Use of nattokinase, a subtilisin-like serine proteinase, to accelerate proteolysis in Cheddar cheese during ripening

Nattokinase, a subtilisin-like serine proteinase produced by Bacillus natto, is present in the traditional fermented Japanese soyabean product "natto". The aim of this study was to investigate the effects of a semi-purified preparation of nattokinase (added to cheesemilk at 80, 160 or 320 μg-mL^-1), on proteolysis in Cheddar cheese during cheese ripening. Levels of pH 4.6-soluble N increased with ripening time and with level of addition of nattokinase. Specifically, increased breakdown of α_s1- and β-caseins was observed in experimental cheeses compared to control cheese. With increasing level of addition of nattokinase, increased production of a peptide with an electrophoretic mobility lower than that of the λ-caseins was observed. Principal component analysis of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-soluble fractions of cheeses clearly differentiated cheeses based on level of addition of nattokinase. Levels of most of the free amino acids were higher in experimental cheeses compared to the control; however, the increase was not proportional to level of addition of nattokinase. Overall, use of nattokinase as an exogenous enzyme accelerated primary proteolysis in Cheddar cheese and increased the pool of most of the free amino acids, which serve as substrates for many catabolic reactions and in turn affects the flavour of cheese.