Polyphenol oxidases are a group of copper containing enzymes found in plants, animals and bacteria. In plants, during fruit maturation, it is thought that polyphenol oxidases are largely responsible for the brown discolouration associated with ripening. This brown discolouration originates from the enzymatic activity of polyphenol oxidase, which is able to catalyse the conversion of polyphenol compounds to quinones. Subsequently the quinone compounds then cross-link to form pigmented compounds of large molecular weights called melanins. The function of polyphenol oxidases in plants are not fully understood, but they are thought to be involved in the protection of the plant from wounds, the scavenging of free radicals and the limitation of infested tissue by pathogens. The quinones formed during this reaction may limit the digestibility of tissues to herbivores by binding proteins, or may form physical barriers to pathogens invasion.
The polyphenol oxidase in tea (Camellia sinensis) is important nutritionally because its activity in the harvested plant can cause alterations in the chemistry of the final product. Black tea is most commonly drunk in the West and its manufacture is possible because a lengthy fermentation process allows the polyphenol oxidase in the tea leaves to metabolise much of the flavan-3-ol content to thearubigens and theoflavins, that give black tea its characteristic taste. This is possible because prior to fermentation, the leaves are crushed which releases the polyphenol oxidase from cellular compartmentalisation and brings it into contact with the flavan-3-ols. In contrast, green tea possesses very low concentrations of the oligomeric theoflavins and thearubigens because the leave are dried following harvest which prevents the release of polyphenol oxidase. Oolong tea is partially fermented and is therefore chemically half-way between green and black tea in chemical composition.
Polyphenol oxidase can be utilised in the manufacture of black tea because the leaves are crushed. Crushing (and other tissue damage) releases the polyphenol oxidase from the vacuoles where the enzyme is generally stored in plants. This then brings the enzyme into contact with the substrate (polyphenols) in the cytoplasm of the cells. The polyphenol oxidases in plants have a broad range of polyphenol substrates such as flavonoids (e.g. anthocyanins, flavones, flavonols, flavan-3-ols), hydroxycinnamic acids (caffeic acid and chlorogenic acid) and proanthocyanidins (e.g. procyanidins, prodelphinidins and propelargonidins). Each polyphenol oxidase enzyme is associated with two atoms of copper at the active site that gives the enzyme the ability to catalyse two types of reaction. Monophenolase activity confers that ability to hydroxylate monophenols to diphenols, and diphenolase activity confers the ability to oxidise o-diphenols to o-quinones, which are subsequently non-enzymatic polymerised to melanins.
The browning of fruit is considered detrimental because it can reduce shelf life and decrease attractiveness. Sulphiting agents are able to inhibit polyphenol oxidase because they release sulphite ions which prevent the formation of melanins, although safety concerns over sodium sulphite have reduced its use. An alternative is cysteine, which can prevents browning by reacting immediately with the quinones, forming colourless compounds. Ascorbic acid is also an effective inhibitor because it reduces the quinine products before they polymerise to melanins. The optimal pH of polyphenol oxidase is around 5 to 7.5, and so acids (e.g. ascorbic acid and citric acid) can inhibit enzyme activity if they lower the pH below this range. Benzoic acid and cinnamic acid (both aromatic carboxylic acids) are also effective inhibitors of fruit browning because they have structural similarities with the polyphenol substrates. Ascorbic acid, cinnamic acid and cysteine in combination show synergistic inhibition of browning.
Figure 1. The reactions catalysed by polyphenol oxidase in plants. First the hydroxylation of a monophenol to a diphenol. Then the oxidation of a diphenol to a quinone.
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