Protein–protein cross-linking Methods, consequences, applications

Protein–protein crosslinking in food:methods,consequences,applications

Juliet A.Gerrard

Department of Plant &Microbial Sciences,University

of Canterbury,Christchurch,New Zealand (Tel.:+64-3366-7001;fax:+64-3364-2083;e-mail:j.gerrard@https://www.360docs.net/doc/ab1825685.html,)

Protein–protein crosslinks play an important role in deter-mining the functional properties of food proteins.Manip-ulation of the number and nature of such protein crosslinks during food processing o?ers a means by which the food industry can manipulate the functional properties of food,often without damaging the nutritional quality.This review discusses advances in our understanding of protein cross-linking over the last decade,and examines current and future applications of this chemistry in food processing.#2003Elsevier Science Ltd.All rights reserved.

Background

Determining the relationship between the structure of any protein and its function is a challenge that bioche-mists struggle to meet in many contexts.For the food technologist,correlating the structure of a food protein with its function,or functionality,within a food is still more di?cult.Food proteins are often denatured during processing,so the food technologist must understand the protein both as a biological entity with a pre-determined function,and as a randomly coiled biopo-lymer.To understand and manipulate food proteins thus 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 poly-meric state may be in?uenced.One type of chemical reaction that has major consequences for protein func-tion in either their native or denatured states is protein crosslinking.It is,therefore,no surprize that protein crosslinking can have profound e?ects on the functional properties of food proteins.

Excellent reviews surveying protein crosslinking in food were published several years ago (Feeny &Whi-taker,1988;Matheis &Whitaker,1987;Singh,1991).This review draws on these earlier works,but focuses on literature published in the last decade.It begins by de?ning the di?erent types of protein crosslinks that can occur in food,before and after processing,and the consequences of these crosslinks for the functional and nutritional properties of the foodstu?.Methods that have been employed to introduce crosslinks into food deliberately are then reviewed,and future prospects for the use of this chemistry for the manipulation of food during processing are surveyed.

The types of crosslinks found in food

Protein crosslinking refers to the formation of covalent bonds between polypeptide chains within a protein (intramolecular crosslinks)or between proteins (inter-molecular crosslinks)(Feeney &Whitaker,1988).In biology,crosslinks are vital for maintaining the correct conformation of certain proteins,and may control the degree of ?exibility of the polypeptide chains.As biolo-gical tissues age,further protein crosslinks may form that often have deleterious consequences throughout the body,and play an important role in the many conditions of ageing (Zarina et al .,2000).Similar chemistry 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 oxidizing conditions and uncontrolled enzyme chem-istry.Such conditions can result in the introduction of protein crosslinks,producing substantial changes in the structure of proteins,and therefore the functional (Singh,1991)and nutritional (Friedman,1999a,1999b,1999c)properties of the ?nal product.A summary of known protein crosslinking in foods is given in Fig.1,in which the information is organised according to the

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amino acids that react to form the crosslink.Not all amino acids participate in protein crosslinking,no mat-ter how extreme the processing regime.Those that react,do so with di?ering degrees of reactivity under various conditions.

Disul?de crosslinks

Disul?de bonds are the most common and well-char-acterized types of covalent crosslink in proteins in biol-ogy.They are formed by the oxidative coupling of two cysteine residues that are adjacent within a food protein matrix.A suitable oxidant accepts the hydrogen atoms from the thiol groups of the cysteine residues,producing disul?de crosslinks.The ability of proteins to form intermolecular disul?de bonds during heat treatment is considered to be vital for the gelling of some food pro-teins,including milk proteins,surimi,soybeans,eggs,meat and some vegetable proteins (Zayas,1997).Gels are formed through the crosslinking of protein molecules,generating a three-dimensional solid-like network,which provides food with desirable texture (Dickinson,1997).

Disul?de 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 lyso-zyme,which has four intramolecular disul?de crosslinks in its native conformation (Masaki et al .,2001).This heat stability in?uences many of the properties of egg white observed during cooking.Similarly,the heat treatment of milk promotes the controlled interaction of denatured -lactoglobulin with k -casein,through the formation of a disul?de bond.This increases the heat stability of milk and milk products,preventing precipitation of -lactoglobulin (Singh,1991).Dis-ul?de bonds are also important in the formation of dough.Disul?de interchange reactions during the mixing of wheat ?our and water result in the pro-duction of a protein network with the viscoelastic properties required for breadmaking (Lindsay &Skerritt,1999).The textural changes that occur in meat during cooking have also been attributed to the formation of intermolecular disul?de bonds (Singh,

1991).

