ABOUT PRESENCE OF FREE PHOSPHOSERINE IN RIPENED CHEESE AND IN ENZYMATIC HYDROLYSATE OF CASEIN

Occurrence of free phosphoserine (SerP) in ripened cheeses was investi gated to clarify whether this amino acid can be directly released from casein by enzymatic attack. Free amino acids extracted from cheese we re separated by IEC or RP-HPLC and the peak of SerP always eluted clos e to unretained material and acidic components. Presence of these inte rferences suggests that the content of free SerP (from 0.8 to 16.4 mmo les/kg cheese) could be highly overestimated. These figures were drama tically lowered (up to 100 times) after a purification step of the che ese extract on cationic column, but the chromatographic separation of SerP was not yet interference-free. However, these values of free SerP are not significant on the basis of the total amount of free amino ac ids (<0.06%). The interfering compounds were characterized in Grana Pa dano cheese by FAB-MS and several low-MW peptides were found, the most abundant of which was the casein phosphopeptide (CPP) ss(16-22)3P. In vitro enzymatic hydrolysis of casein confirmed that accumulation of s hort-chain CPPs having the common structure X-SerP-SerP-SerP-Glu-Glu-X occurs, explaining incomplete recovery of the amino acids. The recove ry increased from 89% up to 96% and the content of Set and Glu approac hed the theoretical value when the enzymatic hydrolysis was performed in presence of alkaline phosphatase. These data are consistent with sp litting of Ser and not of SerP from casein by enzymatic attack and wit h accumulation of only free Ser in cheese ripening. Because of the acc umulation of ''enzyme-resistant'' CPPs, in vitro enzymatic hydrolysis of casein can be completed when alkaline phosphatase is included in th o pool of enzymes.