13 Applications of Oxidoreductases in Foods

Colja Laane
DSM Food Specialties, Delft, The Netherlands

Yvonne Bruggeman
Unilever Research, Vlaardingen, The Netherlands

Chris Winkel
Quest International, Naarden, The Netherlands

I.

INTRODUCTION

Oxidative and reductive processes play an important role in foods. They inuence not only the taste but also the texture, appearance, shelf life, nutritional value, and process tolerance of food products. Both enzymatic and nonenzymatic redox processes are involved. In some cases redox processes lead to undesirable effects such as off-avor, reduced shelf life, or textural problems. In other cases, they contribute in a positive way to the nal aroma, an improved texture, a more desirable appearance, or an increased shelf life. Controlling the redox behavior in food systems during all stages of processing and storage is therefore of utmost importance. Up until now redox reactions in foods were controlled mainly by carefully selecting raw materials, by adapting process conditions, by adding chemicals or antioxidants, or by designing air-tight packaging materials. As yet, little attention has been paid to tailor redox reactions in foods by the addition of oxidoreductases or by changing the prole or content of oxidoreductases in food raw materials by genetic tools. Presumably, the major bottleneck for the application of oxidoreductases to foods is that economically efcient enzyme production is, with a few exceptions, still

not feasible. In addition, public concerns about the use of recombinant enzymes in food products is slowing down their market introduction. In this chapter the current and potential usage of oxidoreductases in controlling the taste, texture, appearance (i.e., color), shelf life, and the nutritional value of food products will be discussed. Increasingly, redox enzymes are being used for biosensor applications in food systems. This topic will not be discussed in this chapter, however. Table 1 lists the most important redox enzymes and their functions in food systems. Most attention will be paid to the market segments bakery, beverages, and dairy and to oxidases, since relatively little is known about role and applications of reductases in food systems. Furthermore, the emphasis will be on added enzymes and not on enzymes already present in the food product constituents. For detailed information on the properties of the individual oxidoreductases the reader is referred to in Sec. IIA of this book. II. TASTE

Many oxidoreductases play a role by inuencing the taste prole of food products. They are involved in the in vivo/in situ biogenesis of desirable aroma compo-

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Table 1 Enzymes

Overview of the Main (Potential) Applications of Oxidoreductases in Food Products Taste In situ and in vitro (off)-avor production In situ and in vitro avor production (ILOX) Removal of cooked avor in UHT-milk In vitro avor production and debittering Debittering of coffee, cacao, and olives GOX, mild acid production Texture Dough extensibility and strength Appearance Flour bleaching Shelf life Nutritional value Destruction vitamin A

Lipoxygenases (LOX)

Alcohol oxidases and dehydrogenases (AO, ADH) Sulfhydryl oxidases (SOX) Peroxidases (POX, LPO) (Poly)phenol oxidases (PPO) Carbohydrate oxidases (GOX, HOX, PYROX) Ascorbic acid oxidase (AAO) Xanthine oxidases (XO) Superoxide dismutases (SOD) Catalases Cholesterol oxidoreductases

AO plus catalase, O2 removal Dough strengthening in combination with POX Crosslinking biopolymers

Assisting PPO in color formation (Dis)coloration; in vitro production of colors

LPO, antimicrobial agent

Laccase, O2 removal GOX plus catalase, O2 removal

Dough strengthening via H2 O2 ; thickening agent Browning

AAO plus catalase, O2 removal Antimicrobial agent Removal reactive O species with catalase Removal excess H2 O2

Destruction vitamin C

Cholesterol reduction

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nents, in the in vitro production of avoring topnotes, and in the endogenous formation of off-avors. The main redox enzymes are discussed below. A. Lipoxygenases

