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Enzymatic browning is one of the major causes in quality loss of vegetables and fruit, beverage and seafood. It is roughly accounted for over 50 percent losses in fruit . The control of browning is very critical for minimizing losses and maintaining economic value to the agriculturist and food processor. In animal tissues, melanosis is the process results in the formation of dark pigments. Although the products of melanosis are not harmful and do not influence flavour or aroma, consumers will not select these products since their brown discoloration connotes spoilage. Browning causes many deleterious changes in the organoleptic and nutritional properties of food products that decrease their market values. However, enzymatic browning is beneficial for some of the food products such as tea, coffee, and cocoa. Nutritional recommendations for the increase of the 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 trigger considerable interest in enzymatic browning.
Enzymatic browning definition
Cell disruption caused by mechanical injury or temperature changes may lead to physiological disorders or even cell death. This loss of cell integrity results in the decompartmentation of phenolic substrates and enzymes . Polyphenol oxidase (PPO) is a critical enzyme catalyzing the oxidation of endogenous phenolic compounds to form grey or brown colors in the enzymatic browning process. PPO is a term which includes a large number of related copper-containing enzymes, including catechol oxidase, tyrosinase and laccase. In the presence of oxygen, PPO oxidizes mono- and di-phenols to o-quinones. Once the products are formed, they polymerize to form high molecular weight compounds or brown pigments as they are highly reactive. The brownish color could be enhanced when they react with amino acids and proteins .
Importance of browning inhibition
It is essential to control enzymatic browning in order to reduce the economic loss during fruit and vegetables processing. It is essential to control enzymatic browning for the better development in the food industry. The most common way used for the inhibition of browning is the addition of sulfating agents to inhibit PPO. However, sulfite is a toxic molecule and can react with a variety of humoral and cellular components and can cause toxicity . Due to the adverse health effects caused by sulfites, alternative compounds are investigated for anti-browning properties. The ideal compound would be the one which is effective, does not cause any quality loss (eg. taste, flavor, nutritional values) and toxicity, as well as low cost. Until now, many approaches are available to inhibit enzymatic browning, which include the use of ascorbic acid, sodium chloride, kojic acid, 4-hexylresorcinol etc. Enzyme inhibitors, reducing agents, acidulant agents, chelating agents and complexing agents are the common chemical treatments of antibrowning . In order to have a better control of enzymatic browning, it is important to understand its mechanism of actions, the property of the enzyme, substrates, products and their interactions. Minimizing the negative sides of enzymatic browning can extend the shelf life and increase the market value of the food products.
Physical treatments are one of the common approaches in the control of enzymatic browning in the food industries. Hot-air drying, freeze-drying, canning and bottling are examples of some preventive methods. Browning is enhanced by wounding of plant tissue as it stimulates phenolic biosynthesis, hence minimizing the mechanical damage of fruit and vegetables reduce the degree of browning. Reducing the temperature can slow down the rate of enzymatic browning as enzymes are generally inactive at low temperature. However, color change still occurs at 0oc, hence it is important to allow the crystallization temperature of water to be reached as rapidly as possible . Color change would be blocked during commercial frozen (-18oC). When the temperature rises again, browning starts and the rate will be greater if the cellular structure of the plant tissues is damaged by freezing, peeling and slicing . Heating or blanching is the simplest method to inhibit browning by denaturing the enzyme PPO. Heating temperature and time are crucial to maintain the quality of the food products as heating will affects the texture, taste, flavor and nutritional value of the food. PPO is completely destroyed at 80oC , but sufficient time is required for the interior part of the fruit or vegetables to reach such temperature. Blanching of green beans at temperatures of 82 oC and above for 3.5 minutes, almost completely inactivated catalase, lipoxygenase, and polyphenol oxidase activities . Protection of the fruit and vegetables from oxygen can prevent enzymatic browning when the plant organs cannot be branched. A variety of methods are used in food industries, including airtight package, partial vacuum and oxygen-poor atmosphere.
