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Principles of Enzymology for the Food Sciences, 2nd edn Marcel Dekker, New York, pp 367–385 P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson 206 March 21, 2012 11:15 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology Wilkinson MG, Kilcawley KN 2005 Mechanism of enzyme incorporation and release of enzymes in cheese during ripening Intl Dairy J 15: 817–830 Wong H, Schotz MC 2002 The lipase gene family J Lipid Res 43: 993–999 Yamada H 1989 Localization in skin, activation and reaction mechanisms of skin sulfhydryl oxidase Nippon Hifuka Gakkai Zasshi 99: 861–869 Yamasaki Y et al 2008 Pullulanase from rice endosperm Acta Biochim Polonica 55(3): 507–510 Yokoyama K et al 2004 Properties and applications of microbial transglutaminase Appl Microbiol Biotechnol 64: 227–454 Yoshio N et al 1986 Purification and properties of d-enzyme from malted barley J Jpn Soc Starch Sci 33: 244–252 Yun JW et al 1995 Continuous production of fructooligosaccharides from sucrose by immobilized fructosyltransferase Biotechnol Tech 9(11): 805–808 P1: SFK/UKS BLBS102-c10 P2: SFK BLBS102-Simpson March 21, 2012 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come 10 Protein Cross-linking in Food – Structure, Applications, Implications for Health and Food Safety Juliet A Gerrard and Justine R Cottam Introduction Protein Cross-Links in Food Disulfide Cross-links Cross-links Derived from Dehydroprotein Cross-links Derived from Tyrosine Cross-links Derived from the Maillard Reaction Age Protein Cross-Links Isolated to Date in Food Health and food safety aspects of MRPs Melanoidins Maillard-Related Cross-Links Cross-links Formed via Transglutaminase Catalysis Other Isopeptide Bonds Manipulating Protein Cross-Linking During Food Processing Chemical Methods Enzymatic Methods Transglutaminase Future Applications of Protein Cross-Linking Acknowledgements References Abstract: The capacity of proteins to cross-link with each other has important implications for food quality Protein cross-linking can influence food texture and appearance, or permit the incorporation of useful nutrients such as essential amino acids or essential oils in foods to accrue crucial benefits Protein cross-linking in foods may be achieved by enzymatic means with transglutaminase or with cross-linking agents like glutaraldehyde This chapter discusses the different cross-linking effects and how they may be exploited by food processor INTRODUCTION Protein cross-links play an important role in determining the functional properties of food proteins Manipulation of the number and nature of protein cross-links during food processing offers a means by which the food industry can manipulate the functional properties of food, often without damaging the nutritional quality This chapter updates the chapter in the previous edition of this book and discusses advances in our understanding of protein cross-linking over the last two decades, as well as examining current and future applications of this chemistry in food processing and its implications for health and food safety It draws on, and updates, two reviews in this area (Gerrard 2002, Miller and Gerrard, 2005) in addition to earlier reviews on this subject (Matheis and Whitaker 1987, Feeney and Whitaker 1988, Singh 1991) The elusive relationship between the structure and the function of proteins presents a particular challenge for the food technologist Food proteins are often denatured during processing, so there is a need to understand the protein both as a biological entity with a predetermined function and as a randomly coiled biopolymer To understand and manipulate food proteins requires a knowledge of both protein biochemistry and polymer science If the protein undergoes chemical reaction during processing, both the ‘natural’ function of the molecule and the properties of the denatured polymeric state may be influenced One type of chemical reaction that has major consequences for protein function in either their native or denatured states is protein cross-linking It is, therefore, no surprise that protein crosslinking can have profound effects on the functional properties of food proteins This chapter sets out to define the different types of protein cross-links that can occur in food, before and after processing, and the consequences of these cross-links for the functional and nutritional properties of the foodstuff Methods that have been employed to introduce cross-links into food deliberately are then reviewed, and future prospects for the use of this chemistry for the manipulation of food