# Trypsin Inhibitor from Soybean Whey Wastewater: Isolation, Purification and Stability
## 3 Results and Discussion
### Stability Analysis of STI
#### 3.4.3 The Effect of Pepsin on the STI Activity  
The gastric environment substantially affects the digestion and absorption of food or oral drugs. After reaching the intestine, STI must maintain sufficient inhibitory activity to realize its functional weight control, anti-cancer, and anti-inflammatory effect. Because recent research has suggested that not all inhibitors are resistant to digestion in mammals, the digestibility of any given inhibitor must be assessed accurately. To investigate the hydrolytic inactivation of STI in a simulated gastric environment, experiments were set up to examine the effect of pepsin treatment on inhibitory STI viability [15].  
\begin{table}
\begin{tabular}{c c c c c} \hline
**pH** & **\(\alpha\)-Helix** & **\(\beta\)-Sheet** & **\(\beta\)-Turn** & **Random Coil** \\ \hline
3.0 & 0.00 \(\pm\) 0.00 & 40.17 \(\pm\) 0.95 a & 15.03 \(\pm\) 0.31 ab & 44.67 \(\pm\) 0.84 b \\
4.0 & 0.00 \(\pm\) 0.00 & 40.13 \(\pm\) 1.67 a & 15.03 \(\pm\) 0.42 ab & 44.83 \(\pm\) 2.07 ab \\
5.0 & 0.00 \(\pm\) 0.00 & 39.57 \(\pm\) 0.94 a & 14.60 \(\pm\) 0.29 b & 45.83 \(\pm\) 1.91 ab \\
6.0 & 0.00 \(\pm\) 0.00 & 39.67 \(\pm\) 0.46 a & 14.93 \(\pm\) 0.17 ab & 45.40 \(\pm\) 0.62 ab \\
7.0 & 0.00 \(\pm\) 0.00 & 39.76 \(\pm\) 0.59 a & 15.10 \(\pm\) 0.22 ab & 45.10 \(\pm\) 0.85 ab \\
8.0 & 0.00 \(\pm\) 0.00 & 38.33 \(\pm\) 1.43 ab & 14.77 \(\pm\) 0.58 ab & 46.87 \(\pm\) 2.04 ab \\
9.0 & 0.00 \(\pm\) 0.00 & 38.60 \(\pm\) 1.53 ab & 15.00 \(\pm\) 0.14 ab & 46.40 \(\pm\) 1.67 ab \\
10.0 & 0.00 \(\pm\) 0.00 & 37.23 \(\pm\) 0.21 b & 14.90 \(\pm\) 0.36 ab & 47.90 \(\pm\) 0.45 a \\
11.0 & 0.00 \(\pm\) 0.00 & 36.90 \(\pm\) 1.19 b & 15.50 \(\pm\) 0.49 ab & 48.60 \(\pm\) 1.43 a \\
12.0 & 0.00 \(\pm\) 0.00 & 35.50 \(\pm\) 0.92 b & 15.60 \(\pm\) 0.64 a & 48.90 \(\pm\) 1.56 a \\ \hline \end{tabular} Results are presented as mean values \(\pm\) standard deviation (\(n\) = 3). Different letters on the same column indicate significant differences.  
\end{table}
Table 2: Secondary structures of STI.  
Figure 7: The effect of pH on the activity of STI **(a)**. The circular dichroism spectra of STI **(b)**. The data of residual trypsin activity in each pH are not significantly different. Residual trypsin inhibitory activity is expressed as the percentage of trypsin inhibitory activity after the treatment accounting for trypsin inhibitory activity of the untreated STI (pH 6.7).  
As shown in Figure 8a, the STI and KTI activity retention gradually decreased with extended popsin treatment time. After 120 min, the STI and BBI inhibition activity retention rates were 44.75% and 33.13%, respectively. The BBI activity retention was 84.93%, which was higher than STI and KTI (\(p<0.05\)) (Figure 8b). In the presence of inactivated popsin and the absence of popsin, the extension of time did not affect the inhibitory trypsin activity. The results indicated that popsin significantly affected STI and KTI, but not BBI, which could be because popsin more likely hydrolyzed large KTI molecules rather than small BBI molecules. Therefore, the trypsin inhibitor displayed excellent resistance to popsin hydrolysis.