Chemicais are the most commonly used for the control of enzymatic browning, but their use in processed food products is restricted to compounds that are nontoxic, wholesome and that do not adversely affect the quality. Inhibition of enzymatic browning can be achieved by hindering the enzyme or by binding to its active site which lower the PPO activity. Chelating agents which remove the copper prosthetic group from PPO can inhibit enzymatic browning. Removing the substrates is another way to prevent enzymatic browning. Substances which undergo complexation with the phenolic substrate and enzymatic modification of phenols are possible ways to inhibit enzymatic browning. Besides substrate, inhibition targeted toward the products, o-quinones, can also be an approach to control enzymatic browning. Chemical inhibitors of enzymatic browning in food will be discussed in detail in this essay.
2. Polyphenol oxidase
PPOs are first discovered in mushrooms by Schoenbein in 1856. They are copper-containing proteins and belong to the group of oxidoreductases. A copper prosthetic group is required for its activity. In the presence of oxygen, polyphenol oxidase catalyses the o-hydroxylation of monophenols to o-diphenols (monophenol oxidase activity). It also oxidises o-diphenols to o-quinones (dipehnol oxidase activity). It is still not clear whether a single enzyme system exhibits both mono- and di- phenol oxidase activities, but it is found that when both monophenol- and diphenol oxidases are present in plants, the ratio of monophenol to diphenol oxidase activity is usually 1:10 to 1:40 . PPO is present in a wide variety of plants and the activity varies from one organ to another and varies inside an organ, depending on the types of tissues or cells. It is thought that PPOs play an important role in the resistance of plants to microbial and viral infections and to adverse climatic conditions. PPOs are also in involved the development of dark pigmentation in animals. The biosynthesis of melanin pigments and other polyphenolic compounds which often provide a protective function . Indeed, there are many studies concerning the prevention of melanin formation on the face by inhibiting tyrosinase activity, so as to develop whitening agents for the cosmetic industry.
Nomenclature of PPOs is sometimes confusing due to their similarity in nature. Jaenicke and Decker write ï¿½ï¿½ï¿½ï¿½Probably there is no common tyrosinase: the enzymes found in animals, plants and fungi are different with respect to their sequences, size, glycosylation and activation ï¿½ï¿½ï¿½ï¿½ PPOs must have a dinuclear copper centre, in which type 3 copper is bound to histidine residue in their active sites, and this structure is highly conserved . PPOs are normally classified into three types for the convenient use of the term:
Catechol oxidase (1,2-benzenediol: oxygen oxidoreductase, EC184.108.40.206) has both monophenol oxidase and diphenol oxidase activity. It is the most common form of PPOs in plants that when people talked about PPOs in food it normally refers to catechol oxidase. Reports on the molecular weight of plant PPO are very diverse and variable and it is estimated the molecular masses of PPOs ranges from 20 to 180kDa . Most of the studies show PPOs have an optimum activity between pH 4 and 7, but some reports show that pH optima varies with the proportions of isoenzymes as well as phenolic substrates used . The optimum temperature of PPOs ranges from 15 to 40 oC and this also depends on the same factors as pH.
Tyrosinase (EC220.127.116.11, monophenol, L-DOPA: oxygen oxidoreductase) refers to as monophenol monooxygenase and corresponds to the same enzymes as EC18.104.22.168 but which always catalyze the hydroxylation of monophenols. In aminals and fungi, PPOs are refered to tyrosinase as L-tyrosine is the major monophenolic substrate. It is very similar to catechol oxidase and sometimes tyrosinase is referred as PPOs. It catalyze two distinct reactions of melanin synthesis: the hydroxylation of tyrosine and the oxidation of 3,4-dihydroxyphenyphenalanine (L-DOPA) to o-dopaquinone. Tyrosinase has been a great concern in humans in the cosmetic industry as it plays a key role in mammalian melanogenesis, which leads to the formation of dark macromolecular pigments, melanin.
