Enzymatic Browning

Appearance, flavour, texture and nutritional value are four attributes considered by consumers when making food choices. Appearance which is significantly impacted by colour is one of the first attributes used by consumers in evaluating food quality. Colour may be influenced by naturally occurring pigments such as chlorophylls, carotenoids and anthocyanins in food, or by pigments resulting from both enzymatic and non-enzymatic reactions. Enzymatic browning is one of the most important colour reactions that affects fruits, vegetables and seafoods. It is catalysed by the enzyme polyphenol oxidase (1,2 benzenediol; oxygen oxidoreductase, EC1.10.3.1) which is also referred to as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase and tyrosinase.

Enzymatic browning is one of the most studied reactions in fruits, vegetables and seafoods. Researchers in the fields of food science, horticulture, plant and postharvest physiology, microbiology, and even insect and crustacean physiology have studied this reaction because of the diversity of its impact in these systems.

When asked to discuss browning in foods, those involved from production to processing, usually reflect on its detrimental influence. Some enzymatic browning reactions are however very beneficial to the overall acceptability of foods. Tea, coffee, and cocoa are important commodities for many developing countries. India, China, Sri Lanka, and Kenya, are major producers of tea (Ensminger et al. 1995). Black, oolong, and green tea are reliant on enzymatic browning for colour and flavour development. Coffee is primarily produced in Brazil and Columbia. Ethiopia, Indonesia, Vietnam, and a number of other developing countries also produce coffee for world markets. Approximately 4.2 million metric tons of coffee beans are produced on an annual basis by the top ten major coffee producing countries. Some discussion occurs as to the contribution of enzymatic browning to colour development during coffee processing.

Cocoa is generally produced by countries located just north and south of the equator with Brazil and West Africa supplying much of the world market. Colour development in cocoa is facilitated by polyphenol oxidase activity during fermentation and drying. Polyphenol oxidases are also responsible for development of the characteristic golden brown colour in dried fruits such as raisins, prunes, dates and figs. Blanching is generally required for inactivation of the enzyme after colour development, in order to minimize discolouration.

Polyphenol oxidases are believed to play key physiological roles both in preventing insects and microorganisms from attacking plants and as part of the wound response of plants and plant products to insects, microorganisms and bruising. As fruits and vegetables ripen, their susceptibility to disease and infestation is increased due to a decline in their phenolic content. Phenoloxidase enzymes endogenous to fruits and vegetables, catalyse the production of quinones from their phenolic constituents. Once formed, these quinones undergo polymerization reactions, leading to the production of melanins, which exhibit both antibacterial and antifungal activity and assist in keeping the fruit and/or vegetable physiologically wholesome.

Research describing the antibacterial, anticancer and antioxidant nature of melanins has triggered considerable interest in enzymatic browning. Nutritional recommendations for increasing the per capita consumption of fruits and vegetables, as well as new technologies of convenience such as the production of precut or minimally processed fruits and vegetables, will provide economic benefit to many countries which market exotic products.

Increases in fruit and vegetable markets projected for the future will not occur if enzymatic browning is not understood and controlled. Enzymatic browning is one of the most devastating reactions for many exotic fruits and vegetables, in particular tropical and subtropical varieties. It is estimated that over 50 percent losses in fruit occur as a result of enzymatic browning (Whitaker and Lee, 1995). Such losses have prompted considerable interest in understanding and controlling phenoloxidase enzymes in foods. Lettuce, other green leafy vegetables, potatoes and other starchy staples, such as sweet potato, breadfruit, yam, mushrooms, apples, avocados, bananas, grapes, peaches, and a variety of other tropical and subtropical fruits and vegetables, are susceptible to browning and therefore cause economic losses for the agriculturist. These losses are greater if browning occurs closer to the consumer in the processing scheme, due to storage and handling costs prior to this point. The control of browning from harvest to consumer is therefore very critical for minimizing losses and maintaining economic value to the agriculturist and food processor. Browning can also adversely affect flavour and nutritional value. Figure 1 shows examples of enzymatic browning in fruits and vegetables.

Aquatic organisms rely on polyphenol oxidases to impart important physiological functions for their development. Polyphenol oxidases are important in hardening of the shell (sclerotization), after moulting in insects and in crustaceans such as shrimp and lobsters. Polyphenol oxidase is also responsible for wound healing. The mechanism of wound healing in aquatic organisms is similar to that which occurs in plants in that the compounds produced as a result of the polymerization of quinones exhibit both antibacterial and antifungal activities. Unfortunately, polyphenoloxidase-catalysed browning of the shell postharvest, adversely affects both the quality and consumer acceptability of these products.

Browning or melanosis in aquatic foods postharvest occurs primarily in crustaceans. These highly prized and economically valuable products are extremely vulnerable to enzymatic browning. Melanosis is usually more severe in lobsters if the head is retained during storage postharvest. If the head is removed, care should be taken to thoroughly wash the tail in order to eliminate proteases that activate latent polyphenol oxidases and promote browning. Although the products of melanosis are not harmful and do not influence flavour or aroma, consumers will not select these products since their brown discolouration connotes spoilage. Severe melanosis on these products can cause tremendous economic losses due to the high value commanded by these aquatic products in the marketplace. There are many examples of imported aquatic products entering the United States, worth millions of dollars, that are reduced markedly or lost completely owing to the severity of melanosis. Unfortunately, a majority of these products originate in developing countries, which lack both the scientific and technical resources, and the processing infrastructure required in order to prevent the occurrence of these devastating losses. Limited susceptibility of a number of crustacean species to melanosis on the other hand, presents the processor with the problem of deciding how to treat the product in order to prevent melanosis.

On the basis of the foregoing discussions, it is clear that browning has both beneficial and deteriorative effects. Control of the deteriorative effects of browning therefore poses a major challenge to the food scientist.

The control of browning in fruits and vegetables hinges upon an understanding of the mechanism(s) responsible for browning in fruits, vegetables and seafoods, the properties of polyphenol oxidase enzyme(s), their substrates and inhibitors, and the chemical, biological and physical factors which affect each of these parameters. Once understood these mechanisms may be applied in either preventing the browning reaction, or slowing its rate, thus extending the shelf life of the product.

In order for the food scientist to better understand how to prevent enzymatic browning, it is important to understand why polyphenol oxidase is present in plant and animal tissues. Despite knowing and hypothesizing some functions of polyphenol oxidase in these tissues, researchers are still trying to piece together the functional puzzle of this enzyme in both plant and animal systems.

Polyphenol oxidases were first discovered in mushrooms and are widely distributed in nature. They appear to reside in the plastids and chloroplasts of plants, although freely existing in the cytoplasm of senescing or ripening plants (Vaughn and Duke, 1984). Cloning and sequencing studies of the copper A binding region of these enzymes shows high conservation between polyphenol oxidases from plants, microorganisms and animals. Polyphenol oxidase is thought to play an important role in the resistance of plants to microbial and viral infections and to adverse climatic conditions. Phenolics, such as chlorogenic acid, caffeic acid and scopolin, etc., which are substrates of this enzyme have been shown to exhibit fungicidal properties.

Polyphenol oxidase catalyses the initial step in the polymerization of phenolics to produce quinones, which undergo further polymerization to yield dark, insoluble polymers referred to as melanins. These melanins form barriers and have antimicrobial properties which prevent the spread of infection or bruising in plant tissues. Plants, which exhibit comparably high resistance to climatic stress, have been shown to posses relatively higher polyphenol oxidase levels than susceptible varieties. Other enzyme systems in plants, such as chitinase, peroxidase, lipoxygenase, phenylalanine ammonia lyase, b-1,3-glucanase, etc., also show increased activity when subjected to stress.

It should be pointed out that the responses of enzymes to stress and infection are dependent on a number of factors, one of which is the host plant itself. Figure 2 for example, shows that two different species of tobacco plants exhibit weak peroxidase and polyphenol oxidase responses when infected with tobacco mosaic virus. A hybrid of the two species however possesses significantly higher levels of both enzymes. Additionally, the two parent species exhibit different isozymes after infection. The hybrid however possesses isozymes of both parent species, whether inoculated or uninoculated with tobacco mosaic virus. The hybrid is also highly resistant to viral and fungal attack when compared to both parent species. Recent evidence suggests that the enzyme may play a role in the growth and metabolism of chloroplasts and in plant growth regulation (Sherman et al. 1995).

Polyphenol oxidase exists in both insects and crustaceans as a zymogen or propolyphenol oxidase form, and is thought to confer disease resistance in animals, insects and crustaceans. The enzyme is also believed to be involved in both immunity and self-recognition. Microbial products such as laminarin (b-1,3-glucan) which initiates phagocytosis have been shown to activate propolyphenol oxidase to the active polyphenol oxidase form. Proteases are also believed to be involved in the activation of the propolyphenol oxidase form. Söderhäll and Unestam (1979) activated crayfish propolyphenol oxidase using b-1,3-glucans, fungal glycoproteins, laminarin pentaose and lipopolysaccharides. Some of these compounds appeared to activate a protease which triggered the activation of propolyphenol oxidase.

Researchers have shown in shrimp and lobster that a latent polyphenol oxidase form was activated either by trypsin or an endogenous enzyme (Savagaon and Sreenivasan, 1978; Ferrer et al. 1989a; Chen et al. 1991a). These researchers proposed that activation in these species was the result of proteolysis, which produced numerous isoenzymes. Other proteases (chymotrypsin and pepsin) did not appear to produce the same level of activation as trypsin, although chymotrypsin was shown to activate polyphenol oxidase in insects.

Figure 3 depicts propolyphenol oxidase activation by host defense mechanisms. Proteases, which activate polyphenol oxidases, are thought to be induced by microbial activity. Secondary metabolites, such as glucans, glycoproteins, laminarins, lipopolysaccharides, etc., produced by microorganisms may also induce the activation of propolyphenol oxidase by proteases. These metabolites are also capable of activating the propolyphenol oxidase even in the absence of proteolytic activity.

Although host-defense has been postulated for polyphenol oxidase activity in animal systems, its major role in crustaceans and insects is most probably related to hardening or sclerotization of the chitin shell during the growth cycle. The enzyme appears to be located both in the cuticle and hemolymph of insects in the propolyphenol oxidase form, which requires an activation process (Figure 3). Since sclerotization or hardening of the shell is associated with the moulting of insects and crustaceans, the activation of propolyphenol oxidase serves as an excellent mechanism for the control of the various stages of moulting. Although polyphenol oxidases have been more widely studied in insects, it is apparent that both crustaceans and insects undergo the sclerotization process by similar mechanisms.

During the sclerotisation process, diphenols are oxidized by polyphenol oxidase to their corresponding quinones. Quinones thus formed react with side groups on adjacent protein molecules thus linking them together for hardening. During the growth process, crustaceans must shed the exoskeleton, which confines them, and replace it with a larger one. Every tissue in the crustacean is affected in some manner by moulting. Each moult cycle is initiated by the shedding of the old exoskeleton (ecdysis). Moulting includes all morphological and physiological changes in preparation for and recovery from ecdysis (Aiken, 1980).

Four stages of moulting (postmoult, intermoult, premoult, moult) were described by Travis (1954). Figure 4 describes the different developmental stages of lobster cuticle formation. Polyphenol oxidase levels correlating to these stages are shown in Figure 5. At stage A, which is representative of a lobster that has already moulted or is at the postmoult stage of its development, the cuticle is absent and only the epidermis is present. The shell has already hardened and polyphenol oxidase activity is very low. At stage B, which would represent an intermoult stage, development of the new cuticle under the old shell begins to take place. At stages C (early premoult) and D (late premoult), the cuticle is more defined, with greater definition at stage D. During the latter stage (stage D), the old shell is ready to be discarded and hardening of the new cuticle is initiated. It is at this stage that the lobster is the most vulnerable to infection, since the new cuticle will not harden until after a few days.

