Abstract
Postbiotics derived from Bacillus species are recognized as promising natural antimicrobial agents. This study aimed to systematically evaluate the inhibitory activity of postbiotics derived from B. velezensis 906 against L. monocytogenes, elucidate the underlying antibacterial mechanisms using agar diffusion assays, broth microdilution, growth kinetics, flow cytometry, phospholipid competition assays, whole-genome mining, and non-targeted metabolomics, and characterize the bioactive metabolites responsible for their antibacterial effects. The postbiotics exhibited significant antagonistic activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. They also inhibited pathogens such as Salmonella and Enterobacter sakazakii. Against L. monocytogenes, the minimum inhibitory concentration was 0.0083 mg/mL. At 1× MIC, the OD600 after 24 h remained at approximately 0.8, compared with 1.3–1.4 in the untreated control, whereas treatment at 4× MIC almost completely inhibited bacterial growth. Mechanistic analyses suggested that the postbiotics interact with membrane phospholipids, resulting in membrane disruption, increased intracellular reactive oxygen species accumulation, and enhanced membrane permeability. Integrated genome mining and non-targeted metabolomics indicated that the antibacterial activity was associated with a coordinated antimicrobial network involving lipopeptides, polyketides, bacteriocin-related compounds, and siderophore-associated metabolites. These findings provide insight into the antibacterial basis of B. velezensis 906 postbiotics and support their potential application in food safety control.