Laccase (p-diphenol oxidase, E.C. 22.214.171.124)(DPO), has the unique ability to oxidase p-diphenols into their corresponding quinones, which allows it to be distinguished from catechol oxidase. The enzyme contains many subunits and there are three types of copper centers in each subunit. They occur mainly in fungi and higher plants while much less frequently in the plant kingdom than PPOs. They are glycoproteins with a carbohydrate content of about 15-41% and their molecular weight range between 60 and 80 kDa. Laccase is also a component for synthesizing lignin in woody tissues .
Phenolic compounds are substrates for PPOs. There is a wide variety of phenolic compounds found in plants, but only a small number serves as direct substrates for PPOs. Degree of enzymatic browning varies widely from one plant to another. Not only content of PPOs contributes to the variation, quantitative and qualitative aspects of their phenolic content also affect enzymatic browning in fruit and vegetables. In the studies of enzymatic browning in various fruits such as apples, grapes, and peaches, the relationship of the rate of browning to phenolic content and polyphenol oxidase activity has been reported [12-14]. Phenolic compounds contain an aromatic ring with one or more hydroxyl groups, together with other substituents. The differences in species, degree of ripening and environmental conditions of growth and storage affect the phenolic composition of plants. Phenolic compounds are essential in fruit and vegetables as they contribute to flavor, color and taste in fruits. Flavonoids are the major phenolic compounds occurred in plants. Caffeic acid derivatives and monomeric flavan-3-ols often appear to be the best substrates . In some cases, phenolic compounds which are not direct substrates can also take part actively in enzymatic browning by coupled oxidation reaction. Tyrosine, an amino acid, is a monohydroxy phenol and it is an important substrate for PPOs in animals. Diagram below shows some of the common PPO substrates:
Caffeic acid derivatives, such as chlorogenic acid are the major o-diphenolic compounds in plants that act as substrates for polyphenol oxidases. Chlologenic acid is the main substrates for enzymatic browning in apples. On the other hand, some phenolic compounds may also act as inhibitors for PPOs. Apple polyphenol oxidases can be inhibited by various cinnamic acids acting as substrate analogues .
2.4 Mechanism of action
PPO catalyzes both the hydroxylation of monophenols to o-diphenols and the dehydrogenation of o-diphenols to o-quinones as shown below:
Figure 2. Action of PPO on monophenols to produce quinines 
Monophenol oxidase activity for the hydroxylation reaction is normally slower than the oxidation reaction required for quinone production and initiation of the browning reaction. Thus it is the rate determining step in the production of brown pigments from monophenols. One mole of oxygen is required for the reaction as shown in Figure 2. When there are only diphenolic substrates, production of the brown pigments will be faster due to their high catalytic rate in the formation of quinines.
The primary products, o-quinones, are reactive and unstable compounds, which can undergo further oxidation to brown melanin pigment (figure 3). O-quinones are strong electrophiles which can suffer nucleophilic attack by water, other polyphenols, amino acids and proteins, leading to Michael-type addition products [16-18]. The final products have more intensive color than the o-quinones. Their color depends on the phenol from which they originate.
