Probiotic-derived extracellular vesicles: Emerging natural inhibitors of foodborne pathogens and biofilms

Foodborne pathogens, Staphylococcus aureus, Escherichia coli, Salmonella and Listeria monocytogenes pose significant health risks to human health and food safety worldwide. Recently, extracellular vesicles (EVs) derived from probiotic lactic acid bacteria (LAB) have emerged as promising natural antimicrobial agents. The aim of this study was to isolate EVs from Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus during stationary growth phase using size-exclusion chromatography (SEC) and to characterize them using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, the antimicrobial activity was accessed using planktonic cell viability assays and antibiofilm activity was evaluated by using crystal violet staining assay. The isolated EVs exhibited spherical shaped morphology, with particle concentrations ranging from 6.7 × 10⁹ to 3.0 x 10⁸ particles/mL and their peak sizes between 90 - 280 nm. EVs significantly inhibited the planktonic cell growth all four tested pathogens in a dose dependent manner. However, a reduction of Salmonella planktonic cells was observed only at 15 µg/mL ( ***P < 0.001 and ****P < 0.0001) for EVs derived from both L. plantarum and L. rhamnosus. Biofilm formation by foodborne pathogens was significantly reduced at all tested concentrations relative to the control for both LAB strains (**p < 0.01). However L. plantarum EVs were the most effective, at reducing Salmonella biofilms by 86% at 5 µg/mL (*P < 0.05). In conclusion, probiotic-derived EVs exhibited significant antimicrobial and antibiofilm activities, highlighting their potential for application in food safety and infection prevention strategies.