Lipoxygenase (LOX, EC 1.13.11.12; see Chapter 43, for detailed information), formerly known as lipoxidase or carotene oxidase, is an iron-containing dioxygenase which catalyzes the oxidation of polyunsaturated fatty acids containing cis, cis-1, 4-pentadiene groups (linoleic, linolenic, and arachidonic acids) to the corresponding conjugated cis, trans dienoic monohydroperoxides. In addition LOX also accepts a relatively broad range of phenolic compounds as substrates (1) and is capable of oxidizing other substrates than the actual substrate. This process is known as co-oxidation and includes compounds like carotenoids and polyphenols (2). LOX is one of those endogenous enzymes that play an important role in both avor generation and offavor formation in virtually all food products derived from plant raw materials. Depending on the nal concentration and the type of food product the same avor component can be a desired aroma component or an off-avor at higher concentrations. For example, C6-aldehydes and alcohols derived by the LOX-catalyzed oxidation of (poly)unsaturated fatty acids have, in most cases, a positive effect on the aroma prole (e.g., wines and juices), but in other beverages, have an undesired avor effect (e.g., beer). Likewise, endogenous LOX is known to generate carbonyl compounds in dough systems and hence inuences bread avor (3). The formation of C6 compounds requires the sequential action of four enzymes of which two are redox enzymes, namely an acylhydrolase, a lipoxygenase, a hydroperoxide lyase, and a yeast-derived alcohol dehydrogenase (4). Typically, off-avor formation is prevented by using crop variants decient in LOX isoenzymes, either by screening or by genetic tools (5) or by controlling the oxygen levels during processing (6), as well as by removing the malodorous oxidation products afterwards. The deodorization of off-avors can be achieved by physical techniques such as adsorption, or enzymatically by converting the undesired aldehydes (e.g., with aldehyde dehydrogenase/oxidase), or alcohols (e.g., with alcohol oxidase) into their corresponding less avorful carboxylic acid. The use of redox enzymes for this purpose has been claimed for several products such as margarine, cream, sh oil, noodles, cooked rice, and soybean products (7, 8).

LOX is also used for the in vitro production of several natural topnote avors, which are mainly added to beverages and dairy products to tailor the avor prole (9, 10). Typical examples include the conversion of polyunsaturated fatty acids into various short to medium-chain aldehydes/alcohols (13), or into S(-)--decalactone (butter avor; 12). Wellknown fatty acidderived avoring aldehydes/alcohols include the above mentioned C5 and C6 compounds, as well as (E2, E6)-nonadienal (cucumber), 1-octen-3one (eld mushroom), (Z5)-octadien-3-one (geranium leaves), (E3, E5)-undecatriene (blasamic), and (E3, Z5, Z8)-undecatetraene (seaweed). Depending on the degree of unsaturation and the regioselectivity of the lipoxygenase, different hydroperoxy compounds are formed, from which the above-mentioned compounds can be derived by subsequent enzymatic reactions. For (Z3)-hexenol, linolenic acid is used as a substrate, while for most of the other aldehydes/alcohols higher unsaturated fatty acids are required. The production of (Z3)-hexenol has recently been commercialized using plant homogenates (e.g., alfalfa sprouts, green peppers) which are relatively rich in hydroperoxide lyase, the enzyme required to split the hydroperoxide fatty acid into smaller fragments. The generated (Z3)-hexenal was converted into (Z3)-hexenol using the reductive enzymatic power of bakers yeast (13). A very elegant approach has been taken recently by Givaudan-Roure. To unify all three enzymes involved in the formation of (Z3)-hexenol, they have cloned and overexpressed both soybean LOX and the hydroperoxide lyase from banana in bakers yeast. In this way they have developed a single-step process which produces (Z3)-hexenol in relatively high yield (14). For the production of lactones a different strategy has to be followed after the formation of the linoleic acid hydroperoxide. It involves the fermentative -oxidation of the hydroperoxide intermediate by the yeast Pichia etchellsii, and the subsequent cyclization of 5hydroxydecanoic acid to the corresponding S(-)--decalactone (10). As shown by Quest International (1), lipoxygenases also accept a relatively broad spectrum of phenolic compounds as substrates. Of interest to the avor industry are the LOX-catalyzed conversions of isoeugenol and coniferyl benzoate from Siam resin into vanillin. At present the commercialization of these biotransformations is hampered by the fact that isoeugenol is not readily available and that coniferyl benzoate is difcult to handle in a reactor. Other well-known reactions of LOX include the cooxidation reaction of carotenoids to yield, among

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others, -ionone (2, 15). More recently is the generation of a toasted or cookie-like avor in starchy materials by a combination of LOX and an -amylase (16).

C.

Sulfhydryl Oxidases

B.