Figure 3. Formation of melanin from tyrosine 
3. Chemical inhibitors of PPO
3.1 Plant Polyphenols
Polyphenols are a diverse group of compounds having multiple phenolic functionalities . Plants are a rich source of polyphenols which are almost free of harm effects. There are continued investigations of tyrosinase inhibitors from plant extracts, aiming to search what bioactive chemicals in plants have significant inhibitory effect. Flavonoids are one of the most common and best studied groups of plant polyphenols. Flavonoids can be further divided into six subgroups, which are flavanones, isoflavones, flavonols, flavanols, flavones and anthocyanidins. Some flavonoids such as catechin act as substrates for PPO while some of them, eg. quercetin and kaempferol have inhibitory effect on PPO [21, 22]. Isao Kubo studies the inhibition kinetics of kaempferol extracted from saffron flower and it is found that to be a competitive inhibitor, its inhibitory activity presumably comes from its ability to chelate copper in the enzyme . This copper chelation mechanism may be applicable for all of the flavonols as long as their 3-hydroxyl group is free . However, 3-o-glycoside derivatives, eg. kaempferol 3-o-glucoside, quercetin 3-o-glucoside, did not inhibit tyrosinase in high concentration . This shows that the free hydroxyl group at C-3 may play an important role in the inhibition. However, some flavones which lack the 3-hydroxyl group, such as luteolin and luteolin-o-glucoside, still have tyrosinase inhibitory effect . (?)-epicatechin 3-O-gallate(ECG), (?)-gallocatechin 3-O-gallate(GCG), and (?)-epigallocatechin 3-O-gallate(EGCG) in tea, especially green tea, are strong inhibitor of tyrosinase and it is found that GCG may act as a competitive inhibitor for the L-tyrosine binding at the active site of the enzyme . It is believed that the flavon-3-ol skeleton with a galloyl moiety at the 3-position is required to inhibit tyrosinase effectively. When the hydrophobicity of gallates increases, the coumpounds are more resistant to be oxidized by tyrosinase as the tertiary structure of the enzyme is disrupted .
Structure-Activity Investigations of stilbenes and related 4-substituted resorcinols, obtained from Artocarpus incisus and other plants suggested that the 4-substituted resorcinol skeleton have potent tyrosinase inhibitory ability . Oxyresveratrol, a stilbene isolated from Morus alba, exhibited 32 times inhibitory effect of kojic acid on tyrosinase and the inhibitors act non-competitively on the enzyme . In the structure analysis of extracts from licorice root, glabridin exerts the strongest inhibitory effect on tyrosinase and the inhibitory effect was more effective against monophenol oxidase activity, the rate-determining step, than diphenol oxidase activity . However, glabridinï¿½ï¿½s analog, glabrene, was 100-fold less active than glabridine while glyasperin C, isolated from the same part of the plant, was two times more active than glabridin [28, 29]. 6-hydroxydaidzein (6,7,4ï¿½ï¿½-trihydroxyisoflavone), one kind of hydroxyisoflavones isolated from soybean koji fermented with Aspergillus oryzae, had 6-fold more than kojic acid acting competitively on the tyrosine binding site of the enzyme . It is found that an isoflavone with hydroxyl groups at both the C6 and C7 positions in the A ring would increase both inhibitory effect and affinity to the enzyme more than 10 times .
Chalcones, with two aromatic rings in trans configuration, have strong inhibitory effects on tyrosinase. Licochalcone A isolated from the roots of glycyrrhiza species competitively inhibit tyrosinase and the effect was 5.4 times more active than kojic acid . Kuraridin, isolated from Sophora flavescens, was 34 times of the activity of kojic acid in inhibiting monophenolase activity of mushroom tyrosinase . It is believed that the 2,4-dihydroxyl groups in the aromatic ring of chalcone structure was the important substituted group to exert strong tyrosinase inhibitory activity as some simple 4-alkylresorcinols were found to exhibit the strong inhibitory effect [26, 33].
3.2 Chelating agents
Chelating agents are chemicals that bind to metal ions and remove them from their sphere of action. EDTA (ethylenediaminetetraacetic acid) is a chelator permitted to be used in the food industry as a chemical preservative. It is believed that EDTA inhibits PPO activity by either binding to the active copper site of PPO or reduces the availability of copper for the enzyme . 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 . EDTA is usually used with other chemicals such as reducing agents (ascorbic acid) and acidulants (citric acid) to prevent enzymatic browning in food. Polyphosphates are another common chelating agent for anti-browning of fresh-peeled fruits and vegetables, but it has low solubility in cold water . Sporix, containing sodium acid pyrophosphate, citric acid, ascorbic acid, and calcium chloride, is a powerful chelator and also an acidulant. Browning prevention in apple juice and cut surfaces was obtained by Sporix . However, it is not approved in U.S. for food use.
