GEL-ELECTROPHORESIS AND IMMUNOBLOTTING FOR THE DETECTION OF CASEIN PROTEOLYSIS IN CHEESE

The whole N fraction of six samples of hard and semi-hard pressed chee ses was analysed using PAGE, polyacrylanzide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha( s1)-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it wa s also possible to determine which casein was the source of each pepti de and which enzymes were active in cheese. Compared with the traditio nal Coomassie staining procedures, immunoblotting is more sensitive an d specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the stat e of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano u sing DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma(3)-casein ( (beta-ca sein(f108-209)) and alpha(s1)-PL1 (alpha(s1)-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin -like enzyme in beta- and alpha(s1)-casein hydrolysis during the ripen ing of this variety of hard pressed cheese.