Proteolysis in rennet casein-based cheese analogues

Rennet caseins manufactured from mid- or late-lactation milk were used to prepare pilot- and commercial-scale Mozzarella-type cheese analogues. Mid-lactation rennet casein and analogues made from it contained low levels of plasmin; late-lactation rennet caseins and analogues made from them had higher levels of plasmin while pilot-scale analogues had higher levels of plasmin than commercial-scale analogues. The cheese analogues were stored at 8 degrees C for 32 weeks during which time total bacterial counts increased from approximately 10(4) to 10(7)-10(8) cfu g-1, with non-starter lactic acid bacteria dominating the microflora. Proteolysis, as reflected by pH 4.6-soluble nitrogen and free amino acids, increased on storage of all the analogues. The increase was greater in the analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in pilot-scale analogues than in commercial-scale analogues, reflecting the differences in plasmin levels as well as slight differences in composition. Polyacrylamide gel electrophoretograms of the cheese analogues showed that beta-casein was hydrolysed more extensively than alphas1-casein and that the concentration of gamma-caseins increased on storage. The degree of hydrolysis of beta-casein was greater in the cheese analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in the pilot-scale analogues than in the commercial-scale analogues reflecting the corresponding plasmin levels. Seventeen peptides in the pH 4.6-soluble extract of the cheeses were isolated and partially sequenced; all originated from the N-terminal region of beta-casein; 9 peptides had Lys29 of beta-casein as the N-terminal, 6 peptides had Arg1 of beta-casein as the N-terminal and the remaining two peptides had Ile30 and Phe33 of beta-casein as the N-terminal, respectively. The pattern of casein hydrolysis in the cheese analogues suggests that plasmin was the primary proteolytic agent contributing to initial hydrolysis of the caseins while microbial proteinases and peptidases may have contributed to the high levels of free amino acids.