Kojic acid (C6H6O4; 5-hydroxy-2-(hydroxymethyl)-4-pyrone), always being use as a standard to compare and study the tyrosinase inhibitory activity of various chemicals in research, is produced by several species of fungi, especially Aspergillus oryzae. Kojic acid possesses both antibacterial and antifungal activities. It is a good chelator and antioxidant that can prevent browning in both plants and seafood. Kojic acid was a competitive inhibitor of the oxidation of chlorogenic acid and catechol by apple polyphenol oxidase . It is commonly found as a food additive and in cosmetic products due to its tyrosinase inhibitory effect. In the phenolic acids tested (caffeic, chlorogenic, cinnamic, coumalic, ferulic, gallic, kojic) for inhibition on PPO, Son et al. reported that kojic acid showed the highest inhibitory activity on apple slice browning . Kojic acid may also act as a reducing agent and inhibitor of the enzyme. . If high dose of kojic acid is given to rodents, the level of tyrosine stimulating hormone increases . However, there is inadequate evidence showing that kojic acid would cause cancer in human. Consumption of kojic acid at levels normally found in food does not present a concern for safety .
3.3 Reducing agent
Reducing agents can act as antibrowning agents by reducing the o-quinones back to o-quinones. Ascorbic acid is a good reducing agent and it also acts as an oxygen scavenger for the removal of molecular oxygen in polyphenol oxidase reactions. As the reaction resulting in the formation of o-quinones is reversible, in the presence of excess reducing agent, o-quinones are reduced to their original o-diphenols. Ascorbic acid is the most widely used antibrowning agent in food due to its reducing property as well as low pH. Since reducing agents act on o-quinones to prevent browning, the effectiveness of ascorbic acids would be decreased if their use is delayed until after the enzymatic reaction has started. In practice, about 0.1-0.3% of ascorbic acid can have protective effect against enzymatic browning. However, one of the drawbacks of ascorbic acid is that it is irreversibly oxidized to dehydroascorbic acid during the reduction process. As a result, it disappears rapidly and browning would occur again upon its depletion (figure 4). To overcome its rapid depletion, some derivatives, such as ascorbic acid-2-phosphate (AAP), or ascorbic acid-triphosphate (AATP), are used as substitutes. They are not reducing agents, but when the action of PPO presents they release ascorbic acids. Therefore they will not be oxidized by oxygen rapidly as AAP and AATP can remain stable in the presence of oxygen. Erythorbic acid (EA) has a similar effect and action as Acorbic acid in the inhibiting enzymatic browning. However, it is destroyed at a faster rate than ascorbic acid.
Figure 4. Prevention of enzymatic browning by ascorbic acid 
Sulphiting agents including sulphur dioxide (SO2) and sulphite are the most widely used inhibitors of enzymatic browning. It is one of the most effective chemical inhibitors of browning and it is effective even in low concentration. Also, it is inexpensive and hence many food manufacturers use it to inhibit microorganisms growth and enzymes. Sulphur dioxide has three actions to inhibit browning. The first one is its reducing property to reduce o-quinones to its original o-diphenols. The second mechanism involves the formation of colorless products with o-quinones and the last one is that SO2 denatures PPO, resulting in the loss of functionality of the enzyme. Madero and Finne  proposed that bisulphite exerted a competitive inhibitory effect on PPO by binding a sulphydryl group at the active site of the enzyme. Ferrer et al.  proposed that bisulphate inhibited PPO irreversibly by forming sulphoquinones when sulphites react with intermediate quinines, causing complete inactivation of the enzyme. In addition, it also inhibits non-enzymatic browning that reduces pigmentation of the fruit and vegetables. Sometimes sulphur dioxide treatment is applied before deep-frozen, drying or freeze-drying of fruit and vegetables. In deep-frozen products of apples and apricots, fruit slices are soaked for 3-4 minutes in 0.4-0.5% sulphur dioxide solution.
