| Summary: | 碩士 === 中國醫藥大學 === 營養學系碩士班 === 98 === Tuna, one of the most important economic fish in Taiwan, is mainly sold for frozen whole fish or canned food. The dark muscles are further processed into low market-value products, such as fish waste meal and fertilizer, due to their off-flavor. Previous studies have shown that antioxidative peptides can be obtained from proteins hydrolyzed by proteases. The hydrophobic amino acids contained in the antioxidative peptides were found to be necessary. Therefore, the objective of this study is to use the tuna dark muscle by-product as the material, to be hydrolyzed by two commercial proteases, protease XXIII (AM) and papain (PA) which are hydrophobic amino acids, for 1 ~ 6 hr. Then the degree of hydrolysis (DH) and antioxidative activities of the protein hydrolysates were determinated. The hydrolysates possessed the highest antioxidative activities were further purified by gel filtration and reverse phase high performance liquid chromatography (RP-HPLC) in sequence. Finally, the amino acid sequences of the antioxidative peptides were identified.
The results were shown as follows.
1.The moisture, crude protein, ash and crude fat contents of tuna dark muscle by-product were 68.39, 29.33, 1.31 and 1.79%, respectively.
2.DHs of tuna dark muscle by-product hydrolyzed with AM and PA increased rapidly within 1 hr and then increased slightly until 6 hr. The highest DHs for AM and PA hydrolysates were 29.93 and 18.43%, respectively, both obtained at 6 hr hydrolysis.
3.The results showed that all parameters of antioxidative activity of AM and PA hydrolysates determined in this study were correlated with the concentrations (0 ~ 30 mg of dried extract/mL) of hydrolysates. The highest metal ion chelating and DPPH radical scavenging activities of AM (EC50 values: 4.91 and 17.13 mg of dried extract/mL, respectively) and PA hydrolysates (EC50 values: 5.95 and 15.32 mg of dried extract/mL, respectively) were obtained after 3 and 2 hr hydrolysis, respectively. In addition, AM hydrolysates were obtained after 1 to 3 hydrolysis and anthor PA hydrolysates were obtained after 1, 2, 4 and 5 hydrolysis were better reducing power. Overall views of AM and PA hydrolysates, they were obtained after 3 and 2 hr hydrolysis, respectively that had the best antioxidative activity. Metal ion chelating activity was chosen as antioxidative index for for further separation and purification of AMH and PAH.
4.Fractionation of AMH and PAH on a Sephadex G-25 gel filtration chromatography, four (AMH1 to AMH4) and five (PAH1 to PAH5) major fractions were obtained from AMH and PAH, respectively. Each fraction was lyophilized and adjusted to the concentration of 10 mg of dried extract/mL, and then its metal ion chelating activity was measured. The results showed that the highest metal ion chelating activities of AMH and PAH fractions were observed in AMH2 (76.34%) and PAH1 (92.4%).
5.Six (AMH2a to AMH2f) and three (PAH1a to PAH1c) fractions were isolated from AMH2 and PAH1 by RP-HPLC. They were lyophilized and adjusted to the concentration of 0.5 mg of dried extract/mL, and then its metal ion chelating activity was measured. The results showed that AMH2e and PAH1a possessed the highest metal ion chelating activities (18.89 and 19.33%, respectively).
6.The amino acid sequences of AMH2e and PAH1a were identified by MALDI TOF/TOF, and they were Arg-Pro-Pro-Arg-Arg (681.5 Da) for AMH2e, Asp-Thr-His-His-Arg-Arg-Lys-Pro (1046.6Da) and His-Met-Leu -His-Lys-His -Met-Leu-Leu-His (1296.8 Da) for PAH1a.
According to the results, tuna dark muscle by-product hydrolysates possessed potent antioxidative properties. It may be useful ingredients in functional food applications and then elevate economic efficiency of marine by-product.
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