Prevention of Enzymatic Browning in Fruits and Vegetables

Pr steadytion of Enzymatic Browning in Fruits and VegetablesPolyphenol oxidase (PPO) is known to be a key enzyme in enzymatic toasting of harvesting, vegetables, beverage and sea sustenance in the fodder assiduity. The toasting of regimen occasions a considerable caliber mischief and stinting loss of nutrition products. Inhibition of dark-brown by physical treatments has wellhead been developed and on that point argon a wide range of chemicals heap as well cut back enzymatic cook. This denomination overviews the various chemical hinderors against enzymatic brown in food.Enzymatic cook is genius of the major builds in quality loss of vegetables and harvest-time, beverage and seafood. It is round accounted for over 50 percent losses in fruit 1. It is influenceually outstanding to get wind enzymatic toasting so as to denigrate losses and main(prenominal)tain the economic values of the food products. In sensual tissues, melanosis is the process results in the makeup of dark keys. The products of melanosis near cartridge holders ca handling alike visual perception as spoilage and this would touch customers non select these products although they ar non harmful and do not influence apprehension sensation of the food. Browning ca usances umteen deleterious changes in the organoleptic and nutritional properties of food products that decrease their marketplace values. However, enzymatic browning is beneficial for both(prenominal) of the food products much(prenominal) as tea, coffee, and cocoa. The consumption of fruits and vegetables has been improverd due to the nutritional recommendations for health maintain. As a result, the market values of minimally neat fruits and vegetables production increases and this will trigger and encourage research in enzymatic browning.Enzymatic browning definitionCell disruption ca utilise by automatic injury or temperature changes may lead to physiological disorders or even cell deat h. This loss of cell integrity results in the decompartmentation of phenop withstand substrates and enzymes 2. Polyphenol oxidase (PPO) is a critical enzyme catalyzing the oxidation of endogenous phenolic resin changes to form grey or brown colors in the enzymatic browning process. PPO is a full term which includes a large number of tie in sloven-containing enzymes, including catechol oxidase, tyrosinase and laccase. In the strawman of oxygen, PPO oxidizes mono- and di-phenols to o-quinones. one time the products ar formed, they polymerize to form tall molecular weight compounds or brown pigments as they argon exceedingly unstable. The brownish color could be enhanced when they react with aminic bitters and proteins 2.Importance of browning crushingIt is congenital to function enzymatic browning in order to reduce the economic loss during fruit and vegetables processing. It is essential to control enzymatic browning for the amend development in the food industry. The some greenness way employ for the suppression of browning is the addition of sulfating agents to inhibit PPO. However, sulfite is a toxic grain and potty react with a variety of humoral and cellular components and whoremaster cause toxicity 3. out-of-pocket to the adverse health essences cause by sulfites, alternative compounds ar investigated for anti-browning properties. The ideal compound would be the one which is core groupive, does not cause any quality loss (eg. taste, flavor, nutritional values) and toxicity, as well as deplorable cost. Until now, many approaches argon available to inhibit enzymatic browning, which include the use of ascorbic harsh, sodium chloride, kojic acid, 4-hexylresorcinol etc. Enzyme inhibitors, reduce agents, acidulant agents, chelating agents and interlinkinging agents are the plebeian chemical treatments of antibrowning 4. In order to boast a better control of enzymatic browning, it is important to understand its machine of actions, the billet of the enzyme, substrates, products and their fundamental interactions. Minimizing the negative sides of enzymatic browning evict extend the shelf life and increase the market value of the food products.Physical inhibitionPhysical treatments are one of the common approaches in the control of enzymatic browning in the food industries. Hot-air drying, freeze-drying, flowerpotning and bottling are examples of some preventive orders. Browning is enhanced by cutting of sow tissue as it stimulates phenolic biogeny, then minimizing the mechanical misuse of fruit and vegetables reduce the degree of browning. cut back the temperature heap dimmed downwardly the rate of enzymatic browning as enzymes are generally lazy at low temperature. However, color change placid occurs at 0oc, t therefore it is important to allow the crystallization temperature of water supply to be reached as promptly as attainable 5. Color change would be blocked during commercial- grade 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 5. high school temperature 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 plant will matchs the