## 2 Results and Discussion  
### Preparation of Protein Hydrolysate from Hairtail (T. japonicus) Muscle (HTP)  
.1.1 Effect of Different Proteases on Protein Hydrolysates from Hairtail (T. japonicus) Muscle (HTP)  
HO\(\bullet\) is a highly reactive radical and can destroy all types of macromolecules such as nucleic acids (mutations), carbohydrates, lipids (lipid peroxidation), proteins and amino acids [24]. DPPH is the traditional and perhaps the most popular standard of the position (g-marker) and intensity of electron paramagnetic resonance (EPR) signals [25]. Therefore, DPPH\(\bullet\) and HO\(\bullet\) has been widely applied to evaluate the antioxidant ability of compounds to act as free radical scavengers or hydrogen donors [7; 25; 26].  
Chemical treatment, enzymatic hydrolysis, and microbial fermentation of food proteins can be used for bioactive peptides production. However, the enzymatic hydrolysis method is preferred in the food and pharmaceutical industries because the other methods may leave residual organic solvents or toxic chemicals in the final products [7]. Treatment of protein substrate with different proteases will produce several types of protein hydrolysates, which exhibit various extents of antioxidant activities against various antioxidant systems [7; 14]. Therefore, the specificity of the enzyme used for the proteolysis is one of the most important factors for the production of bioactive peptides.  
In the experiment, defatted proteins of hairtail muscle were separately hydrolyzed with alcalase, trypsin, neutrale, pepsin, and papain at designed conditions and the antioxidant capacities of the resulted hydrolysates at the concentration of 6.0 mg protein/mL were shown in Figure 1. The data indicated that HO\(\bullet\) scavenging capacities of the protein hydrolysates were significantly influenced by the type of protease (\(p<0.05\)). The DPPH\(\bullet\) scavenging activities of alcalase and papain hydrolysates were 53.45 \(\pm\) 1.05% and 47.75 \(\pm\) 2.34%, respectively, which were significantly higher than those of trypsin (37.05 \(\pm\) 0.97%), neutrale (35.05 \(\pm\) 0.97%), and pepsin (39.85 \(\pm\) 1.28%) hydrolysates (\(p<0.05\)) (Figure 1A). The HO\(\bullet\) scavenging activities of neutrale, alcalase, and papain hydrolysates were 50.61 \(\pm\) 1.16%, 48.76 \(\pm\) 1.64%, and 48.89 \(\pm\) 1.58%, respectively, which were significantly higher than those of trypsin (35.21 \(\pm\) 0.67%) and pepsin (33.29 \(\pm\) 1.01%) hydrolysates (\(p<0.05\)) (Figure 1B). However, there were no significant differences on the HO\(\bullet\) scavenging activities of neutrale, alcalase, and papain hydrolysates at the concentration of 6.0 mg protein/mL (\(p>0.05\)). In addition, the active sites of papain were lie in basic amino acids, particularly Arg- and Lys-; and the active sites of alcalase were lie in Ala-, Leu-, Val-, Tyr-, Phe-, and Try-. The active sites are significantly different between alcalase and papain, which will help to shorten the hydrolysis time and improve the hydrolysis degree (DH) of protein hydrolysates [7; 27; 28].  
Under the designed conditions, the protein hydrolysate of hairtail (_T. japonicus_) muscle was prepared using binary-enzymes hydrolysis process (papain + alcalase) and referred to as HTP, and the antioxidant activity of protein hydrolysates was presented in Figure 2. At the concentration of 6.0 mg protein/mL, DPPH\(\bullet\) and HO\(\bullet\) scavenging activities of HTP were 60.72 \(\pm\) 1.05% and 58.17 \(\pm\) 1.53%, respectively, which was significantly higher than those of the hydrolysates prepared separately using papain and alcalase (\(p<0.05\)). Therefore, papain and alcalase were selected for the preparation of protein hydrolysate of hairtail muscle.  
Purification of Antioxidant Peptides from Protein Hydrolysate from Hairtail (T. japonicus) Muscle (HTP)