Background: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the co-cultivated strains of Aspergillus oryzae QM-6 and Aspergillus niger QH-3, optimization of acid and neutral protease activities was carried out using Box-Behnken design with response surface methodology.

Results: The optimum conditions were found to be as follows: inoculation proportion (X₁), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X₂), 33°C; inoculum size (X₃), 0.5% (w/w); incubation time (X₄), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of co-cultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the co-cultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products.

Conclusions: The optimal conditions of koji-making in PBP were obtained using RSM. The co-cultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content which would thus have potential application in the PBP industry.