The highest level of polyphenol oxidase activity correlates to the moulting stage at which the formation of new cuticle occurs (Stages C and D) (Figure 4). Propolyphenol oxidase is present throughout the moult cycle. Apparently similar propolyphenol oxidase and polyphenol oxidase activities during the late premoult stage, also seems to suggest that the activated enzyme is present during formation of the new cuticle and is required for the initiation of sclerotization. Increased polyphenol oxidase levels with a corresponding increase in quinones at that stage may very well serve to prevent the penetration of microorganisms through the unfinished cuticle thus functioning as part of a host-defense mechanism.

Figure 6 provides an example of a visual scale for the progression of melanosis, while Table 1 shows the scale used to describe the progression of melanosis (black spot) on pink shrimp (Penaeus dourarum).

Table 1. Colour scale used to describe the progression of melanosis (black spot) on pink shrimp. (Adapted from Otwell and Marshall, 1986)

Polyphenol oxidase catalyses two basic reactions: hydroxylation to the o-position adjacent to an existing hydroxyl group of the phenolic substrate (monophenol oxidase activity), and oxidation of diphenol to o-benzoquinones (dipehnol oxidase activity). Both reactions utilize molecular oxygen as a co-substrate. Whether a single enzyme system exhibits both mono- and di- phenol oxidase activities is still unclear. However, when both monophenol- and diphenol oxidases are present in plants, the ratio of monophenol to diphenol oxidase activity is usually 1:10 or as low as 1:40. (Nicolas et al. 1994).

Polyphenol oxidase isozymes were first isolated from mushroom. Subunits of the enzyme were observed to differ with respect to chemical, physical and kinetic properties. These subunit differences were believed to be responsible for relative affinities of the enzymes for both mono- and diphenolic substrates. Polyphenol oxidases isolated from mango possess two isozymes, both of which show specificity for o-diphenols. Gross Michel banana polyphenol oxidase was capable of oxidizing o-diphenols but not monophenols, while other varieties were shown to exhibit both mono- and diphenol activities (Palmer, 1963). In insects, polyphenol oxidase isolated from the cuticle exhibits only diphenol oxidase activity, while polyphenol oxidase from insect hemolymph exhibits monophenol oxidase activity. Polyphenol oxidases associated with shrimp cuticle exhibit both activities.

Monophenol oxidase catalyses the hydroxylation of monophenols to o-diphenols (Figure 7). The enzyme is referred to as tyrosinase in animals, since L-tyrosine is the major monophenolic substrate. In plants, the enzyme is sometimes referred to as cresolase owing to the ability of the enzyme to utilize the monophenolic substrate, cresol. Tyrosinase activity is also used to describe monophenol and diphenol oxidases in plant systems, although L-tyrosine is probably not a major substrate for the enzyme in plant systems, considering the rich abundance of phenolics in plant systems. This rich abundance of phenolics in plants is also the probable reason for referring to the enzyme as a polyphenol oxidase.

Monophenol oxidase activity is generally overlooked in plants since the hydroxylation reaction is dramatically slower than the oxidation reaction required for quinone production and initiation of the browning reaction. Monophenol oxidase (tyrosinase) has been given somewhat more attention in insect and crustacean systems, owing to its physiological significance in conjunction with diphenolase activity, in hardening of the cuticle for sclerotization.

The enzyme is also capable of metabolizing aromatic amines and o-aminophenols, both of which are structurally very similar to mono- and diphenols (Toussaint and Lerch, 1987) (Figure 8).

The oxidation of diphenolic substrates to quinones in the presence of oxygen is catalysed by diphenol oxidase activity (Figure 9). Diphenol oxidases have received much attention owing to their high catalytic rate and their association with the formation of quinones, which lead, to production of the brown pigment, melanin (Figure 10).

Whitaker (1972a) classified the diphenol oxidase catalysed reaction as having an ordered Bi Bi mechanism, owing to the involvement of three substrates: oxygen, diphenol and diphenol. Mechanisms of the diphenol oxidase reaction are still unknown since no free radical intermediates are formed and oxygen binds to the enzyme prior to binding of the o-diphenolic substrate. Figure 11 shows a simplified mechanism for the hydroxylation and oxidation of phenols by polyphenol oxidase. Both mechanisms involve the two copper moieties on the polyphenol oxidase.

Laccase (p-diphenol oxidase, E.C. 1.10.3.2)(DPO) is a type of copper-containing polyphenol oxidase. It has the unique ability of oxidizing p-diphenols, thus allowing it to be distinguished from o-diphenol oxidases such as catechol oxidase (Figure 12). Laccases are deep blue in colour in the pure state, and are remarkably non-specific as to their reducing substrates. They are glycoproteins with molecular weights ranging between 60 and 80 kDa, and a carbohydrate content varying between 15 and 41 percent. The copper content of purified laccases varies between two and four atoms per enzyme molecule or subunit (Thurston, 1994). Several phenolic substrates, including polyphenols, methoxy-substituted phenols, diamines and a considerable range of other compounds serve as substrates for laccase. Substrate oxidation by laccase results in the generation of one free radical (Reinhammar and Malmström, 1981) and is accompanied by the reduction of oxygen to water.

Laccases occur in many phytopathogenic fungi and in certain higher plants (Mayer and Harel, 1991). Laccases from higher plants, and fungal laccases are known to be cytoplasmic. With the exception of peaches (Harel et al. 1970) and apricots (Dijkstra and Walker, 1991), laccases do not occur in fruits and vegetables. Laccase (genus Rhus) was first discovered in lacquer trees by Yoshida (1883). It was later detected in fungi by Laborde (1896). A number of fungi including Lactarius, Polyporus, Aspergillus, Pleurotus, Polystictus, Psalliota, Glomerella, Podospora, Botrytis, Neurospora and Russula possess laccase activity (Franke, 1960). The carbohydrate content of laccase from Rhus varies between 32 and 45 percent, while that of laccase from Podospora has a carbohydrate content of 23 percent and that from Neurospora has a carbohydrate content of 11 percent.

Fungal laccase from Neurospora exists as both an intracellular and an extracellular enzyme. Joel et al. (1978) reported the occurrence of laccase in the cavity of the secretory ducts of all the members of the Anacardiaceae. The presence of a laccase-type phenol oxidase was demonstrated in the bacterium, Azospirillum lipoferum (Givaudan et al. 1993).

Laccase is a component of the lignin synthesizing system in woody tissues (Bao et al. 1993). It is however unrelated to ligninolysis in some fungi. Laccase from Aspergillus nidulans is involved in pigment synthesis (Hermann et al. 1983). Laccase produced by the fungus Botrytis cinerea are also implicated in pathogenic infections which lead to soft rot infections in cucumbers (Viterbo et al. 1993).

Catechol oxidase and laccase are distinguishable both on the basis of their phenolic substrates, and their inhibitor specificities (Table 2). Differences in the reaction mechanisms and the oxidation levels of copper at the active site of catecholase and laccase account for differences in their responses to certain inhibitors. Laccase activity is unaffected by CO, phenylhydrazine or 2,3-naphthalenediol, all of which are inhibitors of catechol oxidase (Keilin and Mann, 1940). The copper chelator diethyldithiocarbamate, cyanide azide and EDTA all serve as inhibitors of laccase activity.

Phenolic compounds are widely distributed in the plant kingdom and are considered to be secondary metabolites. Structurally they contain an aromatic ring bearing one or more hydroxyl groups, together with a number of other substituents. Plants provide nearly all the phenols found in higher animals, since higher animals are incapable of synthesizing compounds with benzonoid rings from aliphatic precursors. The polyphenolic composition of fruits varies in accordance with species, cultivar, degree of ripening and environmental conditions of growth and storage. Phenolics also contribute to colour, astringency, bitterness, and flavour in fruits.

Phenolic compounds occurring in food materials are mostly of the flavonoid type. Of the naturally occurring flavonoid compounds, anthocyanidins, flavonols, and cinnamic acid derivatives occur most frequently in foods. Catechins are also naturally occurring compounds, which are structurally related to other flavonoids having the basic nucleus of 1,3-diphenylpropane (Figure 13). Flavonols, together with flavones and flavanones are light yellow in colour, and are collectively termed anthoxanthin pigments. Quercetin, myricetin, and kaempferol are the most commonly occurring flavonols (Figure 13), and are generally glycosylated. The majority of naturally occurring flavonols possess a B-ring hydroxylation pattern similar to catechol. Catechol can therefore be considered to be the significant o-dihydroxyphenol. It becomes extremely important as a model substrate in enzymatic oxidation studies. Tyrosine on the other hand which is a monohydroxy phenol, is an important amino acid. Hydroxylation of tyrosine leads to the formation of dihydroxyphenylalanine (DOPA).

Benzoic acid derivatives and cinnamic acid derivatives are shown in Figure 13. Caffeic acid derivatives, such as chlorogenic (caffeoylquinic) acid and caftaric (caffeoyltartaric) acid, are often among the major o-diphenolic compounds in plants that serve as substrates for polyphenol oxidases. Chlologenic acid (Fig. 13) is one of the key substrates for enzymatic browning in apples. Enzymatic oxidation of caftaric acid takes place immediately upon crushing grapes (Cheynier and Moutounet, 1992).

Relatively few of the phenolic compounds in fruits and vegetables serve as substrates for polyphenol oxidase. Catechins, cinnamic acid esters, 3,4-dihydroxy phenylalanine (DOPA), and tyrosine (Table 3) are the most important natural substrates of polyphenol oxidase in fruits and vegetables. The main substrates of polyphenol oxidase in certain fruits and vegetables do not however commonly occur as phenolic constituents of plant material. The principal phenolic substrate in banana for example, was identified as dopamine (3,4-dihydroxy phenylethylamine), while that in dates is 3-o-caffeoylshikimic acid (dactylifric acid), and in yam-tuber tissues is a catechin-like substrate known as catecholamine.

Table 3. Phenolic substrates of PPO in fruits, vegetables, and seafoods.

The substrate specificity of polyphenol oxidase varies in accordance with the source of the enzyme. Phenolic compounds and polyphenol oxidase are, in general, directly responsible for enzymatic browning reactions in damaged fruits during postharvest handling and processing. The relationship of the rate of browning to phenolic content and polyphenol oxidase activity has been reported for various fruits such as apples (CoSeteng and Lee, 1987), grapes (Lee and Jaworski, 1988), and peaches (Lee et al. 1990).

In addition to serving as polyphenol oxidase substrates, phenolic compounds serve as inhibitors of polyphenol oxidases. Various cinnamic acids were observed to act as substrate analogues and serve as good inhibitors of apple polyphenol oxidase (Walker, 1995). Their inhibitory action decreased in the following order: cinnamic acid > p-coumaric acid > ferulic acid > benzoic acid.

Enzymatic browning does not occur in intact plant cells since phenolic compounds in cell vacuoles are separated from the polyphenol oxidase which is present in the cytoplasm. Once tissue is damaged by slicing, cutting or pulping, however, the formation of brown pigments occurs. Both the organoleptic and biochemical characteristics of fruits and vegetables are altered by pigment formation. The rate of enzymatic browning in fruit and vegetables is governed by the active polyphenol oxidase content of the tissues, the phenolic content of the tissue, pH, temperature and oxygen availability within the tissue.

As described in Chapter 1, polyphenol oxidase catalyses the oxidation of phenols to o-quinones, which are highly reactive compounds. O-quinones thus formed undergo spontaneous polymerization to produce high-molecular-weight compounds or brown pigments (melanins). These melanins may in turn react with amino acids and proteins leading to enhancement of the brown colour produced. Many studies have focused on either inhibiting or preventing polyphenol oxidase activity in foods. Various techniques and mechanisms have been developed over the years for the control of these undesirable enzyme activities. These techniques attempt to eliminate one or more of the essential components (oxygen, enzyme, copper, or substrate) from the reaction.

i) The elimination of oxygen from the cut surface of fruits or vegetables greatly retards the browning reaction. Browning however occurs rapidly upon exposure to oxygen. Exclusion of oxygen is possible by immersion in water, syrup, brine, or by vacuum treatment.

ii) This copper prosthetic group of polyphenol oxidases must be present for the enzymatic browning reaction to occur. Chelating agents are effective in removing copper.

iii) Inactivation of the polyphenol oxidases by heat treatments such as steam blanching is effectively applied for the control of browning in fruits and vegetables to be canned or frozen. Heat treatments are not however practically applicable in the storage of fresh produce.

iv) Polyphenol oxidase catalyses the oxidation of phenolic substrates such as caffeic acid, protocatechuic acid, chlorogenic acid, and tyrosine. Chemical modification of these substrates can however prevent oxidation.

v) Certain chemical compounds react with the products of polyphenol oxidase activity and inhibit the formation of the coloured compounds produced in the secondary, non-enzymatic reaction steps, which lead to the formation of melanin.

