2.2. Experimental Design

In order to obtain the best conditions for enzyme production (amylase and protease) from koji-making, a sequence of factorial designs was used. Using the STATGRAPHIC Centurion software (version 16.1) to optimize koji-making parameters by the RSM with the CCD 2² + star.

The first experiment was conducted with two factors, including molds addition (X₁) (0.02 ÷ 0.04%) and koji-making time (X₂) (24 ÷ 36 hours). The numbers of treatments were 13, including 5 replications of the central points. Each factor was surveyed with five levels, coded from −α to +α (with α = ± 1.4142). The level of encrypted variables and experimental layout is shown in Table 1.

In the second experiment, RSM and CCD were also used to investigate the effects of two factors, including incubation temperature (X₃) (27 ÷ 33°C) and pH of koji medium (X₄) (5.5 ÷ 6.5). Each factor was coded from −α to +α (with α = ± 1.4142). The design was 13 treatments, including 5 replications of the central points. The level of encrypted variables is shown in Table 2.

Table 1: Variable coding and survey levels of mold addition and koji-making time. [Click here to view]

Table 2: Variable coding and survey levels of incubation temperature and pH of koji. [Click here to view]

2.3. Analysis Methods

2.3.1. Determination of α-amylase activity

The α-amylase activity (U/g dry matter) was measured by an amylase unit expressed by the amount of enzymes that were capable of catalyzing the hydrolysis of 1 g of starch for 1 hour at 30°C and pH = 4.7 ÷ 4.8 [11]. The optical density of the solution was measured on a colorimeter with a liquid layer length of 1 cm and wavelength λ = 676 nm. The optical density difference between the control solution and the test solution is proportional to the amount of starch that has been exposed to α-amylase.

2.3.2. Determination of protease activity

This method was based on casein hydrolysis by enzymes in a research preparation solution [12]. Amino acids formed in hydrolysis reaction were measured by the Folin reagent. The unit of protease activity was the amount of an enzyme that converts an amount of sodium caseinate to a nonprecipitated form by tricloroacetic acid equivalent to 1 μmol of tyrosine at 30°C for 1 minute.

2.3.3. Determination of moisture

The moisture content was measured according to the standard method, as described by [13]. The sample was dried at 105°C to constant weight. The moisture W (%) was calculated using Equation (1):

$\mathrm{W}\mathrm{(\%)}=\frac{({\mathrm{m}}_1-{\mathrm{m}}_2)}{{\mathrm{m}}_1}\times 100(1)$

where m₁ is the initial mass of sample (g) and m₂ is the final mass of sample (g).

2.4. Data Analysis Methods

Data were collected and processed by STAGRAPHICS Centurion 16.1 software for the analysis of variance and least significant difference test to conclude the difference between the averages of experiments at 5% confidence (p = 0.05) and Microsoft Excel software for calculating and graphing.

The appropriateness of the predicted model was assessed through the correlation coefficient R². The equation optimizes the response surface of general form experiments according to Equation (2):

$\mathit{Y}={\mathrm{b}}_{\mathrm{o}}+{\sum }_{\mathrm{i}=1}^{\mathrm{k}}{\mathrm{b}}_{\mathrm{i}}{\mathrm{x}}_{\mathrm{i}}+{\sum }_{\mathrm{i}=1}^{\mathrm{k}}{\sum }_{\mathrm{j}=1}^{\mathrm{k}}{\mathrm{b}}_{\mathrm{ij}}{\mathrm{X}}_{\mathrm{i}}{\mathrm{X}}_{\mathrm{j}}(2)$

where Y is object function, β_o is constant, β_i is the linear coefficient, β_ii is the square coefficient, β_ij is the interaction coefficient, and X_i and X_j are survey variables.

3.1. The Effects of Mold Addition and Koji-Making Time on Amylase and Protease Activities

The effects of molds and koji-making time on the presence of mycelium are shown in Figures 1 and 2. The results showed that the mycelium of 0.016% and 0.02% of A. oryzae developed less than mold with 0.03 ÷ 0.044% after 30 hours of incubation (Fig. 1).

Moreover, when the time of mold culture was during the period of less than 24 hours, there was no presence of mycelium. At 24 hours of koji-making, the mycelium began to grow scattered. The white mycelium covered a fairly thick layer on the medium at 30 and 36 hours of incubation. At 38.5 hours of the process, mycelium gradually turned to a yellow areca flower and appeared to have spores (Fig. 2).

The cycle of A. oryzae growth on bran could be divided into periods: the period of growth and germination of the sporangium (10 ÷ 11 hours of the first), the period of the rapid development of the fibrous system (4 ÷ 18 hours), and the strong enzyme generation period (lasting 10 ÷ 20 hours) [14]. The growth of molds also showed a contrasting relationship with the moisture of the medium [15]. Aspergillus oryzae used water on the surface of the medium to grow and create mycelium, forming spores [16]. The result in Figure 3 showed that the moisture content of the medium decreased with the increasing incubation time. In the early stage of the incubation process (less than 24 hours), the molds had not adapted to the medium, so the growth rate was quite slow and the moisture content of koji was more than 70%. In the period of 24–36 hours, the moisture of koji was 61.85% ÷ 70.39%. This result was also consistent with many research studies [17,18] where suitable moisture and time for A. oryzae to grow and produce enzymes were about 60 ÷ 68% and 30 ÷ 42 hours.