texture, taste, flavor and nutritional value of the food. PPO is breeze throughly destroy at 80oC 6, just sufficient time is demand for the interior part of the fruit or vegetables to reach much(prenominal) temperature. Blanching the green beans for 3.5 minutes at 82 oC and above, inactivated catalase, lipoxygenase, and polyphenol oxidase activities completely6. Protection of the fruit and vegetables from oxygen batch prevent enzymatic browning when the plant organs apprizenot be branched. A variety of methods are employ in food industries, including airt ight package, partial vacuum and oxygen-poor atmo field of force.Chemical inhibitionChemicais are the closely comm merely used for the control of enzymatic browning, however their use in processed food products is restricted to compounds that are nontoxic, hale and that do not adversely affect the quality7. Inhibition of enzymatic browning can be achieved by hindering the enzyme or by medical dressing to its active site which lower the PPO bodily process. Chelating agents which pack the hog prosthetic concourse from PPO can inhibit enzymatic browning. Removing the substrates is anformer(a) 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 detai l in this essay.Polyphenol oxidaseDefinitionPPOs are first discovered in mushrooms by Schoenbein in 1856. They are papal bull-containing proteins and belong to the meeting of oxidoreductases. A copper prosthetic group is required for its drill. In the presence of oxygen, polyphenol oxidase catalyses reception of monophenols to o-diphenols (monophenol oxidase action at law). It also oxidises o-diphenols to o-quinones (dipehnol oxidase act). The enzyme in plants slang both mono-and diphenol oxidase activity and the ratio of monophenol to diphenol oxidase activity is usually 110 to 140. 8. 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. The image of PPOs in plants is believed to resist the infection of microorganisms and virus and to extreme climatic conditions. PPOs are also in involved the development of dark pigmentation in animals. The bio subtraction of mela nin pigments and other polyphenolic compounds which often propagation provide a protective function 1. 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. linguistic processNomenclature of PPOs is some clock conf development due to their similarity in nature. Jaenicke and Decker write likely there is no common tyrosinase the enzymes erect in animals, plants and fungi are different with respect to their sequences, size, glycosylation and activation 9 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 10. PPOs are normally classify into three types for the convenient use of the termCatechol oxidaseCatechol oxidase (1,2-benzenediol oxygen oxidoreductase, EC1.10.3.1) has both monophenol oxidase and diphenol oxidase activity. It is the closely com mon 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 in truth diverse and variable and it is estimated the molecular masses of PPOs ranges from 20 to 180kDa 2. Most of the studies visualise PPOs have an optimum activity between pH 4 and 7, yet some reports show that pH optima varies with the proportions of isoenzymes as well as phenolic substrates used 2. The optimum temperature of PPOs ranges from 15 to 40 oC and this also depends on the said(prenominal) factors as pH.TyrosinaseTyrosinase (EC1.14.18.1, monophenol, L-DOPA oxygen oxidoreductase) refers to as monophenol monooxygenase and corresponds to the aforesaid(prenominal) enzymes as EC1.10.3.1 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 rattling similar to catechol oxidase and sometimes tyrosinas e 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.LaccasesLaccase (p-diphenol oxidase, E.C. 1.10.3.2)(DPO), has the unique king to oxidase p-diphenols into their match 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 high plants while much less oftentimes in the plant estate than PPOs. They are glycoproteins with a carbohydrate gist of about 15-41% and they have a molecular weight of about 60 to 80 kDa.Substrate phenolic compounds are substrates for PPOs. in that respect 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 orchard apple trees, grapes, and peaches, the force plays of phenolic content and polyphenol oxidase on the rate of enzymatic browning have been account 11-13. Phenolic compounds contain an aromatic ring with one or more hydroxyl groups, together with other substituents. The differences in species, ripening and milieual conditions of harvest-tide and storage influence 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 deriva tives and monomeric flavan-3-ols often appear to be the best substrates 2. 