## 4 Discussion  
It is known that resistance to degradation of gastrointestinal enzymes and subsequent adsorption via intestinal cells are required for the orally digested peptides to exert bioactivity in vivo [21]. Hence, this study firstly employed in silico proteolysis of 16 proteins by gastrointestinal enzymes to obtain peptides, and then focused on oligopeptides with 2-5 amino acids only, which are considered to be trans-epitheially absorbable or transportable by specific transport systems, including the PepT1 or PepT2 transporter-mediated transport system for di- and tri-peptides [22] and the SOPT1/SOPT2 system for tetra- and penta-peptides [23]. Based on these facts, a number of oligopeptides (2 \(\leq\) peptide length \(\leq\) 5) from 16 shellfish proteins were investigated for their binding ability to angiotensin-converting enzyme. The present data demonstrated that mussel-derived NADH dehydrogenase could be a good source of potent binders to target protein ACE, with a frequency value (F = 0.0721) of high-affinity oligopeptides. Moreover, razor-clam-derived cytochrome b and ATP synthase were also good selections for strong binders, with F values of 0.0604 and 0.0603, respectively. Thus, it could be helpful for to supplement hypertensive patients with these proteins or protein extracts from razor claims or mussels. The amino acid composition of a peptide is important for its activity [24]. It was reported that N-terminal amino acids with a high frequency of occurrence were leucine (L), valine (V), isoleucine (I), alanine (A), glycine (G), tyrosine (Y), and phenylalanine (F). Among them, L has the highest frequency at 14.70%. The C-terminal amino acids with a higher frequency of occurrence were proline (P), tyrosine (Y), phenylalanine (F), isoleucine (I), and leucine (L). Among them, P has the highest frequency at 23.21% [25]. In this study, 18 top high-affinity oligopeptides were identified as binding to angiotensin-converting enzyme (Table 2). The proportion of these oligopeptides containing hydrophobic N- or C-terminals is 88.9%, suggesting the importance of hydrophobic amino acids. This is consistent with a previous report that hydrophobic amino acids have a good inhibitory activity [26] and are more common clinically than hydrophilic-based amino acids [27]. Moreover, among the identified oligopeptides, there are five abundant residues: hydrophobic valine (V, 22.2%) and isoleucine (I, 22.2%) and hydrophilic serine (S, 33.3%) in the N-terminal, and hydrophobic tryptophan (W, 33.3%) and phenylalanine (F, 38.9%) in the C-terminal. This is partly consistent with the above results and supports previous reports that valine (V) may contribute to the inhibitory activity of antihypertensive peptides [28] and that the presence of aliphatic or aromatic amino acids at the N- or C-terminus can enhance the inhibitory activity [29]. Angiotensin-converting enzyme is a major target for lowering blood pressure and its inhibition will leads to an overall antihypertensive effect. Many angiotensin converting enzyme inhibitory peptides have been identified from food sources, including various marine animals [30]. [PERSON] et al. [8] utilized several enzymes (pepsin, papain, alpha-chymotrypsin, neutrase, and alcalase) to hydrolyze Pacific cod skin and two ACE inhibitory peptides, GASSGMPG and LAVA, were obtained with IC50 values of 6.9 and 14.5 \(\upmu\)M, respectively. [PERSON] et al. [31] found that the peptide CNVPLSP isolated from seahorses (Hippocampus abdominalis) showed a potent ACE inhibitory effect with an IC50 value of 0.088 \(\pm\) 0.002 mg/mL. [PERSON] et al.