell differentiation, and secondly, it inhibits Akt and hence protein synthesis. Myogenin is a transcription factor involved in the coordination of the development and repair of skeletal muscle. No effect was seen on the transcriptional regulation of muscle atrophy F-box (MAFbx) (Hulmi et al., 2009), which is important in muscle proteolysis and atrophy (Bodine et al., 2001). Taken together these results indicate a coordinated response to resistance exercise and WP ingestion which is advantageous for muscle hypertrophy. In older men, WP ingestion before and after exercise was also able to prevent a postexercise decrease in myostatin mRNA levels, however in this case the decrease was observed 48 hours postexercise (Hulmi et al., 2008) as opposed to 1 hour postexercise in the younger age group (Hulmi et al., 2009). Therefore, there appears to be an age-dependent time response to muscle myostatin gene expression in healthy men following exercise, but in both cases protein intake before and after exercise appears to attenuate the effect (Hulmi et al., 2008, 2009). These differences are perhaps not unexpected given that recovery from exercise or injury is known to be slower in older muscle compared to younger muscle (Brooks & Faulkner, 1990). There appear to be other age-related responses to WP ingestion in association with resistance exercise, with older men in the protein group showing increased myostatin binding protein follistatin related gene protein mRNA responses compared to the placebo group (Hulmi et al., 2008), but a similar study in younger men showed no response (Hulmi et al., 2009).

16.9 Mechanisms of Action

In addition to being a building block for protein, leucine is known to be the key AA signaling stimulation of MPS. The mechanism involves leucine binding to Sestrin 2 (leucine sensor; Wolfson et al., 2016), which causes the dissociation of both Sestrin 2 and GATOR2 (a GTPase-activating protein) enabling activation of mTORC1 (rapamycin complex-1). mTOR is a serine/threonine kinase that plays a major role in controlling cell growth by coordinating environmental signals from nutrients, growth factors, and exercise (Morrison, Hara, Ding, & Ivy, 2007). Activation of mTORC1, which in itself involves phosphorylation, leads to subsequent phosphorylation of downstream targets eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and p70 ribosomal S6 kinase 1 (p70S6K) resulting in the inhibition of 4E-BP1 and activation of p70S6K which phosphorylates further proteins involved in the initiation of mRNA translation, ultimately resulting in the stimulation of MPS (Ivy et al., 2008). Given the importance of leucine in this process it has been hypothesized that leucinemia would lead to high intracellular leucine concentrations, which in turn would bind to Sestrin 2 leading to stimulation of MPS and that it is the leucine content which is more important than total protein (Phillips, 2016), with a “leucine threshold” of 2 3 g per meal having to be reached (Churchward-Venne et al., 2014). The addition of 5 g leucine to 6.25 g of WPI was shown to stimulate MPS to the same extent as 25 g dose of WPI (Churchward-Venne et al., 2014). Moreover, Koopman et al. (2005) showed that the addition of leucine to a PRO 1 CHO beverage increased MPS to a greater extent following resistance exercise than a PRO 1 CHO beverage alone, indicating that leucine can enhance the anabolic effect of protein. Therefore, protein supplements that contain a high leucine content appear to optimize MPS. AAs have also been shown to stimulate protein synthesis via an mTOR independent pathway as they can independently phosphorylate 4E-BP1 and p70S6K thus leading to their inhibition and activation, respectively (Kimball & Jefferson, 2004). Exercise also promotes mRNA transcription and protein synthesis, however full activation requires elevated plasma insulin levels (Kuo, Browning, & Ivy, 1999; Kuo, Hunt, Ding, & Ivy, 1999). Insulin’s effects are also via the mTOR pathway, however in this case it occurs via the activation of two kinases known as PI 3-kinase and Akt (Kimball, Farrell, & Jefferson, 2002). Clearly MPS can be stimulated by a variety of compounds via a number of different mechanisms, with the evidence generally supporting the potential use of WP or WPH for increasing MPS.

16.9.3

Repair of Muscle

In order for muscle to recover after damage, two cellular processes must occur: minimization of protein degradation and stimulation of protein

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synthesis which is essential for muscle regeneration and hypertrophy. WP is an excellent source of the AA required for muscle and tissue growth, maintenance, and repair, as well as preventing the catabolic actions which occur during exercise (Hoffman & Falvo, 2004). In fact, it has been suggested that given the very similar AA compositions of WP and skeletal muscle, supplementation with WP may provide the optimal AA profile for muscle remodeling (Cooke et al., 2010). Evidence from the literature supports the ability of WP supplementation to positively impact muscle repair with a study in untrained individuals showing that short-term WPI consumption (14 days) was able to attenuate a decline in strength following eccentric exercise (Cooke et al., 2010), potentially due to increased protein synthesis and decreased muscle damage as indicated by a trend for decreased LDH activity compared to the CHO control group. Nosaka, Sacco, and Mawatari (2006) showed that supplementation with a mixture of AA during recovery days reduced muscle soreness and damage following endurance eccentric exercise, with a further study showing that it was most likely the BCAA responsible for this effect (Jackman, Witard, Jeukendrup, & Tipton, 2010).

16.9.4

Protein Oxidation

The use of BCAA as an energy source is usually associated with endurance exercise, and has been implicated in the central fatigue hypothesis (Davis, 1995). The high BCAA (particularly leucine) of WP coupled with the rapid digestion rate of WP results in a rapid and transient increase in blood AA concentrations which further stimulates AA oxidation and contributes to reduced whole-body protein synthesis. However, ingestion of more slowly digested proteins, such as casein, leads to a slower and more prolonged rise in plasma AA resulting in less oxidation and more protein synthesis (Reidy et al., 2013).

16.9.5

Gene Expression

Muscle protein degradation occurs primarily via the ubiquitin proteasome system which includes three key transfer reactions including ubiquitin ligases (E3). Of the E3 ligases, two key enzymes mediate muscle protein loss particularly in catabolic conditions, namely MAFbx and muscle-specific RING finger-1 (MuRF-1). mTOR kinase is involved in the MPS signaling pathway, with expression ultimately leading to enhanced mRNA translation and MPS. A study in resistance-exercised rats has shown that provision of dietary WP is able to modify the expression of the genes related to protein metabolism (Haraguchi et al., 2014). Specifically, MuRF-1 was reduced by exercise, WP supplementation reduced MAFbx gene transcription independent of

16.9 Mechanisms of Action

exercise, and WP in combination with resistance exercise reversed the reduction in expression of mTOR normally seen with exercise thus resulting in higher muscle weight gain in resistance-exercised WP fed rats (Haraguchi et al., 2014).

16.9.6

Mood and Cognition

During and after sustained exercise the plasma ratio of free tryptophan/BCAA increases (Blomstrand, Hassme
n, Ek, Ekblom, & Newsholme, 1997). As BCAA and tryptophan compete for transport, the relative increase in tryptophan favors its transport into the brain and hence the synthesis and release of serotonin (5-HT), leading to central fatigue (Davis, 1995). The role of serotonin in fatigue and performance is supported by studies in rats and humans; when brain serotonin levels were manipulated by pharmacological means, results showed that elevated serotonin levels impaired performance while decreased levels led to improved running performance (Blomstrand, 2001). Results from studies testing the hypothesis that dietary supplementation with BCAA may attenuate the serotonin response delaying fatigue, and thus improving endurance performance, have been equivocal. In some studies, ingestion of BCAA reduced the perceived exertion and mental fatigue during exercise and improved cognitive performance after exercise (Blomstrand, 2001). For example, during standardized cycle ergometer exercise, participants receiving BCAA supplementation had lower RPE and mental fatigue (Blomstrand et al., 1997). Similarly, participants taking a BCAA supplement during a 30-km cross-country race displayed improved cognitive function after the race than control participants (Hassme
n, Blomstrand, Ekblom, & Newsholme, 1994). In a study investigating the effects of CHO and WP on RPE during football-specific intermittent exercise, RPE levels were lower in the CHO 1 WP group throughout the protocol than in participants receiving placebo or CHO alone (Alghannam, 2011). However, not all studies have shown positive effects. Eight weeks of supplemental WP with leucine failed to improve endurance or cognitive performance although increases in muscular strength and LBM were observed (Walker et al., 2010).

16.9.7

Immune Function

Exercise can negatively impact immune function through a variety of mechanisms; firstly, exercise lowers blood glucose levels and increases the levels of stress hormones such as cortisol that affect immune function. Exercise also leads to generation of reactive oxygen species (ROS) and oxidative stress which can lead to apoptosis of susceptible immune cells. During intense exercise, the demand for glutamine by the muscle, liver, and kidneys

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is higher than what is being produced by skeletal muscle and hence immune organs (spleen, thymus, lymph nodes) are exposed to glutamine deficiency. Therefore, factors that influence plasma glutamine levels and antioxidant levels have the potential to impact on immune function. It is now well accepted that in addition to supplying essential nutrients, some food proteins can confer additional health benefits beyond nutrition including modulation of immune function. WP is a prime example, having been shown to have multiple beneficial effects including bone metabolism, blood pressure (via ACE inhibitory peptides), modulating T and B cell function, suppression of inflammatory responses, and enhancement of phagocytic activity (Rutherfurd-Markwick & Moughan, 2005). WP contains antioxidants, immunoglobulins, and bioactive proteins and peptides (following digestion) with antimicrobial, antiviral, anticancer, and immune-enhancing effects. For example, α-La, which makes up about 25% of WP is reported to have anticancer, immune-, and moodenhancing properties as well as reducing cortisol levels (Bawa, 2007). Milk protein-derived bioactive peptides also help stimulate the uptake of AA after resistance exercise and promote the building of skeletal protein, resulting in a larger increase in lean muscle and muscle strength, which are beneficial to young athletes and the elderly (Phillips, Hartman, & Wilkinson, 2005). The AA composition of WP, being rich in sulfur AA, is able to help maintain the redox status of immune cells thus reducing the oxidative damage to highly susceptible lymphocytes (Cruzat, Krause, & Newsholme, 2014). The importance of L-glutamine as an immunonutrient cannot be underestimated due to its multiple roles, and WP provides an excellent source of this AA. Glutamine can be used as an oxidizable fuel, is a precursor for GSH, a substrate for nucleotide synthesis, a modulator of intermediary metabolism of AA (Cruzat et al., 2014), increases rates of MPS and regulation of muscle protein balance (Paul, 2009), enhances heat-shock protein responses induced by chronic or acute inflammation, and enhances neutrophil and lymphocyte function (Cruzat et al., 2014). Provision of high amounts of BCAA to skeletal muscle will enhance synthesis of glutamine for immune and other metabolic processes (Cruzat et al., 2014). Dietary supplementation with maltodextrin, WP, and glutamine was able to partially prevent exhaustive exercise-induced lymphocyte apoptosis (Cury-Boaventura et al., 2008). From an exercise perspective, on an intracellular level, glutamine improves cell hydration state and volume (via promoting water uptake, increasing Na1 uptake and release of K1) which is important for resistance to injury

16.9 Mechanisms of Action

(Cruzat et al., 2014). Exercise has been reported to decrease plasma L-glutamine levels, lymphocyte numbers via apoptosis (Cury-Boaventura et al., 2008) leading to an increased risk of upper respiratory tract infection (Cruzat et al., 2014). While some studies in athletes have shown that oral supplementation with L-glutamine (0.1 g/kg body weight) or WP enriched with L-glutamine are able to attenuate or reverse these effects (Cury-Boaventura et al., 2008), others have not reported the same results (Cruzat et al., 2014). A study in endurance athletes showed that both acute and chronic BCAA supplementation (about 6 g/day) was able to attenuate a drop in plasma L-glutamine levels and also modified the immune suppression promoted by the exercise (Bassit et al., 2002).

16.9.7.1 Antiinflammatory Effects During exercise the antiinflammatory cytokine IL-6 is released, in part due to muscle damage and glycogen depletion (Cruzat et al., 2014). Consumption of WP has been shown to attenuate this response during 120 minutes prolonged cycling exercise (Schroer et al., 2014). Another study showed that consumption of a CHO 1 WP supplement (ratio 3.5:1) decreased lipid peroxidation, IL-6, and CRP after exhaustive cycling (Kerasioti et al., 2013). The mechanism by which reduction of IL-6 occurs is unclear, although it may be via reduction of muscle damage as following exercise the levels of indirect muscle damage markers (creatine kinase and myoglobin) are generally lower in athletes consuming WP (Schroer et al., 2014).

16.9.7.2 Antimicrobial Effects WP contains approximately 1% 2% of the antimicrobial protein lactoferrin (Lf). Due to their ability to bind iron, Lf and its bioactive peptide lactoferricin have been shown to inhibit the growth of bacteria and fungi, however, they are also able to promote the growth of beneficial bacteria such as Bifidobacteria (Bawa, 2007). Lf and lactoferricin are also able to work synergistically with other WPs such as lysosome and lactoperoxidase against viral and bacterial organisms (Cruzat et al., 2014) (see also Chapter 1).

16.9.7.3 Antioxidant Effects Exercise is known to increase ROS production by neutrophils and this induces cell death of susceptible immune cells (Cury-Boaventura et al., 2008). With heavy exercise the effect is exacerbated as glutathione (GSH) levels are also reduced, consequently the oxidative stress-induced apoptosis in lymphocytes and thymocytes is significantly increased (Cury-Boaventura et al., 2008). WP is frequently used to enhance antioxidant defences (Bartfay, Davis, Medves, & Lugowski, 2003; Kerasioti et al., 2012). The antioxidant activity exhibited by WP occurs via the presence of proteins such as α-La which can chelate heavy metals, leading to the reduction of oxidative

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stress because of its iron-chelating properties but perhaps more importantly through provision of the AA required for GSH synthesis (Kerasioti et al., 2014).

16.9.7.4 WP as a Provision for Increasing Glutathione WP contains an ample supply of cysteine and methionine and also contributes glutamine (which can be converted to glutamate): AA required for GSH synthesis. When levels of cysteine are low, this AA is preferentially used for protein synthesis and hence provision of cysteine through WP is able to enhance GSH biosynthesis. In addition, WP induces the synthesis of glutathione peroxidase, the enzyme which converts GSSG into GSH (Kerasioti et al., 2014). Glutathione has strong antioxidant properties, scavenging ROS. It does this through several mechanisms: firstly, through GSH peroxidase catalyzed reduction of hydrogen- and organic-peroxides; secondly, GSH is able to scavenge OH and singlet oxygen; and finally, GSH is able to directly or indirectly reduce tocopherol radicals by reducing semidihydroascorbate thereby preventing free radical chain reaction and lipid peroxidation (Ji & Leeuwenburgh, 1996). Through these mechanisms, GSH is able to assist the body in combating various diseases (Hoffman & Falvo, 2004) and has the potential to reduce exercise-induced oxidative stress (Cruzat et al., 2014) and associated muscle damage and muscle cell apoptosis. In support of this, a study in young, healthy athletes showed that daily consumption of 20 g of WP for 12 weeks enhanced GSH levels, improved athletic performance, and decreased body fat percentage (Lands, Grey, & Smountas, 1999). Kerasioti et al. (2012) also showed that consumption of a CHO 1 WP supplement (ratio 3.5:1) reduced Thiobarbituric Acid Reactive Substances (TBARS) but not other measured markers of oxidative stress following exhaustive cycling compared to isocaloric CHO intake. Long-term consumption (6 weeks) of WP has also been shown to increase total antioxidant content and GSH levels in resistance-trained subjects (Sheikholeslami & Ahmadi, 2012).

16.10 16.10.1

CONTRAINDICATIONS AND COMPLICATIONS Energy Balance

The satiating effect of protein in the general population is well known and consumption of WP decreases both ratings of hunger prior and caloric intake at subsequent meals (up to 4 hours after consumption) compared to other protein sources such as turkey, egg, or tuna (Akhavan et al., 2010; Devries & Phillips, 2015). A study in nonathletes indicates these short-term satiating effects of WP are most likely due to impact on appetite hormones such as

16.10 Contraindications and Complications

glucagon-like peptide 1 and ghrelin (Lemmens, Martens, Born, Martens, & Westerterp-Plantenga, 2011) and also improved blood glucose control via both insulin-dependent and insulin-independent mechanisms (Akhavan et al., 2010). However, athletes are often already consuming protein at much higher levels than nonathletes and since a high habitual protein intake reduces the effect of gastric emptying and satiety responses, therefore athletes may have different responses to increased protein consumption (MacKenzie-Shalders, Byrne, Slater, & King, 2015). The likelihood of different responses following longterm consumption of WP is supported by work in mice which suggests that the mechanism by which WPI increases satiety following long-term feeding is not via changes in satiety hormones, but by reduction in intestinal length and weight and reduced stomach weight possibly mediated by changes in gastric expression of Wnt5a and Fzd4 (McAllan, Speakman, Cryan, & Nilaweera, 2015). A study in resistance-trained athletes investigating the effects of WP supplementation (20, 40, 60, 80 g) in liquid form on satiety and food intake showed that increasing the WP supplement dose above 20 g did not result in any further increase in satiety or decrease in food intake (MacKenzie-Shalders et al., 2015). However, there are concerns that frequent, excessive consumption of high protein diets by athletes could compromise intake of important nutrients or could, under certain circumstances, lead to issues in meeting high energy requirements. This could be an issue, e.g., in athletes who preload with protein as a preload of 20 g of WP has been shown to reduce subsequent energy intake by 150% (Akhavan et al., 2010).

16.10.2

Lactose Intolerance, Allergies, Dairy-Free Diets

WPC contains lactose (to varying extents, depending on degree of processing) and therefore may not be suitable for consumption by people who are lactose intolerant, nor is it suitable for people who suffer from dairy milk protein allergies. Children constitute a vulnerable group for cow milk protein allergy, with 2% 7% developing this allergy (Patel, 2015), and can lead to atopic dermatitis (eczema). However, enzymatically hydrolyzed WP may be better tolerated as they are less allergenic. Duan, Yang, Li, Zhao, and Huo (2014) showed that mice fed with WPH had lower allergenicity relative to WPC. However, the hydrolysis process produces bitter peptides which make the product less palatable. Although ultrafiltration can be used to remove the larger bitter peptides this process will also result in removal of potentially useful peptides (e.g., bioactive peptides) and protein as well as making the product costlier.

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An alternative for athletes who avoid dairy products for any reason are the many available plant-based protein sources, although a large number contain lower levels of leucine (6% 8%) than animal-based proteins (8% 11%) and therefore may not stimulate MPS to a similar extent as WP (Joy et al., 2013). The most popular plant-based protein source is soy protein as it is a higher quality protein than other vegetable proteins and SPI is the preferred product type, as it contains the highest protein content at 90% (Paul, 2009). A number of studies carried out in resistance-trained subjects have shown that supplementation with either SPI or WP achieve similar LBM gain (Paul, 2009). Rice protein isolate has been shown to support similar changes in strength and body composition as WPI in resistance training subjects (Joy et al., 2013).

16.10.3

Other Health Complications

Consumption of very high levels of any protein can impair gastric emptying and thus lead to gastrointestinal distress, which may in turn impact on sports performance (Schroer et al., 2014), hence excessive consumption of WP may also lead to gastrointestinal issues. Dietary supplementation with WP has been reported to precipitate acne flare in some teenagers. Silverberg (2012) reported five cases of male patients (14 18 years) experiencing the onset of acne shortly after initiation of WP supplementation. Three of the teenagers used the supplement for muscle building in football training and the other two were attempting to gain weight. The condition generally resolved after discontinuing the product and reoccurred if consumption of the product was restarted (Silverberg, 2012). It was suggested that WP may be the fraction of dairy products that promotes acne formation (Silverberg, 2012). Work in nonexercising rats suggests consumption of high levels of WP (252 g/kg diet) may result in adverse effects, with increased apoptosis observed after only 5 days’ consumption and increased inflammatory markers and hepatotoxicity after consumption for 4 weeks (Gürgen et al., 2015). Although consumption of WP can cause issues such as allergies in some individuals, it appears that for most people dietary supplementation with WP will not result in deleterious effects unless intake is excessive.

16.11 LIMITATIONS OF RESEARCH AND FUTURE DIRECTIONS Clearly, not all studies show ergogenic effects of WP ingestion which may relate to the degree of experimental control. On the one hand, strict control

16.11 Limitations of Research and Future Directions

of dietary and training patterns is required to isolate the effect of the treatment, especially for long-term training studies (which makes them expensive and difficult to conduct); however, athletes may also exercise after observing an overnight fast or after suboptimal energy and/or CHO intake. These practices may reduce the generalizability of findings to “real-world” practice (i.e., poor ecological validity), especially as the diet of many sportsmen and women already contains the optimal amount of proteins (Laskowski & Antosiewicz, 2003). Furthermore, applying research findings from elite/well-trained athletes to most recreational exercisers (and vice versa) is problematic. Well-trained individuals may display a “ceiling effect” whereby gains in muscle mass become progressively more difficult as the athlete reaches his/her genetic hypertrophic potential (Schoenfeld et al., 2013). Recreational exercisers (vast majority of population) may be unable to maintain the rigors of homogenous training regimes or may not be as motivated as elite athletes to maintain precise supplementation regimes (Pasiakos et al., 2015). Moreover, neural adaptations are likely to contribute to improvements in muscle strength during the initial few weeks of training in previously untrained people (Narici et al., 1996). Therefore, the results of studies must be placed into context and practitioners and manufacturers should present the findings in appropriate ways for the consumer. Many studies have also used small sample sizes, especially those examining protein turnover or involving long-term training, and therefore may not have been sufficiently powered to detect the effects of WP supplementation (Erskine et al., 2012). Therefore, metaanalyses, which combine the data from several studies, may be used to examine the effectiveness of WP supplementation using a larger subject “pool.” Differences among participants in microRNAs involved in posttranslational control of genes coding for skeletal muscle protein growth are related to the variability observed for changes in LBM and strength performance accompanying training (Pasiakos et al., 2015). Therefore, even though researchers may randomize the subjects into experimental and control groups, it is likely that some or all of the differences observed between groups may be a reflection of those characterized as high or low responders due to differences in microRNA expression rather than to any effect related to WP supplementation. Therefore, finding out whether an individual may or may not respond to training and WP supplementation may potentially be advantageous for the consumer; lifestyle genomic testing may be one way of exploring this further. Moreover, studies involving mRNA expression of other genes, such as upstream and downstream targets of mTOR and E3 ligases, in a larger number of animals could help to clarify the complex network of genes involved in the regulation of muscle growth (Haraguchi et al., 2014).

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The research on WP supplementation, in conjunction with resistance exercise, to attenuate sarcopenia in the ever-growing elderly population is still in its early stages. Furthermore, although protein metabolism is similar between young men and women, older women seem to show reduced capacity and responsiveness to anabolic signaling in response to WP intake (Smith et al., 2010). Due to the aging population worldwide, further research exploring WP ingestion and interactions between age, sex, and exercise is warranted.

16.12

CONCLUSIONS

Several conclusions can be made with regard to WP supplementation for sports and exercise: 1. WP leads to greater improvements in strength and muscle mass in athletes, compared to contrast supplements, when combined with prolonged resistance training. This is due to several potential reasons including higher leucine content, enhanced insulin signaling, and improved mTOR phosphorylation and mTOR mRNA transcription. 2. WP is superior to other proteins due to inclusion of all EAA, high BCAA content, and the highest amount of leucine. For optimal MPS, a “leucine threshold” of 2 3 g must be reached, which can be found in 20 g WP. The addition of leucine to a smaller dose of WP can be as effective as ingesting a larger dose of WP. 3. WP is a fast-digesting protein whereas casein is a slow-digesting protein. Some evidence suggests consuming a protein blend (containing “slow” and “fast” proteins) may stimulate an initial anabolic stimulus (from high-leucine-containing WP) and prolonged anabolism from the slower-releasing casein or soy proteins. 4. There may be an “anabolic window” within the first few hours’ postresistance exercise when athletes should consume WP for optimal MPS. However, when controlling for other covariates, the prevailing view is that the total amount of WP ingested is more important than whether protein is consumed before, during or after exercise. 5. Athletes have a higher recommended protein intake (1.2 2.0 g/kg body weight per day) than nonexercising individuals (0.8 g/kg body weight per day). The optimal dose of WP intake for maximal MPS is 20 g for young adults; any additional protein is oxidized and results in urea production (i.e., “muscle full effect”). Athletes can overcome the “muscle full effect” by consuming 20 g bolus of WP every 3 hours following resistance exercise. 6. WP ingestion can create an enhanced anabolic state within muscle after endurance exercise, which may beneficially affect the recovery of muscle function and muscle repair. However, the addition of WP to CHO drinks during and following endurance exercise is only beneficial

16.12 Conclusions

7.

8.

9.

10.

11.

12.

13.

when the delivery of CHO is less than optimal. Nevertheless, adding WP to CHO may help with repairing damaged muscle and lead to improvements in subsequent endurance performance. Coingestion of WP with other bioactives such as caffeine, creatine, and vitamin D has been shown to benefit anaerobic and aerobic performance, strength performance, and muscle mass, and reduce risk of fractures and falls due to increased muscle mass and strength in elderly individuals, respectively. Changes in muscle mass and muscle strength during the initial weeks of resistance training are not influenced when WP supplements are provided to untrained individuals. This is because initial improvements in muscle strength following resistance exercise is due to neurological changes with muscle hypertrophy-related adaptations leading to strength gains after several weeks of training. For team sport athletes, WP supplementation may improve anaerobic power, increase muscle mass, provide fuel during exercise, and help with muscle repair and muscle glycogen recovery following exercise. WP intake can increase satiety and, in conjunction with resistance exercise, can increase lean muscle mass (thus increase resting energy expenditure) and reduce fat mass, and therefore aid in weight loss and weight management. Older adults’ muscles may be less sensitive to anabolic stimulus (i.e., “anabolic resistance”) and so require a higher WP intake (1.0 1.5 g/kg body weight per day) than younger adults (0.8 g/kg body weight per day). Older people have a higher “leucine threshold” and thus require greater amount of leucine to stimulate MPS at rest and following resistance exercise. To enable better MPS throughout the day (and to attenuate age-related losses in muscle mass and strength) older individuals should consume sufficient WP in regular boluses throughout the day. Young men and women of a similar health status and BMI have similar protein turnover rates. However, older women have smaller muscle mass but the age-associated decrease is slower. Elderly women have an inability to increase MPS in response to protein feeding, possibly due to reduced capacity and responsiveness to anabolic signaling, but more research is needed on the effects of WP intake and the interactions of age, sex, and exercise. Children require adequate protein for normal growth and development which should be met by consuming a balanced diet. However, if adequate protein intake is not achieved then protein may be used as a substrate rather than for synthesis of new tissue. More research is needed on whether WP supplementation may be useful in children and adolescents under certain conditions.

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14. Some types of WP contain lactose and certain proteins which may not be tolerated by all individuals. However, WPH may be better tolerated, as they are less allergenic but produces bitter peptides which make the product less palatable (and the process makes the product much costlier). 15. WP intake during exercise, if provided in too high a quantity (and without CHO), may cause gastrointestinal distress and lead to reductions in performance.

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Pereira, P. C. (2014). Milk nutritional composition and its role in human health. Nutrition, 30, 619 627. Persistence Market Research. (2017). Global market study on sports nutrition: Revenues to exceed US $37 bn by 2019 end, sports drinks continue to fuel market growth. ,http://www.persistencemarketresearch.com/market-research/sports-nutrition-market.asp. Accessed May 2017. Petrie, H. J., Stover, E. A., & Horswill, C. A. (2004). Nutritional concerns for the child and adolescent competitor. Nutrition, 20, 620 631. Peyrollier, K., Hajduch, E., Blair, A. S., Russell, H., & Hundal, H. S. (2000). L-leucine 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-leucine-induced up-regulation of system A amino acid transport. Biochemical Journal, 350, 361 368. Phillips, S. M. (2016). The impact of protein quality on the promotion of resistance exerciseinduced changes in muscle mass. Nutrition & Metabolism, 13, 64. Phillips, S. M., Hartman, J. W., & Wilkinson, S. B. (2005). Dietary protein to support anabolism with resistance exercise in young men. Journal of the American College of Nutrition, 24, S134 S139. Pordesimo, L. O., & Onwulata, C. I. (2009). Whey texturization for snacks. In C. I. Onwulata, & P. J. Huth (Eds.), Whey processing, functionality and health benefits (pp. 169 184). Chichester: John Wiley & Sons. Potgieter, S. (2013). Sport nutrition: A review of the latest guidelines for exercise and sport nutrition from the American College of Sport Nutrition, the International Olympic Committee and the International Society for Sports Nutrition. South African Journal of Sports Nutrition, 26, 6 16. Power, O., Hallihan, A., & Jakeman, P. (2009). Human insulinotropic response to oral ingestion of native and hydrolysed whey protein. Amino Acids, 37, 333 339. Pupovac, J., & Anderson, G. H. (2002). Dietary peptides induce satiety via cholecystokinin-A and peripheral opioid receptors in rats. Journal of Nutrition, 132, 2775 2780. Rasmussen, B. B., Tipton, K. D., Miller, S. L., Wolf, S. E., & Wolfe, R. R. (2000). An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise. Journal of Applied Physiology, 88, 386 392. Reidy, P. T., Walker, D. K., Dickinson, J. M., Gundermann, D. M., Drummond, M. J., Timmerman, K. L., . . . Jennings, K. (2013). Protein blend ingestion following resistance exercise promotes human muscle protein synthesis. The Journal of Nutrition, 143, 410 416. Reidy, P. T., Walker, D. K., Dickinson, J. M., Gundermann, D. M., Drummond, M. J., Timmerman, K. L., . . . Rasmussen, B. B. (2014). Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle. Journal of Applied Physiology, 116, 1353 1364. Rennie, M. J., Bohe, J., Smith, K., Wackerhage, H., & Greenhaff, P. (2006). Branched-chain amino acids as fuels and anabolic signals in human muscle. Journal of Nutrition, 136, S264 S268. Rindom, E., Nielsen, M. H., Kececi, K., Jensen, M. E., Vissing, K., & Farup, J. (2016). Effect of protein quality on recovery after intense resistance training. European Journal of Applied Physiology, 116, 2225 2236. Rodriguez, N. R., Di Marco, N. M., & Langley, S. (2009). American College of Sports Medicine position stand. Nutrition and athletic performance. Medicine & Science in Sports & Exercise, 41, 709 731. Rondanelli, M., Klersy, C., Terracol, G., Talluri, J., Maugeri, R., Guido, D., . . . Perna, S. (2016). Whey protein, amino acids, and vitamin D supplementation with physical activity increases

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CHAPTER 17

Whey Proteins in Functional Foods Ranjan Sharma OzScientific Pty Ltd, Hoppers Crossing, VIC, Australia

17.1

INTRODUCTION

Functional foods, although having no widely recognized definition, refer to foods and ingredients with benefits over and above their nutritional value. The term functional foods originated in Japan, which was the first country to allow health claims for food products under the category Foods for Specific Health Use (FOSHU) in 1991 to combat the increased cost of health care. In the mid-1990s, the International Life Sciences Institute (ILSI) Europe coordinated a functional food project that was known as FUFOSE (Functional Food Science in Europe) involving about 100 European experts in nutrition and medicine who critically assessed the state of the science in functional foods (Ashwell, 2002). These experts then worked on a concept of functional foods and elaborated, for the first time, a global framework that included a strategy for the identification and development of functional foods and for the scientific substantiation of their effects, with the objective of justifying health-related claims. ILSI has defined functional foods as physiologically active food components which provide health benefits. Another global organization, International Food Information Council (IFIC), defines the functional foods as dietary components with a health benefit beyond basic nutrition (Hassler, 1998). The term functional foods is synonymous with nutraceuticals and bioactives which refer to components with perceived health benefits. These are available as powders, drinks, or nutritional supplements and can be administered by consumers themselves. Another related group, so-called medical foods, however, refer to specialized nutrition products which are used in dietary management of a disease or condition with distinct nutritional requirements. Medical foods are administrated under medical supervision in nursing homes and hospitals. Functional foods are available at supermarkets, pharmacies, 637 Whey Proteins. DOI: https://doi.org/10.1016/B978-0-12-812124-5.00018-7 © 2019 Elsevier Inc. All rights reserved.

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online stores, and major retailers while medical foods are available on prescription in hospitals, pharmacies, and online stores. Excessive nutrition, poor lifestyle choices, such as poor diet, lack of physical activity, and inadequate relief of chronic stress are key contributors in the development and progression of preventable chronic diseases, including obesity, Type 2 diabetes, hypertension, cardiovascular diseases, and several types of cancer. According to Health, Nutrition, and Population Family of the World Bank’s Human Development Network (Nikolic, Stanciole, & Zaydman, 2011), these noncommunicable, lifestyle diseases are on the rise not only in developed countries but also in developing and poor countries (Fig. 17.1). In high-income countries, lifestyle diseases have long been the leading cause of mortality and morbidity. However, by 2030, lifestyle diseases are expected to account for three-quarters of the disease burden in middle-income countries, approaching the level of high-income countries. In absolute terms, deaths from lifestyle diseases in middle- and low-income countries are projected to be 43 million by 2030. By 2030, cancer incidence is projected to increase by 70% in middle-income countries, 82% in low-income countries, and 40% in high-income countries (WHO, 2014). As the leading cause of death globally, lifestyle diseases were responsible for 38 million (68%) of the world’s

Percent of total death due to lifestyle diseases 100 90

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FIGURE 17.1 Comparison of deaths caused by noncommunicable lifestyle diseases between 2008 and 2030. Based on Nikolic, I. A., Stanciole, A. E., & Zaydman, M. (2011). Chronic emergency: Why NCDs matter. World Bank Health, Nutrition and Population Discussion Paper.

17.2 Whey Proteins

56 million deaths in 2012. More than 40% (16 million) were premature deaths under the age of 70. Almost three-quarters of all lifestyle-related disease deaths (28 million), and the majority of premature deaths (82%), occur in low- and middle-income countries (WHO, 2014). Interest in functional foods has grown significantly in the past 10 years due to a growing awareness of the correlation between diet and health, and factors contributing to increased health care costs such as aging population, sedentary lifestyle, and associated diseases. The market for functional foods has been growing rapidly since it is now believed that functional foods may aid in prevention of many chronic diseases and help provide benefits in maintaining healthy lifestyles. Fortified/functional packaged food was valued at $159 billion globally in 2016, making it the largest category within the health and wellness industry (Mascaraque, 2016). Whey proteins, once considered waste from cheese manufacture, have proven to be a source of bioactive components, and when used in functional foods can potentially lead to prevention of lifestyle diseases (Madureira, Pereira, Gomes, Pintado, & Malcata, 2007; Marshall, 2004; McIntosh et al., 1998; Patel, 2015; Sharma & Shah, 2010; Solak & Akin, 2012; Steijns, 2001).

17.2

WHEY PROTEINS

Whey proteins, which represent 20% of the total protein content of bovine milk, are commonly sold as a nutritional supplement, especially popular for sports and bodybuilding applications (see also Chapter 16: Sports and Exercise Supplements for more information on these applications). Whey contains five major proteins β-lactoglobulin (β-La), α-lactalbumin (α-La), immunoglobulins (Igs), bovine serum albumin (BSA), and proteose peptone and, if the whey is from cheese manufacture, glycomacropeptide (GMP), which together make up 85% of whey protein (Fig. 17.2) (see Chapter 1 for a description of the major and minor whey proteins). Commercially whey proteins are available in a range of protein-rich ingredients starting from whey powder (with the lowest amount of protein) to over 98% purified individual protein fractions. Fig. 17.3 shows a schematic description of current range of whey-protein-based ingredients with physiological functional activities.

17.2.1

β-Lactoglobulin

β-Lg is the predominant protein in whey representing approximately half of the total whey proteins in bovine milk. Although it can be found in the milk of many other mammals, it is essentially absent in human milk (Guo, 2014).

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Whey proteins in acid and sweet whey Other proteins Enzymes GMP Immunoglobulins Bovine serum albumin α-Lactalbumin β-Lactoglobulin 0

10

20

30

40

50

60

% of total whey protein Sweet whey

Acid whey

FIGURE 17.2 Proportion of individual proteins in whey proteins derived from acid and sweet (cheese) whey. Based on Patel, H., & Patel, S. (2015). Understanding the role of dairy proteins in ingredient and product performance. Technical Report. US Dairy Export Council. ,http:// www.thinkusadairy.org/resources-and-insights/resources-and-insights/application-and-technical-materials/technical-report-understandingthe-role-of-dairy-proteins-in-product-performance. Accessed 05.04.17 (Patel & Patel, 2015).

FIGURE 17.3 Range of whey protein ingredients with increasing commercial potential and physiological functional properties. WPC, whey protein concentrate (35% 80% protein); WPI, whey protein isolate (90% protein); WPH, whey protein hydrolysate (80% 90% protein).

17.2 Whey Proteins

In food and beverage applications, β-Lg can be used as a functional ingredient with excellent gelling properties which can be used as structuring and stabilizer agents in dairy products such as yogurts and cheese spreads (Chatterton, Smithers, Roupas, & Brodkorb, 2006). β-Lg has also been found to be resistant to gastric digestion remaining stable in the presence of acids and proteolytic enzymes present in the stomach (Barros, Ferreira, Silva, & Malcata, 2001; Sawyer & Kontopidis, 2000); hence, it tends to remain intact during passage through the stomach. It is also a rich source of cysteine, an amino acid bearing a key role in stimulating synthesis of glutathione (GSH), which is composed of three amino acids, glutamine, cysteine, and glycine (Hernandez-Ledesma, Recio, & Amigo, 2008). Besides being a source of essential and branched chain amino acids, β-Lg is an important source of biologically active peptides that are inactive within the native sequence of the protein, but can be released by in vivo or in vitro enzymatic hydrolysis. Once released and absorbed, these peptides may play important roles in human health promoting antihypertensive, antioxidant, and antimicrobial activities (Hernandez-Ledesma et al., 2008) (see Chapter 14: Bioactive Peptides for further information on bioactive peptides). While investigating the effects of proteolysis of β-Lg using a range of commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and corolase), Hernandez-Ledesma, Davalos, Bartolome, and Amigo (2005) found that a combination of pepsin, trypsin, and chymotrypsin was the most effective in producing β-Lg hydrolysates with the most antioxidant activity. The evidence for antimicrobial activity (the ability to activate the microbial autolytic system) and immunostimulatory activity (the ability to improve the phagocytic cell functioning) of β-Lg hydrolysate was recently presented by Biziulevicius, Kislukhina, Kazlauskaite, and Zukaite (2006). According to the authors, the hydrolysates acted antimicrobially in vitro by stimulating the autolytic system of all 20 naturally autolyzing and four naturally nonautolyzing microbial (bacterial and fungal) strains tested. When β-Lg hydrolysate was given to mice, it was shown to enhance the phagocytosing capacity of peritoneal macrophages. The authors suggested that immunostimulating activity of β-Lg hydrolysates was a consequence of their antimicrobial activity (Biziulevicius et al., 2006). β-Lg has also been shown to have a potential for binding small hydrophobic molecules such as retinoic acid, which has the potential to modulate lymphatic responses (Guimont, Marchall, Girardet, & Linden, 1997). Other potential functionalities of β-Lg include being a precursor for several opioid and ACE-inhibitory peptides (Pihlanto-Leppälä, 2001) and having hypocholesterolemic activity (Nagaoka et al., 2001).

