3.1. Physical properties
The initial pHs of different treatments were all around pH 6.32, then the pH decreased followed by an increasing after 36 h of fermentation (Fig. 1a). The results showed that the koji sample containing 60% soybean presented the highest increasing of pH to 6.97 after 72 h of cultivation. The increasing of pH was due to the production of various extracellular proteins from A. oryzae S. during soybean koji fermentation [3]. On the contrary, moisture content decreased with the fermentation time. It changed from 35-40% to 19- 22% during 72 h of fermentation in 3 different combinations of substrates (Fig. 1b). The sample contained higher content of soybean showed higher initial moisture content. The decreasing of moisture content was caused by the mycelia growth of A. oryzae S. during fermentation. Moreover, moistening brings a suitable aw for mold growth because of the easy penetration of mycelia into the substrates [9]. It was opposite to the results of Chutmanop et al. [5] that the moisture content of fermented wheat and rice bran increased during fermentation. It might be due to the different substrates. Soybean particle is bigger than rice and wheat bran. It supports air circulation and affect oxygen and heat transfer in the substrate.
3.2. Effect of different combinations of substrates on enzymes production
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].
3.3. Effect of different combinations of substrates on reducing sugar content
The profile of reducing sugar production was similar to the amylase activity. The reducing sugar content increased during the cultivation period. The highest reducing sugar content was 341.01 mM/g dry weight presented in 40% soybean sample (Fig. 1f). The increasing of reducing sugar content is caused by the enzymatic hydrolysis of starch in the substrates during fermentation [9].
3.1. Physical properties
The initial pHs of different treatments were all around pH 6.32, then the pH decreased followed by an increasing after 36 h of fermentation (Fig. 1a). The results showed that the koji sample containing 60% soybean presented the highest increasing of pH to 6.97 after 72 h of cultivation. The increasing of pH was due to the production of various extracellular proteins from A. oryzae S. during soybean koji fermentation [3]. On the contrary, moisture content decreased with the fermentation time. It changed from 35-40% to 19- 22% during 72 h of fermentation in 3 different combinations of substrates (Fig. 1b). The sample contained higher content of soybean showed higher initial moisture content. The decreasing of moisture content was caused by the mycelia growth of A. oryzae S. during fermentation. Moreover, moistening brings a suitable aw for mold growth because of the easy penetration of mycelia into the substrates [9]. It was opposite to the results of Chutmanop et al. [5] that the moisture content of fermented wheat and rice bran increased during fermentation. It might be due to the different substrates. Soybean particle is bigger than rice and wheat bran. It supports air circulation and affect oxygen and heat transfer in the substrate.
3.2. Effect of different combinations of substrates on enzymes production
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].
3.3. Effect of different combinations of substrates on reducing sugar content
The profile of reducing sugar production was similar to the amylase activity. The reducing sugar content increased during the cultivation period. The highest reducing sugar content was 341.01 mM/g dry weight presented in 40% soybean sample (Fig. 1f). The increasing of reducing sugar content is caused by the enzymatic hydrolysis of starch in the substrates during fermentation [9].
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