Alcohol Oxidases and Dehydrogenases

In general, aldehydes are more potent avor compounds than their alcoholic counterparts. Hence, alcohol oxidases are interesting enzymes for the in vitro production of avoring preparations which, among others, can be applied in beverages. Typical examples include the use of methanol oxidase from Pichia, Hansenula, and Candida (17) for the production of natural acetaldehyde from ethanol. This enzyme is induced during growth on methanol. At the end of the logarithmic growth phase cells are harvested and incubated with ethanol. In this way concentrations of $ 1:5% natural acetaldehyde can be achieved, which can be concentrated further to the desired application level. From yeast to yeast the substrate specicity of the alcohol oxidase is different. Hence, this procedure can also be used to convert other alcohols, such as hexenol and other long-chain alcohols, to their corresponding aldehyde (10, 18). As alternatives to alcohol oxidases the corresponding dehydrogenases could in principle be used (19). A severe drawback, however, is that these dehydrogenases require the expensive cofactor NAD(P) instead of (cheap) oxygen as an electron acceptor. Although various sophisticated NAD(P) cofactor regenerating systems have been designed and substantial cost reductions have been realized in this way, it is evident that in commercial applications oxidases are preferred over their dehydrogenase counterparts. The use of dehydrogenases for food purposes seems to be restricted to whole-cell conversions. Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum is a special type of alcohol oxidase. Recently, it has been shown that this stable, avincontaining enzyme has a very broad substrate specicity and readily converts para-substituted phenols into interesting avor precursors or avoring compounds (2022). Apart from natural vanillin and coniferyl alcohol, different vinylphenols (e.g., para-vinylguaiacol) and allylphenols can be produced from cheap raw materials and oxygen as an electron acceptor. VAO can also be used for generation of avor building blocks. To that end a natural mix of phenolic compounds could be treated with VAO to enrich foods or avor preparations with a range of vinylic/allylic and aldehydic substances.

Sulfhydryl oxidase (SOX, no EC number assigned; see Chapter 41 for more details) catalyzes the formation of disulde bonds from (protein) thiols. SOX is an Fe/Cucontaining glycoprotein and has been detected in bovine, human, goat, pig, rabbit, and rat milks (23). The enzyme is capable of oxidizing the sulfhydryl groups of cysteine and glutathione, as well as milk proteins to their corresponding disuldes using molecular oxygen as electron acceptor (24). In practical applications bovine milk SOX may be added to UHT milk to reduce cooked avors. Patents (see Refs. in 24) for this application have been issued. The enzyme has been immobilized on porous glass, and its effectiveness in ameliorating the cooked avor has been demonstrated on a pilot scale using immobilized enzyme columns. D. Peroxidases

Peroxidases (POX, EC 1.11.1.7; see Chapters 28 and 29 for more details) occur widely in nature and is the general name for a group of both highly specic and nonspecic enzymes which use hydrogen peroxide instead of oxygen as an electron acceptor. Peroxidases, especially the heme-containing ones, can catalyze a large number of different reactions including sulfoxidation, N-demethylation, oxidation, and hydroxylation and hence are of potential interest in the production of specic avoring topnotes. A recent example involves the demethylation of methyl Nmethylanthranilate (ex Citrus) to monomethylanthranilate (25). The latter compound is an important topnote avor in Concord grapes. Soybean, horseradish, and microperoxidases were found to be convenient catalysts for this reaction. In addition POX gives rise to a fresh avor prole when added to tomato paste (26). E. Polyphenol Oxidases

Polyphenol oxidases (PPO) are a group of several enzymes. Different activities can be found within this group: tyrosinase (monophenol monooxygenase, EC 1.14.18.1; see Chapter 39) converting a phenol into a catechol group; 1,2-diphenol oxidase or catechol oxidase (EC 1.10.3.1) converting catechol into an o-quinone, and laccase (EC 1.10.3.2; see Chapter 40) converting a 1,4-diphenol into p-quinone. Usually the rst two activities are linked, as catechol is much more readily oxidized than a phenol. Most enzymes that catalyze 1,4-diphenol oxidation also act on 1,2-diphenols.

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The bitter taste in many food products is often the result of the presence of polyphenolic compounds such as guaiacols. PPOs are capable of oxidizing these compounds and hence can be applied to reduce bitterness. At present the debittering of coffee beans (27), Adzuki beans (28), and cacao beans (29) by PPOs have been claimed. Another application involves the use of laccase to debittering olives by treating stoned, chopped olives in the presence of air at increased temperatures (30).

III.

