Zinc oxide nanoparticles (ZnONPs) exhibit antimicrobial, antibiofilm, and antioxidant properties, and their green synthesis using exopolysaccharides (EPS) from probiotic bacteria represents a sustainable alternative to conventional chemical reducing agents. In this study, EPS derived from Limosilactobacillus fermentum was employed as a bioreducing and stabilizing agent for the synthesis of EPS-ZnONPs, which were characterized by zeta potential analysis, UV–Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The nanoparticles presented hydrodynamic diameters ranging from 200 to 400 nm and maintained spectral stability in the UV–Vis region for up to 120 days of storage. Antimicrobial and antibiofilm activities were assessed against Escherichia coli and Staphylococcus aureus using a resazurin-based microtiter assay at concentrations ranging from 25 to 1.56 mg/mL. EPS-ZnONPs reduced biofilm formation by 81.65%, 46.39%, and 62.05% at 1× and 2× MIC, respectively, after 24 h of incubation. SEM analysis revealed membrane discontinuities in treated cells, while both microorganisms exhibited increased production of reactive oxygen species, indicating the induction of oxidative stress. Cytotoxicity assays demonstrated that none of the EPS-ZnONPs components exerted toxic effects on Vero CCL-81 cells. Collectively, the eco-friendly synthesis, lack of cytotoxicity, and functional biocompatibility of EPS-ZnONPs support their potential application as antimicrobial agents against foodborne pathogens.
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