Figure 1: Effect of mold addition on the presence of mycelium (a) initially, (b) at 0.016%, (c) at 0.02%, (d) at 0.03%, (e) at 0.04%, and (f) at 0.044%. [Click here to view]

Figure 2: Koji-making time in the presence of mycelium (a) initially, (b) at 21.5 hours, (c) at 24 hours, (d) at 30 hours, (e) at 36 hours, and (f) at 38.5 hours. [Click here to view]

In addition, the ability to produce more or less enzymes of molds means the development of mycelium [17]. The response surface models showing the effect of the mold addition and koji-making time on the activities of amylase and protease were built up (Fig. 4). The results showed that, as the addition of molds and incubation time increased, the activities of enzyme production increased to an optimal value and then decreased gradually. The higher the molds addition does not mean the higher the enzyme activities at the end of the koji process [19]. Additionally, mycelium formation and enzyme activities were closely related. Amylase and protease activities increased strongly when the medium began to form mycelium (from 24 to 30 hours) and decreased when spores appeared. This result is similarly demonstrated in the study of [20]. This was due to either the asexual cycle or the spore formation of A. oryzae that was involved in the production of secondary metabolites, such as enzymes or organic acids. Spore formation is undesirable because of reducing enzyme activities. The process of maximizing enzyme production usually ends when molds begin to form spores [17]. In addition, the lack-of-fit values of the models that were 0.06 and 0.43 (>0.05), respectively, showed the model’s compatibility with the experiment. The optimal amylase activity was 61.77 U/g dry matter when 0.03% molds and 30.75 hours of incubation time were applied. Protease activity was optimal at 12.37 U/g dry matter when the percentage of molds was 0.03 and it was fermented for 30.41 hours.

Figure 3: Changes in the moisture content of koji according to incubation time. [Click here to view]

Figure 4: The response surface and contour plots showing the effect of mold addition and koji-making time on (a) Amylase activity and (b) Protease activity. [Click here to view]

Regression equations showing the relationship of the molds addition and koji-making time to amylase and protease activities were described in Table 3. All equations met the conditions with a high coefficient of determination (R² > 0.84). The compatibility between the predictive and experimental data of amylase and protease activities was shown in Figure 5 and there was a high correlation between predictive and experimental data (R² > 0.84).

The results of simultaneous optimization of multiple response surfaces showed optimal amylase and protease activities when 0.03% of mold and 30 hours for incubation time were applied (Fig. 6). Amylase and protease activities were 61.62 and 12.52 U/g dry matter, respectively.

Thus, the parameters selected for the next studies were the addition of molds at the rate of 0.03% and the time of koji making was 30 hours.

3.2. The Effects of Temperature and pH of Koji-Making on Enzyme Activities

The response surface models showing the correlation between the incubation temperature and pH of the medium to amylase and protease activities were built up (Fig. 7). The temperature and pH of koji-making affected the quadratic on enzyme activities. When the pH of the medium increased from 5.5 to 6.0, the amylase and protease activities increased; however, the pH continued to increase to 6.5 and the enzyme activities decreased. Likewise, amylase and protease activities increased with increasing temperature (up to 30°C) and a further increasing in the koji-making temperature decreased. The results showed that amylase production with optimal activity was 61.43 U/g dry matter when it was fermented at 30.14°C and pH 6.02. Meanwhile, the optimal protease activity was 12.37 U/g dry matter when the temperature and pH of koji making were 27.76°C and 5.89°C, respectively.

Table 3: Predictive regression equations of the relationship between the molds and koji-making time. [Click here to view]

Figure 5: Correlation between experimental and theoretical data (from Y1 and Y2) for (a) Amylase activity and (b) Protease activity. [Click here to view]

Figure 6: Overplay plots of enzyme activities according to molds addition and koji-making time. [Click here to view]

The temperature of koji-making is a strong influence on the reaction rate. The speed of reaction will increase rapidly under the effect of temperature. If the optimal threshold is exceeded, it will inhibit the incubation [21]. Temperature not only affects the growth of microorganisms but also affects their biological activity. The effects of incubation temperature on mold growth and enzyme activities need to be studied to understand the role of temperature control in koji-making. In addition, the initial pH of the koji medium is one of the factors that have an important influence on the ability of enzyme biosynthesis. The initial pH of the medium affects the metabolism of microorganisms, leading to a change in the absorption of food. The influence level of pH also depends on the substrate, buffer properties, temperature, and so forth [22].

Figure 7: The response surface and contour plots showing the effect of temperature and pH of koji on (a) Amylase activity and (b) Protease activity. [Click here to view]

Table 4: Predictive regression equations of the relationship between temperature and pH on enzyme activities. [Click here to view]

Figure 8: Correlation between experimental and theoretical data (from Y3 and Y4) for (a) amylase activity and (b) protease activity. [Click here to view]

Figure 9: Overplay plots of enzyme activities according to temperature and pH of koji. [Click here to view]

Table 5: Results of the experiment and optimization model. [Click here to view]

In addition, the lack-of-fit values were not statistically significant (p > 0.05) and correlation coefficients of regression equations showing the relationship of temperature and pH to amylase (Y₃) and protease (Y₄) were high (R² > 0.85) (Table 4), so it confirmed the more significance and reliability of the regression models established. The compatibility between the predictive and experimental data of amylase and protease activities was determined with the high correlation coefficient (R² > 0.85) (Fig. 8). Furthermore, the results of optimization of multiple response surfaces showed amylase and protease activities reached the highest values at 29.94°C and pH 5.97 (Fig. 9). The experimental results of incubating at 30°C and pH 6.0 were equivalent to the results predicted from the model (Table 5). The previous studies had shown that incubating at 30°C and pH 6.0 was the optimal temperature and pH for A. oryzae to grow and produce enzymes with high activities on solid-state fermentation (SSF) [23–26].