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17.2.2

α-Lactalbumin

α-La is the second most abundant protein in whey representing approximately 20% of whey proteins. It is found in considerable quantity in human breast milk (Guo, 2014). α-La is relatively heat-stable and generally has poor gelling capacity; however, it can be used as a source of essential amino acids. α-La is commercially used in supplements for infant formulae, because of its similarity in structure and composition to the human milk protein (Lönnerdal, 2014). A recent study suggested that the protein content of infant formula may be reduced if the formulation was enhanced with α-La as an amino acid composition similar to human milk can be achieved (Sandström, Lönnerdal, Graverholt, & Hernell, 2008). Table 17.1 shows approximate composition of a commercial α-La powder. α-La has been shown to improve brain function and helps in alleviating stress and depression. As α-La is a tryptophan-rich protein, a diet enriched with α-La increases the ratio of tryptophan to the other large neutral amino acids, which may in turn increase brain serotonin content (Markus et al., 2005). Increased brain serotonin may improve the ability to cope with stress, whereas a decline in serotonin activity is involved in depressive mood (Merens et al., 2005). A diet rich in α-La has been found to increase the ratio of tryptophan/large neutral amino acids, and to improve cognitive functioning in individuals with high neuroticism scores (Markus et al., 2000). A study by Booij, Merens, Markus, and Van der Does (2006) investigated the effects of an α-La-enriched diet on cognition in recovered depressed patients. Mood, cognitive function, and plasma amino acids were assessed before and after dietary intake. The results suggested that although α-La had no effect on mood, it improved abstract visual memory and impaired simple motor performance.

Table 17.1 Approximate Composition of Commercial α-La Powder Component

Amount

Moisture, % Protein, % α-La, % of total protein Tryptophan, % Fat, % Lactose, %

5 90 90 5 0 0

Based on commercial ingredient specification sheets.

17.2 Whey Proteins

17.2.3

Bovine Serum Albumin

BSA is a large molecular weight protein that makes up approximately 10% of total whey proteins in milk. Because of its size and higher levels of structure, BSA can bind free fatty acids and other lipids, as well as flavor compounds; however, this feature is severely hampered upon denaturation (Kinsella & Whitehead, 1989). Like other milk proteins, BSA is a source of essential amino acids but its applications in functional foods have not been explored. There are limited studies investigating the therapeutic potential of BSA (Krissansen, 2007). Laursen, Briand, and Lykkesfeldt (1990) showed that several commercial BSA preparations had an inhibitory potential on growth of the human breast cancer cell line, MCF-7. Serorphin (Tyr-Gly-Phe-Gln-AsnAla) (f399 404), a peptide from BSA, has been shown to have opioid agonist activity (Meisel, 2005). Another BSA peptide, albutensin A or serokinin (Ala-Leu-Lys-Ala-Trp-Ser-Val-Ala-Arg) (f208 216), has been found to be an ACE-inhibitor, and is reported to have ileum-contracting and ileum-relaxing activities (Meisel, 2005).

17.2.4

Immunoglobulins

Igs, which are derived from bovine blood serum, represent 8% 10% of total whey proteins in milk. They are made up of three distinct classes: IgM, IgA, and IgG (IgG1 and IgG2). In bovine milk and colostrum, IgG1 is the major immunoglobulin while in human milk it is IgA (Fox & McSweeney, 1998). The physiological function of Ig is to provide various types of immunity in the body (El-Loly, 2007). Immunoglobulins are transferred from mother to child in utero via cord blood and by breastfeeding, and serves as a child’s first line of immune defence. Immunoglobulins are partially resistant to proteolytic enzymes, and are not inactivated by gastric acids (Korhonen, Marnila, & Gill, 2000). An in vitro study demonstrated bovine milk-derived IgG suppresses human lymphocyte proliferative response to T cells at levels as low as 0.3 mg/mL of IgG (Kulczycki & MacDermott, 1985). A number of controlled clinical studies have shown that oral administration of hyperimmune milk preparations containing high levels of specific antibodies can provide effective protection, and to some extent may also be of therapeutic value, against gastrointestinal infections in humans (Korhonen et al., 2000; Lilius & Mamila, 2001).

17.2.5

Glycomacropeptide

GMP is a hydrophilic peptide (amino acid residue 102 169) of κ-casein that provides stability to casein micelles in milk (Brody, 2000). When rennet acts on κ-casein during cheese manufacture, GMP is released into the whey. GMP makes up about 15% 20% of the whey proteins. Recent advances in

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fractionation have allowed separation of GMP from cheese whey into commercial GMP-enriched ingredients. Due to the highly negative charge of GMP at low pH where whey proteins are positively charged, GMP can be isolated by an ion exchange process. When whey at pH 3 is treated with a cation exchanger, the GMP is not adsorbed and may be concentrated and desalted by ultrafiltration. Alternatively, GMP from whey at pH less than 4 can be bound to an anion exchanger while the rest of the whey proteins are not bound. Pure GMP can then be eluted from the ion exchanger. The approximate composition of a commercial GMP is shown in Table 17.2. GMP is unique among the whey proteins in that it is a glycoprotein and thus, has an oligosaccharide chain attached to it. It also is unique because it contains no phenylalanine, tryptophan, or tyrosine. GMP also has high levels of the branched-chain amino acids, leucine, isoleucine, and valine. This composition of GMP gives it some unique characteristics that can be utilized in a variety of interesting applications. A small proportion of the population has phenylketonuria (PKU), meaning they are unable to digest phenylalanine. GMP is one of the few amino acid sources PKU patients can tolerate because pure GMP does not contain phenylalanine (Van Calcar & Ney, 2012). Published research has linked GMP with many physiological functions, including: promotion of growth of bifidobacteria; suppression of gastric secretions; inhibition of bacterial and viral adhesion; modulation of immunesystem responses; and prevention of binding of cholera and Escherichia coli enterotoxins. In simpler terms, GMP offers potential benefits to intestinal health, appetite control, reduced dental caries, enhanced immunity, and protection against diarrhea. Some of the bioactive properties of GMP are: antiinflammatory (Daddaoua et al., 2005), toxin binding (Kawasaki et al., 1992); inhibition of bacterial and viral adhesion (Neeser, Chambaz, Vedovo, Prigent, & Guggenheim, 1988); immune modulation and protection against diarrhea (Otani, Monnai, Kawasaki, Kawakami, & Tanimoto, 1995); and as a source of amino acids for people suffering from phenylketonuria (Ney et al., 2009).

Table 17.2 Approximate Composition of Commercial GMP Powders Component

Amount

Moisture, % Protein, % GMP, % of total protein Sialic acid, %

5 80 90 4

Based on commercial ingredient specification sheets.

17.2 Whey Proteins

17.2.6

Lactoferrin

Lf is an iron-binding glycoprotein present in colostrum, milk, and whey. Lf exists as a single peptide chain with a molecular weight of approximately 80,000 Da (Adlerova, Bartoskova, & Faldyna, 2008). It is folded into two globular units with each unit able to bind 1.4 mg of iron per gram of protein. Bovine Lf is somewhat similar in structure to the human form, having approximately 70% of the same amino acids. The iron-binding ability of Lf is responsible for many biological functions such as its bacteriostatic effect, growth-promoting effect on certain cell lines, prevention of lipid peroxidation, and promotion of iron absorption in the body (Levay & Viljoen, 1995). Lf is one of few proteins in whey that is positively charged at pH 7.0 (isoelectric point of approximately pH 7.9) while most other proteins are negatively charged. This feature of Lf has been exploited in commercial isolation of Lf. Using cation-based resins and selective salt solutions, Lf can be separated from other positively charged proteins attached to the resin. Further concentration of Lf is carried out using ultrafiltration and spray drying. When reduced to its purest form, it is pink in color. Commercially, Lf is available in a range of protein concentrations. Due to the low amount present in milk and whey, the cost of separation is high and therefore, ingredient cost is high. Table 17.3 shows the approximate composition of commercial Lf powders. Lf can provide several physiological functional (bioactive) properties, which are mainly derived from its ability to bind iron. Lf inhibits the growth of pathogenic bacteria and fungi, due to its ability to bind large quantities of iron (Arnold, Brewer, & Gauthier, 1980; Kirkpatrick, Green, Rich, & Schade, 1971). Lf binds iron very strongly, thus rendering this essential nutrient unavailable to support microbial growth. Lf also disrupts bacterial digestion of carbohydrates, further limiting bacterial growth. In addition, the action of Table 17.3 Approximate Composition of Commercial Lf Powders Component

Amount

Moisture, % Protein, % Lf, % of total protein Iron, % Ash, % Fat, % Lactose, %

5 95 90 13 1 ,1 ,1

Based on commercial ingredient specification sheets.

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pepsin in the stomach converts Lf into lactoferricin, which has broadspectrum activity against pathogenic bacteria and yeast (Madureira et al., 2007). Lf also can bind to parasites and the outer membrane of Gramnegative bacteria, making the cell wall more permeable and, thus improving the efficiency of antibiotics (Omata et al., 2001). Lf contributes to the defence against pathogens by activation of cells involved in the antiinflammatory response during the course of microbial infection, thus enhancing self-immunity (Levay & Viljoen, 1995). Commercial Lf is suitable for applications in health supplements, functional foods and drinks, infant formulas, cosmetics, and oral care products, as well as for animal feed. Examples of potential markets for Lf are: supplements for the elderly or immune-compromised patients; supplements for recovery from gastrointestinal infections; products used to stimulate the body’s immune system to help deal with toxic environments, disorders, or treatments; and prophylactic products for travelers’ diarrhea.

17.2.7

Lactoperoxidase

Lactoperoxidase (EC 1.11.1.7) (Lp) is an enzyme present in colostrum and milk, with a molecular weight of approximately 77.5 kDa (Sharma et al., 2013). Bovine colostrum and milk contain about 11 45 and 13 30 mg/L Lp respectively (Pakkanen & Aalto, 1997). In whey, Lp constitutes approximately 0.5% of whey proteins (de Wit & van Hooydonk, 1996). The biological significance of Lp is its involvement in the natural host defence system against invading microorganisms. Separation of Lp from whey is based on the same principle as used for isolation of Lf. Lp is positively charged at the normal pH of whey (isoelectric point in the pH range 9.0 10.0) and can be bound to cation exchange resins and fractionated from the rest of the whey proteins. Lp inactivates a wide spectrum of microorganisms through an enzymatic action. This reaction involves two cofactors, hydrogen peroxide and thiocyanate ions, which, together with Lp, constitute the LP system (Bafort, Parisi, Perraudin, & Jijakli, 2014). Activation of the enzyme results in the formation of hypothiocyanite ions, which are responsible for the antimicrobial action. The reaction of LP system relies on the production of short-lived intermediary oxidation products of the thiocyanate ion (OSCN ) that reacts with bacterial cytoplasmic membranes, and impairs the function of metabolic enzymes. As addition of H2O2 is not permitted in certain countries, in situ development of H2O2 is carried out by the addition of glucose oxidase (a permitted additive). The thiocyanate ion can be either naturally present (as in the case of animal tissues and plant), or added as sodium or

17.3 Health Benefits of Whey Proteins

Table 17.4 Approximate Composition of Commercial Lp Powders Component

Amount

Moisture, % Protein, % LP, % of total protein LP activity (ABTS method), U/mg protein Ash, %

6.8 91 83 270 2

Based on commercial ingredient specification sheets.

potassium thiocyanate. Commercially, Lp is isolated from either skim milk or whey using an ion-exchange process similar to that used for isolation of Lf. The basic principle underlying the process is the fact that Lp has an isoelectric pH in the alkaline range (9.0 9.5) which means that it is positively charged at the normal pH of cheese whey (6.0 6.6) while the rest of the proteins are negatively charged. This difference in isoelectric pH is used to adsorb Lp to an anion exchange column to separate it from other proteins. The gross composition of a commercial Lp is shown in Table 17.4. Lp when used in the form of the LP system has a broad spectrum of antibacterial activity, having a bacteriostatic effect against Gram-positive bacteria and a bactericidal effect against Gram-negative microorganisms, e.g., pseudomonads, coliforms, Salmonella, and Listeria (Reiter & Harnulv, 1984). Potential applications of Lp include its use with the LP system activating ingredients (thiocyanate and hydrogen peroxide) in toothpaste formulations to protect against oral streptococci (Reiter & Harnulv, 1984). The LP system has shown protection against contamination of poultry with Campylobacter jejuni during slaughter (Borch, Wallentin, Rosen, & Bjorck, 1989). The Lp system has also been found to be effective in the bacterial clearance of airways, indicating a possible application in patients suffering from cystic fibrosis (Gerson et al., 2000; Wijkstrom-Frei et al., 2003).

17.3 17.3.1

HEALTH BENEFITS OF WHEY PROTEINS Cancer

A number of epidemiological and experimental studies have indicated that milk proteins exert an inhibitory effect on the development of several types of cancers. Some recent experiments in rodents indicate that the antitumor activity of the dairy products is in the protein fraction and more specifically in the whey protein component of milk. The impact of whey proteins upon

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cancer prevention has been thoroughly reviewed by Bounous, Batist, and Gold (1991) and Gill and Cross (2000). Several studies have demonstrated that whey protein feeding of mice specifically enhances immune response to sheep red blood cells (Bounous & Kongshavn, 1985, 1989; Bounous, Kongshavan, & Gold, 1988; Bounous, Batist, & Gold, 1989; Bounous, Gervais, Amer, Batist, & Gold, 1989). Rats with colon cancer fed with whey protein hydrolysate developed significantly less macroscopic and microscopic tumors compared to the group fed with untreated whey protein (Attaallah, Yilmaz, Erdoğan, Yalçin, & Aktan, 2012). Studies have shown that whey protein is superior to other dietary proteins for suppression of tumor development (Parodi, 2007). In the presence of Lf, colon cancer induced in rats showed reduced tumor expression (Sekine et al., 1997) and metastasis of primary tumors in mice was inhibited (Yoo et al., 1998). Benefits of BSA in inhibition of growth in human breast cancer have been demonstrated in an in vitro study (Laursen et al., 1990). Whey protein’s anticancer potential is believed to derive largely from the antioxidant, detoxifying, and immune-enhancing effects of GSH and Lf (Bounous et al., 1991; Marshall, 2004). Bounous et al. (1991) hypothesized that cysteine in whey proteins helps in replenishment of GSH during its depletion in immune deficiency states. Cysteine is considered a crucial limiting amino acid for intracellular GSH synthesis. Whey proteins, thus have the ability to increase GSH concentration in relevant tissues, and may exert an antitumor effect via stimulation of immunity through the GSH pathway. It is considered that oxygen radical generation is frequently a critical step in carcinogenesis; hence the effect of GSH on free radicals, as well as carcinogen detoxification, could be important in inhibiting carcinogenesis induced by a number of different mechanisms (Bounous et al., 1991).

17.3.2

Immune Health

Whey proteins are rich in free cysteine which enhances the production of GSH which is important in immune regulation. Cell culture studies and in vivo studies have demonstrated that whey proteins may enhance nonspecific and specific immune responses (Gomez, Ochoa, Herrera-Insua, Carlin, & Cleary, 2002). Studies on the immunomodulatory properties of dietary whey proteins in mice have been reported by several researchers (Bounous et al., 1988; Wong & Watson, 1995). Ingestion of bovine milk whey proteins, either as a supplement in an adequately balanced commercial diet or as the only protein source in a balanced diet, consistently enhanced secondary humoral antibody responses following systemic immunization with ovalbumin, when

17.3 Health Benefits of Whey Proteins

compared with other protein sources such as soybean protein isolate and ovine colostrum whey proteins (Wong & Watson, 1995). Whey protein concentrates enhance innate mucosal immunity during early life and have a protective role in some immune disorders (Pérez-Cano et al., 2007). The ability of whey proteins to modulate immune response in children with atopic asthma was studied by Lothian, Grey, and Lands (2006). The authors hypothesized that a whey-based oral supplement (HMS90) given to children with atopic asthma, a Th2 cytokine disease, would improve lung function and decrease atopy. Whey-based infant formula may also protect infants from atopic dermatitis (Alexander, Schmitt, Tran, Barraj, & Cushing, 2010). It has been reported that as whey proteins can enhance glutathione levels they may help in combating incidences of psoriasis (Prussick, Prussick, & Gutman, 2013) and HIV (Micke, Beeh, Schlaak, & Buhl, 2001). Psoriasis is chronic autoimmune disease causing thick skin, dry scales, and red patches. It was investigated whether bioactive whey protein isolate can increase glutathione levels and resultantly combat the severity of systemic inflammation due to psoriasis. The intake of 20 g/day whey protein isolate improved the condition of the patients (Prussick et al., 2013). HIV infection is characterized by an enhanced oxidant burden and a systemic deficiency of the GSH. The semiessential amino acid cysteine, which is present in whey proteins, is the main source of the free GSH. In a study by Micke et al. (2001), 30 subjects with HIV received a daily dose of 45 g whey protein from one of two sources—Protectamin or Immunocal. These formulas contained cysteine-rich whey protein isolates. After two weeks of oral supplementation, the Protectamin-supplemented group demonstrated significantly elevated glutathione levels, while the Immunocal group had statistically nonsignificant elevations.

17.3.3

Weight Management

Whey protein has potential as a functional food ingredient that can contribute to the regulation of body weight by providing satiety signals which affect both short-term and long-term food intake regulation (Luhovyy, Akhavan, & Anderson, 2007). As whey is an inexpensive source of high nutritional quality protein, the utilization of whey as a physiologically functional food ingredient for weight management is of current interest. Whey proteins are widely used by the weight-loss industry for its protein content alone. The essential and nonessential amino acids in whey act as substrates for protein synthesis and may improve body mass index in individuals participating in exercise programs (Burke et al., 2001).

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Whey proteins appear to have a greater effect on short-term food intake in humans than casein, soy protein, and egg albumin (Anderson, Tecimer, Shah, & Zafar, 2004). The satiety effect of proteins is affected by factors such as dose, form (solid vs liquid), duration to next meal, the formulation of the treatment, the presence or absence of other macronutrients, and in the case of whey, the amount of GMP. For example, whey proteins (45 g, 15% GMP) suppressed food intake more than egg albumin and soy protein at a pizza meal 60 minutes later in young men when the proteins were provided alone in the form of sweetened beverages (Anderson et al., 2004). While investigating the effect of whey proteins and GMP on satiety in adult humans, Lam, Moughan, Awati, and Morton (2009) found some evidence that whey proteins and their components enhanced satiety over a short-term period compared to carbohydrate but there was no consistent effect of either whey proteins alone or GMP. A study by Chungchunlam, Henare, Ganesh, and Moughan (2014) found that GMP is not responsible for the satiety effect of whey proteins. In a 12-week clinical trial, Frestedt, Zenk, Kuskowski, Ward, and Bastian (2008) investigated the effect of a specialized whey fraction (Prolibra, high in leucine, bioactive peptides, and calcium) in reducing body weight. The study results showed that subjects taking Prolibra lost significantly more body fat (6.1% of their body fat) mass and showed a greater preservation of lean muscle compared to subjects consuming the control beverage. This product has, however, failed to receive a health claim from the European Food Safety Authority (EFSA). The EFSA panel concluded that a cause and effect relationship cannot be established between the consumption of Prolibra and “helps to reduce body fat while preserving lean muscle” in the context of energy restriction for weight loss (EFSA, 2012). The effect of whey proteins on satiety may be mediated by its effect on the release of satiety hormones insulin and cholecystokinin (CCK) (Luhovyy et al., 2007). Whey proteins stimulate the release of insulin modifying the glycemic response and plasma concentrations of insulin which are strongly associated with short-term satiety and decreased food intake (Samra, Wolever, & Anderson, 2007). CCK is a well-established satiety hormone; CCK and its A subtype receptor are involved in protein-induced food intake suppression (Miesner, Smith, Gibbs, & Tyrka, 1992). Milk proteins increase CCK concentrations in plasma, with whey proteins showing more effect than caseins (Hall, Millward, Long, & Morgan, 2003).

17.3.4

Osteoporosis

Milk and dairy products are widely recognized for their benefits in osteoporosis due to their high level of bioavailable calcium. A Japanese company,

17.5 Conclusions

Snow Brand Milk has been successful in producing and marketing a whey protein-based ingredient, Milk Basic Protein (MBP), that has shown ability to stimulate proliferation and differentiation of osteoblastic cells as well as suppress bone resorption (Takada, Aoe, & Kumegawa, 1996; Takada et al., 1997; Toba et al., 2000). MBP is prepared from fractionated whey through a cation exchange resin. The total protein concentration of MBP is 98% and contains Lf, Lp, and other minor components. According to Snow Brand Milk, MBP is a multifunctional protein that acts directly and indirectly on bone cells to reinforce the bone itself, making it more receptive to calcium while at the same time preventing excessive calcium from being dissolved from the bones. Several clinical trials support MBP’s positive effects in both men and women, the latter ranging in age from young to postmenopausal (Toba et al., 2001; Uenishi et al., 2007). Daily doses of MBP 40 mg (equivalent to 400 800 mL of milk) appear to be sufficient to produce significantly increased bone mineral density and reduced bone resorption.

17.3.5

Stress and Mental Health

Among the whey proteins, α-La has been shown to improve cognitive performance and mood in stress-vulnerable subjects (Markus, Olivier, & de Haan, 2002; Markus et al., 2000). The main reason for the benefits of α-La is attributed to its high level of tryptophan that acts as a substrate to increase serotonin levels which may be vulnerable to depletion by chronic stress. After the studies, the subjects all showed higher ratios of plasma Trp-LNAA (the ratio of plasma tryptophan to the sum of the other large neutral amino acids), believed to be an indirect indication of brain serotonin function (Markus et al., 2000).

17.4 COMMERCIAL FUNCTIONAL FOODS AND SUPPLEMENTS CONTAINING WHEY PROTEIN INGREDIENTS The health benefits of whey proteins have been commercialized in a range of functional foods and nutritional supplements. A summary of some products containing whey proteins is presented in Table 17.5. Information in Table 17.5 is sourced from the manufacturers’ websites.

17.5

CONCLUSIONS

Whey proteins are versatile nutritional ingredients with a range of health benefits. Besides being the source of essential amino acids, each of the whey proteins has the potential to be used as a food ingredient in functional foods

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Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Commercial Product

Whey Protein and Health Benefits

ThinkitDrinkit

A whey protein isolate powder containing 95% protein with an enhanced level of α-La. The claimed benefit is cognitive support for positive mood As α-La is a tryptophan-rich protein, a diet enriched with α-lactalbumin increases the ratio of tryptophan to the other large neutral amino acids, which may in turn increase brain serotonin content It has a high content (26%) of branched chain amino acids and contains high levels of tryptophan, a precursor to serotonin. Serotonin is regarded by some researchers as a chemical that is responsible for maintaining mood balance, and that a deficit of serotonin may lead to depression (Markus et al., 2005) http://thinkitdrinkit.com/product/lacprodan-alpha-20-cogntive/ Accessed 12 March 2017

bioZzz

bioZzz α-lactalbumin is promoted as a nighttime whey protein. Studies have shown that evening consumption of α-La can improve sleep quality, morning alertness, cognitive performance under stress, and mood under stress (Markus et al., 2000, 2005). According to the manufacturer, a natural sleep aid with 20 g of protein per serving, BioZzz is ideal for those seeking better sleep or a protein supplement for nighttime workouts https://www.biprousa.com/shop/all/biozzz-nsf-certified Accessed 23.03.17

Immunocal

Immunocal is a specially formulated whey protein concentrate derived from whey. It contains glutathione building blocks and is marketed for the treatment of glutathione deficiency, high oxidative stress, and immune deficiency. It also appears to deplete tumor cells of glutathione and render them more vulnerable to chemotherapy (Kennedy, Konok, Bounous, Baruchel, & Lee, 1995). It increases glutathione concentrations in relevant tissues which may combat oxidant-driven tumor growth. Immunocal may enhance immunity and increase plasma glutathione concentrations in individuals infected with hepatitis B (Watanabe et al., 2000) http://www.immunocal.com Accessed 16.03.17

Continued

17.5 Conclusions

Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Continued Commercial Product

Whey Protein and Health Benefits

acneadvance

acneadvance is a bioactive protein complex containing Lf. Lf is a natural, biologically active milk protein isolated from whey. Clinical studies have shown that Lf significantly reduced blemishes, with visual results seen within 2 weeks. According to the manufacturer, acneadvance is an all-natural ingredient that promotes a healthier complexion by enhancing the body’s natural defences to assist in fighting bacteria, and helping to repair damaged cells caused by blemishes http://www.futurebiotics.com/acneadvance.html#description Accessed 18.03.17

ImmunPlex

ImmunPlex is an undenatured whey protein isolate containing α-La, Lf, and β-Lg and is promoted as a source of glutathione. Glutathione is considered to promote immune health. The whey protein isolate used contains 51% β-Lg, 23% α-La, 18% GMP, 5% BSA and Igs, and 2% Lf https://www.prohealth.com/shop/product.cfm/product__code/PH143 Accessed 03.04.17 http://www.boxingscene.com/supplements/50263.php Accessed 03.04.17

IMUPlus

IMUPlus is a pharmaceutical grade ( . 99%) nondenatured whey protein isolate formula: a functional food that provides bioactive precursors for the intracellular production of glutathione, a critical constituent for the immune system and a vital antioxidant and detoxifying agent. IMUPlus utilizes a proprietary process to attain a product containing over 99% nondenatured whey protein, including bioactive Lf, lysozyme, Lp, GMP, α-La, and BSA Glutathione has been found to enhance immune function, eliminate toxins and carcinogens, increases antioxidant and ionizing radiation protection, and supports DNA synthesis and repair, protein prostaglandin and leukotriene synthesis, amino acid transport, and enzyme activity and regulation https://www.drugs.com/drp/whey-protein-isolate.html Accessed 26.03.17

Continued

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Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Continued Commercial Product

Whey Protein and Health Benefits

Prolibra 290

Prolibra 290 is a whey protein isolate-based powder manufactured by Glanbia Nutritionals. It is promoted as a weight management ingredient reducing fat and promoting lean muscle mass Prolibra 290 IP is an all-natural dairy derived ingredient designed to be used as part of a weight management program. Prolibra 290 IP undergoes an agglomeration process that allows it to disperse easily in beverages and solutions. The soya lecithin used is identity preserved This product has, however, failed to receive health claim from European Food Safety Authority (EFSA). The EFSA panel concluded that a cause and effect relationship cannot be established between the consumption of Prolibra and “helps to reduce body fat while preserving lean muscle” in the context of energy restriction for weight loss (EFSA, 2012) http://www.sportsingredients.com/products/prolibra-290-ip?t 5 0&c 5 0 Accessed 04.04.17

Milk Basic Protein

According to Snow Brand Milk, MBP is a multifunction protein that acts directly and indirectly on bone cells to reinforce the bone itself, making it more receptive to calcium while at the same time preventing excessive calcium from being dissolved from the bones (Aoe et al., 2001) According to the manufacturer MBP helps calcium adhere to bones and prevents calcium from dissolving from bones http://www.meg-snow-mbp.com/english/about/work.html Accessed 03.04.17

Tylactin RTD (ready-to-drink)

Tylactin RTD is a medical food formula which contains Tylactin, the proprietary, advanced formulation of glycomacropeptide (GMP) and essential amino acids without added tyrosine and phenylalanine. Phenylalanine is not added because phenylalanine naturally converts into tyrosine. It contains 15 g protein equivalent per carton http://www.cambrooke.com/products/tylactin/rtd/#.WOVz3qIlGM8 Accessed 06.04.17

Tylactin RESTORE Powder

Tylactin RESTORE Powder is a hydration beverage for the dietary management of tyrosinemia (TYR) All Tylactin RESTORE medical food formulas contain Tylactin, the proprietary, advanced formulation of GMP and essential amino acids http://www.cambrooke.com/products/tylactin/restore-powder/#.WOV076IlGM8 Accessed 06.04.17

Continued

17.5 Conclusions

Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Continued Commercial Product

Whey Protein and Health Benefits

Tylactin RESTORE

Tylactin RESTORE is a great tasting hydration beverage for the dietary management of TYR types I, II, III. RESTORE comes as a ready-to-drink beverage in a clear, recyclable bottle. RESTORE is a medical food formula that contains Tylactin, the proprietary, advanced formulation of glycomacropeptide and essential amino acids without added phenylalanine and tyrosine. Phenylalanine is not added because phenylalanine naturally converts into tyrosine http://www.cambrooke.com/products/tylactin/restore/#.WOV0aqIlGM8 Accessed 06.04.17

BetterMilk

BetterMilk (powdered formula) is a mix-with-liquid powdered metabolic formula for the dietary management of phenylketonuria (PKU) that makes a creamy, milk-like beverage with a hint of sweetness BetterMilk medical food formula contains Glytactin, the proprietary, advanced formulation of glycomacropeptide and essential amino acids. BetterMilk is for use in the dietary management of phenylketonuria in people aged 2 years and older. BetterMilk is intended for adults and children who are under medical supervision for proven PKU. Protein in prescribed amounts must be supplemented to completely meet phenylalanine requirements http://www.cambrooke.com/products/glytactin/bettermilk/#.WOVu4KIlGM8 Accessed 06.04.17

Glytactin RESTORE Powder

Glytactin RESTORE Powder is a hydration beverage for the dietary management of PKU in a convenient powder packet to take on-the-go All RESTORE medical food formulas contain Glytactin, the proprietary, advanced formulation of GMP and essential amino acids http://www.cambrooke.com/products/glytactin/restore-powder/#.WOVwCqIlGM8 Accessed 06.04.17

Continued

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Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Continued Commercial Product

Whey Protein and Health Benefits

Glytactin BUILD

Glytactin BUILD is a versatile powdered medical food for the dietary management of PKU. Glytactin BUILD was designed to enhance the availability of the specialized Glytactin protein for those on a low phenylalanine diet seeking more protein and fewer calories than traditional PKU Medical Foods It contain Glytactin, the proprietary, advanced formulation of GMP and essential amino acids http://www.cambrooke.com/products/glytactin/build/#.WOV1a6IlGM8 Accessed 06.04.17

Glytactin SWIRL

Glytactin SWIRL is a powdered formula that can be mixed with 3 4 ounces cold water to make a creamy pudding or 6 8 ounces cold water to make a shake/ smoothie. SWIRL is a metabolic formula for ages 1 year and older for the dietary management of PKU SWIRL contains Glytactin, the proprietary, advanced formulation of glycomacropeptide and essential amino acids http://www.cambrooke.com/products/glytactin/swirl/#.WOV1t6IlGM8 Accessed 06.04.17

Glytactin RTD

Glytactin RTD is a whole protein PKU formula in a ready-to-drink format for the dietary management of PKU http://www.cambrooke.com/products/glytactin/rtd/#.WOV2BaIlGM8 Accessed 06.04.17

UltraMeal Whey

UltraMeal Whey is a nutritional drink mix containing whey protein isolate and whey protein hydrolysate. This formula is promoted as a nutritional Support for Healthy Body Composition http://www.metagenics.com/mp/medical-foods/ultrameal-whey Accessed 06.04.17

Continued

17.5 Conclusions

Table 17.5 Commercial Functional Food and Nutritional Supplement Applications of Whey Proteins Continued Commercial Product

Whey Protein and Health Benefits

Pro-Stat MAX

A sugar-free, hydrolyzed whey-based liquid protein medical food for critical illness providing 11 g of high quality protein and 80 calories in 1 fluid ounce Pro-Stat MAX is indicated for increased protein needs in low volume related to: pressure ulcers, wounds, critical illness, unintentional muscle loss, protein-energy malnutrition, low serum proteins, pre- and postsurgery, sarcopenia, and cancer https://shop.specializedadultnutrition.com/p-68-pro-stat-max.aspx? Accessed 06.04.17

ProSource NoCarb Liquid Protein

ProSource NoCarb Liquid Protein is specifically formulated to provide the nutrients necessary for the dietary management of protein-energy malnutrition and protein deficiency ProSource NoCarb Liquid Protein combines the efficiency of hydrolyzed collagen and whey protein for a sugar-free, concentrated, low-volume, high-protein supplement. According to the manufacturer, ProSource NoCarb Liquid Protein is clinically proven in a double-blind, peer-reviewed, published study to raise albumin levels in hemodialysis patients http://www.medtrition.com/products/prosource-nocarb-liquid-protein-sugar-free/ nutritional-information/ Accessed 06.04.17

Travelan

Travelan capsules made from colostrum contain high levels of immunoglobulins and other antibodies Travelan reduces your risk of getting Travelers’ Diarrhea (TD) rather than having to treat the symptoms after the condition has begun. Travelan has been shown in medical clinical trials to significantly decrease the risk of TD and to decrease the symptoms of minor gastrointestinal disorders, whereas treatment products aim to slow diarrhea symptoms after TD has begun http://www.travelan.com.au/ Accessed 06.04.17

and can potentially lead to prevention of lifestyle diseases. One of the key components of whey proteins is cysteine, a semiessential amino acid that plays a key role in stimulating synthesis of glutathione, which is involved in many processes in the body, including tissue building and repair, making

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chemicals and proteins needed in the body, and for the immune system. Whey proteins are also a reservoir of bioactive peptides that can be released by enzymatic hydrolysis of protein. Health benefits of whey proteins include inhibitory effect on cancers, enhancement of immune response, regulation of body weight by providing satiety signals, and improved cognitive performance in stress-vulnerable subjects. Several functional food products have been commercialized to exploit these benefits.

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Hall, W. L., Millward, D. J., Long, S. J., & Morgan, L. M. (2003). Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. British Journal of Nutrition, 89, 239 248. Hassler, C. M. (1998). Functional foods: Their role in disease prevention and health promotion. Food Technology, 52(11), 63 70. Hernandez-Ledesma, B., Davalos, A., Bartolome, B., & Amigo, L. (2005). Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS. Journal of Agriculture & Food Chemistry, 53, 588 593. Hernandez-Ledesma, B., Recio, I., & Amigo, L. (2008). Beta-lactoglobulin as source of bioactive peptides. Amino Acids, 35, 257 265. Kawasaki, Y., Isoda, H., Tanimoto, M., Dosako, S., Idota, T., & Ahiko, K. (1992). Inhibition by lactoferrin and kappa-casein glycomacropeptide of binding of cholera toxin to its receptor. Bioscience, Biotechnology & Biochemistry, 56, 195 198. Kennedy, R. S., Konok, G. P., Bounous, G., Baruchel, S., & Lee, T. D. (1995). The use of a whey protein concentrate in the treatment of patients with metastatic carcinoma: A phase I-II clinical study. Anticancer Research, 15, 2643 2649. Kinsella, E., & Whitehead, D. M. (1989). Proteins in whey: Chemical, physical and functional properties. Advances in Food and Nutrition Research, 33, 343 438. Kirkpatrick, C. H., Green, I., Rich, R. R., & Schade, A. L. (1971). Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: Relation to host-defense mechanisms in chronic mucocutaneous candidiasis. The Journal of Infectious Diseases, 124, 539 544. Korhonen, H., Marnila, P., & Gill, H. (2000). Milk immunoglobulins and complement factors, a review. British Journal of Nutrition, 84, 75 80. Krissansen, G. W. (2007). Emerging health properties of whey proteins and their clinical implications. Journal of the American College of Nutrition, 26, 713S 723S. Kulczycki, A., Jr., & MacDermott, R. P. (1985). Bovine IgG and human immune responses: Con A-induced mitogenesis of human mononuclear cells is suppressed by bovine IgG. International Archives of Allergy & Applied Immunology, 77, 255 258. Lam, S. M., Moughan, P. J., Awati, A., & Morton, H. R. (2009). The influence of whey protein and glycomacropeptide on satiety in adult humans. Physiology & Behaviour, 96, 162 168. Laursen, I., Briand, P., & Lykkesfeldt, A. E. (1990). Serum albumin as a modulator on growth of the human breast cancer cell line, MCF-7. Anticancer Research, 10, 343 351. Levay, P. F., & Viljoen, M. (1995). Lactoferrin: A general review. Haematologica, 80, 252 267. Lilius, E. M., & Mamila, P. (2001). The role of colostral antibodies in prevention of microbial infections. Current Opinion in Infectious Diseases, 14, 295 300. Lönnerdal, B. (2014). Infant formula and infant nutrition: Bioactive proteins of human milk and implications for composition of infant formulas. American Journal of Clinical Nutrition, 99, S712 S717. Lothian, J. B., Grey, V., & Lands, L. C. (2006). Effect of whey protein to modulate immune response in children with atopic asthma. International Journal of Food Science & Nutrition, 57, 204 211. Luhovyy, B. L., Akhavan, T., & Anderson, G. H. (2007). Whey proteins in the regulation of food intake and satiety. Journal of the American College of Nutrition, 26, S704 S712. Madureira, A. R., Pereira, C. I., Gomes, A. M. P., Pintado, M. E., & Malcata, F. X. (2007). Bovine whey proteins Overview on their main biological properties. Food Research International, 40, 1197 1211. Markus, C. R., Jonkman, L. M., Lammers, J. H., Deutz, N. E., Messer, M. H., & Rigtering, N. (2005). Evening intake of alpha-lactalbumin increases plasma tryptophan availability and

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Uenishi, K., Ishida, H., Toba, Y., Aoe, S., Itabashi, A., & Takada, Y. (2007). Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women. Osteoporosis International, 18, 385 390. Van Calcar, S. C., & Ney, D. M. (2012). Food products made with glycomacropeptide, a low phenylalanine whey protein, provide a new alternative to amino acid-based medical foods for nutrition management of phenylketonuria. Journal of the Academy of Nutrition and Dietetics, 112, 1201 1210. Watanabe, A., Okada, K., Shimizu, Y., Wakabayashi, H., Higuchi, K., Niiya, K., . . . Kohri, H. (2000). Nutritional therapy of chronic hepatitis by whey protein (non-heated). Journal of Medicine, 31, 283 302. Wijkstrom-Frei, C., El-Chemaly, S., Ali-Rachedi, R., Gerson, C., Cobas, M. A., Forteza, R., . . . Conner, G. E. (2003). Lactoperoxidase and human airway host defense. American Journal of Respiratory & Cell Molecular Biology, 29, 206 212. de Wit, J. N., & van Hooydonk, A. C. M. (1996). Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Netherlands Milk & Dairy Journal, 50, 227 244. Wong, C. W., & Watson, D. L. (1995). Immunomodulatory effects of dietary whey proteins in mice. Journal of Dairy Research, 62, 359 368. World Health Organization (WHO). (2014). Global status report on noncommunicable diseases 2014. Geneva: WHO. Yoo, Y. C., Watanabe, S., Watanabe, R., Hata, K., Shimazaki, K., & Azuma, I. (1998). Bovine lactoferrin and lactoferricin inhibit tumor metastasis in mice. Advances in Experimental Medicine & Biology, 443, 285 291.