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.Caffeic acid derivatives, such as chlorogenic acid, the major diphenolic compounds in plants, 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 1. apparatus of actionMonophenol oxidase activity for the hydroxylation reaction is normally slower than the oxidation reaction for the production of quinine, which leads to the initiation of the polymerization reaction. Thus it is the rate determining gait in the production of bro wn 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 steadfast electrophiles which can suffer nucleophilic attack by water, other polyphenols, amino acids and proteins, leading to Michael-type addition products 14-16. The final products have more intensive color than the o-quinones. Their color depends on the phenol from which they originate.Chemical inhibitors of PPOPlant PolyphenolsPolyphenols are a diverse group of compounds having multiple phenolic functionalities 18. 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 meaning(a) repressing effect. Flavonoids are one of the most common and best canvass groups of plant polyphenols. Flavonoids can be further divided into sextuplet 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 restrictive effect on PPO 19, 20. Isao Kubo studies the inhibition kinetics of kaempferol extracted from orange yellow flower and it is found that to be a belligerent inhibitor, its repressive activity presumably comes from its ability to chelate copper in the enzyme 20. This copper chelation mechanism may be applicable for all of the flavonols as long as their 3-hydroxyl group is free 20. However, 3-o-glycoside derivatives, eg. kaempferol 3-o-glucoside, quercetin 3-o-glucoside, did not inhibit tyrosinase in high dumbness 20. This shows that the free h ydroxyl 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 21. ()-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 prevailing at the active site of the enzyme 22. 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 23.Structure-Activity Investigations of stilbenes and related 4- superceded resorcinols, obtained from Artocarpus incisus and other plants suggested that the 4-substituted resorcinol skelet on have potent tyrosinase inhibitory ability 24. 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 25. 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 26. However, glabridins 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 26, 27. 6-hydroxydaidzein (6,7,4-trihydroxyisoflavone), one figure of hydroxyisoflavones isolated from soybean koji fermented with genus Aspergillus oryzae, had 6-fold more than kojic acid acting competitively on the tyrosine binding site of the enzyme 28. It is found that an isoflavone with hydroxyl groups at both the C 6 and C7 positions in the A ring would increase both inhibitory effect and affinity to the enzyme more than 10 times 28.Chalcones, with two aromatic peal 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 29. Kuraridin, isolated from Sophora flavescens, was 34 times of the activity of kojic acid in inhibiting monophenolase activity of mushroom tyrosinase 30. 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 24, 31.Chelating agentsChelating agents are chemicals that bind to metal ions and remove them from their sphere of action. EDTA (ethylenediaminetetraacetic acid) is a chemical preservative permitted for food industry use. It is a chelator and 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 7. the United States Food and Drug Administration sanctioned the use of Calcium disodium EDTA and disodium EDTA to be used as a food additive 32. EDTA is usually used with other chemicals such as reduce 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 7. Sporix, containing citric acid, ascorbic acid, sodium acid pyrophosphate and calcium chloride, is a powerful chelator and also an acidulant. Browning prevention in apple juice and cut fall outs was obtained by Sporix 33. However, it is not sanctioned in U.S. for food use.Kojic acid (C6H6O4 5-hydroxy-2-(hydroxymethyl)-4-pyrone), always macrocosm use as a standa rd 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. 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 34. Kojic acid may inhibit the enzyme acting as a reduce agent. If high dose of kojic acid is plantn to rodents, the level of tyrosine stimulating endocrine gland increases 35. However, there is inadequate evidence showing that kojic acid would cause genus Cancer in human. Consumption of kojic acid at levels normally found in food does not pres ent a concern for invulnerablety 36.Reducing agentReducing agents can act as antibrowning agents by trim down the o-quinones back to o-quinones. Ascorbic acid is a good reducing agent and antioxidant which removes 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 bring down 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 potency of ascorbic acids would be decreased if their use is slow down 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 can be oxidized to dehydroascorbic acid irreversibly during the reaction. As a result, it disappears quickly an d 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.Sulphiting agents including mho dioxide (SO2) and sulphite are real popular browning inhibitors used in the food industry. 