臺灣博碩士論文加值系統

摘 要

海龍鬚菜是本國常見養殖海藻，常用來當做九孔飼料。近來發現海龍鬚菜具有保健活性成分，為了提高海龍鬚菜的利用價值，本研究針對其蛋白質水解物進行機能性評估。本研究將新鮮海藻以木瓜酵素於37℃進行15小時酵素水解，所得粗水解物進行實驗分為二部分。第一部分實驗以凍乾水解物進行SDS-PAGE分析，了解水解蛋白質分子量大小分布。第二部分實驗將粗水解物進行硫酸銨蛋白質鹽析(salting out)，分別收集0-20％、20-40％、40-60％、60-80％、80-100％硫酸銨濃度共5區間的鹽析物，再以透析(dialysis)方式將硫酸銨完全移除。將5區間的蛋白質鹽析物進行抗自由基活性之分析，包括DPPH自由基清除力、氫氧自由基清除力、清除超氧陰離子效應、DNA氧化傷害保護力四項。實驗結果顯示，20-40％、40-60％、60-80％、80-100％硫酸銨區間的蛋白質鹽析物之DPPH自由基清除能力，隨著樣品濃度的增加而提高，具有濃度相依效應（dose-dependent effect）。其中80-100％硫酸銨區間的鹽析物（800 ppm），其DPPH自由基清除能力效果最佳（36.02％），而20-40％硫酸銨區間的鹽析物（800 ppm）、40-60％硫酸銨區間的鹽析物（800 ppm）、60-80％硫酸銨區間的鹽析物（400 ppm）三者之清除率依序為30.19％、30.79％、30.05％。在DNA氧化傷害保護效力試驗中，結果顯示5個區間硫酸銨濃度的鹽析物中，以20-40％硫酸銨區間的鹽析物（5 mg/ml）最高（96.32％），而同濃度之40-60％硫酸銨區間的鹽析物與80-100％硫酸銨區間的鹽析物在DNA氧化傷害保護效力分別為49.66％及59.81％，然而在0-20％硫酸銨區間的鹽析物與60-80％硫酸銨區間的鹽析物無明顯對DNA氧化傷害保護效力，此外，將20-40％硫酸銨區間的鹽析物進行DNA氧化傷害保護效力之濃度梯度試驗，研究結果顯示，隨著樣品濃度的增加而有提高現象，具有濃度相依效應。在氫氧自由基清除力試驗中，結果顯示5個區間硫酸銨濃度的鹽析物中並無明顯清除能力。在清除超氧陰離子效應測定，研究結果得知20-40％硫酸銨區間的鹽析物（50 ppm）有較明顯的清除超氧陰離子效力（91.98％），而於同濃度之0-20％硫酸銨區間的鹽析物、40-60％硫酸銨區間的鹽析物、60-80％硫酸銨區間的鹽析物、80-100％硫酸銨區間的鹽析物對清除超氧陰離子效力依序為80.60％、80.56％、66.24％、67.13％。綜合上述，硫酸銨蛋白鹽析物具有抗自由基與保護DNA的氧化傷害活性，具有開發天然抗氧化劑潛力。

