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研究生:楊定山
研究生(外文):YANG,TING-SHAN
論文名稱:甲魚(中華鱉)中分離生物活性胜肽及抗氧化活性之研究
論文名稱(外文):Study on isolation and antioxidant activity of Trionychidae (Pelodiscus sinensis) bioactive peptide
指導教授:張瑞璋張瑞璋引用關係
指導教授(外文):CHANG,REY-CHANG
口試委員:朱惠鈴丁俞文張瑞璋鄭至玉
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:115
中文關鍵詞:甲魚生物活性胜肽抗氧化
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近年來,許多動物和植物來源的蛋白質水解物已被發現具有抗氧化活性,本研究將中華鱉 (Pelodiscus sinensis)蛋白質分別以5%三種不同蛋白酶 (Alcalase, Neutrase,及Flavourzyme)進行水解,發現以Flavourzyme 之水解產物具有最高的抗氧化活性,包括DPPH清除能力 (22.5 ± 1.2 %)、TEAC總抗氧化能力 (55.0 ± 1.01 %)、而還原力則測定A620 nm其吸光值為 (0.17±0.01)。進一步以Flavourzyme探討水解條件,發現以4%酵素於pH 7.0反應1小時,其DPPH清除能力達29.91 ± 2.86 %,而水解率則相當於 121.32 ± 2.11 nM之leucine的胺基酸含量。將水解產物以Sephadex G-25進行分子篩層析,得到主要3個劃分 (P1–P3)其中P2劃分具有最高的DPPH清除能力,因此將P2劃分進行細胞生物活性探討,並且將P2劃分進一部以逆向高效液相層析儀 (RP-HPLC)進行分離純化,得到五個劃分 (P2-1 ~ P2-5),因此將具有較高的抗氧化活性P2-2與P2-4劃分進行ESI-MS/MS分析,依質譜數據進行比對分析,預測七條胜肽胺基酸序列分別為:SGVGF、TAAGF、AGINPA、APAGPQ、LGKGY、VAKGY、THAAPN,進行胜肽人工合成後並進行抗氧化活性測定,實驗結果發現LGKGY、VAKGY這兩條胜肽具有抗氧化活性,並且進行RAW小鼠巨噬細胞實驗,結果發現具有抑制NO生成及抑制發炎效果。由本研究結果發現中華鱉蛋白質水解物具有生物活性功能,能提供鱉養殖產業應用性發展的參考。
In recent years, hydrolyzed proteins from many animal and plant sources had been found to have strong antioxidant activity. Protein derived from Trionychidae (Pelodiscus sinensis) was hydrolyzed using three kinds of proteases (5%) including Alcalase, Neutrase, and Flavourzyme. Protein hydrolysate produced from Flavourzyme was discovered to have the highest antioxidant activity, which is determined by its DPPH radical scavenging activity (22.5±1.2%), Trolox equivalent antioxidant capacity (55.0±1.01%), and reducing power(A620) (0.17±0.01). To find out the optimum parameters for hydrolysate production, Flavourzyme hydrolysis conditions was further investigated. Under pH 7 environment, 4% Flavourzyme was able to produce hydrolysate with 29.91 ± 2.86% DPPH radical scavenging activity within 1 hour. After reaction with o-Phthaldialdehyde, the one-hour fluorescent intensity of Flavourzyme hydrolysate was equivalent to 121.32 ±2.11nM Leucine content. Hence, Flavourzyme hydrolysate produce from optimized condition was further fractionated by size exclusion chromatography on Sephadex G-25 into three major fractions (P1–P3). Since only P2 retain DPPH scavenging activity, it was then again fractionated by reversed-phase high performance liquid chromatography (RP-HPLC) into five major fractions (P2-1–P2-5), among which P2-2 and P2-4 have better DPPH radical scavenging activities. In addition, both Trionychidae protein hydrolysates and purified Trionychidae protein hydrolysates reduced inflammatory reaction through diminishing NO production in RAW mouse macrophage cell. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. The peptide sequences were identified as SGVGF, TAAGF, AGINPA, APAGPQ, LGKGY, VAKGY, and THAAPN. In future, isolated bioactive peptides can be used as potent antioxidants to be used as additives in food or pharmaceutical products.