Fig.1.A summary of the crosslinking reactions that can occur during food processing (Feeney &Whitaker,1988;Friedman,1999a;Singh,

1991).Further details are given in the text.

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Crosslinks derived from dehydroprotein

Alkali treatment is used in food processing for a number of reasons,such as the removal of toxic con-stituents and the solubilization of proteins for the pre-paration of texturised products.However,alkali treatment can also cause reactions that are undesirable in foods,and its safety has come into question(Fried-man,1999a,1999c;Savoie et al.,1991;Shih,1992). Exposure to alkaline conditions,particularly when cou-pled to thermal processing,induces racemization of amino acid residues and the formation of covalent crosslinks,such as dehydroalanine,lysinoalanine and lanthionine(Friedman,1999a,1999c).Dehydroalanine is formed from the base-catalyzed elimination of per-sul?de from an existing disul?de crosslink.The forma-tion of lysinoalanine and lanthionine crosslinks occurs through -elimination of cysteine and phosphoserine protein residues,thereby yielding dehydroprotein resi-dues.Dehydroprotein is very reactive with various nucleophilic groups including the"-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 resi-dues may also react(Singh,1991).The resulting intra-and intermolecular crosslinks are stable,and food pro-teins that have been extensively treated with alkali are not readily digested,reducing their nutritional value. Mutagenic products may also be formed(Friedman, 1999a,1999c).

Crosslinks derived from tyrosine

Various crosslinks formed between two or three tyr-osine residues have been found in native proteins and glycoproteins,for example in plant cell walls(Singh, 1991).Dityrosine crosslinks have recently been identi-?ed in wheat,and are proposed to play a role in for-mation of the crosslinked protein network in gluten (Tilley et al.,2001).They have also been formed indir-ectly,by treating proteins with hydrogen peroxide or peroxidase(Singh,1991),and are implicated in the for-mation of caseinate?lms by gamma irradiation(Mez-gheni et al.,1998).Polyphenol oxidase can also lead indirectly to protein crosslinking,due to reaction of cysteine,tyrosine,or lysine with reactive benzoquinone intermediates generated from the oxidation of phenolic substrates(Feeney&Whitaker,1988;Matheis&Whi-taker,1987).

Crosslinks derived from the Maillard reaction

The Maillard reaction is a complex cascade of chemi-cal reactions,initiated by the deceptively simple con-densation of an amine with a carbonyl group,often within a reducing sugar or fat breakdown product (Fayle&Gerrard,2002).During the course of the Maillard reaction,reactive intermediates,such as -dicarbonyl compounds and deoxysones,are generated and lead to the production of a wide range of com-pounds,including polymerized brown pigments called melanoidins,furan derivatives,nitrogenous and hetero-cyclic compounds(e.g.pyrazines)(Fayle&Gerrard, 2002).Protein crosslinks form a subset of the many reaction products,and the crosslinking of food proteins by the Maillard reaction during food processing is well established(Ames,1992;Easa et al.,1996a,1996b; Fayle et al.,2000,2001;Gerrard et al.,1998a,1999, 2002a,2002b,2002c;Hill&Easa,1998;Hill et al.,1993; Mohammed et al.,2000).The precise chemical struc-tures of these crosslinks in food,however,are less well understood.

In biology and medicine,where the Maillard reaction is important during the ageing process,several crosslink structures have been identi?ed,including those shown in Fig.2(Brinkmann et al.,1995;Lederer&Buhler,1999; Monnier et al.,1999;Nagaraj&Sady,1996).One of the ?rst protein-derived Maillard reaction products isolated and characterized was the crosslink pentosidine(Dyer et al.,1991;Sell&Monnier,1989),a?uorescent moiety that is believed to form through the condensation of a lysine residue with an arginine residue and a reducing sugar.The exact mechanism of formation of pentosi-dine remains the subject of considerable debate(Biemel et al.,2001a;Chellan&Nagaraj,2001).Compared to the extensive literature on the Maillard chemistry in vivo,relatively little has been reported on the existence of pentosidine in food.However,building on the results in the medical arena,Henle et al.(1997)have

