## 3 Results
### Optimization of the Fermentation Medium
**} & \multicolumn{10}{c}{**Variables**} & \multicolumn{1}{c}{**NM Potency**} \\ \cline{2-13}  & \(X_{1}\) & \(D_{1}\) & \(X_{2}\) & \(D_{2}\) & \(X_{3}\) & \(D_{3}\) & \(X_{4}\) & \(X_{5}\) & \(X_{6}\) & \(X_{7}\) & \(X_{8}\) & \(X_{9}\) & \(X_{10}\) & \(D_{4}\) & **(U/mL)** \\ \hline
1 & + & \(-\) & + & \(-\) & + & + & + & \(+\) & \(-\) & \(-\) & + & + & \(-\) & + \\
2 & + & + & \(-\) & + & + & \(-\) & \(-\) & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + \\
3 & + & \(-\) & + & + & \(-\) & \(-\) & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + & \(7594\) & \(100\) \\
4 & + & \(-\) & + & + & + & + & \(-\) & \(-\) & + & + & \(-\) & + & \(+\) & \(-\) & \(7280\) & \(87\) \\
5 & \(-\) & \(-\) & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + & + & \(+\) & \(+\) & \(-\) & \(-\) & \(7782\) & \(111\) \\
6 & + & + & + & + & \(-\) & \(-\) & + & + & \(-\) & + & + & \(-\) & \(-\) & \(-\) & \(6652\) & \(\pm\) 80 \\
7 & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + & + & + & \(+\) & \(-\) & \(-\) & + & \(+\) & \(8849\) & \(\pm\) 115 \\
8 & + & + & \(-\) & \(-\) & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + & \(+\) & \(+\) & \(+\) & \(8033\) & \(\pm\) 72 \\
9 & \(-\) & + & + & \(-\) & \(-\) & \(-\) & \(-\) & + & \(-\) & + & \(-\) & + & \(+\) & \(+\) & \(6088\) & \(\pm\) 60 \\
10 & \(-\) & + & + & + & + & \(-\) & \(-\) & + & + & \(-\) & + & \(+\) & \(+\) & \(-\) & \(-\) & \(7656\) & \(\pm\) 97 \\
11 & \(-\) & + & + & \(-\) & + & + & + & \(-\) & \(-\) & \(-\) & + & \(-\) & \(+\) & \(-\) & \(-\) & \(9602\) & \(\pm\) 108 \\
12 & \(-\) & + & + & \(-\) & + & + & + & + & \(-\) & \(-\) & + & \(+\) & \(-\) & \(+\) & \(-\) & \(6903\) & \(\pm\) 76 \\
13 & + & + & + & + & \(-\) & \(-\) & + & + & \(-\) & + & \(+\) & \(-\) & \(-\) & \(-\) & \Furthermore, a second-order polynomial equation, which reflected the impact of all terms (linear, quadratic, and interactive) on the response appropriately, was used to predict the NM potency of _Sf_6-2 after fermentation:  
\[ \begin{split} Y=10,608.00+934.13\times X_{1}+988.50\times X_{2}-194.63\times X_{3}+404.75\times X_{1}\times X_{2}-31.00\times X_{1}\times X_{3}+762.75 \times\\ X_{2}\times X_{3}-1295.00\times{X_{1}}^{2}\ -\ 2431.75\times{X_{2}}^{2}-1186.50 \times{X_{3}}^{2},\end{split}  \tag{13}\]  
The optimal concentration of each factor (soluble starch, peptone, and (NH4)2 SO4) was generated using this second-order polynomial equation; concentrations were 73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively, and the corresponding NM potency of _Sf_6-2 was 10,910 U/mL. Finally, S/6-2 was fermented under these optimal conditions to verify model validity. This generated an NM potency (practical response) of 10,849 \(\pm\) 141 U/mL, which was approximately 99% of the predicted value and indicated perfect agreement with the model. After this optimization, \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Trial No.** & \(X_{1}\) **(Soluble Starch)** & \(X_{2}\) **(Peptone)** & \(X_{3}\) **((NH4)2 SO4)** & **NM Potency** \\
1 & \(-1\) & \(-1\) & 0 & 5169 \(\pm\) 62 \\
2 & 1 & \(1\) & 0 & 9403 \(\pm\) 132 \\
3 & 0 & \(-1\) & \(-1\) & 6725 \(\pm\) 89 \\
4 & \(-1\) & 1 & 0 & 7286 \(\pm\) 87 \\
5 & 0 & 0 & 0 & 10,611 \(\pm\) 153 \\
6 & 0 & 1 & \(-1\) & 6227 \(\pm\) 75 \\
7 & 0 & 0 & 0 & 10,608 \(\pm\) 138 \\
8 & \(-1\) & 0 & 1 & 6040 \(\pm\) 54 \\
9 & 0 & 1 & 1 & 8780 \(\pm\) 86 \\
10 & 1 & \(-1\) & 0 & 5667 \(\pm\) 72 \\
11 & \(-1\) & 0 & \(-1\) & 7784 \(\pm\) 87 \\
12 & 0 & 0 & 0 & 10,602 \(\pm\) 116 \\
13 & 1 & 0 & 1 & 8407 \(\pm\) 75 \\
14 & 0 & \(-1\) & 1 & 6227 \(\pm\) 50 \\
15 & 1 & 0 & \(-1\) & 10,275 \(\pm\) 134 \\
16 & 0 & 0 & 0 & 10,611 \(\pm\) 118 \\
17 & 0 & 0 & 0 & 10,608 \(\pm\) 105 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Response surface method (RSM) arrangements and results.