TEXTURE

The use of oxidoreductases in texturizing food is based on their ability to crosslink proteins and/or polysaccharides. This property is of particular interest to improve the texture of dough and paste products (softness, volume, elasticity, crunchiness) or dairy products (mouth feel, appearance), and of sh and meat pastes. Basically, enzymatic crosslinking can occur via two routes: 1. Indirect via enzymatically produced hydrogen peroxide (e.g., glucose oxidase, hexose oxidase, ascorbic acid oxidase), or via the enzymatic production of radicals (e.g., peroxidase, lipoxygenase). 2. Direct via the oxidation of functional groups in the protein. Examples include the linkage of tyrosine and ferulic acid residues by tyrosinase and laccases, linkages of lysine residues by lysyl oxidase, or the formation of disulde bridges by sulfhydryl oxidase. An alternative, nonredox procedure for direct crosslinking is being explored using transglutaminases (see Chapter 51). These enzymatic routes are being explored to replace chemical oxidizers, such as dehydroascorbic acid and potassium bromate. Replacement of the nonspecic chemical compounds by redox enzymes could have the benet of more specic and better-controlled oxidation processes. Other disadvantages of the chemical substances are related to safety and labeling issues. One of the major challenges in the baking industry is to nd a good replacer for potassium bromate, which is a difcult task since bromate is still more effective and cheaper than the enzymatic alternative. The most important texturizing redox enzymes will be discussed below. A. Lipoxygenases

been isolated from seeds of commercial cultivars and have been well characterized (31, 32). Recent studies indicate that L2 has the greatest effect among LOX isoenzymes on dough extensibility and strength (33) and is also mostly responsible for the production of undesirable aroma compounds in bread doughs (34; see Sec. II, Chapter 43). For L3 an increase in foaming activity has been reported, as well as an overall improvement in breadmaking quality of wheat our (35). The bakery yeast Saccharomyces cerevisiae also contains LOX. Recently, this enzyme was partially puried (36), but its potential, if any, on breadmaking remains to be established. Nowadays, it is feasible to change the prole and content of LOX (iso)enzymes in plants either by classical means (5), or potentially by genetic modication. For example, by appropriate crosses, near-isogenic soybean seeds have been developed that lack either isoenzymes L1 and L3, or isoenzymes L2 and L3. These LOX-minus mutants still grow well in the eld (37). In principle, transgenic plants lacking or overexpressing one or more LOX isoenzymes could be constructed and tailored to specic applications (38). To that end the heterologous expression of one or more soybean LOX isoenzymes in wheat could be of interest. B. Sulfhydryl Oxidases

The action of SOX (see Sec. II, Chapter 41, for general information) may be the same as those of chemical oxidizing agents, provided the formation of disulde bonds is the primary mode by which these agents function. In extensive testing (39, 40) it was found that SOX alone has no inuence on loaf volume, dough strength, or mixing tolerance. Also, relatively high concentrations using recombinant SOX from Aspergillus awamori did not have any signicant positive effect (40). One reason could be that SOX has only a limited afnity for thiol groups in gluten proteins and as a result its application in the food industry seems to be limited to the removal of small off-avor molecules (see Sec. II, Chapter 41). C. Peroxidases

A rich source of LOX is soybeans. Soybean LOX contains three distinct lipoxygenase isoenzymes, designated as L1, L2, and L3. These isoenzymes have

For detailed information about POX see Section II, Chapters 28 and 29. Wheat our contains a peroxidase that can crosslink phenolic constituents such as ferulic acid and vanillic acid. However, the pH optimum of the wheat enzyme is 4.5, and in the pH range of 56 of wheat doughs the activity is considerably lower than at pH 4.5 (41). Although the dough-improving effect of