developed

methods to detect the compound,and reported low levels in roasted co?ee and bakery products.They con-cluded that pentosidine does not have a major role in the polymerisation of food proteins.Iqbal et al.(1997, 1999a,1999b)have investigated the role of pentosidine in meat tenderness in broiler hens and there is a report of increased pentosidine under conditions of high pres-sure,which could be relevant to some areas of food processing(Schwarzenbolz et al.,2000).Other Maillard crosslinks have recently been detected in food(Biemel et al.,2001b),but our understanding of crosslinking of food proteins by the Maillard reaction,and how this crosslinking relates to the functional properties of foods,remains in its infancy(Gerrard et al.,2002b). Although not strictly classi?ed under the heading of Maillard chemistry,animal tissues such as collagen and elastin contain complex heterocyclic crosslinks,formed from the apparently spontaneous reaction of lysine and derivatives with allysine,an aldehyde formed from the oxidative deamination of lysine catalyzed by the enzyme lysine oxidase(Feeney&Whitaker,1988).The extent to which these crosslinks occur in food has not been well-studied,although their presence in gelatin has been dis-cussed,along with the presence of pentosidine in these systems(Cole&Roberts,1996,1997).

Crosslinks formed via transglutaminase catalysis

An enzyme that has received extensive recent atten-tion for its ability to crosslink proteins is transglutami-nase.Transglutaminase catalyses the acyl-transfer reaction between the -carboxyamide group of peptide-bound glutamine residues and various primary amines. As represented in Fig.1,the"-amino groups of lysine residues in proteins can act as the primary amine, yielding inter-and intramolecular"-N-( -glutamyl)ly-sine crosslinks(Motoki&Seguro,1998).The formation of this crosslink does not reduce the nutritional quality of the food as the lysine residue remains available for digestion(Seguro et al.,1996).

Transglutaminase is widely distributed in most animal tissues and body?uids,and is involved in biological processes such as blood clotting and wound healing."-N-( -Glutamyl)lysine crosslinks can also be produced by severe heating(Motoki&Seguro,1998),but are most widely found where a food is processed from material that contains naturally high levels of the enzyme.The classic example here is the gelation of?sh muscle in the formation of surumi products,a natural part of traditional food processing of?sh by the Japa-nese suwari process,although the precise role of endo-genous transglutaminase in this process is still under debate(An et al.,1996;Motoki&Seguro,1998)."-N-( -Glutamyl)lysine bonds have been found in various raw foods including meat,?sh and shell?sh.Transglu-taminase-crosslinked proteins have thus long been ingested by man(Seguro et al.,1996).The increasing applications of arti?cially adding this enzyme to a wide range of processed foods are discussed in detail below. Other isopeptide bonds

In foods of low carbohydrate content,where Maillard chemistry is inaccessible,severe heat treatment can result in the formation of isopeptide crosslinks during food processing,via condensation of the"-amino group of lysine,with the amide group of an asparagine or glutamine residue(Singh,1991).This chemistry has not been widely studied in the context of food. Manipulating protein crosslinking during processing—methods and consequences

So far,we have seen that reactions during processing, storage,and cooking of food products may involve changes in existing cross linkages,as well as the forma-tion of new ones of types not found in untreated mate-rial,and discussed the various crosslinks that have been characterized in processed foods.In this section,the application of this knowledge in the deliberate mod-i?cation of food proteins during processing is con-sidered.

A major task of modern food technology is to gen-erate new food structures with characteristics that please the consumer,using only a limited range of ingredients.Proteins are one of the main classes of molecule available to confer textural attributes,and the crosslinking and aggregation of protein molecules has been cited as one of the most important mechanisms for engineering food structures with desirable mechanical properties(Dickinson,1997).The crosslinking of food proteins can in?uence many properties of food,includ-ing texture,viscosity,solubility,emulsi?cation and gel-ling properties(Kuraishi et al.,2000;Motoki& Kumazawa,2000).Many traditional food textures are derived from a protein gel,including those of yoghourt, cheese,sausage,tofu and surimi.Crosslinking provides an opportunity to create gel structures from protein solutions,dispersions,colloidal systems,protein-coated emulsion droplets or protein-coated gas bubbles and create new types of food,or improve the properties of traditional ones(Dickinson,1997).In addition,judi-cious choice of starting proteins for crosslinking can produce food proteins of higher nutritional quality through crosslinking of di?erent proteins containing complementary amino acids(Zhu et al.,1995). Chemical methods

An increasing understanding of the chemistry of pro-tein crosslinking opens up opportunities to control these processes during food processing.Many commercial crosslinking agents are available,for example from Pierce(2001).These are usually double-headed reagents derived from molecules developed to derivatize the side chains of proteins(Feeney&Whitaker,1988;Singh,

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1991),and generally exploit the lysine and/or cysteine residues of proteins in a speci?c manner.Doubt has recently been cast as to the accuracy with which reac-tivity of these reagents can be predicted(Green et al., 2001),but they remain widely used for biochemical and biotechnological applications.

