## 3 Results
### Stability of ACEI Peptides against ACE  
Based on the interaction with ACE during the pre-incubation test, ACE inhibitory peptides can be organized into the following three categories: true inhibitors, real substrates, and pro-drugs [23; 37]. True inhibitors are not hydrolyzed and maintain their ACE inhibitory activity. Both real substrates and pro-drugs are hydrolyzed by the ACE, leading to the release of inactive or less active fragments for the former, and highly active fragments for the latter. Following 3 h of incubation with ACE at 37 \({}^{\circ}\)C, the ACE inhibitory activity of the following peptides WLQL, LPLF, VPGLAL, LVGLPL, and LPSW was compared with samples without pre-incubation. The inhibitory activity (%) for LPSW increased from 48.93% (without pre-incubation) to 68.19% (after pre-incubation with ACE), compared with the same concentration of 20 uM (Figure 10). Moreover, the IC\({}_{50}\) value of LPSW changed significantly following pre-incubation with ACE (without pre-incubation: 20.80 \(\pm\) 0.79 uM; after pre-incubation: 13.39 \(\pm\) 0.88 uM) (Table 1). Based on the LC-MS analysis, two small fragments, LP and SW, were recognized as the hydrolysis products of LPSW by ACE (Figure 2), which suggests that LPSW is a pro-drug. Similar to LPSW, both WLQL and VPGLAL were classified as pro-drugs of ACE. As expected, WLQL was cleavaged into small fragments, such as WL and QL (Figure 3), while VPGLAL was hydrolyzed into two peptides, AL and VPGL (Figure 10). In contrast, the ACE inhibition activity (%) of VPGLAL was notably enhanced from 51.03% to 65.27% after the pre-incubation test (at a concentration of 500 uM) (Figure 10). The tetrapeptide WLQL produced an IC\({}_{50}\) value that showed a downward trend from 16.87 \(\pm\) 0.54 uM without ACE pre-incubation to 8.5 \(\pm\) 0.86 uM after pre-incubation with ACE (Table 1). According to the results of the LC-MS spectrum and stability analysis via pre-incubation with ACE, it was confirmed that LPLF was a real substrate, while LVGLPL was a true inhibitor (Figures 10, 11 and 12). A similar situation occurred with the pro-drug-type LPLF, which was also affected by ACE, resulting in the release of small fragments of LP and \begin{table}
\begin{tabular}{c c c c c} \hline
**Peptide** & **Catalytic Parameter * & \multicolumn{3}{c}{**Concentration (\(\mu\)M)**} \\ \hline  & & 0 uM & 17 uM & 23 uM \\ LPSW & V\({}_{\text{max}}\) & 6.99 & 4.65 & 3.76 \\  & K\({}_{\text{m}}\) & 5.06 & 5.84 & 5.13 \\  & & 0 uM & 15 uM & 25 uM \\ WLQL & V\({}_{\text{max}}\) & 1.47 & 1.44 & 1.42 \\  & K\({}_{\text{m}}\) & 1.57 & 2.48 & 3.24 \\  & & 0 uM & 485 uM & 630 uM \\ VPGLAL & V\({}_{\text{max}}\) & 7.19 & 8.06 & 7.17 \\  & K\({}_{\text{m}}\) & 7.02 & 19.64 & 20.83 \\  & & 0 uM & 250 uM & 320 uM \\ LPLF & V\({}_{\text{max}}\) & 7.52 & 6.15 & 7.59 \\  & K\({}_{\text{m}}\) & 4.83 & 9.19 & 13.99 \\ \hline \end{tabular}  
* V\({}_{\text{max}}\) is the maximum reaction velocity; K\({}_{\text{m}}\) is the Michaelis constant.  
\end{table} 
Table 2: Kinetic constants of enzyme-catalyzed reactions at different peptide concentrations. LF (Figure A3). However, after pre-incubation, the new cleavage products of 300 \(\upmu\)M LPLF showed reduced inhibitory activity that fell from 58.23% (without pre-incubation) to 26.6% (Figure A2), which implies that LPLF is a real substrate. As observed in Figures A2 and A5, LVGLPL was not hydrolyzed by ACE and its ACEI activity was not significantly changed (from 25.27% to 31.30% at the level of 2000 \(\upmu\)M).