## 2 Results and Discussion
### In Silico Simulated Gastrointestinal (GI) Digestion of Bioactive Peptides
In addition, there is a possibility of identifying novel peptides. Therefore, toxicity evaluation has been suggested as a mandatory approach prior to further development of identified motifs [33]. The software ToxinPred was used for predicting the toxicity of peptides as well as their physicochemical properties including hydrophobicity, hydrophilicity, charge, isoelectric point and molecular weight of the released dipeptides. The toxicity of peptides was investigated based on hybrid model of dipeptide composition and motif scanning [37]. According to the model developed using the machine-learning techniques support vector machine (SVM) based on amino acid compositional analysis, residue preferences combined with quantitative matrix-based and motif-based predictions, ToxinPred creates the platform to assess realistic toxicity of peptides [38]. The developed method reveled that valine (V), threonine (T), arginine (R), glutamine (E), methionine (M), leucine (L), lysine (K), isoleucine (I), phenylalanine (F) and alanine (A) were abundant in non-toxic peptides, whereas cysteine (C), histidine (H) and asparagine (N) were predominant in toxic peptides [38]. Predictions obtained following toxicity analysis show that none of the dipeptides (PL, GF, GP, DF, PR) released from sea cucumber-derived peptide simulated digestion have any potential toxicity. Thus, these findings indicate that sea cucumber-derived peptides can be used as functional food ingredients or nutraceutical products. According to the predictions of ToxinPred, molecular weight of the peptides ranged from 172.20 to 280.29 Da. Out of the five identified ACE inhibitory potent dipeptides obtained from sea cucumber protein hydrolysates, three (PL, GP and AF) had an isoelectric point (pI) of 5.88 with 0 net charge (Table 9). The remaining peptides, DF and PR, had negative charge (\(-1\)) and positive charge (\(+1\)) with a pI of 3.88 and 10.11, respectively. Structural properties, including charge, peptide sequence and low molecular size have been identified as influential characteristics for the bioavailability of food-derived peptides [39]. It has been reported that short chain peptides produced from gelatin hydrolysates have the potential to enter blood stream by crossing the intestinal barriers of humans and exert their bioactivity [40]. Generally, small peptide sequences are less prone to further degradation and possess high bioavailability [41]. In addition, most of the reported potent ACE inhibitory peptides are di- or tripeptides with the ability of binding to the buried active site of ACE [41]. Limited information is available in the literature that discusses correlation between the isoelectric point of peptides and their ACE inhibitory activity. Hydrophobicity values of identified bioactive dipeptides ranged from \(-0.05\) to \(0.43\), while hydrophilicity values were in the range of \(-0.9\) to \(0.25\). PL showed the highest hydrophobicity value (\(0.23\)) and lowest hydrophilicity value (\(-0.9\)). Therefore, the varying physicochemical parameters such as hydrophobicity and hydrophilicity indicate the potential of utilizing the predicted short-chain peptides derived from sea cucumber in a wide range of food systems, including water soluble, lipid soluble as well as in emulsions. In general, ACE inhibitory peptides are expected to exert their functionality in both hydrophilic and hydrophobic systems [42]. In Silico Analysis of Absorption, Distribution, Metabolism and Excretion (ADME) Profile of Bioactive Peptides Derived from Sea Cucumber Hydrolysates  
The trend of using virtual screening for identifying the potential bioactive compounds has become an asset in drug discovery process [43]. Drug development assessments generally include absorption, distribution, and excretion (ADME) parameters. The web-based tool SwissADME provides robust predictive models and primary information for individual parameters important for drug development process [44].