Many techniques are applied in the prevention of enzymatic browning. Relatively new techniques, such as the use of killer enzymes, naturally occurring enzyme inhibitors and ionizing radiation, have been explored and exploited as alternatives to heat treatment and the health risks associated with certain chemical treatments. Processing technologies applied in the control of enzymatic browning in fruits and vegetables are now reviewed.

Heat treatment is the most widely utilized method for stabilizing foods because of its capacity to destroy microorganisms and to inactivate enzymes. Steam blanching is one of the most effectively applied methods of heat treatment for controlling enzymatic browning in canned or frozen fruits and vegetables. Steam blanching is not however practical for the prevention of browning in fresh foods. Temperatures applied in steam blanching treatments vary in accordance with the thermostability of the enzyme to be inactivated as well as with the nature of the food product. Pasteurization is generally conducted at temperatures ranging between 60 ^oC and 85 ^oC, while blanching techniques are often operated at temperatures ranging between 70^oC and 105 ^oC or higher. In general, exposure of polyphenol oxidases to temperatures in the 70-90 ^oC range, results in the destruction of their catalytic activity (Vámos-Vigyázó, 1981). Blanching of green beans in an automatic rotary hot water blancher at temperatures of 82 ^oC and above for 3.5 minutes, almost completely inactivated catalase, lipoxygenase, and polyphenol oxidase activities (Lee et al. 1988). Thermal inactivation profiles of important enzymes such as peroxidase, polyphenol oxidase, and lipoxygenase in fruit and vegetable processing, follow first-order reaction kinetics. The thermal inactivation profile of the thermostable fraction of several potato enzymes is shown in Figure 14 (Svensson, 1977). From this Figure it is obvious that at lower temperatures much longer times are required to accomplish a 90 percent reduction in enzyme activity.

Thermal process characteristics for enzymatic reactions are described in accordance with kinetic parameters such as decimal reduction times (D), inactivation rate constant (k), z-values (z), and activation energies (Ea). The D value, or decimal reduction value, is defined as the time required to inactivate 90 percent of the original enzyme activity at a given temperature. An inactivation reaction, which follows first-order kinetics, has a D value equivalent to 2.303/k. Temperature dependence of the D-value is given by the z-value, which represents the temperature increase required in order to obtain a ten-fold (1-log cycle) decrease in D-value. For a first-order decay process, the D value is equivalent to ln (10)k. Similar to the z-value is the activation energy (Ea), which expresses the temperature dependence of the k-value as indicated in the Arrhenius relationship:

The Q₁₀ value is the change in the rate of a reaction that occurs with a 10 ^oC change in temperature.

Rate T

Q₁₀ = -----------------------

Rate T+10^oC

The Q₁₀ value can be related to the Arrhenius equation as,

Q₁₀ = e^10Ea/RT

Where Ea is independent of temperature, provided that conditions are appropriate and Q₁₀ is dependent on temperature.

Steam and water blanching are widely applied in the inactivation of enzymes and for stabilizing frozen vegetables against off-flavour development, nutritional loss and discolouration. Blanching also prevents chlorophyll and carotenoid pigments from undergoing enzymatic degradation. Time and temperature must both be controlled during blanching. The time taken for the complete heat inactivation of polyphenol oxidases varies considerably with the food product. Water blanching prevents darkening in frozen sweet potatoes by significantly decreasing the polyphenol oxidase activity (Ma et al. 1992). A blanch treatment at 100^oC for 3 min or 94 ^oC for 5 min is required to obtain products with minimal darkening.

Blanching is nutritionally disadvantageous in that it results in losses in vitamins, flavours, colours, texture, carbohydrates and other water-soluble components. Requirements for large amounts of water and energy, coupled with waste disposal problems make blanching technically disadvantageous. Blanched products generally require a cooling step prior to freezing. Additional leaching takes place if this cooling step is carried out by immersion of the product in cold water.

3.1.2 Refrigeration

The rate of enzyme-catalysed reactions is controlled to a great extent by temperature. For every 10^oC temperature increase (in biological important ranges), there is a two-fold increase in the rate of an enzyme-catalysed reaction, referred to as the temperature coefficient (Q₁₀). On the other hand, for every 10 ^oC reduction in temperature a similar decrease in the rate of biological activity occurs. At low temperatures, reduced kinetic energy of the reactant molecules results in a decrease in both mobility and "effective collisions" necessary for the formation of enzyme-substrate complexes and their products.

Chilling involves the temporary storage of foods at temperatures above freezing. Some vegetables (broccoli, berries, spinach, peas, etc.) are either pre-cooled or stored at chilling temperatures. Other commodities (bananas, mangoes, avocados, tomatoes, etc.) are susceptible to chill injury and should therefore not be stored below their respective critical temperatures (Fennema, 1975a). The chilling process is generally accomplished either with the use of moving air, water (hydrocooling), ice, or vacuum (vacuum cooling).

Cold preservation and storage during distribution and retailing are necessary for the prevention of browning in fruit, vegetables, and seafood, since refrigerated temperatures are effective in lowering polyphenol oxidase activity.

3.1.3 Freezing

Freezing temperatures of -18 ^oC or below are often used for the long-term preservation of food. Mechanisms of enzyme inactivation at freezing temperatures can be explained by several hypotheses. Tappel (1966) proposed that solutes, such as salt, sugars, and other carbohydrates, are effective enzyme inhibitors at high concentrations. Increased solute concentrations attained in the semi-frozen state would therefore enhance both substrate and product inhibition. Fennema (1975b) attributed the effects of freezing to changes in pH. According to his theory, changes in buffer concentration and composition could cause changes in pH and thus acidity, due to enhanced mobility of the hydrogen ion in ice as compared to that in water. Another factor involved in enzyme inactivation at freezing temperatures is the perturbation of sulphydryl groups essential for the activity of some enzymes. Perturbation of sulphydryl groups is attributed to increased propensity for SS-SH interchange as a result of 1) increased concentration of disulfide groups, 2) conformational changes, and 3) accelerated oxidation of sulphydryl groups due to increased oxygen concentrations within the ice (Fennema, 1975b). Recent discussions have attributed enzyme inactivation on freezing to the influence of water removal during the freezing process. Removal of water during freezing causes changes that eventually result in an alteration of the microenvironment of the enzyme, in a manner similar to dehydration.

As with high temperature inactivation, low-temperature inactivation has its drawbacks. Freezing causes changes in texture and other freshness characteristics. Freezing can also lead to the decompartmentalization of certain enzymes, substrates, and/or activators as a result of cell disruption thus facilitating enzyme activity in the frozen state, which is enhanced upon thawing of the food (Ashie and Simpson, 1996).

3.1.4 Dehydration
Physical Methods

Water generally exerts an enormous influence on enzyme activity in that it behaves both as a solvent and a reactant (Ashie and Simpson, 1996). Water activity (a_w) is defined as the ratio of the partial pressure of the water vapour above a sample to that of saturated water vapour at the same temperature. Water activity can affect reactions in solid media either by restricting the mobility of the reactants or by altering the active conformation of substrate and enzyme. In general, enzymatic activities decrease with a decrease in a_w. Water activity can be controlled either by partial drying or through the addition of water binding agents, such as polyols, sugars and salts, to increase stability and inhibit microbial growth. Dehydration has varied effects on enzymes because of their differing responses to the concentrations of various solutes, inhibitors, or activators. Solutes such as sugars and proteins often have a protective effect against enzyme inactivation.

Freeze-drying is another dehydration process, which involves the removal of moisture from foods in the frozen state by sublimation under high conditions of high vacuum. Low temperatures applied in the process inhibit undesirable chemical and biochemical reactions while minimizing the loss of volatile aromatic compounds. Rapid freezing during the process minimizes enzymatic browning and generally yields a product of excellent quality, while slow freezing is somewhat less effective in minimizing browning. Freeze-dried mushrooms prepared by a slow-freezing process were observed to have higher polyphenol oxidase activity than those prepared by the Freon-12 dipping process (Fang and Chiang, 1975). Other physical methods of dehydration include spray-drying, radiative-, solar- and microwave drying.

Chemical Methods

Salts can also have a dehydrating effect in that high salt concentrations remove water from the reaction medium (i.e. reduce a_w), making it unavailable for water-protein interactions, thus resulting in reduced enzyme activity. Sodium chloride, sucrose, and other sugars, glycerol, propylene glycol and modified corn syrups are some of the solutes used for dehydration. Salts are also capable of modifying the thermodynamics of enzyme catalysed reactions. Polyvalent anions, such as SO₃^2-and PO₄^2-, in general tend to stabilize enzymes, while polyvalent cations, such as Ca²⁺ and Mg²⁺, tend to have a destabilizing effect on enzymes (Adams, 1991).

3.1.5 Irradiation

Food irradiation is increasingly recognized as a method for reducing postharvest food losses, ensuring hygienic quality, and facilitating wider trade in foodstuffs. Food irradiation is unique in that it makes use of ionizing radiation for improving the shelf life or wholesomeness of the product. Food irradiation is effective in securing the long-term preservation of foods through the inactivation of microorganisms. Meats, seafoods, fruits, vegetables, and cereal grains can be preserved by irradiation. Food irradiation can extend the shelf life of those foods which, after harvest, continue to be physiologically active, by delaying their maturation or sprouting.

Gamma rays (from cobalt-60 or cesium-137), X-rays, and accelerated electrons (electron beams) are the forms of ionizing radiation applied in food processing. Food irradiation has been approved for both preservation and processing applications in the United States. The effects of varied doses of ionizing radiation on fresh fruits and vegetables are summarized in Table 4 (Kader, 1986).

Table 4. Effects of ionizing radiation on fresh fruits and vegetables.

Dose (kGy)	Observed effects
0.05-0.15	Sprout inhibition in tuber, bulb, and root vegetables; inhibition of growth in asparagus and mushroom
0.15-0.75	Insect disinfestation
0.25-0.50	Delayed ripening of some tropical fruits such as banana, mango, and papaya
>1.75	Control of postharvest disease
1.00-3.00	Accelerated softening; development of off-flavors in some commodities
>3.00	Excessive softening; abnormal ripening; incidence of some physiological disorders; impaired flavor

1 kilogray (kGy) = 1000 gray (Gy), which is the SI unit of energy absorbed (1 joule/kg) from ionizing radiation. 1 Gy = 100 rad (1 rad = 100 erg/g), and 1 kGy=100 krad

Low-dose gamma irradiation of fresh shrimp decreased melanosis and significantly increased the shelf life of shrimp stored on ice (Novak et al. 1967). However low-dose irradiation of shrimp after the onset of melanosis, resulted in acceleration of the rate of melanosis. Browning in potato tubers and tropical fruits after gamma-irradiation was extensively reviewed by Thomas (1984; 1986). Gamma irradiation up to 1 kGy inhibited sprouting in potato tubers and caused a reduction in both total phenolic and chlorogenic acid contents (Pendharkar and Nair, 1987). Mondy and Gosselin (1989) reported that a radiation dose of 1 kGy caused less darkening in potatoes than higher doses. Increased blackspot formation in potatoes at higher irradiation doses may occur due to the rupture of lipid membranes and the liberation of polyphenol oxidase from mitochondria, which in turn oxidize phenolic substrates in the vacuole, and cause browning. Browning might therefore be minimized by controlling the dosage level of the ionizing radiation applied.