663

Author Index

A

Aalto, J., 646 Aarsland, A., 583 Abargouei, A. S., 562 Abbaspourrad, A., 521 Abd El Salam, M. H., 358 Abd El-Aziz, M., 29 30 Abdul-Fattah, A. M., 125 126 Ábel, M., 105 Abete, I., 560 Abou El-Nour, A., 366 367 Aboul-Enein, H. Y., 168 169 Aboumahmoud, R., 219 Abrahamsen, R. K., 109 Acharya, M. R., 360 Ackatia-Armah, R. S., 556 Acree, T. E., 383 384 Actor, J. K., 443t, 451 452 Adair, L. S., 562 Adamcik, J., 137 138, 196, 205 206 Adamopoulos, K. G., 472 Adams, A., 253 254 Adams, M. C., 108 Adams, S. L., 355 356, 496 Addeo, F., 166 167 Adel-Patient, K., 448 Adhikari, B., 135 136 Adjonu, R., 223 224 Adler-Nissen, J., 462 463 Adlerova, L., 645 Aebersold, R., 170 174 Aekplakorn, W., 559 Agboola, S., 223 224 Agboola, S. O., 462 463, 469 470 Agerstam, H., 520 Agterof, W., 383 Aguilera, J., 471 472 Aguilera, J. M., 189 196 Aguirre-Mandujano, E., 350

Agyare, K. K., 218 220 Ahmadi, K. G. F., 586t, 618 Ahmed, N. S., 35 Ahmed, T., 565 Aihara, K., 535 536 Ainsworth, S., 306 Ait-Ameur, L., 140 142 Ait-Oukhatar, N., 530 531 Akashi, S., 10 Akhavan, T., 495 496, 528, 611, 618 619, 649 Akhtar, M., 249 250, 263, 265 266 Akin, N., 639 Akino, A., 524 Akkanen, S., 535 536 Akkerman, C., 474 475 Akkerman, M., 170 171 Ako, H., 35 36 Ako, K., 196, 204 205 Aktan, A. O., 648 Akuzawa, R., 34 Al-Attabi, Z., 185 Al-Hanish, A., 256 257 Al-Mashikhi, S., 14, 449 Al-Saadi, J. M. S., 132 Alderman, H., 550 551 Aleixandre, M. A., 524 525 Alewood, P. F., 21, 24 25, 140 142, 160 161, 165 167, 170, 174 175 Alexander, D. D., 442 445, 553, 585 592, 649 Alexiadis, A., 104 105 Alghannam, A. F., 596, 615 Ali, A., 603, 607 Ali, I., 168 169 Alichanidis, E., 34 Allain, A.-F., 284 Allelein, S., 273 274 Allen, D. G., 592 593

Allen, J. C., 6 7, 189 196 Alleyne, M. C., 361 362 Alli, I., 199, 217, 470 471 Alluwaimi, A. M., 458 Almaas, H., 532 533 Almécija, M. C., 103 104, 113 Alpert, M. A., 559 Altic, L. C., 318 Alting, A. C., 201 202, 215, 508 Altinkaya, S. A., 417 418 Alvarez, G., 189 196, 298 Alvarez, I., 313 Aly, E., 451 452 Amaya-Llano, S., 528 Amer, V., 648 Ames, J. M., 251 252, 506 507 Ametani, A., 261 Ametani, M., 414 Amigo Rubio, J. M., 350 351 Amigo, L., 24, 167, 522t, 524 525, 641 Amiot, J., 462 463 Amiot, S., 465 466 Anborgh, P. H., 443t, 453 Andersen, M. H., 454 Anderson, G. H., 528, 611, 649 650 Anderson, H., 495 496 Anderson, M., 22 Andersson, G., 128 Andersson, J., 169 Andreasen, P. A., 162 Andrén, A., 166 167 Andrewes, P., 388 Andrews, A. T., 34 Anema, S. G., 187, 189 196, 201 202, 203f, 283 284, 287 288, 305 306, 472 474, 473t, 508 Angersbach, A., 228 229 Anjum, F. M., 30 Anker, M., 416

665

666

Author Index

Añón, M. C., 414 Antony, A. C., 455 456 Antosiewicz, J., 603, 620 621 Anugu, A. K., 320 321 Anwar, S., 272 Aoe, S., 650 651, 652t Aoi, W., 594, 606 Aoustin, E., 427 428 Aparnathi, K., 128 Apenten, R. K. O., 187 189, 470 471 Appel, R. D., 446 447 Aragon, A. A., 598 Aranberri, I., 466 469 Aranda, P., 451 452 Arefi-Oskoui, S., 107 Arena, S., 157, 160, 173 174, 252 253 Areta, J. L., 598, 604 605 Arias, M., 283 284, 286 287 Armitt, J. D., 382 383 Armstrong, H. J., 269 270 Armstrong, J. L., 610 611 Arnaudov, L. N., 205 206 Arnberg, K., 445 446 Arnold, R., 456 Arnold, R. R., 451 452, 645 646 Aronson, A., 520 Arosio, P., 292 293 Arrighi, V., 208 Arsenault, J., 555 Artym, J., 451 452 Arunkumar, A., 106, 273 274 Arya, S. S., 17 Aryana, K. J., 361 363 Aryastami, N., 551 Ashbrook, J. D., 451 Ashkar, S., 443t, 453 Ashokkumar, M., 213 214, 302 304, 306 311, 308t Ashton, L., 295 Ashwell, M., 637 Asselin, J., 462 463 Assendelft, W. J., 442 445 Astbury, N. M., 354 355 Astrup, A., 553, 560 Atan, S. A., 603 Atherton, P. J., 604 Athira, S., 522t, 526 527, 534 535 Atkinson, S. A., 463 Atri, H., 207 208 Attaallah, W., 648

Augustin, M. A., 135, 138 139, 261 262, 265 268 Auty, M. A., 224 225 Auty, M. A. E., 201 202, 253 254, 265f, 266f, 349 Avena-Bustillos, R. J., 419 Awati, A., 650 Axelos, M. A., 211 Axelos, M. A. V., 138 140 Aylward, E., 380t Azhar, M. E., 269 270 Azuma, N., 414 Azza, A. I., 35

B

Baba, A., 34 Badia, J. D., 426 427 Badii, F., 207 208 Bae, E. K., 155 156 Baer, D. J., 561 Baer, R. J., 358, 360 Báez, G. D., 135, 138, 263, 267 269 Baeza, R., 139 140 Bafort, F., 27, 646 Baginski, M. A., 170 Bagnall, W., 302 Bahadir Saltik, M., 106 Bahwere, P., 554, 557 Baier, S., 223 Baier, S. K., 199, 223 Bains, H. S., 553 Bairoch, A., 446 447 Bajaj, R., 524 525 Bakman, S., 443t Balabani, S., 295 Balci, A. T., 34 Baldasso, C., 106 Baldwin, K. A., 364 Ballard, F. J., 36 Ballerini, G. A., 138 Balmer, S. E., 445 Balny, C., 283 284 Banach, J. C., 500, 510 513 Banerjee, D., 104 105 Bangsbo, J., 594 Banks, J. M., 201, 364, 409 410 Banon, S., 419 Bansal, B., 185 Bansal, N., 29 30, 35, 158, 187, 201 202, 216 217, 260 261, 337 338, 348 349, 465 466 Bao, J., 104 105

Barac, M., 581 582 Barac, M. B., 202 Barbano, D. M., 108, 377, 382 383, 389 392, 391t, 392t, 396f Barboza, M., 451 452 Barbut, S., 196, 289 Bareuther, C., 34 35 Barile, D., 173 174, 459 460 Barker, D. J., 553 Barkholt, V., 443t Barlow, G. H., 4 5 Barone, J. R., 407 408 Barraj, L. M., 442 445, 649 Barrantes, E., 350 Barrer, R. M., 419 Barreto, P. L., 219 220 Barreto, P. L. M., 353 354 Barros, R. M., 641 Barros, T. C., 106 Barry, K., 129 130 Barsotti, L., 228 229, 314, 315t Bartfay, W. J., 617 618 Bartolome, B., 641 Bartoskova, A., 645 Baruchel, S., 652t Barukˇciˇc, I, 103 104, 446 447 Barzyk, W., 443t, 457 Bassette, R., 382 Bassit, R. A., 616 617 Bastian, E., 335 336, 377, 379 381, 393 395, 398 400, 399f Bastian, E. D., 164 165, 520, 562, 650 Batema, R. P., 451 452 Bates, D. M., 302 Batist, G., 647 648 Batra, P., 556 557 Batterham, E. S., 270 271 Bauer, J. M., 601, 609 Bauer, W. J., 359 Bauman, D. E., 382 383 Baur, D. A., 595 Baussay, K., 189 196, 190t, 204 205 Bawa, S., 155 156 Bawa, S. E., 579, 616 617 Bayless, K. J., 22 23 Baynes, J. W., 506 507 Beatriz, M., 526 Beaudart, C., 610 Beaulieu, M., 196 197 Beavers, K. M., 561

Author Index

Becker, G., 602 Bede, M. J., 448 Bee, J. S., 292 Beeh, K. M., 520, 649 Beelen, M., 598 Behrman, J. R., 550 551 Belcourt, L. A., 500 501, 505 Belem, M. A. F., 531 532 Belitz, H. D., 408 409 Bell, K., 7 8 Bell, R., 213 214 Bellmann, S., 212 Belloque, J., 284 Belmar-Beiny, M., 185 Bendall, J. G., 382 383 Bendicho-Porta, S., 314 Benfeldt, C., 31 32, 156 157, 159t, 162, 164 165 Benjamas, C., 528 529 Benjamin, H. J., 603 Bennett, S. H., 443t Beresford, T. P., 283 284 Berg, T. H. A., 102 103 Bering, S. B., 448 Berkhout, B., 447 448 Bernal, V., 199 Bernard, C., 209 210 Berner, L., 602 Bernetti, A., 564 Berrios, J. D. J., 412 413 Berry, G. P., 465 466 Bertenshaw, E. J., 495 496 Bertoldo-Pacheco, M. T., 528 529 Berton-Carabin, C., 210, 469 470 Bertrand-Harb, C., 462 463 Bertucco, A., 230 Besner, G. E., 443t Beszédes, S., 105 Betts, J. A., 606 Betz, M., 155 156 Beuchat, L. R., 443t, 455 Beyrer, M., 115 116 Bhandari, B., 124 126, 135 136, 140 142, 157, 166 167, 174 175, 306, 505 506 Bhaskaracharya, R., 213 214, 302, 308t Bhatt, H., 250 251 Bhattacharjee, C., 112, 169 Bhattacharjee, S., 112, 169 Bhattacharjeek, C., 108 Bhattacharya, A., 104 105

Bhattacharya, C., 104 105 Bhattacharya, S. D., 7 8 Bhushan, S., 106 Bhutta, Z. A., 555 Bianchi, L., 3 4 Bibby, B. M., 594 Billeaud, C., 445 446 Bingham, E. W., 16t, 35 36, 38t Binks, B. P., 466 469 Biolo, G., 584 585, 598 Birlouez-Aragon, I., 140 142, 252 253 Birnie, L. M., 583 Bischoff-Ferrari, H. A., 609 Bischur, G., 424 Bishop, J. R., 318 Bissonnette, N., 22 23, 159t Biziulevicius, G. A., 641 Bizzozero, J., 354 355 Bjerkvig, R., 520 Björck, L., 159t, 164, 456, 647 Blair, A. S., 611 Blakeborough, P., 456 Blanc, B. A., 199 Blanch, E. W., 295 Bland, A. P., 349 Blayo, C., 295 296 Blecker, C., 21 22 Block, J. D., 156 157 Blomstrand, E., 615 Blumenfeld, O. O., 216 217 Bode, L., 443t, 459 460 Bodine, S. C., 612 Boesman-Finkelstein, M., 30 Bogahawaththa, D., 200 201 Boguth, G., 165 166 Bohe, J., 594 Bohl, M., 563 Boirie, Y., 211, 582 Boisen, A., 32, 165 Bojsen, A., 449 Boland, M., 4, 582 Bolisetty, S., 137 138 Bomser, J. A., 228 229 Bon, C. L., 189 196 Bonavaud, S. M., 610 Bond, M. D., 37 38 Bönisch, M. P., 353 354 Bonnaillie, L. M., 549 550 Bonomi, F., 189 196 Bonsmann, G., 529 Booij, L., 642 Boom, R., 496 497, 512 513

Borch, E., 647 Bordignon-Luiz, M. T., 219 220, 353 354 Bordin, G., 156 157 Born, J. M., 618 619 Borreani, J., 357 358 Børsheim, E., 605 Boskey, A. L., 453 Bosse, J. D., 559 560 Both, P., 530 Bottenus, R. E., 417 Böttger, F. H., 271 272 Bottomley, R. C., 5 Bouaouina, H., 214, 414 Bouhallab, S., 130 132, 140 142, 205 206, 254 255, 471 472, 530 531 Bouman, S., 7 Bounous, G., 533, 647 649, 652t Boutin, Y., 532 533 Boutrou, R., 212 Bouvier, F., 10 Bouyer, C., 303 304 Bovay, C., 203 204, 341 342 Bovenhuis, H., 7 8 Bovetto, L., 189 196 Boye, J. I., 185, 199, 216 217, 470 471 Boˇzanic, R., 103 104, 446 447 Braat, P., 383 Bramaz, N., 447 448, 531 Bramley, P. M., 393 394 Brand-Miller, J., 450 Brandelli, A., 525 526, 531 533 Brandon, D. L., 581 Brandt, N., 427 428 Brantl, V., 531 532 Brantsaeter, A., 551 552 Bräuer, S., 415 416 Brault, D., 415 Braun, D., 412 413 Breen, L., 567, 606 607 Bremer, M. G., 200 Brennan, J. G., 414 415 Brennand, C. P., 382 Brew, K., 7 9, 199, 470 471 Brewer, M., 451 452, 645 646 Briand, P., 643 Briard-Bion, V., 131 Briend, A., 555 Briggs, D., 127 128 Britten, M., 187 189, 225 226, 364 365, 413

667

668

Author Index

Brncic, S. R., 306 Broadhurst, M. K., 166 167 Brodkorb, A., 19, 196 197, 201 202, 210 212, 255, 259, 269 270, 466 472, 641 Brody, E. P., 156 157, 450, 643 644 Broersen, K., 135 137 Bromley, E. H. C., 196, 204 205 Bronlund, J. E., 135 136 Brooks, S. V., 612 Brotons, G., 196 Brown, E., 201 202 Brown, K. H., 555 Brown, P. H., 528, 611 Brown, R. J., 164 165, 364 Browning, K. S., 613 Brownlow, S., 4 5, 189 196 Bruce, B., 309 Bruce, B. D., 414 415 Brück, W. M., 443t, 447 448, 450, 463 Brulé, G., 98 99, 414 Brun, J. M., 466 469 Bruni, N., 443t, 451 452 Brunstrom, J. M., 500 Brüssow, H., 14 Bryant, C., 223 Bu, G., 271 272 Buchheim, W., 366 367 Buckley, J. D., 155 156 Buclet, N., 427 428 Buecking, M., 584 Buera, M. del P., 125 126 Bugaud, C., 382 383 Buggy, A. K., 210, 466 469, 471 472 Bugnicourt, E., 411, 419, 423f, 424 425, 425f, 427 428 Buhl, R., 520, 649 Bund, T., 273 274 Burd, N. A., 567, 595, 600, 602 603 Burgain, J., 124, 127 129, 128f, 250 251, 348 349 Burke, D., 593, 608 Burke, D. G., 649 Burke, L. M., 604 Burnett, B. P., 449 Burova, T., 135, 220 221 Burrington, K. J., 336, 338 339, 360 Burton, H., 318 Busnel, J.-P., 189 196

Busti, P., 133 Busti, P. A., 135, 138, 263 Butler, J. E., 449 Buys, E., 27 Buys, E. M., 185 Byers, C. H., 168 Bylund, G., 378f, 390f Byrne, E. P., 294, 300 Byrne, N. M., 619

C

Cadwallader, K. R., 377 381, 380t, 384 385, 497 Caetano-Silva, M. E., 522t, 528 530 Caffin, J. P., 7 Cahalin, L. P., 559 Cai, B., 268 269 Cai, W., 562 Cai, X., 522t Caillet, S., 418 Cairoli, S., 189 196 Çakir-Fuller, E., 116 117, 226 227 Caldeo, V., 466 469 Callaghan, M., 555, 557 Callanan, M. J., 230 Caltagirone, J. P., 303 304 Calvo, M., 13, 448, 451 453 Calvo, M. M., 201, 383, 409 410 Cameron-Smith, D., 568 Camirand, W. M., 415, 581 582 Campagna, S., 20, 22, 443t, 457 Campanella, O. H., 200 201, 205 206 Campbell, B., 209, 466 469 Campbell, L., 208 Campbell, M., 356, 498 Campbell, R., 396f Campbell, R. E., 377, 379 386, 380t, 384t, 389 397 Campbell, W. W., 495 496, 561 562 Campos-Giménez, E., 140 142 Cândido, L. M. B., 537 538 Candow, D. G., 593 Cao, J. X., 155 156 Capeless, E. L., 35 Capellas, M., 283 284, 349 Cardador-Martínez, A., 528 Carey, M. F., 579 Carlin, L. G., 648 Carlsson, A., 159t, 164 Carneiro, E. M., 610 Caroli, A. M., 3 4, 157 Carr, B. T., 378 379

Carraro, E., 595 Carrascosa, A. V., 285 Carrera Sanchez, C., 139, 537 538 Carroll, T., 14 Carter, D. C., 10 11 Cartier, P., 22 Cartland-Glover, G. M., 300 Cartoni, G., 167 Carunchia Whetstine, M. E., 377 386, 380t, 384t, 389 391, 391t Carunchia Whetstine, M. E. C., 497 Carver, J. A., 227 Casadonte, D. J., 414 415 Castellino, F. J., 8 Catherine, B. H., 501 502 Catignani, G. L., 417, 524 Cattaneo, S., 25, 270 271 Caudle, A. D., 497 Cavaggioni, A., 5 6 Cavallieri, A. L. F., 6 7 Cebo, C., 3 4, 7 8 Ceolín, M., 285 Cermak, N. M., 566, 585 592 Chabance, B., 450, 524 Chale, A., 565 566, 586t Chambaz, A., 644 Chambers, A. F., 443t Champagne, C. M., 560 Champion, D., 125 126 Chan, E. Y. C., 357 Chanasattru, W., 223 Chand, A., 168 169 Chandan, R. C., 30 Chandrapala, J., 200, 302, 308 309 Chang, W. H., 533 Chang, Y. K., 594 Chaplin, L. C., 199 Chapron, L., 414 Charbonneau, S., 209 210 Charissou, A., 140 142 Charm, S. E., 291 292 Chase, H. A., 168 169 Chassenieux, C., 201 202 Chatterton, D. E. W., 448 449, 453, 455, 457 458, 641 Chatzidiakou, Y., 563 Chaud, M. V., 528 531 Chaufer, B., 111 Chaurin, V., 356, 500, 509 Cheang, B., 111 112 Cheeseman, G. C., 472 474 Cheftel, J., 284 286, 408

Author Index

Cheftel, J. C., 115, 228 229, 283 286, 298 300, 314, 414 Cheison, S. C., 9, 443t Chen, C. H., 533 Chen, G. W., 535 536 Chen, H., 427 Chen, H. L., 169 Chen, H. M., 525 526 Chen, L. W., 553 Chen, P., 501 Chen, V. V. Y. T., 169 Chen, W., 9, 443t Chen, W. C., 594 595 Chen, W. L., 169, 443t Chen, X., 221 Chen, X. D., 127 130, 185, 306, 385 Chen, Y., 221, 443t, 452 453 Chen, Y. J., 500 501, 503 504 Chen, Y. Y., 443t Chena, X., 343 344 Chena, Y., 343 344 Cheng, H., 250 253 Cheng, K. J., 36 Cheol-Hyun Kim, C.-H., 337 338 Chessa, S., 157 Chevalier, F., 166 167, 220 221, 258 259, 261 Chevallier, M., 124, 130, 132 133, 136 137, 140 143 Chikuni, K., 11 Childs, J. L., 377, 498 Chilliard, Y., 22 Chin, C.-J., 293 294 Chirife, J., 125 126 Chiu, C. C., 594 Cho, C. E., 528, 611 Cho, Y.-H., 337 338, 345 Chobert, J., 220 221 Chobert, J. M., 135, 462 463 Chobert, J.-M., 215, 220 221, 228 229 Chock, P. B., 525 Choi, D. Y., 169 Choi, K. Y., 136 137 Choiset, Y., 220 221 Cholette, H., 385 Choo, C. L., 291 292 Choudhary, R., 320 321 Chowanadisai, W., 7 8 Christensen, B., 22 24, 159t, 453 454

Chung, C., 357 Chung, M.-S., 125 126 Chungchunlam, S. M., 650 Churchward-Venne, T. A., 567, 613 Chuyen, N. V., 220 Cialdella-Kam, L. C., 611 Ciawi, E., 303 Cifuentes, A., 167 Cilliers, F. P., 318, 321 Cilliers, T., 318 Cinelli, P., 407, 421 422, 427 428 Ciron, C. I. E., 349 Civille, G. V., 378 379, 380t, 497 Claeys, W., 189 196 Claeys, W. L., 17, 20 Clare, D. A., 255, 417, 524, 531 532 Clark, A., 199 Clark, D., 551, 553 Clark, S., 284 285, 500, 511 513 Clarke, A. E., 185 Clarke, D. T., 132 133 Clarke, S., 130 Cleary, T. G., 648 Clément, J., 283 284 Clevenger, C. V., 162 163 Clifton, P., 551, 560 Clifton, V., 551 Clint, J. H., 466 469 Cocchi, D., 3 4 Coccioli, F., 167 Coddeville, B., 21 Cogan, T. M., 185 Cohen Stuart, M. A., 138 139 Coker, R. H., 561 562 Colker, C. M., 586t Colonetti, T., 566 Coltelli, M. B., 427 428 Coltelli, M.-B., 410 413, 425 427 Comerford, K. B., 563 564 Comparin, D., 21 22 Comstock, S. S., 443t, 459 460 Conde, J. M., 210 Condés, M. C., 414 Condon, S., 313 Conesa, C., 453 Connolly, P., 352 Considine, T., 187 189, 216 217, 225 226, 283 287, 352 Consortium, N., 552f Contarini, G., 379 381, 383 Conte-Junior, C. A., 443t Contreras-Lopez, E., 125 126

Contreras, M. M., 522t Cooke, M. B., 586t, 592 594, 613 614 Cooke, P. H., 298 300 Cooney, M., 206 207 Cooney, S., 33 Cooper, R., 597 Cordoni, D., 423f, 425f Cornacchia, L., 210, 469 470 Cornec, M., 209 210, 268 Corr, S. C., 445 Corradini, C., 21 22 Correa, A. P. F., 525 Corredig, M., 107, 196 197, 202, 318 Corrigan, B., 226 227 Corvalan, C., 206 Corzo-Martinez, M., 273, 500 501, 503 Cosette, P., 457 Costantino, H. R., 502 Costanzo, P., 559 Côté, G. L., 138 139, 221 222 Cotter, M. P., 347 348 Coulon, J., 382 383 Coulson, E. J., 35 36 Coulter, S. T., 472 474 Coupland, J., 209 210 Coupland, J. N., 340, 466 469 Courthaudon, J. L., 22, 443t Cowan, S., 106 Cox, D. M., 174 175 Coyle, E. F., 605 606 Cozzolino, A., 365 Craven, H. M., 313 Creamer, L. K., 3 6, 158, 186 187, 199 201, 203 204, 216 217, 283 285, 297 298, 355 356, 448, 465 471, 496 Creighton, T. E., 187 Crelier, S., 302 Creuzenet, C., 415 416 Cribb, P. J., 579, 585, 586t, 592 593, 602, 611 612 Croguennec, T., 19, 130 132, 186 189, 205 206, 255, 471 472 Croissant, A. E., 377, 382 383, 382f, 391t, 393 395, 397, 497 Cronin, D. A., 304, 315t Crook, J. A., 318 Cross, M. L., 155 156, 532 533, 647 648

669

670

Author Index

Crowley, S. V., 466 469, 471 472 Cruzat, V. F., 616 618 Cryan, J. F., 619 Cullor, J. S., 318, 458 Cunsolo, V., 3 4 Cuppett, S. L., 421 Cuq, B., 420 421 Cuq, J., 408 Currell, K., 595 Cury-Boaventura, M. F., 616 618 Cushing, C. A., 442 445, 649 Cutler, C. L., 605 606 Cuvelier, G., 360

D

Da Costa, A. R., 109 da Cunha, R. L., 6 7 Da Silva Pinto, M., 136 137 Daddaoua, A., 644 Dalaly, B. K., 35 Dalgalarrondo, M., 220 221 Dalgleish, D., 206 207, 223 224 Dalgleish, D. G., 200 202, 249 250, 462 463, 465 471, 508 Dallal, G., 559 Dallmann, K., 423f, 425f Dalsgaard, T. K., 160 Dambmann, C., 416 417 D’Ambrosio, C., 252 253 Damodaran, S., 138, 209 211, 218 222, 252 253, 259, 271 272 Danesh, E., 360 Dangaran, K., 408 Dangin, M., 211, 567 568 Daniels, M. J., 560 Danielsen, M., 174 Dannenberg, F., 197 198 D’Aprano, G., 415 D’Arcy, B. R., 185 Dario, C., 3 4 Daroit, D. J., 525 Das, G., 448 449 Datta, N., 9 10, 31, 472 Datta, S., 112, 169 Daubert, C., 205 206 Daubert, C. R., 206 207, 255, 281 Daufin, G., 111 Davalos, A., 641 Davey, M., 104 105 Davie, E. W., 417 Davies, M. J., 553

Davis-Fleischer, K. M., 443t Davis, A. M., 440 441, 446 447 Davis, G. E., 22 23 Davis, J., 211 Davis, J. M., 614 615 Davis, J. P., 138, 158, 255 256 Davis, M. T., 617 618 Davison, G., 580 Dawson, K. A., 196 De Almeida, F. N., 270 271 de Antoni, G., 346 347 De Block, J., 7, 140 142 De Frutos, M., 167 de Graaf, C., 500 de Groot, L. C., 566 de Haan, E., 651 de Jong, P., 7, 474 475 de Jongh, H. H., 263 de Jongh, H. H. J., 6 7, 135, 138 139, 189 196, 215 De Kimpe, N., 253 254 De Kock, H. L., 185 de Kruif, C. G., 202, 212, 465 466, 508 de Kruif, K., 470 471 de Kruif, K. G., 187 189, 190t, 196 198, 201 202, 286 287, 416 de la Calle, B., 156 157 de la Fuente, M. A., 185 de la Hoz, L., 383 de Lima Zollner, R., 220 de Luis, R., 313 314, 315t, 317 De Meulenaer, B., 264 de Noni, I., 25, 270 271 De Saris, W. H. M., 585 592 De Simone, C., 533 de Vries, R., 205 206 de Wit, J. N., 4 5, 186, 190t, 258, 409, 646 De, B. J., 167 Dean, L. L., 382, 382f Dean, L. O., 389 391 Debeaufort, F., 425 426 DeBoer, R., 1 2, 26, 28 DeCastro, M., 366 367 Decker, E. A., 199, 223, 379 381 Deeth, H. C., 7, 9 10, 21, 24 25, 31, 124, 132, 140 142, 157 158, 160 161, 165 167, 170, 172f, 174 175, 174f, 185, 187, 201 202, 216 217, 260 261,

306, 337 338, 348 349, 465 466, 472, 505 506 Deglon, D. A., 293 294, 294t Degner, B., 357 DeGregori, B., 189 196 DeJong, G., 417 DeKruif, K. G., 189 196 Del Aguila, E. M., 443t Del Coso, J., 607 Del Rio, M., 418 Delahaije, R. J. B. M., 189 196, 190t Delahunty, C. M., 379 381 Delaney, C., 129 130 Delaplace, G., 6 7, 196 Delgado-Andrade, C., 273 Della Gatta, P. A., 568 Delorenzi, N., 209 210 Delorenzi, N. J., 133, 135, 138, 263 Demers-Mathieu, V., 522t Demetriades, K., 209 210, 466 469 Demirci, A., 319 den Breeijen, H., 552f den Hartog, G., 449 Deng, Y., 160 Denhardt, D. T., 453 Denkov, N. D., 209, 466 469 Derse, P. H., 395 Desai, N., 362 Desobry-Banon, S., 133 134 Desobry, S., 124, 133 134, 419 Desorby, S., 440 Desrumaux, A., 214, 414 Dettling, C., 531 Deutsch, E. W., 171 172 Deutz, N., 561 Deutz, N. E., 565 Devreese, B., 320 Devries, M. C., 520, 560, 601, 618 619 Dewettinck, K., 264 Dewey, K. G., 441, 449 Dewit, J. N., 409 410 Deysher, E., 213 214 Di Gioia, L., 420 Di Liddo, R., 37 38, 38t Di Luccia, A., 166 167 Di Marco, N. M., 583 Di Noto, V., 230 Diaz, M. D. F., 228 229, 314 Dickinson, E., 209 210, 249 250, 263, 265 266, 283 285, 466 469, 501 502 Dierckx, S., 264

Author Index

Diez-Masa, J. C., 167 Dijksterhuis, G. B., 500 Dillard, C. J., 451 452, 519 Dincer, T. D., 302 Ding, Y., 254 Ding, Z., 611, 613 Diniz, R. S., 267 268 Dionysius, D. A., 17 19 Dissanayake, M., 116 117, 189 196, 190t, 209 210, 213 214, 298, 301, 344 345 Dixon, B. M., 559 560 Djalali, M., 169 Doi, E., 206 207 Doi, J., 602 Dolan, L. C., 454 Dombrowski, J., 268 Dominguez, E., 13 Dominguez, H., 525 526 Domininghaus, H., 412 413 Domon, B., 173 174 Donald, A. M., 196 Donato-Capel, L., 230 Donato, L., 189 196, 190t, 201 202, 367, 465 466 Donella-Deana, A., 465 466 Dong, A., 4 Dong, J. Y., 560 Donkor, O., 535 536 Donkor, O. N., 189 196 Donovan, M., 187 Donovan, S. M., 443t, 451 454, 459 460 Doran, G., 223 224 Dos Santos, R. C., 586t Dosako, S. I., 7 Doublier, J.-L., 196 197 Doucet, D., 158, 255 256 Doull, F., 532 533 Douwes, A. C., 442 445 Dowling, A. P., 466 469 Downey, B., 530 Drake, M., 224 225, 253 254, 260f, 335 336 Drake, M. A., 255, 377 385, 380t, 382f, 384t, 387f, 389 400, 389f, 391t, 392t, 396f, 399f, 497 498 Drapala, K. P., 224 225, 251f, 253 254, 256 257, 264, 265f, 266f Drioli, E., 169 Drouet, L., 524

Drouin, R., 532 533 Drusch, S., 268 D’Souza, N. M., 102 103 Duan, C., 619 Duchoslav, E., 174 175 Dudemaine, P. L., 22 23, 159t Duerasch, A., 174 175 Duersch, J. W., 385 Duggan, E., 366 367 Duk, M., 451 452 Dumay, E., 115, 228 229, 283 286, 295 296, 298 300, 314 Dumay, E. M., 283 286, 414 Dumoulin, E., 398 Dunker, A. K., 284 285 Dunlap, C. A., 138 139, 221 222 Dunn, J. M., 115, 298 300 Dunnill, P., 189 196, 190t Dunstan, D. E., 207 208, 474 475 Dupont, D., 19, 212, 382 383 Durand, D., 187 196, 205 206, 465 466 Durham, R. J., 383 384, 386 387 Durier, C., 414 Duringer, C., 520 Dusting, J., 295 Dutta, B., 448 449 Dutta, S., 108 Dyer, A. R., 36 Dyer, J. M., 270 271 Dzwolak, W., 285, 392 393

E

Earnshaw, R. G., 303 Easa, A. M., 132 Eckerskorn, C., 160 161 Ecroyd, H., 227 Eder, B., 251 252 Edsall, J. T., 474 Edwards, S., 5 6 Ehlers, G., 158 Einhorn-Stoll, U., 263 Eisenstein, J., 559 Eissa, A. S., 417 Eitenmiller, R. R., 30, 35 Ek, S., 615 Ekblom, B., 615 El Soda, M., 385 El-Bakry, M., 225 226 Elgar, D. F., 170 171 El-Garawany, G. A., 358 Eliot, K. A., 601, 608 609

Ellis, R. J., 256 257 Ellison, R. T., 455 El-Loly, M. M., 643 Elmnasser, N., 321 Elofsson, U., 200 Eloualia, Z., 398 El-Sayed, M. M. H., 168 169 El-Shabrawy, S. A., 534 535 Elsner, P., 412 413 Elvassore, N., 230 Embuscado, M. E., 407, 410 411 Emmons, D. B., 385 Ena, J. M., 448 Endres, H.-J., 424 Engels, M., 443t Enyeart, J. A., 448 Erabit, N., 189 196, 190t, 298 Erbersdobler, H. F., 160, 272 Ercili-Cura, D., 218 Erdman, J. W. Jr., 117 Erdman, K. A., 604 Erdogan, N., 648 Erhardt, G. J., 157 Eriksen, E. K., 532 533 Ernstrom, C. A., 364, 366 367, 385 Errington, A. D., 508 Erskine, R. M., 585, 586t, 600, 621 ËSanli, T., 353 354 Eshpari, H., 377 378, 387f Esmaillzadeh, A., 562 Esmarck, B., 599 Esteban, M. M., 392 393 Etling, N., 451 Etzel, M. R., 106, 261, 271 274, 339, 341, 415 Euston, S., 209 210 Euston, S. R., 208 Evans, J., 377, 379 381, 389 392, 391t, 392t, 398 Evans, M., 263 Evans, M. T. A., 5 Evans, R. W., 155 156 Everett, D. W., 317 Eyerer, P., 412 413

F

Faber, W., 103 104 Fabrucini, B., 586t Fairbanks, A. J., 388 Faist, V., 272 Faldyna, M., 645 Falvo, M. J., 613 614, 618

671

672

Author Index

Fameau, A.-L., 138 Famelart, M.-H., 202, 414 Fan, F., 125 126 Fane, A. G., 109 Fang, Y., 306 Fang, Y. Z., 525 Farias, M., 536 538 Farías, M. E., 200, 463, 470 471 Farkas, B., 335 336, 398 400, 399f Farkye, N. Y., 29 30, 35 Farnaud, S., 155 156 Farrell, H., 201 202 Farrell, H. M., 3 4, 3f, 7 8, 10, 157, 159t, 448, 580 Farrell, P. A., 613 Farsijani, S., 567 Fasman, G. D., 124 Faulkner, J. A., 612 Fauquant, J., 98 99 Favaro, A., 230 Fechner, A., 139 Fee, C. J., 168 169 Feeney, S., 450 Fekete, A. A., 563 Felipe, X., 283 284, 286 288 Fenaille, F., 140 142 Fenelon, M. A., 199, 210, 361 362, 466 469, 471 472, 476f Fenimore, P. W., 290 291 Fennema, O., 136, 138 Ferguson, L. C., 10 Fernandes, P., 459 Fernandez-Martin, F., 474 Fernández, A., 522t Ferrando, A. A., 583 Ferraz, H., 138 Ferreira, C. A., 641 Ferreira, I. M. P. L. V. O., 7 8 Ferry, J. D., 125 126 Fersht, A., 290 291 Fertsch, B., 283 284 Fetzer, R. W., 414 Feys, G., 284 Fife, R. L., 361 362 Filonzi, E. L., 455 456 Finckenberg, P., 523 524 Finkelstein, R. A., 30 Finlayson, J. S., 218 Finnigan, S., 209 210 Fischer, J., 460 462 Fischer, P., 204 205, 208 209 Fisher, M. C., 496, 510 Fiszman, S., 352 353

Fitzgerald, G. F., 519 521, 524 FitzGerald, R. J., 392 393, 470 471, 521, 522t, 524, 530 532, 534 535, 582 Fitzpatrick, J. J., 129 130, 294, 300 Fitzsimons, S. M., 196 197, 281, 357 Fletcher, G., 585 Fletcher, J. E., 11, 451 Fletcher, P. D. I., 466 469 Fleury, Y., 20, 457 Flick, D., 189 196, 298 Fliss, I., 532 533 Floch, F., 532 533 Flores, R., 550 551 Flosi Paschoalin, V. M., 443t Flynn, A., 5 Foegeding, E., 205 206, 211 Foegeding, E. A., 138, 158, 160 161, 199, 206 207, 225 227, 255 256, 281, 340, 342 343, 345, 366 367, 385, 396f, 469 470, 508 Folk, J. E., 218 Folland, J. P., 585 Fonseca, L., 459 Ford, J. E., 455 456 Forsum, E., 551 Foskett, A., 603 Foster, K. D., 135 136 Fountoulakis, M., 165 166 Fox, A. J., 395 396 Fox, K. M., 399 400 Fox, M., 474 475 Fox, P. F., 3 5, 7 8, 20, 34, 123, 155, 163 165, 185 186, 283 284, 286 288, 460, 463, 465 466, 470 471, 580, 643 Francis, G. L., 36 Francois, S., 470 471, 508 Frankel, E. N., 381 382, 460 462 Frauenfelder, H., 290 291 French, S. J., 139, 354 355, 366 367 Frestedt, J. L., 520, 562, 650 Friedman, M., 132, 220, 383, 581 Friedrich, J., 290 291 Friend, B. A., 30, 35 Frokiaer, H., 443t Frongillo, E. A., 550 551 Frontela, C., 451 452 Froy, O., 563 564 Fruhbeck, G., 211

Fruton, J. S., 216 217 Fry, C. S., 565 Fryer, P., 185, 294, 300 Fryer, P. J., 189 196, 414 Fu, D., 320, 415 Fujimoto, K., 525 526 Fujio, Y., 285 Fujita, Y., 186 Fukuda, D. H., 598, 602 603, 607 608 Fukuhara, K.-I., 10 Fukumoto, L. R., 13, 113 Fukushima, D., 581 Fung, D. Y. C., 382 Funtenberger, S., 283 285 Furlanetti, A. M., 1 2, 24 26 Furuya, M., 10 Fweja, L. W. T., 28 29

G

Gagnaire, V., 33, 166 167 Gaiani, C., 127 129, 306, 348 349 Gaillard, J. L., 20, 457 Gajendragadkar, C. N., 108 Galani, D., 187 189, 470 471 Galazka, V. B., 283 285 Galietta, G., 420 Gallardo-Escamilla, F. J., 379 381, 383 387, 387f Ganesh, S., 650 Ganss, B., 453 Ganzevles, R. A., 138 139, 263 Gao, Y., 265 266 Gapper, L. W., 455 456 Garbis, S., 165 166, 170 172 García-Garibay, M., 5 García-Risco, M. R., 287 Garg, A. P., 443t Gasilova, N., 167 Gaspard, S. J., 201 202, 228 Gasparetto, C. A., 259 Gassner, A. L., 167 Gatti, C., 209 210 Gatti, C. A., 133 Gauche, C., 219 220, 353 354 Gaucher, I., 33 Gaucheron, F., 33, 286 287 Gaudel, C., 527 528 Gautam, S., 496, 500, 509 510 Gauthier, J. J., 451 452, 645 646 Gauthier, S., 532 533 Gauthier, S. F., 2, 346, 443t, 457 458, 462 463, 469 470, 532 533

