Adaptive laboratory evolution of Lactobacillus helveticus enhances acid tolerance for food-grade, non-GMO...

Lactobacillus helveticus (L. helveticus) is widely used in fermented dairy products and probiotic formulations, but its growth and functionality are impaired under acidic conditions encountered during food fermentation and gastrointestinal passage. To enhance acid tolerance, adaptive laboratory evolution (ALE) was performed by gradually lowering the pH from 5.0 to 4.0, yielding two evolved strains, LH_1E_113-5 and LH_2E_117-4, with improved growth, cell viability, and lactic acid production under low-pH conditions. Whole-genome resequencing revealed mutations in genes linked to central carbon metabolism (ldh_2, pfkA, and pox5), oxidative stress response (rclA_1), and amino acid metabolism (metG and bmr3_2). Metabolomic profiling showed increased TCA cycle activity, enhanced NADPH production, and shifts in amino acid and lipid metabolism, indicating coordinated metabolic reprogramming. Importantly, ALE provides a non-GMO strategy to improve robustness in food-grade lactic acid bacteria, offering a practical alternative where genetically modified organisms are restricted. These findings provide a foundation for development of L. helveticus strains with enhanced stability for industrial fermentation and food biotechnology applications.