關鍵字：海龍鬚菜Gracilaria tenuistipitata、蛋白質水解物、抗自由基、氧化傷害、抗氧化劑。

Abstract

Gracilaria tenuistipitata, as human food or abalone feed in Taiwan, is a edible red seaweed in aquaculture. It is recently discovered that Gracilaria tenuistipitata contains certain functional ingredients for health. In order to increase the use value of Gracilaria tenuistipitata, the functional assessment of the protein hydrolysates was carried out in this study. Treated with the papain, the fresh seaweed was hydrolyzed at 37 ℃for 15 h. The obtained hydrolysates derived from the process above were divided into two parts. The first part was carried out freeze-drying and then SDS-PAGE analysis to determine their molecular weight distributions. The second part was precipitated by various concentration ranges of ammonium sulfate (AS) including 0-20%, 20-40%, 40-60%, 60-80% and 80-100% (salting out) and then AS was removed via dialysis. The precipitants of the five protein hydrolysates were evaluated their DPPH, superoxide anion, and hydroxyl radical scavenging ability. In addition, the protective effect of DNA oxidative damage derived from hydroxyl radical was also investigated. DPPH scavenging activity of the protein hydrolysate precipitants from 20-40%, 40-60%, 60-80%, and 80-100% AS showed a dose-dependent effect. The protein hydrolysate precipitant from 80-100 % AS (800 ppm) carried the best capacity of the DPPH radical scavenging (36.02%), while the protein hydrolysate precipitants from 20-40% (800 ppm), 40-60 % (800 ppm), and 60-80% (400 ppm) AS showed DPPH
scavenging rates of 30.19%, 30.79%, and 30.05%, respectively. The results of protective effects of DNA oxidative damage showed that the protein hydrolysate (5 mg/ml) from 20-40% AS precipitation was the highest efficacy (96.32%), while those of 40-60% and 80-100% AS precipitation were 49.66% and 59.81%, respectively. However, protein hydrolysate precipitants from 0-20% and 60-80% AS performed no significant effect on DNA oxidative damage protection. In addition, the protein hydrolysate precipitant from 20-40% AS showed a dose-dependent trend of DNA oxidative damage protection effect. Moreover, it showed that the five protein hydrolysate precipitants demonstrated no significant effect on DPPH scavenging. In the determination of the superoxide anion scavenging, it indicated that protein hydrolysate precipitant from 0-20% AS (50 ppm) showed the most significant scavenging activity (91.98%), while the those from of 0-20%, 40-60%, 60-80%, and 80-100% AS exhibited scavenging ability of 80.60%, 80.56%, 66.24%, and 67.13%, respectively. To summarize, the protein hydrolysates from AS precipitation with noticeable protective effect of DNA oxidative damage and antiradical activity may have a great potential for developing natural antioxidant products.


Keywords: Gracilaria tenuistipitata, protein hydrolysate, antiradical activity, oxidative damage, antioxidant

目錄

中文摘要 Ⅰ
英文摘要 Ⅲ
誌謝 Ⅴ
壹、前言 1
貮、文獻回顧 4
一、藻類簡介 4
二、海龍鬚菜（Gracilaria）簡介 5
三、自由基與活性氧對生物體影響之探討 8
（一）自由基與活性氧之定義 8
（二）自由基與活性氧對生物體所造成之傷害 11
（三）生物體內抗氧化防禦系統 11
四、活性胜肽 13
(一) 蛋白質酵素水解物之性質 13
(二) 生物活性胜肽(Bioactive peptides) 13
（1）降高血壓活性 (Antihypertensive activity) 14
（2）抗氧化活性 (Antioxidative activity) 14
（3）降膽固醇活性 (Hypochdesterolemic activity) 15
（4）抗菌活性 (Antibacterial activity)) 16
（5）免疫調節 (Immunoregulatory) 17
（6）礦物質結合(Mineral binding) 17
参、材料與方法 19
一、實驗材料 19
(一) 海龍鬚菜原料 19
(二) 實驗試藥 19
(三) 實驗儀器設備 22
二、實驗方法 24
(一) 實驗架構設計流程圖 24
(二) 海龍鬚菜前處理儲存 25
(三) 海龍鬚菜蛋白水解製備 25
（四）海龍鬚菜蛋白粗水解液硫酸銨鹽析與透析之製備 25
（五）SDS-PAGE (SDS-polyacrylamide gel electrophoresis)蛋白質膠體電泳-不連續聚丙烯胺膠體溶液調配法 26
(六) 蛋白質電泳分析方法 29
1. SDS-PAGE膠體試劑配製 29
2. 樣品的製備與電泳條件 30
3.膠體染色及脫色 30
4.膠體-玻璃紙三明治組合包紮乾燥法 31
(七) DPPH 自由基清除能力之分析 32
(八) 氫氧自由基清除能力之分析. 33
(九) 超氧陰離子清除能力之分析 35
(十) DNA保護試驗 36
肆、結果與討論 38
一、海龍鬚菜粗水解物與鹽析物之產率 38
二、海龍鬚菜水解物凍乾及以硫酸銨沈澱各區間凍乾之15％SDS-PAGE
40
三、抗自由基活性分析 42
(一) DPPH自由基清除能力之分析 42
(二) 氫氧自由基清除能力之分析 47
(三) 清除超氧陰離子能力之測定 54
(四) DNA保護試驗 60
伍、結論 63
六、參考文獻 65


