Pyroglutamyl-γ3-casein as marker of proteolysis and ripening period of Grana Padano and Parmigiano Reggiano cheeses

Grana Padano (G.P.) and Parmigiano Reggiano (P.R.) are extra-hard, long ripened PDO cheeses and, during maturation, they undergo to extensive proteolysis. Different chemical indices related to proteolysis and ageing period have been investigated in G.P. (n=155) and P.R. (n=27) cheese samples aged from 7 to 38 months, by means of ion exchange chormatography and capillary electrophoresis (CE). The levels of intact caseins, γ-caseins and free amino acids were measured but showed low correlation with the ripening period as they varied among samples of the same age. A peptide, identified by means of HPLC/electrospray ionization-mass spectrometry as pyroglutamyl-γ3-CN (pγ3-CN), resulted from cyclization of N-terminal GLU of γ3-CN into pyroglutamic acid. According to CE electropherograms, this peptide accumulated as a function of cheese age. Based on the peak area ratio of pγ3-CN and γ3-CN, an algorithm suitable to determine the cheese age was proposed and adopted to verify the typicalness and the ripening period of the two G.P. categories “G.P. ripened over 16 months” and “G.P. Riserva” (i.e. ripened over 20 months).