Ionizing radiation at doses exceeding 1 kGy can introduce various types of physiological disorders in food products. Free radicals produced during the treatment of food with ionizing radiation, are capable of reacting with various food constituents and inducing undesirable side effects, such as tissue darkening, lipid oxidation and decreased vitamin content. Nonenzymatic browning reactions of free amino acids and proteins with reducing sugars, such as glucose, may be responsible for this discolouration.

The sensitivity of enzymes to ionizing radiation is measured by D₃₇, which is defined, as the dose required to inactivate 63 percent of the original activity of the enzyme. Where food systems are concerned, sensitivity is often expressed as D_E, which is the dose required for 90 percent inactivation of the enzyme. D₃₇ values of some enzymes in model systems measured in the dry state are shown below (Roozen and Pilnik, 1971).

Enzyme D₃₇ (kGy)

Alkaline phosphatase 40-50

Pectin esterase 60

Peroxidase 30-70

Enzymes exhibit a high degree of stability in the absence of oxygen, presumably due to formation of the superoxide anion (O₂^-), which can interact to raise the energy level of other active molecules and residues. Combined treatments using both irradiation and heat or other methods have demonstrated a synergistic effect in preventing enzymatic browning.

3.1.6 High pressure treatment

Enzyme denaturation is caused by rearrangement and/or destruction of noncovalent bonds such as hydrogen bonds, hydrophobic interactions, and ionic bonds of the tertiary protein structure. Pressure can influence biochemical reactions by reducing molecular spacing and increasing interchain reactions. High-pressure treatment is a potentially viable technique for preserving food quality through the inactivation of endogenous food enzymes. Pressures exceeding 5 kbar generally cause irreversible denaturation of enzymes due to the weakening of hydrophobic interactions and the breaking of intramolecular salt bridges (Cheftel 1992; Masson 1992). High-pressure treatments can result in either reversible or irreversible changes in protein structure. Loss of catalytic activity under high-pressure conditions however varies in accordance with the enzymes, the nature of the substrates, the temperature and the duration of high pressure processing (Cheftel, 1992; Kunugi, 1992).

High-pressure treatment is known to preserve quality carriers, such as flavour, taste, and vitamins in foods (Cheftel, 1992). Depending on the nature of the food, it may also induce quality changes in the food. A proper combination of pressure and temperature might be used to enhance enzyme inactivation in certain foods. This combination would prevent off-flavour development, and changes in colour, nutritional value, and structure (Farkas, 1987).

Polyphenol oxidase is highly pressure resistant. Pressures of 5 and 7 kbar are required for the inactivation of apple polyphenol oxidase at pH 4.5 and 5.4, respectively (Nicoli et al. 1994). Polyphenol oxidases from different sources show different pressure-temperature behaviours (Table 5). Weemaes et al. (1998) showed that inactivation of polyphenol oxidase from apple, grape, avocado and pear at room temperature (25 ^oC) became noticeable at »600, 700, 800 and 900 Mpa, respectively. However, plum polyphenol oxidase was not inactivated at room temperature by pressures of up to 900 Mpa.

Table 5. Pressure stability of PPO derived from various sources.

Origin of PPO	Medium	Treatment	Activation/Inactivation	Reference
Apple	crude extract (pH 4.5) crude extract (pH 5.4) crude extract (pH 7)	#300 Mpa/25^oC/1min >500 Mpa/25 ^oC/1 min #300 Mpa/25^oC/1min >700 Mpa/25 ^oC/1min #100 Mpa/25^oC/1min	activation inactivation activation inactivation inactivation	Anese et al. (1995)
Avocado	partially purified	800 Mpa/25 ^oC	inactivation	Weemaes et al. (1998)
Onion	purified enzyme in Tris buffer (pH 6.5, 0.01M)	> 700 Mpa/25 ^oC/10 min	activation, which is maximal at 500 MPa	Butz et al., (1994)
Pear	in slices partially purified	400 Mpa/25^oC/10 min 900 Mpa/25 ^oC	activation slight inactivation	Asaka and Hayashi (1991) Weemaes et al. (1998)
Plum	partially purified	900 Mpa/25 ^oC	no inactivation	Weemaes et al. (1998)
Potato	in cubes	400 Mpa/<50^oC/15 min	no inactivation	Knorr (1993)
White grapes	partially purified	700 Mpa/25 ^oC	inactivation	Weemaes et al. (1998)

Pressure inactivation of polyphenol oxidase in Tris buffer at 45 ^oC required treatments of 900 Mpa for a period exceeding 30 min (Figure 15). The inactivation of pure enzymes by pressure is dependent on the immersion medium, the pH, as well as the temperature and duration of the treatment. Food constituents can however have a protective effect on enzymes during high pressure treatment. Mushroom polyphenol oxidase shows very high pressure stability, although it is a thermosensitive enzyme which is readily inactivated by temperatures exceeding 50 ^oC (Weemaes et al. 1997).

Figure 15. Effect of high hydrostatic pressure on relative activity of polyphenoloxidase in Tris buffer (pH 7) at 45 oC. (From Seyderhelm et al., 1996).

3.1.7 Treatment with supercritical carbon dioxide (SC-CO₂)

A pure substance exists in a state that exhibits both gas- and liquid-like properties at temperatures and pressures above its critical point (Kiran and Zhuang, 1997). A supercritical fluid (SC) on the other hand exists in the fluid state above its critical temperature and pressure, i.e. it has a density similar to that of a liquid, surface tension close to zero, and its diffusivity and viscosity range between that of a liquid and a gas. Carbon dioxide has a low critical temperature and pressure (Rizvi et al. 1986), which makes it ideal in use as a SC fluid. Its nontoxicity, nonflammability, low cost and availability (Hardardottir and Kinsella, 1988) coupled with its environmentally acceptable nature make it highly desirable for food processing applications. SC carbon dioxide treatments are effective in destroying microorganisms, as well as in inactivating unwanted enzymes in foods. Their effectiveness in food preservation is due to the fact that upon dissolution in water, high pressure CO₂ produces carbonic acid, which effects a temporary reduction in pH thus inactivating enzymes and microorganisms. SC carbon dioxide has been reported to inactivate PPO (Zemel, 1989). Purified Florida spiny lobster, brown shrimp, and potato polyphenol oxidases exhibited a time-related decline in activity following treatment at 43 ^oC with high pressure carbon dioxide at 58 atm (Chen et al. 1992). Kinetic studies showed that crustacean polyphenol oxidases were more vulnerable to SC carbon dioxide treatment than potato polyphenol oxidase.

3.1.8 Ultrafiltration (UF)

UF is a widely used membrane separation technology which has been shown to be effective in stabilizing the colour of white wines and other fruit juices (Flores et al. 1988; Sims et al. 1990). The use of ultrafiltration as an alternative to sulphiting for the control of enzymatic browning has been studied (Sims et al. 1989; Goodwin and Morris, 1991). UF is believed to remove polyphenol oxidase, but not lower-molecular-weight polyphenols or Maillard-reaction precursors, which could undergo nonenzymatic browning during storage of the wine.

Banana PPO fractions are reported to have molecular weights in excesses of 30 kDa (Galeazzi et al. 1981), which is well within the range of molecular weight cut-offs for UF membranes. UF therefore offers potential for improving the colour stability of banana juice without the application of heat, which is known to alter the flavour of banana juice.

3.1.9 Ultrasonication

Heat treatment is capable of altering the organoleptic properties of foods and reducing the bioavailability of certain nutrients. The combined use of heat and ultrasonic waves can contribute to enzyme inactivation. When applied to a liquid, ultrasonic waves promote acoustic cavitation leading to bubble formation. Bubble-liquid interfaces continuously undergo changes in shape and size. Acoustic streams occurring within the liquid in the vicinity of the bubble, often cause severe shear stresses which can promote enzyme denaturation. Ultrasonication also promotes chemical reactions involving H× and OH× free radicals formed by the decomposition of water within the oscillating bubbles (El. piner et al. 1965). These free radicals could be scavenged by amino acid residues of the enzymes participating in structure stability, substrate binding, or catalytic functions (Gebicki and Gebicki, 1993).

3.2 Inhibitors

Enzymatic browning can be inhibited by targeting the enzyme, the substrates (oxygen and polyphenols) or the products of the reaction.

i) Inhibition targeted toward the enzyme

Mayer and Harel (1979) classified the inhibitors which act directly on polyphenol oxidase into two groups. The first group, which consists of metal ion chelators, such as azide, cyanide, carbon monoxide, halide ions and tropolone, is well documented for the inhibition of polyphenol oxidase from various sources. The chloride ion was shown to be noncompetitive for apple polyphenol oxidase, while other halide ions were observed to have a competitive inhibitory effect (Janovitz-Klapp et al. 1990). The second group of inhibitors, which consists of aromatic carboxylic acids of the benzoic and cinnamic series, has been widely studied (Janovitz-Klapp et al. 1990). Compounds of this group behave as competitive inhibitors of polyphenol oxidase, owing to their structural similarity with phenolic substrates.

ii) Inhibition targeted toward the substrate

Enzymatic browning can be controlled by removal of either the oxygen or phenolic substrates, from the reaction medium. Elimination of oxygen is perhaps the most satisfactory methodology for preventing phenol oxidase catalysed phenolic oxidation. The removal of oxygen can however result in metabolic deviations since excessive reduction of oxygen induces anaerobic metabolism, leading to breakdown and off flavour development in foods (Ballantyne et al. 1988).

Vacuum packaging of pre-peeled potatoes to exclude oxygen, was observed to extend their shelf life (Langdon, 1987). Vacuum packaged products however rapidly undergo browning upon exposure to air. Anaerobic conditions created by vacuum packaging are a cause for safety concern in that they are potentially capable of supporting the growth of Clostridium botulinum and the production of its toxin (Tamminga et al. 1978).

Specific adsorbents, which undergo complexation with the phenolic substrate may be applied in the physical elimination of phenolic compounds from food systems. The use of cyclodextrins for the removal of phenolic compounds from raw fruit and vegetable juices has been patented in the United States (Hicks et al. 1990). Cyclodextrins are thought to inhibit polyphenol oxidase activity through the formation of inclusion complexes with polyphenols (Sapers et al. 1989). Sulphated polysaccharides also have an inhibitory effect on browning (Tong and Hicks, 1991). Apart from possible complexation, sulphate groups are believed to exert their inhibitory effect through chelation of the copper prosthetic group of the polyphenol oxidase (Tong and Hicks, 1991).

Enzymatic modification of phenolic substrates may serve to inhibit polyphenol oxidase activity. O-methyltransferase for example converts o-dihydroxy phenolics to the corresponding methoxy derivatives, which do not serve as substrates for polyphenol oxidase (Finkle and Nelson, 1963). Similarly, protocatechuate 3,4-dioxigenase purified from Pseudomonas aeruginosa prevents the browning of Gravenstein apple juice (Kelly and Finkle, 1969) due to substrate modification. These enzyme modification techniques are not however feasible in commercial use, owing to the high cost of these enzymes.

iii) Inhibition targeted toward the products

O-quinones, which are the products of diphenol oxidation, are capable of reacting with each other, resulting in the formation of dimers of the original phenol. These dimers, which possess an o-diphenolic structure, undergo re-oxidation resulting in the formation of larger oligomers of varying colour intensities. Ascorbic acid (Hsu et al. 1988), thiol compounds (Henze, 1956), sulphites (Sayavedra-Soto and Montgomery, 1986), and amino acids (Kahn, 1985) are however capable of inhibiting dimer formation and re-oxidation either by reducing o-quinones to o-diphenols, or through the formation of colourless addition products.

