Integrative metabolomic and genomic characterization of the high antimicrobial activity of Lacticaseibacillus paracasei and its potential for juice preservation.

Microbial contamination remains a significant challenge for fruit-based products, and natural preservatives from lactic acid bacteria (LAB) present a promising solution. In this study, we explored Lactiplantibacillus paracasei strains isolated from traditional Tibetan fermented dairy products for their broad-spectrum antimicrobial activity and underlying mechanisms. Among 150 LAB isolates, strains 104-3, A-2, and 26-4 exhibited potent inhibition of both Gram-negative and Gram-positive bacteria, including multidrug-resistant ESKAPE pathogens, and yeast species such as Candida tropicalis and Saccharomyces cerevisiae. Metabolomic analysis revealed strain-specific patterns, with fatty acids, small peptides, and aromatic organic acids, including phenyllactic acid, enriched in the most active strains. Functional assays showed that 2-ethyl-2-hydroxybutyric acid (2-EHBA), 2-hydroxyisocaproic acid (HICA), and phenyllactic acid exhibited stronger antibacterial activity than the other tested compounds, suggesting that these metabolites may be the primary contributors to the antibacterial activity of the LAB fermentation supernatant. The activities of 2-EHBA and HICA were modulated by pH, ionic strength, and temperature. Comparative genomics revealed strain-specific variations in gene content, carbohydrate-active enzymes, and central metabolic pathways, which correlated with differences in antimicrobial metabolite production. The application of 1.5% (w/v) freeze-dried fermentation supernatants from strain 104-3 in a pitaya juice model significantly inhibited S. aureus and S. cerevisiae, without affecting juice quality. These results suggest the potential of LAB metabolites for clean-label preservation. Collectively, these findings demonstrate the link between LAB metabolism and antimicrobial efficacy, indicating L. paracasei metabolites as promising candidates for natural food preservation.