Natural selection of Lacticaseibacillus paracasei mutant: Weaken the post-acidification of fermented milk

Probiotic fermented milk has emerged as a prominent functional food for its substantial role in promoting intestinal health and overall wellness. However, a persistent challenge in the industry is post-acidification, a process where lactic acid bacteria continue fermenting during storage, leading to excessive acidity, textural degradation, and flavor alterations. This study focuses on the identification and characterization of a naturally mutated strain of Lacticaseibacillus paracasei capable of significantly attenuating post-acidification in fermented milk. A novel mutation was discovered in the α-glucosidase gene (AF91_03255), wherein the substitution of arginine at position 477 with glycine altered the enzyme's binding dynamics. This mutation enhanced binding stability between the enzyme and its substrate by approximately 16.8%, indirectly modulating acid production without directly limiting glucose metabolism. Through a combination of multi-omics techniques, including metabolomics and genomic sequencing, this study revealed significant shifts in the glycerophospholipid metabolic pathway, suggesting alterations in cell membrane composition as a core mechanism. Additionally, comparative analyses between the wild-type and mutant strains demonstrated notable differences in acid production, viable cell counts, and storage stability. The mutant strain maintained a higher pH and reduced titratable acidity during extended storage while preserving bacterial viability, distinguishing it from traditional approaches reliant on additives or processing modifications. The findings underscore the potential of α-glucosidase as a pivotal target for post-acidification regulation in fermented milk products. Furthermore, this work contributes valuable insights into the strain improvement strategies essential for advancing the stability and sensory qualities of fermented milk.