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. Th e first one is its reducing property to reduce o-quinones to its original o-diphenols. The second mechanism involves the formation of pale 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 37 reported that bisulphite could bind to the sulphydryl group at the active site of the enzyme so as to exert competitively inhibit the enzyme. Ferrer et al. 38 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 restricte d in food products as it may cause potentially adverse effects on health. There are many reports showing that sulfites are genotoxic in vitro but not in vivo 39. In the report of the safety evaluation of sulphites as food additives by orbit 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 consumption as high level of sulfites in the body may cause life-threatening adverse reactions 39.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 40. 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 by forming stable complexes with copper 40. 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 major power inhibit enzymatic browning 41. The MRPs derived from glucose with sulfhydryl amino components (cysteine or the tripeptide, glutathione) could be considered as potential lifelike inhibitors and this was supported by Billuad et al.s study 42. 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 imitation derived from cysteine/ribose model system 43. In a study examining various types of MRPs for their tyrosinese inhibitory effect, monosaccharide-glutathione was more active than glutathione 44. However, MRPs are unstable and they may negatively affect the sensory quality of the food products as there was unpleasant flavor formed from the apple slices hardened with MPRs 44.AcidulantsMost enzymes especially oxidative enzymes activity is rattling 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 alter and there may be conformational chan ge that decreases the binding of substrate and catalytic activity. A pH below 3 can effectively inhibit PPO activity 45. In addition, under extreme pH, substrates may be profligate or change its conformation as a result, the degraded substrates parting the molecular features of the substrates that they may act as enzyme inhibitors 46. 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 an acidulants which widely used 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 cabal can be a good way applied to control litchi fruit quality 47. 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 to chelate the copper 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.Complexing agentsComplexing 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 put 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 using chlo rogenic acid as a substrate to compare -, - and -cyclodextrins in the formation of inclusion complexes, -cyclodextrins are more effective in inhibiting browning in apple juice 48. The internal cavity of -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. Cyclodextrins are insoluble in water, so it is better to be used in volatile or insoluble food ingredients in the food industry. The use of cyclodextrins in fruit and vegetable juice are patented, but it have not been ap turn up to use in food by the FDA 49.Chitosan, a naturally abundant polymer of -(1-4)-linked -D-glucosamine, is obtained 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 enzymatic browning was inhibited by adding 200 ppm chitosan to Mcintosh apple juice 50. When 2% of chitosan was treated with shrimp during storage, the incidence of melanosis was reduced 51. Another study showed that chitosan polish can inhibit enzymatic browning in longan fruits and it can improve shelf life and quality of the fruit 52. The effect mightiness 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.Enzyme inhibitors4-hexylresorcinol is the one of the most potential PPO inhibitors apply to fresh 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 7. 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 53. 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 54. Everfresh, a patented product containing 4-hexylresorcinol as the active ingredients and sodium chloride, showed better stability to blackspo t formation than sulphites and it is proposed to be use for the inhibition of enzymatic browning in fruit and vegetables 55.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 saltiness on PPO and peroxidase activity, increase levels of salinityreduced both oxidases activities immediately after cutting and passim 7days of storage 56. Samples cultivated under highsalinity had also the lowest change in colour and showed the lowest decrease in total phenolic content and antioxidant capacity after 3days of storage 56. 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 e ffect 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 the copper at the active site of the enzyme. Sodium chloride at a concentration of 2-4% is usually used to inhibit browning in the food industry. At 1-2% concentration soaking for less than 1 minute, it can bear temporary protection from surface browning of sliced peeled apples for freezing, or ready-to-use fresh food.Other chemical inhibitorsSodium 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 gr