Peptic digestion of β-casein: Time course and fate of possible bioactive peptides

Numerous peptides obtained by enzymatic digestion of food proteins have been reported to exhibit biological activities. In this study, the focus was placed on peptides of β-casein from bovine milk after a gastro-analogous in vitro digestion with pepsin, a protease with broad specificity. In order to study the time course of the digestion, the process was stopped after specific times and the samples were subjected to HPLC separation followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and nanoelectrospray (nanoESI) quadrupole time-of-flight (qTOF) mass spectrometry. A combined sequencing approach using de novo interpretation and databases was employed. Overall, 100% of the β-casein sequence was covered by identifying 125 peptides of 4–84 residues in length, including 3 phosphorylated species. The results show that the peptic hydrolysis starts at the C-terminus of the protein. The release of known bioactive peptides from β-casein following the peptic digestion under simulated gastric conditions is unlikely with a few exceptions. Furthermore, an amino acid variation was found, providing evidence for the existence of an additional genetic variant of β-casein.