Sulphites are regulatory restricted in food products due to its potentially adverse effects on health. Many reports have shown that sulfites are genotoxic in vitro but not in vivo . In the report of the safety evaluation of sulphites as food additives by World Health Organization (WHO), it concluded that when a suitable alternative method of preservation exists, its use should be encouraged, particularly in the applications in the food industry that the use of sulfites is extensive which may lead to its high intake as high level of sulfites in the body may cause life-threatening adverse reactions .
Thiol containing (sulfhydryl) compounds are compounds that an oxygen atom in the compound has been replaced by a sulphur atom. Many studies showed these compounds such as cysteine, N-acetyl-L-cysteine and reduced glutathione are able to inhibit enzymatic browning in fruits and vegetables . In this group, cysteine is the most effective anti-browning agent. The proposed mechanism was that it reacted with the quinone intermediates to form stable colorless compounds. The cysteine-quinone adducts are also the competitive inhibitors of PPO. Nicolas et al. showed that cysteine directly inhibits PPO through the formation of stable complexes with copper . However, use of cysteine is not so encouraged in the food industry because concentration of cysteine used to achieve significant inhibitory effect on PPO would have negative effects on taste. It has also been shown that Maillard reaction products (MRPs) are potential inhibitors enzymatic browning. Amadori rearrangement products, key intermediates in the first states of the Maillard reaction, has chelating, reducing and oxygen-scavenging properties and it might inhibit enzymatic browning . The MRPs derived from glucose with sulfhydryl amino components (cysteine or the tripeptide, glutathione) could be considered as potential natural inhibitors and this was supported by Billuad et al.ï¿½ï¿½s study . In a further study conducted by Hesham A. et al., the inhibitory effect of the thiol containing compounds was comparable with 4-hexyl resorcinol and being significantly (p < 0.05) higher than ascorbic acid. The MRPs derived from cysteine/glucose model system were more active than their counterpart derived from cysteine/ribose model system . In a study examining various types of MRPs for their tyrosinese inhibitory effect, monosaccharide-glutathione was more active than glutathione . However, MRPs are unstable and they may negatively affect the sensory quality of the food products as there was unpleasant odor formed from the apple slices treated with MPRs .
Most enzymes especially oxidative enzymesï¿½ï¿½ activity is very pH-dependent. Extreme pH can denature the enzymes that lead to the loss of the catalytic function. The groups in the active site of the enzymes are ionizable. In the environment of high proton concentration, the structure of the active site may be affected and there may be conformational change that decreases the binding of substrate and catalytic activity. A pH below 3 can effectively inhibit PPO activity . In addition, substrates may be degraded or change its chemical property in extreme pH and the degraded substrates often behave as enzyme inhibitors, since they share the molecular features of the substrate . Common acidulants use for PPO inhibition are citric acid, organic acids such as malie, tartaric and malonie acids and inorganic acids such as phosphoric and hydrochloric acids. Citric acid is one of the most widely used acidulants in the food industry as it is cost effective and highly available. 0.5-2% of it has protective effects against enzymatic browning in fruit and vegetables. The use of 100mmol/L citric acid combined with 10mmol/L glutathione was found to give a great control of browning in litchi fruits and it is recommended this combination can be a good way applied to control litchi fruit quality . Since it is difficult to achieve efficient browning inhibition through pH control solely, citric acid is frequently used in combination with other anti-browning agents, such as ascorbic acid for chelating the copper at the active site of the enzyme. However, when the pH is lowered to a level the taste of the food products may be affected and it only can be applied in acidic foods.
