Pancreatic hydrolysis of bovine casein: Identification and release kinetics of phosphopeptides

Bovine casein was digested with pancreatin at pH 8.0 in a batched stirred tank reactor. In total, 52 casein phosphopeptides (CPPs) in the time-course samples were separated and identified using liquid chromatography-tandem mass spectrometry by means of neutral loss scanning and database searching. Of these peptides, 23, 7, 8, 10 and 4 are singly, doubly, triply, quadruply and quintuply phosphorylated, respectively. Furthermore, 14 peptides contained the cluster sequence, S(P)S(P)S(P)EE, providing the mineral binding sites; these peptides were all formed after 10 min of hydrolysis, but none of them survived after 6 h of pancreatic digestion. A study of the release kinetics of CPPs allowed determination of the degrees of hydrolysis for the preparation of target peptides with high yields. The discrimination of pancreatic attack on different phosphorylated regions of bovine casein was also analyzed in terms of the release of CPPs.