during processing are surveyed, including a consideration of health and food safety aspects Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 207 P1: SFK/UKS BLBS102-c10 P2: SFK BLBS102-Simpson March 21, 2012 208 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology PROTEIN CROSS-LINKS IN FOOD Protein cross-linking refers to the formation of covalent bonds between polypeptide chains within a protein (intramolecular cross-links) or between proteins (intermolecular cross-links) (Feeney and Whitaker 1988) In biology, cross-links are vital for maintaining the correct conformation of certain proteins and may control the degree of flexibility of the polypeptide chains As biological tissues age, further protein cross-links may form, which often have deleterious consequences throughout the body and play an important role in the many conditions of ageing (Zarina et al 2000, Ahmed 2005, Nass et al 2007, GuI et al 2009) Chemistry similar to that which occurs during ageing may take place if biological tissues are removed from their natural environment – for example, when harvested as food for processing Food processing often involves high temperatures, extremes in pH, particularly alkaline, and exposure to oxidising conditions and uncontrolled enzyme chemistry Such conditions can result in the introduction of protein cross-links, producing substantial changes in the structure of proteins, and therefore the functional (Singh 1991) and nutritional (Friedman 1999a, 1999b, 1999c) properties of the final product A summary of protein crosslinking in foods is given in Figure 10.1, in which the information is organised according to the amino acids that react to form the cross-link Not all amino acids participate in protein crosslinking, no matter how extreme the processing regime Those that react so with differing degrees of reactivity under various conditions Disulfide Cross-links Disulfide bonds are the most common and well-characterised types of covalent cross-link in proteins in biology They are formed by the oxidative coupling of two cysteine residues that are close in space within a protein A suitable oxidant accepts the hydrogen atoms from the thiol groups of the cysteine residues, producing disulfide cross-links The ability of proteins to form intermolecular disulfide bonds during heat treatment is considered to be vital for the gelling of some food proteins, including milk proteins, surimi, soybeans, eggs, meat and some vegetable proteins (Zayas 1997) Gels are formed through the cross-linking of protein molecules, generating a three-dimensional solid-like network, which provides food with desirable texture (Dickinson 1997) Disulfide bonds are thought to confer an element of thermal stability to proteins and are invoked, for example, to explain the stability of hen egg white lysozyme, which has four intramolecular disulfide cross-links in its native conformation (Masaki et al 2001) This heat stability influences many of the properties of egg white observed during cooking Similarly, the heat treatment of milk promotes the controlled interaction of denatured β-lactoglobulin with κ-casein through the formation of a disulfide bond This increases the heat stability of milk and milk products, preventing precipitation of β-lactoglobulin (Singh 1991) Disulfide bonds are also important in the formation of dough Disulfide interchange reactions during the mixing of flour and water result in the production of a protein network with the viscoelastic properties required for bread making (Lindsay and Skerritt 1999) The textural changes that occur in meat during cooking have also been attributed to the formation of intermolecular disulfide bonds (Singh 1991) Cross-links Derived from Dehydroprotein Alkali treatment is used in food processing for a number of reasons, such as the removal of toxic constituents and the solubilisation of proteins for the preparation of texturised products However, alkali treatment can also cause reactions that are undesirable in foods, and its safety has come into question (Savoie et al 1991, Shih 1992, Friedman 1999a, 1999c) Exposure to alkaline conditions, particularly when coupled to thermal processing, induces racemisation of amino acid residues and the formation of covalent cross-links, such as dehydroalanine, lysinoalanine and lanthionine (Friedman 1999a, 1999b, 1999c) Dehydroalanine is formed from the base-catalysed elimination of persulfide from an existing disulfide cross-link The formation of lysinoalanine and lanthionone cross-links occurs through β-elimination of cysteine and phosphoserine protein residues, thereby yielding