## 3 Results  
### Identification of Angiotensin-Converting Enzyme (ACE) Inhibitory Activity  
Five DPP-IV inhibitory peptides discovered in our previous study (LPSW, VPGLAL, WLQL, LPLF and LVGLPL) were utilized to identify their ACEI activities [18]. The synthesized peptides were evaluated for their ACE inhibitory activity using an in vitro ACEI assay and an RP-HPLC system. The IC\({}_{50}\) value of each peptide was determined using Graphpad Prism 5.0 (GraphPad Software, Inc.) by nonlinear regression of ACEI activities (%) at various concentrations, as indicated in Figure 11, and the results are shown in Table 1. Captopril (1.7 uM), used as the positive control in the in vitro assay, demonstrated an ACEI effect of 99.6% (the result is not shown here). Among these five peptides, WLQL exhibited the highest ACEI effect with the IC\({}_{50}\) value of 16.87 \(\pm\) 0.54 uM, as compared to the remaining peptides (Table 1). The tetrapeptide LPSW (IC\({}_{50}\) value of 20.80 \(\pm\) 0.79 uM) also displayed high ACEI activity. The other peptides, LPLF and VPGLAL, showed moderate IC\({}_{50}\) values of 300.08 \(\pm\) 17.30 uM and 573.00 \(\pm\) 54.10 uM, respectively, while the peptide LVGLPL showed the lowest ACEI effect with the highest IC\({}_{50}\) value of more than 2000 uM (Table 1). Moreover, their IC\({}_{50}\) values with the DPP-IV enzyme are also provided in Table 1 [18]. The mode of ACE inhibition was defined using the Lineweaver and Burk analysis. Peptide stability was examined during the pre-incubation assay with ACE for 3 h at 37 \({}^{\circ}\)C. After preincubation, the resulting solution was injected into LC-MS to monitor the remaining amount of peptide and the formation of hydrolysis products. The IC\({}_{50}\) value (with and without ACE preincubation) was determined using RP-HPLC. According to previous studies [4; 21; 22], the activity of ACEI peptides is commonly affected by the following three main interrelated factors: the presence of several specific amino acids, their hydrophobic or hydrophilic nature, and the length of the sequences. Specifically, the highly potent ACEI peptides have aromatic amino acids (tryptophan, tyrosine, and phenylalanine) or hydrophobic amino acids at their C-terminal, which play an important role in ACE inhibitory activity [20; 21]. Comparatively, these peptides seem to display promising inhibitory activities against ACE, especially WLQL and LPSW. In a similar manner to our material, eggs from chickens are the focal point in biological activity research due to their good nutritional value \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Peptide** & **Molecular Mass** & **Length** & **DP-IV IC\({}_{50}\) (uM)** & **ACE IC\({}_{50}\) (uM)** & **Mode of ACE** & **Stability of Peptide** & **ACEI IC\({}_{50}\) (uM) after** \\  & **(Da)** & **Length** & **(uM)** [18] & **ACE Inhibition** & **with ACE** & **with ACE** & **Pre-incubation with ACE** \\ \hline WLQL & 599 & 4 & \(432.5\pm 11.85\) & \(16.87\pm 0.54\) & Competitive & Hydrolyzed & \(8.5\pm 0.86\) \\ LPSW & 502 & 4 & \(269.7\pm 15.91\) & \(20.80\pm 0.79\) & Non-competitive & Hydrolyzed & \(13.39\pm 0.88\) \\ LPLF & 489 & 4 & \(463.6\pm 5.52\) & \(300.08\pm 17.30\) & Competitive & Hydrolyzed & nd \\ VPGLAL & 569 & 6 & \(289.2\pm 11.85\) & \(573.00\pm 54.10\) & Competitive & Hydrolyzed & nd \\ LVGLPL & 611 & 6 & \(>\)2000 & \(>\)2000 & nd & Not hydrolyzed & nd \\ \hline \hline \end{tabular}
\end{table} 
Table 1: DPP-IV and ACE inhibitory activity of five peptides derived from SSTY protein hydrolysate. and the fact that they are a popular and cheap product. [PERSON] et al. reported that nine ACEI peptides were identified in chicken egg white that contained two to five residues in their sequences, with IC\({}_{50}\) values ranging from 1.30 mM to 5.47 mM [10]. Moreover, with an excellent ACE inhibitory effect, the peptide LKYAT (IC\({}_{50}\) value of 0.09 \(\upmu\)M) identified from chicken egg white is considered as a promising ingredient for functional food [11].