In vitro digestion of milk proteins including intestinal brush border membrane peptidases. Transepithelial transport of resistant casein domains

[Display omitted] •Simulated digests with BBM increase free amino acids and reduce peptide size.•Exclusive cleavages could be ascribed to the BBM inclusion in the intestinal phase.•Human jejunum peptides comprise BBM- and non BBM-derived sequences.•β-casomorphin-7 and related resistant sequences show different apparent permeability. The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six β-casein derived peptides resistant to gastrointestinal digestion, including the opioid β-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides.