# A Method of Reducing Salt Content in Fermented Soy Sauce Improves Its Flavor and Quality
## 3 Results and Discussion  
### Determination of the Optimal Koji Collection Time and the Types and Ratios of Raw Materials  
The growth of _A. oryzae_ 3.042 during the preparation of koji largely determines the quality of the fermented soy sauce [26]. Here, the activities of protease, glucoamylase, and cellulase in the culture at 36 h, 40 h, and 44 h were determined to find out the optimal koji collecting time and the appropriate type and proportion of the raw materials. During the initial stage of soy sauce fermentation, _A. oryzae_ secretes a significant number of enzymes, resulting in the highest utilization rate of the raw materials. Protease [27] is the most crucial enzyme system secreted in koji, as it fully hydrolyzes protein substances into nutrients. These nutrients contribute to the rich flavor and high quality of a soy sauce. Figure 1a-c show the results of different protease activities. During the period of 36-44 h, the protease activity of the koji increased gradually. The activities of the neutral protease, alkaline protease, and acid protease of the koji reached their peaks at 44 h, at which time the overall protease activity in group 5 was the highest, with neural, alkaline, and acid protease activities of 1422.53 U/g, 1122.53 U/g, and 500.61 U/g, respectively. The alkaline and neutral proteases were the most active in the initial system due to the generally high pH at the beginning of the fermentation. As the fermentation progressed, the pH of the system gradually decreased, leading to an increase in acid protease activity [21]. The raw soybean meal material was defatted of extruded soybeans, because after extrusion treatment, it is distributed in flakes, without the three-dimensional particles of soybeans [28]. Moderate deformation can increase the contact point between the raw material and protease to improve efficiency. And the moderate amount of the raw fried wheat material hydrolyzed by amylase produces glucose, which can provide energy for _A. oryzae_, so the ratio of the raw material in group 5 had a higher protease activity. In the fermentation process of soy sauce, glucoamylase is crucial to the hydrolysis of the raw materials, which directly affects the utilization rate of the raw materials and the quality of soy sauce. Glucoamylase hydrolyzes starch from the nonreducing, nonterminal a-1.4 glucosidic bond to produce glucose and also slowly hydrolyzes the a-1.6 glucosidic bond to convert it into glucose. Glucoamylase hydrolyzes starch raw materials into monosaccharides such as glucose, which provide carbon sources for microorganisms during soy sauce fermentation and participate in the Maillard reaction, thus affecting the flavor and taste of soy sauce [29]. As shown in Figure 1d, the glucoamylase activity of koij generally showed an upward trend during 36-44 h of koij culture. When cultured for 44 h, the glucoamylase activities of all groups reached the maximum. Moreover, the glucoamylase activity of group 5 was 2461.55 U/g, which is higher than that of the other experimental groups. Therefore, it was confirmed that the time of the koji collection was the 44 th hour. Cellulose is a compound enzyme that catalyzes the hydrolysis of the \(\beta\)-1,4 glucosidic bond within cellulose to cellobiose and glucose and improves the quality of fermented soy  
Figure 1: Enzyme activities of koji: (**a**) neutral protease; (**b**) alkaline protease; (**c**) acid protease; (**d**) glucoamylase; (**e**) cellulase. (Numbers 1–6 indicate different proportions of the raw materials: no. 1—soybean:fried wheat = 5:5; no. 2—soybean:fried wheat = 6:4; no. 3—soybean:fried wheat = 7:3; no. 4—soybean meal:fried wheat = 5:5; no. 5—soybean meal:fried wheat = 6:4; and no. 6—soybean meal:fried wheat = 7:3). sauce by accumulating these substances. However, if _A. oryzae_ overproduces cellulose, it can lead to excessive hydrolysis of cellobiose and glucose.