Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Fructose-induced topographical changes in fructophilic, pseudofructophilic and non-fructophilic lactic acid bacterial strains with genomic comparison.

Fructophilic Lactic Acid Bacteria (FLAB), Fructobacillus fructosus DPC7238 and pseudofructophilic Leuconostoc mesenteroides DPC7261 and non-FLAB Limosilactobacillus reuteri DSM20016 strains were studied for their growth and morphological evolution as a function of increased fructose concentrations (0, 25, and 50% w/v) in the media. A comparison of the genomics of these strains was carried out to relate observed changes and understand fructose-rich adaptations. The viability of FLAB strains were reduced by approx. 50% at a 50% fructose concentration, while the Limosilactobacillus reuteri strain was reduced to approx. 98%. Electron microscopy demonstrated that FLAB strain, Fructobacillus. fructosus and pseudofructophilic Leuc. mesenteroides, were intact but expanded in the presence of high fructose in the medium. Limosilactobacillus reuteri, on the other hand, ruptured as a result of excessive elongation, resulting in the formation of cell debris when the medium contained more than 25% (w/v) fructose. This was entirely and quantitatively corroborated by three-dimensional data obtained by scanning several single cells using an atomic force microscope. The damage caused the bacterial envelope to elongate lengthwise, thus increasing width size and lower height. The cell surface became comparatively smoother at 25% fructose while rougher at 50% fructose, irrespective of the strains. Although Fructobacillus fructosus was highly fructose tolerant and maintained topological integrity, it had a comparatively smaller genome than pseudofructophilic Leuc. mesenteroides. Further, COG analysis identified lower but effective numbers of genes in fructose metabolism and transport of Fructobacillus fructosus, essentially needed for adaptability in fructose-rich niches.