## 4 Discussion  
Proteins in seafoods and their by-products have a high structural diversity and represent a massive resource for the mining of bioactive peptides [1]. Bioactive peptides are released from the sequence of proteins by different hydrolysis methods, such as solvent extraction, microbial fermentation, enzymatic hydrolysis and chemical treatment [5]. Nevertheless, the proteolytic hydrolysis process is the popular method in health care food industries to avoid toxic chemicals or residual organic solvents in the final products. In addition, the specificity and parameters of the proteases significantly affect the MW and amino acid composition of bioactive peptides [47; 48; 49]. In some cases, specific enzyme combinations are used to enhance the DH of protein and prepare hydrolyses enriched with low MW APs [44; 48; 50]. The structural activity specialties can provide guides to speculate the activity of isolated APs. The amino acid composition and sequence, MW or molecular size, and spatial structures are deemed  
Figure 9: EC\({}_{50}\) values of P2, P4 and P6 on hydroxyl radical scavenging activity when treated at different temperatures (**A**) and pH values (**B**). The data are expressed as the mean \(\pm\) SD (\(n=3\)). \({}^{\mathrm{a-d}}\) Values with same letters of same sample indicate no significant difference (\(p>0.05\)). to have a vital role in the activity of APs. In general, APs with a low MW have more opportunities to interact with target molecules to terminate free radical reactions and prevent unsaturated lipid peroxidation [44]. The conclusion was confirmed by the literature that the antioxidant capacities of protein hydrolyses or APs were inversely related to the logarithm of their average MW [29; 35]. In addition, small APs have a high likelihood to exploit new drugs because they easily pass through the blood-brain barrier to cause effects in human bodies [32]. The six isolated APs (P1 to P6) are tripeptides to octapeptides with MWs varying from 375.39 to 861.95 Da (Table 1), which shows that they have a high chance of interacting with ROS to prevent lipid peroxidation. Previous literature indicated that the kinds of amino acids in the APs were regarded as key factors for their activity [5]. The hydrophobic groups of hydrophobic amino acid residues such as Pro, Met, Ala, Leu, and Ile, can highly react with hydrophobic polyunsaturated fatty acids (PUFAs) to inhibit lipid peroxidation in lipid-rich foods [6; 8]. [PERSON] et al. confirmed that the inhibiting ability of Pro-Met-Arg-Gly-Gly-Gly-Gly-Tyr-His-Tyr (PMRGGGGYHY) on free-radical chain reactions was attribute to Met residue because it could serve as a reactive site for formatting a sulfoxide structure to scavenge oxidants [51]. The pyrrolidine ring of the Pro residue can increase the antioxidant ability of Pro-His-His (PHH), Leu-Asp-Glu-Pro-Asp-Pro-Leu (LDEPDPL), Gly-Pro-Gly-Gly-Phe-Ile (GPGGFI), and Phe-Ile-Gly-Pro (FIGP), and to scavenge ROS due to its low ionization potential [52; 53]. [PERSON] et al. [8] and [PERSON] et al. [44] reported that the Ala residues contribute to the ability of Gly-Ile-Glu-Trp-Ala (GIEWA) and Ala-Lys-Gly (AKG) on radical-scavenging and lipid peroxidation inhibition. Moreover, the aromatic groups of Trp, Phe, and Tyr residues can provide protons to stabilize electron-deficient radicals [54; 55]. [PERSON] et al. reported that the sequence of APs with Trp residues (Trp-Asn-Glu-His and His-Glu-Trp) and Tyr residues (Arg-Tyr, Lys-Tyr, Tyr-Asp, Tyr-Tyr, Arg-Tyr-Asn, Tyr-Asp-Tyr, Tyr-Glu-Gly, and Tyr-Glu-Asn) showed high antioxidative ability [54]. The Phe residue in KHNRGDEF can serve as a radical scavenger through providing protons to quench unpaired radicals [55]. In consequence, hydrophobic/aromatic amino acid residues in WMFDW (Trp, Met, and Phe), WMGPY (Trp, Met, and Pro), and EMGPA (Met, Pro and Ala) are vital factors for their activity. The carboxyl and amino groups in polar amino acid residues are important for the hydroxyl radical scavenging and metal ion chelating capacities of APs [8; 30].