Multi-omics and phenotypic analysis of acid stress adaptation in Lactiplantibacillus plantarum isolated from milk kefir

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