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peroxidase is well documented, the reactions involved in the dough are only poorly understood. Model studies with peroxidase, glutathione, and cysteine indicate that sulfhydryl and/or lysyl groups may be involved (42). Peroxidase-catalyzed reactions result in a decreased amount of lysine recovered from proteins after acid hydrolysis. Peroxidases, or the quinones formed by the enzyme, oxidatively deaminate lysyl residues to form lysylaldehydes, resulting in the formation of protein polymers, as was revealed by gel ltration. These aggregates could not be dissociated by detergents, which indicates that covalent bonds were formed. In addition, the dough-strengthening effect of peroxidases is also ascribed to the crosslinking of carbohydrates and the coupling of carbohydrates to proteins. The gelation of wheat pentosans is due to the oxidative dimerization of ferulic acid moieties, which are covalently linked to pentosans (43). Recently, a study has been described showing the effect of different peroxidases on the baking performance of German Kaiser rolls (41). Deliberately sticky doughs were prepared in order to demonstrate the effects of added peroxidases. Especially the addition of soybean POX led to strengthening of wheat doughs as determined by the stability of the dough after shaking the dough for 1 min in a laboratory shaker. This improving effect was observed at both short and long fermentation times. Dough volume increased after application of these peroxidases. Compared with (GOX), the results of peroxidases are better or at least equal in terms of stability and volume. In bread dough, peroxidases seem to act without the extra addition of hydrogen peroxide in spite of their peroxide dependence. This may indicate that peroxide is present in dough at sufcient amounts, or that it is generated as a result of the peroxidase reaction. In this way, substrate radicals formed by peroxidase can react with oxygen to form hydrogen peroxide. When this reaction is occurring, catalytic amounts of peroxide present in dough are sufcient to get the cycle started, and may explain why no hydrogen peroxide has to be added together with a peroxidase. An alternative explanation is that wheat contains endogenous oxidases, which are active enough to produce some hydrogen peroxide in situ. Indeed, the addition of hydrogen peroxideproducing enzymes such as GOX has a benecial effect on baking (4446). In other systems than doughs, POX has been claimed as a thickening and stabilizing agent in, for example, ice creams, deserts, sauces, and jams and jellies (47). There is also a recent patent application (48) on the POX-catalyzed gelling of hemicelluloses to form

gels or viscous media for application as fat or gelatin replacer, as well as for avor delivery, coating, or glazing. D. Glucose Oxidases

Glucose oxidase (GOX, EC 1.1.3.4; see Chapter 30 for detailed information) catalyzes the conversion of glucose into the mild-tasting gluconic acid via glucono-lactone and hydrogen peroxide. GOX complies with the FAO/WHO and GRAS requirements for food grade enzymes and is one of the few commercially available oxidases from Aspergillus niger and Penicillium strains at relatively low costs. Especially, the generation of hydrogen peroxide is believed to give the antiweakening effect in bread doughs (40). In a recent study by Hilhorst et al. (49) the effect of GOX on Dutch rusk dough was dough stiffening with a clear loss in extensibility. This made the overall effect quite undesirable. For comparison, they also tested peroxidase (see Sec. III.C above). This enzyme gave a dough-stiffening effect without loss in extensibility. The authors explain the difference by the fact that hydrogen peroxide from the GOX-catalyzed reaction oxidizes randomly and links the gluten network with the arabinoxylan network, whereas the peroxidase only increases the amount of crosslinks in the arabinoxylan fraction without affecting the gluten network or the coupling between the networks. It has been found that synergistic effects occur when using a combination of oxidative enzymes like the combination of GOX and SOX (50). Furthermore, the dough has an increased stability. E. Hexose Oxidases

A newcomer in the eld of redox enzymes for bakery products is hexose oxidase (HOX, EC 1.1.3.5) from red seaweeds. This glycosylated avoprotein is related to GOX but has a broader substrate specicity toward hexose sugars, including oligomers. Like GOX, it acts on the C1-position of the sugar moiety (51). Recently, the enzyme from Chondrus crispus has been isolated, cloned, and overexpressed in several recombinant organisms such as Pichia pastoris, Saccharomyces cerevisiae, and E. coli (51, 52). However, the production levels of active enzyme are still poor at this moment and have to be improved. For this reason, and because C. crispus has a long standing tradition as an edible organism, efforts have been made to develop a large-scale production method with the use

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of carrageenase, which reduces the viscosity of the crude extract (51). For HOX similar applications can be envisaged as for GOX (see above). In particular, its application in bakery products is foreseen (53), since HOX is able to generate more H2 O2 owing to its broader substrate specicity, and hence should be more effective than GOX in dough strengthening. Likewise, combinations of HOX and H2 O2 -consuming enzymes, such as POX, may be envisaged. Another enzyme with similar properties as HOX, isolated from Acremonium strictum T1, has been reported in the literature (54). The enzyme is called an oligosaccharide oxidase and is able to oxidize several di- and oligosaccharides at the anomeric site, yielding the corresponding di- and oligobionic acids. A detailed comparison between HOX and oligosaccharide oxidase performance in dough applications has not been made yet. F. Pyranose Oxidases

A.