Unfortunately,these reagents are expensive and not often approved for food use,so their use has not been widely explored(Singh,1991).They do,however,prove useful for‘proof of principle’studies to measure the possible e?ects of introducing speci?c new crosslinks into food.If an improvement in functional properties is seen after treatment with a commercial crosslinking agent,then further research e?ort is merited to?nd a food approved,cost e?ective means by which to intro-duce such crosslinks on a commercial scale.Such‘proof of principle’studies include the use of glutaraldehyde to demonstrate the potential e?ects of controlled Maillard crosslinking on the texture of wheat-based foods(Ger-rard et al.,2002b,2002c).The crosslinking of hen egg white lysozyme with a double-headed reagent has also been used to show that the protein is rendered more stable to heat and enzyme digestion,with the foaming and emulsifying capacity reduced(Feeney&Whitaker, 1988;Singh,1991).Similarly,milk proteins crosslinked with formaldehyde showed greater heat stability(Singh, 1991).

For many years,the baking industry has been chemi-cally modifying?our to improve the consistency,tex-ture,and strength of many baked goods.Since most ?our improvers are oxidising agents,their improving mechanism is thought to occur by the introduction of additional disul?de bonds to the dough network(Lind-say&Skerritt,1999).

Enzymatic methods

The use of enzymes to modify the functional proper-ties of foods is an area which has attracted considerable interest,since consumers perceive enzymes to be more ‘natural’than chemicals.Enzymes are also favoured as they require milder conditions,have high speci?city,are only required in catalytic quantities,and are less likely to produce toxic products(Singh,1991).Thus enzymes are becoming commonplace in many industries for improving the functional properties of food proteins (Chobert et al.,1996;Poutanen,1997).

Due to the prominence of disul?de crosslinkages in food systems,enzymes that regulate disul?de inter-change reactions are of interest to food researchers.One such enzyme is protein disul?de isomerase(PDI).PDI catalyses thiol/disul?de exchange,rearranging‘incor-rect’disul?de crosslinks in a number of proteins of bio-logical interest(Hillson et al.,1984;Matheis& Whitaker,1987).The reaction involves the rearrange-ment of low molecular sulfhydryl compounds(e.g.glu-tathione,cysteine and dithiothreitol)and protein sulfhydryls.It is thought to proceed by the transient breakage of the protein disul?de bonds by the enzyme, and the reaction of the exposed active cysteine sulfhy-dryl groups with other appropriate residues to reform native linkages(Singh,1991).

PDI has been found in most vertebrate tissues and in peas,cabbage,yeast,wheat and meat(Singh,1991).It has been shown to catalyse the formation of disul?de bonds in gluten proteins synthesized in vitro.Early reports suggested that a high level of activity corre-sponded to a low bread making quality(Grynberg et al., 1977).The use of oxidoreduction enzymes,such as PDI, to improve product quality is an area of interest to the baking industry(van Oort,2000;Watanabe et al.,1998) and the food industry in general(Hjort,2000).The potential of enzymes such as protein disul?de isomerase to catalyse their interchange has been extensively reviewed by Shewry and Tatham(1997).

Sulfhydryl(or thiol)oxidase catalyses the oxidative formation of disul?de bonds from sulfhydryl groups and oxygen and occurs in milk(Matheis&Whitaker, 1987;Singh,1991).Immobilised sulfhydryl oxidase has been used to eliminate the‘cooked’?avour of ultra high temperature treated milk(Swaisgood,1980).It is not a well studied enzyme,although the enzyme from chicken egg white has received recent attention in biology (Hoober et al.,1999).Protein disul?de reductase cata-lyzes a further sulfhydryl–disul?de interchange reaction and has been found in liver,pea and yeast(Singh,1991). Peroxidase,lipoxygenase and catechol oxidase occur in various plant foods and are implicated in the dete-rioration of foods during processing and storage.They have been shown to crosslink several food proteins, including bovine serum albumin,casein, -lactoglobulin and soy,although the uncontrolled nature of these reactions casts doubt on their potential for food improvement(Singh1991).Lipoxygenase,in soy?our, is used in the baking industry to improve dough prop-erties and baking performance.It acts on unsaturated fatty acids,yielding peroxy free radicals and starting a chain reaction.The crosslinking action of lipoxygenase has been attributed to both the free radical oxidation of free thiol groups to form disul?de bonds and to the generation of reactive crosslinking molecules such as malondialdehyde(Matheis&Whitaker,1987).

