Metabolomics‐Based Functional Characterization of <i>Levilactobacillus Brevis</i> : Insights Into Probiotic and Antimicrobial Potential

Food contamination remains a significant public health issue, often resulting in foodborne illnesses caused by pathogenic microorganisms. Lactic acid bacteria (LAB), particularly Levilactobacillus brevis, have garnered increasing interest for their antimicrobial capabilities in enhancing food safety. This study investigates the antimicrobial activity and secondary metabolite production of cell-free supernatant extract in ethyl acetate of L. brevis MTCC 1750, emphasizing its potential application in food bio-preservation. The strain demonstrated key probiotic attributes, including acid tolerance, strong cell surface hydrophobicity, and 48% auto-aggregation after 12 h, indicating good survivability and colonization potential. L. brevis MTCC 1750 exhibited broad-spectrum antibacterial and antifungal activity, forming clear inhibition zones. Additionally, it significantly inhibited biofilm formation and quorum sensing (QS)-regulated pigment production, reducing violacein, prodigiosin, and pyocyanin by 52.05%, 90.43%, and 43.73%, respectively. Importantly, metabolomics-guided analyses (FTIR, HPTLC, and HR-LCMS) provided a detailed chemical fingerprint of diverse bioactive metabolites, including peptides, fatty acids, and phenolic compounds, which has not been previously much reported for this strain. Furthermore, the study uniquely demonstrates the dual functionality of this strain, combining probiotic attributes with strong anti-QS and antibiofilm activities, thereby highlighting its novelty compared to earlier investigations. The nonhemolytic activity of the strain further supports its safety. These findings highlight Levilactobacillus brevis MTCC 1750 as a promising natural biopreservative with potent antimicrobial and antibiofilm properties. The integration of metabolomics-guided secondary metabolite profiling with functional assays represents a novel contribution, positioning this strain as a safe, multifunctional candidate for food safety enhancement and shelf-life extension.