Author Index

Gavaldà-Navarro, A., 457 458 Gaye, P., 8 Gebler, J. C., 443t Gee, V. L., 349, 466 469 Geer, D., 291 292, 300, 414 Geerts, B. F., 528 Geerts, M. E. J., 18 Gehin-Fouque, F., 451 Geiger, T., 130 Gelfi, C., 167 Gendreau, S., 209 210 Geneix, N., 10 Gennadios, A., 320, 411, 415 416, 425 426 Gentès, M.-C., 340 Gerard, A., 303 304 Gerard, P., 377 Gerard, P. D., 377 378, 384t, 395, 497 Gerberding, S. J., 168 German, J. B., 451 452, 459 462, 519 Gerrard, J. A., 130, 260, 388, 500 501 Gerson, C., 647 Gersovitz, M., 495 496 Gervais, F., 648 Gervilla, R., 283 284 Gésan-Guiziou, G., 97, 102 Geurts, T. J., 297 298 Gezimati, J., 200 201 Ghaly, A. E., 319 Ghorpade, V. M., 415 416 Ghosh, S., 108, 112, 555 Gibala, M. J., 606 Gibbs, B. F., 531 532 Gibbs, J., 650 Gibson, G. R., 443t Gidley, M. J., 306 Giehl, T. J., 455 Gil-Castell, O., 426 Gilar, M., 443t Giles, H. F., 412 413 Gill, H., 443t, 643 Gill, H. S., 155 156, 532 533, 647 648 Gill, M. H. M., 353 354 Gilliland, S. E., 395 Gillman, M. W., 552f Gilsdorf, M., 449 Gimel, J. C., 189 Giner, J., 228 229 Girardet, J. M., 20 22, 160 161, 443t, 457, 641

Girault, H. H., 167 Giromini, C., 563 Giroux, H. J., 225 226 Giuliano, G., 393 395 Giuseppin, M. L. F., 189 Givens, D. I., 563 Gjesing, B. O. O., 462 463 Gleinser, M., 445 Glomb, M. A., 157 Glow, K. M., 603 Goddick, L., 388 Goddik, L. M., 388 389 Godfrey, K. M., 553 Goehring, K. C., 443t, 459 460 Goff, H. D., 359 360, 466 469 Gogate, P. R., 108 Gold, P., 647 648 Goldberg, A. L., 583 Golding, M., 388, 582 Goldman, A. S., 14 Golinelli, L. P., 443t, 449, 455 Golkar, A., 155 156 Golowczyc, M., 459 Gomes, A. M. P., 520, 639 Gomes, D., 346 347 Gomes, D. M. G. S., 353 354 Gomez-Zavaglia, A., 346 347 Gomez, H. F., 648 Gontard, N., 421 422 González de Mejía, E., 528 González-Martínez, C., 352 González-Tello, P., 103 104 Goodall, S., 169 Goodison, A., 465 466 Gorbe, S. B., 264 Gordon, L., 310 Gordon, W. G., 7 8 Görg, A., 165 166 Gorissen, T., 212 Gorris, L. G., 421 422 Gorry, C., 361 362 Goss-Sampson, M., 597 Gotham, S. M., 189 196, 190t Goto, Y., 216 217 Gottlöber, R. P., 415 416 Goudarzi, M., 360, 528 Gouws, P., 318 Grácia-Juliá, A., 294 295 Grahl, T., 313, 315t, 317 Grandison, A. S., 28 29, 169 Grant, I. R., 318 Grant, J. E., 301 Graverholt, G., 443t, 446 447, 642

Green, D. W., 189 196, 190t Green, I., 645 646 Green, M. L. H., 414 415 Greenberg, J. A., 559 Greenhaff, P., 594 Greenhaff, P. L., 608 609 Greiter, M., 460 462 Grey, V., 649 Grey, V. L., 618 Grieger, J., 551 Grieser, F., 303 Griesser, H. J., 128 Grieve, P. A., 17 18 Griffin, M. C. A., 4 5 Griffin, W. G., 4 5 Griffiths, M. W., 16t, 27, 31, 34 35 Grijspeerdt, K., 7 Grimm, R., 459 460 Grinberg, V. Y., 283 284 Grinfeld, E., 606 607 Groot, L. C., 585 592 Grosch, W., 408 409 Groves, M. L., 35 36, 38t Gruppen, H., 160, 189 196, 190t, 260, 413 Grygorczyk, A., 342 343 Guadix, A., 103 104, 113 Guadix, E., 113 Guadix, E. M., 103 104 Guamis, B., 283 284 Guan, Y. G., 314, 315t Gubbels, J. W., 442 445 Guerrero, A., 173 174 Guggenheim, B., 644 Guilbert, S., 420 422 Guillet, J., 445 446 Guimont, C., 641 Guinee, T. P., 185, 347 348, 361 363, 365 367 Guingant, A., 283 284 Gulseren, I., 309 Gülseren, I., 414 415 Gulzar, M., 130 132, 131f, 134 135, 143, 205 206 Gumá, A., 610 Guo, H. Q., 155 156 Guo, L., 528 529 Guo, M., 639, 642 Guo, M. F., 496 Guo, M. R., 5 Guo, Q., 206 207 Guo, T. L., 221 Guob, T. L., 343 344

673

674

Author Index

Gupta, G., 443t, 451 Gupta, N., 35 Gupta, P., 417 Gupta, R., 160 161 Gupta, S., 443t Gupta, V. K., 366 367 Gürgen, S. G., 620 Gurr, M., 440t Gurtler, J. B., 443t, 455 Gutman, J., 649 Guy, E. J., 472 474, 473t Guy, P. A., 140 142 Guyomarc’h, F., 132, 134 135, 143, 189 196, 201 206, 367, 465 466 Guzey, D., 309, 414 415

H

Ha, E., 155 156, 409, 507 Ha, E. Y. W., 99 100, 131 132, 377, 383 384, 580 Ha, J. K., 382 Habicht, J. P., 550 551 Haboubi, N., 565 Haddad, L., 550 551 Haertlé, T., 135, 220 221, 283 284 Hagen, S. J., 292 Hagiwara, T., 414 Hahn, R., 168 169 Haigh, B. J., 166 167 Hajduch, E., 611 Hajibeygi, M., 107 Hakansson, A., 9 Hall, W. L., 650 Halldorsson, T. I., 552f, 553 Hallihan, A., 528, 610 Halpin, H., 563 Halton, T. L., 495 496, 559 Hambling, S. G., 186 Hamel, U., 524 Hamer, R. J., 135, 508 Hammann, F., 320, 408, 411 412, 415 Hamouz, F., 421 Handa, A., 415 Handschin, S., 137 138 Hanna, M. A., 320, 415 416, 425 426 Hansen, E., 168 169 Hansen, M., 594 595 Hansen, P. S., 132 133 Hansen, S. I., 455 456 Hanson, B., 585

Hanssens, I. A., 320 Haque, M. K., 136 Haque, Z. U., 361 363 Hara, D., 613 Harada, E., 452 453 Haraguchi, F. K., 586t, 614 615, 621 Hardin, C. C., 225 226 Hardman, M. J., 186, 470 471 Hardy, J., 419 Hargreaves, C., 130 Hargrove, R. E., 385 Harland, H. A., 472 474, 473t Harmon, R. J., 10 Harnulv, B. G., 28, 396 397 Harnulv, G., 647 Harper, W., 220 221 Harper, W. J., 139, 366 367 Harrington, D., 381 Harris, B. J., 440 441 Harris, R. C., 608 Harstad, O. M., 583 Hartel, R. W., 360 Hartman, J. W., 616 Hartmann, R., 520 521 Hartwig, A. C., 607 Harwalker, V. R., 385, 392 393 Hascoet, J. M., 440 441, 445 Haselmann, K. F., 454 Hashemi, M., 417 418 Hassannia-Kolaee, M., 417 418 Hassler, C. M., 637 Hassmén, P., 615 Hata, I., 450 Hata, Y., 524 Hattori, M., 221 222, 261 Haug, A., 583 Hauway, A., 382 383 Havea, P., 128, 130, 196 197, 200 201, 283 284, 301, 470 471 Havenaar, R., 212 Hayakawa, I., 283 287 Hayaloglu, A. A., 361 362 Hayasawa, H., 455 Hayashida, K., 452 453 Hayes, A., 579, 592 593, 606 607 Hayes, K. D., 164 165 Hayes, M., 32 33, 521, 524, 533 Hazizaj, A., 318 He, J., 267 268 He, K., 562 He, L., 258

He, S. H., 22 He, Y., 343 344 Hebert, J., 462 463 Heck, J. M. L., 7 8, 449 Heegaard, C. W., 453 Heenan, S., 498 Heffernan, S. P., 300 Heine, W. E., 7 8 Heinig, M. J., 449 Heino, A., 103 Held, J., 320, 415 Hem, S. L., 303 Hemar, Y., 185, 508 Henare, S. J., 650 Hendrickx, M., 189 196 Henle, T., 34 35, 136 137, 174 175, 251 252 Hennigs, C., 125 126 Henriksson, A., 535 536 Henriques, M., 346 347 Henriques, M. H. F., 353 354 Heppe, D. H., 551 552, 552f Herbig, A. L., 6 7, 196 Herceg, I. L., 155 156, 306 Herceg, Z., 155 156, 213 214, 306 Heremans, J. F., 17 18 Heremans, K., 286 Hering, N. A., 443t, 458 459 Hermansson, A.-M., 207 208, 416 Hernandez-Izquierdo, V. M., 412 413 Hernández-Ledesma, B., 522t, 524 527, 534 535, 641 Hernández-Sánchez, H., 5 Hernando, I., 352 353, 357 358 Hernell, O., 446 447, 642 Herrera-Insua, I., 648 Herrero-Martínez, J. M., 167 Herse, J. B., 18 Herskovi, T. T., 186 Hess, S. J., 255 Hetherington, M. M., 565 Heydari, I., 563 Heymann, H., 378 379 Heymsfield, S. B., 559 Hickey, R. M., 449 450 Hicks, K. B., 249 250 Hiddink, J., 7 Higuchi, M., 599, 606 Hii, M. J. W., 301 Hill, A. R., 366 367 Hill, C., 519 520 Hill, C. A., 524

Author Index

Hill, K. M., 606 607, 610 Hill, R. L., 8 Hill, S. E., 269 270 Hiller, B., 135, 268 269, 271 Hillier, R. M., 472 474, 473t Hindmarsh, J. P., 355 356, 496 Hines, M. E., 199 Hinrichs, J., 283 284, 286 287, 361 362 Hintoiu, A., 223 224 Hinz, L.-V., 416 417 Hiraoka, Y., 7 8, 288 289 Hirata, H., 535 536 Hirayama, K., 10 Hirs, C. H. W., 35 36 Hirst, R., 209 210 Hirth, T., 412 413 Hirtz, C., 166 167 Hite, B. H., 282 Ho, J. X., 10 11 Hoare, R. J. T., 11 12 Hochstrasser, D. F., 446 447 Hoddinott, J., 550 551 Hodge, J. E., 251 252 Hodnett, M., 129 130 Hoffman, B., 451 Hoffman, J. R., 599, 613 614, 618 Hoffmann, M. A. M., 133, 189 197, 190t Hoffmann, P., 227 Hoffmann, R., 157 Hofman, A., 552f Hofman, Z., 596 597 Hogan, S. A., 199, 356, 471 472, 476f, 500 501, 509 Hogenboom, J. A., 25, 270 271 Hogenkamp, P. S., 500 Højrup, P., 454 Holland, B., 107 Holland, J. W., 21, 24 25, 124, 140 142, 157, 160 161, 165 167, 170, 174 175, 505 506 Holm, G. E., 213 214 Holm, J., 455 456 Holm, L., 560, 610 Holmes, D. G., 385 Holowachuk, E. W., 11 Holsinger, V. H., 335 336 Holt, C., 5 6, 189 196, 443t, 460 462, 524 525 Hong, J., 522t Hong, Y.-H., 199

Hongsprabhas, P., 196 Hooijdonk, A. C. M., 27 Hoover, D. G., 283 284 Hoppe, C., 445 446, 556 557 Hopper, K. E., 7 8 Horswill, C. A., 603 Horton, S., 550 551 Hoshino, M., 216 217 Hosri, C., 348 349 Hosseini-nia, T., 285 Høstmark, A. T., 583 Houck, K., 318 Hourigan, J. A., 383 384 Howard Zhang, Q., 228 229 Howes, T., 125 126, 135 136 Hsieh, W. C., 230 Hsu, K.-H., 136, 138 Hu, F. B., 495 496, 559 Hu, Z., 345 Hu, Z. X., 218 Huang, C. C., 594 Huang, G. R., 528 529 Huang, P., 520 Huang, S., 522t Huang, W. C., 594 Huber, K. C., 407, 410 411 Huffman, L. M., 381, 389 Hulmi, J. J., 560 561, 586t, 598, 600, 612 Hultman, E., 608 Hundal, H. S., 611 Hunt, D. G., 613 Hunt, J. A., 466 469 Hunter, R. J., 508 Hunziker, P., 447 448, 531 Huo, G., 619 Huppertz, T., 201 202, 283 288, 580 Hurley, L. S., 451 Hurley, M. J., 31 33 Hurley, W., 200 Hurley, W. L., 8, 12 13, 449 450 Hurrell, R., 529 Huss, M., 115, 213 214, 298 300, 353 354 Hussain, M., 30 Huth, P., 409 410, 416 Huyghebaert, A., 283 284, 286 Hwang, F. F., 443t Hwang, H., 611 Hwang, S. A., 443t Hyman, C. R., 355 356 Hyun, C. K., 524 525

I

Iametti, S., 189 196, 190t, 284 285 Ibáñez, R., 113 Ibanoglu, E., 6 7, 9 10 Ibanoglu, S., 6 7, 9 10 Ichinose, A., 417 Ikeda, S., 218, 225 226, 268 269 Ikeguchi, M., 288 289 Imomoh, E., 295 Imoto, T., 130 Imtiaz, S. R., 356, 498, 500 501, 503 Inagaki, M., 443t, 457 Indyk, H., 455 456 Innocente, N., 21 22 Ipsen, R., 132 133, 226 227, 283 284, 298, 350 351, 414 Iqbal, T., 129 130 Irudayaraj, J. M., 319 Isidra, R., 526 Ismail, A., 185 Ismail, A. A., 217, 285, 470 471 Ismail, B., 258, 261 Itoh, T., 25 Ivanov, I. B., 209, 466 469 Ivy, J. L., 599, 605 606, 611, 613

J

Jackman, S. R., 614 Jackson, J. G., 7 Jacob, M., 136 137 Jacobs, M., 106 Jacquier, J. C., 393 Jacquot, A., 532 533 Jaddoe, V. W., 552f Jafary, F., 27 Jägerstad, M., 455 456 Jakeman, P., 528, 610 Jakubowicz, D., 563 564 Jalali, A., 107 Jambrak, A. R., 155 156, 213 214, 306 309 James, A. E., 109 Jan, G., 166 167 Janer, C., 450 Jang, H. D., 201 202 Janghorbani, M., 562 Janhøj, T., 350 351 Janjarasskul, T., 418 Janssen, I., 564 565 Jansson, T., 252 253 Jardin, J., 131 Jasion, V. S., 449

675

676

Author Index

Jasionowska, R., 167 Jaspe, J., 292 Jauhiainen, T., 535 536 Jauregi, P. J., 169 Javidipour, I., 398 Jayaraman, P., 300 Jaziri, M., 447 448 Je, J. Y., 525 526 Jean, M. C., 501 502 Jeantet, R., 131 132 Jefferson, L. S., 613 Jegouic, M., 283 285 Jelen, P., 9 10, 199 Jenness, R., 472 474 Jensen, E., 583 Jensen, H. B., 165 166 Jensen, J., 594 Jervis, M., 394 396 Jervis, S., 377 378, 381, 394 397, 396f Jervis, S. M., 383 384, 384t, 389f, 393 394, 399 400 Jeukendrup, A. E., 595, 606, 614 Jeurink, T. J. M., 465 466 Jeyarajah, S., 6 7, 189 196, 190t Jhaveri, A., 17 Ji, L. L., 618 Jia, M., 228 229 Jia, X., 228 229, 257 Jian, W., 267 269 Jiang, J. X., 528 529 Jiang, R., 443t, 453 Jiang, R. L., 22 23 Jiang, Y., 421 422 Jiang, Z., 259 Jijakli, M. H., 27, 646 Jiménez-Castaño, L., 221 222, 228 229, 258 259, 264 Jiménez-Flores, R., 350 351 Jiménez-Guzmán, J., 5 Jimenez, A., 597 Jiménez, M., 451 Jin, M., 228 230 Jo, Y., 379 381 Joerger, R. D., 418 Johansen, A.-G., 109 Johler, F., 268 John, K., 11 Johnson, R. L., 383 384 Johnston, D. B., 249 250 Johnston, W. H., 453 Jolles, P., 532 533 Jones, O. G., 137 138

Jong, S., 211 212 Jongen, W., 383 Jongen, W. M., 254 Jongh, H. H. d., 413 Jooyandeh, H., 360 Joshi, L., 449 450 Jou, K., 220 221 Jousse, F., 383 Joutsjoki, V. V., 536 Jovanovi´c, S., 409 410, 581 582 Jovanovic-Malinovska, R., 459 Joy, J. M., 585, 586t, 620 Joyce, A. M., 471 472 Joyce, P., 33 Juillard, V., 524 Juliano, L., 523 524 Juliano, P., 302, 304 Jung, G., 160 161 Jung, J.-M., 203 204 Jung, W. K., 525 526 Jungbauer, A., 168 169

K

Kackmar, J., 107 Kadla, J. F., 417 Kahala, M. M., 536 Kaiman, D. S., 586t Kaiser, W., 412 413 Kajihara, J., 285 Kajimoto, O., 535 536 Kaláb, M., 350, 366 367 Kalaivani, S., 113 Kalan, E. B., 35 36, 38t Kalantzopoulos, G., 385 Kalichevsky, M. T., 283 284, 414 Kaliszewska, A., 12 Kalonia, D. S., 125 126 Kamat, D., 12 Kamau, S. M., 9, 443t, 446 447 Kamemori, N., 452 453 Kaminarides, S., 366 367 Kaminogawa, S., 32 33, 261 Kananen, A., 224 225 Kanda, A., 599, 606 Kanda, Y., 227 Kane, M., 449 Kane, M. D., 606 Kanekanian, A., 582 583 Kang, E. J., 377, 393 397 Kankanamge, R., 346 Kanno, C., 414 Kansal, S., 553 Karaca, O. B., 361 362

Karagül-Yüceer, Y., 379 381, 380t, 497 Karam, M. C., 348 349 Karande, A. A., 448 449 Karasu, K., 104 105 Karel, M., 125 126, 135 136 Karmas, R., 135 136 Kashanian, S., 27 Kaska, J., 443t Kasperson, K. M., 360 Kassem, J. M., 446 447, 451, 473t Kato, A., 221 222 Kato, M., 285 Kato, Y., 215 216 Katsanos, C. S., 568 Katz, A. L., 605 606 Katzmarzyk, P. T., 564 565 Kaul, P., 346, 509 510 Kavanagh, G., 199 Kavas, G., 361 362 Kawai, Y., 215 216 Kawakami, H., 644 Kawanaka, K., 599, 606 Kawasaki, Y., 25, 537 538, 644 Kaya, A., 361 362 Kazlauskaite, J., 641 Kee, H. J., 447 448 Kehoe, J. J., 196 197, 227, 255, 343, 471 472 Kelleher, S. L., 443t Kelly, A. L., 31 33, 155, 162 167, 201 202, 283 284, 286 288, 349, 379 381, 462 463, 466 469, 471 472, 580 Kelly, G. M., 462 463, 469 470 Kelly, J., 99 100, 381, 399 Kelly, M. L., 382 383 Kelly, P., 459 460 Kelly, P. M., 100 101, 111 112, 226 227, 349, 356, 366 367, 381, 470 471, 500, 509 Kelly, S. M., 283 284 Kendall, K. L., 598, 602 603 Kennedy, J. W., 610 Kennedy, R. S., 652t Kentish, S., 213 214, 302 303, 308t Kentish, S. E., 302, 308 309 Keogh, M. K., 6 7, 129 130, 206 207 Keowmaneechai, E., 209 210 Keppler, J. K., 215 216 Kerasioti, E., 617 618

Author Index

Kerche, F., 115 116 Kessler, H. G., 197 198, 283 284 Khaldi, M., 7 Khan, S. A., 417 Khanal, R. C., 382 383 Kharb, S., 440 441, 440t, 460 Khataee, A., 107 Khodaiyan, F., 417 418 Khokhar, S., 187 189 Khorshid, M. A., 35 Khwaldia, K., 419 Kielwein, G., 524 Kieseker, F. G., 132 133 Kilara, A., 581 582, 584 Kilcoyne, M., 450 Kilpi, E. R., 536 Kim, B. C., 443t Kim, D. A., 209 211, 268 Kim, D. J., 450 Kim, E. H.-J., 127 129 Kim, J., 294 Kim, J. I., 169 Kim, M. N., 134 Kim, S., 443t Kim, S. B., 529 530 Kim, S. K., 525 526 Kimball, S. R., 613 Kindstedt, P. S., 5 King, N. A., 619 Kingshott, P., 128 Kinik, O., 361 362 Kinsella, E., 643 Kinsella, J., 209 210 Kinsella, J. E., 6 7, 218 219, 297 300, 508 Kirkpatrick, C. H., 645 646 Kislukhina, O. V., 641 Kitabatake, N., 186 Kitamoto, N., 215 216 Kitts, D. D., 520 521, 531 Klaiber, R. G., 157, 503 Klaning, E., 454 Klarenbeek, G., 199, 409 410 Klein, G. A., 422 Klein, P. D., 7 8 Klein, S., 598 Kleinschmidt, T., 103 104 Klibanov, A. M., 355 356, 502 Kline, J. B., 162 163 Klip, A., 610 Klostermeyer, H., 34 35, 201 202, 251 252, 359 Klotz, I. M., 4 5

Knaggs, G. S., 456 Knezevic, J., 443t Knight, M. I., 36, 38 39, 38t Knol, E. F., 449 Knorr, D., 228 229 Knoth, A., 139 Knudsen, J. C., 209 210 Knudsen, V. K., 552f Kobashigawa, Y., 288 289 Kobayashi, I., 284 285 Kobayashi, K., 200 201, 221 222 Koca, N., 361 363 Koch, G., 531 532 Kocie ba, M., 451 452 Kockel, T. K., 125 126 Koga, J., 599, 606 Koh, L. A., 304 305 Koh, L. L. A., 213 214 Koletzko, B., 441 Kolhouse, J. F., 455 456 Kolodziejczyk, E., 203 204, 341 342 Kolver, E. S., 382 383 Kondo, T., 414 415 Kong, B. H., 526 527 Kong, J., 296 297 Kongshavn, P. A. L., 648 Konigsberg, W., 216 217 Konok, G. P., 652t Konrad, G., 103 104, 223 224 Konstance, R. P., 289, 306 Kontopidis, G., 5 6, 443t, 448, 641 Koopman, R., 613 Koppelman, S., 417 Korchowiec, B., 443t Korhonen, H., 12 14, 17, 443t, 449, 521, 524 525, 532 536, 643 Korhonen, H. J., 155 156 Korpela, R., 523 524, 535 536 Koshy, R. R., 418 Kosikowski, F. V., 377, 388 389 Koskinen, P., 524 525 Kosters, H. A., 6 7, 189 196 Kostevich, V. A., 162 163 Kouaouci, R., 19 Koudelka, T., 215 216, 227 Koutchma, T., 318 Kovacs, E. M. R., 495 496 Kovanen, V., 612 Koyasu, A., 284 285 Kramer, T. A., 294 Kramski, M., 14, 449 450

Krause, I., 536 Krause, M., 616 Krauß, S., 103 104 Krebs, M. R. H., 196 Kresic, G., 306 309 Kreuß, M., 537 538 Krieger, J. W., 560, 598 Krishnamurthy, K., 319 Krissansen, G. W., 443t, 450, 643 Kristiansen, K. R., 284 Kristo, E., 318 320 Krochta, J., 418 Krochta, J. M., 410 413, 416 422 Kruzel, M. L., 443t, 451 452, 520 Kubow, S., 285 Kuhn-Sherlock, B., 356, 498 Kuhn, K. R., 6 7 Kujibida, G. W., 392 393 Kulbe, K. D., 460 462 Kulczycki, A. Jr., 449, 643 Kulkarni, B., 558 Kulmyrzaev, A., 223 Kulozik, U., 103 104, 116 117, 155 156, 168 169, 186, 189 196, 190t, 268, 298, 300, 310, 353 354, 414, 470 472, 536 538 Kumar, R., 526 527 Kumegawa, M., 650 651 Kunin, A. S., 35 36 Kunugi, S., 284 285 Kunz, C., 443t, 459 460 Kunzek, H., 263 Kuo, C. H., 613 Kurpad, A. V., 495 496 Kurth, L. B., 425 426 Kuskowski, M. A., 520, 562, 650 Kussendrager, K. D., 27, 159t, 163, 396 397 Kussy, D., 380t Kuwajima, K., 7 8, 288 289 Kvistgaard, A. S., 443t, 449, 454, 583 Kwan, L., 113 Kwon, D. Y., 524 525

L

Laaksonen, T. J., 256 257 Laborde, J. L., 303 304 Labuza, T. P., 127, 134 135, 217, 258, 339, 355 356, 495 496, 500 503, 505, 508 510 LaClair, C. E., 339, 341 Lacroix, I. M., 522t, 527 528

677

678

Author Index

Lacroix, I. M. E., 527 528 Lacroix, M., 212, 415, 418 Lad, M., 206 207 Lagu, A. L., 167 Lai, C. J., 291 292 Lakemond, C. M., 413 Lalande, M., 7 Lalchandani, S., 302 Lam, H., 171 172 Lam, R. S., 210 Lam, R. S. H., 466 469 Lam, S. M., 650 Lambers, T. T., 211 212 Lamsal, B. P., 500, 511 513 Lan-Wei, Z., 107 108, 365 Landel, R. F., 125 126 Lands, L. C., 618, 649 Lang, G., 157 Langer, R., 355 356, 502 Langevin, D., 138 140, 211 Langkamp-Henken, B., 560 Langley, S., 583 Langrish, T. A. G., 125 128 Langsrud, T., 532 533 Larco, J. I., 392 393 Larick, D. K., 377 378, 381 382 Larnkjær, A., 445 446 Larsen, L. B., 31 33, 156 157, 159t, 160 162, 164f, 165 167, 172f, 174f Larumbe-Zabala, E., 585 592, 597, 600, 602 603, 610 Laskowski, R., 603, 620 621 Lassalle, M. W., 288 289 László Kiss, Z., 105 Latella, J., 298 300 Laudadio, V., 3 4 Launay, B., 6 Laursen, I., 643, 648 Lavie, C. J., 559 Lavin, E., 383 384 Law, A. J. R., 283 284 Law, J., 385 Lawless, H. T., 378 379, 383 384 Layman, D. K., 155 156, 583 Lazarevic, D., 427 428 Lazennec, F., 295 296 LazzariKarkle, E. N., 537 538 Lazzeri, A., 423f, 425f, 427 428 Le Floch-Fouéré, C., 127, 130, 133, 135, 138, 140 143 Le Meste, M., 125 126

Le, T. T., 124, 130, 133 136, 140 142, 157, 160 161, 166 167, 172f, 174 175, 174f, 505 506 Lea, T., 532 533 Leardi, R., 379 381 Leaver, J., 134, 201, 409 410 LeBlanc, J. G., 533 Lebrilla, C. B., 173 174, 459 460 Lebrun, I., 523 524 Lechevalier, V., 205 206 Ledda, L., 166 167 Lederer, M. O., 157, 503 Ledl, F., 220 Ledward, D. A., 283 285 Lee-Huang, S., 443t, 455 Lee, A. P., 382 383 Lee, B. H., 531 532 Lee, D., 136 138 Lee, F. Y., 359 Lee, J., 303, 308t Lee, J. R., 524 525 Lee, K. D., 383 Lee, K. M., 366 367 Lee, S., 529 Lee, S. J., 155 156 Lee, S.-Y., 418 Lee, T. D., 652t Lee, V. W. K., 519 520 Lee, W. J., 352 Lee, Y. K., 291 292 Lee, Y. Z., 314 Leeuwenburgh, C., 618 Lefebvre, J., 4 5 Legrand, J., 214, 414 Leidy, H., 564, 564f Leidy, H. J., 495 496, 559 560 Leinberger, D., 124 126 Leiva, G. E., 140 142 Lejeune, M. P. G. M., 495 496 Leksrisompong, P., 377, 392 393 Leksrisompong, P. P., 392 393 Lelas, V., 155 156, 213 214, 306 Lelieveld, J., 212 Lemmens, S. G., 560, 618 619 Lent, L. E., 408, 411 Leon-Sicairos, N., 455 Léonil, J., 135, 140 142, 166 167, 170, 220 221, 254, 530 531 Lerno, L., 173 174 Leroy, J. L., 550 551 Leslie, W. D., 559

Lesmes, U., 264 265, 267 Leutenegger, C. M., 458 Levay, P. F., 645 646 Levi, C. S., 212 Levieux, D., 10 Levin, M. A., 360 Levy, E., 451 452 Lewis, M. J., 7, 9 10, 28 29, 472 Li-Chan, E., 113, 127, 440 441 Li-Chan, E. C., 522t, 527 528 Li-Chan, E. C. Y., 527 528 Li, A., 619 Li, E. W. Y., 533, 537 538 Li, H., 415 416 Li, J., 427 Li, S. Q., 228 229, 314, 315t Li, W., 107 Li, X. E., 393 394 Li, Y., 501, 511 Li, Y. M., 201 202, 203f Liang, L., 500 501 Liang, M., 169 Liaw, I. W., 377 378, 383 386, 384t, 387f, 388, 389f Lieberman, H. R., 579, 600 Lien, E. L., 440 441, 443t, 446 448, 460 462 Lieske, B., 116 117 Lievonen, S. M., 256 257 Lilius, E. M., 643 Lillard, J. S., 255, 259 Lim, L. T., 410 411 Lin, S.-Y. D., 421 422 Lin, X. Y., 501 Linden, G., 22, 443t, 457, 641 Lindmark-Månsson, H., 166 167 Lindner, R. A., 227 Lindsay, R. C., 382 Lindshield, B. L., 556 Lindström, P., 200 Ling, Y. F., 522t Link, A. J., 170 171 Linkmark-Mansoon, H., 200 Linko, P., 283 284 Linko, Y.-Y., 283 284 Linse, S., 9 Lisak Jakopovic, K., 446 447 Lisko, I., 612 Lister, I. M. B., 21 Listiyani, M. A. D., 383 384, 384t, 389 391, 389f, 394 396 Liu, B., 443t, 451 452

Author Index

Liu, D. K., 35 Liu, D. S., 503 Liu, D. Z., 474 475 Liu, F., 18, 265 267 Liu, G., 136 137, 259, 261 262, 340 Liu, H. L., 230 Liu, H. S., 230 Liu, J., 254, 259 Liu, Q., 526 527 Liu, T. X., 6 Liu, W. R., 355 356, 502 Liu, X. M., 9, 443t, 496 497, 500 503 Liu, Y., 125 126 Livney, Y. D., 200 201 Lix, L. M., 559 Liyanaarachchi, S., 116 117, 298, 344 345 Liyanaarachchi, W. S., 227 228, 301, 343 Llorca, E., 352 353, 357 358 Lloyd, M. A., 255 Lluch, A., 495 496 Lo, C. G., 383 Lobato-Callerosa, C., 350 Lobley, G. E., 583 Loch, J. I., 225 226 Lockwood, C. M., 560 561 Loenneke, J. P., 560 561 Loisel, C., 214, 414 Lollo, P. C. B., 526 527, 596 597 Lomax, M. A., 583 Lonchamp, J., 208 Long, S. J., 650 Longland, T. M., 560 Lonnerdal, B., 22 23 Lönnerdal, B., 7, 17, 19, 440 441, 443t, 446 448, 451 455, 463, 642 Loomis, A. L., 302 Lopetcharat, K., 377 Lopez, C., 504 505 Lopez de Romaña, D., 555 Lopez-Buesa, P., 302 López-Fandiño, R., 24, 221 222, 258 259, 283 288, 533, 536 Lorenzen, P. C., 135, 268 269, 271, 536 Lorient, D., 22, 408, 443t Lothian, J. B., 649 Lottspeich, F., 160 161

Loupil, F., 284 Lourdes, A., 526 Loveday, S. M., 3, 349, 355 356, 496 497, 500 501, 505 Lovegrove, J. A., 563 Lowe, E. K., 283 284 Løwenstein, H., 462 463 Loye, S., 22, 443t Lu, F. J., 533 Lu, H., 341 342 Lu, J., 271 272 Lu, L., 562 Lu, N. Y., 498 502, 499f, 504 505 Lu, R. R., 9, 443t, 522t Lu, W., 520 Luan, B. B., 466 469 Lubec, G., 165 166 Lubran, M. B., 383 384 Lucassen, P. L., 442 445 Lucey, J. A., 221 222, 252 253, 259, 271 274, 352, 361 362, 460 462 Luck, P. J., 138 Luden, N. D., 595 Lugowski, S., 617 618 Lugt, R. V., 596 597 Luhovyy, B. L., 495 496, 528, 611, 649 650 Luo, Y., 271 272 Lutter, P., 174 175 Luyten, H., 584 Luz Sanz, M., 273 Lykkesfeldt, A. E., 643 Lyng, J. G., 304, 315t Lyngbye, J., 455 456 Lyster, R. L., 199, 472 474

M

Ma, C., 265 266 Ma, C.-Y., 185, 218 219 Maas, A. J. R., 7 MacDermott, R. P., 449, 643 Macdonald, I. A., 354 355 MacDougal, D. P. G., 349 Macej, O., 581 582 MacGibbon, J., 53 MacKenzie-Shalders, K. L., 619 Mackereth, A. R., 130 Mackey, K. L., 362 Mackie, A., 268 269 Mackie, A. R., 211 212 Madadlou, A., 521, 528

Maddalena, J., 35 36 Madsen, K., 594 Madureira, A. R., 520, 639, 645 646 Maes, W., 523 524 Maffulli, N., 600 601, 609 Maga, E. A., 29 30 Magboul, A. A. A., 162 Maggi, S. P., 584 585 Mahajan, S. S., 388 389 Mahe, S., 211 Mahmoodreza, G., 563 Mahmoud, N. S., 319 Mahmoud, R., 411 Mahoney, A. W., 529 Maillard, L. C., 251 252 Majorek, K. A., 11 Majumdar, S. R., 559 Malcata, F. X., 520, 639, 641 Malcolm, G., 448 Malcolm, P., 211 212 Malec, L. S., 140 142 Malessa, R., 284 285 Malin, E., 201 202 Maluccio, J. A., 550 551 Mamila, P., 643 Mamontov, E., 158 Manary, M., 555 556 Manderson, G. A., 186, 470 471 Mangone, M., 564 Mann, B., 24, 155, 443t, 522t, 524 527, 536 Mann, J., 560 Mann, M., 170 172 Manninen, A. H., 611 612 Manoj, P., 349 Manso, M. A., 166 167, 173 174, 533, 536 Mansouri, N. E., 398 Mantha, V. R., 382 Manzoni, P., 451 453 Mao, X. Y., 348 349 Marangoni, A., 196 Marangoni, A. G., 289 Marcell, P. D., 455 456 Marchall, E., 641 Marcone, M., 289 Marcus, S. L., 39 Marella, C., 107 Marie, G. N., 501 502 Märkl, H., 313, 315t, 317 Markofski, M. M., 602 Markus, C. R., 642, 651, 652t

679

680

Author Index

Markworth, J. F., 568 Marnila, P., 12 14, 17, 443t, 449, 643 Marquina, P., 302 Mars, M., 500 Marshall, K., 165 166, 449 450, 525 526, 639, 648 Marsili, R. T., 379 381 Martens, E. A., 618 619 Martens, M. J., 618 619 Marth, E. H., 382 Martín-Álvarez, P. J., 522t Martin-Belloso, O., 314 Martin-Moreno, J., 563 Martin, G. J. O., 302, 474 475 Martin, L., 533 Martin, P., 3 4, 7 8 Martinez-Alvarenga, M. S., 126 127, 132 134, 138 139 Martinez-Ferez, A., 103 104 Martínez-Maqueda, D., 531 532 Martinez, J. A., 560 Martínez, K. D., 268 Martinez, M., 536 Martinez, M. J., 200, 470 471, 537 538 Martini, S., 213 214, 304, 339 340 Martins, J., 264 Martins, S. I., 254 Martorell, R., 550 551 Mary, S. K., 418 Mascaraque, M., 645 646 Masci, M., 167 Maskan, M., 126 Masle, I., 10 Maslova, E., 553 Mason, J. B., 456 Mason, T. J., 155 156, 213 214, 302 303, 306 Masotti, F., 25, 270 271 Massaro, S., 500 Masson, P., 283 284 Masson, P. L., 17 18 Matar, C., 533, 535 536 Mate, J. I., 419 Matheson, A. R., 465 466 Mathot, A. G., 20, 457 Mathur, M. P., 35 Matijevic, T., 443t Matsudomi, N., 6 7, 200 201, 227, 508 Matsumura, Y., 209 Mattas, J., 354

Mattiasson, B., 169 Matulka, R. A., 454 Maubois, J. L., 2, 98 99, 391 392, 530 531 Mauri, A. N., 414 Mawatari, K., 614 Mawson, R., 302 Mayeur, S., 451 453 Mazzali, M., 22 Mazzeo, M. F., 160 Mc Carthy, N., 210, 466 469 McAinch, A. J., 281, 606 607 McAllan, L., 619 McAlpine, A. S., 186 McArthur, S. L., 128 McCarthy, K. L., 418 McCarthy, N. A., 466 469 McCarthy, O., 206 McCarthy, O. J., 206 McClements, D., 209 210 McClements, D. J., 199, 206 207, 209 210, 223, 264 267, 357, 379 381, 466 469 McClenaghan, N., 527 528 McDaniel, M. R., 388 389 McDermott, J., 174 175 McDermott, R. L., 466 469 McDonald, K. F., 319 McDonough, F. E., 385, 393 394 McElroy, S. J., 443t, 457 458 McGauley, S. E., 289 McGivern, P. L., 449 McGlory, C., 597 McGregor, G. D., 11 12 McGuffey, M. K., 158, 206 207, 255 256, 285, 469 470 McHugh, T. H., 412 413, 419 McIntosh, G. H., 519 520, 639 McKee, M. D., 453 McKellar, R. C., 385 McKenna, A. B., 189 196 McKenna, B. M., 472 474, 473t McKenzie, H. A., 4 5, 7 8, 186 McLellan, T. M., 579, 595, 599 600 McMahon, B. H., 290 291 McMahon, D. J., 355 356, 361 363, 496 497, 500 501, 505 506, 509, 511 McManus, J. J., 210, 466 469, 471 472 McManus, W. R., 355 356, 496 McMorrow, A. M., 561 562 McNerney, O., 419