Classification of Inhibitors

The use of browning inhibitors in food processing is restricted by considerations relevant to toxicity, wholesomeness, and effect on taste, flavour, texture, and cost. Browning inhibitors may be classified in accordance with their primary mode of action. Six categories of polyphenol oxidase inhibitors are applicable in the prevention of enzymatic browning (Table 6). These include (1) reducing agents; (2) acidulants; (3) chelating agents; (4) complexing agents; (5) enzyme inhibitors; (6) enzyme treatments. Each category of inhibitor is now discussed:

Table 6. Representative inhibitors of enzymatic browning.(Adapted from McEvily et al. 1992)

Reducing agents	sulphiting agents ascorbic acid and analogs cysteine glutathione
Chelating agents	phosphates EDTA organic acids
Acidulants	citric acid phosphoric acid
Enzyme inhibitors	aromatic carboxylic acids aliphatic alcohol anions peptides substituted resorcinols
Enzyme treatments	oxygenases o-methyl transferase proteases
Complexing agents	cyclodextrins

3.2.1 Reducing agents/Antioxidants

Reducing agents play a role in the prevention of enzymatic browning either by reducing o-quinones to colourless diphenols, or by reacting irreversibly with o-quinones to form stable colourless products. Reducing compounds are very effective in the control of browning. Sulphiting agents are the most widely applied reagents for the control of browning in the food industry.

a. Sulphiting agents

Sulphites are the most widely used inhibitors of enzymatic browning. Sulphiting agents include sulphur dioxide (SO) and several forms of inorganic sulphite that liberate SO₂ under the conditions of their use.

SO₂: sulphur dioxide

SO₃^2-: sulphite

HSO₃^-: bisulphite

S₂O₅^2-: metabisulphite

SO₂ and sulphite salts form sulphurous acid (H₂SO₃) and exist as a mixture of the ionic species, bisulphite (HSO₃^-) and sulphite (SO₃^2-) anions in aqueous solution. The predominant ionic species varies in accordance with pH, ionic environment, water activity, presence of non-electrolytes, and concentration of the medium in which they are dissolved. Maximum HOS3^- concentrations exist at pH 4, while at pH 7, both SO₃^2- and HSO₃^- exist in approximately equivalent concentrations (Green, 1976). Increased concentrations of sulfite at pHs of less than 5 were observed to enhance the inhibition of polyphenol oxidase-catalysed browning (Sayavedra-Soto and Montgomery, 1986). The dibasic acid undergoes ionization according to the following reaction scheme:

SO₂×H₂O -> (H₂SO₃)^- -> HSO₃^- + H⁺

HSO₃^- ->SO₃^2- + H⁺

with pKa values of 1.89 and 7.18 (25 ^oC, zero ionic strength) for the first and second ionizations, respectively (Figure 16).

Figure 16. The distribution of species of sulfurous acid at various pH values. (From Ough, 1984).

Sulphites serve a multifunctional role in foods. They possess antimicrobial activity and inhibit both enzymatic and non-enzymatic browning reactions. Madero and Finne (1982) proposed that bisulphite exerted a competitive inhibitory effect on polyphenol oxidase, by binding a sulphydryl group at the active site of the enzyme. Ferrer et al. (1989b) on the other hand, proposed that bisulphate inhibition was due to the reaction of sulphites with intermediate quinones, resulting in the formation of sulphoquinones, which irreversibly inhibited polyphenol oxidase, causing complete inactivation. Mechanisms involved in the control of enzymatic browning by sulphites are shown in Figure 17.

Figure 17. The primary role of reducing agents such as sulphiting agents in the inhibition of enzymatic browning is to reduce the pigment precursors (quinones) to colourless, less-reactive diphenols. (Adapted from Walker, 1977).

Although sulphites are very effective in controlling browning, they are subject to regulatory restrictions owing to their potentially adverse effects on health. Many reports have described allergic reactions in humans, following the ingestion of sulphite-treated foods by hypersensitive asthmatics. The use of sulphiting agents in food processing is based on sulfur dioxide equivalence (Modderman, 1986). Table 7 gives a list of sulphiting agents and their theoretical yields of sulfur dioxide. The Joint Expert Committee on Food Additives (JECFA) of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) recommend an acceptable sulphite daily intake of 0-0.7 mg sulphur dioxide per kg of body weight.

Table 7. Chemicals yielding sulfur dioxide which are currently allowed for use in food as preservatives. (Adapted from Green, 1976)

Chemical	Formula	Theoretical Yield (%)	Solubility (mg/100 mL)
Sulphur dioxide	SO₂	100.0	11 at 20 ^oC
Sodium sulphite anhydrous	Na₂SO₃	50.8	28 at 40 ^oC
Sodium sulphite (heptahydrate)	Na₂SO₃ @7H₂O	25.4	24 at 25 ^oC
Sodium hydrogen sulphite	NaHSO₃	61.6	300 at 20 ^oC
Sodium metabisulphite	Na₂S₂O₅	67.4	54 at 20 ^oC
Potassium metabisulphite	K₂S₂O₅	57.6	25 at 0 ^oC

Sulphites are currently applied for the inhibition of melanosis (blackspot) in shrimp, potatoes, mushrooms, apples, and other fruits and vegetables. Sulphites are also applied in stabilizing the flavour and colour of wines. Sulphite concentrations necessary for controlling enzymatic browning vary widely in accordance with the food material and the time required for inhibition of the browning reaction (Taylor et al., 1986). Where only monophenolic substrates, such as tyrosine are present, as in the case of potatoes, relatively low levels of sulphite are effective in inhibiting browning. On the other hand, where diphenols are present, as is the case in avocados, much higher sulfite concentrations are required for the control of browning.

Sulphites no longer have "Generally Required as Safe Status" (GRAS) status for use on fruits and vegetables served raw, sold raw or presented to the consumer as raw in the United States. According to the United States Federal Register (1988) foods containing detectable levels of a sulphiting agent, at 10 ppm regardless of source, must declare the sulfite and its content on the ingredient label. More regulatory restrictions are likely to be globally applied to the use of sulphites in foods since sulphite allergies pose a health risk in many populations. Regulations enacted by the United States Food and Drug Administration (FDA) in 1995 prohibit the use of sulphites in salad bars. As a result, there has been a considerable focus on identifying appropriate sulphite substitutes for use in foods. The FDA has proposed maximum residual sulphur dioxide levels for certain foods. In accordance with these proposed limits, residual sulphur dioxide levels for fruit juices, dehydrated potatoes, and dried fruit, are 300, 500, and 2000 ppm respectively (Federal Register, 1988). Shrimp products having residual sulphite levels in excess of 100 ppm are considered adulterated, since these levels are considered unsafe (Federal Register, 1985).

b. L-Ascorbic acid

Ascorbic acid is a moderately strong reducing compound, which is acidic in nature, forms neutral salts with bases, and is highly water-soluble. L-ascorbic acid (vitamin C) and its various neutral salts and other derivatives have been the leading GRAS antioxidants for use on fruits and vegetables and in fruit juices, for the prevention of browning and other oxidative reactions (Bauernfeind and Pinkert, 1970).

Ascorbic acid also acts as an oxygen scavenger for the removal of molecular oxygen in polyphenol oxidase reactions. Polyphenol oxidase inhibition by ascorbic acid has been attributed to the reduction of enzymatically formed o-quinones to their precursor diphenols (Walker, 1977). Ascorbic acid is however irreversibly oxidized to dehydroascorbic acid during the reduction process, thus allowing browning to occur upon its depletion (Figure 18). More stable forms of ascorbic acid derivatives, such as erythrobic acid, 2- and 3-phosphate derivatives of ascorbic acid, phosphinate esters of ascorbic acid, and ascorbyl-6-fatty acid esters of ascorbic acid, have however been developed to overcome these problems (Seib, 1985; Sapers and Hicks, 1989). Ascorbic acid esters release ascorbic acid upon hydrolysis by acid phosphatases (Liao and Seib, 1988). Their relative effectiveness as browning inhibitors varies in accordance with the food product (Bauernfeind and Pinkert, 1970). Compounds containing reactive amino or thiol groups can greatly affect the reactivity of o-quinones.

Figure 18. Mechanism of prevention of colour formation by ascorbic acid.

Ascorbic acid causes a distinct yellow off-colour, when used in the prevention of melanosis in shrimp (Otwell and Marshall, 1986). It is usually applied in conjunction with citric acid in order to maintain a more acidic pH level. In addition, it is also believed to have a chelating effect on the copper prosthetic group of polyphenol oxidase (Whitaker, 1972b).

c. Erythorbic acid

Erythorbic acid and its salt, sodium erythorbate, are strong reducing agents with GRAS status. They both act as oxygen scavengers, thus eliminating oxygen as a substrate for browning reactions. Erythorbic acid is the D-isomer of ascorbic acid but does not have vitamin C activity. Its use in conjunction with citric acid has often been suggested as a substitute for sulphites in the control of enzymatic browning. Current research suggests that L-ascorbic acid and erythorbic acid both possess equivalent antioxidant properties. A combination of both acids is applied at the retail level for inhibiting both oxidative rancidity and discolouration in vegetables, salads, apples, and frozen seafood. Erythobic acid or sodium erythorbate can suppress browning reactions in frozen fruits.

d. Cysteine

Cysteine is an effective inhibitor of enzymatic browning. It is reported to be more effective than sodium bisulphite as an antibrowning agent (Kahn, 1985). Concentrations of cysteine and other thiols required for the achievement of acceptable levels of browning inhibition have however been shown to have negative effects on taste. The inhibition of melanosis by cysteine is thought to be due to the formation of colourless thiol-conjugated o-quinones (Pierpoint, 1966). Cysteine has also been shown to reduce o-quinones to their phenol precursors (Walker, 1977; Cilliers and Singleton, 1990).

A mode of action for cysteine and cysteinyl addition in the control of browning proposed by Richard-Forget et al. (1992) is illustrated in Figure 19. Cysteine-quinone adducts serve as competitive inhibitors of polyphenol oxidase. Sulphydryl (thiol) compounds N-acetyl-L-cysteine (NAC) and reduced glutathion (GSH) are also excellent inhibitors of browning of potato powder (Figure 20, Friedman et al. 1992).

Figure 19. Effect of cysteine and cyteinyl addition compounds with o-quinones on the enzymatic oxidation of o-diphenols. (From Richard-Forget et al., 1992).

Figure 20. Inhibition of potato browning by N-acetyl-L-cysteine (5% potato in water; inhibitor 0.8 mM; 5 h; room temperature). (From Friedman et al., 1992).

e. Phenolic antioxidants

Antioxidants are defined by the United States Food and Drug Administration (FDA) as substances, which may be applied in preserving food by retarding deterioration, rancidity or discolouration due to oxidation. Antioxidants inhibit oxidative processes by reacting with free radicals, through metal chelation, and by scavenging singlet oxygen.

Both synthetic and naturally occurring phenolic antioxidants are used in food applications. Several synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiarybutyl hydroxyquinone (TBHQ) and propyl gallate (PG), are permitted for use in food. Their structures are shown in Figure 21. Plant phenolic compounds such as tocopherols, flavonoid compounds, cinnamic acid derivatives, and coumarins are naturally occurring compounds, which have an antioxidant effect, that renders them inhibitory to polyphenoloxidase, and thus browning.

Figure 21. Major antioxidants used in food.

3.2.2 Acidulants

Ionizable groups of the protein structure of enzymes are affected by the pH of the food medium. These groups must be in the appropriate ionic form in order to maintain the conformation of the active site, bind substrates, or catalyse the enzymatic reaction (Segel, 1976). Changes in the ionization status of enzymes are generally reversible. Irreversible denaturation can however occur under conditions of extreme pH. The stability of the substrate is also affected by changes in pH, since substrates can undergo chemical breakdown under extreme conditions of pH. Degraded substrates often behave as enzyme inhibitors, since they share the molecular features of the substrate (Tipton and Dixon, 1983).

Acidulants are generally applied in order to maintain the pH well below that required for optimum catalytic activity of an enzyme. Acidulants such as citric, malic, and phosphoric acids are capable of lowering the pH of a system, thus rendering polyphenol oxidase polyphenol oxidase inactive (Richardson and Hyslop, 1985). Acidulants are often used in combination with other antibrowning agents.

a. Citric acid

Citric acid is the one of the most widely used acidulants in the food industry. It is typically applied at levels ranging between 0.5 and 2 percent (w/v) for the prevention of browning in fruits and vegetables. In addition, it is often used in combination with other antibrowning agents such as ascorbic or erythorbic acids and their neutral salts, for the chelation of prooxidants and for the inactivation of polyphenol oxidase. Recommended usage levels for citric acid typically vary between 0.1 and 0.3 percent (w/v) with the appropriate antioxidant at levels ranging between 100 and 200 ppm (Dziezak, 1986). Citric acid exerts its inhibitory effect on polyphenol oxidase by lowering the pH as well as by chelating the copper at the active site of the enzyme.

