Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment-mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, \u3b2-CNf(7-28)4P, \u3b1(s1)-CNf(61-79)4P and \u3b1(s2)-CNf(7-21)4P. In total, 45 phosphopeptides were identified: 24 from \u3b1-CN, 16 from \u3b1(s1)-CN and 5 from \u3b1(s2)-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed