Milk digestion peptidomics: Tracking caseinophosphopeptides in simulated gastrointestinal digestion

The dynamics of caseinophosphopeptides (CPP) in simulated gastrointestinal digestion was studied. CPP in the undigested milk and in vitro simulated gastrointestinal hydrolysates were enriched using immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) beads and measured using liquid chromatography-tandem mass spectrometry. There were 62, 80 and 38 CPP identified from undigested milk, simulated gastric and pancreatic hydrolysates, respectively. In total, 142 unique CPP and 27 casein phosphorylation sites were identified. Only 5 of 62 endogenous CPP in undigested milk survived in the simulated gastrointestinal hydrolysates and these 5 CPP were semi-quantified. Most of the CPP in undigested milk came from beta-casein. Further, 13 CPP with phosphothreonine were found in simulated gastrointestinal hydrolysates. Seven CPP with the conserved sequence SpSpSpEE were identified from simulated gastrointestinal hydrolysates. Only 2 of the 6 CPP with the conserved sequence SpSpSpEE in undigested milk survived in the simulated gastric digestion, and all of them were digested by pancreatin during simulated pancreatic digestion. The results showed that most of the CPP were released from large endogenous CPP or caseins in simulated gastrointestinal hydrolysates. However, most of them were degraded after simulated pancreatic digestion while the content of some persistent CPP increased at the same time