Casein and whey protein in vitro digestion in comparison with duodenal and jejunal in vivo digests

The COST Action INFOGEST has developed a harmonized static in vitro digestion protocol based on physiologically relevant conditions1. This method has already proved to produce comparable results in inter-laboratory trials2. Since peptides generated during protein digestion might be biologically relevant for different physiological functions, it is important to directly compare the in vitro results towards in vivo data at peptide level. Therefore, the objective of this work was to assess the casein and whey proteins in vitro digestion according to the harmonized protocol and to match the outcomes with in vivo digests from pig duodenum (with in vitro gastric phase), and human jejunum (with the in vitro intestinal phase). Protein degradation was followed by SDS-PAGE and the HPLC-MS/MS sequenced peptides were processed using peptidomic and statistic tools. In pig duodenum, small amounts of intact caseins were present in all samples, while intact caseins were observed up to 60 minutes of gastric in vitro digestion. Regarding whey proteins, β-lactoglobulin could be detected in human jejunal digests taken at 60 minutes after ingestion, and similarly, this protein was hydrolysed after 60 min into contact with the simulated intestinal juice. The peptide profiles generated after in vitro and in vivo digestions showed clear similarities with characteristic overrepresented regions in all milk proteins. For instance, N-terminal and C-terminal domains of β-casein, and αs1-casein regions 23-30 and 183-193, were the source of abundant peptides, in both, in vitro and in vivo digests. Based on the peptide profiles, the correlation coefficients calculated between in vitro digests and the human jejunal effluents were within the range found for the different volunteers of the study. Results indicate that the harmonized in vitro protocol is a robust and simple model and constitutes a good approximation to the physiological digestion of milk proteins.