An agar-based bioassay for accurate screening of the total antioxidant capacity of lactic acid bacteria cell-free supernatants

This study aims to develop a new, simple, and efficient method for estimating the total antioxidant capacity of lactic acid bacteria-cell free supernatant. The bioassay is based on diffusion and reduction of permanganate in agar medium under acidic conditions where the Mn7+ ions are quantitatively oxidized to Mn2+ and shift from an intense purple color to colorless. Hence, the reaction enables fast detection of the bleaching diameter during diffusion of the sample in permanganate agar. This bleaching diameter is correlated to the reducing power of the substance tested. The method was tested and validated to quantify the total antioxidant capacity of culture supernatants of probiotic strains (Lacticaseibacillus rhamnosus LGG and Lactiplantibacillus plantarum 299v) and 25 lactic acid bacteria isolated from a human intestinal origin and compared to the PRAC and DPPH methods. The results were treated statistically by analysis of variance. This method proved to be linear (R² in the linear experiment of ascorbic acid was 0,99), precise with repeatability intraday RSD of 2.07 to 5.5% and intermediate precision RSD of 2.95 to 5.53%, and accurate (100.29 to 108.58%) at 30 min, 1 h, and 4 h in the selected range of 1.5-5.5 mM of ascorbic acid. The developed permanganate agar reduction bioassay is a fast, reliable, and cost-effective technique for the prescreening and detecting the total antioxidant capacity of supernatants of lactic acid bacteria and possibly other sources of natural antioxidants.