圖目錄

圖一、Gracilaria tenuistipitata 海龍鬚菜 6
圖二、主要的活性氧種類 10
圖三、氧分子的氧化還原及激發狀態 10
圖四、抗氧化劑（RH）與DPPH自由基之反應機制 32
圖五、Nitroblue tetrazolium 與超氧陰離子自由基之反應機制 35
圖六、海龍鬚菜水解物與不同區間硫酸銨沈澱海龍鬚菜水解物之15％SDS-PAGE 41
圖七、20-40％硫酸銨沈澱海龍鬚菜水解物在不同濃度與50ppm Vit C對DPPH清除力變化 43
圖八、40-60％硫酸銨沈澱海龍鬚菜水解物在不同濃度與50ppm Vit C對DPPH清除力變化 44
圖九、60-80％硫酸銨沈澱海龍鬚菜水解物在不同濃度與50ppm Vit C對DPPH清除力變化 45
圖十、80-100％硫酸銨沈澱海龍鬚菜水解物在不同濃度與50ppm Vit C對DPPH清除力變化 46
圖十一、0-20％硫酸銨沈澱海龍鬚菜水解物在不同濃度對氫氧自由基清除力變化 48
圖十二、20-40％硫酸銨沈澱海龍鬚菜水解物在不同濃度對氫氧自由基 清除力變化 49
圖十三、40-60％硫酸銨沈澱海龍鬚菜水解物在不同濃度對氫氧自由基清除力變化 50
圖十四、60-80％硫酸銨沈澱海龍鬚菜水解物在不同濃度對氫氧自由基清除力變化 51
圖十五、80-100％硫酸銨沈澱海龍鬚菜水解物在不同濃度對氫氧自由基清除力變化 52
圖十六、透析後五區間之硫酸銨沈澱物溶液 53
圖十七、0-20％硫酸銨沈澱海龍鬚菜水解物在不同濃度對超氧陰離子清除力變化 55
圖十八、20-40％硫酸銨沈澱海龍鬚菜水解物在不同濃度對超氧陰離子清除力變化 56
圖十九、40-60％硫酸銨沈澱海龍鬚菜水解物在不同濃度對超氧陰離子清除力變化 57
圖二十、60-80％硫酸銨沈澱海龍鬚菜水解物在不同濃度對超氧陰離子清除力變化 58
圖二十一、80-100％硫酸銨沈澱海龍鬚菜水解物在不同濃度對超氧陰離子清除力變化 59
圖二十二、（A）不同區間硫酸銨沈澱海龍鬚菜水解物Fe2＋/H2O2誘導
DNA傷害之保護效力。（B）SC保護程度。 61
圖二十三、20~40％區間硫酸銨沈澱海龍鬚菜水解物在不同濃度下以Fe2＋/H2O2誘導DNA傷害之保護效力 62














表目錄

表一、各百分飽和濃度硫酸銨添加量 43
表二、常用SDS-PAGE 膠體溶液 (單位mL) 43
表三、海龍鬚菜水解物凍乾及以硫酸銨沈澱各區間凍乾之產率（0/00）
43