3.5 Complexing agents
Complexing agents are able to form complexes with PPO substrates or reaction products, so as to reduce the browning effects. Cyclodextrins and cyclic nonreducing oligosaccharides of six or more D-glucose residues are examples of complexing agents. The hydrophobic central core of cyclodextrins can form inclusion complexes with phenol compounds, leading to the depletion of PPO substrates. Due to the limited space in the core of cyclodextrins, larger guest molecules only partially bind to it and form relatively weak complexes. However, chemical modification of cyclodextrines increases the binding and hence increases the inhibitory effect on PPO. In a study comparing ï¿½\-, ï¿½]-, ï¿½^-cyclodextrins in the formation of inclusion complexes with chlorogenic acid as a substrate of apple polyphenol oxidase. ï¿½]-cyclodextrins are more effective in inhibiting browning in apple juice . The internal cavity of ï¿½]-Cyclodextrins is non-polar that it induced inclusion complex formation with the phenolic substrates of polyphenol oxidases, preventing them oxidizing to quinones and hence stop the subsequent polymerization to brown pigments. However, complex formation by cyclodextrins is not specific and it may remove the desirable color and flavor compounds of the food product. As cyclodextrins are insoluble in water, it is better to be used in the molecular encapsulation of insoluble or volatile food ingredients in the food industry . Cyclodextrins have not yet been approved for food use by the United States FDA, although the applicability of cyclodextrins in fruit and vegetable juices has been patented .
Chitosan, a naturally abundant polymer of ï¿½]-(1-4)-linked -D-glucosamine, is derived from the chitin of shellfish. It is widely used in agricultural and horticultural as natural biocontrol and elicitor, water filtration and biomedical. Chitosan is a kind of dietary fibre that have an effect in reducing the absorption of bile salts in the small intestine, hence lowering the blood cholesterol level. It also helps to stop bleeding and has anti-bacterial effects. It is safe and non-toxic, and may even gain health benefits to the body. It has been reported addition of 200 ppm chitosan to Mcintosh apple juice can inhibit enzymatic browning . The mechanism of the inhibition by chitosan is not known, but it may be due to its ability 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 . Another study showed that chitosan coating can inhibit enzymatic browning in lychee fruits and can improve the storability of fruits . The effect might be due the formation of protective barrier on the surface of the fruit, preventing the supply of oxygen to PPO. Therefore it is applicable that chitosan can be used in the combination with other agents to control browning and improve quality in fruit and vegetables during post-harvesting and storage.
3.6 Enzyme inhibitors
4-hexylresorcinol is the one of the most potential PPO inhibitors apply to fresh-cut products. It has obtained the GRAS status for use in the prevention of shrimp melanosis and it proved to be more effective than sulphites in the inhibition of melanosis . Due to its specific mode in the inhibition of enzymatic browning and effectiveness at low concentration, it may be a potential substitute to sulphites. The combination of it with ascorbic acid improved browning control in apple slices . In a study investigating the combination effect of cysteine, kojic acid and 4-hexylresorcinol on the inhibition of enzymatic browning in Amasya apple juice, the interaction of kojic acid and 4-hexylresorcinol was found to have a positive effect on the inhibition of enzymatic browning . Everfresh, a patented product containing 4-hexylresorcinol as the active ingredients and sodium chloride, showed better stability to blackspot formation than sulphites and it is proposed to be use for the inhibition of enzymatic browning in fruit and vegetables .