第一章 前言 1
第二章 文獻回顧 2
2.1甲魚簡介 2
2.1.1甲魚分佈 2
2.1.2甲魚生態習性 2
2.1.3甲魚成分 3
2.2自由基 (Free radical) 之定義 5
2.2.1自由基的生成 7
2.3非酵素性抗氧化劑及抗氧化酵素 12
2.3.1非酵素性抗氧化劑 13
2.3.2 抗氧化酵素 15
2.4 生物活性胜肽的研究 17
2.5 蛋白質水解物的分離方法 21
2.5.1 膠過濾層析 21
2.5.2 高效能液相層析法 23
2.5.3串聯式質譜儀 24
2.6 抗氧化活性測定方法 26
2.6.1 DPPH自由基清除能力測定 26
2.6.2亞鐵離子螯合能力測定 26
2.6.3 TEAC 總抗氧化能力 28
2.6.4還原能力測試 28
2.6.5脂質過氧化抑制能力測定 28
2.7 胜肽含量測定 30
2.8 細胞活性測定方法 31
2.8.1細胞之培養 31
2.8.2 細胞存活率分析 31
2.8.3 一氧化氮測定 31
2.8.4 胞內活性氧分析 32
2.9 發炎反應 33
2.10 黑色素 34
第三章 材料與方法 36
3.1 實驗架構 36
3.2 實驗材料 37
3.2.2 使用藥品與溶劑 37
3.2.3儀器設備 38
3.2.4細胞株 39
3.3 樣品製備 39
3.3.1 甲魚各個部位前處理 39
3.3.2 酵素水解 40
3.3.3 甲魚肉蛋白酶水解條件的探討 40
3.4 抗氧化活性測定試驗 42
3.4.1 DPPH自由基清除能力測定 42
3.4.2 TEAC (Trolox equivalent antioxidant capacity) 總抗氧化能力測定 43
3.4.3 還原力能力測定 43
3.5 酪胺酸酶活性抑制測定 43
3.6 OPA胜肽含量測定 44
3.7 膠過濾層析法 44
3.7.1 管柱充填 44
3.7.2 樣品製備 45
3.7.3 管柱層析 45
3.8 高效能液相層析法 45
3.8.1 樣品前處理 45
3.8.2高效能逆向層析最適條件 46
3.9 串聯式質譜儀 46
3.10 胜肽合成 47
3.11 細胞實驗 47
3.6.1 細胞毒性測試 47
3.6.2 一氧化氮 (NO) 測定 47
3.6.3 B16胞外黑色素測定 48
3.6.4 B16胞內黑色素測定 48
第四章 結果與討論 49
4.1 甲魚不同部位水解物抗氧化活性的探討 49
4.2 甲魚肉蛋白酶水解條件的探討 50
4.3 甲魚肉水解物抑制酪胺酸酶活性的探討 53
4.4 甲魚肉水解物的分離純化膠過濾層析 53
4.4.1 膠過濾層析 53
4.4.2 高效能液相層析 (HPLC) 的純化 54
4.5胜肽胺基酸序列分析 54
4.7 細胞生物活性分析 58
4.7.1 RAW 264.8 小鼠巨噬細胞生物活性分析 58
4.7.2 B16 小鼠黑色素瘤細胞生物活性分析 59
第五章 結論 60
第六章 參考文獻 104

丁克祥(1996),SOD 生物醫學淺論,藝軒圖書出版社。
李敏雄(2004),酵素在食品加工的應用,藝軒圖書出版社。
柯以侃(2002),儀器分析,新文京開發出版有限公司。
孫存普、張建中、段紹瑾(1999),自由基生物學導論,中國科學技術大學出版社。
徐壽山、黃立峰、沈百林(1996),鰻鱉養殖實用新技術,中國農業出版社,中國北京,p64~66
張孟文、宗愉、馬積藩(1998),中國動物誌 爬行綱 第一卷 總論 龜鱉目,科學出版社,中國北京,p66~71
郭智宏(2001)。腸道吸收-胺基酸與胜肽。食品工業,33(5):15-23。
陳石根(2002),酶學,九州圖書文物出版社。
陳國城(1999),酵素工程學,藝軒圖書出版社。
廖大修、陳靜瑩(2004),蛋白質體學,醫藥基因生物技術教學資源中新。
趙保路(1999),氧自由基和天然抗氧化劑,科學出版社。
劉興鑑、孫逸民、陳玉舜、趙敏勳、謝明學(2010),儀器分析,權威圖書出版社。
鄭文騰、王俊昇、陳建初(1998),市售不同性狀甲於人工飼料對甲魚之成長試驗,中國水產月刊,548:3-13
鄭榮梁、魏耀揮、趙崇義、黃中洋、陳建春(2013),自由基生物醫學,藝軒圖書出版社。
鞠長增(1997),鱉的養殖與疾病防治,柯南科學技術出版社,中國鄭州,p27~32
龐戰軍、陳玫、陳瑗(2000),自由基醫學研究方法,人民衛生出版社。
Arathi, B.P., Raghavendra-Rao Sowmya, P., Vijay, K., Baskaran, V., and Lakshminarayana, R. Metabolomics of carotenoids: The challenges and prospects - A review. Trends in Food Science & Technology.
