Nh

Leuco compound

+ Protein

Indole 5,6-Quinone

Indole 5,6-Quinone

+ Protein

Melanin - Protein sufficient amounts of acidulants, such as citric, malic, or phosphoric acids to yield a pH of 3 or lower. Oxygen can be eliminated by vacuum or by immersing the plant tissues in a brine or syrup. Phenolic substrates can be protected from oxidation by reaction with borate salts, but these are not approved for food use.

For products such as vegetables, which ultimately end up cooked, heat inactivation by steam or hot water blanching is the most practical method of inactivating PPOs. Overblanching should be avoided due to loss of firmness and nutrients. Most commercial blanching of vegetables has been based on the residual activity of the peroxidase because peroxidase is one of the most stable enzymes in plants. It has been generally accepted that if peroxidase is destroyed it is quite unlikely that other enzymes including PPOs will have survived. There are, however, problems associated with the use of peroxidase as an indicator of an adequate blanch in that several enzymes other than peroxidase have been shown to be largely responsible for quality deterioration during frozen storage of different vegetables (67,68). Therefore, optimization of blanching of individual products has to be established in order to tailor the process to each type of raw material and product desired. Heat inactivation of PPO in fruit products has been applied to fruit juices and purees and to fruit intended for such products. Because the enzyme is very labile to heat at 85┬░C and above, the higher the temperature, the shorter the time required for inactivation. To minimize the undesirable changes due to excess heating, optimum temperature-time requirements for PPO must be established. Rapid cooling after enzyme inactivation is also necessity for best quality retention.

Sulfur dioxide is a very effective inhibitor of PPO and has been used for many years. It readily reacts with compounds such as aldehyde and other carbonyl-containing molecules. These reaction products are ineffective against PPO, and therefore, a sufficient amount of free S02 must be maintained. In order to be effective in preventing PPO activity, S02 must penetrate throughout the tissues. Sul-furous acid penetrates better than the bisulfite form. The use of excess S02 produces both undesirable flavors and an excessively soft product. In recent years the safety of sulfites in foods has been questioned because of their hazard to certain asthmatics. Since August 9, 1986, the U.S. FDA has banned the use of sulfur dioxide in fresh fruits and vegetables and has required a label declaration on those food products (such as dehydrated fruits, frozen potato products, wine) containing more than 10 ppm of sul-fiting agent. Because of these restrictions, food processors have turned to a number of sulfite alternatives, mostly formulations effective against enzymatic browning, with varying success. The demand for more effective browning inhibitors has stimulated considerable research activity in this area during the last decade.

Ascorbic acid and its isomer, erythorbic acid, and derivatives such as ascorbic acid-2-phosphate, ascorbic acid-triphosphate, ascorbic acid-6-fatty acid esters are reported to control browning. Ascorbic acid is a very effective reducing agent, as it reduces the o-quinones formed by PPO to the original o-dihydroxy phenolic compounds. Ascorbic acid alone or in combination with citric acid have been used widely by the food industry. Its prevention of browning lasts as long as any residual ascorbic acid remains, and, therefore, stabilized forms of ascorbic acid should be effective sulfite substitutes. One should keep in mind that the excess amounts of oxidized ascorbic acid can produce brown pigments by nonenzymatic browning reactions. Browning inhibitor penetration can be enhanced by vacuum infiltration, which also removes air from the products' void spaces.

Cinnamic acid and benzoic acid were found to be effective for apple products, and carbon monoxide has been applied to control browning in mushrooms. 4-Hexyl-resorcinol (Everfresh®) is known to be effective on shrimp. Kojic acid and salicylhydroxamic acid were also known as PPO inhibitors. Compounds that bind or complex PPO substrates also may be potential inhibitors. Poly(vinylpolypyrollidone ) and cyclodextrins can bind polyphenols and prevent their participation in enzymatic browning. Glutathione and AT-acetylcystein were also reported to be effective in controlling browning in apple, potato, and fresh juices. Protease enzymes as well as low molecular weight peptides were claimed to be effective browning inhibitors (69,70). Selecting raw materials for processing that have a low tendency to brown is another way to control browning. Empire and Granny Smith apples and Atlantic potato are examples of cultivars that brown slowly (21,71).

Another approach involves enzymes that transform the substrates of PPOs by methylation or oxidative cleavage of the benzene nucleus. o-Methyl transferase methylates the 3-position of 3,4-dihydroxy aromatic compounds in the presence of a methyl donor, converting PPO substrates into inhibitors of PPOs such as caffeic acid to ferulic acid. The exclusion of oxygen as a means of controlling enzymatic browning is generally used in combination with other methods. For example, retail packs of frozen peach slices maintain their high quality when packaged in ascorbic acid-containing syrup combined with hermetically sealed containers wherein the oxygen has been removed from the head space. Oxygen concentrations in the atmosphere of packaged produce can be controlled by modified atmosphere packaging. Although this method can delay browning, low oxygen may induce an anaerobic condition that entails a risk of undesirable quality change and anaerobic microbial growth. Edible coating materials such as xan-than were reported to prevent enzymatic browning and extend the shelf life of fruits and vegetables used in salad bars and prepared salads. Ultrafiltration removes PPO and prevents browning. Osmotic dehydration using sugar or syrup also inhibits enzymatic browning and protect flavors.

Most of chemicals mentioned here are not fail-safe, not acceptable to some consumers, and cannot be used to prevent browning in intact fruits and vegetables. Through better understanding of the mechanism of action of PPO and its essential metabolic roles in plants, it is expected that genetic engineering techniques will be the most important tools in preventing unwanted enzymatic browning. A molecular biology approach will provide a more precise method of decreasing PPO expression, while retaining the desirable genetic traits of plants.

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