## 4 Discussion
Some artificial antioxidants such as propyl gallate (PG), butylated hydroxyapatite (BHA), and butylated hydroxytoluene (BHT) exhibit strong antioxidant activity and have been used to scavene free radicals in biological and food systems, but their application is strictly regulated [68; 69]. Studies on antioxidant peptides have been reported. These antioxidant peptides were found to have relatively low molecular weight, simple structure, high activity, easy absorption, better stability in different situations, and no harmful immune reactions [70]. In this study, we tested the antioxidant activities of hydrolyses and peptide fractions of collagen from the skin of big eye tuna (_Thununus obesus_) using a scavenging technique against 2,2-difenil-1-picrylhydrazil (DPPH) radicals. This scavenging technique against DPPH radicals was chosen because it is one of the commonly used techniques in analyzing the antioxidant activity of a compound in vitro. The principle is the DPPH provides maximum absorbance at a wave-length of 517 nm. The presence of antioxidant properties in the hydrolyses and peptide fractions was shown by a decrease in absorbance or a decrease in the intensity of the purple color. In the ascorbic acid solutions of 0.00, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00 mg/L, which were used as positive controls, the color change was clearly visible, as purple turned into yellow. Meanwhile, the color change was not as intensive for the hydrolyses and peptide fractions. The two highest antioxidant activities were produced by the PBN fraction <3 kDa and PPS fraction <3 kDa (wherein these fractions consisted of peptides <3 kDa). This result is consistent with previous studies, which also found that small peptides gave better results than large peptides [32; 33]. In addition, for future research, several test methods can be applied to produce more comprehensive observations of the antioxidant activity study. The selection of the analytical method, the solvent used, the concentration of the test compound, the concentration of the radical compound, and the incubation time also need to be considered. The antioxidant activities obtained by scavenging technique against DPPH radicals were studied statistically and expressed as profile plots. The factors observed were the collagen types, the enzymes used to hydrolyze collagen into hydrolyses and peptide fractions, and the molecular sizes. The types of collagens used in this study were bromelain soluble collagen (BSC) and pepsin soluble collagen (PSC). These collagens were obtained from the skin of bigeye tuna (_Thununus obesus_) using the same method, except for the enzymes used, namely bromelain for BSC and pepsin for PSC. The two collagens gave different effects on antioxidant activity even though they were obtained from the same skin, namely from bigeye tuna skin. The estimated marginal average value of the antioxidant activity of PSC is higher than that of BSC. This shows that in producing antioxidant compounds, overall, PSC gives better results than BSC. Alcalase, neutrale, and savinase are enzymes used to further hydrolyze the collagen. These enzymes work specifically to produce specific peptides, which in turn affect the antioxidant activity produced. Savinase overall showed the best results compared to neutrale and alcalase in producing antioxidant activity. Antioxidant activities were also studied as they affected the molecular sizes. The samples studied were the hydrolysate (a mixture of peptides of various molecular sizes), the peptide fraction >10 kDa (i.e., the fraction containing peptides with a molecular weight greater than 10 kDa), the peptide fraction 3-10 kDa (i.e., the fraction containing peptides with a molecular weight between 3 and 10 kDa), and the peptide fraction <3 kDa (i.e., the fraction containing peptides smaller than 3 kDa). The results of statistical analysis expressed as profile plots showed that the peptide fraction <3 kDa gave the highest antioxidant activity, which was shown from the estimated marginal average value.