Impact of the in vitro gastrointestinal digestion protocol on casein phosphopeptide profile of Grana Padano cheese digestates

•Release of casein phosphopeptides during Grana Padano cheese digestion was studied.•Two in vitro gastrointestinal digestion (GID) protocols were compared.•Similar number of CPPs containing the amino acid motif sssEE were generated.•Importance of the in vitro GID protocol to prove cheese bioactivity.

The release of casein phosphopeptides (CPPs) during in vitro gastrointestinal digestion (GID) of Grana Padano (GP) cheese samples aged 13, 19 and 26 months was studied applying two protocols. The first one (PePan) was chosen as a common simple in vitro GID method, which uses only pepsin and pancreatin as digestive enzymes. The second protocol (COST) represented a more physiologically relevant one, which has recently received an international scientific consensus as a standard in vitro method for food digestion. The nature of released CPPs was assessed by UPLC coupled to High Resolution Mass Spectrometry. After pepsin attack, pancreatin proteolysed stronger, and a double number of CPPs generated in PePan digestates in comparison to the COST ones, despite of the cheese aging period. Upon the two GIDs, CPPs released almost from the same domains of parent caseins. A similar number and type of triphosphorylated CPPs containing the motif SerP-SerP-SerP-Glu-Glu (sssEE), the most effective Ca binding amino acid sequence, generated. Overall, the results emphasize the role of the in vitro digestion protocol when the best proof of cheese bioactivity related to CPPs has to be assessed.