CHARACTERIZATION OF PRATO CHEESE RIPENING BY MASS SPECTROMETRY AND CAPILLARY ELECTROPHORESIS

Among all biochemical changes that occur during cheese ripening, proteolysis is considered the most complex event, characterized by the hydrolysis of the caseins to peptides, amino acids, and their degradation products. The aim of this study was to characterize the proteolysis of Prato cheese during 120 days of ripening via hydrolysis profile of caseins by capillary electrophoresis (CE) and peptide profile by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Cheeses were produced in triplicate according to traditional technology and were evaluated after 1, 15, 30, 60, 90 and 120 days of ripening by CE and MALDI-MS. The electropherograms revealed the initial hydrolysis of αs1-casein at Phe23-Phe24 bond, by the action of the residual chymosin, releasing αs1-CN (f24-199) (αs1-I-CN), which accumulated at the beginning of ripening and was practically totally degraded after 120 days of ripening. Degradations of β- and κ-caseins, although slower, were observed by the reduction of the peak intensities of κ-casein and the β-CN A1 and β-CN A2 variants. The score plot obtained by principal component analysis (PCA) of mass spectral data revealed a clear separation of the cheeses in different stages of ripening. At the beginning of ripening, cheeses were characterized by high abundance of the αs1-CN f1-16 (m/z 1877) peptide, which showed a mean relative intensity of 96.12% on day 1 and 0.72% after 120 days of ripening. Cheeses with longer ripening times showed a higher abundance of the αs1-CN f1-13 (m/z 1536) peptide, which mean relative intensity increased from 12.23% to 90.91% from the 1st to the 120th day of ripening, respectively.