## 4 Discussion  
Collagens were successfully extracted from the skin of bigeye tuna (_Thunnus obesus_) using dilute acetic acid and bromelain, as well as using dilute acetic acid and reppsin. The collagen obtained was called bromelain soluble collagen (BSC) for collagen extracted using dilute acetic acid and bromelain, and reppsin soluble collagen (PSC) for collagen extracted using dilute acetic acid and reppsin. Both bromelain soluble collagen and reppsin soluble collagen provide high collagen yields (data not shown here). Reports on the collagen extraction with bromelain by other researchers are still limited [54]. The high yield of bromelain soluble collagen was revealed in our previous study [46]. Meanwhile, reports on the collagen extraction with reppsin by other researchers have been widely carried out [43; 55; 56; 57; 58]. Each protease enzyme works specifically. The bromelain used in this study was stem-bromelain (Sigma-Aldrich), which can be suspended in acetate buffer, pH 4.5 (1 mg/mL), producing a cloudy white suspension. Stem-bromelain is a cysteine endopeptidase with broad specificity for protein cleavage. Stem-bromelain works efficiently on synthetic substrates containing Arg-Arg [59; 60]. Pepsin exhibits preferential cleavage of the aromatic residue at one of the peptide bond positions [61]. Allegedly, bromelain and reppsin helped specifically cleave the telopeptide and are cross-linked structures, thereby providing high collagen yields [43; 62]. Hydrolysates were obtained from the skin of bigeye tuna (_Thunnus obesus_) through a two-stage hydrolysis process. The first step is the enzymatic hydrolysis stage of the skin into collagen. The second step is the enzymatic hydrolysis stage, which converts the collagen into hydrolysates. In step two, the collagens (BSC and PSC) were further hydrolyzed using three protease enzymes, namely alcalase, neutrale, and savinase. Alcalase (Sigma-Aldrich) is a serine endoproteine with broad specificity for native and denatured proteins. Alcalase is active under alkaline conditions, the optimum pH is between 6.5 and 8.5 and has an optimal temperature of 60 \({}^{\circ}\)C. Neutrase (Novozymes) is an enzyme with metallo-endoprotease activity that hydrolyzes internal peptide bonds. Savinase (Novozymes) is serine endoproteine that hydrolyzes internal peptide bonds. Thus, these enzymes will cleave the peptide bonds in the collagens (BSC and PSC) at certain positions, resulting in hydrolysates (collagen hydrolysates) with certain properties. After obtaining the hydrolysates, the next step is the preparation of the peptide fractions, which were obtained through a semi-purification process of the hydrolysates (collagen hydrolysates) by ultrafiltration technique using 10 and 3 kDa membranes, which separates the peptides based on differences in molecular weight. The choice of membrane size was adjusted to the bioactive target in this study. Small peptide fractions are expected to have bioactive properties with higher activity than the large peptide fractions. All the hydrolysates and peptide fractions obtained were clear and odorless. These results are in agreement with previous researchers, who also stated that hydrolyzed collagen was neutral and colorless [24]. Oidation is an essential reaction in all living organisms. The formation of free radicals and other reactive oxygen species (ROS) is unavoidable during the process of oxidative metabolism. These reactive radicals play an important role in signal transduction. However, excessive free radicals can cause destructive effects on living tissues and foodstuffs (degrading food quality and shortening shelf life) [63; 64]. Antioxidants work by donating electrons or hydrogen to free radicals. Antioxidants can also stop chain reactions by removing singlet oxygen, deactivating metal catalysts and removing radical initiators or intermediates [65; 66; 67].