Enhancing oxidative tolerance and fermentation performance of Lactobacillus casei through adaptive laboratory evolution

The ability to withstand oxidative stress is crucial for the industrial performance of Lactobacillus casei , a key starter culture in milk fermentation. In this study, adaptive laboratory evolution was applied to enhance its oxidative tolerance. The evolved strain exhibited a 3.38-fold increase in superoxide dismutase activity, with 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and superoxide anion radical scavenging rates increasing by 1.58-, 1.35-, and 1.5-fold, respectively. Compared to the ancestral strain, it exhibited over twofold greater tolerance to acidic conditions and bile salts, enhanced biofilm formation ability, and improved viability during milk fermentation and refrigerated storage, maintaining viable cell counts of 1.63 × 10 7 CFU/mL versus 1.4 × 10 6 CFU/mL. Furthermore, evolved strain conferred enhanced antioxidant properties to the fermented milk. Untargeted metabolomics coupled with partial least squares-discriminant analysis (PLS-DA) confirmed that the evolved L. casei played a critical role in shaping the metabolic profile of fermented milk compared to the ancestral strain, with 540 metabolites being differentially enriched. Further functional analysis revealed that milk fermented with the evolved strain was enriched in antioxidant-related metabolites, particularly those involved in amino acid metabolism and alkaloid biosynthesis. This study highlighted the potential of oxygen-tolerant L . casei in enhancing the functional and preservative qualities of fermented milk. • Adaptive laboratory evolution (ALE) improves the oxygen tolerance of Lactobacillus casei. • The evolved strain exhibits enhanced production of antioxidant enzymes. • Fermented milk produced by the evolved strain retains greater antioxidant activity. • Metabolomic analysis identifies alkaloids as key metabolites in fermented milk.