Selective Protein and Lipid Cargo Sorting into Extracellular Vesicles of Lacticaseibacillus paracasei subsp. paracasei.

Extracellular vesicles (EVs) are nanoscale lipid bilayer particles that transport bioactive cargo. EVs from lactic acid bacteria show health benefits and drug-delivery potential, yet cargo encapsulation remains understudied. Here, EVs were isolated, purified, and characterized from the bacteriocin-producing strain Lacticaseibacillus paracasei subsp. paracasei YCL29, which was isolated from fermented Yucha. Proteomic and lipidomic analyses were subsequently conducted to compare the EVs with their parental L. paracasei YCL29 cells. Nanoflow cytometry indicated EVs with mean diameter 86.0 ± 18.8 nm at 4.83 × 109 particles/mL. Proteomic analysis identified 2,120 high-confidence proteins and revealed selective cargo sorting, with 313 proteins enriched in EVs and 446 downregulated relative to the parent cells. EV-enriched proteins were associated with stress tolerance, nutrient transport/metabolism, nucleic acid metabolism, catalysis, and antibiotic biosynthesis, including cold-shock and antioxidant proteins were detected under non-stress conditions. Lipidomic analysis showed enrichment of phosphatidylethanolamine and triglycerides, defining a distinct EV lipid signature. These findings provide insights into potential cargo-sorting mechanisms and functions of L. paracasei EVs.