Inhibition of residual coagulant in cheese using pepstatin

Pepstatin A, an inhibitor of acid proteases, was added (7.5, 15 or 30 μmol L^-1) to the curds/whey mixture at the start of cooking to inhibit residual coagulant in miniature (20 g) Cheddar-type cheeses. No degradation of α(s1)-casein was observed by urea-polyacryl amide gel electrophoresis (PAGE) in the pepstatin-treated cheeses, indicating that all the concentrations of pepstatin used in this study effectively inhibited residual coagulant throughout ripening. The level of water-soluble N (WSN) as % of total N increased very slowly in the pepstatin-treated cheeses, while there was a steady increase in WSN in the control cheeses; after 4 months of ripening, the level of WSN in the control cheese was nearly three times as high as in the cheese treated with 30 μmol L^-1 pepstatin. Urea-PAGE of water-soluble fractions (WSF) showed marked differences between pepstatin-treated cheeses and their respective controls throughout ripening. Reverse-phase HPLC of the WSF of the cheeses showed that the peptides α(s1)-CN f1-9/13, which are formed from the chymosin-produced peptide, α(s1)-CN f1-23, by the action of the cell envelope-associated proteinase of Lactococcus, were not present in pepstatin-treated cheeses. Levels of total free amino acids (as determined by the Cd-ninhydrin method) were higher in controls than in pepstatin-treated cheeses throughout ripening. The results of this study demonstrated that pepstatin is a very effective inhibitor of residual coagulant in cheese.