Acid tolerance is essential for the probiotic efficacy of lactic acid bacteria during food processing and gastrointestinal transit. Among 21 Lactiplantibacillus plantarum isolates from milk kefir, isolate KY4-11 exhibited superior acid resistance (85.3% survival after 2 h at pH 3.0). Integrated multi-omics and physiological analyses revealed a multilayered adaptive strategy. Acid stress triggered intracellular acidification (pHin = 5.40), reduced ATP and reactive oxygen species levels, and induced membrane damage (extracellular β-galactosidase increased to 53.69 U/mL). KY4-11 counteracted these effects by enhancing H + -ATPase activity (1.97-fold), promoting biofilm formation, and activating antioxidant systems. Genomic analysis identified 111 probiotic-associated genes (including cysE , tuf , dps ). Transcriptomic and metabolomic data showed coordinated upregulation of amino acid metabolism, including glutamate conversion to γ-aminobutyric acid, aspartate degradation to ammonia, and activation of cysteine/methionine-dependent antioxidant pathways. DNA and protein repair systems, along with cell envelope remodeling, were significantly enhanced. Concurrently, energy-intensive processes like glycolysis and nucleotide synthesis were downregulated to conserve resources. Differentially expressed small RNAs suggest post-transcriptional regulation of stress responses. Together, these findings demonstrate that KY4-11 survives acid stress through a systemic network integrating metabolic reprogramming, redox homeostasis, membrane integrity, and genome maintenance, providing a mechanistic foundation for developing robust probiotics from traditional fermented dairy sources. • Isolate KY4-11 survives pH 3.0 via multilayered acid tolerance mechanisms • Ammonia from amino acid deamination buffers intracellular acidification • Integrated omics reveals coordinated metabolic and DNA repair responses • sRNAs potentially regulate acid-stress genes (e.g., mutS , tcyB ) • Kefir-derived KY4-11 is a robust candidate for probiotic applications
Yuan et al. (Tue,) studied this question.