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

Mol Nutr Food Res. 2024 May;68(10):e2300620. doi: 10.1002/mnfr.202300620. Epub 2024 May 6.


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.

Keywords: INFOGEST; extracellular vesicles; gastrointestinal digestion; infant; milk.

MeSH terms

  • Animals
  • Bile* / metabolism
  • Cattle
  • DNA-Binding Proteins
  • Digestion* / physiology
  • Endosomal Sorting Complexes Required for Transport
  • Extracellular Vesicles* / metabolism
  • Milk* / chemistry
  • Transcription Factors


  • Tsg101 protein
  • DNA-Binding Proteins
  • Transcription Factors
  • Endosomal Sorting Complexes Required for Transport