## 2 Results and Discussion
### In Silico Simulated Gastrointestinal (GI) Digestion of Bioactive Peptides
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Sample** & **Peptide** & **Results of Enzyme Action** & **Location of Released Peptides** & **Active Fragment Sequence** & **Bioactivity of Identified Peptide** \\ \hline Internal organs & GPPGPQWPLDF & GPPGPQW—PL—DF & (1–7), (8–9) (10–11) & PL & (8–9) (10–11) & ACE inhibitor No Antioxidant or ACE inhibitory activity \\  
Calculated quantitative parameters of proteolysis include theoretical degree of hydrolysis (DH\({}_{\text{t}}\)), the frequency of the release of fragments with a given activity by selected enzymes (A\({}_{\text{E}}\)), and the relative frequency of the release of fragments with a given activity by selected enzymes (W). The DH\({}_{\text{t}}\) values for peptides varied from 11.11 to 33.33%. Among them, GPGMMGP showed the highest efficiency (33.33%) on releasing the bioactive enzymes and GPPGPGNAF was the lowest (11.11%). Descriptors like A\({}_{\text{E}}\) and W directly analyzed the potential bioactivity of sea cucumber derived peptides. The higher A\({}_{\text{E}}\) values suggest the possibility of having higher number of peptides with specific activity. The highest A\({}_{\text{E}}\) value (0.25) was observed in APDMAFPR from flower and internal organs [32]. The other remaining peptide sequences, namely GPPGPQWPLDF and GPGMMGP, were common for all samples and their calculated A\({}_{\text{E}}\) values were 0.18 and 0.14, respectively. The highest relative frequency of the release of fragments with a given activity by selected enzymes (W) was 0.33 in APDMAFPR, followed by GPPGPQWPLDF (0.25). Similar W value (0.14) was observed for GPGMMGP and GPPGPGNAF. It was suggested that W values are considered complimentary to A\({}_{\text{E}}\) values [19]. In addition, W values can be correlated with catalytic specificities and the number of recognition sites in each enzyme [33]. Most of the bioactive fragments remaining after in silico simulated digestion of sea cucumber derived peptides possess either P (proline) or phenylalanine (F) in their dipeptide sequences. The resultant bioactive motifs contain PL (proline-leucine), DF (aspartic acid-phenylalanine), GP (glycine-proline), AF (alanine-phenylalanine) and PR (proline-arginine). Similar findings were reported by [PERSON] et al. [32] in their in silico study of identifying biopeptides from collagen derived from various sources including cow, pig, sheep, chicken, duck, horse, salmon, rainbow trout, goat, rabbit and turkey. Authors reported that all the identified motifs which exhibited ACE inhibitory activity contained proline, phenylalanine, glycine, leucine and arginine. As described earlier, previous studies on ACE inhibitory peptides also revealed that proline residues appear to provide resistance to digestive enzymes. The dipeptide sequences with a C-terminal proline and hydroxyproline were reported to be more bioavailable compared to other amino acid sequences [34]. Specifically, the presence of proline is well documented for its distinct ability for binding to ACE and interestingly, most commercially existing inhibitors bear the proline residue in their sequences [35]. Furthermore, some studies have identified that the presence of hydrophobic amino acids at the penultimate position of C-terminus as being a favorable feature for ACE inhibitory potency [36]. Thus, in silico prediction of generating potent ACE inhibitory peptides from sea cucumber samples may contribute to a better understanding of peptide efficiency before performing in vitro and in vivo experiments. Nevertheless, these findings can be used as a preliminary information on the stability of the sea cucumber derived peptides as well as to help determining the relationship between amino acid composition of peptides and their GI digestion. Prediction of Toxicity and Physicochemical Properties of Released Bioactive Peptide Fractions after In Silico Digestion  
In silico proteolysis does not guarantee that the resultant peptides with bioactivities are always safe for use in food and pharmaceutical products.