Arts, M.J.T.J., Sebastiaan Dallinga, J., Voss, H.-P., Haenen, G.R.M.M., and Bast, A. (2003). A critical appraisal of the use of the antioxidant capacity (TEAC) assay in defining optimal antioxidant structures. Food Chemistry 80, 409–414.
Bae, S.-H., and Suh, H.-J. (2007). Antioxidant activities of five different mulberry cultivars in Korea. LWT - Food Science and Technology 40, 955–962.
Bamdad, F., Wu, J., and Chen, L. (2011). Effects of enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein. Journal of Cereal Science 54, 20–28.
Blair, I.A. (2001). Lipid hydroperoxide-mediated DNA damage. Experimental Gerontology 36, 1473–1481.
Blokhina, O., Virolainen, E., and Fagerstedt, K.V. (2003). Antioxidants, Oxidative Damage and Oxygen Deprivation Stress: a Review. Ann Bot 91, 179–194.
Bougatef, A., Nedjar-Arroume, N., Manni, L., Ravallec, R., Barkia, A., Guillochon, D., and Nasri, M. (2010). Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita) by-products proteins. Food Chemistry 118, 559–565.
Cai, L., Wu, X., Zhang, Y., Li, X., Ma, S., and Li, J. (2015). Purification and characterization of three antioxidant peptides from protein hydrolysate of grass carp (Ctenopharyngodon idella) skin. Journal of Functional Foods 16, 234–242.
Canabady-Rochelle, L.L.S., Harscoat-Schiavo, C., Kessler, V., Aymes, A., Fournier, F., and Girardet, J.-M. (2015). Determination of reducing power and metal chelating ability of antioxidant peptides: Revisited methods. Food Chemistry 183, 129–135.
Chang, T.-S. (2009). An Updated Review of Tyrosinase Inhibitors. Int J Mol Sci 10, 2440–2475.
Chen, Z.-T., Chu, H.-L., Chyau, C.-C., Chu, C.-C., and Duh, P.-D. (2012). Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress. Food Chemistry 135, 2119–2127.
Chi, C.-F., Wang, B., Deng, Y.-Y., Wang, Y.-M., Deng, S.-G., and Ma, J.-Y. (2014). Isolation and characterization of three antioxidant pentapeptides from protein hydrolysate of monkfish (Lophius litulon) muscle. Food Research International 55, 222–228.
Chi, C.-F., Wang, B., Hu, F.-Y., Wang, Y.-M., Zhang, B., Deng, S.-G., and Wu, C.-W. (2015). Purification and identification of three novel antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) skin. Food Research International 73, 124–129.
Chu, H.-L., Chien, J.-C., and Duh, P.-D. (2011). Protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells. Food Chemistry 129, 871–876.
Church, F.C., Swaisgood, H.E., Porter, D.H., and Catignani, G.L. (1983). Spectrophotometric Assay Using o-Phthaldialdehyde for Determination of Proteolysis in Milk and Isolated Milk Proteins1. Journal of Dairy Science 66, 1219–1227.
Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science & Technology 11, 254–262.
Erbaş, M.S.e., H.( 2011)Importance of free radicals and occurring during food processing. GIDA - Journal of Food.349-356
Fenton, H. J. H. (1894).Oxidation of tartaric acid in presence of iron. Journal of the Chemical Society, Transactions, 899-910
Food enzymes: industrial potential and scientific challenges. In Chemical Aspects of Food Enzymes. A.T. Andrews (ed.) Andrews A.T.Royal Society of Chemistry, London, pp. 289-314
Fox, O. F., Morriesey, P. A. and Mulvihill, D. M. (1982). Chemical and enzymatic modification of food proteins. Elsevier Appl. Sci., NJ, U.S.A Gerontology, 2001. 1473-1481.
Goodwin, J.S., and Webb, D.R. (1980). Regulation of the immune response by prostaglandins. Clinical Immunology and Immunopathology 15, 106–122.
Griendling, K.K., and FitzGerald, G.A. (2003). Oxidative Stress and Cardiovascular Injury Part I: Basic Mechanisms and In Vivo Monitoring of ROS. Circulation 108, 1912–1916.