McSweeney, P. L. H., 31 34, 162, 185 186, 460, 465 466, 470 471, 643 McSweeney, S. L., 209 210, 465 466 McSwiney, M., 205 McVeagh, P., 450 McWilliams, M., 449 Mediwaththe, A., 296 297 Medves, J. M., 617 618 Meehan, S., 227 Mehalebi, S., 187 189, 204 Mehra, R., 6 7, 111 112, 186, 459 460, 583 Meilgaard, M. C., 378 379 Meininger, G. A., 22 23 Meisel, H., 155 156, 520 521, 530 533, 536, 643 Meisel, M., 524 Meister, F., 415 416 Mejia, E. G. D., 525 526 Melgar, P., 550 551 Melton, L. D., 269 270 Meltretter, J., 173 175 Mendis, E., 525 526 Mercadante, D., 4 5 Mercedes, R., 526 Merchiers, M., 140 142, 167 Mercier, A., 532 533 Merens, W., 642 Merin, U, 111 Merin, U., 10 Mero, A. A., 598, 612 Mersch, E., 300, 414 Mert, B., 319 320, 415, 420 Messens, W., 283 284 Metin, M., 361 363 Metsämuuronen, S., 113 Metz, T. O., 506 507 Metzger, L. E., 107, 513 Meyer, C. J., 293 294, 294t Meyer, D. H., 35 36, 38t Meyer, W. L., 35 36 Mezzenga, R., 137 138, 203 205, 208 209 Michaelsen, K. F., 445 446 Michel, M., 230, 283 284, 581 Micke, P., 520, 649 Middleman, S., 291 292 Miesner, J., 650 Migliore-Samour, D., 447 448, 532 533 Miguel, M., 524 525

Author Index

Mihindukulasuriya, S. D. F., 410 411 Mikkelsen, B. Ø., 350 351 Mikkelsen, T. B., 552f Mikkelsen, T. L., 443t, 457 Milkovska-Stamenova, S., 157 Miller, P. E., 553, 561 562, 585 593 Miller, S., 561 Miller, S. L., 602 603 Mills, O. E., 189 196, 190t Mills, S., 519 521 Millward, D. J., 155 156, 650 Milne, J. M., 17 19 Mimouni, A., 306 Min, D. B., 228 229 Mine, Y., 533, 537 538 Minekus, M., 212, 500 Miracle, R. E., 377, 379 381, 380t, 383 385, 384t, 389 393, 389f Miralles, B., 24, 167, 531 532 Miranda, G., 3 4, 7 8 Miroljub, B., 409 Mistry, V. V., 377, 388 389 Mitchell, C. J., 560, 568 Mitchell, J. R., 269 270 Mittal, G. S., 31 Miura, T., 34 Mizuno, S., 534 535 Mizushima, S., 535 536 Mleko, S., 357, 366 367 Moakes, R., 207 208 Moatsou, G., 33 Modak, M. J., 39 Modi, R., 128 Moeckel, U., 174 175 Moerkens, A., 512 Mohammadreza, V., 563 Moitzi, C., 204 Mølgaard, C., 445 446 Molinaro, S., 414 Molitor, M. S., 106 Mollé, D., 33, 140 142, 170 171, 254 Molle, G., 457 Møller, H. S., 165 166 Møller, L., 457 Møller, R. E., 284 285 Mollerup, J., 168 169 Momany, F. A., 448 Monahan, F. J., 421 Monma, M., 11 Monnai, M., 644

Montella, J., 348 349 Montoni, A., 532 533 Moor, U., 199 Moore, D., 603 Moore, D. R., 392 393, 566, 595, 601, 604 Moore, V. M., 553 Moorthy, D., 556 557 Mora-Gutierrez, A., 201 202 Morales-Suárez-Vaerla, M., 563 Morales, R., 268 Moraru, C. I., 415 416, 421 Morato, P. N., 610 611 Morbidelli, M., 292 293 More, G. R., 474 Moreau, A., 6 7, 196 Morell, P., 352 353 Moreno, F. J., 273, 500 501 Morgan, A. J., 10 Morgan, D. J., 304, 315t Morgan, F., 10, 134 135, 140 142, 170 171, 254 Morgan, L. M., 650 Morgan, P. E., 227 Morifuji, M., 527 528, 596, 599, 606, 610 611 Morikawa, K., 285 Morin, P., 364 365 Moriwaki, H., 227 Morley, J. E., 565 Moro, A., 138, 209 210 Morr, C., 409, 470 471 Morr, C. V., 99 100, 131 132, 310, 377, 383 384, 580 Morris, E. R., 196 197, 281, 357 Morrison, M., 455 Morrison, P. J., 611, 613 Morrissey, P. A., 470 471 Mortier, L., 7 Morton, H. R., 650 Morton, R. W., 597 599, 604 606 Moscovici, A. M., 271 Moskovitz, J., 525 Mossberg, A. K., 9 Motil, K., 495 496 Moughan, P. J., 616, 650 Mounsey, J. S., 6, 226 227, 343, 366 367 Mount, E. M., 412 413 Mourad, W., 462 463 Moure, A., 525 526 Mousan, G., 12 Mousavi, M., 417 418

Mozaffarian, D., 563 Mrukowicz, J. Z., 442 445 Mu, T. H., 228 229, 314 Mu, T.-H., 414 Mudgal, P., 205 206 Muir, D. D., 134, 350, 364, 463 Mukhopadhyay, D., 448 449 Mulcahy, E. M., 224 225, 251f, 253 257, 259 262, 260f Mulet-Cabero, A.-I., 211 212 Mullally, M., 522t, 524 525 Mullen, J. E. C., 34 35 Muller, A., 111 Müller, K., 407 408, 412 413, 417 419 Muller, L. D., 382 383 Müller, M., 283 284 Mullins, C. G., 347 348 Mulsow, B. B., 136 137 Mulvaney, S., 230 Mulvihill, D. M., 187, 196 197, 209 210, 224 225, 251f, 253 254, 256 257, 260f, 261 262, 265f, 266f, 281, 357, 465 466 Muñoz-Guerra, J., 607 Muñoz, F. C., 443t Muñoz, G., 607 Munro, H. N., 495 496 Munro, P. A., 185, 206, 462 463 Murai, N., 7 8 Muramoto, K., 525 526 Murphy, E. G., 199, 210, 465 466, 469 472, 475 476, 476f Murphy, P. M., 283 284 Murray, B. A., 470 471, 534 535 Muschiolik, G., 139 Muthukumaran, S., 302 Muthukumarappan, K., 107 Mutilangi, W., 345 Mutrie, J. C., 443t

N

Nacka, F., 135, 220 221 Naclerio, F., 585 592, 597, 600, 602 603, 610 Nagamatsu, A., 216 217 Nagaoka, S., 641 Nagasawa, K., 261 Naik, L., 524 525 Nair, R. R., 443t, 457 458 Naito, H., 530

681

682

Author Index

Najim, N. H., 14, 449 Nakai, S., 13, 113, 440 441 Nakajima, K., 284 Nakamura, S., 221 222 Nakamura, T., 215 216 Nakamura, Y., 535 536 Nakano, T., 168 169 Narang, A. P., 553 Naranjo, G. B., 140 142 Narici, M. V., 585, 600 601, 609, 620 621 Narin, C., 528 529 Narine, S. S., 289 Narong, P., 109 Narsimhan, G., 209 210, 268 Nasirpour, A., 155 156, 440, 440t Nasu, K., 11 Navarro, M. P., 273 Nayak, K. K., 417 Nechwatal, A., 415 416 Needs, E. C., 283 284, 286 288, 349 Neelima, S. R., 155 156 Neelima, Sharma, R., 443t, 450, 463 Neelima., 24, 536 538 Neeser, J. R., 644 Neirynck, N., 225 226, 264 Nelson, B. K., 391 392 Netto, F. M., 220, 528 529 Neves, L. X., 586t Newbold, M., 377, 389 391, 391t, 392t Newburg, D. S., 443t, 451 452 Newman, J., 393 Newsholme, E. A., 615 Newsholme, P., 527 528, 616 Newton, A. E., 388 Newton, M., 592 593 Ney, D. M., 26, 644 Neyestani, T. R., 169 Ng, T. B., 18, 36, 38t, 168 169 Nguyen, B. T., 201 202 Nguyen, D. N., 443t, 448, 451 453 Nguyen, N. H. A., 305 306 Nickels, J. D., 158 Nickerson, M. T., 210, 221 223, 466 469 Nicolai, T., 187 196, 201 206, 226, 413, 465 466 Nicolas, M. G., 220 221, 462 463 Nicorescu, I., 413 Nicoud, L., 292 293 Nielsen, B. R., 142

Nielsen, J. H., 160 Nielsen, M. S., 454 Nielsen, P. M., 416 417 Nielsen, S. S., 164 165 Nieuwenhuizen, A., 559, 593 Nieuwenhuizen, W. F., 565 Nijdam, J. J., 127 128 Nikolic, I. A., 638 639, 638f Nikolov, A., 138 Nikolov, A. D., 138 Nilaweera, K. N., 619 Nipo, W. K., 35 36 Nishimura, T., 528 529 Nitta, K., 288 289 Nnanna, I. A., 392 393 Noci, F., 304, 315t Nokihara, K., 525 526 Noller, K., 320, 411, 415 417, 423f, 424, 425f Nommsen-Rivers, L. A., 449 Nongonierma, A. B., 392 393, 521 Nono, M., 465 466 Noppe, P., 225 226 Norde, W., 200 Noriega, K. E., 556 Noronha, N., 366 367 Norris, G. E., 203 204 Norris, R., 522t Norton, B. W., 270 271 Norton, C., 561 Norton, E. J., 11 Norton, I., 207 208 Norwood, E.-A., 124, 127, 129 130, 132 144, 146, 250 251 Nosaka, K., 592 593, 614 Novales, B., 138 140, 211 Novalin, S., 460 462 Novi, G., 161t Nualpun, S., 528 529 Nuijens, J. H., 18 Nurminen, M. L., 523 524 Nursten, H., 252 253, 272 273 Nygren-Babol, L., 455 456 Nylander, T., 200 Nyström, M., 113

O

Obeid, J., 603 Oberg, C. J., 361 362 Obermaier, C., 165 166 Oboroceanu, D., 213 214 O’Callaghan, D. J., 226 227, 462 463

O’Callaghan, D. M., 209 210, 460 462, 465 466 Ochoa, T. J., 648 Oda, M., 285 Oddy, W. H., 443t, 458 459 O’donnellO’Donnell, R., 170 171 Odriozola-Serrano, I., 314, 315t Oevermann, A., 443t, 447 448 Oey, I., 317 O’Flaherty, F., 164 165 Oftedal, O. T., 3, 5 6 Ogasawara, M., 11 Ogasawara, M. A., 520 Ogge, G., 448 449 Ogliari, P. J., 353 354 Ognjen, M., 409 Oh, H. E., 216 217 Oh, N. S., 273 Oh, S., 443t, 450 Oikawa, S. Y., 560 Okawa, T., 460 463 O’Kennedy, B. T., 6 7, 186, 226 227, 343, 348 349, 356, 366 367, 500, 583 Okunuki, H., 158 Okur, O. D., 353 354 Olafsdottir, A., 551 Olano, A., 221 222, 273, 283 287, 500 501 Oldfield, D. J., 187 189, 472 474, 473t Olieman, C., 167 Oliveira, K. M. G., 4 Oliver, C. M., 269 270 Olivier, B., 651 Ollier, A., 10 O’Loughlin, I. B., 200, 212, 356, 470 471, 509 Olsen, C. E., 284 Olsen, K., 283 284, 414 Olsen, S., 551 Olsen, S. F., 551 553, 552f Oltman, A. E., 377 O’Mahony, J. A., 3 5, 7 8, 20, 28 31, 34, 201 202, 224 225, 251f, 253 254, 256 257, 260f, 265f, 266f, 462 463, 466 469, 471 472 Omar, M. M., 366 367 Omata, Y., 645 646 Onwulata, C., 409 410, 416 Onwulata, C. I., 185, 249 250, 289, 298 300, 306, 389, 582 583

Author Index

Opdahl, L. J., 358 Oria, R., 302 O’Riordan, D., 393 O’Riordan, E. D., 421 O’Riordan, E. F., 366 367 O’Riordan, N., 449 O'Riordan, N., 18, 20 Osawa, T., 215 216 Oseguera-Toledo, M., 528 Oshita, T., 200 201 O’Sullivan, A. C., 472 474, 473t O’Sullivan, M., 366 367, 393, 421 Otani, H., 450, 644 Otte, J., 283 284, 414, 534 535 Otter, D. E., 469 470 Ou, K., 529 530 Oussalah, M., 418 Outinen, M., 103 Owczarz, M., 292 293 Owens, G. F., 35 Oymaci, P., 417 418 Oysun, G., 361 362 Ozimek, L., 168 169 Özkan, L., 106 Özkan, N., 129 130

P

Paddon-Jones, D., 564, 564f, 583 Padwal, R., 559 Paes-Leme, A. F., 528 529 Pagan, R., 313 Pagani, M. A., 260 Paik, S. R., 136 137 Pakkanen, R., 646 Palacín, M., 610 Palani, K., 215 216 Pallansch, M. J., 472 474 Palmano, K. P., 170 Palmer, M., 306, 308 309, 308t Palmio, J., 564 565 Pan, D. D., 155 156 Pang, J., 267 268 Pang, Z., 348 349 Panick, G., 284 285, 294 295 Paniwnyk, L., 213 214 Paoloni, M., 564 Papiz, M. Z., 4, 186, 448 Pappas, C. P., 6 7 Paques, M., 508 Paquet, D., 20 22 Paquin, P., 6, 284, 469 470 Paquot, M., 21 22 Parajo, J. C., 525 526

Parak, F. G., 290 291 Parisi, O., 27, 646 Parisod, V., 174 175 Park, C. W., 136 137, 224 225, 253 256, 259 260, 260f, 262, 335 336, 377 378, 381, 384 385, 397 400, 399f Park, R. J., 382 383 Parker, J. D., 377 378 Parker, M., 381 Parkinson, C. J., 5 Parkinson, E. L., 209 210 Parko, J., 318 Parodi, P. W., 520, 648 Parris, N., 170, 249 250, 413 Parry, D. A., 158, 448, 466 469 Parry, R. M., 30 Pas, M., 230 Pascual, C. Y., 392 393 Pasiakos, S. M., 550, 579, 600, 620 621 Pasin, G., 563 564 Patel, H., 128, 217, 640f Patel, H. A., 187, 201 202, 216 217, 283 289 Patel, K. N., 128 Patel, M. R., 360 Patel, S., 155 156, 158, 619, 639, 640f Paterson, A. H. J., 135 136, 443t, 459 Patino, J. M. R., 210 Patocka, G., 9 10 Patrick, J. S., 167 Patterson, S. D., 171 172 Paul, G. L., 583, 592, 598, 616, 620 Paulsson, M., 200 Pavelic, J., 443t, 457 458 Pavlath, A. E., 415, 581 582 Pearce, D., 127 129 Pearce, K. N., 187 189 Pedersen, L., 168 169 Pedersen, L. R., 457 Pedrosa, M., 392 393 Pedrosa, M. L., 586t Pelaez, C., 450 Pelegrine, D. H. G., 259 Pellegrini, A., 443t, 447 448, 522t, 531 Pellegrino, L., 25, 31, 260, 270 271 Pelosi, P., 5 6 Peng, X. Y., 526 527

Pennings, B., 567 568, 601 Penny, M., 555 Penttila, I. A., 443t, 458 459 Perani, L., 458 Perdigón, G., 533 Pereira Alcântara, L. A., 113 Pereira, C., 346 347 Pereira, C. I., 520, 639 Pereira, C. J. D., 353 354 Pereira, P. C., 582 Pérès, J. M., 530 531 Perez-Alvarado, G. C., 320 321 Pérez-Cano, F. J., 649 Pérez-Gago, M., 418 Pérez-Gago, M. B., 410 411, 416, 418 420 Perez-Hernandez, G., 341 Perez, C., 419 Perez, M. D., 13, 186, 448, 451 452 Perez, O. E., 228 229 Perez, V., 553, 585 592 Perpetuo, E. A., 523 524 Perraudin, J. P., 27, 646 Perreault, V., 364 365 Perticaroli, S., 158 Perusko, M., 256 257 Pesic, M. B., 202 Peteresen, T. E., 32 Peters, T., 10 Petersen, A. C., 568 Petersen, D., 416 417 Petersen, K., 422 423 Petersen, P. H., 284 Petersen, T. E., 20 23, 31 32, 156 157, 162, 165, 453 454, 457 Petit, J., 6 7, 189 196, 190t Petocz, P., 450 Petrie, H. J., 603 Petropakis, H. J., 472 Petschow, B. W., 451 452 Pettersson, J., 9 Peugeot, M.-L., 138 Peyrollier, K., 611 Pezeshki, M., 169 Pham, T. V., 314 Phan-Xuan, T., 137 138, 196, 203 204 Phillips, J. G., 298 300 Phillips, L., 209 210 Phillips, L. G., 414 Phillips, S. M., 392 393, 520, 560, 595, 597, 601 602, 613, 616 Picciano, M., 456

683

684

Author Index

Picciano, M. F., 456 Pihlanto-Leppälä, A., 522t, 524 525, 531 532, 641 Pihlanto, A., 19, 521, 525, 532 536 Pihlanto, A. M., 536 Piilola, K., 524 525 Pikal, M. J., 125 126 Pikus, S., 357 Pilar Buera, M., 135 136 Pilosof, A., 310, 536 Pilosof, A. M., 228 229 Pilosof, A. M. R., 125 126, 139, 200, 268, 470 471, 537 538 Pinder, D. N., 203 204 Pintado, M. E., 520, 639 Piot, M., 414 Piringer, O. G., 418 Pischetsrieder, M., 173 175 Pizones Ruiz-Henestrosa, V. M., 268 Plackett, D., 409 411, 417 Plans, M., 345 Plummer, T. H., 35 36 Poon, S., 185 Popineau, Y., 220 221 Porapakkham, Y., 559 Pordesimo, L. O., 582 583 Potanin, A. A., 294 Potgieter, S., 609 Pothan, L. A., 418 Potier, M., 124 Potter, R., 213 214, 339 340 Pouliot, Y., 2, 346, 364 365, 443t, 451 452, 457 458, 469 470 Poulsen, N. A., 165 166 Pourahmad, R., 417 418 Poussa, T., 535 536 Poutrel, B., 7 Pouzot, M., 189, 203 204 Povolo, M., 379 381 Power, O., 522t, 528, 610 Powers, J., 284 285 Prado, C. M., 559 Prakash, D., 443t Prakash, J., 346, 509 510 Prakash, S., 348 349 Pramuk, K., 440 441 Prasad, S., 474 Prasartkul, P., 559 Prata, L. F., 1 2, 24 26 Prell, C., 443t Price, J., 169 Price, N. C., 283 284 Price, W. E., 227

Price, W. V., 395 Prigent, M. J., 644 Prinz, T., 407 408 Pripp, A. H., 534 535 Pritchard, A. M., 189 196 Prussick, L., 649 Prussick, R., 649 Ptashkin, S. M., 413 Puhl, C., 417 Pupovac, J., 611 Purcell, J. M., 413 Purwandari, U., 352 Purwanti, N., 226, 289, 496 497, 512 513 Puyol, P., 186, 448

Q

Qi, P. X., 185, 296 300 Qi, X. L., 189 196 Qian, M. C., 383, 388, 398 Qian, Z. J., 525 526 Qin, B. Y., 158 Qin, L. Q., 560 Qin, S., 228 229 Queguiner, C., 298 300 Quezada-Gallo, J. A., 425 426 Quiles, A., 357 358 Quiros, A., 524 525, 534 536 Qvist, K. B., 284

R

Raat, H., 552f Rade-Kukic, K., 215 216, 268 Rademacher, B., 283 284, 287 Radha, C., 346, 509 510 Rae, J., 303 Rafiee, H., 211 212 Rahaman, T., 160 Rai, D., 443t, 451 452 Raikos, V., 185 Rainard, P., 7 Rajapakse, N., 525 526 Rajput, Y. S., 24, 155, 443t, 536 Rakchanyaban, U., 559 Ralston, G. B., 4, 186 Ramachandran, K. S., 414 Ramanujam, K. S., 454 Ramchandran, L., 160, 189 196, 227 228, 301, 343 Ramis-Ramos, G., 167 Rammer, P., 520 Ramos, M., 24, 167, 284 285, 287, 524 525, 531 532

Ramos, Ó. L., 407, 419 422 Rangavajhyala, N., 460 462 Rangel, A. H. D., 12 Rantamäki, P., 103 Rao, K. R., 8 Rao, Q. C., 496, 510 Rapeanu, G., 223 224 Raposo, F. C., 156 157 Rasmussen, B. B., 565, 602 603 Rasmussen, J. T., 162, 453 Rasmussen, L. K., 31 32, 156 157, 457 Raso, J., 313 Rastall, R. A., 273 Rastogi, V., 410 411 Ratcliffe, I., 263 Rauch, D. J., 418 Rauh, V. M., 164 165 Rawel, H. M., 215 216, 268 Recio, I., 24, 167, 522t, 524 525, 531 532, 641 Rector, D., 6 7, 508 Reddy, I. M., 529 Reeds, P. J., 7 8 Regester, G. O., 36, 519 520 Regnault, S., 209 210 Regupathi, I., 113 Reichert, K., 411 412 Reid, D. S., 412 413 Reidy, P. T., 586t, 592, 614 Reif, M., 290 291 Reinemann, D. J., 318 Reiter, B., 28, 396 397, 647 Relkin, P., 6, 209 210, 360 Remondetto, G. E., 6 Ren, D., 296 297 Renard, D., 4 5 Renkema, J. M., 413 Rennie, M. J., 594 Rennie, P. R., 130 Renzone, G., 160, 161t, 252 253 Requena, T., 450 Res, P. T., 566, 585 592, 599 Resch, J. J., 281 Resmini, P., 260 Reuter, H., 366 367 Reville, W. J., 347 348 Reynoso-Camacho, R., 528 Rheem, S., 443t Rhim, J. W., 320 Rhim, J.-W., 415 Rhoads, D. D., 94 Ribes-Greus, A., 426

Author Index

Rich, R. R., 645 646 Richardson, T., 158 Richter, R. L., 310 Riedel, C. U., 445 Riener, J., 304, 315t Riera, F. A., 522t Riezs, P., 414 415 Rigby, N. M., 211 212 Rigby, P., 565 Righetti, P. G., 167 Rigo, J., 450 Rindom, E., 586t, 593 Ritchie, S., 106 Rittmanic, S., 338 339 Rivera, J. A., 550 551 Rizvi, S., 230 Roberts, C. J., 292 293, 293f Roberts, S. B., 559 Robinson, J. S., 553 Robinson, R. K., 352, 581 Robitaille, G., 22 23, 159t Rocca-Smith, J. R., 496 Rodrigues, L. R., 168 169 Rodríguez Patino, J. M., 139, 537 538 Rodriguez-Saona, L., 345 Rodriguez, A. R., 156 157 Rodriguez, N. R., 583 Roefs, S., 189 196, 416 Roefs, S. P., 204 Roefs, S. P. F. M., 187 189, 190t, 196 198, 470 471 Rogalska, E., 443t Roginski, H., 228 229, 314 317 Romano, M., 169 Rondanelli, M., 566 567, 609 610 Rong, Y., 450 Ronn, B., 560 Roos, Y. H., 125 126, 136, 199, 256 257, 472, 476f Ros, G., 451 452 Rosati, C., 393 394 Rose, D., 465 466 Rosen, M., 647 Rosenberg, I., 564 Rosenberg, I. H., 443t, 456, 556 557 Rosenberg, M., 206 207, 389 Ross-Murphy, S., 199 Ross, A. I. V., 31 Ross, R. P., 519 521, 524 Rossitto, P. V., 318 Rothwell, J., 6 7 Roubenoff, R., 564 565

Roudaut, G., 125 126 Roupas, P., 641 Rouvet, M., 189 196, 203 204, 341 342 Roux, S., 252 253 Row, K. H., 169 Rowe, M. T., 318 Roy, A., 104 105 Roy, N., 448 449 Roychoudhury, A. K., 7 8 Ru, Q., 254 Ruan, R., 501 Ruck, M., 174 175 Rudloff, S., 443t, 459 460 Ruegg, M., 199 201 Ruel, M., 550 551 Ruger, P. R., 360 Rullier, B., 138 140, 211 Rumball, S. V., 448 Russell, H., 611 Russell, S., 383 Russell, T. A., 377, 497 Rustad, P. I., 596 Rutherfurd-Markwick, K. J., 607, 616 Rutherfurd, K. J., 532 533 Rvan, R., 167 Ryan, J. T., 450 Ryan, K., 205, 226 Ryan, K. N., 226, 341 342 Ryan, M. P., 457 459 Rybalka, E., 592 593

S

Sabato, S. F., 415 Saboya, L. V., 391 392 Sacco, P., 592 593, 614 Sadat, L., 522t Sadek, C., 127 Saeed, F., 30 Sagis, L. M. C., 266 267 Sah, B. N. P., 281 Sahan, N., 361 363 Saiga, A., 528 529 Saini, P., 530 Saint-Jalmes, A., 138 Saint-Sauveur, D., 532 533 Saito, K., 526 Saito, M., 11 Saito, T., 25 Sakai, K., 216 217 Sakai, M., 216 217 Sakamoto, K., 592 593 Sakuma, T., 174 175

Sakurai, K., 216 217 Sakurai, M., 288 289 Saldo, J., 283 284 Salehi-Marzijarani, M., 562 Salmiéri, S., 418 Salonen, A., 138 Salou-Cavalier, C., 298 300 Salt, L., 211 212 Salter, D. N., 455 456 Saltmarch, M., 127, 134 Saltzman, E., 559 Salzano, A. M., 252 253 Samra, R. A., 650 Samudrala, R., 361 362 Samygina, V. R., 162 163 Samyn, P., 410 411 Sanchez, L., 451 452 Sánchez, L., 453 Sanchón, J., 448 Sandler, B., 445 446 Sandoval-Castilla, O., 350 Sandström, O., 446 447, 450, 642 Sängerlaub, S., 407 408, 412 413, 417, 419 Sangild, P. T., 448 Sangwan, R., 524 525 Santilli, V., 564 Santos, J. M., 167 Santos, L. H. L. M. L. M., 7 Santos, M. J., 168 169 Sarikus, G., 353 354 Sarioglu, H., 160 161 Saris, W. H., 566 Sarkar, A., 264 265 Sarkar, P., 108 Sarker, A., 349 Satue-Gracia, M. T., 460 462 Saucier, L., 418 Sauer, G., 268 Saunders, M. J., 595, 606 607 Sava, N., 189 196 Savelkoul, H. F. J., 449 Savello, P., 219 Savello, P. A., 366 367, 411 Savin, G., 203 204 Savre, M., 344 Sawada, J., 158 Sawin, E. A., 443t, 451 Sawyer, L., 3 6, 186, 443t, 641 Sawyer, W., 196 197 Sawyer, W. H., 4 5, 216 217 Scaloni, A., 160, 161t, 252 253 Scampicchio, M., 268

685

686

Author Index

Schaafsma, G., 155 156, 211 Schack, L., 22, 443t, 453 Schade, A. L., 645 646 Schaffter, S., 206 Schanbacher, F. L., 10 Scharnagl, C., 290 291 Schaschke, C. J., 283 284, 414 Schaupp, C., 168 169 Schebor, C., 125 126 Schenkel, P., 361 363 Scher, J., 124, 133 134, 348 349, 440 Scheraga, H. A., 448 Scherze, I., 139 Schieberle, P., 408 409 Schlaak, J. F., 649 Schleicher, E., 220 Schlemmer, D., 320, 415 Schlimme, E., 530 Schmelzle, H., 445 Schmid, M., 320, 407 408, 411 413, 415 417, 419, 421 425, 423f, 425f, 427 428 Schmidt, D. G., 530 Schmidt, S. J., 129 Schmidt, W. F., 407 408 Schmitt, C., 136, 140 142, 187 196, 201 204, 215 216, 268, 341 342, 413 Schmitt, C. J. E., 230 Schmitt, D. F., 442 445, 649 Schnepf, M., 421 Schoenfeld, B. J., 598 599, 620 621 Schokker, E. P., 125 126, 200 201, 203 204, 470 471 Scholl, S. K., 129 Schols, H. A., 160 Scholten, E., 266 267 Scholz, C., 448 449 Schonewille, A. J., 458 459 Schreier, P., 185 Schröder, A., 210, 469 470 Schroder, J. B., 583 Schroer, A. B., 595, 597, 617, 620 Schrooyen, P., 269 270 Schuck, P., 123, 131 132 Schuler, L. A., 8 Schulman, W., 209 210 Schultz, C. J., 185 Schulz, P. M., 168 169 Schumann, J., 116 117 Schurer, G. J., 366 367

Schutzler, S., 561 Schwartz, B., 462 463 Schwarz, K., 215 216 Schwing, J., 103 104 Scollard, P. G., 283 288 Scot, E. D., 94 Scott, K. J., 455 456 Scott, P. H., 445 Scotter, M., 394 395 Scrimshaw, N. S., 495 496 Seawright, G. L., 449 Segawa, T., 7 8 Seiquer, I., 273 Sekine, K., 648 Selänne, H., 598, 612 Selhub, J., 443t, 456 Sellers, P., 189 196 Selomulya, C., 306 Selvaggi, M., 3 4 Semba, R. D., 553 Sen, A., 7 8 Sen, D., 104 105, 108 Senaratne, V., 470 471, 508 Seppo, L., 535 536 Serra, M., 418 Setarehnejad, A., 583 Sethumadhavan, G. N., 138 Setiowati, A. D., 264 Sever, S., 263 Severin, S., 29, 531 532 Seydim, Z. B. G., 353 354 Sforza, S., 160 Shah, D., 650 Shah, N., 639 Shah, N. P., 535 536 Shahabi-Ghahfarrokhi, I., 417 418 Shahani, K. M., 30, 35 Shahedi, M., 155 156 Shahid, M., 30 Shalaby, S. M., 534 535 Shams-White, M. M., 561 562 Sharieat, S. Z. S., 27 Sharma, B., 168 169 Sharma, P., 315t, 317 Sharma, R., 24, 187, 201 202, 216 217, 524 527, 536, 639 Sharma, S., 646 Shaw, D. C., 4, 186 Shaw, M., 366 Shaw, N. B., 421 Sheehy, P. A., 10, 22 23, 29 30 Sheffield-Moore, M., 583 Sheikholeslami, V. D., 586t, 618

Sheldrake, R. F., 11 12 Shepard, D. S., 564 565 Sherman, M. P., 443t, 451 452 Sherman, W. M., 605 606 Shi, Q., 586t Shi, Y. D., 460 Shidfar, F., 563 Shimizu, A., 285 Shimizu, M., 531 Shin, H. K., 524 525 Shin, I.-S., 337 338 Shin, K., 455 Shockravi, A., 107 Shoemaker, C., 206 207 Shojaei-Rami, S., 203 204, 341 342 Shrestha, A., 135 136 Siciliano, R. A., 160 Siebert-Raths, A., 424 Sigma., 28 Silva, J. L., 283 284 Silva, J. T., 443t Silva, M. E., 586t Silva, S. V., 641 Silverberg, N. B., 620 Simatos, D., 125 126 Simmons, M., 300 301 Simó-Alfonso, E. F., 167 Simon, L., 496, 500, 509 510 Simons, J. F. A., 6 7 Simons, J.-W. F. A., 189 196, 190t, 215 Sindayikengera, S., 155 156 Singer, N. S., 115, 298 300 Singh, A., 223 224 Singh, A. M., 249 250, 462 463, 469 470 Singh, D., 553 Singh, H., 158, 185, 187 189, 196 197, 200 201, 203 207, 216 217, 264 265, 283 285, 349 352, 355 356, 462 463, 465 466, 469 471, 474 475, 496, 582 Singh, L., 555 Singh, P., 168 169 Singh, R., 168 169 Singh, T. K., 377 385 Sinha, N. K., 7 8 Sinha, R., 346, 509 510 Sipola, M., 523 524 Sithole, R., 388 389, 398 Sitren, H. S., 560

Author Index

Siuzdak, G., 172 173 Skeie, S. B., 109 Skibsted, L. H., 142, 283 285, 414 Skingle, D. C., 270 271 Skov, A. R., 560 Skura, B. J., 13 Slangen, K. J., 530 Slater, G. J., 619 Sleigh, R. W., 383 384 Smeets, R., 596 597 Smiddy, M. A., 286 287 Smith, A. E., 598, 602 603, 607 608, 622 Smith, A. K., 360, 366 367 Smith, A. M., 456 Smith, D., 283 284 Smith, E., 94 Smith, G. I., 602 603 Smith, G. M., 284 Smith, G. P., 650 Smith, K., 594 Smith, K. L., 10 Smith, R. D., 506 507 Smith, S., 379 381, 383 384, 386 387, 387f Smith, T. J., 377 381, 380t, 383 387, 387f, 389 395, 397 Smith, W. L., 458 Smithers, G., 641 Smithers, G. W., 91, 93 94, 156 158, 519 520 Smolenski, G. A., 166 167 Smoluchowski, M., 293 294 Smountas, A. A., 618 Smulders, P. E. A., 221 Smyth, D. G., 216 217 Sobhaninia, M., 155 156 Sodek, J., 453 Soderlund, K., 608 Sodini, I., 348 349, 354 Soenen, S., 559, 593 Sokhey, A., 230 Sokmen, B., 607 Sokolov, A. P., 158 Sokolov, A. V., 162 163 Solak, B. B., 639 Somers, M. A. F. J., 221 Sommerer, N., 166 167 Somoza, V., 160 Song, K., 529 Sørensen, E. S., 20 24, 159t, 443t, 453 454, 457 Sorensen, J., 24

Sothornvit, R., 416 417, 419, 421 Soukoulis, C., 358 Sourdet, S., 360 Sousa, A., 21 Spahis, S., 451 452 Speakman, J. R., 619 Spector, A. A., 11, 451 ˇ Spehar, I. D., 32 33 Spiegel, T., 115, 213 214, 298 300 Spotti, M. J., 155 156, 206, 221 222, 270 Sprague, R. C., 599 Sprong, R. C., 458 459 St Laurent, T. G., 606 St-Gelais, D., 340 Stäbler, A., 411 413 Stachtiaris, S., 366 367 Stading, M., 416 Stafleu, A., 500 Stanciole, A. E., 638 639, 638f Stanciu, S., 223 224 Stanciuc, N., 223 224 Stanic-Vucinic, D., 256 257 Stanley, R. A., 269 270 Stanojevic, S. P., 202 Stanton, C., 519 521, 524 Stapelfeldt, H., 142, 284 285 Staples, A. W., 586t, 605 Star, D. P., 382 383 Stathis, C. G., 592 593, 606 607 Steele, J. L., 536 Steele, W. F., 455 Steijns, J. M., 639 Stein, A. D., 550 551 Steinhauer, T., 103 104 Stelwagen, K., 166 167 Stensballe, A., 165 166 Stephani, R., 140 142 Sternesjo, Å., 456 Stevens, G. W., 302 Stevens, H., 35 36 Stevenson, R. J., 385 Stobaugh, H. C., 554, 556 558 Stockmann, R., 283 284 Stoinska, B., 442 445 Stojadinovic, M., 168 169 Stone, A. K., 221 223 Storcksdieck, S., 529 Stout, J. R., 560 561, 598, 602 603 Stout, M. A., 377 378 Stover, E. A., 603 Straatsma, H., 474 475 Streaker, C. B., 228 229

Streuper, A., 7 Striolo, A., 230 Struik, L. C. E., 125 126 Strydom, D. J., 37 38, 38t Stuart, M. A. C., 263 Studenski, S., 561 Suarez Weise, A., 550 551 Subirade, M., 6, 284, 295 Suchin, S., 382 383 Sudfeld, C. R., 554 555 Sugahara, T., 443t, 457 Sugai, S., 7 8, 288 289 Sugawara, T., 288 289 Sui, Q., 228 229, 314 317, 315t Sui, Q. A., 315t, 317 Sullivan, L. M., 212 Sullo, A., 207 208 Sumner, I. G., 285 Sun Pan, B., 535 536 Sun, C. C., 510, 512 513 Sun, W. W., 257, 270 Sun, W.-W., 136 137, 228 229 Sun, Y., 94, 267 268 Sung-Moon Hong, S.-M., 337 338 Surel, O., 202 Suri, D., 555 Suri, D. J., 556 557 Suslick, K. S., 414 415 Sutariya, S., 217 Sutariya, S. G., 201 203 Suzuki, Y. A., 17, 19 Svanborg, C., 9, 520 Svanborg, S., 109 Svendsen, I., 455 456 Svensson, M., 9 Swain, M. A., 586t Swaisgood, H. E., 201 202, 206 207, 417, 465 466, 524, 531 532 Swanson, B. G., 284 285 Sweetsur, A. W. M., 463 Syrbe, A., 359 Szajewska, H., 442 445 Szlachetka, K., 501

T

Taboada-Serrano, P., 293 294 Tahavorgar, A., 563 Taheri-Kafrani, A., 271 272 Tai, C. S., 443t, 448 Taiwo, K., 228 229 Takada, Y., 650 651 Takagi, K., 158

687

688

Author Index

Takahashi, K., 261 Takahashi, R., 535 536 Takano, T., 524 Takase, M., 453 Takeuchi, T., 452 453 Talbott, R. D., 451 452 Taleb, H., 453 Talens, P., 418 Tamehana, M., 185 Tamime, A. Y., 350, 352, 581 Tamm, F., 268 Tammineedi, C. V. R. K., 320 321 Tamopolsky, M. A., 392 393 Tamura, T., 456 Tan, S.-Y., 345 Tanabe, S., 528 529 Tanaka, N., 284 285 Tang, C. Y., 107 Tang, C.-H., 218 219, 421 422 Tang, J. E., 353 354, 392 393, 595 Tang, Q., 206 Tani, F., 206 207 Taniguchi, Y., 285 Tanimoto, M., 644 Tanimoto, S. Y., 218 219, 218f Tao, N., 459 460 Tarhan, Ö., 206 Tarnopolsky, M., 594 Tarnopolsky, M. A., 579, 604 Tawa, N. E., 583 Tawfeek, H. I., 14, 449 Taylor, M. A., 354 355 Taylor, M. W., 187 189 Taylor, S., 294, 300 Taylor, S. M., 414 Tcholakova, S., 209, 466 469 Te Morenga, L., 560 Tecimer, S. N., 650 Tedford, L.-A., 283 284, 414 Teixeira, J. A., 168 169 Teply, L. J., 395 Teschemacher, H., 524, 531 532 Teshima, R., 158 Tessaro, I. C., 106 Tessier, H., 465 466 Testin, R. F., 411 Theil, P. K., 12 13, 200, 449 450 Thibault, C., 22 23, 159t Tholey, A., 215 216 Thomä-Worringer, C., 24, 26, 26f Thoma, C., 536 Thomas, C. R., 291 292, 300, 414 Thomas, D. T., 604

