## 3 Results
### Optimization of the Fermentation Medium  
As the growth properties of mutant strains may have changed, and the original fermentation medium was no longer suitable for target product synthesis, it therefore required optimization. We used PB designs (Table 3) and regression analysis (Table 4) to screen for the three key components vital for NM potency in _Sf_6-2 from the original fermentation medium. These were soluble starch, peptone, and (NH4)2 SO4 (\(p\)-value \(<\) 0.05). Response coefficients also indicated that these variables had huge effects on NM potency (Table 4) [43]. These factors were then optimized by Box-Behnken design (Tables 5 and 6) and ANOVA analysis (Table 7). Model precision and reliability were both demonstrated by the correlation coefficient, \(R^{2}\) = 0.9026, and the coefficient of variation = 11.57%. The adjusted \(R^{2}\) = 0.78 was also close to the actual \(R^{2}\), indicating predictive model responses were adequate. In addition, the _Prob_ \(>\)\(F\) (\(p\)-value) of the model was calculated as 0.0082 \(<\) 0.01, meaning the regression model was extremely significant. Next, the interaction of these factors was assessed. According to regression analysis of the experimental design, the interaction terms (\(X_{1\times 2}\), \(X_{1\times 3}\), and \(X_{2\times 3}\)) were not significant (\(p\)-value \(>\) 0.05), while the linear terms (\(X_{1}\) and \(X_{2}\)) and second order terms (\(X_{1}\)\({}^{2}\), \(X_{2}\)\({}^{2}\), and \(X_{3}\)\({}^{2}\)) were significant, especially for the \(X_{2}\)\({}^{2}\) s order term (\(p\)-value \(<\) 0.01). On the other hand, a three-dimensional view (Figure 6) displayed clearly in a contour plot of the response surface and showed that along with the increasing soluble starch, peptone, and (NH4)2 SO4, the NM potency was improved and reached to maximum at the optimal condition of the three factors. However, NM potency decreased sharply with increases in the three components in the fermentation medium. Figure 5: Iterative ARTP mutagenesis and investigations of genetic stability. (**A**) A _S. fradiue_ lethality plot by ARTP mutagenesis; (**B**) The distribution of mutant strains with different NM potencies in each ARTP round; (**C**) The cumulative effects of iterative ARTP mutagenesis on NM potency in mutant strains; (**D**) Genetic stability of the high-producing NM mutant strain, _Sf_6-2. WT: wild-type strain (_S. fradiue_ GC 6010); \(R_{P}\): positive mutation rate; \(R_{M}\): mutation rate; \(R_{P/M}\): \(R_{P}\) and \(R_{M}\). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Variables**} & \multicolumn{2}{c}{**Terms**} & \multicolumn{2}{c}{**Values**} & \multicolumn{1}{c}{**Coefficient**} & \multicolumn{1}{c}{**\(t\)-Value**} & \multicolumn{1}{c}{**\(p\)-Value**} & \multicolumn{1}{c}{**Confidence Level (\%)**} \\ \cline{2-7}  & **Components (g/L)** & **Low (–)** & **High (+)** & **Coefficient** & \(t\)**-Value** & \(p\)**-Value** & **Confidence Level (\%)** \\ \hline \(X_{1}\) & NaCl & 3.5 & 5.5 & \(-\)38 & \(-\)0.13 & 0.902 & 9.8 \\ \(X_{2}\) & (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & 5.0 & 7.0 & 967 & 3.31 & 0.021 & 97.9 \\ \(X_{3}\) & **Peptone** & 8.0 & 10 & 1092 & 3.74 & 0.013 & 98.7 \\ \(X_{4}\) & **Peant meal** & 18 & 38 & 753 & 2.58 & 0.052 & 94.8 \\ \(X_{5}\) & Soluble starch & 60 & 80 & \(-\)992 & \(-\)3.40 & 0.019 & 98.1 \\ \(X_{6}\) & Soybean meal & 4.0 & 6.0 & 351 & 1.21 & 0.282 & 71.8 \\ \(X_{7}\) & Glucose & 10 & 30 & \(-\)615 & \(-\)2.11 & 0.089 & 91.1 \\ \(X_{8}\) & Soybean oil & 2.0 & 4.0 & \(-\)163 & \(-\)0.56 & 0.600 & 40.0 \\ \(X_{9}\) & Corn steep liquor & 1.5 & 3.5 & \(-\)188 & \(-\)0.65 & 0.547 & 45.3 \\ \(X_{10}\) & Yeast extract & 5.0 & 7.0 & \(-\)452 & \(-\)1.55 & 0.182 & 81.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Plackett–[PERSON] regression analysis. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Trial No.