3.2.3 Chelators

Enzymes generally possess metal ions at their active sites. Removal of these ions by chelating agents can therefore render enzymes inactive. Chelating agents complex with prooxidative agents, such as copper and iron ions, through an unshared pair of electrons in their molecular structures. Chelators have been applied in various food processing applications, for enzyme inactivation (McEvily et al. 1992). Chelators used in the food industry include sorbic acid, polycarboxylic acids (citric, malic, tartaric, oxalic, and succinic acids), polyphosphates (ATP and pyrophosphates), macromolecules (porphyrins, proteins), and EDTA.

Other non-GRAS chelating agents which are capable of inhibiting polyphenol oxidase include cyanide, diethyldithiocarbonate, sodium azide and 2-mercaptobenzothiazole, carbon monoxide, mercaptobenzthiazol, dimercaptopropanol, and potassium methyl xanthate.

Ascorbic acid also has a chelating effect on the prosthetic group of polyphenol oxidase.

a. EDTA

EDTA is a chelating agent permitted for use in the food industry as a chemical preservative. Calcium disodium EDTA (21 CFR 172.120) and disodium EDTA (21 CFR 172.135) have been approved for use as food additives by the United States Food and Drug Administration (Anon, 1992). Highly stable complexes are formed by the sequestering action of EDTA compounds on iron, copper, and calcium. Maximum chelating efficiency occurs at the higher pH values where carboxyl groups exist in a dissociated state (Dziezak, 1986). EDTA is generally used in combination with other chemical treatments for the prevention of enzymatic browning in foods. It not very effective as an inhibitor of peach polyphenol oxidase (Wong et al. 1971).

A typical combination of anti-browning agents might consist of a chemical reducing agent (ascorbic acid), an acidulant (citric acid) and a chelating agent (EDTA).

b. Phosphates

Polyphosphates, sodium acid pyrophosphate, and metaphosphate are chelating agents of limited cold water solubility. They have been used as antibrowning agents for fresh-peeled fruits and vegetables at concentrations as low as 0.5 to 2 percent (final concentration in the dip solution) (McEvily et al. 1992). Sporix^TM, an acidic polyphosphate mixture (sodium acid pyrophosphate, citric acid, ascorbic acid, and calcium chloride), has been observed to delay the onset of oxidation and enzymatic browning in fruits and vegetables (Gardner et al. 1991).

c. Maltol

Maltol (3-hydroxy-2-methyl-4H-pyran-4-one), a g-pyrone derivative, is a relatively weak inhibitor of the formation of pigmented products. Structurally, it contains an a, b-unsaturated keto-enol constituent, which makes it a good chelator. Maltol does not however chelate copper at the active site of polyphenol oxidase. It prevents browning either through its ability to conjugate o-benzoquinones back to o-dihydroxyphenols or through irreversible inactivation of polyphenol oxidase (Kahn, 1995).

d. Kojic acid

Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one), a g-pyrone derivative, is a fungal metabolite produced by many species of Aspergillus and Penicillium. It is a good chelator of transition metal ions such as Fe (III) and Cu (II) (Beélik, 1956; Wiley et al. 1942). Kojic acid occurs in many fermented Oriental foods (Kinoshita et al. 1968), and possesses both antibacterial and antifungal activities. A mixture of ascorbic acid and kojic acid has been patented for use as an anti-browning agent in foods (Fukusawa et al. 1982). Kojic acid has potential applicability in the prevention of melanosis in both plant and seafood products. Saruno et al. (1979) demonstrated that kojic acid from Aspergillus albus inhibited mushroom PPO activity. Kojic acid was also shown to inhibit melanosis in pink shrimp (Applewhite et al. 1990). Chen et al. (1991b) determined that kojic acid was a competitive inhibitor of the oxidation of chlorogenic acid and catechol by apple polyphenol oxidase.

Kojic acid inhibits the rate of formation of pigmented products, as well as the rate of oxygen uptake, when various o-dihydroxy- and trihydroxy phenols are oxidized by tyrosinase (Kahn, 1995). Tyrosinase inhibition by kojic acid was thought to be due to the ability of kojic acid to bind copper at the active site of the enzyme. Although kojic acid is a good inhibitor of polyphenol oxidase, its toxicity is of concern. Wei et al. (1991) reported weak mutagenic activity of kojic acid in a Salmonella typhimurium assay.

e. Polysaccharides

Various sulfated polysaccharides, including carrageenans, amylose sulfate, and xylan sulfate, were determined to be effective browning inhibitors in both apple juice and diced apples (Tong and Hicks, 1991). Pectin, a naturally occurring anionic polysaccharide at a concentration of 0.5 percent, gave between 5 and 10 percent inhibition of apple juice browning (Tong et al. 1995). Carboxyl groups present in pectin are believed to be capable of chelating the copper moiety of polyphenol oxidase, thus preventing browning.

f. Carbon monoxide (CO)

Carbon monoxide (CO) is a known inhibitor of many copper-containing oxidases and behaves as a noncompetitive inhibitor of phenolic substrates. It has been studied in preventing the discolouration of Shitake mushrooms (Fujimoto et al. 1972). Polyphenol oxidase activity extracted from freeze-dried mushroom powder was inhibited by CO (Albisu et al.1989). This inhibition was however reversible, and removal of CO led to restoration of the initial activity. A two-step gas treatment of potato strips with SO₂ followed by CO, resulted in 93.7 percent and 99.9 percent inactivation of prophenol oxidase and polyphenol oxidase respectively, after 60 days at room temperature (Kramer et al. 1980). A number of safety problems are however associated with the use of carbon monoxide gas.

3.2.4 Complexing agents

a. Cyclodextrins

The cyclodextrins (CDs) are a class of cyclic oligosaccharides produced by the action of cyclomaltodextrin glucanotransferase (CGTase) on liquified starches. Industrially produced CDs contain 6-8 glucose units per macrocycle, linked by a-(1,4)-glycosidic bonds and include cyclomaltohexose (a-CD, 6 units), cyclomaltoheptaose (b-CD, 7 units), and cyclomaltooctose (g-CD, 8 units). The chemical structure and a diagrammatic representation of the functional structure of beta-CD are depicted in Figure 22. Structurally, all of the C-6 (primary) hydroxyl groups project from one side of the torus of the CD, while the C-2, 3 (secondary) hydroxyl groups project from the other. The central cavity of CD is hydrophobic while the outer region of the oligosaccharide is hydrophilic due to the presence of primary and secondary hydroxyls at both the narrow and wide bases. CD's are highly insoluble. Their solubility can however be increased by the action of de-branching enzymes such as isoamylase and pullulanase (Okada et al., 1988).

Figure 22.Schematic and chemical structure of a ß-cyclodextrin molecule.

The most important functional property of cyclodextrins is their ability to behave as clathrate-like compounds in the formation of inclusion complexes with a range of guest molecules. If the guest molecule is of suitable size and conformation that allow it to bind within the hydrophobic core, complex formation takes place. Larger guest molecules form relatively weak complexes due to partial binding. Greater inclusion activity of these larger guest molecules can however be obtained by suitable chemical modification of the CD. This application is of particular interest to the food industry for the molecular encapsulation of insoluble or volatile food ingredients (Pagington, 1986).

The use of CD has been proposed for the control of enzymatic browning in apple products (Billaud et al. 1995; Sapers et al. 1989). CD inhibits juice browning through the binding of polyphenol oxidase substrates. Polyphenols can be removed by b-CD and by insoluble polyvinyl polypyrrolidone or polyethylene glycol (Osuga et al. 1994). The thermodynamics of inclusion complexes of a-CD, b-CD, g-CD, and polymerized b-CD with chlorogenic acid as a substrate of apple polyphenol oxidase were studied in order to elucidate a mechanism for the inhibition of juice browning. Alpha -CD and g-CD were less effective than the b-CD in the inhibition of browning in apple juice (Irwin et al. 1994). The internal cavity of b-CD is slightly apolar, thus allowing it to induce inclusion complex formation with guest molecules such as phenolic substrates of polyphenol oxidases, thereby preventing their oxidation to quinones and subsequent polymerization to brown pigments. The adsorption of flavour or colour compounds by cyclodextrins poses a major drawback to their use in food systems. Although the applicability of cyclodextrins in fruit and vegetable juices has been patented (Hicks et al. 1990), cyclodextrins have not yet been approved for food use by the United States FDA.

b. Chitosan

Chitosan, a naturally abundant polymer of b-(1®4)-N-acetyl-D-glucosamine, is derived from the chitin of shellfish. Chitosan has antimicrobial properties, is soluble in dilute organic acids and is capable of forming films or membranes. Chitosan is non-toxic, biodegradable, and a naturally occurring product in our food supply. It has been shown to inhibit enzymatic browning in apple and pear juices (Sapers, 1992). The addition of 200 ppm chitosan to McIntosh apple juice, resulted in the inhibition of browning. Although the mechanisms by which chitosan inhibits browning are not known, its inhibitory effect is probably a consequence of the ability of the positively charged polymer to adsorb suspended polyphenol oxidase, its substrates, or products. Treatment of shrimp with 2 percent chitosan resulted in a consistently reduced incidence of melanosis during storage (Simpson et al. 1997). Chitosan also exhibited strong antimicrobial properties inhibiting several microorganisms at concentrations ranging between 0.0075 - 0.01 percent (Simpson et al. 1997).

A research study conducted by Zhang and Quantick (1997) indicated that chitosan coating had potential inhibitory activity on polyphenol oxidase and peroxidase activity in lychee (Litchi chinensis Sonn.) fruit. Chitosan has been shown to improve the storability of fruits. Its effectiveness in this respect is therefore thought to be due to the formation of a protective barrier on the surface of fruit, which reduces the supply of oxygen for the enzymatic oxidation of phenolics. Chitosan is non-toxic and is biologically safe (Hirano et al. 1990). Thus, the application of a chitosan coating for the control of browning and quality improvement in fruits and vegetables might be accomplished in combination with other methods such as low temperature and suitable packaging.

3.2.5 Enzyme inhibitors

a. 4-Hexylresorcinol

Substituted resorcinols, which are m-diphenolic compounds that are structurally related to phenolic substrates, have a competitive inhibitory effect on polyphenol oxidase activity (McEvily et al. 1991, 1992). Hydrophobic substitution with hexyl, dodecyl, and cyclohexyl groups at the 4-position of the aromatic resorcinol ring increases the effectiveness of their competitive inhibitory effect on polyphenol oxidase (McEvily et al. 1992). Studies conducted by McEvily et al. (1992) revealed cyclohexyl-substituted resorcinols to have the lowest I_50, i.e. the inhibitor concentration that resulted in 50 percent inhibition of polyphenoloxidase activity, at a concentration of 0.2 mM of the substituted resorcinol. Both the monophenolase and diphenolase activities of tyrosinase are inhibited by 4-hexylresorcinol (4-HR). Four-hexylresorcinol has a long history of use in pharmaceuticals and is considered to be safe and effective in use as an anti-browning agent (Frankos et al. 1991).

Four-hexylresorcinol has several advantages over sulphites when applied in the control of browning in foods. These include its specific mode of inhibitory action, effectiveness at low concentrations, inability to bleach preformed pigments, and chemical stability. It has a synergistic effect with ascorbic acid in the prevention of browning. Ascorbic acid reduces quinones generated by polyphenoloxidase while 4-HR specifically interacts with polyphenol oxidase, and renders it incapable of catalysing the enzymatic reaction (Kahn and Andrawis, 1985) (Figure 23).