Lipoxygenases

It is interesting to note that the use of soybean lipoxygenase was described in the 1930s as a means to bleach the our in preparation of white bread. More recent experiments have shown that carotenoids present in wheat our are destroyed by co-oxidation. Wheat our itself contains little LOX activity, but LOX is abundantly present in, for example, soybeans. To that end wheat our is often fortied with up to 0.5% enzyme-active soy our (34, 60). Other applications of LOX include the bleaching of noodles, whey products, rice, and wheat bran (6164). B. (Poly)phenol Oxidases and Peroxidases

Pyranose oxidase (PYROX, EC 1.1.3.10; see Refs. 5557, 59 for more detailed information) catalyzes the oxidation of several monosaccharides at the C2position and is therefore different from GOX, which oxidizes glucose at the C1-position. While GOX is very specic, PYROX is able to catalyze other substrates such as maltose and pentoses (e.g., xylose). As a result, the PYROX oxidation product of glucose is 2-ketoglucose and not gluconic acid. At present, this enzyme has been puried from several white rot fungi and a Bacidiomycetous fungus (5557). PYROX has also been reported to show signicant activity toward Dglucono-1,5-lactone, which is produced by the GOXcatalyzed oxidation of glucose (58). So if PYROX is combined with GOX, there will be more substrate available for PYROX, thereby prolonging the activity of PYROX and enhancing the total amount of hydrogen peroxide produced (59). The claimed effects in baking are gluten strengthening, reduced dough stickiness, and increased volume and crumb structure for bread (59).

IV.

APPEARANCE/COLOR

Apart from texture, the appearance of food products is determined to a large extent by their color. Several redox enzymes are known to inuence the color of foodstuffs. The most important ones will be discussed below.

PPOs (see Sec. II, Chapter 39 for general information) play an important role in the browning of fresh fruits and vegetables (65, 66), in the coloring and avoring of tea (67, 68), and in improving the quality of coffee (69). A major concern in the food industry is to prevent the development of enzymatic browning prior to the processing of fruits and vegetables (70). This is accomplished by removing oxygen or by inhibiting PPO activity using inhibitors such as metal chelating agents, inorganic ions (e.g., halide anions), benzoic acid and some substituted cinnamic acids, reducing agents (e.g., L-cysteine, glutathione, sulte, SO2 , ascorbic acid), small natural peptides, and combinations thereof (68). In contrast, during the fermentation of black tea PPO is used to initiate browning by oxidation of polyphenolic substances such as catechins to theaavins. The quality of tea, based on sensory evaluation of color and bitterness, has been correlated with total theaavin content (71). Theaavins, thearubigins, and caffeine are all essential ingredients in high-quality teas. The addition of microbial laccases and/or peroxidases to green tea has shown that no higher theaavin levels can be obtained than with endogenous PPO. Addition of exogenous laccase/peroxidase to black tea, however, did yield a very signicant increase in the color intensity of the tea (72). Polyphenol oxidases also play an important role in coffee processing (69). The activity of PPO in green coffee beans has been consistently related to the quality of the coffee beverage. The exact role of PPO in cocoa beans is less well understood. There are, however, indications that it plays a role in browning during the curing of the cocoa beans (73). In cereals, PPO is responsible for the darkening of the breadcrumb, particularly in whole-grain and rye

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breads. The rate of browning is greatest in sourdough baking. Excessive discoloration can be prevented by the addition of sodium metabisulte or ascorbic acid (41). Exogenous PPO is also reported to have a positive inuence on the rheological properties of bread (74). PPO can also be used for the in vitro production of colors. For example, Pruidze (75) reported the production of a whole range of natural colors by treating waste streams of beet and tea with PPO. Furthermore, it was claimed that safower pigments become darker red when safower petals were sprayed with a dilute laccase solution (76). The role of endogenous POX in (dis)coloration is not fully understood. It seems that the oxidative role of peroxidases is useful in assisting PPOs in oxidizing the polyphenols and ultimately contributing toward the color and avor of tea. The same seems to be true for coffee and cocoa. C. Ascorbic Acid Oxidases

Ascorbic acid oxidase (E.C.1.10.3.3; see Ref. 77 for detailed information) plays a role in beverages such as lemon and grapefruit juices, where it is responsible for the initiation of browning and loss of vitamin C activity during storage (77). The extent of browning can be minimized by steam blanching or by the exclusion of oxygen. The rate of ascorbic acid oxidation increases markedly in the presence of metallic ions, especially copper and iron. Hence, food processed in tin cans and processing equipment should be copper free. While the loss of ascorbic acid cannot be prevented completely, it can be reduced to a minimal level during processing.

