Integrated proteomic characterization of extracellular vesicles from breast-milk–derived Limosilactobacillus fermentum

Introduction: Bacterial extracellular vesicles (BEVs) are lipid-based nanoparticles mediating microbe-host communication. While Lactic Acid Bacteria (LAB) produce BEVs, molecular data on strains from human milk remain limited. This study characterizes physicochemical and proteomic features of BEVs from three Limosilactobacillus fermentum strains from human milk and evaluates their compatibility with intestinal epithelial cells.Materials and Methods: BEVs were isolated from cultures of L. fermentum DSM 34871, 34872, and 34873 via ultracentrifugation. Vesicles were analyzed by nanoparticle tracking, ζ-potential, nucleic acid and protein quantification, and LC–MS/MS proteomics. Protein localization and function were annotated using GO and KEGG analyses. Cytocompatibility was tested on Caco-2 cells via MTT assay.Results: BEVs averaged 133–135 nm in diameter, carried negative surface charge, and showed strain-dependent nucleic acid content. Proteomics identified 851 vesicle-associated proteins, including a conserved core of 495, mainly cytoplasmic and involved in translation, metabolism, and amino acid biosynthesis. Several moonlighting proteins were detected, including factors implicated in host-microbe interactions such as adhesion and immune modulation. BEVs showed no cytotoxicity toward cells.Conclusions: This study provides a comprehensive profile of L. fermentum BEVs from human milk, revealing their complex proteome, potential roles in host interaction, and compatibility with intestinal epithelium, supporting future exploration of their impact on maternal-infant health.