臺灣博碩士論文加值系統

中文摘要
本研究探討吳郭魚(Oreochromis niloticus x O. aureus)、烏魚(Mugil cephalus) 及虱目魚 (Chanos chanos)等國內大宗養殖魚類魚鱗之一般成分、膠原蛋白萃取與去灰分條件、膠原蛋白類型與特性、胺基酸組成及其酵素水解物之抗氧化性與細胞增生效果。吳郭魚、烏魚、養殖及野生虱目魚魚鱗之蛋白質含量分別為48、54、61及56%，灰分則為45、41、36及38%，養殖與野生虱目魚魚鱗以0.2 M HCl處理30分鐘，吳郭魚及烏魚以0.3 M HCl處理40分鐘去灰分，粗膠原蛋白收率皆在45%以上。四種魚鱗膠原蛋白均含有相異的α-chain，α1及α2，為TypeⅠ類型，養殖與野生魚間無差異，而其SDS-PAGE圖譜與牛皮Type I膠原蛋白類似，胺基酸組成以Gly、Pro、Hyp及Ala等含量較高。吳郭魚魚鱗粗膠原蛋白以2% Alcalase、3% Protease Type ⅩⅣ及1% Collagenase進行一次及二次水解0、1、2、3、4及5小時後，評估水解物清除DPPH (α,α-diphenyl-β-picrylhydrazyl)自由基、還原力及螯合銅離子能力，結果顯示以2% Alcalase 在50℃下水解4小時之抗氧化性較強。水解1小時後游離及複合胺基酸含量分別增加3.5與2倍，似與抗氧化性增強有關。經超過濾依分子量將水解液劃分，其中5~10 kDa及< 5 kDa之清除DPPH能力較強，還原力則隨分子量增加而有增加的趨勢，分子量5~10 kDa 及< 5 kDa之螯合銅離子能力高於> 10 kDa之水解物，水解物小分子胜肽類具較強抗氧化性。吳郭魚魚鱗純膠原蛋白、>10 kDa、5~10 kDa及< 5 kDa之酵素水解物對人類角質細胞株細胞存活率分別為113%、149%、136%及133%，均高於控制組，顯示水解物在適當濃度下具有促進細胞生長能力。

Abstract
　　The objectives of this study are to investigate the proximate composition, amino acid constituents, decalcifying condition for extraction of collagen, collagen type and characteristics of fish scales from tilapia (Oreochromis niloticus x O. aureus), striped mullet (Mugil cephalus), and milkfish (Chanos chanos). The antioxidant activities and cell viability of collagen hydrolysates were also studied. The crude protein contents of tilapia, striped mullet, cultured and wild milkfish scales were 48, 54, 61 and 56%, and ash contents were 45, 41, 36 and 38%, respectively. The decalcification condition for milkfish scales (cultured and wild) was 0.2 M HCl treatment for 30 min, and tilapia and striped mullet were 0.3 M HCl for 40 min. The yield of collagen extracted from all fish scales was higher than 45%. Four kinds of fish scale collagen were heterotrimers with α-chain, α1 and α2, indicating that these scale collagen were Type I collagen. No difference was observed between cultured and wild fish. The profiles of SDS-PAGE were also similar to that of calf skin collagen Type I. The dominant amino acids of collagen were Gly, Pro, Hyp and Ala. The crude fish scale collagen of tilapia was treated with 2% Alcalase (w/w), 3% Protease type ⅩⅣ and 1% Collagenase for 0, 1, 2, 3, 4 and 5 hour. The antioxidant activites of the hydrolysates were measured using the methods including the scavening effect on α,α-diphenyl- β-picrylhydrazyl (DPPH) radical, reducing power and chelating ahilities of metal ion Cu2+. Results showed the highest antioxidant activity was found in 2% Alcalase hydrolysates at 50℃ for 4 hour. Free and combined amino acids increased by 3.5 and 2 times after 1 hour hydrolysis. The increased free and combined amino acids might be associated with the increase of antioxidant activity. The Alcalase hydrolysates were then separated by ultrafiltration, and the fraction with MW 5~10 kDa and <5 kDa were observed to have the highest scavening effect on DPPH radical. Reducing power increased with increasing MW of the UF fraction. The chelating of Cu2+ for MW 5~10 kDa and <5 kDa were higher than that of >10 kDa. Results indicated the peptides of hydrolysates possessed antioxidant activities. The cell viability of human keratinocye treated with tilapia scale collagen and it hydrolysate with MW >10 kDa, 5~10 kDa and <5 kDa were 113%, 149%, 136% and 133%, respectively. Results revealed that tilapia scale collagen and its hydrolysate with proper concentration could enhance the growth of human keratinocyte.