dehydroprotein residues Dehydroprotein is very reactive with various nucleophilic groups, including the ε-amino group of lysine residues and the sulfhydryl group of cysteine In severely heat- or alkali-treated proteins, imidazole, indole and guanidino groups of other amino acid residues may also react (Singh 1991) The resulting intra- and intermolecular cross-links are stable, and food proteins that have been extensively treated with alkali are not readily digested, reducing their nutritional value Mutagenic products may also be formed (Friedman 1999a, 1999c) Cross-links Derived from Tyrosine Various cross-links formed between two or three tyrosine residues have been found in native proteins and glycoproteins, for example in plant cell walls (Singh 1991) Dityrosine crosslinks have recently been identified in wheat and are proposed to play a role in the formation of the cross-linked protein network in gluten (Tilley et al 2001) They have also been formed indirectly by treating proteins with hydrogen peroxide or peroxidase (Singh 1991) and are implicated in the formation of caseinate films by gamma irradiation (Mezgheni et al 1998) Polyphenol oxidase can also lead indirectly to protein cross-linking, due to reaction of cysteine, tyrosine, or lysine with reactive benzoquinone intermediates generated from the oxidation of phenolic substrates (Matheis and Whitaker 1987, Feeney and Whitaker 1988) Such plant phenolics have been used to prepare crosslinked gelatin gels to develop novel food ingredients (Strauss and Gibson 2004) Cross-links Derived from the Maillard Reaction The Maillard reaction is a complex cascade of chemical reactions, initiated by the deceptively simple condensation of an amine with a carbonyl group, often within a reducing sugar or fat P1: SFK/UKS BLBS102-c10 P2: SFK BLBS102-Simpson March 21, 2012 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come 209 10 Protein Cross-linking in Food – Structure, Applications, Implications for Health and Food Safety Maillard cross-links see figure 10.2 Glutamyl-lysine cross-links (an isopeptide bond) O N H Severe heat/ transglutaminase Lysine NH3 Uncharacterised maillard cross-links? NH2 H2N Isopeptide bond O Arginine NH2 N H Glutamine O N H Severe heat O Histidine NH Proline +Lysine O Aspartate (+glutamate) HN Food protein, either native or denatured Disulfides [O] SH Cysteine S S N Alkali OH Serine (and derivatives) Tryptophan Dehydroalanine O H N Alkali NH Dehydroprotein Hydrocarbon Tyrosine +other amino acids OH +Lysine Largely unreactive, except perhaps radical reactions? or Histidinoalanine, ornithinoalanine, arginine derivative +Cysteine [O] Lanthionine S Lysinoalanine H N Dityrosyl cross-links, e.g Quinone structures, e.g HO O OH +Lysine, cysteine proline, etc Poorly defined cross-link structures O Figure 10.1 A summary of the cross-linking reactions that can occur during food processing, from Gerrard 2002 Further details are given in the text breakdown product (Fayle and Gerrard 2002, Friedman 1996a) During the course of the Maillard reaction, reactive intermediates, such as α-dicarbonyl compounds and deoxysones, are generated and lead to the production of a wide range of compounds, including polymerised brown pigments called melanoidins, furan derivatives, nitrogenous, and heterocyclic compounds (e.g pyrazines; Fayle and Gerrard 2002) Protein cross-links form a subset of the many reaction products, and the cross-linking of food proteins by the Maillard reaction during food processing is well established (Gerrard 2002, Miller and Gerrard, in press, 2006) The precise chemical structures of these cross-links in food, however, are less well understood Thus, surprisingly little is known about the extent of Maillard cross-linking in processed foods, the impact of this process on food quality, and how the reaction might be controlled to maximise food quality In biology and medicine, where the Maillard reaction is important during the ageing process, several cross-link structures have ... and food safety It draws on, and updates, two reviews in this area (Gerrard 2002, Miller and Gerrard, 2005) in addition to earlier reviews on this subject (Matheis and Whitaker 1 987 , Feeney and. .. understand the protein both as a biological entity with a predetermined function and as a randomly coiled biopolymer To understand and manipulate food proteins requires a knowledge of both protein biochemistry. .. this chemistry for the manipulation of food during processing are surveyed, including a consideration of health and food safety aspects Food Biochemistry and Food Processing, Second Edition Edited