All enzymes discussed so far have been over-sha-dowed in recent years by the explosion in research on the enzyme transglutaminase.Due largely to its ability to induce the gelation of protein solutions,transgluta-minase has been investigated for uses in a diverse range of foods and food-related products.The use of this enzyme has been the subject of a series of recent reviews,covering both the scienti?c and patent litera-ture(Kuraishi et al.,2001;Motoki&Seguro,1998; Nielsen,1995;Zhu et al.,1995).These are brie?y high-lighted below.

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Transglutaminase

The potential of transglutaminase in food processing was hailed for many years before a practical source of the enzyme became widely available.Early work was carried out on transglutaminase derived from animal sources,particularly guinea pig liver,but this was obviously impractical on a commercial scale!Thus the production of a microbially derived enzyme by Ajino-moto Inc.proved pivotal in paving the way for indus-trial applications(Zhu et al.,1995).In addition,the transglutaminases that were discovered in the early years were calcium ion dependent,which imposed a barrier for their use in foods that did not contain a suf-?cient level of calcium.The commercial preparation is not calcium dependent,and thus?nds much wider applicability(Zhu et al.,1995).The production of microbial transglutaminase,derived from Streptoverti-cillium mobaraense,is described by Zhu et al.(1995)and methods with which to purify and assay the enzyme are reviewed by Wilhelm et al.(1996).The commercial enzyme operates e?ectively over the pH range4–9,from 0to50 C(Motoki&Seguro,1998).

There is a seemingly endless list of foods in which the use of transglutaminase has been successfully used:sea-food,surimi,meat,dairy,baked goods,sausages(as a potential replacement for phosphates and other salts), gelatin(Kuraishi et al.,2001),noodles and pasta(Larre et al.,1998,2000).It is?nding increasing use in restructured products,such as those derived from scal-lops and pork(Kuraishi et al.,1997).In all cases, transglutaminase is reported to improve?rmness,elas-ticity,water-holding capacity,and heat stability(Kur-aishi et al.,2001).It also has potential to alleviate the allergenicity of some proteins(Watanabe et al.,1994). Dickinson reviewed the application of transglutaminase to crosslink di?erent kinds of colloidal structures in food and enhance their solid-like character in gelled and emulsi?ed systems,controlling rheology and stability (Dickinson,1997).These applications are particularly appealing in view of the fact that crosslinking by trans-glutaminase is thought to protect nutritionally valuable lysine residues in food from various deteriorative reac-tions(Seguro et al.,1996).Furthermore,the use of transglutaminase potentially allows production of food proteins of higher nutritional quality,through cross-linking of di?erent proteins containing complementary amino acids(Zhu et al.,1995).

The use of transglutaminase in the dairy industry has been explored extensively.The enzyme has been trialed in many cheeses,from Gouda to Quark,and the use of transglutaminase in icecream is reported to yield a pro-duct that is less icy,and more easily scooped(Kuraishi et al.,2001).Milk proteins form emulsion gels which are stabilized by crosslinking,opening new opportunities for protein-based spreads,desserts and dressings(Dick-inson&Yamamoto,1996).

Soy products have also bene?tted from the introduc-tion of transglutaminase,with the enzyme providing manufacturers with a greater degree of texture control. The enzyme is reported to enhance the quality of tofu made from old crops,giving a product with increased water holding capacity,a good consistency,a silky and ?rmer texture and one that is more robust in the face of temperature change(Kuraishi et al.,2001).

New foods are being created using transglutaminase, for example,imitation shark?n for the South East Asian market has been generated by crosslinking gelatin and collagen(Zhu et al.,1995).Crosslinked proteins have also been tested as fat substitutes in products such as salami and yoghourt(Nielsen,1995)and the use of transglutaminase-crosslinked protein?lms as edible ?lms has been patented(Nielsen,1995).

Not surprisingly,the rate of crosslinking by transglu-taminase depends on the particular structure of the protein acting as substrate.Most e?cient crosslinking occurs in proteins that contain a glutamine residue in a ?exible region of the protein,or within a reverse turn (Dickinson,1997).Casein is a very good substrate,but globular proteins such as ovalbumin and -lactoglobu-lin are poor substrates(Dickinson,1997).Denaturation of proteins increases their reactivity,as does chemical modi?cation by disruption of disul?de bonds,or by adsorption at an oil–water interface(Dickinson,1997). Many of the reported substrates of transglutaminase have actually been acetylated and/or denatured with reagents such as dithiothreitol under regimes that are not food approved(Nielsen,1995).More work needs to be done to?nd ways to modify certain proteins in a food-allowed manner in order to render them amenable to crosslinking by transglutaminase in a commercial setting.

