Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities.

Bacterial membrane vesicles (BMVs) are bi-layered nanostructures derived from Gram-negative and Gram-positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density-gradient centrifugation, size-exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large-scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field.

Bacterial extracellular vesicles and associated functional proteins in fermented dairy products with Lacticaseibacillus paracasei.

Cells of all kingdoms produce extracellular vesicles (EVs); hence, they are present in most environments and body fluids. Lacticaseibacillus paracasei produces EVs that have attached biologically active proteins (P40 and P75). In this study, EV and functional proteins were found in five different commercial dairy-fermented products carrying L. paracasei. Strains present in those products were isolated, and with one exception, all produced small EVs (24-47 d.nm) carrying P40 and P75. In order to winnow bacterial EV from milk EV, products were subjected to centrifugal fractionation at 15,000 × g (15 K), 33,000 × g (33 K), and 100,000 × g (100 K). P75 was present in all supernatants and pellets, but P40 was only found in two products bound to the 15 and 33 K pellets, and 16S rDNA of L. paracasei could be amplified from all 100 K EVs, indicating the presence of L. paracasei EV. To investigate the interactions of bacterial EV and proteins with milk EV, L. paracasei BL23 EV was added to three commercial UHT milk products. Small-size vesicles (50-60 d.nm) similar to L. paracasei BL23 EV were found in samples from 100 K centrifugations, but intriguingly, P40 and P75 were bound to EV in 15 and 33 K pellets, containing bovine milk EV of larger size (200-300 d.nm). Sequencing 16S rDNA bands amplified from EV evidenced the presence of bacterial EVs of diverse origins in milk and fermented products. Furthermore, L. paracasei 16S rDNA could be amplified with species-specific primers from all samples, showing the presence of L. paracasei EV in all EV fractions (15, 33, and 100 K), suggesting that these bacterial EVs possibly aggregate and are co-isolated with EV from milk. P40 and P75 proteins would be interacting with specific populations of milk EV (15 and 33 K) because they were detected bound to them in fermented products and milk, and this possibly forced the sedimentation of part of L. paracasei EV at lower centrifugal forces. This study has solved technically complex problems and essential questions which will facilitate new research focusing on the molecular behavior of probiotics during fermentation and the mechanisms of action mediating the health benefits of fermented products.