Research has demonstrated that the presence of synthetic preservatives tends to disrupt the balance of the gut microbiota, thereby posing a significant threat to food safety and human health. The present study investigates the antibacterial activity of a novel bacteriocin-like produced by Latilactobacillus sakei (L. sakei) against Escherichia coli (E. coli), as well as its mechanism of action. This study aims to validate the significant antibacterial effect of bacteriocin-like YY-1 produced by L. sakei W-1 through dialysis combined with Tricine-SDS-PAGE analysis, and to determine its molecular weight. The results of the study indicate that the molecular weight of the bacteriocin-like is less than 2.7 kDa. Moreover, this bacteriocin-like YY-1 exhibits broad-spectrum antimicrobial properties, demonstrating antibacterial activity against E. coli, S. Typhimurium, A. baumannii, P. mirabilis, S. aureus, L. monocytogenes. Furthermore, bacteriocin-like YY-1 exhibits optimal antibacterial activity at a pH of 4.0, with its activity gradually diminishing as pH increases. It is completely inactivated by trypsin treatment, while papain and proteinase K treatments significantly reduce its antibacterial activity. Additionally, this bacteriocin-like YY-1 has been demonstrated to inhibit the growth of E. coli and disrupt its normal development. The Viable/Necrotic Cell Stain and SEM observations confirmed that the bacteriocin-like compound induces cell death by forming distinct pores through the disruption of the cell membrane’s structural integrity. In summary, YY-1—a bacteriocin-like substance characterised by its low molecular weight (<2.7 kDa), broad-spectrum activity, and pore-forming mechanism—is a highly promising natural alternative to synthetic preservatives, capable of mitigating the damage caused by chemical additives to the gut microbiota.

Aeromonas hydrophila is a globally distributed aquatic pathogen. Antibiotic overuse in traditional management practices has intensified problems such as antimicrobial resistance and environmental pollution, creating an urgent need for safe alternatives. This study characterized a BLIS produced by Leuconostoc falkenbergense SBL-85-2, which shows potential as an antibiotic alternative in aquaculture. L. falkenbergense SBL-85-2 exhibited no hemolytic activity, lacked typical high-risk virulence factors, and only low-identity vancomycin-related intrinsic genes (vanT and vanY) were identified by the CARD database, which may preliminarily indicate its potential biosafety for aquatic applications. The BLIS exhibited marked activity against A. hydrophila, with an inhibition zone of 39.14 ± 0.65 mm, as well as other critical pathogens including Aeromonas rivipollensis, Aeromonas salmonicida, Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, and Vagococcus fluvialis. Furthermore, the BLIS exhibited remarkable thermal stability (retained 87.18 ± 0.14% activity after treatment at 100 °C for 10 min) and UV stability (92.67 ± 0.75% residual activity after 3 h exposure) coupled with sensitivity to proteases, confirming its proteinaceous nature. Molecular weight determination indicated that the BLIS is a low-molecular-weight peptide (<2.7 kDa). Mechanistically, the BLIS disrupted bacterial cell membrane integrity, ultimately resulting in the leakage of cellular contents and a time-dependent reduction in intracellular ATP levels (reduced by 69.16 ± 4.10% at 2 h), exerting its antimicrobial effect. Collectively, our results indicate that this BLIS combines potent antibacterial efficacy with a favorable biosafety profile, suggesting its significant potential for the biological control of A. hydrophila in aquaculture.