Sodium chloride, or saline water, is widely used in daily life as an anti-browning agent in freshly cut apple. It can also slow the microbial growth in the food products. Its effects on PPO increase when concentration increases. In an experiment examining the effects of salinity on PPO and peroxidase activity, increasing levels of salinity reduced both oxidases activities immediately after cutting and throughout 7 days of storage. Samples cultivated under high salinity had also the lowest change in colour and showed the lowest reduction in total phenolic content and antioxidant capacity after 3 days of storage . However, due to its inadequate protective effect at low concentration and unwanted salty flavor at high concentration, its application in the food industry is limited. When it is used with ascorbic acid and acidulants such as citric acid, the inhibitory effect is enhanced and prolonged. However, as the pH is getting higher, the inhibitory effect would decrease. Its inhibition to PPO might be achieved by interacting with copper at the active site of the enzyme. Sodium chloride at a concentration of 2-4% is commonly used in the food industry for the inhibition of browning. At 1-2% concentration soaking for less than 1 minute, it can afford temporary protection from surface browning of sliced peeled apples for freezing, or ready-to-use fresh food.
3.7 Other chemical inhibitors
Sodium chlorite is an effective sanitizer inhibiting microbial growth. It can generate chlorine dioxide in acidic environment. Chlorine dioxide is a very powerful oxidizing agent and found to have inhibition in browning reaction. The anti-browning effect of sodium chlorite was greater than that treated with citric acid and the effect of their combination was even better . Sodium Chlorite had a strong inhibition of apple PPO and the extent of inhibition was influenced by its concentration and pH. Further studies indicated that sodium chlorite is a mixed type of inhibitors for PPO activity . The FDA has approved its use on raw fruits and vegetables in the range of 0.5-1.2 g/L followed by a potable water rinse.
Honey has been found to inhibit enzymatic browning. The browning of apple slices was inhibited to a greater extent by 10 percent honey, than by a sucrose solution containing same sugar content, although sugaring alone can inhibit enzymatic browning . Honey contains antioxidants such as tocopherols, alkaloids, ascorbic acid, flavonoids, and phenolics and this antioxidant contents may participate in inhibiting polyphenol oxidase activity. It is also believed that a small peptide isolated from honey exerts its inhibitory effect. Some kinds of proteins, peptides, and amino acids can exert an inhibitory effect on polyphenol oxidase activity by chelating copper at the active site of PPO to form stable complexes .
Treatment with protease is another way to inhibitor enzyme browning and PPO is protein in nature and it can be broken down by protease. The plant proteases ficin, papain and bromelain are very effective browning inhibitors. Pineapple juice was found to be effective in inhibiting browning in apple rings and it is believed than bromelain, organic acids and sulphydryl compounds in pineapple juice are responsible for the inhibitory effect . However, application of protease treatment in the food industry is not so encouraged due to its high cost.
3.8 Combined treatment
More effective anti browning effect can frequently achieved by using combination of the anti-browning agents. A common combination to control browning in apple slice is the use of ascorbic acid and calcium chloride. There are many studies showing the combination of the inhibitors is more effective than using the inhibitor alone in preventing enzyme browning. However, it is noted that the choice of combination should not affect the original properties of the food products or causing toxicity.
The consumption of fruits and vegetables may increase in the coming future since people are more concern about their health and the freshness of the food. Fruits and vegetables are one of the nutritious food and the undiscovered nutritional and medical values of plant sources are being discovered today and also in the future. Browning of plant products, including fruit, vegetables and beverage, and animal materials such as seafood by enzymatic oxidation of phenolic compounds by PPO can cause the loss of quality, nutritional values and even the formation of toxic or unpleasant compounds. It is still not known whether todayï¿½ï¿½s prevention methods and technologies are enough for the demand in the future. Although there are many kinds of enzymatic inhibitors investigated and tested, much work in this field is still needed. When applying in food products, safety is the first concern while good quality needed to be achieved by using the inhibitors. Effectiveness and cost are also evaluated for commercial use. In addition, method of treatment is another consideration. Most of the physical treatments to inhibit browning are quite complicated and not cost-effective. Using chemical inhibitors is considered to be simple and fast. It is hoped that in the near future, more and more efforts can be put on this field for searching potential inhibitors of enzymatic browning, as well as using the existing information to integrate and formulate a product, overcoming the disadvantages of the existing PPO inhibitors, which can be widely used in the food industry.