以商業蛋白酶水解鮪魚血合肉加工副產品製備抗氧化活性胜肽之研究

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Title:	以商業蛋白酶水解鮪魚血合肉加工副產品製備抗氧化活性胜肽之研究 The Study on Development of Antioxidative Peptides Derived from Protein Hydrolysates of Tuna Dark Muscle By-product by Commercial Proteases
Authors:	曾瑜菀;Yu-Wan Tseng
Contributors:	健康照護學院營養學系碩士班
Keywords:	鮪魚血合肉加工副產品;蛋白質水解物;抗氧化活性;胜肽 tuna dark muscle by-product;protein hydrolysate;antioxidative activity;peptide
Date:	2010
Issue Date:	2010-09-29 12:09:40 (UTC+8)
Abstract:	鮪魚是我國最重要的經濟魚類之一，一般以冷凍全魚或罐頭方式販售，其血合肉由於具特殊腥臭味，因此被製成經濟價值不高的魚粉或魚溶漿。近年來研究指出，酶水解蛋白質後可產生之具抗氧化活性胜肽，且發現此胜肽之胺基酸組成常為疏水性胺基酸。因此本實驗利用鮪魚血合肉加工副產品為原料，以作用於疏水性胺基酸之市售商業蛋白酶 protease ΧXIII (AM) 及 papain (PA) 於最適條件下進行 1 ~ 6 hr 水解，測定其鮪魚蛋白質水解物之水解度 (degree of hydrolysis; DH) 及抗氧化活性，將具最佳抗氧化活性之水解物藉凝膠過濾法 (gel filtration) 及高效能液相層析法 (RP-HPLC) 分離純化，並鑑定此抗氧化活性胜肽之胺基酸序列。實驗結果如下, 1.鮪魚血合肉加工副產品之基本成分含量為水分占 68.39%、粗蛋白占29.33%、灰分占 1.31%、粗脂肪占 1.79%。 2.AM 或 PA 蛋白質水解物皆在水解 1 hr 內的 DH 會顯著上升，爾後則隨水解時間增加而逐漸上升。當水解 6 hr 時，AM 及 PA 蛋白質水解物 DH 分別為 29.93 和 18.43%。 3.在抗氧化活性測定方面，皆發現 AM 和 PA 蛋白質水解物之抗氧化活性與濃度 (0 ~ 30 mg of dried extract/mL) 呈正相關。且 AM 和 PA 蛋白質水解物分別在水解 3 hr 和 2 hr 皆具有最佳的亞鐵離子螯合能力 (EC50 分別為 4.91和 5.95 mg of dried extract/mL) 以及清除 2,2-diphenyl-1-picrylhydrazyl (DPPH) 自由基能力 (EC50 分別為 17.13 和 15.32 mg of dried extract/mL)。此外， AM 及 PA 蛋白質水解物分別以水解 1 ~ 3 hr 及水解 1、2、4 和 5 hr還原力效果較佳。就整體而言，以水解 3 hr 之 AM蛋白質水解物及 2 hr 之 PA 蛋白質水解物 (AMH 及 PAH) 最具有較佳抗氧化活性。故未來再分離純化則選用 AMH 和 PAH 時並以亞鐵離子螯合能力作為抗氧化活性指標。 4.利用 Sephadex G-25 凝膠過濾法區分 AMH 和 PAH，其主要可分別分離出 4 個 (AMH1 ~ AMH4) 和 5 個 (PAH1 ~ PAH5) 區分物，並將各區分物冷凍濃縮後，濃度調整至 10 mg of dried extract/mL 並測定其亞鐵離子螯合能力，結果以 AMH2 (76.34%) 以及 PAH1 (92.4%) 為最佳。 5.將 AMH2 以及 PAH1 以 RP-HPLC 法分離純化，分別分離出 6 個 (AMH2a ~ AMH2f) 和 3 個波峰 (PAH1a ~ PAH1c)，並將各區分物冷凍濃縮後，濃度調整至 0.5 mg of dried extract/mL 並測定其亞鐵離子螯合能力，結果以 AMH2e (18.89%) 以及 PAH1a (19.33%) 為最佳。 6.AMH2e 和 PAH1a 經 MALDI TOF/TOF 經分析鑑定後，分別可得 1 及 2 個胜肽之胺基酸序列：AMH2e 為 Arg-Pro-Pro-Arg-Arg (681.5 Da)；PAH1a 為 Asp-Thr-His-His-Arg-Arg-Lys-Pro (1046.6 Da) 和 His- Met-Leu -His-Lys-His-Met-Leu-Leu-His (1296.8 Da)。綜合以上結果所述，鮪魚血合肉加工副產品之蛋白質水解物具有抗氧化活性且有利於機能性食品之開發並提升副產品的經濟價值。 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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