陸、參考文獻

范繼中、李雅琳、盧雅雯、吳純衡(2009).龍鬚菜之洋菜膠萃取與藻渣糖化處理。水產研究，17(1)：81-91
許博揚(2007).龍鬚菜藻類前列腺素生成之探討。國立海洋大學食品科學系博士論文。
黃淑芳(2001).臺灣海藻資源之研究。臺灣文獻，52(3): 171-211。
黃淑芳(1989).認識藻類。台灣省立博物館出版。台北市。pp. 1-54。
黃淑芳(2000).臺灣東北角海藻圖錄。國立臺灣博物館。臺北。
黃麗月(2004).龍鬚菜養殖要點-養殖漁業管理手冊技述篇。行政院農業委員會水產詴驗所。
黃任瑰(1993).龍鬚菜吸收及累積鋅之研究。台灣大學碩士論文。
鄒兆佳 (2001). Paclobutrazol 前處理對甘藷淹水耐受性及抗氧化系統之影響。中國文化大學生物科技研究所 碩士論文。
馬盈(2003).龍鬚菜多醣及其水解產物生理活性之探討。國立屏東科技大學食品科學系碩士論文。
吳全耀、吳黃素月、張恬瑞、鍾仕偉、李孟頎、繆自昌、鄭昌家(1997).龍鬚菜加工應用－綠化保鮮試驗。中國水產月刊。532：27-42。
吳全耀(1998).巧味芽（龍鬚菜）的養殖管理、綠化及一般成份。農委會漁業署出版品。144：21-26。
吳全耀(2001).巧味芽(龍鬚菜)為保健食品的利用。漁業推廣，173: 46-52。
吳全耀、吳黃素月、陳維君、江鯤芝、曾美滿、王至俊(2000).巧味芽（龍鬚菜）餅乾對預防骨質疏鬆症的研究。科技新知，37-44。
吳柏青(2003).海洋蔬菜~海龍鬚。農業世界雜誌，243: 74-75。
吳伯宏 (2004) 自由基、老化與抗氧化配方。食品工業 36(2)：45-51。
王佑民、林孟萱、吳政宗、粟隆瑋、韓建國。龍鬚粉添加對硬質餅乾影響。台灣食品科學技術學會第 39 次年會。
王佑民、林孟萱、吳政宗、粟隆瑋、韓建國。龍鬚粉添加對餐包品質影響。台灣食品科學技術學會第 39 次年會。
任雅馨(2005).細基龍鬚菜萃取產物之免疫與抗癌活性分析。國立臺灣大學海洋研究所碩士論文。
劉錦芬(2001).臺灣養殖龍鬚菜萃取物之免疫生理活性探討。國立臺灣海洋大學食品科學系碩士論文。
莊 榮 輝（2005）.酵素化學實驗。
楊海寧、江永棉(1982).臺灣產龍鬚菜（Gracilaria）之分類研究。臺灣水產學會刊。9：55-71。
漁業年報(2008).行政院農委會漁業署出版。
陳茂松、陳武雄(1970).1968 年本省外銷龍鬚菜之品質調查研究。台灣省水產詴驗所詴驗報告，16：175-180。
陳惠英、顏國欽（1998).自由基、抗氧化防禦與人體健康。中華民國
營養學會雜誌。23: 105-121。
周國慶(1973).本省龍鬚菜洋菜成份之研究。台灣水產學會刊。2：40–44。
Aruoma, O. I. (1998) Free radical, oxidative stress and antioxinants in human health and disease. Journal of the American Oil Chemists′ Society. 75: 119-213.
Bartosz, G. (2003) Generation of reactive oxygen species in biological
systems. Comments on Toxicology. 9: 5-21.
Bird, N. L., Chen, L. C. M. and McLachlan, J. (1979). Effect of temperature, light and salinity on growth in culture of Chondrus crispus, Furcellaria lumbricalis, Gracilaria tikvahiae (Gigartinales,
Rhodophyta), and Fucus serratus (Fucales, Phaeophyta). Botanica
Marina. 22: 521–527.
Bird, T. K. and Benson, P. H. (1987). Seaweed Cultivation for Renewable
Resources. Development in Aquaculture and Fisheries Science, Elsevier. 16: 200-201.
Cheng, L. H., Karim, A. A. and Norziah, C. C. (2002). Modification of
microstructral and physical properties of konjac glucomannan-based
films by alkali and sodium carboxymethylcellulose. Food Research
International. 35: 829-836.
Cao W., Chen WJ., Suo ZR and Yao YP (2008). Protective effects of ethanolic extracts of buckwheat groats on DNA damage caused by hydroxyl radicls. Food Research International. 41: 924-929.
Cacciuttolo, M. A., Trinh, L., Lumpkin, J. A. and Rao, G. (1993). Hyperoxia induces DNA damage in mammalian cells. Free Raducal Biology and Medicine. 14: 267-276.
Chedekel, M. R., Smith, S. K., Post, P. W., Pokora, P. and Vessell, D.L. (1978) photodestruction of pheomelanin: Role of oxygen. Proceedings of the National Academy of Sciences of the United States of America. 75: 5395-5399.
Cross, K. J., Huq, N. L., and Reynold, E. C.( 2007). Casein phosphopeptides in oral health chemistry and clinical applications. Current Pharmaceutical Design. 13: 793-800.
Deutscher MP ed (1990) Guide to protein purification (Methods in Enzymology, Vol. 182) Academic Press, Inc.