目錄 頁 次
中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅱ
壹、研究背景與目的 1
貳、文獻整理 3
一、魚鱗化學組成與應用 3
(一)化學組成 3
1.一般成分 3
2.胺基酸組成 3
(二)應用 4
1.生醫材料 4
(1).膠原蛋白 4
(2).氫氧基磷灰石 4
2.食品 4
3.化妝品 5
二、魚鱗、魚皮、魚鰭及魚骨之膠原蛋白 5
(一)含量與分佈 5
(二)型態與種類 6
(三)變性溫度之比較 8
三、脂質氧化作用 8
(一)自氧化作用 (autooxidation) 8
(二)熱氧化作用 (thermal oxidation) 9
(三)光氧化作用 (photosensitized oxidation) 9
(四)酵素催化氧化作用 (enzymatic oxidation) 10
四、抗氧化劑作用原理 10
(一)自由基終止劑 (free radical terminator) 10
(二)還原劑或氧清除劑 (reducing agent or oxygen scavengers) 11
(三)金屬螯合劑 (chelating agent) 11
(四)單重氧的抑制劑 (singlet oxygen inhibitor) 11
五、蛋白質水解物之抗氧化特性 12
參、實驗材料與方法 14
一、實驗材料 14
二、實驗項目與方法 16
(一)魚鱗去灰化條件之探討 16
(二)魚鱗膠原蛋白之萃取 16
(三)魚鱗膠原蛋白型態鑑定 16
(四)酵素水解液的製備 17
(五)水解液抽出物之調製 17
(六)分析方法 17
1.一般成分 17
2.胺基酸組成 18
3.游離胺基酸之分析 18
4.複合胺基酸(combined amino acid ) 18
5.抗氧化測定 18
(1)清除��,��-diphenyl-��-picrylhydrazyl (DPPH)自由基能力之測定 18
(2)還原力測定 19
(3)螯合銅離子能力 19
6.細胞培養條件...................................................................19
7.細胞存活率 (MTT assay)之測定 20
(七)統計分析 20
肆、結果與討論 21
一、魚鱗一般成分 21
二、鹽酸處理對魚鱗粗膠原蛋白收率之影響 21
三、魚鱗粗膠原蛋白及純膠原蛋白之SDS-PAGE圖譜 22
四、魚鱗膠原蛋白之胺基酸組成 23
五、吳郭魚魚鱗粗膠原蛋白酵素水解液之抗氧化特性 24
(一)不同酵素水解液抗氧化性之比較 24
(二)二次酵素水解液抗氧化性之比較 25
(三)游離胺基酸與複合胺基酸在水解期間之變化與抗氧化之關係 26
六、吳郭魚魚鱗粗膠原蛋白酵素水解物分子量劃分及其抗氧化 性 27
(一)清除DPPH自由基之能力 27
(二)還原力 28
(三)螯合銅離子能力 28
七、吳郭魚魚鱗膠原蛋白與酵素水解物對角質細胞株存活率之 影響 29
伍、結論 30
陸、參考文獻 31




表目錄

表一、魚鱗一般成分 38
表二、吳郭魚、烏魚、養殖虱目魚及野生虱目魚魚鱗膠原蛋白之 胺基酸組成 39
表三、吳郭魚魚鱗粗膠原蛋白之 2% Alcalase與3% Protease Type ⅩⅣ水解間對清除DPPH自由基、還原力及螯合銅離子能 力之比較 40
表四、吳郭魚魚鱗粗膠原蛋白經2% Alcalase與3% Protease Type ⅩⅣ二次水解對清除DPPH自由基、還原力及螯合銅離子 能力之比較 41
表五、吳郭魚魚鱗粗膠原蛋白經2% Alcalase與3% Protease Type ⅩⅣ及1% Collagenase二次水解對清除DPPH自由基、還 原力及螯合銅離子能力之比較 42
表六、吳郭魚魚鱗粗膠原蛋白之2%Alcalase水解物游離胺基酸在 水解期間之變化 43
表七、吳郭魚魚鱗粗膠原蛋白之2 %Alcalase水解物複合胺基酸在 水解期間之變化 44








