In order to improve the enzyme activity of Pixian horsebean paste, fungus with high protease production ability from traditional fermented soy sauce products were isolated and identified. The enzyme activities of two Koji making processes were studied using single strain and multi-strain co-cultivation. Two strains, QM-6 and QH-3, with high-protease production ability were isolated. They were further identified as Aspergillus oryzae and Aspergillus niger, respectively, using morphology and biological analysis. The broad bean koji produced by co-cultivation with A. oryzae QM-6 and A. niger QH-3 had shorter fermentation period and more fragrance, compared with that using single strain cultivation. Moreover, activities of neutral protease, alkaline protease, and aminopeptidase were higher in koji produced by co-cultivation in comparison to those produced by A. oryzae QM-6 alone, with the highest activities of 1 532 U/g, 1 164 U/g, and 151 U/g, respectively. The highest activities of acid protease, cellulase, and amylase were also obtained using co-cultivation (513 U/g, 121 U/g, and 574 U/g, respectively), which were about twice as high as that of single strain fermentation. Based on the changes in mycelium, spores, color, flavor, texture of koji making, and enzyme activities, co-cultivation of A. oryzae QM-6 and A. niger QH-3 in broad bean koji making could effectively improve enzyme activities and qualities of broad bean koji to compensate the shortages of producing koji by single strain fermentation.
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