Summary The objective of this study was to clarify the roles of copper, ascorbic acid, and xanthine oxidase in the spontaneous development of oxidized flavor in milk. At a mole ratio as low as 3.5 moles of neocuproine to one of copper, the cuprous chelator neocuproine effectively prevented oxidized flavor. Consequently, of the metals in milk, copper must have been the only one affected, indicating that it is an essential metal catalyst in lipid oxidation. Destruction of ascorbic acid by the relatively specific catalyst ascorbic acid oxidase prevented spontaneous peroxidation; but when the original ascorbic acid level was restored, spontaneous oxidized flavor developed. With less specific light and hydrogen peroxide treatments to destroy ascorbic acid, some anomalies were noted. It was concluded that at the copper concentrations normally present in milk, ascorbic acid is an essential reactant in spontaneous, as in copper-induced, oxidized flavor. At copper levels above 1μg/g, ascorbic acid was not necessary for lipid peroxidation, presumably because at the higher copper-protein ratios, protein substituted for ascorbic acid in reducing cupric copper to cuprous. In milk, in washed milk, and even in a model system containing only copper and a reducing agent in buffered linoleate, ascorbic acid was a more effective promoter of lipid oxidation than any other reducing agent tested. It is postulated that the pro-oxidant activity of ascorbic acid depends on its ability to reduce copper from cupric to cuprous, and on a specific association between copper and ascorbic acid that in some unexplained manner increases its pro-oxidant properties. A number of experiments was directed toward establishing a role for xanthine oxidase in lipid oxidation reactions, but no consistent results were obtained to support the hypothesis that spontaneous development of oxidized flavor depends on the activity of xanthine oxidase in milk.
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