Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4–10) and temperature (60–100 °C). Furthermore, the proteolytic treatment reduced the biomolecule’s antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

The escalating global concern regarding bacterial antibiotic resistance in animal production has intensified the search for sustainable and effective alternatives to conventional antimicrobials. In this study, two L. reuteri strains (LBM-Ti195 and LBM-Ti173) are isolated from broiler cecal microbiota that were characterized through an integrated approach, combining phenotypic assays with comparative genomic analysis. Both strains exhibited antibacterial activity against relevant veterinary and foodborne pathogens, including Listeria monocytogenes CECT 934, Staphylococcus aureus CECT 239, Clostridium perfringens Type A, and Campylobacter jejuni CCAMP 162. The inhibitory activity anti-S. aureus increased by more than 10% modifying cultivation conditions, while comparative genomic analysis identified an M23-family metallopeptidase as a potential candidate for pathogen inhibition. Phenotypically, both strains produced folate and metabolized fructooligosaccharides (FOS) and inulin, supporting their potential compatibility with synbiotic formulations. Genome reconstruction reinforces these functional findings by revealing a complete predicted de novo folate biosynthesis pathway. In addition, CAZyme annotation identified higher copy numbers of glycosyltransferases GT2 and GT4 compared with the reference strains, suggesting differences in cell-surface carbohydrate metabolism and exopolysaccharide (EPS)-associated traits. Safety profiling revealed no hemolytic activity or conserved virulence factors under the tested conditions. However, phenotypic tetracycline resistance was detected, and in silico analysis identified an acquired tetW gene in a putative plasmid-associated context, highlighting the importance of an in-depth evaluation of strains with probiotic potential. Collectively, these findings position LBM-Ti195 and LBM-Ti173 as promising avian-derived L. reuteri candidates for next-generation zootechnical probiotic development, while highlighting antimicrobial resistance (AMR) mitigation and further functional validation as essential steps toward safe application.