Optimizing Prebiotic Saccharides in Culture Medium for the Bifidobacteria-Derived Extracellular Vesicles as...

Extracellular vesicles (EVs) are cell-derived membrane particles that function in intracellular communication. Recently, the secretion of EVs from bacterial cells has been reported, and extensive studies aimed at elucidating their characteristics have been conducted. EVs from probiotics, such as Bifidobacteria, can be used as biomaterial immunoadjuvants by stimulating innate immunity in the host. When cultivating Bifidobacteria, prebiotic saccharides are present in the culture medium as nutritional substances that promote growth. However, little is known about the effect of the type of prebiotic saccharide on the characteristics of Bifidobacteria-derived extracellular vesicles (B-EVs). In this study, we optimized the saccharides for B-EVs as biomaterial immunoadjuvants. Preferable growth of bacteria was observed when cultured in a medium containing glucose (GLU), galactooligosaccharides, and fructooligosaccharides, but very poor bacterial growth was observed without saccharides. The production levels of B-EVs varied with the prebiotic saccharides, and cultivation using a GLU-containing medium resulted in the largest EV yield. Additionally, irrespective of the saccharide types in the culture medium, each of the B-EVs contained peptidoglycan and nucleic acids. Moreover, these B-EVs facilitated the release of tumor necrosis factor-α as well as the expression of co-stimulatory molecules (CD40) in mouse macrophage-like RAW264.7 cells via a Toll-like receptor-2 pathway. These findings indicate that cultivation of Bifidobacteria in a GLU-containing medium is an optimal condition for obtaining desirable EVs for use as a biomaterial immunoadjuvant.