Halliwell, B., Murcia, M.A., Chirico, S., and Aruoma, O.I. (1995). Free radicals and antioxidants in food and in vivo: What they do and how they work. Critical Reviews in Food Science and Nutrition 35, 7–20.
Harnedy, P.A., and FitzGerald, R.J. (2012). Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods 4, 6–24.
Hernández-Ledesma, B., García-Nebot, M.J., Fernández-Tomé, S., Amigo, L., and Recio, I. (2014). Dairy protein hydrolysates: Peptides for health benefits. International Dairy Journal 38, 82–100.
Hu, Z.-M., Zhou, Q., Lei, T.-C., Ding, S.-F., and Xu, S.-Z. (2009). Effects of hydroquinone and its glucoside derivatives on melanogenesis and antioxidation: Biosafety as skin whitening agents. Journal of Dermatological Science 55, 179–184.
Huang, M.-H., Chu, H.-L., Juang, L.-J., and Wang, B.-S. (2010). Inhibitory effects of sweet potato leaves on nitric oxide production and protein nitration. Food Chemistry 121, 480–486.
Kleekayai, T., Harnedy, P.A., O’Keeffe, M.B., Poyarkov, A.A., CunhaNeves, A., Suntornsuk, W., and FitzGerald, R.J. (2015). Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes. Food Chemistry 176, 441–447.
Koppenol, W. H. (2001). The Haber-Weiss cycle –70 years late. Redox Report, 29-234
Korde (Choudhari), S., Sridharan, G., Gadbail, A., and Poornima, V. (2012). Nitric oxide and oral cancer: A review. Oral Oncology 48, 475–483.
Lee, J., Chung, H., Chang, P.-S., and Lee, J. (2007). Development of a method predicting the oxidative stability of edible oils using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Food Chemistry 103, 662–669.
Libby, P. (2002). Inflammation in atherosclerosis. Nature 420, 868–874.
Lochmann, D., Stadlhofer, S., Weyermann, J., and Zimmer, A. (2004). New protamine quantification method in microtiter plates using o-phthaldialdehyde/N-acetyl-l-cysteine reagent. International Journal of Pharmaceutics 283, 11–17.
Mahind Senevirathne, and Se-Kwon Kim (2012). Marine Medicinal Foods: Implications and Applications: Animals and Microbes (Academic Press).
Mann, M., Hendrickson, R.C., and Pandey, A. (2001). Analysis of Proteins and Proteomes by Mass Spectrometry. Annual Review of Biochemistry 70, 437–473.
Miller, S.B. (2006). Prostaglandins in Health and Disease: An Overview. Seminars in Arthritis and Rheumatism 36, 37–49.
Miyamoto, S., Martinez, G.R., Medeiros, M.H.G., and Di Mascio, P. (2014). Singlet molecular oxygen generated by biological hydroperoxides. Journal of Photochemistry and Photobiology B: Biology 139, 24–33.
Moncada, S., Palmer, R.M., and Higgs, E.A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142.
Nikoo, M., Benjakul, S., and Xu, X. (2015). Antioxidant and cryoprotective effects of Amur sturgeon skin gelatin hydrolysate in unwashed fish mince. Food Chemistry 181, 295–303.
Nimalaratne, C., Bandara, N., and Wu, J. (2015). Purification and characterization of antioxidant peptides from enzymatically hydrolyzed chicken egg white. Food Chemistry 188, 467–472.
Noori, S. (2012). An Overview of Oxidative Stress and Antioxidant Defensive System. Journal of Clinical & Cellular Immunology 01.
Pospíšil, P., and Prasad, A. (2014). Formation of singlet oxygen and protection against its oxidative damage in Photosystem II under abiotic stress. Journal of Photochemistry and Photobiology B: Biology 137, 39–48.
Rajapakse, N., Mendis, E., Byun, H.-G., and Kim, S.-K. (2005). Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems. The Journal of Nutritional Biochemistry 16, 562–569.
Ranathunga, S., Rajapakse, N., and Kim, S.-K. (2005). Purification and characterization of antioxidative peptide derived from muscle of conger eel (Conger myriaster). Eur Food Res Technol 222, 310–315.
Righelato, R.C. and Rodgers, (1987) Food Enzymes: Industrial Potential and Scientific Challenges. In Chemical Aspects of Food Enzymes ed. Andrews A. T, Royal Society of Chemistry, London 298-314
Robak, J., and Gryglewski, R.J. (1988). Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology 37, 837–841.
