Determination of Peroxyl Radical-Scavenging Activity in Food by Using Bactericidal Action of Alkyl Peroxyl Radical

A novel bioassay for antioxidant activity based on bactericidal actions of alkyl peroxyl radicals (ROO^•) was developed. ROO^• was generated by the reaction of heme irons and various alkyl hydroperoxides. A Gram-positive bacterium, Staphylococcus aureus, was subjected to cytotoxic treatment with ROO^• with or without various antioxidants. This bacterial suspension was serially diluted, on a 96-well multiplate, in mannitol broth containing phenol red as a pH indicator. After overnight incubation, the growth of ROO^•-treated bacteria produces acid, resulting in a change in color of the broth, which permits easy quantitation of viable bacteria protected by antioxidants. Accordingly, ROO^• scavenging by various antioxidants could be reproducibly quantitated. Among 22 antioxidants tested, cytotoxicity was substantially inhibited by α-tocopherol, glutathione, l-cysteine, probucol, butylated hydroxyanisole, and butylated hydroxytoluene. Furthermore, the antibactericidal activity of various teas and nonalcoholic drinks showed very high correlation with their antioxidant activities determined by luminol-dependent chemiluminescence assay (r = 0.91). By this simple bioassay, a large number of samples can be analyzed simultaneously to determine ROO^•-scavenging activity.