Isolation and Physicochemical Characterization of Extracellular Vesicles (EVs) from Lactiplantibacillus plantarum Strain CIDCA 83114 Grown in a Minimal Synthetic Medium (MSM)

Abstract

Lactobacilli are Gram-positive fermentative bacteria with GRAS (“Generally Recognized As Safe”) status. Since 2017, it has been reported that many lactobacilli strains can form extracellular vesicles. Extracellular vesicles (EVs) are spherical bilayered membrane structures of nanometric size that carry cytoplasmic components. Bacterial EVs are considered important mediators between host and commensal bacteria, but their biological effects have not been widely studied. The purification of EVs involves methodological difficulties. Due to their nanometric size, EVs are prone to being co-purified with media compounds, which interfere with their characterization. The present work aimed to obtain high-purity EVs and to determine their physicochemical properties. To achieve the objectives, we developed a minimal synthetic medium (MSM) suitable for the growth of Lactiplantibacillus plantarum CIDCA 83114 and also designed a protocol to concentrate and obtain its EVs. The medium was free of high-molecular-weight compounds and surfactants, and the purification protocol included EV concentration via a tangential flow filtration step. The EVs obtained had an average size of 110 nm ± 40 nm and a negative zeta potential. They exhibited a mean protein content of 100 µg/mL with a clear protein profile absent in the culture media. EVs also contain RNA and scarce DNA. Our work contributed to improving the purification of lactobacilli EVs by replacing the traditional medium used in lactic acid bacteria growth (MRS) with a synthetic minimal medium (MSM). The design of the MSM allows for high-purity vesicles to be obtained and accurately characterized.