In order to identify the most influencing parameters considered in the ongoing work, an analysis of variance was performed on the results. ANOVA was used to evaluate the validity and adequacy of the model effects of the operating parameters and their interactions. The effects of three factors on response were found by RSM using three-dimensional plots and contour plots.
The effects of variables on the F-value could be observed from the response surface plots. As illustrated in
Figure 7, F-value as the response variable investigated the impact of changing two out of three factors on it. All the response surfaces exhibited concave shapes with both center and edges falling within the studied domain. This behavior indicates that there is an optimum response for the F-value.
Figure 7 indicates that the impact of reaction temperature and enzyme ratio on the F-value is greater than that of residence time, emphasizing the significant influence of reaction temperature and enzyme ratio on F-value.
Thus, when the residence time was between 22 min and 34 min and the enzyme ratio was between 0.5–0.9, the F-value was significantly increased (
Figure 7b). At the reaction temperature of 38–50 °C, α-amylase did not reach the optimal reaction temperature, and the residence time was short, so the decomposition effect of amylase on starch particles was unsatisfactory [
22]. At temperatures exceeding 50 °C during mashing, β-glucanase activity was quickly diminished, while the solubilization of β-glucans from intact cell walls persisted [
23,
24,
25]. This could be explained by the presence of carboxypeptidase and other highly thermoresistant enzymes in barley malt [
26]. As the thermal insulation temperature in this study was within the effective range, the effects of such enzymes were not considered. There was a two-step degradation of β-glucan, first by a lichenase to β-glucooligosaccharides and finally by a β-glucosidase to fermentable glucose units [
27]. Because of the short residence time, β-glucan may only have been decomposed into β-oligosaccharides, but it had no significant effect on the turbidity of the clarified solution. The decomposition of β-glucan in malt was limited, and the remaining β-glucan will cause turbidity in less solution, which can also be compared and predicted from the side of different filtration performances between malts. Apart from beta glucanase, Arabian xylanase was also present. This enzyme hydrolyzes arabinoxylan and reduces the viscosity of wort [
28]. The reduction in viscosity had a certain effect on the fermented liquid filtration but did not affect the turbidity of the clarifying liquid. The activity of the neutral protease peaked at 30–55 °C within 10–30 min of reaction time [
29]. This experiment achieved the optimal activity temperature and reaction time for neutral protease, resulting in its highest level of activity. Therefore, the efficiency of proteolytic hydrolysis was higher than that of amylase and glucanase. In case the proteins are not decomposed, they can give rise to turbid particles resulting in turbid wort [
30]. Thus, the filtration efficiency of malt can be evaluated and predicted based on this observation. The optimal proportion of the enzymes with the highest F-value was between 0.5 and 0.9, which shows the proportion of β-glucanase was gradually increased and the proportion of amylase and protease decreased. However, since the temperature and time of protease and glucanase action were within the optimal range, it was speculated that because of the differences among different malts, only limited starch, β-glucan, and protein could be hydrolyzed, while the remaining substances would cause turbidity, so as to quickly detect and predict the filtration performance of malt.
Figure 8a shows the effects of reaction temperature, residence time, and enzyme ratio on the differentiation between samples. Enzyme ratio and reaction temperature had significant influence on the degree of differentiation. When the reaction temperature was 38–50 °C and the enzyme ratio was 0.5–1.3, the differentiation was greater than 130 °C. For greater differentiation, an enzyme ratio of 0.5 and an insulation temperature of 38–47 °C are recommended. In addition, as shown in the
Figure 7, although the differentiation between samples was large within the experimental range, higher α-amylase ratio and reaction temperature would result in sufficient enzymatic hydrolysis reaction, and fewer colloidal particles would enter the wort, which will reduce the differentiation between samples. As shown in
Figure 8b, c, holding time and holding temperature affect sample differentiation in a small range. If the holding time is 27–38 min and reaction time is 38–47 °C, then the differentiation will be greater than 130 °C. On the other hand, when the residence time is 22–27 min, then temperature change will have a very minute effect on the degree of differentiation. Therefore, only when the residence time is kept above 27 min can a higher degree of differentiation be obtained. According to these results, both high enzyme ratio and low residence time lead to decreased differentiation between samples. The reasons for the decrease in differentiation could be the uneven content of various substances between the given samples or the effects of holding time and holding temperature on enzyme activity that cause excessive decomposition, and the uniformity of turbidity between samples.