Thomas, M. E. C., 124 126, 133 134 Thomas, S., 418 Thomas, U., 443t, 447 448, 531 Thompkinson, D. K., 440 441, 440t, 460 Thompson, M. E., 414 415 Thompson, N. A., 159t, 163 Thorn, D. C., 227 Thorsdottir, I., 551, 560 Tian, Q., 348 349 Tiernan, D., 33 Timasheff, S. N., 4 5, 186 Timmons, B. W., 603 Tipton, K., 598 Tipton, K. D., 155 156, 584 585, 586t, 597, 602 603, 606, 614 Tirelli, A., 31 Tirrell, M., 291 292 Tittsler, R. P., 385 Toba, Y., 650 651 Tobin, J. T., 115, 472 Todd, M. K., 606 Tolkach, A., 186, 189 196, 310, 470 472, 537 538 Tolstoguzov, V., 504 505 Tomaino, R. M., 381 382 Tomasula, P. M., 289, 296 297, 306, 549 550 Tomazi, T., 353 354 Tome, D., 155 156, 559, 593 Tomizawa, H., 130 Tong, P. S., 348 349, 354, 377 378, 380t, 387f, 408, 497 Toole, B. J., 610 Toone, R. J., 606 Toppino, P. M., 379 381 Torley, P., 223 224 Toro-Sierra, J., 116 117 Torres, I. C., 350 351 Torres, J. A., 383 Toubro, S., 560 Townend, R., 4 5, 186 Trabulsi, J., 440 441 Trachootham, D., 520 Tracton, A. A., 410 411 Tran Le, T., 225 226 Tran, A., 355 356, 496 Tran, H., 9 10, 472 Tran, N. L., 442 445, 649 Trauger, S. A., 172 173 Trevizan, G., 220 Treweek, T. M., 227

Trujillo, A. J., 283 284 Truong, V. D., 417 Tsai, J.-S., 535 536 Tsai, W. Y., 533 Tsakalidou, E., 385 Tsouris, C., 293 294 Tsurui, Y., 285 Tuck, A. B., 443t Tufarelli, V., 3 4 Tulipano, G., 3 4 Tullio, L. T., 537 538 Tunick, M. H., 124, 127, 129 130, 142, 298 300, 549 550, 581 582 Tupasela, T., 524 525 Turchiuli, C., 398 Turcotte, O., 364 365 Turgeon, S., 469 470 Turgeon, S. L., 196 197, 340 Turner, L. G., 381 382 Turner, S. A., 159t, 163 Tuttle, R. W., 448 Twigg, P. D., 11 Twomey, M., 6 7 Twomey, T., 129 130 Tyrka, A., 650 Tzia, C., 358 Tzioumis, E., 562

U

Uauy, R., 555 Udd, B., 564 565 Udechukwu, M. C., 530 Udenigwe, C. C., 530 Uenishi, K., 650 651 Ulaganathan, V., 211 Ulbrich, M., 263 Uniacke, T., 34, 288 Upadhyay, V. K., 286 287 Uribarri, J., 252 Ustundag, B., 443t, 458 Ustunol, Z., 319 320, 415, 420 Utley, C. S., 455 456 Utracki, L. A., 417 Uysal, H., 361 362

V

Vaghela, M. N., 581 582, 584 Valentine, R., 606 Valle, N. R. D., 520 Vallee, B. L., 37 38 van Aken, G., 211 212 van Amburgh, M. E., 382 383

Author Index

van Arendonk, J. A. M., 7 8 Van Barneveld, R. J., 270 271 Van Beeumen, J., 320 van Berkel, P. H. C., 18 Van Boekel, M. A. J. S., 126, 134, 253 254, 260 Van Calcar, S. C., 644 Van Camp, J., 283 284, 286, 534 535 Van Cong, N., 448 449 van Dam, R. M., 552f van den Berg, G., 223 224, 365 van den Berg, L. E., 412 413, 416 417 van den Bosch, W. G., 211 212 Van der Does, A. J., 642 van der Goot, A. J., 496 497, 512 513 Van der Linden, H. J. L. J., 7 van der Meer, R., 458 459 Van der Meeren, P., 225 226, 264 van der Padt, A., 106 Van der Plancken, I., 189 196 van der Veen, E., 512 513 van der Vusse, G. J., 451 van Eijk, J. T., 442 445 Van Essen, M., 606 van Hooijdonk, A. C. M., 159t, 163, 396 397, 646 Van Lancker, F., 253 254 Van Loey, A., 315t, 317 van Loon, L. J., 566 Van Loon, L. J. C., 585 592 van Mil, P., 189 196 van Mil, P. J. J. M., 133, 196 197 van Neerven, R. J. J., 449 Van Renterghem, R., 7, 140 142 van Rooijen, P. J., 530 van Valenberg, H. J. F., 7 8 van Veen, H. A., 18 19 van Vliet, T., 138 139, 263, 413 van Zanten, J. H., 469 470 Vanaman, T. C., 8 Vanasupa, L. S., 408 Vanhee, K., 320 Vanhooren, A., 320 Vapaatalo, H., 523 524 Vapattanawong, P., 559 Vardhanabhuti, B., 206 207, 340 Vasbinder, A. J., 201 202, 212 Vasiljevic, T., 116 117, 160, 189 196, 200, 209 210,

213 214, 227 228, 281, 298, 301, 343 345, 535 536 Vasilyev, V. B., 162 163 Vatanpour, V., 107 Vaz, M., 495 496 Vazquez-Landezverde, P. A., 383 Vecchio, G., 189 196 Vedovo, S. D., 644 Vegarud, G. E., 532 533 Veldhorst, M., 495 496 Velickovic, T. C., 256 257 Venema, K., 443t Venema, P., 210, 469 470 Verbeek, C. J., 412 413, 416 417 Vercet, A., 302 Verdini, R., 135, 263 Vereijken, J., 496 497, 584 Verespej, E., 200 201 Verhasselt, V., 458 459 Verheul, M., 187 196, 204, 416, 470 471 Verlaan, G., 596 597 Vermeer, A. W., 200 Vermeir, L., 264 Vermeirssen, V., 534 535 Verreijen, A. M., 610 Verstappen, P. A., 596 597 Versteeg, C., 31, 228 229, 314 317 Verstraete, W., 534 535 Vettel, H. E., 472 474 Vetvicka, V., 32 Vidal, V., 215 Vidcoq, O., 295 296 Vidigal, M. C. T. R., 358 Vieco, E., 98 99 Vieira, K. P., 220 Viljoen, M., 645 646 Villamiel, M., 221 222, 258 259, 500 501 Villas-Boas, M. B., 220 Vincent, D., 173 174 Violleau, F., 202 Virtanen, T., 535 536 Visker, M. H. P. W., 7 8 Visschers, R. W., 6 7, 189 196, 215, 508 Visser, S., 530 Vogt, E. S., 593 Voilley, A., 425 426 Volek, J. S., 560 561, 598 Volpi, E., 602 Volterman, K., 603

von Fellenberg, R., 447 448, 531 Voragen, A. G. J., 135 Vorwerg, W., 415 416 Voswinkel, L., 168 169 Vrvic, M. M., 202

W

Waanders, J., 216 217 Wackerhage, H., 594 Wada, K., 130 Wada, R., 186 Wagner, J. R., 412 413 Wagoner, T. B., 345 Wahn, U., 462 463 Wakabayashi, H., 453 Walisinghe, N., 129 130 Walk, T., 160 161 Walkenström, P., 207 208 Walker, T. B., 586t, 615 Wallace, J. M., 458 Wallentin, C., 647 Wallingford, J. C., 460 462 Walsh, D. J., 534 535, 582 Walsh, G., 457 459 Walsh, M., 213 214 Walsh, M. K., 304, 339 340 Walstra, P., 289 291, 294 295, 297 298 Walter, H., 251 252 Walzem, R. L., 451 452, 519 Wan, J., 228 229, 314 317 Wan, L. P., 196 Wan, Y., 562 Wang, B., 450 Wang, H. X., 18, 37 Wang, K. X., 453 Wang, L., 196 197 Wang, P. F., 528 529 Wang, P. Y., 560 Wang, Q., 258, 261, 310, 414 415 Wang, R., 107 Wang, S., 522t Wang, S. S., 155 156 Wang, T., 345, 460 462 Wang, W., 218, 222 223, 262, 339 340, 345, 525 526 Wang, X. L., 348 349 Wang, Y.-N., 107 Ward, L., 345 Ward, L. S., 520, 562, 650 Warin, F., 134 Warme, P. K., 448 Warncke, M., 268

689

690

Author Index

Warner, B. B., 443t Warner, B. W., 443t Warthesen, J. J., 383 Wasan, D., 138 Washburn, S. P., 382, 382f Wasinger, V. C., 171 172 Watanabe, A., 652t Watson, D., 320 321 Watson, D. L., 648 649 Web, E. C., 35 36 Webb, B., 213 214 Webb, H. A., 36 Webb, W., 172 173 Weber, G., 283 284 Weber, J., 557 Wedholm-Pallas, A., 166 167 Weenen, H., 565 Wege, L., 412 413 Weiler, K., 520 521, 531 Weinberger, L., 4 5 Weinheimer-Haus, E. M., 561 562 Weisgarber, K. D., 586t, 593, 600 Weiss, J., 309, 414 415 Weiss, W., 165 166 Weitl, K., 353 354 Weiz, A., 174 175 Weller, C. L., 320, 411, 415, 421 Wen-Qiong, W., 107 108, 365 Wendland, M., 460 462 Wenshui, X., 29, 531 532 West, C. E., 453 454 Wester, T. J., 583 Westergaard, V., 474 476 Westermann, C., 445 Westerterp-Plantenga, M., 593 Westerterp-Plantenga, M. S., 495 496, 559 560, 618 619 Westerterp, K., 593 Westerterp, K. R., 559 560 Weterings, M., 115 116 Weymuth, H., 174 175 Wharton, B. A., 445 Wheeler, T. T., 166 167 Whetstine, M. E. C., 377 White, C. H., 359 White, S. S., 399 400 Whitehead, D. M., 643 Whitehead, N. P., 592 593 Whitfield, F. B., 377, 388 Whitlock, G., 559 Whitson, M. E., 379 381, 386, 389 391, 397

Whittaker, A. K., 306 Wicker, L., 196 197 Widzer, M. O., 599 Wieclaw, K., 443t Wierenga, P. A., 160, 189 196, 190t Wihodo, M., 415 416, 421 Wijayanti, H. B., 158, 187, 201 202, 216 217, 260 261, 337 338, 465 466 Wijkstrom-Frei, C., 647 Wilbey, R. A., 34 Wilborn, C. D., 579, 586t, 602 Wild, F., 411, 416 417, 419, 423f, 424, 425f Wilde, P., 268 269 Wiles, P. G., 301 Wiley, D. E., 102 105, 109 Wilkins, M. R., 171 172 Wilkinson, C., 500 Wilkinson, S. B., 616 Willemsen, S. P., 552f Willett, W. C., 552f Williams, A. D., 579 Williams, B. D., 584 585 Williams, M. L., 125 126 Williams, P. A., 263 Williams, R. P., 228 229 Williams, R. P. W., 314 317 Willson, K. J., 553 Windhab, E., 207 208 Winkelhausen, E., 459 Winkler, H., 415 416 Winter, R., 284 285 Wirote, Y., 528 529 Wise, M., 602 Wisniewski, J. R., 172 173 Witard, O. C., 604, 614 Wnorowski, A., 142 Wojciechowski, K. L., 396f Wolcott, R., 94 Wolever, T. M., 354 355, 650 Wolf, S. E., 602 603 Wolfe, R. R., 561, 565, 583 585, 598, 602 603 Wolfson, R. L., 613 Wolz, M., 189 196, 190t, 298, 300 301, 414 Womack, C. J., 595 Wong, B. L., 291 292 Wong, C. W., 648 649 Wong, D. W. S., 415, 581 582 Wong, P. T. T., 286

Wong, S. S., 249 Wood, B., 135 136 Wood, R. W., 302 Wooster, T. J., 135, 138 139, 261 262, 265 268 Worobo, R. W., 443t Worsley, A., 553 Wouters, J. T. M., 297 298 Wright, B. J., 377, 379 381, 398 Wright, C. S., 561 562 Wright, J. M., 377, 379 381, 380t, 393 Wroblewska, B., 12 Wrodnigg, T. M., 251 252 Wu, C., 392 393 Wu, G., 525 Wu, H., 522t Wu, S., 459 460 Wu, S. Y., 186 Wu, Y., 421 Wüst, J., 173 175 Wyman, J., 474 Wynn, P. C., 10, 22 23, 29 30 Wyrobnik, T., 407 408

X

Xia, H. S., 414 415 Xia, W. S., 155 156 Xia, X. F., 526 527 Xiao, Q., 341 342 Xiao, Y., 296 297 Xiaoming, L. X., 339 Xiong, Y. L., 190t, 196 Xu, D., 228 230 Xu, W., 553 Xue, H., 107 108, 365 Xun, P., 562

Y

Yadav, M. P., 249 250, 264 266 Yalçin, A. S., 648 Yamada, H., 130 Yamaguchi, S., 34 Yamamoto, N., 524 Yamauchi, F., 525 526 Yamauchi, K., 32 33, 453 Yang, B., 29 30 Yang, J., 284 285 Yang, L., 619 Yang, S., 525 Yang, X. Q., 228 229, 257, 270 Yang, Y., 567, 586t, 601 602

Author Index

Yang., 524 525 Yarasheski, K. E., 602 Yasar, K., 361 362 Yaspelskis, I. I. I., 605 606 Yates, M. D., 377, 498 Yaylayan, V. A., 142 Ye, A., 196 197, 206 207, 350 351 Ye, A. Q., 466 469 Ye, X. Y., 18, 36, 38t, 168 169 Yeaman, S. J., 610 Yee, K. W. K., 104 105 Yeom, H. W., 228 229 Yeomans, M. R., 495 496 Yeung, E. W., 592 593 Yi, L., 107 108, 365 Yiacoumi, S., 293 294 Yilmaz, A. M., 648 Yim, M. B., 525 Yong, Y. H., 227, 342 343 Yoo, Y. C., 648 Yoon, Y., 497 Yoshida, S., 168 169 Yoshika, Y., 227 Young, V. R., 495 496 Younus, H., 272 Yu, C., 443t Yu, S., 296 297 Yu, S. J., 228 229, 257, 270 Yu, Y. Q., 443t, 455 456 Yucel, U., 340

Z

Zabbia, A., 185 Zafar, T. A., 650

Zahoor, T., 30 Zakahrova, E. T., 162 163 Zakora, M., 534 535 Zappacosta, F., 166 167 Zasypkin, D. V., 286 Zavaleta, N., 455 Zawadzki, K. M., 605 606 Zaydman, M., 638 639, 638f Zehetner, G., 34 35 Zemel, M. B., 155 156, 507 Zeng, X. A., 257 Zeng, X.-A., 228 229 Zenk, J. L., 520, 562, 650 Zevchak, S. E., 377 Zhang, J., 174 175, 230 Zhang, J. W., 563 Zhang, L., 174 Zhang, Q. B., 506 507 Zhang, Q. H., 228 229, 314 Zhang, Q. X., 522t Zhang, W., 213 215 Zhang, X., 498 Zhang, Z., 208, 359 Zhang, Z. L., 560 Zhao, B., 155 156 Zhao, L., 522t, 528 529 Zhao, L. L., 348 349 Zhao, R., 619 Zhao, Z., 341 342 Zheng, Z., 271 272 Zhong, Du, M., 174 175 Zhong, J. Z., 295

Zhong, Q., 136 137, 213 215, 218 220, 218f, 222 223, 228 230, 259, 261 262, 340, 345 Zhou, J., 528 529 Zhou, P., 134 135, 221, 339, 355 356, 496, 500 503 Zhou, W., 125 126 Zhoua, P., 343 344 Zhu, D., 217, 221 222, 252 253, 256 259, 261, 271 272, 495 496, 508 509 Zhu, H., 138, 209 211 Ziajka, S., 392 393 Zidane, F., 530 531 Zillinger, W., 407 408 Zimecki, M., 451 452, 520 Zimmerman, J. K., 4 5 Zink, J., 407 408, 414 416 Zisu, B., 213 214, 302, 304 311, 308t Zittle, C. A., 16t, 35 36, 38t Zobrist, M. R., 288 Zolfi, M., 417 418 Zorzano, A., 610 Zubel, J., 392 393 Zukaite, V., 641 Zulet, M. A., 560 Zulewska, J., 108, 377, 389 391, 391t, 392t Zumaeta, N., 294, 300 Zuniga, R., 471 472 Zuniga, R. N., 189 196, 190t, 204 Zydney, A. L., 111 112 Zylberman, V., 125 126

691

Subject Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A

AAs. See Amino acids (AAs) Abcor, 62 63 Abcor/KMS, 64 66 plant, 60 tubular design, 59 60 UF300S, 62 Absorbance at 500 nm (Abs500), 261 Absorption index, 118 processes, 113 114 rate, 611 612 ABTS method. See 2,2ʹ Azino-di[3ethylbenzthiazoline-6sulphonic acid] method (ABTS method) ACE. See Angiotensin-convertingenzyme (ACE) Acetic acid, 537 538 Acetyl-2-furan, 498 Acid casein whey, 62 63, 87 88, 99 100, 386 387 Acid phosphatase, 33 35 characteristics, 33 34 significance, 34 35 Acid phosphomonoesterase, 33 Acid whey, 66 Acid-gel-based cheeses, 99 Acidic amino groups, 254 Acidification of UF-prepared whey protein retentates, 335 336 Acidified whey beverages, 338 Acids formation, 254 Acneadvance, 652t Acoustic cavitation, 303, 303f, 308 309 Acoustic field, 303

Activity based/enzymatic assays, 163 165 Adenosine antagonist, 607 Adhesion maps of aged WPI powder, 127 Adolescents, resistance training for, 603 ADPI. See American Dairy Products Institute (ADPI) Adsorption, 101 Advanced Maillard reaction products (AMPs), 250 251 Advanced Membrane Technologies Inc. (AMT), 70 Advanced MS techniques, 174 Aeration, 359 Affinity microfiltration membranes, 92 Agglomeration of powdered whey protein ingredients, 398 Agglomeration/instantization and whey protein in sports nutrition, 72 78 agglomerating spray dryer, 79f designer protein, 72 75 growth of instantized whey protein powders, 75 77 photomicrograph of agglomerated dairy powder, 79f removing need for rewet agglomeration, 78 Aggregates, 138 139 Aggregation, 298 300 of pure WP fractions in model systems, 284 286 theoretical approach of whey thermal denaturation and, 186 198

kinetics and thermodynamics of β-Lg unfolding and aggregation, 196 198 molecular properties influence of β-Lg, 186 196 prevention of denaturation/ aggregation of whey proteins, 213 230 Air water interface (A/W interface), 139 140, 267 268 AITC. See Allyl isothiocyanate (AITC) Akt phosphorylation, 610 613 Albutensin A, 643 Alcohols, 59 60, 382 Aldehydes, 379 382, 388 Allergies, 619 620 Allyl isothiocyanate (AITC), 215 216 α-lactalbumin (α-La), 7 10, 78 81, 131, 155, 185, 199, 255, 285, 290f, 408 409, 446 448, 460, 470 474, 503, 519 520, 580, 617 618, 639, 642, 651 α-La-enriched whey protein ingredients, 460 462 characteristics, 7 8 composition of commercial α-La powder, 642t function, 9 genetic variants, 157 significance, 9 10 structure, 8 α-lactophorin, 155 156 αs1-casein, 466 469 αS2-casein (αS2-CN), 158 Amadori compounds, 252 253, 272 American Dairy Products Institute (ADPI), 97 98

693

694

Subject Index

AMF. See Anhydrous milk fat (AMF) Amide groups, 160 161 Amino acids (AAs), 440, 579 aromatic, 320 composition, 25, 281 of bovine milk lysozyme, 30 of whey proteins, 582 583 of WP, 616 cysteine, 649 instability under heating, 130 sequence of bovine α-La, 8 sequence of lysozyme, 30 sulfur, 158 transporter gene expression, 592 Amino groups, 215 217 AMPs. See Advanced Maillard reaction products (AMPs) AMT. See Advanced Membrane Technologies Inc. (AMT) Amyloid fibrils, 205 206 Anabolic/anabolism, 592 resistance, 601 response, 597 window, 598 Analytical chromatography, 170 171 Analytical methods, 116 117, 157 158, 161 171 activity based/enzymatic assays, 163 165 capillary electrophoresis, 167 gel electrophoresis, 165 167 immunological techniques, 161 163 liquid chromatography, 168 171 process-induced molecular changes affecting whey proteins analysis, 158 161 proteomics approaches, 171 175 Analytical tests, 378 379 Ancestral β-Lg, 5 6 Anemia, 528 529 Angiogenesis, 37 38 Angiogenins, 37 39 Angiotensin I-converting enzyme, 523 524 Angiotensin-converting-enzyme (ACE), 155 156, 534 535 ACE-inhibitory peptides, 523 524 inhibitor β-lactophorin, 160 Anhydrous milk fat (AMF), 350 8-Anilino-1-naphthalenesulfonate (ANS), 225 226 Anion-exchange

chromatography, 168 169 membrane, 169 ANS. See 8-Anilino-1naphthalenesulfonate (ANS) Anthocyanins, 155 156 Antidiabetic peptides, 527 528 Antihypertensive effect, 535 536 Antihypertensive peptides, 523 525 Antiinflammatory effects, 617 Antimicrobial activity, 641 effects, 617 peptides, 160, 531 Antioxidant(s), 616 effects, 617 618 levels, 615 616 peptides, 525 527 power assay, 526 APV LeanCreme process development, 117 118 Arg-Gly-Asp sequence, 23 Arginine (Arg), 157 Arla process, 92 93 Aromatic amino acids, 320 Ash, 377 Asparagine (Asn), 157 Aspergillus oryzae (A. oryzae), 534 535 Association/dissociation of β-Lg monomers, 4 5 Athletes, training status of, 600 Atrophy, 612 Australian sugar industry, 66 67 Autocrine action, 612 Aveka CCE Technologies mill, 511 512 Avocado oil, 155 156 Avonmore Dairies in Ireland, 68 Avonmore Foods, 69 70 Avonmore’s Ballyragget site, 60, 68 Avonmore’s plant at Richfield, Idaho, 69 70 A/W interface. See Air water interface (A/W interface) 2,2ʹ Azino-di[3-ethylbenzthiazoline6-sulphonic acid] method (ABTS method), 28, 163

B

Babcock & Wilcox, 55 56 Bactericidal effect, 647 Bacteriostatic effect, 645, 647 Bar formulations, 356

Batch rewet agglomeration system, 75 77 BCAAs. See Branched-chain amino acids (BCAAs) Beany flavor compounds, 498 Benzoyl peroxide, 395 β-carotene, 155 156 β-casein, 343, 466 469 β-casomorphins, 531 532 β-endorphins, 607 β-lactoglobulin (β-Lg), 3 7, 78 81, 106, 130, 135 136, 155, 185, 209, 211, 220 221, 255, 284 285, 408, 448 449, 470 471, 503, 519 520, 580, 639 641 β-Lg dextran conjugate-stabilized emulsions, 267 characteristics, 3 4 chemical modification, 216 217 enriched fraction, 336 function, 5 6 hydrolysate, 641 influence of molecular properties, 186 196 pathways during heat-induced denaturation and aggregation, 188f reports on factors influence molecular properties, 190t kinetics and thermodynamics of β-Lg unfolding and aggregation, 196 198 distribution of protein, 198f model systems, 506 507 molecular structure, 187f potential functionalities, 641 significance, 6 7 structure, 4 5 variants, 157 β-lactophorin, 155 156 β-mercaptoethanol (2-Me), 508 BetterMilk, 652t BI. See Browning index (BI) Bifidobacteria, 445 Bifidobacterium lactis (B. lactis), 535 536 Bio-based polyethylene (Bio-PE), 429 Bio-based polyethylene terephthalate (Bio-PET), 429 Bio-isolates group, 69 Bio-PE. See Bio-based polyethylene (Bio-PE)

Subject Index

Bio-PET. See Bio-based polyethylene terephthalate (Bio-PET) Bioactive peptides, 155 156, 530, 641 derived from whey proteins, 521 533, 521f, 522t antidiabetic peptides, 527 528 antihypertensive peptides, 523 525 antimicrobial peptides, 531 antioxidant peptides, 525 527 cytomodulatory peptides, 533 immunomodulatory peptides, 532 533 mineral-binding peptides, 528 531 opioid peptides, 531 532 GMP, 536 538 production and enrichment, 534 536 enzymatic hydrolysis, 534 535 microbial fermentation, 535 536 Bioactive(s) coingestion with, 605 610 caffeine, 607 608 carbohydrate, 605 607 creatine, 608 609 vitamin D, 609 610 components in whey, 459 460 lactose, 459 oligosaccharides, 459 460 compounds, 418 proteins, 616 Bioactivity of whey proteins, 91 Biochemical oxygen demand (BOD), 52 Biodegradation, 426 Biological value (BV), 336 337, 580 Biopolymers, 407 bioZzz, 652t BiPRO, 69, 87 88 Bleaching chemical, 395 396 enzymatic, 396 397 general, 394 395 impact on whey, 393 394 Blends, 417 418 bLf. See Bovine lactoferrin (bLf) Blocking reagents, 217 Blood glucose homeostasis, 520 BM. See Buttermilk (BM) BMI. See Body mass index (BMI)

BMP. See Buttermilk powder (BMP) BOD. See Biochemical oxygen demand (BOD) Body composition, 560, 593 whey protein and optimal, 560 562 Body mass index (BMI), 559 “Bottom-up” approaches, 171 174 Bovine α-La, 7 8 β-Lg, 4 colostrum, 17 commercial protein, 162 Lf, 645 milk, 639 protein, 580 osteopontin, 23 protein, 17 pseudocathepsin D, 32 Bovine lactoferrin (bLf), 18, 451 452 Bovine liver ribonuclease (RNase BL4), 37 38 Bovine milk, 3, 7, 27, 35, 439 440, 440t β-Lg in, 470 471 cathepsin D, 32 enzyme, 30 FBP in, 455 456 Igs class in, 449 lysozyme, 30 PP3, 20 21 proteins in infant’s diet, 462 463 RNase-A, 35 36 Bovine serum albumin (BSA), 10 12, 103 104, 155, 185, 199, 255, 285, 309, 451, 470 471, 503, 580, 639, 643, 648 Brain serotonin, 642 Branched-chain amino acids (BCAAs), 158, 336 337, 520, 580, 614, 644 Breastfeeding, 442 445 Brown rice syrup, 496 Brownian motion, 293 294, 294t Browning index (BI), 126 BSA. See Bovine serum albumin (BSA) Bubble bubble coalescence, 303 2,3-Butanedione, 254 Buttermilk (BM), 109 Buttermilk powder (BMP), 347 348

Buttery flavors, 379 381 Butylated hydroxyanisole, 525 Butylated hydroxytoluene, 525 BV. See Biological value (BV) By-products of WPI production, 71 72

C

CA. See Cellulose acetate (CA) Caffeine, 607 608 Caking, 129 130 Calcium, 6 7, 196 binding, 7 8 Calcium caseinate (CaCas), 115 116, 500 Calcium dichloride (CaCl2), 101 102, 205 Calf milk replacer, 56 Calorimetric technique, 196 197 Camel colostrum, 10 Campylobacter jejuni (C. jejuni), 647 Canadian Beer Company, 114 Candida albicans (C. albicans), 451 452 Capillary electrophoresis (CE), 140 142, 156 157, 167 Caprine PP3, 21 Carbery plant, 60 at Ballineen in County Cork, 59 60 Carbery system, 71 Carbohydrate (CHO), 249, 254, 377, 496, 581 582 endurance exercise, 605 607 interaction with, 220 223 moieties, 263 resistance exercise, 605 substrates in conjugation, 255 256 Carboxyl groups, 215 217 Carboxymethyllysine (CML), 140 142 “Cardboard”, 304 Cardiorespiratory endurance exercise, 594 Cardiovascular diseases (CVDs), 523 524, 563 Carrageenan, 221 222 Carrick-on-Suir plant, 68 Casein (CN), 6, 99 100, 263, 337 338, 341, 348 349, 407, 465 466, 580, 614

695

696

Subject Index

Casein (CN) (Continued) casein-based ingredients, 336 337 casein-predominant formulas, 445 446 components, 462 463 IF emulsions, 472 micelles, 359 Casein hydrolysates (CNHs), 528 Casein phosphopeptides (CPPs), 521, 530 function, 530 Caseinomacropeptide (CMP), 25, 160 161 Casein whey protein coprecipitates (CWPCPs), 367 Casoxins, 531 532 Catching up phenomenon, 143 Cathepsin D, 162, 165 characteristics, 31 33 significance, 33 Cathepsins B, 162 Cation exchange chromatography, 30, 37, 168 169 Cation exchange process, 69 CCK. See Cholecystokinin (CCK) CD. See Circular dichroism (CD) cdk2. See Cell-cycle kinase (cdk2) CE. See Capillary electrophoresis (CE) Ceiling effect, 620 621 Cell culture studies, 648 Cell-cycle kinase (cdk2), 612 Cellulose acetate (CA), 53 54 Centrifugation method, 169 Ceramic membranes, 69 70 microfiltration, 70 systems, 71 Cereals, 580 CFR. See Complete Filtration Resources (CFR) Chaperone proteins, 6, 227 228 effects, 342 345 Charged membranes, 106 Cheddar cheese, 67 68, 85, 364 Red Cheddar, 108 ripened low-fat, 363 salt whey, 67 68 trained panel flavor profiles, 387f whey production with, 386

Cheese, 51, 360 367 contribution of commercial whey protein-based fat replacers, 361 363 denatured whey protein concentrates, 364 365 incorporation of whey proteins into pasteurized PCP and ACP, 365 367 protein cross-linking and whey protein incorporation, 365 type and influence of culture on whey flavour, 385 387 trained panel cardboard flavor intensities of fluid wheys, 387f trained panel flavor profiles of fluid whey from mozzarella cheese, 387f yield extension techniques, 85 Cheese whey, 346 347 proteins and advances in cheese making technology, 82 85 increasing cheese output, 83 84 increasing cheese yield, 85 Cheesemaking process, 381, 391 392 Chemical bleaching, 395 396, 396f Chemical oxidizing agents, 377 378 Chemical reactions, 125 126, 415 416 Chewiness, 358 Children, resistance training for, 603 Chitosan, 341 342 Chlorine, 55 р-Chlormercuribenzoate, 508 CHO. See Carbohydrate (CHO) Cholecystokinin (CCK), 528, 611, 650 Chondral plate growth, 553 Chromatographic method, 157 158, 196 197 Chronic diseases, whey protein and, 563 564 cardiovascular diseases, 563 diabetes, 563 564 Chymosin, 155, 385 Chymotrypsin, 346, 641 CIE L*a*b* system, 422 CIE LCH, 422 CIE XYZ, 422 CIP. See Cleaning-in-place (CIP)

Circular dichroism (CD), 196 197, 314 Cleaning-in-place (CIP), 102 103, 464 465 Clostridium difficile (C. difficile), 449 Clostridium tyrobutyricum (C. tyrobutyricum), 30 31 CML. See Carboxymethyllysine (CML) CML. See Nε-carboxymethyllysine (CML) CMP. See Caseinomacropeptide (CMP) CMPA. See Cows’ milk protein allergy (CMPA) CN. See Casein (CN) CNHs. See Casein hydrolysates (CNHs) Coarse emulsions, 466 469 Coating, 411 merging food packaging requirements and properties, 424 425 protein-based, 411 wet, 410 412 whey protein films, 425 426 whey protein-based, 421 422, 426 427 Coca Cola, 61 63 Cognition, 615 Coingestion with bioactives, 605 610 caffeine, 607 608 carbohydrate, 605 607 creatine, 608 609 vitamin D, 609 610 Cold-set gelling whey proteins, 357 Colloidal modifications of proteins, 291 294 Colloidal stability, 292 293, 345 Colon cancer, 648 Color changes, 505 507, 506f measurements, 126 of stored powder, 126 127 Colorimeter, 252 253 Commercial Ambion RNaseAlert QC system, 38 39 Commercial functional foods, 651 Commercial proteases, 641 Commercial RO systems, 56 Commercial WPI (WPIC), 132

Subject Index

Common Market, 61 Complete Filtration Resources (CFR), 71 Composites, 417 418 Composition of whey ingredients, 388 391 Compressive rheology of fouling layers, 104 105 Concanavalin A-Sepharose affinity chromatography, 32 Concentration polarization, 101 Conduction, 289 290 Conformational modifications of proteins, 291 294 Conjugate-stabilized emulsions, 266 267 Conjugate-stabilized systems, 264 Conjugated individual whey protein fractions, 258 259 Conjugated milk protein systems, 269 Conjugated protein, 249 Conjugated whey proteins nutritional properties, 270 273 techno-functional properties, 257 270 emulsification, 263 267 foaming, 267 269 gelation and textural properties, 269 270 heat stability, 260 262 solubility, 258 260 Conjugation of food proteins, 249 of whey protein, 268 Continuous rewet agglomeration process, 77 “Continuous SEParation” Technology (CSEP Technology), 81 Continuous ultrafiltration plants, 104 Conventional wet and dry heating, 257 Coprecipitated casein whey protein ingredients, 367 Coproduct, 81 Corn syrup, 496 Corn syrup solids (CSSs), 256 Corolase, 641 Cortisol, 615 616 Cottage cheese, 99

curds, 386 387 whey, 99 100 Counter Diffusion, 66 67 Cows’ milk protein allergy (CMPA), 12 CP Kelco, 114 CPPs. See Casein phosphopeptides (CPPs) Creatine, 608 609 Cronobacter sakazakii (C. sakazakii), 455 Cross-flow velocity, 102 103 Cryptosporidium parvum (C. parvum), 449 Crystalline sugars, 496 Crystallization, 135 136 sugar, 505 CSEP Technology. See “Continuous SEParation” Technology (CSEP Technology) CSSs. See Corn syrup solids (CSSs) CVDs. See Cardiovascular diseases (CVDs) CWPCPs. See Casein whey protein coprecipitates (CWPCPs) Cyclization, 131, 131f Cysteine (Cys), 157, 216 217, 441, 446 447, 508, 520, 583, 618, 648 protein, 158 residues, 158 sulfonic acid, 217 Cytokines, 458 459 Cytomodulatory peptides, 533

D

DAD. See Diode array detector (DAD) Dairy, 65 dairy-based UHT, 250 251 dairy-free diets, 619 620 dairy-like flavours, 379 381 powders, 125 126 products, 379 381, 555, 641, 650 651 proteins, 352, 495, 580 streams, 66 Dairy-Lo, 353 354, 361 362 Dairy-Lo based MWP, 361 362

D’Arcy’s law, 100 101 DATEM. See Diacetyl tartaric acid esters of mono-and diglycerides (DATEM) De Danske Sukkerfabrikker (DDS), 55 56, 59 DDS plate-and-frame UF system, 64 DE value. See Dextrose equivalent value (DE value) Deamidation, 130, 157, 160 161 Defatted WPC, 262 Deformation, 300 Degree of hydrolysis (DH), 253 254, 346, 392 393, 416 417 Delactosed whey, 66 Demin 90. See 90-Demineralized Whey (Demin 90) Demineralized whey powder (DWP), 440 441, 459 Demineralized whey protein, 78 80 Denaturation, 185, 255, 410 and aggregation of pure WP fractions in model systems, 284 286 α-La, 285 β-Lg, 284 285 BSA, 285 mixed whey protein solutions, 286 kinetics and thermodynamics of β-Lg unfolding and aggregation, 196 198 prevention of denaturation/ aggregation of whey proteins physicochemical modifications, 213 230 theoretical approach of whey thermal denaturation and aggregation, 186 198 molecular properties influence of β-Lg on, 186 196 Denaturation temperature (Td), 475 Denatured whey protein concentrates, 364 365 Denatured WPC (DWPC), 364 365 Dephosphorylation of phosphocaseins, 34 Desalination Systems Inc. (Desal), 65 Descriptive analysis, 378 379

697

698

Subject Index

Descriptive analysis (Continued) of fluid whey and whey proteins, 380t Designer protein, 72 75, 76f Desserts fresh dairy desserts, 357 358 frozen desserts, 358 Detector, 172 173 DEW. See Dried hen egg white (DEW) Dextran, 221 222, 256 Dextrose equivalent value (DE value), 256 DF. See Diafiltration (DF) 1-DGE. See One-dimensional gel electrophoresis (1-DGE) 2-DGE. See Two-dimensional electrophoresis (2-DGE) DH. See Degree of hydrolysis (DH) DHLA. See Dihydrolipoic acid (DHLA) DIAAS. See Digestible Indispensable Amino Acid Score (DIAAS) Diabetes, 527, 563 564. See also Insulin Diacetyl tartaric acid esters of monoand diglycerides (DATEM), 225 226 Diafiltration (DF), 98 99, 106 for WPC80 production, 389 391 Dietary protein, 559 560 Dietary supplementation, 620 Differential scanning calorimetry (DSC), 196 197, 261 Diffusion, 289 290 Diffusion NMR techniques, 225 226 Diffusion-controlled process, 294 Digestability of whey proteins, 211 212 Digestible Indispensable Amino Acid Score (DIAAS), 550 Digestive enzymes, 272 Dihydro-2-methyl-3-furanone, 498 Dihydrolipoic acid (DHLA), 216 217 2,5-Dimethylpyrazine, 498 2,6-Dimethylpyrazine, 498 Diode array detector (DAD), 167 Dipeptidyl peptidase IV (DPP-IV), 527 528 2,2-Diphenyl-1-picrylhydrazyl assay, 526

Direct heat treatment, 472 Direct steam injectors, 472 Discrimination testing, 378 379 Disulfide bonds, 281, 348 349 disulfide bond-induced protein aggregation, 502 inhibition of disulfide bond formation, 508 509 splitters, 508 bridges, 132, 409 410 Dithio(bis)-p-nitrobenzoate (DTNB), 216 217 Dithiothreitol (DTT), 216 217, 219, 508 DM. See Dry matter (DM) Door Oliver, 63 Dorr Oliver system membrane cartridges, 58 Dose-response, 604 605 Double burden of malnutrition, 562 Downstream processing, 296 297 DPP-IV. See Dipeptidyl peptidase IV (DPP-IV) Dried hen egg white (DEW), 510 Dried high-protein whey products, 398 Dried proteins, 381 382 Drop-in solutions, 429 Dry heating approach, 205 206, 256, 258 Dry matter (DM), 353 354, 472 474 Drying fluid dairy products, 398 of WPI droplets, 127 DSC. See Differential scanning calorimetry (DSC) DTNB. See Dithio(bis)-pnitrobenzoate (DTNB) DTT. See Dithiothreitol (DTT) Durasan outer wrap, 65 DWP. See Demineralized whey powder (DWP) DWPC. See Denatured WPC (DWPC) Dynamic light scattering, 196 197 Dynamic membrane. See Surface layer Dyslipidemia, 563

E

4E (eIF4E)-binding protein 1 (4EBP1), 613 EAAs. See Essential amino acids (EAAs) ED. See Electrodialysis (ED) EFSA. See European Food Safety Authority (EFSA) EGF. See Epidermal growth factor (EGF) Egg lysozyme, 31 Elasticity, 358 Electrodialysis (ED), 66, 78 80, 460 462 Electrophoresis, 162 Electrophoretic mobility, 196 197 Electrospray ionization (ESI), 172 173 Electrospray ionization MS technique (ESI-MS technique), 170 171, 216 217 Electrostatic interaction, 283 284 Electrostatic repulsion, 264 ELISA. See Enzyme-linked immunosorbent assay (ELISA) Emulsification, 263 267, 357 emulsified triglyceride substrate, 22 emulsifiers, 358 359, 466 469 emulsifying properties, 139, 281 Emulsifying salts (ES), 365 366 Emulsion stability, 264, 338 347 chaperone-protein effects, 342 345 formation and role of soluble whey protein aggregates, 341 342 novel fermented whey-based drinks, 346 347 storage stability, 341 whey protein hydrolysates for improved heat and storage stability, 346 End-of-life options for whey proteinbased films and multilayer laminates, 426 428 biodegradation of whey proteinbased films and laminates, 426 427 recyclability of whey protein-based multilayer laminates, 427 428

