Adaptive laboratory evolution under oxidative stress in lactococcus lactis subsp. lactis LM1009: transcriptomic...

This study aimed to improve the oxidative stress resistance of Lactococcus lactis subsp. lactis LM1009 through adaptive laboratory evolution. L. lactis subsp. lactis LM1009 was exposed to gradually increasing hydrogen peroxide concentrations over 50 days, resulting in an adapted strain that tolerated up to 5 mM hydrogen peroxide. Compared to the original strain, L. lactis subsp. lactis LM1009-A (adapted strain) exhibited enhanced resistance to oxidative, acid, and heat stress, along with increased surface hydrophobicity, auto-aggregation, and intestinal adhesion capacity. The antioxidant activity was significantly improved, as demonstrated by elevated 2,2-diphenyl-1-picrylhydrazyl radical scavenging, stronger linoleic acid peroxidation inhibition, and reduced intracellular reactive oxygen species (ROS) levels. Transcriptomic analysis revealed widespread reprogramming, including the downregulation of genes related to membrane biosynthesis and energy metabolism and the upregulation of genes involved in oxidative defense and redox balance. Real-time quantitative PCR analysis revealed a metabolic shift from glucose-based to alternative sugar utilization pathways, suggesting decreased nicotinamide adenine dinucleotide production and increased nicotinamide adenine dinucleotide phosphate availability, contributing to redox stabilization. In lipopolysaccharide-stimulated macrophages, the L. lactis subsp. lactis LM1009-A suppressed nitric oxide release and downregulated pro-inflammatory markers, including interleukin (IL)-6, IL-1β, tumor necrosis factor-α, cyclooxygenase-2, and inducible nitric oxide synthase. These effects were associated with reduced activation of the ROS-mediated mitogen-activated protein kinase and nuclear factor-kappa signaling pathways. In conclusion, the adapted L. lactis subsp. lactis LM1009-A exhibited enhanced stress resistance and functional bioactivity, supporting its potential as a next-generation probiotic for oxidative and inflammatory stress management and for industrial applications.