About presence of free phosphoserine in ripened cheese and in enzymatic hydrolysate of casein

Occurrence of free phosphoserine (SerP) in ripened cheeses was investigated to clarify whether this amino acid can be directly released from casein by enzymatic attack. Free amino acids extracted from cheese were separated by IEC or RP-HPLC and the peak of SerP always eluted close to unretained material and acidic components. Presence of these interferences suggests that the content of free SerP (from 0.8 to 16.4 mmoles/kg cheese) could be highly overestimated. These figures were dramatically lowered (up to 100 times) after a purification step of the cheese extract on cationic column, but the chromatographic separation of SerP was not yet interference-tree. However, these values of free SerP are not significant on the basis of the total amount of free amino acids (<0.06%). The interfering compounds were characterized in Grana Padano cheese by FAB-MS and several low-MW peptides were found, the most abundant of which was the casein phosphopeptide (CPP) β(16-22)3P. In vitro enzymatic hydrolysis of casein confirmed that accumulation of short-chain CPPs having the common structure X-SerP-SerP-SerP-Glu-Glu-X occurs, explaining incomplete recovery of the amino acids. The recovery increased from 89% up to 96% and the content of Ser and Glu approached the theoretical value when the enzymatic hydrolysis was performed in presence of alkaline phosphatase. These data are consistent with splitting of Ser and not of SerP from casein by enzymatic attack and with accumulation of only free Ser in cheese ripening. Because of the accumulation of "enzyme-resistant" CPPs, in vitro enzymatic hydrolysis of casein can be completed when alkaline phosphatase is included in the pool of enzymes.