Subject Index

Endogenous antioxidant defence mechanisms, 525 Endopeptidases, 164 165, 416 417 Endurance capacity and performance, 594 595 Endurance exercise, 579, 594 596, 605 607. See also Resistance exercise endurance capacity and performance, 594 595 muscle protein synthesis, 595 596 and training, 599 600 Energy balance, 618 619 density, 304 305 Enhanced protein functionality, 264 Enthalpic gain, 208 209 Entropic gain, 208 209 Enzymatic bleaching, 396 397 Enzymatic cross-linking, 218 220 Enzymatic hydrolysis, 160 161, 223 225, 534 535 Enzyme glycogen synthase, 610 Enzyme-linked immunosorbent assay (ELISA), 31, 140 142, 156 157, 161 162, 174 175, 252 253 Epidermal growth factor (EGF), 457 458 EPS. See Exopolysaccharides (EPS) ES. See Emulsifying salts (ES) Escherichia coli (E. coli), 449, 451 452 enterotoxins, 644 ESI. See Electrospray ionization (ESI) ESI-MS technique. See Electrospray ionization MS technique (ESIMS technique) ESL milk. See Extended shelf-life milk (ESL milk) ESPEN. See European Society for Clinical Nutrition and Metabolism (ESPEN) Essential amino acids (EAAs), 579, 642, 649, 651 658 Esters, 382 Ethylene vinyl alcohol copolymers (EVOH), 424, 427 430 European Food Safety Association, 318

European Food Safety Authority (EFSA), 346 347, 650 European Society for Clinical Nutrition and Metabolism (ESPEN), 565 European Union (EU), 61, 425 426 EVOH. See Ethylene vinyl alcohol copolymers (EVOH) Excess creatine, 609 Exercise, 615 616. See also Sports and exercise supplements endurance exercise, 594 596, 605 607 resistance exercise, 584 593, 605 Exogenous peroxide, 396 397 Exopeptidases, 164 165 Exopolysaccharides (EPS), 351 352 Extended shelf-life milk (ESL milk), 27 28 Extrinsic environmental factors, 289 Extrinsic factors, 142 Extrude, 510 511 Extrusion process, 412 413

F

Factorial statistical design, 466 469 FAO. See Food Agriculture Organization (FAO) “Fast” digesting protein, 592 Fat, 340, 377 concentration, 389 391 contribution of commercial whey protein-based fat replacers, 361 363 fat-free WPIs, 75 removal, 389 391 residual, 98 99 Fat-in-dry matter (FDM), 362 363 Fatty acids, 225 226, 385 FBFs. See Fortified blended foods (FBFs) FBP. See Folate binding proteins (FBP) FDA. See US Food and Drugs Administration (FDA) FDM. See Fat-in-dry matter (FDM) Feed concentration, 102 103 Female exercisers, 602 603 FEP. See Fluorinated Ethylene Propylene (FEP) Fermented whey beverage (FWB), 345

Ferriprotoporphyrin IX, 27 FGF. See Fibroblast growth factor (FGF) Fibroblast growth factor (FGF), 457 458 Fick’s law, 418 Filamentous network, 503 Filler, 87 Film model, 101 102 modifications, 417 418 technologies for processing films and coatings, 410 413 extrusion, 412 413 wet coating, 410 412 Film formation, 409 410 and performance, protein modification for optimizing, 413 418 biochemical modifications, 416 417 chemical modifications, 415 416 film modifications, 417 418 physical modifications, 413 415 protein structure related properties and preconditions for, 408 409 FilmTec, 56, 67 68 3838 NF membranes, 68 Filter cake. See Surface layer Filtration Engineering Inc., 67 68 Fizzique carbonated protein water, 89, 90f 2ʹ-FL. See 2ʹ-Fucosylactose (2ʹ-FL) Flavor, 497 aspects of whey protein ingredients bleaching, 393 397 composition of whey ingredients, 388 391 flavor aspects of liquid whey, 383 387 flow diagram of general whey processing from raw milk, 378f fluid milk, 382 383 fluid whey processing, 388 hydrolysates, 392 393 origin of flavors in whey, 379 382

699

700

Subject Index

Flavor (Continued) sensory analysis, 378 379 serum protein, 391 392 spray drying and dry storage, 398 400 storage, 397 in whey, 379 382 Flavourzyme, 346, 529 530 Flow properties of whey protein, 129 130 Flowability, 363 Fluid Cheddar wheys, 386 trained sensory panel profiles of, 384t Fluid milk, 382 383, 382f Fluid viscosity, 414 Fluid whey, 381 382, 397 processing pasteurization, 388 separation, 388 Fluorescence assays, 252 253 Fluorescence technique, 196 197 Fluorinated Ethylene Propylene (FEP), 319 320 Fluorophos Test System, 34 FO. See Forward osmosis (FO) Foam formation, 138 Foaming, 138 139, 267 269, 281 Folate binding proteins (FBP), 455 456 FBP-bound folate, 456 Folded conformations of proteins, 290 291 Folded monomeric protein, 292 293 Food applications, 281, 392 393, 581 industry, 298 issues, 584 packaging applications, 407 408 merging food packaging requirements and properties of whey protein-based films and coatings, 424 425 packaging requirements of food products, 422 423 potential applications of whey protein based films and coatings in, 422 425 requirements of food products, 422 423

safety, 425 426 shelf life, 419 Food Agriculture Organization (FAO), 155 156, 550 Foods for Specific Health Use (FOSHU), 637 Force 1. See Peak force Force 2. See Maximum negative force Formula milks, 78 80 Formulation dynamics, 464 465 of high-protein nutrition bars, 496 497 Fortified blended foods (FBFs), 554 Fortified/functional packaged food, 639 Forward osmosis (FO), 107 FOSHU. See Foods for Specific Health Use (FOSHU) Fourier transform ion cyclotron resonance (FTICR), 172 173 Fractal analysis, 350 351 Fractional synthetic rate (FSR), 567 Fragmentation, 300 “Fraunhofer” theory, 118 Free energy of change (ΔG), 290 291 Free radicals, 381 Free thiol group blocking reagents, 508 Freeze drying, 398 Freeze-dried material, 511 512 Frequency critical, 302 304 Fresh cheeses, 361 Fresh dairy desserts, 357 358 Fresh sweet whey, 335 336 Friesland Campina, 80 81 Frozen desserts, 358 Frozen yogurts, 358 FSR. See Fractional synthetic rate (FSR) FT 40, 67 68 FTICR. See Fourier transform ion cyclotron resonance (FTICR) FTIR spectroscopic analysis, 342 343, 356 FTIR-ATR spectra, 345 2ʹ-Fucosylactose (2ʹ-FL), 459 460 Functional Food Science in Europe (FUFOSE), 637 Functional foods, 637 639

comparison of deaths caused by noncommunicable lifestyle diseases, 638f health benefits of whey proteins, 647 651 and supplements containing whey protein ingredients, 651 whey proteins, 639 647 Functional properties impact on, 136 140 emulsifying properties, 139 foaming properties, 138 139 heat-induced aggregation properties, 136 138 interfacial properties, 139 140 solubility, 136 of whey proteins, 258 Functionality, 269 enhanced protein functionality, 264 Furans, 388 389 Furfuryl methanol, 498 Furosine, 160 FWB. See Fermented whey beverage (FWB)

G

Galactooligosaccharides (GOS), 273, 459 4-O-β-D-Galactopyranosyl-D-glucose. See Lactose γ-irradiation, 415 Gas chromatography-mass spectrometry, 252 253 Gastric inhibitory polypeptide, 528 Gastrointestinal (GI), 346 distress, 620 Gastrointestinal tract (GIT), 272, 450, 598 GEA manufacturers, 118 Gel chromatography, 116 117 Gel electrophoresis, 156 157, 165 167 1D PAGE and 2D PAGE separation of proteins, 166f Gel filtration (GF). See Gel permeation chromatography (GPC) Gel layer. See Surface layer Gel permeation chromatography (GPC), 169 170

Subject Index

Gel-based method, 157 158 “Gel-based” proteomics, 171 172 “Gel-free” proteomics, 171 172 Gelling, 281 Gene expression, 614 615 Genetic polymorphism of α-La, 7 8 GI. See Gastrointestinal (GI) GIT. See Gastrointestinal tract (GIT) Gln. See Glutamine (Gln) Global cheese manufacture, 99 Globular proteins, 209, 412 413 GLP-1, 528 Glucose, 503 504 serum, 527 528 syrup, 496 transport, 605 606 Glucose transporters (GLUT-4), 610 Glutamine (Gln), 157, 615 616 Glutathione (GSH), 216 217, 353 354, 617 618, 641, 648 WP as provision for increasing, 618 Glycans, 18, 21 Glycation, 249 of β-Lg, 271 273 of WPI, 136 137 Glycerol, 496 Glycodelin A, 448 449 Glycoforms, 140 142 Glycogen synthase activity, 605 606 Glycogen synthase kinase 3 (GSK3), 610 Glycogen synthesis, 610 611 Glycomacropeptide (GMP), 1, 81, 91, 106, 155 156, 168 169, 200, 336 337, 450 451, 463, 519 520, 536 538, 639, 643 644 analysis, 26 characteristics, 24 26 composition of commercial GMP powders, 644t isolation of GMP from whey, 536 538 reversed phase-HPLC chromatogram of bovine GMP, 26f significance, 26 Glycosylation, 165 166 Glyoxal-derived lysine dimer (GOLD), 157 Glytactin BUILD, 652t Glytactin RESTORE Powder, 652t

Glytactin RTD, 652t Glytactin SWIRL, 652t GMP. See Glycomacropeptide (GMP) GOLD. See Glyoxal-derived lysine dimer (GOLD) Golden Cheese in Corona, 73 GOS. See Galactooligosaccharides (GOS) Gouda cheese, 99, 364 GPC. See Gel permeation chromatography (GPC) Gram-negative bacteria, 29, 645 646 Gram-positive bacteria, 29 Growing Up in Singapore Towards health Outcomes study (GUSTO study), 553 Growth-promoting effect, 645 GSH. See Glutathione (GSH) GSK3. See Glycogen synthase kinase 3 (GSK3) Gumminess, 358, 361 362 GUSTO study. See Growing Up in Singapore Towards health Outcomes study (GUSTO study) Gut hormones, 528, 611

H

HAMLET. See Human α-La made lethal to tumor cells (HAMLET) Hard cheeses, 85 Hardening solutions to avoid adding protein hydrolysates, 509 510 creation of heterogeneity by pretexturization, 510 513 inhibition of disulfide bond formation, 508 509 texture, 500 505 moisture migration, 501 502 protein aggregation, 502 505 sugar crystallization, 505 Havens modules, 63 Hawera installation, 64 HB-EGF. See Heparin-binding EGF (HB-EGF) HCl. See Hydrochloric acid (HCl) Health benefits of high-protein diets, 495 496

complications, 620 of whey proteins cancer, 647 648 immune health, 648 649 osteoporosis, 650 651 stress and mental health, 651 weight management, 649 650 Heat-denaturation, 466 469 Heat(ing), 305 absence particle size distribution, 297f shear impact on properties of WP in, 294 297 coagulation, 281 convection, 289 290 heat-and shear-treatment of WPCs, 115 heat-induced aggregation properties, 136 138 changes in whey proteins, 199 200 deamidation, 160 161 gels, 206 oxidative deamination, 388 heat-stabilizing effect, 344 345 heat-stable whey powder, 311 312 whey proteins, 214 215 on properties of WP microparticulation, 297 301 stability, 260 262, 338 347 chaperone-protein effects, 342 345 effect, 311 formation and role of soluble whey protein aggregates, 341 342 novel fermented whey-based drinks, 346 347 storage stability, 341 whey protein hydrolysates for improved heat and storage stability, 346 treatment, 348 349, 472 Helicobacter pylori (H. pylori), 449 Henry’s law, 418 419 Heparin-binding EGF (HB-EGF), 457 458 HEW. See Hydrolysates of egg white (HEW) HFCS. See High-fructose corn syrup (HFCS)

701

702

Subject Index

Hi-gelling WPC, 357 High blood pressure, 523 524 High pressure homogenization (HPH), 349, 363 High pressure processing (HPP), 281, 296 297, 349 High solubility of WP, 281 High-biological-value proteins, 580 High-fructose corn syrup (HFCS), 496 High-molecular-weight polymers, 135 136 High-performance liquid chromatography (HPLC), 163, 170 172, 252 253 High-pressure processing (HP processing), 282 denaturation and aggregation of pure WP fractions in model systems, 284 286 effect on proteins, 282 284 HP-induced changes in milk, 286 289 High-protein bars, 356 dairy powders, 132, 145 diets, 558 560 powders, 75 WPCs, 98 99, 460 462 High-protein nutrition bars, 495 497. See also Human milk; Protein formulation, 496 497 health benefits of high-protein diets, 495 496 quality, 497 507 sensory properties, 497 498 stability, 500 texture, 498 500 texture hardening, 500 505 quality losses color Changes, 505 507 nutrition Loss, 507 solutions to avoid hardening adding protein hydrolysates, 509 510 creation of heterogeneity by pretexturization, 510 513 inhibition of disulfide bond formation, 508 509 texture hardening, 500 505 moisture migration, 501 502 protein aggregation, 502 505 sugar crystallization, 505

High-quality protein, 550, 561, 579 580 High-value whey fractions, 168 169 Higher-molecular-weight peptides, 469 470 HiTrap SP, 168 169 HIV, 649 HLB. See Hydrophilic lipophilic balance (HLB) hLf. See Human lactoferrin (hLf) HMF. See 5-Hydroxymethyl-2furfuraldehyde (HMF) HMO. See Human milk oligosaccharides (HMO) Hollow-fiber UF design, 58 Homogenization, 466 469 effect, 295 HP. See Hydrostatic pressure (HP) HP processing. See High-pressure processing (HP processing) HPH. See High pressure homogenization (HPH) HPLC. See High-performance liquid chromatography (HPLC) HPP. See High pressure processing (HPP) Human lactoferrin (hLf), 451 452 Human milk, 78 80, 439 441, 440t, 445 446. See also Highprotein nutrition bars; Milk cytokines, 458 EGF delivery, 457 458 FGF21 in, 457 458 Igs class in, 449 LP in, 455 procathepsin, 32 Human milk oligosaccharides (HMO), 459 460 Human α-La made lethal to tumor cells (HAMLET), 9, 520 Humidity, 142 effect, 133 134 HWP. See Hydrolyzed whey protein (HWP) HWPI. See Hydrolyzed whey protein isolate (HWPI) Hybrid mass analyzers, 172 173 Hydrochloric acid (HCl), 68 Hydrocolloids, 281, 357 358

Hydrodynamic radius (Rh), 204 Hydrodynamic-shear-controlled process, 294 Hydrogen bonding interaction, 283 284 Hydrogen peroxide (H2O2), 395, 646 647 Hydrolases, 455 Hydrolysates, 392 393, 524 525 Hydrolysates of egg white (HEW), 510 Hydrolysis, 611 612 process, 619 of whey protein molecules, 255 256 Hydrolyzed proteins, 510 Hydrolyzed whey protein (HWP), 462 463 Hydrolyzed whey protein isolate (HWPI), 421, 505 506 Hydrophilic filler, 107 peptide, 643 644 Hydrophilic lipophilic balance (HLB), 340 Hydrophobic addition of hydrophobic compounds, 225 226 interactions, 281, 283 284 molecules, 155 156, 641 Hydrophobic component of proteose peptones, 21 Hydrostatic pressure (HP), 282, 414 Hydroxyacetone, 254 5-Hydroxymethyl-2-furfuraldehyde (HMF), 140 142, 252 253 5-Hydroxymethylfurfural. See 5Hydroxymethyl-2furfuraldehyde (HMF) Hygroscopic powder, 129 130 Hyperaminoacidemia, 605 Hypocyanous acid, 28 Hypothiocyanate, 28, 455 Hypothiocyanite ions, 646

I

ICAT technology. See Isotope-coded affinity tag technology (ICAT technology) Ice creams, 358 360 fresh dairy desserts, 357 358 frozen desserts, 358 IE. See Ion exchange (IE)

Subject Index

IEC. See Ion exchange chromatography (IEC) IEMC. See Ion-exchange membrane chromatography (IEMC) IF. See Infant formula (IF) IFIC. See International Food Information Council (IFIC) IGF. See Insulin-like growth factor (IGF) Igs. See Immunoglobulins (Igs) IL. See Interleukin (IL) ILSI. See International Life Sciences Institute (ILSI) IMAC. See Immobilized metal affinity chromatography (IMAC) Imidazole group, 526 Immobilized metal affinity chromatography (IMAC), 113 Immobilized pH gradient strips (IPG strips), 165 166 Immune blotting, 156 157 Immune cell functions, 532 533 Immune function, 615 618 antiinflammatory effects, 617 antimicrobial effects, 617 antioxidant effects, 617 618 WP as provision for increasing glutathione, 618 Immune health, 648 649 Immunoaffinity CE, 167 Immunoblotting, 162 Immunocal, 649, 652t Immunoglobulins (Igs), 12 14, 12t, 103 104, 155, 185, 200, 449 450, 519 520, 580, 616, 639, 643 characteristics, 12 13 in colostrums, 14 IgA, 12 13, 200, 449, 643 IgD, 200 IgE, 200, 271 272 IgG antibodies, 12 13, 449, 533, 643 IgG1, 13 14, 200, 643 IgG2, 13 14, 200, 643 IgM, 12 13, 200, 449, 643 significance, 14 structure, 13 14 Immunologic method, 157 158 Immunological techniques, 161 163

Immunomodulatory functions, 533 peptides, 532 533 Immunoprecipitation, 161 163 Immunostimulatory activity, 641 ImmunPlex, 652t IMUPlus, 652t In vitro digestion models, 212 In vitro gastric model, 271 In vivo studies, 643, 648 Indirect heat treatment, 472 Indole, 382 383 Industrial applications, biopolymers, 407 408 Industrial chromatography, 109 111 Industrially-sourced WPCs, 111 Infancy, malnutrition managing during, 554 Infant formula (IF), 439 440, 466 469 composition, 440t incorporation of whey protein ingredients, 460 466 manufacturers, 82 minimum levels of indispensable and conditionally indispensable amino acids, 441t physiological function of components in whey protein ingredients, 442 460 whey protein fractionation and hydrolysates for, 78 82 functionality impacts on IF stability, 466 476 Initiation, lipid oxidation process section, 381 INRA. See Institut National de la Recherche Agronomique (INRA) Instantization of whey protein, 398 Instantized whey protein powder, 72 75 growth of, 75 77 Institut National de la Recherche Agronomique (INRA), 69 70 Insulin, 610 611 insulin-dependent mechanisms, 618 619

insulin-independent mechanisms, 618 619 insulin-induced GLUT-4 translocation, 610 611 secretion, 605 606 signalling, 610 611 Insulin-like growth factor (IGF), 457 458 IGF-1, 609 610 Interleukin (IL), 458 IL-1β, 458 IL-2, 454, 458 IL-6, 458 IL-8, 458 Intermolecular disulfide bonds, 339, 502 International Food Information Council (IFIC), 637 International Life Sciences Institute (ILSI), 637 Intralipid, 22 Intrinsic factors, 142 143 2-Iodoacetamide, 508 Iodoacetic acid, 508 Ion exchange (IE), 66 technology, 69 72 Ion exchange chromatography (IEC), 168 170, 460 462, 537 538 Ion source, 172 173 Ion-exchange membrane chromatography (IEMC), 112, 169 Ionizing irradiation, 415 IPG strips. See Immobilized pH gradient strips (IPG strips) Iron-binding ability of Lf, 645 Iron-free form of Lf, 451 452 Irradiation of foods with UV-C light, 318 Irreversible aggregation, 199, 286, 293 294 Iso-Asp, 160 161 Isobaric tagging for relative and absolute quantification (iTRAQ), 174 Isoelectric point (pI), 168 169, 196 Isoleucine, 158, 644 Isomerases, 455 Isotope-coded affinity tag technology (ICAT technology), 174

703

704

Subject Index

Isotypes, 200 iTRAQ. See Isobaric tagging for relative and absolute quantification (iTRAQ)

J

J chain, 13 Jet-milling, 511 512

K

κ-Carrageenan, 357 κ-casein (κ-CN), 24, 155, 287 288, 465 466, 643 644 Kerry Foods, 77, 82 2-Ketone, 498 Ketones, 379 381, 383 Kiwi Dairies’ factory in Hawera, 64 Klebsiella pneumoniae (K. pneumoniae), 532 533 Knowledge-based hybrid neural network, 104 105

L

LAB. See Lactic acid bacteria (LAB) Labatt invention, 115 Lacprodan OPN-10, 454 Lactate dehydrogenase (LDH), 592 593 “Lacteen” ingredient, 60 61 Lactic acid bacteria (LAB), 335, 524, 534 Lactobacillus acidophilus (L. acidophilus), 535 536 Lactobacillus casei (L. casei), 535 536 Lactobacillus helveticus (L. helveticus), 524 Lactobacillus plantarum (L. plantarum), 345 Lactococcus lactis (L. lactis), 345 Lactoferricins, 19, 155 156, 451 452, 645 646 Lactoferrin (Lf), 78 80, 92, 112, 155, 185, 255, 451 453, 519 520, 531, 580, 645 646, 645t characteristics, 17 19 potential markets for, 646 significance, 19 20 Lactogenin, 37 38 Lactoperoxidase (LP), 18, 80, 155, 163, 185, 317, 396 397, 455, 519 520, 531, 617, 646 647

characteristics, 27 28 significance, 28 29 Lactophoricin, 22, 457 Lactophorin, 20 Lactoribonuclease, 35 36 Lactose, 61, 115, 126, 398, 459 content, 143 effect, 134 and fat concentration, 389 391 intolerance, 619 620 lactose/protein molar ratio, 133 134 molecules bound per protein, 134 related changes, 132 136 change of state, 135 136 correlation between lactosylation and aggregation, 133f effect on protein structure, 135 Maillard reaction and storage conditions, 132 135 state, 142 Lactose Company of NZ Ltd, 63 Lactosylated protein, 134, 139 142 Lactosylation, 160 sites of whey proteins, 161t LAL. See Lysinoalanine (LAL) Laminates, biodegradation of, 426 427 Large-for gestational-age births (LGA births), 553 LC. See Liquid chromatography (LC) LCMS techniques. See Liquid chromatography-mass spectrometry techniques (LCMS techniques) L/D ratio. See Length-to-diameter ratio (L/D ratio) LDH. See Lactate dehydrogenase (LDH) “Leaf” UF system, 58 Lean body mass (LBM, 556 558, 593 LeanCreme reference process, 117 118 Legumes, 580 Length-to-diameter ratio (L/D ratio), 412 413 Leucine (Leu), 158, 520, 583, 613, 644 concentration, 567 threshold, 601 602 Lf. See Lactoferrin (Lf)

LFC. See Low-fat Gouda-type cheeses (LFC) LGA births. See Large-for gestationalage births (LGA births) Ligases, 455 Lipid(s), 407, 418, 496, 504 505 oxidation, 377, 379 381, 397 compounds, 388 process, 381 Lipolysis, 382, 385 Liquid chromatography (LC), 156 157, 168 171 analytical chromatography, 170 171 preparative chromatography, 168 169 Liquid chromatography-mass spectrometry techniques (LCMS techniques), 31 Liquid whey, 383 385 flavor aspects cheese type and influence of culture, 385 387 chymosin and proteolysis, 385 trained sensory panel profiles of fluid cheddar wheys, 384t Liquid WPC (LWPC), 353 354 Live microorganisms, 335 Loading buffer pH, 112 Locust bean gum (LBG), 348 349 Logging decay data, 197 198 Low birth weight, 551, 558 Low-acid/neutral whey protein-based RTDs, 89 91 Low-fat Gouda-type cheeses (LFC), 363 Low-frequency power ultrasound processing/treatment, 281 power ultrasound, 302 304 effect on solubility, 306 307 effect on viscosity, 307 309 effect on whey protein gelation, 309 312 temperature and sonication of WP solutions, 305 306 ultrasound and WP, 302 Low-frequency sonication, 309, 311 312 Low-heat-processed WPC, 88 Low-molecular-weight emulsifiers, 358 359 Lp, potential applications of, 647 LP. See Lactoperoxidase (LP)

Subject Index

LTmax. See Maximum loss tangent (LTmax) LWPC. See Liquid WPC (LWPC) Lyases, 455 Lysine (Lys), 157, 446 447, 507 damage, 140 142 glycation at, 157 of high-protein nutrition bar model systems, 507f Lysinoalanine (LAL), 132, 157, 260, 270 271 Lysosome, 617 Lysozyme, 18, 164, 185, 455, 531 characteristics, 29 30 significance, 30 31

M

MAFbx. See Muscle atrophy F-box (MAFbx) Maillard browning, 505 506 Maillard reaction (MR), 125 126, 129, 132 135, 138 142, 157, 220, 249, 355 356, 498, 506 507 chemistry, 251 253 in dairy-based products, 251f MR-induced protein aggregation, 503 Maillard reaction products (MRPs), 250 251, 272 273 Maillard-induced conjugation, 271 Major whey proteins and peptides, 446 451 α-La, 446 448 β-Lg, 448 449 GMP, 450 451 Igs, 449 450 serum albumin, 451 MALDI. See Matrix assisted laser desorption ionization (MALDI) Malnutrition double burden, 562 managing during infancy, 554 Maltitol, 496 Maltodextrin (MD), 221 222, 255 256 MAM. See Moderate acute malnutrition (MAM) Mammalian target of rapamycin (mTOR), 609, 613 615

Manufacture of whey protein products whey protein concentrates and isolate by ultrafiltration, 97 109 general classification of whey types, 100f membrane and process developments, 106 109 membrane performance, 100 104 membrane separation operation and protein selectivity, 104 106 proximate composition of commercially traded WPC, 98t salient features of spiral-wound UF membranes, 110t UF spiral-wound membrane manufacturers and state of art, 109 whey protein fractions by membrane-based separations, 109 114 MAP. See Modified atmosphere packaging (MAP) Marron Foods, 77 Mascot, 171 172 Mass analyzer, 172 173 Mass spectrometer, 167 Mass spectrometry (MS), 156 157 coupled with electrospray ionization, 140 142 methodologies, 165 166 MS-based methods, 163, 173 174 Mastitic milk, 33 Mathematical model, 104 105 Matrix assisted laser desorption ionization (MALDI), 160 161, 172 173 MALDI-TOF-MS, 173 174 Maximal albumin protein synthesis, 604 Maximum negative force, 356 Maximal protein synthesis, 604 Maximum loss tangent (LTmax), 363 MBP. See Milk Basic Protein (MBP) MCT. See Medium-chain triglyceride (MCT) MD. See Maltodextrin (MD)

MD Foods. See Mejeriselskabet Danmark Foods (MD Foods) Meal(s), 583 replacement beverages, 584 Mechanistic Target Of Rapamycin (mTOR), 568, 611, 613 Medical foods, 637 Medium-chain triglyceride (MCT), 73 Mejeriselskabet Danmark Foods (MD Foods), 59 Melanoidins, 220, 252 Membrane filtration, 52, 123, 581 performance, 100 104, 107 pretreatment effect, 103 104 UF processing parameters, 102 103 permeability, 531 pore size, 111 112 and process developments, 106 109 charged membranes, 106 nanolayered membrane, 107 protein cross-linking, 107 109 processing, 53 separation operation and protein selectivity, 104 106 continuous operation and control, 104 105 diafiltration, 106 multistage spiral-wound ultrafiltration plant configuration, 105f separation technologies, 169 whey protein fractions by membrane-based separations, 109 114 absorption processes, 113 114 Membrane resistance (Rm), 100 101 Mental health, 651 Mercaptoethanol, 30, 216 217 Merging food packaging requirements and properties of whey protein-based films, 424 425 Met. See Methionine (Met) Metal-chelating peptides, 528 529 Methacrylate copolymer cationexchange chromatography, 168 169 Methionine (Met), 157 158, 583 Methyl ketones, 388

705

706

Subject Index

5-Methyl-2-furfuryl methanol, 498 Methylglyoxal-derived lysine dimer (MOLD), 157 5-Methyltetrahydrofolic acid, 456 MF. See Microfiltration (MF) MFGM. See Milk fat globule membrane (MFGM) Mg-Al double hydroxide (Mg-Al NLDH), 107 Micellar casein whey, 439 440 Microbial fermentation, 535 536 Microbial proteolysis, 535 536 Microbial transglutaminase (mTGase), 353 354 Microbubbles, 303 Micrococcus luteus (M. luteus), 29 Microfiltered whey permeate, 107 Microfiltration (MF), 52, 69 72, 439 440 MF retentate, 71 72 MF SM permeate, 100 process, 391 392 Microparticulated whey protein (MWP), 114 118, 226 227, 344 345, 350 351 examples of commercial initiatives, 117 118 spray drying effect on microparticulate functionality, 116 117 types, 350 351 Microparticulated WPC emulsions, 116 117 Microparticulation, 298 300, 363 efficiency, 301 Mid-upper arm circumference (MUAC), 556 Mild thermal pasteurization, 338 Milk, 103 104, 318, 455, 531. See also Human milk; Protein fermentation, 335 HP-induced changes in, 286 289 immunoglobulins, 14 milk-based beverages, 335 oligosaccharides, 92 93 osteopontin, 23 products, 650 651 proteins, 580, 582, 650 function, 358 359 hydrolysates, 532 533 ingredients, 366 367

milk protein-derived bioactive peptides, 616 serum, 1, 84 solids-not-fat portion of ice cream, 359 types, 445 446 Milk Basic Protein (MBP), 650 651, 652t Milk fat globule membrane (MFGM), 33, 92 93, 457 Milk protein concentrate (MPC), 1, 337 338, 498 bars, 500 MPC70, 360 MPC80, 228, 510 511 MPC85, 511 512 Mineral mineral-binding peptides, 528 531 precipitation, 101 102 Minor whey proteins and peptides, 451 459 cytokines, 458 459 enzymes, 455 FBP, 455 456 growth factors, 457 458 Lf, 451 453 OPN, 453 454 PP3, 457 Mitchelstown Co-operative Creamery, Ireland, 69 Mixed whey protein solutions, 286 Moderate acute malnutrition (MAM), 550, 554 Modified atmosphere packaging (MAP), 423 Moisture content, 496 497 migration, 501 502 moisture-induced protein aggregation, 502 Molar mass, 139 140 MOLD. See Methylglyoxal-derived lysine dimer (MOLD) Molecular mobility, 125 126 Molecular weight (MW), 12 Molecular weight cutoffs (MWCOs), 97 Monitoring powder evolution, 140 142 lactose state, 142 Maillard reaction, 140 142

Monomerization of β-Lg dimmers, 284 285 Monosaccharides, 21, 25, 253 254 Monovalent ions, 68 Mood and cognition, 615 “Mother Country” of United Kingdom, 61 MPB. See Muscle protein breakdown (MPB) MPC. See Milk protein concentrate (MPC) MPS. See Muscle protein synthesis (MPS) MR. See Maillard reaction (MR) MRM. See Multiple reaction monitoring (MRM) MRPs. See Maillard reaction products (MRPs) MS. See Mass spectrometry (MS) mTGase. See Microbial transglutaminase (mTGase) mTOR. Mammalian target of rapamycin (mTOR);. See Mechanistic Target Of Rapamycin (mTOR) MUAC. See Mid-upper arm circumference (MUAC) Multiangle light scattering, 196 197 Multilayer laminates, end-of-life options and, 426 428 biodegradation of whey proteinbased films and laminates, 426 427 recyclability of whey protein-based multilayer laminates, 427 428 Multiple atomization nozzles, 78 Multiple reaction monitoring (MRM), 140 142, 174 175 Multiple-sprint sports, 596 597 Muramidase, 29 MuRF-1. See Muscle-specific RING finger-1 (MuRF-1) Murray Goulburn’s plant at Leongatha in Australia, 81 Muscle atrophy F-box (MAFbx), 612, 614 615 “Muscle full effect”, 598, 604 605 Muscle glycogen resynthesis, 605 606 Muscle mass, 585 592 increasing, 611 613

Subject Index

Muscle protein balance, 604 degradation, 614 615 Muscle protein breakdown (MPB), 584 585 Muscle protein synthesis (MPS), 568, 579, 595 596 Muscle proteolysis, 612 Muscle repair, 596, 613 614 Muscle-specific RING finger-1 (MuRF-1), 614 615 MW. See Molecular weight (MW) MWCOs. See Molecular weight cutoffs (MWCOs) MWP. See Microparticulated whey protein (MWP) Myofibrillar protein synthesis, 592 Myogenin, 612 Myostatin, 612

N

Nanofibrils, 340 Nanofiltration (NF), 52, 66 68, 99 100. See also Ultrafiltration (UF) first NF/UO for salt whey, 67f Nanolayer technology, 107 Nanolayered membrane, 107 “Native whey”, 84, 439 440 proteins, 83 84, 360 361, 537 538 Native β-Lg, 203 204, 409, 448 Natural cheeses, 361 362 intended for cooking, 362 363 proportion, 366 367 NEC. See Necrotizing enterocolitis (NEC) Necrotizing enterocolitis (NEC), 452 453, 457 458 NEM. See N-ethylmaleimide (NEM) Net protein balance (NPB), 598 Net protein synthesis, 583 Net protein utilization (NPU), 582 N-ethylmaleimide (NEM), 216 217, 508 Neural function, 520 Neuregulin 4 (NRG4), 457 458 Neutral pH beverages, 337 338 New Zealand (NZ), 51 52 New Zealand Dairy Board (NZDB), 61 Newtonian behavior, 206

Newtonian fluids, 289 290 NF. See Nanofiltration (NF) N-glycosylneuraminic acid, 18 90-Demineralized Whey (Demin 90), 97 98, 98t Nitrogen-containing components, 51 NMR. See Nuclear magnetic resonance (NMR) N-terminal sequencing methods, 163 Non-GI tract enzymes, 346 Noncovalent bonds, 116 117 Noncovalent interactions, 283 284 Nonenzymatic lactosylation, 160 Nonfat dry milk solids, 358 359 Nongelling polysaccharide guar gum, 357 Nonprotein nitrogen components (NPN components), 51, 463 Nonreducing sugars, 223 Nonthermal preservation process, 282 Novel fermented whey-based drinks, 346 347 Novel processing technologies HP processing, 282 289 low-frequency power ultrasound treatment, 302 312 pulsed electric field technology, 312 318 shear-based processing, 289 301 ultraviolet irradiation, 318 321 Novel products, 93 NPB. See Net protein balance (NPB) NPN components. See Nonprotein nitrogen components (NPN components) NPU. See Net protein utilization (NPU) NRG4. See Neuregulin 4 (NRG4) Nuclear magnetic resonance (NMR), 196 197 NMR spectra of model highprotein nutrition bars, 501 502 NutraSweet, 114 Nutrition(al). See also High-protein nutrition bars bars, 88 89, 354 355 beverages, 584 and functional protein source, 289 functionality, 250 251

interventions, 555 loss, 507 nutritionally functional applications, 88 91 properties of conjugated whey proteins, 270 273 supplements, 496 497 Nutritive and therapeutic aspects of whey proteins age-dependence of rate of leg muscle loss, 564f difference in birth weight, 552f whey protein, 550 and chronic diseases, 563 564 potential role in helping to reducing overnutrition, 558 562 potential role in helping to reducing undernutrition, 550 558 and sarcopenia, 564 569 NZ. See New Zealand (NZ) NZ Dairy Research Institute (NZDRI), 62 NZDB. See New Zealand Dairy Board (NZDB) NZDRI. See NZ Dairy Research Institute (NZDRI) Nε-carboxymethyllysine (CML), 174 175

O

O-glycosylated threonine sites, 23 O-phthalaldehyde method (OPA method), 136 137, 140 142 O-phthaldialdehyde, 252 253 O/W interface. See Oil/water interface (O/W interface) Obesity, 558 559. See also Fat 2-Octanone, 498 Off-flavor production, 395 Off-flavor reduction, mechanism for, 335 336 Oil/water interface (O/W interface), 263 Oils, 340 Older adults, 565, 608 609 muscles, 623 Older age adults, 600 602 Oligomers, 136 138 Oligosaccharides, 459 460 OMSSA, 171 172

707

708

Subject Index

One-dimensional gel electrophoresis (1-DGE), 156 157, 165 166 One-dimensional LC-MS (1D LCMS), 170 171 OPA method. See O-phthalaldehyde method (OPA method) Operating parameters, 102 103 Opioid peptides, 160, 531 532 OPN. See Osteopontin (OPN) Optical properties, 422 Optimal body composition, whey protein and, 560 562 Orthokinetic aggregation, 293 294, 294t Orthokinetic growth, 294 Osmotic effects, 101 Osteopontin (OPN), 92 93, 453 454 characteristics, 22 23 significance, 23 24 Osteoporosis, 650 651 Overnutrition, whey protein potential role in helping to reduce double burden of malnutrition, 562 higher protein diets and weight loss, 558 560 whey protein and optimal body composition, 560 562 Overweight, 558 559. See also Obesity Owatonna Riverbrands of Owatonna MN, 73 Oxidation, 377 oxidation-sensitive components, 266 267 protein, 614 Oxidative bleaching, 394 395 Oxidative stress, 525, 615 616 Oxidizable fuel, 616 Oxidizing agents, 55 Oxidoreductases, 455 Oxygen, 422 423 oxygen permeability, 419 420 oxygen-sensitive products, 423

P

PA. See Polyamide (PA) Packaged ingredient solutions, 351 352 Packaging, 429 PAGE. See 1D-Polyacrylamide gel electrophoresis (PAGE)

Pall membranes, 69 70 Para-κ-CN, 155 Partial denaturation technology, 208 Partial hydrolysis, 210 Partially denatured whey protein concentrates (PDWPCs), 208, 364 Partially Glycated Whey Protein (PGWP), 344 Particle microstructure, 127 modifications at particle scale, 126 130 caking and flow properties, 129 130 color of stored powder, 126 127 particle microstructure, 127 effect of particle size, 129 particle surface chemistry, 127 128 SEM images of fresh and aged powders, 128f size, 130, 142 effect, 129 surface chemistry, 127 128 Particulate gels, 503 Pasilac systems, 59 Pasilac/DDS, 63 Pasteurization, 388 Pasteurized ACP. See Pasteurized analogue cheese products (Pasteurized ACP) Pasteurized analogue cheese products (Pasteurized ACP), 365 367 Pasteurized PCPs. See Pasteurized processed cheese products (Pasteurized PCPs) Pasteurized processed cheese foods, 366 Pasteurized processed cheese products (Pasteurized PCPs), 365 367 Pasteurized processed cheese spreads, 366 Patterson Candy International (PCI), 55 56, 60 61 PC. See Phosphatidylcholine (PC) P:C ratio. See Protein:carbohydrate ratio (P:C ratio) PCA. See Principal component analysis (PCA)