Sailaja Rao, P., Kalva, S., Yerramilli, A., and Mamidi, S. (2011). Free Radicals and Tissue Damage: Role of Antioxidants. Free Radicals and Antioxidants 1, 2–7.
Sampath Kumar, N.S., Nazeer, R.A., and Jaiganesh, R. (2012). Purification and identification of antioxidant peptides from the skin protein hydrolysate of two marine fishes, horse mackerel (Magalaspis cordyla) and croaker (Otolithes ruber). Amino Acids 42, 1641–1649.
Scandalios, J. G.(1997)Oxidative stress and the molecular biology of antioxidant defenses:Molecular genetics of superoxide dismutase in plants. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. p.527-568.
Scheibmeir, H.D., Christensen, K., Whitaker, S.H., Jegaethesan, J., Clancy, R., and Pierce, J.D. (2005). A review of free radicals and antioxidants for critical care nurses. Intensive and Critical Care Nursing 21, 24–28.
Sen, S., Chakraborty, R., Sridhar, C., Reddy, Y.S.R., and De, B. (2010). Free radicals, antioxidants, diseases and phytomedicines: current status and future prospect. International Journal of Pharmaceutical Sciences Review and Research 3, 91–100.
Sheikhpour, R. (2013). Diabetes and Oxidative Stress: The Mechanism and Action. Iranian Journal of Diabetes and Obesity 5, 40–45.
Shimada, K., Fujikawa, K., Yahara, K., and Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40, 945–948.
Singh, M., Kapoor, A., and Bhatnagar, A. Oxidative and reductive metabolism of lipid-peroxidation derived carbonyls. Chemico-Biological Interactions.
Szabó, C., Ischiropoulos, H., and Radi, R. (2007). Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6, 662–680.
Tanzadehpanah, H., Asoodeh, A., and Chamani, J. (2012). An antioxidant peptide derived from Ostrich (Struthio camelus) egg white protein hydrolysates. Food Research International 49, 105–111.
Tavano, O.L. (2013). Protein hydrolysis using proteases: An important tool for food biotechnology. Journal of Molecular Catalysis B: Enzymatic 90, 1–11.
Torres-Fuentes, C., Alaiz, M., and Vioque, J. (2012). Iron-chelating activity of chickpea protein hydrolysate peptides. Food Chemistry 134, 1585–1588.
Torres-Fuentes, C., Contreras, M. del M., Recio, I., Alaiz, M., and Vioque, J. (2015). Identification and characterization of antioxidant peptides from chickpea protein hydrolysates. Food Chemistry 180, 194–202.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.D., Mazur, M., and Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology 39, 44–84.
Van den Berg, R., Haenen, G.R.M.M., van den Berg, H., and Bast, A. (1999). Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chemistry 66, 511–517.
Wang, B., Li, L., Chi, C.-F., Ma, J.-H., Luo, H.-Y., and Xu, Y. (2013). Purification and characterisation of a novel antioxidant peptide derived from blue mussel (Mytilus edulis) protein hydrolysate. Food Chemistry 138, 1713–1719.
Wang, B.-S., Chang, L.-W., Wu, H.-C., Huang, S.-L., Chu, H.-L., and Huang, M.-H. (2011). Antioxidant and antityrosinase activity of aqueous extracts of green asparagus. Food Chemistry 127, 141–146.
Wang, J., Qi, Z., and Yang, Z. (2014). Evaluation of the protein requirement of juvenile Chinese soft-shelled turtle (Pelodiscus sinensis, Wiegmann) fed with practical diets. Aquaculture 433, 252–255.
Xia, Y., Bamdad, F., Gänzle, M., and Chen, L. (2012). Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis. Food Chemistry 134, 1509–1518.
Yates, J.R. (1998). Mass spectrometry and the age of the proteome. J Mass Spectrom 33, 1–19.
Yen, W.-J., Chyau, C.-C., Lee, C.-P., Chu, H.-L., Chang, L.-W., and Duh, P.-D. (2013). Cytoprotective effect of white tea against H2O2-induced oxidative stress in vitro. Food Chemistry 141, 4107–4114.
You, L., Zhao, M., Regenstein, J.M., and Ren, J. (2010). Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry 120, 810–816.
Zeinali, F., Homaei, A., and Kamrani, E. (2015). Sources of marine superoxide dismutases: Characteristics and applications. International Journal of Biological Macromolecules 79, 627–637.
Zhuang, H., Tang, N., and Yuan, Y. (2013). Purification and identification of antioxidant peptides from corn gluten meal. Journal of Functional Foods 5, 1810–1821.

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