Antioxidant Activity and Metabolomic Characterization of Lactiplantibacillus plantarum MCS1903 Isolated from Naturally Fermented Tofu Whey

Naturally fermented tofu whey is a nutrient-rich byproduct of tofu production that harbors diverse lactic acid bacteria (LAB) with potential probiotic properties. However, the antioxidant mechanisms of these LAB, particularly the roles of different cellular fractions and their metabolic basis, remain unclear. This study aimed to isolate LAB from naturally fermented tofu whey and evaluate their antioxidant activities across cellular fractions, combining in vitro assays, 16S rDNA-based identification, metabolomic profiling, and cellular validation to elucidate the underlying mechanisms. Six LAB strains were isolated and screened for 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging capacity and environmental stress tolerance. Among the identified isolates, Lactiplantibacillus plantarum MCS1903 exhibited the highest extracellular antioxidant activity. Non-targeted metabolomic analysis of cell-free supernatant revealed distinct metabolic profiles compared with the MRS control, with significant enrichment of antioxidant-related metabolites and pathways. In Caco-2 cells, MCS1903 supernatant (<5%, v/v) showed no significant cytotoxicity and effectively alleviated H2O2-induced oxidative stress by modulating the Nrf2/Keap1-HO-1 signaling pathway. These findings indicate that tofu whey is a valuable source of functional LAB, and MCS1903 represents a promising candidate for probiotic and functional food applications, supporting the valorization of tofu whey and development of natural antioxidant probiotics derived from fermented food byproducts.