PCI. See Patterson Candy International (PCI) PCN. See Phosphocasein (PCN) PCPs. See Processed cheese products (PCPs) PCr. See Phosphocreatine (PCr) PD. See Protein digestibility (PD) PDCAAS. See Protein Digestibility Corrected Amino Acid Score (PDCAAS) PDWPCs. See Partially denatured whey protein concentrates (PDWPCs) PE. See Polyethylene (PE) Peak force, 356 Peebles instantizer, 77, 77f PEF. See Pulsed electric field (PEF) PEG. See Polyethylene glycol (PEG) Pentosidine, 157 Pepsin, 346, 534 535, 641 Pepstatin, 32 Pepstatin-Sepharose affinity chromatography, 32 Peptides, 1, 155 156, 393, 532 533, 616 antidiabetic, 527 528 antihypertensive, 523 525 antimicrobial, 531 antioxidant, 525 527 cytomodulatory, 533 fragments of whey proteins, 157 immunomodulatory, 532 533 mineral-binding, 528 531 opioid, 531 532 peptide I, 19 peptide II, 19 peptide III, 19 self-aggregation, 346 Peptidoglycans, 29 PER. See Protein efficiency ratio (PER) Performance bars, 354 355 Perikinetic growth, 294 Permeate flux, 100 101 Permeate recycle ratio, 104 105 Permeation, 418 Peroxidation process, 381 PES. See Polyethersulfone (PES) PET. See Polyethylene terephthalate (PET) PGWP. See Partially Glycated Whey Protein (PGWP)

Subject Index

pH, 102 103, 298 300, 416 of fluid product, 399 400 PHAs. See Polyhydroxyalkanoates (PHAs) Phase separation-induced protein aggregation, 504 505 Phenylalanine, 320, 441, 644 Phenylketonuria (PKU), 91, 644 Phenyx, 171 172 PHF-W. See Whey-protein partially hydrolyzed formulae (PHFW) Phosphate salts, 338 339 Phosphatidylcholine (PC), 225 226 Phosphocasein (PCN), 34, 475 Phosphocreatine (PCr), 608 Phosphodiesterases, 35 Phospholipids (PLs), 109 Phosphorylation, 24 25, 165 166 Physical aging, 125 126 Physical degradations, 125 126 Physical instability, 249 250 Physical linkages, 409 410 Physical modifications, 413 415 of whey protein, 213 215 Physically functional applications, 87 88 Physicochemical modifications of whey proteins, 213 230 chemical modification addition of hydrophobic compounds, 225 226 chaperone proteins, 227 228 derivatization of amino, carboxyl, and sulfhydryl groups, 215 217 enzymatic cross-linking, 218 220 enzymatic hydrolysis, 223 225 interaction with carbohydrates, 220 223 soluble whey protein aggregates and microparticulated whey proteins, 226 227 physical modification, 213 215 structural modification, 228 230 Physiological functional properties, 645 646 Physiological functions, 644 pI. See Isoelectric point (pI) PI 3-kinase, 613 Pilot plant, 59, 62

PKU. See Phenylketonuria (PKU) PLA. See Polylactic acid (PLA) Plasma glutamine levels, 615 616 Plasma Trp-LNAA, 651 Plasminogen-derived activities, 165 Plate-and-frame device, 55, 60 61 PLs. See Phospholipids (PLs) 1D-Polyacrylamide gel electrophoresis (PAGE), 18 2D-Polyacrylamide gel electrophoresis (PAGE), 286 Polyamide (PA), 55, 424 Polyclonal antibodies, 161 162 Polyesters, 421 422 Polyethersulfone (PES), 54 membranes, 106 Polyethylene (PE), 427 428 Polyethylene, 419 420 Polyethylene glycol (PEG), 113, 257 Polyethylene terephthalate (PET), 427 428 Polyhydroxyalkanoates (PHAs), 429 Polylactic acid (PLA), 429 Polymer(s), 417 blending, 417 cohesion, 421 422 coil, 290 films, 411 polymer-based multilayer packaging materials, 427 428 Polyolefins, 421 422 Polyphosphates, 338 339 Polysaccharides, 221 222, 407 Polysorbate 80, 358 359 Polysulfide-amide (PSA), 107 Polysulfones (PS), 54 Polyvalent ions, 68 Polyvinyl pyrrolidone (PVP), 107 Polyvinylidene difluoride MF membranes (PVDF MF membranes), 70 “Popcorn whey”, 52 Pore blocking, 101 Posttranslational modifications (PTM), 157, 165 166 Powder particle surface, 129 Powder stability, 125 126 Power ultrasound, 302 304 effect on solubility, 306 307 effect on viscosity, 307 309 effect on whey protein gelation, 309 312 PP. See Proteose peptone (PP)

PP8f. See Proteose peptone 8 (fast) (PP8f) PP8s. See Proteose peptone 8 (slow) (PP8s) PPX. See Protein polydextrose (PPX) Precipitation method, 169 precipitation/gel layer formation, 101 Pregelatinized starch, 281 Pregnancy, inadequate nutrition of women during, 551 Premeal consumption of WP, 611 Preparative chromatography, 163, 168 169 Prepurification of α-La, 111 Pressing-by-centrifugation method, 365 Pressure, 102 103 pressure-driven membrane processes, 97 treated milk yogurt, 349 Pretexturization, heterogeneity creation by, 510 513 extrude and toast, 510 511 jet-milling, 511 512 protein cross-linking by transglutaminase, 513 superfine grinding, 512 513 Principal component analysis (PCA), 144, 145f Pro-Stat MAX, 652t Probiotics, 335 Procathepsin D system, 31 32, 165 Process factors, 313 Process-induced molecular changes affecting whey proteins analysis, 158 161 characteristics of whey proteins and methods, 159t lactosylation sites of whey proteins, 161t Processed cheese products (PCPs), 365 366 Processed cheeses, 361, 365 366 Product constituents, 101 102 Product factors, 313 Product fouling, 101 Product shelf life, 337 Proenzymes, 161 162 Prolac, 63 Prolibra, 520, 650

709

710

Subject Index

Prolibra 290, 652t ProOptibol, 72 73, 74f Propagation section, 381 Propyl gallate, 525 ProSource NoCarb Liquid Protein, 652t Protease(s), 164 165 protease A, 346 protease M, 346 protease S, 346 Protectamin, 649 Protein, 1, 165 166, 290 291, 407 408, 496 497, 584, 604. See also High-protein nutrition bars adsorption, 208 209 aggregates formation, 341 aggregation, 502 505 allergenicity, 462 463 bars, 354 356 content, 495 control, 611 cross-linking, 107 109, 131 132, 349, 365 of skim milk, 353 354 by transglutaminase, 513 databases, 171 172 degradation, 613 614 denaturation, 292, 300 301 deposition, 101 102 fortification of yogurts, 352 353 hydrolysates, 59 60 adding, 509 510 hydrolysis, 416 417 ingredients, 581 582 intake, 612 sarcopenia and, 565 lactosylation, 139 140 modifications, 160 161 nanofibrils, 262 oxidation, 614 particle formation via heatinduced aggregation, 298 protein-based coatings, 411 protein-based FWB, 345 protein-based plastics, 412 413 protein-fortified citrus beverages, 584 protein-structure perturbing agents, 216 217 protein polysaccharide complexes, 340 conjugates, 271 272

protein protein interactions, 269 protein protein thiol disulfide interchange reactions, 508 509 quality, 582 related changes, 130 132 protein crosslinking, 131 132 protein structure modifications, 130 131 satiety effect, 650 solubility, 260f sources, 498, 580 581 dairy (milk) proteins, 580 soy protein, 581 stability, 124 structure effect on, 135 modifications, 130 131 supplements, 579 synthesis, 604 turnover, 602 Protein Data Bank, 11 Protein digestibility (PD), 582 Protein Digestibility Corrected Amino Acid Score (PDCAAS), 336 337, 556, 582 Protein efficiency ratio (PER), 582 Protein:carbohydrate ratio (P:C ratio), 553 Protein polydextrose (PPX), 354 355 Proteolysis, 382, 385 of β-Lg, 641 of whey proteins, 167 Proteolytic cleavage, 162 Proteolytic enzymes, 163 165, 346 Proteomics, 157, 171 173 approaches, 157 158 proteomics, 171 173, 172f proteomics in whey protein analysis, 173 175, 174f in whey protein analysis, 173 175 Proteose peptone (PP), 20, 519 520, 639 fraction of milk, 20 PP3, 20 22, 457 characteristics, 20 21 significance, 21 22 PP5, 20 Proteose peptone 8 (fast) (PP8f), 20 Proteose peptone 8 (slow) (PP8s), 20 PS. See Polysulfones (PS)

PS-UF membranes, 59 PSA. See Polysulfide-amide (PSA) Pseudocathepsin D, 31 32 Pseudomonas aeruginosa (P. aeruginosa), 427, 451 452 Psoriasis, 649 PTM. See Posttranslational modifications (PTM) Pulsed electric field (PEF), 228 229, 281 technology, 312 318, 315t effect on whey proteins, 314 318 “Pulsed-UV” light, 319, 321 PVDF MF membranes. See Polyvinylidene difluoride MF membranes (PVDF MF membranes) PVP. See Polyvinyl pyrrolidone (PVP) Pyrazine derivatives, 498 Pyrroles, 388 389

Q

Quadruple ion trap (Q-IT), 172 173 Quadrupole 1 (Q1), 174 175 Quadrupole time-of-flight (Q-TOF), 172 173

R

Radial immunodiffusion, 13 Raman spectroscopy, 142 Ratings of perceived exertion (RPE), 596 Rats, 648 resistance exercised WP fed, 614 615 spontaneously hypertensive, 524 525 work in nonexercising, 620 Reactive nitrogen species, 525 Reactive oxygen species (ROS), 525, 615 616 Ready-to-drink (RTD), 335, 652t dairy-based beverages, 335 Ready-to-use therapeutic foods (RUTFs), 554 Reconstituted WP powders, 306 307 Recovery from resistance exercise, 592 593 Recycling, 426 “Red Cheddar”, 108 Refractive index, 118

Subject Index

Refueling with CHO immediately postexercise, 605 606 Regulate glycogen synthesis, 599 Regulatory aspect, 425 426 Relative humidity (RH), 142, 256 Relaxation enthalpy, 125 126 Renin angiotensin system, 523 524 Rennet casein whey, 99 100 Renneting process, 84 Residual fat in cheese whey, 98 99 Resistance exercise, 579, 584 593, 602, 605 athletes, 604 605 body composition and weight loss, 593 publications on, 586t recovery, 592 593 strength performance and muscle mass, 585 592 and training, 597 599 Resistance training, 592 593 high-intensity, 593 intense, 593 programme, 602 Resistance-trained athletes investigating effects, 619 Retinoic acid, 641 Reverse osmosis (RO), 52, 55 56, 346 347 Reversed-phase HPLC, 196 197 Reversed-phased chromatography (RPC). See Gel permeation chromatography (GPC) Reversible aggregation, 293 294 Rewet agglomeration, 73 removing for, 78 Rh. See Hydrodynamic radius (Rh) RH. See Relative humidity (RH) Rheological characterization of liquid solid boundary, 356 properties, 206 208, 348 349 tests, 350 351 “Rhobust” expanded bed adsorption technology, 81 Ribonucleases (RNases), 35 39, 38t characteristics, 35 39 RNase-A, 35 36 RNase-B, 35 36 RNase-C, 35 36 RNase-D, 35 36 significance, 39

Ribonucleic acid (RNA), 35 RNase BL-4. See Bovine liver ribonuclease (RNase BL-4) RNase-5. See Angiogenins RNases. See Ribonucleases (RNases) RO. See Reverse osmosis (RO) Roller dryers, 52 Romicon hollow-fiber design, 64 65 ROS. See Reactive oxygen species (ROS) Rotating disk membrane, 112 RP, 170 RP-HPLC, 170 in combination with MS for protein or peptide analyses, 170 171 RPE. See Ratings of perceived exertion (RPE) RTD. See Ready-to-drink (RTD) Ructose systems, GC-MS peaks of, 498 RUTFs. See Ready-to-use therapeutic foods (RUTFs)

S

SA. See Serum albumin (SA); Sialic acid (SA) Saccharomyces cerevisiae (S. cerevisiae), 524 Salt whey, 66, 67f SAM. See Severe acute malnutrition (SAM) Sanders’ larger fluid-bed agglomerators, 73 75 Sarcopenia and economic impact, 564 565 hypotheses about mechanism of action of whey protein intake on, 567 569 and protein intake, 565 whey protein supplementation and sarcopenia in elderly, 565 567 Sarcopenic elderly, 609 610 Satiating effect of protein, 618 619 Satiety effect of proteins, 650 Scanning electron micrographs, 350 ScCO2. See Supercritical carbon dioxide (ScCO2) Schiff bases, 251 252, 272

Scraped-surface heat exchanger (SSHE), 115 SD-MWP-pH3. See Spray-dried, denatured MWP produced at pH 3 (SD-MWP-pH3) SDS. See Sodium deodecyl sulfate (SDS) SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Seawater, 53 Secondary/tertiary structures, 130 Selective reaction monitoring (SRM). See Multiple reaction monitoring (MRM) Semihard cheese varieties, 99 Semispherical particle shape, 127 Sensitive HPLC method, 31 Sensory analysis, 378 379, 380t properties of high-protein nutrition bars, 497 498 quality, 419 SeparaTech. See Separation Technology Inc. (SeparaTech) Separation, 388 methods, 169 techniques, 123 in proteomics, 166 167 trained panel cardboard flavor intensities of cheddar, 389f Separation techniques, 168 Separation Technology Inc. (SeparaTech), 68, 70 Sephadex G-50, 30 SEQUEST, 171 172 Serious nausea symptoms, 595 Serokinin, 643 Serorphin, 643 Serotonin, 615 Serum 25-hydroxyvitamin D, 609 Serum albumin (SA), 10 12, 451 characteristics, 10 function, 11 significance, 11 12 structure, 11 Serum glucose, 527 528 Serum protein, 1, 391 392, 392t Serum protein concentrate (SPC), 391 392 Serum TGF-β1 secretion, 532 533

711

712

Subject Index

Severe acute malnutrition (SAM), 550, 554 SGA births. See Small for gestational age births (SGA births) Shear accompanying preprocess or simultaneous application, 297 301 conformational and colloidal modifications of proteins due to, 291 294 effects of rotating disk UF, 108 impact on properties of WP in absence of heat, 294 297 processing, 281 Shear-based processing, 289 301 conformational and colloidal modifications of proteins, 291 294 general principles, 289 291 impact of shear accompanying preprocess or simultaneous application, 297 301 impact of shear on properties of WP in absence of heat, 294 297 Shigella flexneri (S. flexneri), 449 Short-chain fatty acids, 379 381 Sialic acid (SA), 18, 450 Sialyllactose, 92 93 Simple sugar protein systems, 504 505 Simplesse, 116 117, 361 362 Simplesse-based MWP, 361 362 trademark, 114 Simulated milk ultrafiltrate (SMUF), 317, 342 343 Simulated moving-bed technology, 169 Simulated processing protocol, 358 Single-step heating method, 206 207 Size exclusion method, 196 197 Size-exclusion chromatography (SEC). See Gel permeation chromatography (GPC) Skatole, 382 383 Skeletal muscle, 611 Skim milk (SM), 99 100, 358 359, 465 466 acid phosphatase in, 34

Skim milk powder (SMP), 98 99, 347 348, 353 354, 459, 549 550 addition, 347 Skim-based formula, 88 Slow protein aggregation process, 339 “Slow” digesting protein, 592 SM. See Skim milk (SM) Small for gestational age births (SGA births), 551 552 Small-angle neutron scattering, 203 204 SME input. See Specific mechanical energy input (SME input) SMP. See Skim milk powder (SMP) SMUF. See Simulated milk ultrafiltrate (SMUF) Snack bars, 354 355 Snow Brand Milk, 650 651 Soccer-specific running test, 596 Sodium caseinate (NaCas), 465 466, 498 502 Sodium chloride (NaCl), 204, 340 Sodium deodecyl sulfate (SDS), 216 217, 225 226 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2, 3f, 116 117, 165 166, 196 197, 205 Sodium percarbonate, 28 Sodium polyacrylate, 113 Sodium stearoyl-2 lactylate (SSL), 225 226 Sol-to-gel transition temperature, 363 Solid lipid system, 504 505 Solubility, 136, 258 260, 298 300 power ultrasound effects on, 306 307 protein solubility, 260f Soluble aggregates, 205 Soluble milk proteins, 1 Soluble proteins, 51 Soluble whey protein aggregates, 208, 226 227 formation and role, 341 342 Solvent casting, 410 411 Sonication at low frequency, 307 of WP solutions, 305 306

Sorbitol, 223, 496 GC-MS peaks, 498 Sound waves, 302 303 Soy, 419 420 protein, 407, 495, 581 bars, 498 Soy protein concentrate (SPC), 497, 581 SPC34, 391 392 SPC80, 391 392 Soy protein isolate (SPI), 497, 581, 620 bars, 498 500 system, 501 502 Spacers, 109 SPC. See Serum protein concentrate (SPC); Soy protein concentrate (SPC) Specific mechanical energy input (SME input), 412 413 Spectrophotometry, 26, 252 253 Spectroscopic technique, 196 197 SPI. See Soy protein isolate (SPI) Spiral applications development, 71 Spiral format, 65 Spiral MF conditions optimizing for WPI production, 71 system, 71 Spiral systems, 64 Spiral UF membranes, 57 Spiral-wound membrane domination, 64 66 organic MF membranes, 98 99 Sports beverages, 584 drink, 337 Sports and exercise supplements. See also Exercise coingestion with bioactives, 605 610 contraindications and complications, 618 620 energy balance, 618 619 health complications, 620 lactose intolerance, allergies, dairy-free diets, 619 620 different sources of protein, 580 581 dairy (milk) proteins, 580 soy protein, 581 different user groups, 600 603 children and adolescents, 603

Subject Index

female exercisers, 602 603 older age adults, 600 602 training status of athletes, 600 dose-response, 604 605 limitations of research and future directions, 620 622 mechanisms of action, 610 618 gene expression, 614 615 immune function, 615 618 increase muscle mass, 611 613 insulin signaling and glycogen synthesis, 610 611 mood and cognition, 615 protein oxidation, 614 repair of muscle, 613 614 timing of whey protein ingestion, 597 600 types of whey protein products, 581 584 whey protein effect on exercise performance, 584 597 SPR spectroscopy. See Surface plasmon resonance spectroscopy (SPR spectroscopy) Spray dryers, 78 Spray drying, 341 342 and dry storage, 398 400 trained sensory panel profiles, 399f effect on microparticulate functionality, 116 117 IF formulations, 474 475 processes, 160 Spray-dried, denatured MWP produced at pH 3 (SD-MWPpH3), 116 117 Spray-dried powder, 222 223 SPX, 118 cautions, 118 microparticulation plant, 85, 86f SPX Flow Technology’s engagement, 117 118 SSHE. See Scraped-surface heat exchanger (SSHE) SSL. See Sodium stearoyl-2 lactylate (SSL) Stability of high-protein nutrition bars, 500 during storage, 124 Stabilizers, 358 359 Stabilizing system, 337 338 Starches, 357

Storage, 397 conditions, 132 135 stability, 341 of protein bars, 510 whey protein hydrolysates for improved heat and, 346 Strain, 289 290 Strands, 205 206 Streamline Direct CST-1, 113 114 Strecker degradation process, 388 Strength performance, 585 592 Streptococcus mutans (Str. mutans), 449, 451 452 Streptococcus pneumonia (Str. pneumonia), 451 452 Stress, 651 hormones, 615 616 vector, 289 290 Strong cation-exchange chromatography, 168 169 Structural modification of whey protein, 228 230 Stunting process, 550 551 Sucrose, 496 Sugar(s), 221 alcohols, 496 crystallization, 505 syrups, 496 Sulfhydryl groups, 215 217 Sulfitolysis, 224 225 Sulfopropyl, 168 169 Sulfur amino acids, 158 high content, 5 Super Cereal Plus, 556 Supercritical carbon dioxide (ScCO2), 230 Superfine grinding, 512 513 Supplementation, 599 Supplements, 583 containing whey protein ingredients, 651 Surface chemical modifications, 128 hydrophobicity, 204 layer, 101 102 properties, 421 422 structure of powder particles, 127 surface-enhanced Raman spectroscopy, 258 Surface plasmon resonance spectroscopy (SPR spectroscopy), 342 343, 455 456

Sweet fluid whey, 388 389 Sweet whey, 99, 339, 463, 581 Sweet whey powder (SWP), 388 389 Syrup phase, 504 505

T

“Tailor” protein functionality, 302 Tandem time-of-flight (TOF-TOF), 172 173 Taurine, 446 447 TBARS. See Thiobarbituric Acid Reactive Substances (TBARS) TCA. See Trichloroacetic acid (TCA) Te Aroha Thames Valley, Waikato, 63 Technical University of Munich (TUM), 117 118 Techno-functional properties of conjugated whey proteins, 257 270 TEM. See Transmission electron microscopy (TEM) Temperature, 102 103, 142, 298 300 effect, 132 133 and sonication of WP solutions, 305 306 Tensile strength, 420 Termination sections, 381 Tert-butylhydroquinone, 525 Texture extrinsic appearance changes, 499f gelation and textural properties, 269 270 hardening of high-protein nutrition bars, 500 505 moisture migration, 501 502 protein aggregation, 502 505 sugar crystallization, 505 of high-protein nutrition bars, 498 500 measurement technique, 356 textural components, 496 497 TFC membranes. See Thin-film composite membranes (TFC membranes) TGase. See Transglutaminase (TGase) TGF-β. See Transforming growth factor beta (TGF-β) Thermal instability of WP, 310 Thermal load reduction on whey proteins in IF manufacture, 472

713

714

Subject Index

Thermal processing, 209 whey proteins behavior aggregate size and structure of thermally denatured whey proteins, 203 206 effects on whey protein functionality, 206 212 heat-induced changes in whey proteins, 199 200 whey protein casein interactions, 201 203 whey protein whey protein interactions, 200 201 Thermal stability of IF containing whey proteins, 470 472 Thermal-induced state, 309 Thermally denatured whey proteins, aggregate size and structure of, 203 206 Thermocalcic process, 98 99 Thermolysin, 641 Thermomechanical coagulation of WPC, 115 Thermoplastic behavior, 412 413 Thermoquarg, 361 Thermoset behavior, 412 413 Thin-film composite membranes (TFC membranes), 55 ThinkitDrinkit, 652t Thiobarbituric Acid Reactive Substances (TBARS), 618 Thiocyanate, 28 Thiocyanate ion (OSCN ), 646 647 Thiol-p-nitrobenzoate (TNB), 216 217 Thiol disulfide interchange reaction, 502 Thomas Technical Services Inc. (TTS), 57 58, 65 Thomas’s early UF plants, 57 Three-dimensional chromatography, 170 171 Time derivative of strain, 289 290 Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS), 128 Time trial performance (TT performance), 594 595 Time-of-flight MS (TOF MS), 160 161 Time-to-exhaustion (TTE), 594 Timing of whey protein ingestion, 597 600

endurance exercise and training, 599 600 resistance exercise and training, 597 599 tests, 595 TMP. See Trans-membrane pressure (TMP) TMR. See Total mixed ration (TMR) TNB. See Thiol-p-nitrobenzoate (TNB) TNBS. See Trinitrobenzenesulfonic acid (TNBS) TNF-α. See Tumor necrosis factoralpha (TNF-α) Toast, 510 511 TOF MS. See Time-of-flight MS (TOF MS) ToF-SIMS. See Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) TOF-TOF. See Tandem time-of-flight (TOF-TOF) Top-down approaches, 171 174 “Tortuous path”, 417 418 Total mixed ration (TMR), 382 383 Total solids (TS), 466 469 Toxic compounds, 271 272 Traditional analytical methods, 157 Traditional precipitation-based processes, 364 Training status of athletes, 600 and whey protein supplementation, 586t Trans-membrane pressure (TMP), 100 101 Transferases, 455 Transferrin, 17 Transforming growth factor beta (TGF-β), 451, 458 459, 612 Transglutaminase (TGase), 107 108, 218, 339 340, 349, 417, 513, 537 538 enzymatic activity, 219 220 protein cross-linking by, 513 TGase-catalyzed cross-linking of β-Lg, 218f of whey protein, 365 transglutaminase-induced crosslinking effects, 365 Transition, 291 Transmission electron microscopy (TEM), 204, 362

Transparent beverage applications, 336 337 Travelan, 652t Trichloroacetic acid (TCA), 20, 463, 537 538 Trinitrobenzenesulfonic acid (TNBS), 252 253 Triple quadrupole, 172 173, 174f Trypsin, 170 171, 346, 534 535, 641 Tryptophan, 446 447, 644 tryptophan-rich protein, 642 TS. See Total solids (TS) TT performance. See Time trial performance (TT performance) TTE. See Time-to-exhaustion (TTE) TTS. See Thomas Technical Services Inc. (TTS) Tubular design, 55 56 TUM. See Technical University of Munich (TUM) Tumor necrosis factor-alpha (TNF-α), 458 “Tween 80”. See Polysorbate 80 Twin-screw extrusion technology, 115 116 Two-dimensional chromatography, 170 171 Two-dimensional electrophoresis (2DGE), 156 157, 160 161, 165 166 Two-phase aqueous extraction, 113 Two-stage tangential flow UF system, 111 112 Two-stage UF, 112 Two-stage valve-type homogenization, 466 469 Two-step heating method, 206 207 Tylactin RESTORE Powder, 652t Tylactin RTD, 652t TYR. See Tyrosinemia (TYR) Tyrosine, 320, 441, 644 Tyrosinemia (TYR), 652t

U

UF. See Ultrafiltration (UF) UHT. See Ultra-high temperature (UHT) Ultra Osmosis (UO), 68 Ultra-high temperature (UHT), 160, 250 251 milk, 9 10

Subject Index

processing, 296 297 technology, 337 UHT-treated milk products, 250 251 Ultrafiltration (UF), 52, 56 66, 97, 168, 335 336, 389, 439 440, 549 550. See also Nanofiltration (NF) of cheese milk, 83, 85 domination of spiral-wound membrane, 64 66 in Europe, 58 61 Frank Thomas’ first UF System, 57f multistage high-protein UF system, 59f in New Zealand, 61 64 PCI tubular UF system, 60f permeate, 58 processing parameters, 102 103 spiral-wound membrane manufacturers and state of art, 109 in United States, 56 58 whey protein concentrates and isolate by, 97 109 UltraMeal Whey, 652t Ultrasonic processing (USP), 349 Ultrasonically induced state, 309 Ultrasonically-assisted UF, 105 Ultrasound, 108, 302, 311 312 Ultraviolet (UV), 167 irradiation, 318 319 effect on whey proteins, 319 321 processing, 281 radiation, 415 Undernutrition, 550 558 linear growth, 554 556 managing malnutrition during infancy, 554 whey protein, weight gain and lean body mass, 556 558 Unhealthy diet, 563 UniProt/Swiss-Prot databases, 171 172 Unmodified whey proteins, 337 338 UO. See Ultra Osmosis (UO) Urea, 216 217, 463 US Department of Agriculture in (USDA), 554 US Food and Drugs Administration (FDA), 114, 346 347, 365 366

USDA. See US Department of Agriculture in (USDA) USP. See Ultrasonic processing (USP) UV. See Ultraviolet (UV)

V

Vacuum packaging, 423 Valine, 158, 520, 644 Valorization techniques, 426 Van der Waals interaction, 283 284, 466 469 VCF. See Volume concentration factor (VCF) Vegetable proteins, 610, 620 Vibrio cholera (V. cholera), 451 452 Virgin whey, 84 Viscosity, 290, 353 354 power ultrasound effect on, 307 309, 308t Vistec process, 98 99 Vitamin D, 566 567, 609 610 Vitamins, 267, 495 Vivapure Q Mini-H column, 112 Volac International in United Kingdom, 88 Volac’s WPI business, 88 Volatile compounds, 379 381, 498 Volatile lipid oxidation compounds, 381 Volatile Strecker degradation products, 252 253 Volatile sulfhydryl compounds, 383 Volume concentration factor (VCF), 97, 106 Volume reduction, 282 283

W

Water, 53, 125 126, 282 283, 388 389, 501 binding, 281, 357 holding capacity, 350 vapor, 418, 422 423 permeability, 419 Water activity (aw), 124, 495, 501 Waxes, 418 Weight gain, 556 558 Weight loss, 558 560 bars, 354 355 body composition and, 593 Weight management, 92, 649 650 Western blotting, 156 157, 161 163 Western Blotting method, 32 Wet coating, 410 412

Wet heating approach, 256 257, 259 Wet processing techniques, 410 411 Wheat gluten, 407, 419 420 Whey, 51, 103 104, 519 bleaching impact on, 393 394 from cheese manufacturing, 549 550 cheese type and influence of culture on whey flavor, 385 387 fractions, 155 156 isolation of GMP from, 536 538 solids, 123 theoretical approach of whey thermal denaturation and aggregation, 186 198 whey-based IF, 442 446 whey-based infant formula, 649 whey-based oral supplement, 649 whey-dominant formulas, 439 440 whey-hydrolysate formulas, 445 446 whey-predominant formulas, 445 446 Whey ingredients composition whey process from cheese vat to, 390f whey protein concentrates and isolates, 388 391 Whey protein (WP), 1, 51, 155 157, 160, 185, 254, 281, 302, 310f, 348 349, 407 408, 422, 445, 531 532, 579 580, 582 583, 592, 609 610, 639 647 α-La, 642 amino acid composition, 582 583 anticancer potential, 648 behavior in thermal processing, 199 206 β-Lg, 639 641 bioactive peptides derived from, 521 533, 521f, 522t BSA, 643 characteristics and methods for measurement or detection, 159t commercial functional food and nutritional supplement applications, 652t

715

716

Subject Index

Whey protein (WP) (Continued) commercial thermal processing effects on WP functionality, 206 212 contribution of commercial whey protein-based fat replacers, 361 363 digestability, 211 212 effect, 314 321 endurance exercise, 594 596 on exercise performance, 584 597 multiple-sprint sports, 596 597 resistance exercise, 584 593 films, 408 410, 415, 417 418 and coatings, 425 426 protein structure related properties and preconditions for film forming, 408 409 steps for film formation, 409 410 fractionation for infant formula, 78 82 fractions by membrane-based separations, 109 114 functionality impacts on IF stability thermal load reduction on whey proteins in IF manufacture, 472 thermal stability of IF containing whey proteins, 470 472 whey proteins and in-process emulsion stability, 466 470 whey proteins behavior in concentration of IF emulsions, 472 476 GMP, 643 644 health benefits, 647 651 high-quality protein, 550 hydrolysis, 82 Igs, 643 incorporation, 365 into pasteurized ACP, 365 367 into pasteurized PCPs, 365 367 ingestion timing, 597 600 Lf, 645 646 Lp, 646 647 mechanism of action of WP intake on sarcopenia, 567 569 microparticulation, 117

minor whey proteins, 14 39, 15t, 451 459 acid phosphatase, 33 35 cathepsin D, 31 33 GMP, 24 26 Lactoferrin, 17 20 LPO, 27 29 lysozyme, 29 31 osteopontin, 22 24 PP3, 20 22 RNases, 35 39 summary of bovine milk soluble (whey) enzymes, 16t summary of reported bovine milk ribonucleases, 38t mixtures, 319 320 nanoparticles, 214 215 and peptides, 446 451 α-La, 7 10 β-Lg, 3 7 Igs, 12 14, 12t SA, 10 12 power ultrasound effect on viscosity of WP solutions, 307 309 power ultrasound effect on whey protein gelation, 309 312 composition of formulation closely resembling infant formula, 311t high-temperature processing of infant formula, 311f prevention of denaturation/ aggregation, 213 230 proportion of individual proteins, 640f proteomics in WP analysis, 173 175 as provision for increasing glutathione, 618 range of whey protein ingredients, 640f refinery, 81 structure and texture of WP gels, 502 supplementation, 585 surface hydrophobicity, 307 308 temperature and sonication of WP solutions, 305 306 whey protein-based coatings, 421 422, 426 427 whey protein-fortified beverages, 335 347

acidified whey beverages, 338 heat and emulsion stability, 338 347 neutral pH beverages, 337 338 whey protein-fortified RTDs, 89 denaturation and aggregation of pure WP fractions, 284 286 shear impact on properties of WP in absence of heat, 294 297 whey protein carbohydrate conjugates, 250 251, 268 approaches to achieve conjugation, 256 257 emulsifiers, 267 factors affecting Maillardinduced conjugation, 253 254 Maillard reaction chemistry, 251 253 nutritional properties of conjugated whey proteins, 270 273 techno-functional properties of conjugated whey proteins, 257 270 whey protein and carbohydrate substrates, 255 256 whey protein casein interactions, 201 203 casein micelle changes as influenced by pH, 203f whey protein casein interactions in IF, 465 466 whey protein whey protein interactions, 200 201 WP microparticulation, 297 301 Whey protein concentrate (WPC), 57 60, 109, 124, 170, 200 201, 255, 335 336, 345, 439 440, 497, 526 527, 549 550, 581 calcium content, 117 118 films, 427 and isolates, 388 391 trained sensory panel profiles of rehydrated, 391t by ultrafiltration, 97 109 WPC-based ingredients, 97 WPC-caffeine-containing supplement, 607 608 WPC35, 60, 97 98 WPC50, 60

Subject Index

WPC55, 63 WPC75, 60, 63, 73 WPC80, 63, 97 98, 117, 497, 549 550 WPC80 1 , 58 59 WPC82, 63, 360 Whey protein concentrates produced containing 34% protein (WPC34), 549 550 Whey protein functionality, commercial thermal processing effects on digestability of whey proteins, 211 212 rheological properties, 206 208 surface properties, 208 211 Whey protein hydrolysate (WPH), 82, 210, 223 224, 262, 345, 356, 392 393, 394f, 439 440, 524 525, 528, 581 582 for improved heat and storage stability, 346 for infant formula, 78 82 Whey protein ingredients applications cheese, 360 367 desserts and ice creams, 357 360 protein bars, 354 356 whey protein-fortified beverages, 335 347 yogurt, 347 354 flavor aspects bleaching, 393 397 composition of whey ingredients, 388 391 flavor aspects of liquid whey, 383 387 flow diagram of general whey processing from raw milk, 378f fluid milk, 382 383 fluid whey processing, 388 hydrolysates, 392 393 origin of flavors in whey, 379 382 sensory analysis, 378 379 serum protein, 391 392 spray drying and dry storage, 398 400 storage, 397 incorporation into IF

formulation dynamics, 464 465 whey protein ingredients development for IF, 460 463 whey protein casein interactions in If, 465 466 physiological function of components in, 442 460 major constituents of whey and functions, 443t major whey proteins and peptides, 446 451 minor whey proteins and peptides, 451 459 other bioactive components in whey, 459 460 whey-based IF, 442 446 supplements containing, 651 Whey protein isolate (WPI), 59 60, 97 98, 124, 200, 250 251, 283 284, 336 337, 344, 360, 388 389, 427, 439 440, 497, 504 505, 526 527, 549 550, 581 bars, 498 502 development, 69 72 by ultrafiltration, 97 109 WPI powders, 170 WPI dextran conjugates, 257 WPI ι-carrageenan, 222 223 WPI κ-carrageenan, 222 223 WPI λ-carrageenan, 222 223 Whey protein phospholipid concentrates (WPPC), 360 Whey protein powder, 123, 352 measures to overcoming changes during storage, 140 147 actions on key influencing factors, 142 143 extrinsic factors, 142 intrinsic factors, 142 143 monitoring powders evolution, 140 142 prediction of whey protein powder changes, 143 147 theoretical correspondences, 147t physicochemical changes during storage, 125 140 impact on functional properties, 136 140 lactose related changes, 132 136

modifications at particle scale, 126 130 molecular mobility, 125 126 powder stability, 125 126 protein related changes, 130 132 Whey protein products, 51 52, 377, 392 393 current whey protein products and applications, 86 91 bioactivity of whey proteins, 91 filler, 87 nutritionally functional applications, 88 91 physically functional applications, 87 88 developments, 91 94 new areas of application, 93 94 new markets, 92 new technologies or processes, 92 93 novel products, 93 key developments, 72 82 agglomeration/instantization and whey protein in sports nutrition, 72 78 whey protein fractionation and hydrolysates for infant formula, 78 82 membrane development, 53 55 CA, 53 54 PA, 55 PES, 54 PS, 54 systems and applications development, 55 72 ion exchange technology, 69 72 microfiltration, 69 72 NF, 66 68 RO, 55 56 UF, 56 66 types, 581 584 amino acid composition of whey proteins, 582 583 food issues, 584 meals versus supplements, 583 whey proteins and advances in cheese making technology, 82 85 Whey protein-based films, 418 and coatings, 429 430 end-of-life options for, 426 428

717

718

Subject Index

Whey protein-based films (Continued) biodegradation of whey proteinbased films and laminates, 426 427 recyclability of whey proteinbased multilayer laminates, 427 428 merging food packaging requirements and properties, 424 425 Whey protein-based nutrition bars high-protein nutrition bars, 495 497 quality of high-protein nutrition bars, 497 507 solutions to avoid hardening of high-protein nutrition bars, 508 513 Whey protein-based packaging films and coatings end-of-life options for whey protein-based films and multilayer laminates, 426 428 food safety and regulatory aspect, 425 426 industrial perspectives, 429 430 packaging relevant properties, 418 422 barrier properties, 418 420 mechanical properties, 420 421 optical properties, 422 surface properties, 421 422 potential applications of whey protein based films and coatings, 422 425

merging food packaging requirements and properties, 424 425 packaging requirements of food products, 422 423 protein modification for optimized film formation and performance, 413 418 biochemical modifications, 416 417 chemical modifications, 415 416 film modifications, 417 418 physical modifications, 413 415 technologies for processing films and coatings, 410 413 whey protein film formation, 408 410 Whey-protein partially hydrolyzed formulae (PHF-W), 82 WheyUP, 337 Whipping, 359 WHO. See World Health Organization (WHO) Williams, Landel, & Ferry equation (WLF equation), 125 126 Wisconsin companies, 75 77 WLF equation. See Williams, Landel, & Ferry equation (WLF equation) Word Health Organization, 155 156 World Health Organization (WHO), 550 551, 600 601 WP. See Whey protein (WP)

WPC. See Whey protein concentrate (WPC) WPC34. See Whey protein concentrates produced containing 34% protein (WPC34) WPH. See Whey protein hydrolysate (WPH) WPI. See Whey protein isolate (WPI) WPI with hydrocolloid blend of xanthan (X) and locust bean gum (L) (WPI-X/L), 348 349 WPIC. See Commercial WPI (WPIC) WPPC. See Whey protein phospholipid concentrates (WPPC)

X

X!Tandem, 171 172 Xanthan, 348 349, 358 Xanthine oxidase, 27 Xenobiotic cross-linking, 132 X-Ray Photoelectron Spectroscopy (XPS), 128

Y

Yogurt, 114, 347 354 manufacturing, 347 milk, 354

Z

Zein, 407 Zymogens, 161 162