Extracellular vesicles of <i>Lactiplantibacillus plantarum</i> PCM 2675 and <i>Lacticaseibacillus rhamnosus</i> PCM 489: an introductory characteristic

<b>Aim:</b> Extracellular vesicles (EVs) are involved in intercellular and interkingdom communication in the complex communities that constitute the niche-specific microbiome of the colonized host. Therefore, studying the structure and content of EVs produced by resident bacteria is crucial to understanding their functionality and impact on the host and other microorganisms. <b>Methods:</b> Bacterial EVs were isolated by differential centrifugation, their size and concentration were measured by transmission electron microscopy and nanoparticle tracking analysis, and the cargo proteins were identified by liquid chromatography coupled to tandem mass spectrometry. The cytotoxicity of bacterial EVs was tested using the human epithelial cell line A549 and an <i>in vivo</i> model of <i>Galleria mellonella</i> larvae. <b>Results:</b> The isolation and preliminary characteristics of EVs from two strains of lactic acid bacteria - <i>Lactiplantibacillus plantarum</i> PCM 2675 and <i>Lacticaseibacillus rhamnosus</i> PCM 489 - were presented, confirming the production of vesicular structures with sizes in the range of 50-170 nm for <i>L. plantarum</i> and 80-250 nm for <i>L. rhamnosus</i>. In addition, various proteins were identified within EVs cargo, with distinct locations of origin, including membrane, cytoplasmic and extracellular proteins, and with diverse functions, including enzymes with confirmed proteolytic activity. Furthermore, bacterial EVs did not show statistically significant cytotoxicity to the host under the tested conditions. <b>Conclusions</b>: A better understanding of the composition and functionality of bacterial EVs may contribute to their future effective use in supporting human health.