Figure 23.The inhibitory effect of 4-hexylresorcinol on PPO. (Adapted from Kahn and Andrawis, 1985).

Four-hexylresorcinol is applicable in the control of browning in fresh and hot-air dried apple slices as well as in apple juice (McEvily et al. 1992). Several studies have shown the effectiveness of 4-HR in controlling enzymatic browning in shrimp (Otwell et al. 1992; McEvily et al. 1991), mushroom (Osuga et al. 1994) and apple slices (Monsalve-Gonzalez et al. 1993). EverFresh^TM a patented product (United States patent # 5,049,438) which consists of 4-HR as the active ingredient and sodium chloride as the carrier agent has been studied as an alternative to sulphites in the control of enzymatic browning, or blackspot, in crustaceans (Lambrecht, 1995). Raw headless brown shrimp dipped in 4-HR for 1 min, exhibited greater stability to blackspot formation for a longer period of time than shrimp dipped in fresh water (controls) or 1.25 percent sodium metabisulphite. After 7 days of storage at 2 ^oC, raw headless brown shrimp treated with water showed 54 percent blackspot; sulphite-treated shrimp showed 11 percent blackspot, while 4-HR-treated shrimp had only 3.6 percent blackspot (Figure 24). At day 14, blackspot on control and sulphite-treated shrimp increased to 75 percent and 25 percent, respectively. The 4-HR treated shrimp did not however show an increase in blackspotting. This compound has been proposed for use on various fruits and vegetables by McEvily et al. (1991). Figure 25 shows inhibition of enzymatic browning by 4-HR in star fruit.

Figure 24. Blackspot (%) in raw head-off brown shrimp treated with 4-HR and 1.25% sulfite, and stored at 2 oC. (From Lambrecht, 1995).

Figure 25. 4-HR treatment (0.02% for 2 min dip) of star fruit. A. Treated B. Non-treated.

Four-hexylresorcinol is a chemically stable, water-soluble compound. Toxicological, mutagenic, carcinogenic, and allergenic studies have shown that there are no risks associated with the levels of 4-HR used in the treatment of shrimp (Frankos et al. 1991). Four-hexylresorcinol has obtained GRAS status from the United States Food and Drug Administration, for use on shrimp (Federal Register, 1992). Its use in the inhibition of shrimp melanosis has no effect on taste, texture, or colour at residual levels of less than 1 ppm (Iyengar et al. 1991; King et al. 1991).

b. Halide salts

Inorganic halides are well-known inhibitors of polyphenol oxidases (Vámos-Vigyázó, 1981). Janovitz-Klapp et al. (1990) determined that NaF was the most potent inhibitor of apple polyphenol oxidase, followed by NaCl, NaBr, and NaI. The inhibition of enzymatic browning by halides decreases with increasing pH. Sodium chloride and calcium chloride at concentrations of ranging between 2 and 4 percent (w/v) are most commonly used in the food industry for the inhibition of browning (Steiner and Rieth, 1989). Poplyphenol oxidase activity was observed to decrease with increasing concentrations of NaCl for peach (Luh and Phithakpol, 1972), eggplant and avocado (Knapp, 1965). Sodium zinc chloride was shown to be a highly effective browning inhibitor when used in combination with calcium chloride, ascorbic acid, and citric acid (Bolin and Huxsoll, 1989).

c. Honey

Honey has been shown to inhibit enzymatic browning. The use of honey as a natural browning inhibitor is therefore of great consumer interest. Honey was shown to inhibit browning in apple slices, grape juice and in model systems (Oszmianski and Lee, 1990). The browning of apple slices was inhibited to a greater extent by 10 percent honey, than by a sucrose solution containing an equivalent sugar concentration. Purification of honey by Sephadex G-15 column chromatography revealed the compound in honey, responsible for the inhibition of polyphenol oxidase, to be a small peptide of approximately 600 Da molecular weight. Proteins, peptides, and amino acids exert an inhibitory effect on polyphenol oxidase activity by chelating the essential copper at the active site of polyphenol oxidase, thus forming stable complexes with Cu²⁺(Kahn, 1985). The honey peptide is thought to exert its inhibitory effect through a similar mechanism.

Honey has been shown to contain antioxidants: tocopherols, alkaloids, ascorbic acid, flavonoids, and phenolics. The antioxidant content and the efficacy of honeys in inhibiting polyphenol oxidase activity vary in accordance with the type of honey (Chen et al, 1998). The effect of commercial browning inhibitors (ascorbate and sodium metasulphite) and various honeys were compared in a study on polyphenol oxidase activity and browning. Antioxidant content showed a positive correlation to honey colour. The addition of various honeys to fresh potato homogenates, resulted in a 0-50 percent reduction in polyphenol oxidase activity and a decrease of 0-7 units in the browning index.

d. Amino acids, peptides and proteins

Amino acids, peptides or proteins can affect polyphenol oxidase-catalysed browning either through direct inhibition of the enzyme or by reacting with the quinone products of polyphenol oxidase catalysis (McEvily et al. 1992). Proteins, peptides and a-amino acids are capable of forming stable complexes with Cu²⁺ (O. Sullivan, 1969). In addition, they are also capable of chelating copper at the active site of polyphenol oxidase. Histidine and cysteine have particularly high affinities for Cu²⁺ since, apart from having NH₂ and COOH groups, histidine possesses an imidazole ring and cysteine, a thiol group, both of which have metal binding capacity (Bell, 1977).

Kahn (1985) studied the effects of proteins, protein hydrolysates, and amino acids on o-dihydroxyphenolase activity in mushroom, avocado and banana. Mushroom PPO was weakly inhibited by mM concentrations of L-lysine, glycine, L-histidine and L-phenylalanine. L-cysteine was the most effective amino acid in inhibiting o-dihydroxyphenolase activity. Amino acids inhibit polyphenol oxidase activity through the formation of stable complexes with copper. In addition, thiol containing inhibitors form sulfur adducts with the o-quinone, thus blocking polymer formation and preventing browning (Figure 26). Mason and Peterson (1965) showed that N-terminal primary amino groups, aliphatic amino groups (secondary amines in amino acids) and thiol-containing amino acids react with o-benzoquinones and 4-methyl-o-benzoquinone, while only thiol-containing compounds and aromatic amines react with oxidation products of DOPA (dihydroxyphenylalanine).

Figure 26. Postulated mechanism for the inhibition of PPO induced enzymatic browning by thiols. (From Friedman and Molnar-Perl, 1990.

e. Aromatic carboxylic acids

Aromatic carboxylic acids of the benzoic acid and cinnamic acid series are polyphenol oxidase inhibitors, owing to their structural similarity to phenolic substrates (Krueger, 1955). Undissociated forms of these acids are capable of inhibiting polyphenoloxidase, through complexation with copper at the active site of the enzyme. The degree of polyphenol oxidase inhibition by carboxylic acids is pH dependent, and increases with a decrease in pH.

Cinnamic acid and its analogues, p-coumaric, ferulic, and sinapic acids were found to be potent inhibitors of potato (Macrae and Duggleby, 1968) and apple polyphenol oxidases (Pifferi et al. 1974; Walker and Wilson, 1975). Cinnamic acid at levels of 0.01 percent was observed to be effective in providing long-term inhibition of polyphenol oxidase in apple juice (Walker, 1976). Benzoic acid and its derivatives had an inhibitory effect on polyphenol oxidase activity in mushrooms (Kermasha et al. 1993) and grapes (Gunata et al. 1987).

f. Aliphatic alcohols

Although the inhibition of polyphenol oxidase by ethanol has been reported (Kidron et al. 1978), there are no extensive studies which describe the effect of aliphatic alcohols on polyphenol oxidase. Valero et al. (1990) studied the effects of natural aliphatic alcohols on grape polyphenoloxidase. Inhibition was observed to increase with increasing chain length of the aliphatic alcohol.

3.3 Other inhibitors

3.3.1 Killer enzymes

As paradoxical as it may seem, enzyme action can be exploited for the control of undesirable enzyme activities. This is achievable in three ways: (1) substrate and/or product modification by enzymes other than the target enzymes; (2) direct inactivation of the target enzyme by other enzymes and (3) inactivation by secondary reactions of highly reactive products. The activities of "killer enzymes" or "anti-enzyme enzymes" which inactivate other enzymes via direct proteolytic activity have been demonstrated. The use of enzymes to control other enzyme-related processes has also has been reported in the control of enzymatic/nonenzymatic browning.

a. Ring-cleaving oxygenases

Kelly and Finkle (1969) proposed irreversible modification of phenolic substrates by enzymes as one mechanism for the control of browning. Apple juice treated with the bacterial enzyme protocatechuate-3, 4-dioxygenase in combination with ascorbic acid prevented browning due to the oxidative ring-opening reaction and ortho-fission of catechols by the enzyme. The enzyme deprived polyphenol oxidase enzymes of required substrates (Kelly and Frinkle, 1969).

b. Catechol transferase

Finkle and Nelson (1963) proposed the use of catechol transferase (EC 2.1.1.6) for the prevention of browning in apple juice. O-methyl transferase, an enzyme capable of methylating the 3-position of 3,4-dihydroxy aromatic compounds was observed to cause irreversible modification of phenolic substrates, thus preventing them from serving as substrates of the browning reaction. Treatment of apple juice with o-methyl transferase and s-adenosyl methionine resulted in conversion of chlorogenic and caffeic acids, to feruloylquinic and ferulic acids respectively, both of which are polyphenol oxidase inhibitors (Finkle and Nelson, 1963).

c. Proteases

The plant proteases ficin, papain and bromelain are sulphydryl enzymes of broad specificity (Labuza et al. 1992; Taoukis et al. 1990) which are very effective browning inhibitors. Ficin was observed to be effective in preventing black spot formation in shrimp under refrigerated conditions (Taoukis et al. 1990). This inhibitory effect is thought to be due to either binding or hydrolysis at specific sites necessary for polyphenol oxidase activity.

Pineapple juice was found to be effective in inhibiting browning in apple rings (Lozano-De-Gonzalez et al. 1993). Bromelain, organic acids, sulphydryl compounds and certain metallic constituents of pineapple juice are thought to be responsible for this inhibitory effect. Polyphenol oxidase activity in plum juice was significantly reduced when the juice was passed through a column containing immobilized proteases (Arnold et al. 1992).

Commercial application of enzyme treatments in the control of enzymatic browning is precluded by their high cost. Combinations of anti-browning agents of a chemical nature are however more affordable and effective in commercial use.

3.3.2 Edible Coatings

The use of edible coatings to minimize undesirable changes due to minimal processing has been reported for several commodities (Baldwin et al., 1995). The coating of fruits and vegetables with semi-permeable films has been shown to retard ripening through modification of endogenous CO₂, O₂ and ethylene levels. Coatings are also useful as carriers of antioxidants and preservatives (Cuppett, 1994). Edible coatings have the potential to retard water loss, to form a barrier to oxygen, and to retain antioxidants, as well as preservatives, on the surface of cut tissue in order to control discolouration. A polysaccharide/lipid bilayer formulation was observed to reduce respiration in cut apples (Wong et al. 1994) through modification of the gas exchange between the processed tissue and the external environment. Sucrose fatty acid esters reduced browning of shredded cabbage (Sakane et al. 1990) which was attributed to reduction of oxygen at the cut surface. Zhang and Quantick (1997) determined that an edible coating based on sucrose esters of fatty acids significantly delayed pericarp browning of lychee fruit. Carboxymethyl cellulose/soy protein coating formulations containing 0.5 percent ascorbic acid applied to freshly cut apples were more effective in antibrowning activity than aqueous solutions of 0.5 percent ascorbic acid alone (Baldwin et al. 1996). The cellulose matrix may well have a protective effect in preventing the degradation of ascorbic acid by oxygen.