V.

SHELF LIFE

enzyme in bovine milk, where it is found in concentrations around 30 mg/L. It is a glycoprotein with a single, covalently bound heme group (78). Lactoperoxidase requires hydrogen peroxide and thiocyanate (SCN ) for antibacterial activity. All three components are referred to as the LP system. The growth inhibitory effect of the LP system is mediated by the generation of SCN oxidation products, mainly hypothiocyanate ions (OSCN ), which attack sulfhydryl groups of vital metabolic enzymes of the microorganisms. Mammalian cells are not affected by the LP system. Only 1020 ppm of lactoperoxidase is required for an effective system. The cofactor requirements are also very low: 1025 ppm for thiocyanate and 1015 ppm for H2 O2 . Without the enzyme H2 O2 is also bactericidal, but at much higher concentrations: 300900 ppm (79). Therefore LPO is often applied in combination with H2 O2 -generating enzymes. From a toxicological point of view, the levels of the cofactors in the LP system as well as the oxidation products are reported to be harmless. The envisaged applications of the LP system are food products (e.g., liquid milk, cheese, meat, sh and poultry products, and functional foods), feed and veterinarian products (e.g., milk replacers, and antidiarrhea and antimastitis preparations), and dental products (7880). Typically, applications have been claimed for Lactobacillus fermented milk products (81), pickled foods (82), sh products (LPO in combination with GOX; 83), and white mold cheeses such as Camembert (84). Also of interest is the effect of LPO on yogurt. By adding LPO to yogurt the excessive acid production of lactic acid bacteria in the yogurt is suppressed (85). These applications are becoming within reach now that it is possible to isolate lactoperoxidase from milk with high purity on an industrial scale (79). Currently, LPO is already commercially available at relatively low costs. B. Xanthine Oxidases

The application of redox enzymes for improving the shelf life of food products includes mainly enzymes which are capable of removing oxygen or reactive oxygen species (e.g., H2 O2 and superoxide anion), as well as enzymes that are able to generate antimicrobial agents. In this way the stability of foods can be increased signicantly with respect to taste, appearance, and microbial spoilage. A. Lactoperoxidases

Lactoperoxidase (LPO; see Ref. 78 and Chapters 19 and 20 for detailed information) is the most prominent

Xanthine oxidase (XO, EC 1.2.3.2; see Chapters 19 and 42 for detailed information) is widely distributed in animals, plants, and microorganisms. It catalyzes the oxidation of hypoxanthine to xanthine and xanthine into uric acid. In addition XO is able to oxidize a wide range of purines, aldehydes, and pteridines with concomitant reduction of O2 to H2 O2 . Under certain conditions XO also produces the highly reactive superoxide anion. Bovine milk is very rich in XO ($ 35 mg=L; 86). XO has been implicated in the oxidative deterioration of milk and dairy products via the

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production of superoxide anion during oxidation of its substrates (87). However, Bruder et al. (88) found no evidence to support a role for native bovine milk XO in lipid oxidation. The results of Weihrauch (89) seem to indicate that in the presence of purines, XO activity generates H2 O2 for the lactoperoxidase system in milk, making it a bactericidal or bacteriostatic agent in milk (23).

VI.

NUTRITIONAL VALUE

C.