Erba, D., Ciappellano, S., and Testolin, G. (2002). Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats. Nutrition. 18: 743-746.
Fitzgerald, R. J., and Murray, B. A. (2006). Bioactive peptides and lactic fermentations. International Journal of Dairy Technology. 59: 118-125.
Foltz, M., Meynen, E. E., Bianco, V., Platerink, C. V., Koning, T. M. M. G., and Kloek, J. (2007). Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched milk beverage are absorbed intact into the circulation. The Journal of Nutrition. 137: 953-958.
Goldstein, S., Meyerstein, D. and Czapski, G. (1993) The Fenton reagents. Free Radical Biology and Medicine. 15: 435-445.
Gill, H. S., Doull, F., Rutherfurd, K. J., and Cross, M. L. (2000). Immunoregulatory peptides in bovine milk. British Journal of Nutrition. 84: Suppl. 1, S111-S117.
Godfrey, T. and West, S. (1996). Industrial Enzymology. Macmillan Press, London.
Horiguchi, N., Horiguche, H., and Suzuki, Y. (2005). Effect of wheat gluten hydrolysate on the immune system in healthy human subjects. Bioscience Biotechnology & Biochemistry. 69: 2445-2449.
Halliwell, B. (1994). Free radicals and antioxidants: a personal view. Lancet 52: 253-265.
Hartmann, R., and Meisel, H. (2007). Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology. 18: 163-169.
Halliwell, B. (1991) Drug antioxidant effects. A basis for drug selection？
Drugs. 42: 569-605.
Jacob, R. A. (1994) Nutrition, health and antioxidants. International News on Fats, Oils and Related Materials. 5: 1271-1275.
Khotimchenko, S. V. and E. V. Levchenko. (1997). Lipids of the red alga Gracilaria verrucosa (Huds.) Carbohydrate Research 40: 541-545.
Kaur, C., and H. C. Kapoor (2001) Antioxidants in fruits and vegetables – the millennium’s health. International Journal of Food Science and Technology. 36: 703-725.
Kim, S. K., Kim, Y. T., Byun, H. G., Nam, K. S., Joo, D. S. and Shahidi, F. (2001a). Isolation and characterization of antioxidative peptides from gelatin hydrolysate of alaska pollack skin. Journal of Agricultural and Food Chemistry. 49: 1984-1989.
Kitts, D. D., and Weiler, K. (2003). Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Design. 9: 1309-1323.
Kirkinezos, I. G. and Moraes, C. T.( 2001). Reactive oxygen species and
mitochondrial diseases. Seminar in Cell and Developmental Biology.
12: 449-457.
Lahaye, M. (1991). Marine algae as sources of fibres: determination of
soluble and insoluble dietary fiber contents in some sea vegetables.
Journal of the Science of Food and Agriculture. 54: 587-594
Lee, T. M., Chang, Y. C. and Lin, Y. H. (1999). Differences in physiological responses between winter and summer Gracilaria tenuistipitata (Gigartinales, Rhodophyta) to varying temperature. Botanical Bulletin of Academia Sinica. 49: 93-100.
Liao, I. C., Huang, T. S., Tsai, W. S., Hsueh, S. L. and Leano, E. M. (2004). Cobia culture in Taiwan: current status and problems. Aquaculture. 237: 155-165.
Li, Y. and Trush, M. A. (1993). Oxidation of hydroquinone by copper：chemical mechanism and biological effects. Archives of Biochemistry and Biophysics. 300: 346-355.
