The protease activities in various combinations of substrates were similar at the beginning of fermentation.
After 24 h, neutral protease activity of 60% soybean sample increased rapidly and reached the highest activity
of 84.38 U/g dry weight at 48 h of fermentation, then it declined (Fig. 1c).
The highest alkaline protease activity was 41.35 U/g dry weight at 72 h of fermentation in 60% soybean
sample (Fig. 1d). Whereas both neutral and alkaline protease activities were lower in the 40% and 50%
soybean sample. The previous study reported that production of enzymes required amino acids by digesting
proteins in substrate. A. oryzae can utilize a pool of nitrogen sources in soybean to produce amino acids, it
decreases the total protein content of soybean koji during cultivation [3, 5].
Soybean is a good substrate containing high nitrogen content to produce proteolytic enzymes. The result
indicated that substrate contained high content of soybean (60%) was the most optimal condition for protease
production in soybean koji fermentation.
However, higher content of wheat bran in the substrate showed higher amylase activity (Fig. 1e). The
highest amylase activity of 731.53 U/g dry weight was obtained in the 40% soybean sample. It is clear that the
high carbon source contained substrate encourages the amylase production. The digestion of carbohydrates in
substrates is required for the metabolism of A. oryzae S. Therefore, amylase production was accompanied
with the growth of fungi [5].