Superoxide Dismutases and Catalases

Superoxide dismutase (SOD; EC 1.15.1.1) and catalase (EC 1.11.1.6; see Chapters 27 and 38 for detailed information) are present in milk and are able to remove reactive oxygen species generated by other (bio)chemical processes (90). SOD catalyzes the reduction of superoxide anion, as produced, for example, by XO, to H2 O2 and O2 . In turn, catalase is able to neutralize H2 O2 to water and oxygen. A low level of exogenous SOD, coupled with catalase, is a very effective antioxidant in dairy products (91). Recently, SOD has been shown to protect beer against free radical damage (92). Obviously, the commercial feasibility of SOD as an antioxidant depends on cost, particularly compared to chemical antioxidants, if permitted. As far as is known, SOD is not used commercially as an antioxidant in food systems. Catalase is used for the cold-sterilization of milk in regions lacking refrigeration and could in principle be applied in developed countries for the treatment of cheese milk (90). Good sources for catalase are beef, liver, Aspergillus niger, and sweet potato. There is also interest in using immobilized catalase reactors for milk pasteurization or for glucose oxidasecatalase reactions (93). Besides the removal of reactive oxygen species by SOD and catalase, other enzymes can be applied to remove the less but still reactive oxygen. Typical examples include glucose oxidase, D-amino acid oxidase, alcohol oxidase, and ascorbic acid oxidase. The disadvantage of these oxidases is that they produce H2 O2 , which by itself is a powerful oxidant. Catalase can be added to remove H2 O2 , but then oxygen is produced again. More recently, polyphenol oxidases such as laccase have been proposed as a deoxygenation tool for beer (94) and juices (95, 96). These enzymes have the advantage that they do not produce H2 O2 , and thus the combination with catalase is not necessary. As a result PPOs allow a more efcient oxygen removal.

Redox enzymes can have both pro- as well as antinutritional effects. For example, lipoxygenase, apart from all its other functions in food products (see Table 1), is involved in the oxidative destruction of liposoluble vitamins (provitamin A) and essential fatty acids (3). Likewise, ascorbic acid oxidase has an antinutritional effect since it oxidizes vitamin C (97). Increasingly, redox enzymes are being claimed to improve the healthiness of especially beverages. For example, peroxidase and catalase have been claimed for the removal of unhealthy hydrogen peroxide in coffee and tea (98). Other examples include the use of cholesterol oxidase, epicholesterol dehydrogenase and cholesterol reductase to lower the cholesterol level in foods such as meat, sh, milk, and egg products (99101).

VII.

CONCLUDING REMARKS

Compared to the usage of hydrolytic enzymes such as proteases, carbohydrates, and lipases, the application of oxidoreductases as a tool to improve the processability and quality of food products is still in its infancy. Like hydrolytic enzymes, oxidoreductases are capable of tailoring the taste, texture, appearance, shelf life, nutritional value, and process tolerance of foods and the properties of all major food constituents (e.g., proteins, carbohydrates, oils, fats, avors; see Table 1). In both cases, they can exert a positive as well as a negative effect on the food quality parameters, which can be reduced or eliminated by careful selection of the raw materials, by properly controlling the process condition, by the addition of counteracting ingredients, and by genetic tools. Likewise, hydrolytic and redoxactive enzymes often have more than one effect. A typical redox example is the multifunctional enzyme lipoxygenase, which can be applied to inuence either taste, texture, appearance, and/or nutritional value of food products. The key difference resides in the types of reactions they catalyze. As a result, redox enzymes can be combined with hydrolytic enzymes in a variety of food products to create improved or even new functionalities, which cannot be realized by either one of them. At present, major bottlenecks for the large-scale application of oxidoreductases include their limited availability, their safety status (not GRAS), their stability, and the fact that they often initiate radical reac-

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tions, which propagate via redox-active food constituents such as metal ions and quinones and hence are difcult to control. In addition, many oxidoreductases require expensive cofactors for catalysis. Despite these disadvantages over hydrolytic enzymes, certain oxidoreductases are increasingly nding a home in food processing. The rst generation (see Table 1) includes enzymes which are cofactor independent and thrive on oxygen (e.g., oxidases) and hydrogen peroxide (e.g., peroxidases). Owing to advances in recombinant DNA technology the low-cost, large-scale production of these enzymes in GRAS host microorganisms is becoming within reach, and the tools are ready to tailor redox enzymes to specic needs by site-directed mutagenesis and directed evolution. Also feasible will be the in planta overexpression of desired redox enzymes in food raw materials and the deletion of undesirable redox traits. The usage of cofactor-dependent oxidative enzymes seems tentatively to be conned to whole-cell systems, in which the cofactor can be regenerated, and hence to the in vitro as well as the in situ production of functional food ingredients such as avors. The same seems to be true for reductases, since most if not all require reducing equivalents in the form of a small protein, hydrogen, and/or NAD(P)H. Clearly, the usage of oxidoreductases in food processing is emerging. The benets and limitations are becoming known, which allows a promising and focused search for new opportunities.

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