Mahmoud, M. I. (1994). Physicochemical and functional properties of protein hydrolysates in nutritional products. Food Technology. 48: 89-95.
Mendis, E., Rajapakse, N. and Kim, S. K. (2005). Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. Journal of Agricultural and Food Chemistry. 53: 581-587
Nagaoka, S., Miwa, K., Eto, M., Kuzuya, Y., Hori, G., and Yamamoto, K. (1999). Soy protein peptic hydrolysate with bound phospholipids decreases micellar solubility and cholesterol absorption in rats and Caco-2 cells. Journal of Nutrition. 129: 1725-1730.
Nagaoka, S., Futamura, Y., Miwa, K., Awano, T., Yamauchi, K., Kanamaru, Y., Tadashi, K., and Kuwata, T. (2001). Identification of novel hypocholesterolemic peptides derived from bovine milk β-lactoglobulin. Biochemical and Biophysical Research Communications. 281: 11-17.
Ondetti, M. A., and Cushman, D. W. (1982). Enzymes of the renin-angiotensin system and their inhibitors. Annual Review of Biochemistry. 51: 283-308.
Recio, I., and Visser, S. (1999). Identification of two distinct antibacterial domains within the sequence of bovine αs2-casein. Biochimica et Biophysica Acta. 1428: 314-326.
Robak, J. and Gryglewski, I. R. (1988) Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology. 37: 837 - 841.
Suja, K. P., Jayalekshmy, A., Arumughan, C. (2004). Free radical scavenging behavior of antioxidant compounds of sesame (Sesamum indicum L.) in DPPH system. Journal Agricultural and Food Chemistry. 52 : 912–915.
Shiomi, K., Yamanaka, H. and Kikuchi, T. (1981). Purification and
physiochemical properties of a hemagglutinin (GAV-1) in the red
alga Gracilaria verrucosa. Bulletin of the Japanese Society of
Scientific Fisheries. 47: 1079-1084.
Silva, S. V., and Malcata, F. X.( 2005). Casein as source of bioactive peptides. International Dairy Journal. 15: 1-15.
Sipola, M., Finckenberg, P., Santisteban, J., Korpela, R., Vapaatalo, H. and Nurminen, M. L. (2001). Long-term intake of milk peptides attenuates development of hypertension in spontaneously hypertensive rats. Journal of Physiology and Pharmacology. 52: 745-754.
Simic, M. G. (1988) Mechanisms of inhibition of free-radical processes in mutagenesis and carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms. of Mutagensis. 202: 377-386.
Temple, A. J. (2000) Antioxidants and disease: more questions than answers. Nutrition Research. 20: 449-459.
Wickens, A. P. (2001) Ageing and the free radical theory. Respiration Physiology. 128: 379-391.
Yamaguchi, T., takamura, H., Matoba, T. and Terao, J. (1998) HPLC
method for evaluation of the free radical-scavenging activity by
foods by using 1, 1-diphenyl-2-picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry. 62: 1201-1204.
Zhong, F., Liu, J., Ma, J. and Shoemaker, C. F. (2007a). Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice. Food Research International. 40: 661-667.
Zhong. F., Zhong, X., Ma, J. and Shoemaker, C. F.( 2007b). Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates. Food Research International. 40: 756-762.