To develop a novel type of cheese using Aspergillus oryzae, four strains (AHU 7139, AHU 7140, AHU 7141 and AHU 7146) were tested with respect to their extracellular protease production on solid-state media containing heatdenatured whey protein under various temperatures and pH levels. Proteolytic activity of the crude enzyme extracts was evaluated under acidic condition considering the circumstance of cheese ripening. Zymographic analysis revealed that several kinds of proteases were secreted, which were dependent on the strain, incubation temperature and initial pH value. Using three strains, six types of culture products were selected as adjunctive materials in combination with an incubation temperature of 15 or 20ºC and an initial pH condition of 4.0 or 6.5. Gouda-type cheese curd was prepared, and the curd particle received 1% (w/w) of the culture product, followed by pressing, cooling, salting and ripening for 3 months at 11.5ºC. An increase in water extractable nitrogen and free fatty acid was observed in some experimental cheeses, depending on the strain and culture history. In particular, incorporation of culture products from A. oryzae AHU 7139, incubated at pH 6.5 and 20ºC affected these parameters. As a novel dairy technology, this study propose new insights into the potential use of A. oryzae for providing an enzyme cocktail to develop palatable cheese flavor when suitable strains and culture conditions offer products with an optimum balance of proteases and lipases.

The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.