Functional and proteomic analysis of Lactobacillus rhamnosus-derived extracellular vesicles with antioxidant and anti-inflammatory activity

Extracellular vesicles (EVs) derived from lactic acid bacteria have gained attention as mediators of host-microbe interaction. However, their molecular characteristics compared to cell-derived vesicles (CDVs) remain poorly understood. In this study, we isolated EVs and CDVs from Lactobacillus rhamnosus and characterized them throughout proteomic and functional analyses. Both vesicle types displayed spherical morphology as observed by nanoparticle tracking analysis and transmission electron microscopy. Western blotting revealed distinct protein profiles, with HSP70 specifically detected in EVs. Proteomic profiling using LC-MS/MS identified a total of 1,342 proteins, including 114 unique to CDVs. Differential expression proteins analysis showed 70 proteins upregulated and 81 downregulated in EVs. Gene Ontology (GO) analysis indicated that EVs were enriched in proteins related to signal transduction, environmental adaptation, and transmembrane transport, while CDVs showed enrichment in ribosomal and nucleic acid metabolism-related proteins. Functional assays further demonstrated that EVs exhibited significantly stronger antioxidant and anti-inflammatory activities than CDVs. These findings suggest that L. rhamnosus-derived EVs possess distinct proteomic signatures and superior bioactivity, supporting their potential role in probiotic-based therapeutic strategies, particularly for skin health.