Whilst the applications of transglutaminase have been extensively reported in the scienti?c and patent litera-ture,the precise mode of action of the enzyme in any one food processing situation remains relatively unex-plored.The speci?city of the enzyme suggests that in mixtures of food proteins,certain proteins will react more e?ciently than others,and there is value in understanding precisely which protein modi?cations exert the most desirable e?ects.Additionally,transglu-taminase has more than one activity:as well as cross-linking,the enzyme may catalyze the incorporation of free amines into proteins by attachment to a glutamine residue.Furthermore,in the absence of free amine, water becomes the acyl acceptor and the -carboxamide groups are deamidated to glutamic acid residues(Ando et al.,1989).The extent of these side reactions in foods, and the consequences of any deamidation to the func-tionality of food proteins,has yet to be fully elucidated. Perhaps the most advanced understanding of the spe-ci?c molecular e?ects of transglutaminase in a food product is seen in yoghourt,where the treated product

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has been analysed by gel electrophoresis and speci?c functional e?ects correlated to the loss of -casein,with the -casein remaining.The speci?city of the reaction was found to alter according to the exact transglutami-nase source(Kuraishi et al.,2001).The speci?c e?ects of transglutaminase in baked goods(Gerrard et al.,1998b, 2000)have also been analyzed at a molecular level.In particular,the enzyme produced a dramatic increase in the volume of croissants and pu?pastries,with desir-able?akiness and crumb texture(Gerrard et al.,1998b). These e?ects were later correlated with crosslinking of the albumins and globulins and high molecular weight glutenin fractions by transglutaminase(Gerrard et al., 2000).Subsequent research suggested that the dominant e?ect was attributable to crosslinking of the high mole-cular weight glutenins(Gerrard et al.,2002c).

Future applications of protein crosslinking

As the extensive recent use of transglutaminase dra-matically illustrates,protein crosslinking has huge potential for the improvement of traditional products and the creation of new ones.Transglutaminase itself will no doubt?nd yet more application as its precise mode of action becomes better understood,especially if variants of the enzyme are found with a broader sub-strate speci?city.Transglutaminase also provides a con-venient tool with which to begin to monitor how the extent of protein crosslinking in?uences food properties. To date,little research has been undertaken in this arena,although data(unpublished)from the author’s laboratory suggests that the number of crosslinks required to induce substantial changes to baked pro-ducts is relatively small.

Whether other enzymes,which crosslink by di?erent mechanisms,can?nd applications that equals the broad range seen for transglutaminase remains open to debate. The thiol exchange enzymes,such as protein disul?de isomerase,may o?er advantages to food processors if their mechanisms can be unravelled,and then controlled within a foodstu?.Other enzymes,such as lysyl oxidase, have not yet been used,but have potential to improve foods(Dickinson,1997)especially in the light of recent work characterizing this class of enzymes(Bu?oni& Ignesti,2000),which may allow their currently unpre-dictable e?ects to be better understood.

Although protein crosslinking is often considered to be detrimental to the quality of food,it is increasingly clear that it can also be used as a tool to improve food properties.The more we understand of the chemistry and biochemistry that take place during processing,the better placed we are to exploit it—minimizing deleter-ious reactions and maximizing bene?cial ones. Furthermore,the stage is now set for a new generation of crosslinked food ingredients,for example hetero-logous biopolymers,combining the foaming properties of one protein with the foam-stabilizing characteristics of another(Dickinson,1997).Beyond this,genetic engineering ultimately allows us to create the food pro-teins of our choice.For example,if the chemistry of crosslinking is su?ciently well understood,proteins with amino acids in precisely the correct position for speci?c crosslinking could be engineered(Seguro et al.,1996). The bioethics debate surrounding the acceptability of GM-food will have to be fully resolved before such new angles can be explored and exploited.In the meantime, crosslinking enzymes seem set to occupy centre stage for food processors wishing to modify their products using protein crosslinking technology. Acknowledgements

I should like to thank all the postgraduates that have worked with me on the protein crosslinking of food,in particular Dr Sia n Fayle,Paula Brown and Indira Rasiah.I thank Peter Steel and Antonia Miller for proof reading this manuscript.

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