Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese

Abstract

SUMMARYThe whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immuno blotting with polyclonal antibodies against β- and αs1-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 thein vitrohydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4·6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as γ3-casein (β-casein(f108–209)) and αs1-PL1 (αs1-casein(f80–199)). In both cases, a Lys–X bond was hydrolysed, indicating the action of a trypsin-like enzyme in β- and αs1-casein hydrolysis during the ripening of this variety of hard pressed cheese.