4. MOLECULAR BIOLOGY OF PHENOXIDASES

Polyphenol oxidases occur in the chloroplasts of almost all higher plants. Cloning of the genes which code for polyphenol oxidase offers the potentials for determining the physiological role of polyphenol oxidase within the chloroplast and for manipulating polyphenol oxidase levels within specific organs. Polyphenol oxidase genes are encoded within the nucleus and undergo translation within the cytoplasm. Once formed, propolyphenol oxidase is transported to the chloroplast where it undergoes proteolytic cleavage, to produce the active polyphenol oxidase form (Vaughn et al., 1988). Predicted molecular weights for polyphenol oxidase in plants, range between 57 and 62 kDa (Hunt et al. 1993; Newman et al. 1993).

Polyphenol oxidase is generally present in low concentrations in all organisms. The enzyme is difficult to obtain in a pure form due to pigment contamination and the occurrence of multiple forms. With the advent of recombinant DNA technology, numerous amino acid sequences of polyphenol oxidases have become available. Primary structures of polyphenol oxidases from Streptomyces glaucescens (Huber et al. 1985), Streptomyces antibioticus (Bernan et al. 1985) and Neurospora crassa (Lerch, 1982), tomato (Shahar et al. 1992; Newman et al. 1993), broad bean (Cary et al. 1992) potato (Hunt et al. 1993), mice (Shibahara et al. 1986) and humans (Kwon et al. 1987; Giebel et al. 1991) have been determined using cDNA sequencing techniques. Polyphenol oxidases of closely related plants, such as tomato and potato, show approximately 91 percent exact homology, while those of tomato and fava bean show only 40 percent exact homology (Wong, 1995).

Polyphenol oxidases from different sources exhibit molecular weight differences. Molecular weights predicted for mature polyphenol oxidases on the basis of cDNA sequences were 58 kDa for mouse, ~63 kDa for human and 128 kDa for mushroom. Mushroom polyphenol oxidase is thought to contain four subunits having a total molecular weight of 128 kDa. Monomeric through octameric forms of mushroom polyphenol oxidases are known to exist (Whitaker and Lee, 1995). Plant polyphenol oxidases are nuclear-encoded copper metalloproteins having a molecular mass of approximately 59 kDa and are localized in the membranes of plastids. Plant genes encoding polyphenol oxidase have recently been cloned and characterised. Although the sequences of plant polyphenol oxidase genes are very similar, only the putative copper binding sites are conserved when plant genes are compared to mammalian, bacterial, or fungal tyrosinases. One possible approach to lowering polyphenol oxidase activity and resultant enzymatic browning reactions is to characterize and inactivate the genes which code for polyphenol oxidase. Inactivation can be accomplished by generating antisense RNAs specific for polyphenol oxidase.

4.1 New approaches for the control of PPO

The involvement of polyphenol oxidase in browning has been studied for a long time. Many questions still remain about the enzyme itself, as well as the mechanism of browning. Chemical and physical methods for controlling enzymatic browning have been reviewed in Chapter 3. Current approaches to understanding and controlling enzymatic browning are however specifically focused on the use of antisense RNA. Antisense RNA techniques have several applications in plant research. They are applicable in studying the in vivo function of particular genes and their biochemical modes of action. Antisense genes have been successfully used for the alteration of plant processes, such as flower pigmentation, fruit ripening and photosynthesis, and to determine the function of cryptic genes. They may also be put to practical use in crop improvement. Antisense RNAs were recently observed to selectively block the gene expression of other plant enzymes such as polygalacturonase and peroxidase in tomatoes.

4.2 Antisense RNA approach

One of the most successful methods developed in recent years for the inhibition of gene expression in plants has been the expression of introduced antisense genes. Antisense technology is based on blocking information flow from DNA via RNA to protein by the introduction of an RNA strand complementary to the sequence of the target mRNA. It is generally assumed that the antisense RNA basepairs to its target mRNA thereby forming double-stranded RNA. Duplex formation may impair mRNA maturation and/or translation or alternatively may lead to rapid mRNA degradation.

Technically, it involves the insertion of a gene or a significant part of it, into the cell in a reverse orientation. Messenger RNA encoded by this antisense gene undergoes hybridization with that encoded by the endogenous gene, precluding production of the protein product (Figure 27). Gene silencing or the elimination of expected phenotypic characteristics, through antisense techniques has received much attention in recent years. The expression of a transgene (i.e. a gene that has been introduced into plant cells through molecular biology techniques) or an endogenous gene appears to be affected by the presence of a homologous transgene. Antisense-mediated control has been observed in bacteria, fungi, plants and mammalian systems. The biological function of naturally occurring antisense RNAs, if any, remains to be determined. Complementary mRNA levels can be reduced in the presence of antisense genes.

Figure 27. Simplified schematic showing how antisense RNA can be used to control gene expression at the translational level (P represents the promoter). (From Martinez and Whitaker, 1995).

Polyphenol oxidase genes have been characterized in broad bean, tomato, potato, and grape. Seven genomic polyphenol oxidase genes were identified in tomato (Newman et al. 1993). Characterization of polyphenol oxidase genes in various plant species has shown that these genes are present in the plant genome as gene families. Such transcriptional regulation offers potential for the design of antisense constructs. Antisense constructs would be required for maintenance of the enzyme if polyphenol oxidase is required for chloroplast metabolism or for resistance to pathogens or predation.

In addition to improving a fundamental knowledge of biological processes, the antisense approach has been applied, to increasing the shelf life of fruit (Fray and Grierson, 1993). Commercial applications of antisense technology now include alterations of flower colour, virus resistance and fruit ripening. The application of antisense technology also extends to improving food quality.

Bachem et al. (1994) determined that the expression of polyphenol oxidase in potatoes was decreased through the use of vectors carrying antisense polyphenol oxidase cDNAs. Approximately 70 percent of the transformed plants had lower polyphenol oxidase activity than controls, and on visual scoring, a significantly low level of discolouration. Insertion of polyphenol oxidase in the sense orientation resulted in very high polyphenol oxidase activity in the lines expressing the construct.

Breeders have been working to decrease polyphenol oxidase levels in apples, bananas, mushrooms, peaches and other plants for many years. Lack of bruising sensitivity in transgenic potatoes, and the absence of any apparent detrimental side effects, opens up the possibility for preventing enzymatic browning in a wide variety of food crops, without resorting to chemical and physical treatments. Quite possibly, browning-resistant varieties may be developed in the future through the insertion of antisense genes that prevent the production of polyphenol oxidase.

5. CONCLUSION

New approaches to control enzymatic browning are under study at universities, government research laboratories, and industry. These alternatives must be evaluated on the basis of effectiveness, cost, and regulatory status. Inhibitors of enzymatic browning must not affect product flavour, texture, and colour. The choice of a particular anti-browning agent will depend on these factors, as well as the method of treatment.

A variety of methodologies can be applied in the control of enzymatic browning. In addition to physical treatment, a wide range of chemicals inhibit polyphenol oxidase activity. Only a limited number of browning inhibitors are considered acceptable with respect to consumer safety and/or cost and act as potential alternatives to sulphites. Whether chemical treatments of food products are achievable or not will depend on their effectiveness and cost relative to that of alternative approaches and on the regulatory status of their use as food additives.

Melanosis Scale	Description
0	Absent
2	Slight, noticeable on some shrimp
4	Slight, noticeable on most shrimp
6	Moderate, noticeable on most shrimp
8	Heavy, noticeable on most shrimp
10	Heavy, totally unacceptable

Test	Catecholase (o-DPO)	Laccase (p-DPO)
Substrate specificity:
o-Dihydroxyphenols	oxidized	-
p-Dihydroxyphenols	nil or slow	oxidized
p-Cresol	oxidized
Guiacol	-	oxidized
1-Naphthol	-	oxidized
p-Phenylene-diamine	-	oxidized
Syringaldazine	-	oxidized
Inhibitor specificity:
Cinnamic acid, p-coumaric acid and ferulic acid	inhibition	nil
Polyvinylpyrrolidone (PVP)	inhibition	nil
Salicylhydroxamic acid (SHAM)	inhibition	nil
4-Hexyl-resorcinol	inhibition	nil
Quaternary ammonium compounds (QACs)	nil	inhibition

Source	Phenolic substrates
Apple	chlorogenic acid (flesh), catechol, catechin (peel), caffeic acid, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxy benzoic acid, p-cresol, 4-methyl catechol, leucocyanidin, p-coumaric acid, flavonol glycosides
Apricot	isochlorogenic acid, caffeic acid, 4-methyl catechol, chlorogenic acid, catechin, epicatechin, pyrogallol, catechol, flavonols, p-coumaric acid derivatives
Avocado	4-methyl catechol, dopamine, pyrogallol, catechol, chlorogenic acid, caffeic acid, DOPA
Banana	3,4-dihydroxyphenylethylamine (Dopamine), leucodelphinidin, leucocyanidin
Cacao	catechins, leucoanthocyanidins, anthocyanins, complex tannins
Coffee beans	chlorogenic acid, caffeic acid
Eggplant	chlorogenic acid, caffeic acid, coumaric acid, cinnamic acid derivatives
Grape	catechin, chlorogenic acid, catechol, caffeic acid, DOPA, tannins, flavonols, protocatechuic acid, resorcinol, hydroquinone, phenol
Lettuce	tyrosine, caffeic acid, chlorogenic acid derivatives
Lobster	tyrosine
Mango	dopamine-HCl, 4-methyl catechol, caffeic acid, catechol, catechin, chlorogenic acid, tyrosine, DOPA, p-cresol
Mushroom	tyrosine, catechol, DOPA, dopamine, adrenaline, noradrenaline
Peach	chlorogenic acid, pyrogallol, 4-methyl catechol, catechol, caffeic acid, gallic acid, catechin, Dopamine
Pear	chlorogenic acid, catechol, catechin, caffeic acid, DOPA, 3,4-dihydroxy benzoic acid, p-cresol
Plum	chlorogenic acid, catechin, caffeic acid, catechol, DOPA
Potato	chlorogenic acid, caffeic acid, catechol, DOPA, p-cresol, p-hydroxyphenyl propionic acid, p-hydroxyphenyl pyruvic acid, m-cresol
Shrimp	tyrosine
Sweet potato	chlorogenic acid, caffeic acid, caffeylamide
Tea	flavanols, catechins, tannins, cinnamic acid derivatives

1.	Introduction
1.1	General overview of enzymatic browning
1.2	Economic benefits of browning in fruits and vegetables
1.3	Economic losses due to browning in fruits and vegetables
1.4	Economic benefits of browning in aquatic foods
1.5	Economic losses due to browning in aquatic foods
2.	Physiological role of enzymatic browning
2.1	Plants
2.2	Animals
2.3	Characteristics of polyphenol oxidase
2.3.1	Monophenol oxidase
2.3.2	Diphenol oxidase
2.3.3	Laccase
2.4	Phenolic substrates
3.	Control of browning
3.1	Processing
3.1.1	Heating
3.1.2	Refrigeration
3.1.3	Freezing
3.1.4	Dehydration
3.1.5	Irradiation
3.1.6	High pressure treatment
3.1.7	Treatment with supercritical carbon dioxide
3.1.8	Ultrafiltration
3.1.9	Ultrasonication
3.2	Inhibitors
3.2.1	Reducing agents/ Antioxidants
a.	Sulphiting agents
b.	L-Ascorbic acid
c.	Erythorbic acid
d.	Cysteine
e.	Phenolic antioxidant
3.2.2	Acidulants
a.	Citric acid
3.2.3	Chelators
a.	EDTA
b.	Phosphates
c.	Maltol
d.	Kojic acid
e.	Polysaccharides
f.	Carbon monoxide
3.2.4	Complex agents
a.	Cyclodextrins
b.	Chitosan
3.2.5	Enzyme inhibitors
a.	4-Hexylresorcinol
b.	Halide salts
c.	Honey
d.	Amino acids, peptides and proteins
e.	Aromatic carboxylic acids
f.	Aliphatic alcohols
3.3	Other inhibitors
3.3.1	Killer enzymes
a.	Ring-cleaving oxygenases
b.	Catechol transferase
c.	Proteases
3.3.2	Edible coatings
4.	Molecular biology of phenoloxidases
4.1	New approaches for the control of PP
4.2	Antisense RNA approach
5.	Conclusion