圖目錄

圖一、鹽酸處理1小時對魚鱗粗膠原蛋白收率之影響 45
圖二、鹽酸處理1小時對魚鱗灰分含量之影響 46
圖三、塩酸處理時間對魚鱗粗膠原蛋白收率之影響 47
圖四、塩酸處理時間對魚鱗灰分含量之影響 48
圖五、吳郭魚與烏魚魚鱗粗膠原蛋白及純膠原蛋白之SDS-PAGE 圖譜 49
圖六、養殖與野生虱目魚魚鱗粗膠原蛋白及純膠原蛋白之 SDS-PAGE圖譜 50
圖七、魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下水解4小 時）不同分子量劃分物對清除DPPH自由基之能力比較 51
圖八、魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下水解4小 時）不同分子量劃分物對還原力之能力比較 52
圖九、魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下水解4小 時）不同分子量劃分物對螯合銅離子之能力比較 53
圖十、吳郭魚魚鱗膠原蛋白對人類角質細胞存活率之影響 54
圖十一、吳郭魚魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下 水解4小時）分子量 >10 kDa劃分物對人類角質細胞存活 率之影響 55
圖十二、吳郭魚魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下 水解4小時）分子量5~10 kDa劃分物對人類角質細胞存活 率之影響 56
圖十三、吳郭魚魚鱗粗膠原蛋白水解物（經2% alcalase於50℃下 水解4小時）分子量 < 5 kDa劃分物對人類角質細胞存活 率之影響 57
圖十四、人類角質細胞以吳郭魚魚鱗純膠原蛋白(10 μg/mL) 培養 後 之貼附圖 58
圖十五、人類角質細胞以吳郭魚魚鱗粗膠原蛋白水解物( >10 kDa, 2.5mg/mL) 培養後之貼附圖 59
圖十六、人類角質細胞以吳郭魚魚鱗粗膠原蛋白水解物(5~10 kDa, 2.5mg/mL)培養後之貼附圖 60
圖十七、人類角質細胞以吳郭魚魚鱗粗膠原蛋白水解物( < 5 kDa, 0.16mg/mL)培養後之貼附圖 61
圖十八、魚鱗去灰分前後之顯微圖片..................................................62

















附圖表目錄

附表一、各種型態之膠原蛋白在醫學上之應用 623
附表二、魚類膠原蛋白種類與分佈 64
附表三、電泳片及相關試劑之配製 65

陸、參考文獻

王隸，1994。利用豬皮膠原蛋白酵素水解物試製機能性配料之研究，東海大學碩士論文，台中。
江晃榮，2002。膠原蛋白的驚人療效，世茂出版社，台北。
吳清熊，1997。水產利用化學，國立編譯館，台北。
呂博文，2004。低免疫性的生醫材料膠原蛋白，科學發展，380：6-11。
周照仁，2005。水產膠原蛋白之研究與利用，水產生物科技新知研討會，國立高雄海洋科技大學出版，高雄。
周照仁、朱玉灼、蕭靖寰，2003。海驪魚皮天然膠原蛋白的精製，台灣水產學會九十二年度學術論文發表會，高雄。
洪雅萍，2004。膠原蛋白產品的功效，科學發展，380：30-35。
陳世輝，2004。膠原蛋白讓你水噹噹，科學發展，380：12-17。
湯家潤，2004。組織工程中的膠原蛋白，科學發展，380：18-23。
黃婉君、朱玉灼、周照仁，2003。鹼前處理對吳郭魚皮製取明膠的影響，台灣水產學會九十二年度學術論文發表會，高雄。
黃富彥、湯正明、徐善慧，2003。揭開膠原蛋白的神秘面紗，科學發展，362：44-47。
黃穎斐，2004。生醫敷料及人工皮膚，科學發展，380：24-29。
楊炯琳、陳瑞祥，1999。膠原蛋白生物材料及應用，化工資訊，2:33-38。
葉明熙、 張原嘉，2002。淺談生醫材料--膠原蛋白，化工科技與商情，39：13-17。
漁業署，2004。中華民國台灣地區漁業年報，台北。
坂口守彥、平田孝，2005。水產資源先進的有效利用法，NTS，日本。
AOAC. 1995. Official Methods of Analysis, 14th ed. Association of Official Analytical Chemists, Arlington, VA.
Brand –Williams, W., Cureliver, M. E. and Berest, C. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technology. 28:25-30.
Chen, H. M., Muramoto, K. and Yamauchi, F. 1995. Structural analysis of antioxidative peptides from soybean β-conglycinin. Journal of Agricultural and Food Chemistry. 43(3): 574-578.
Dosio, F., Arpicco, S., Canevari, S., Figini, M. and Gastaldi, D. 1999. Single-step　purification of immunotoxins containing a high ionic charge ribosome inactivatingprotein clavin by carboxymethyl high-performance membrane chromatography. Journal of Chromatography A. 830:329-335.
Dziezak, J. D. 1986. Preservatives: antioxidant. Food Technology. 40:94-102.
Fahmi, A., Morimura, S., Guo, H. C., Shigematsu, T., Kida, K. and Uemura, Y. 2004. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry. 39:1195-1200.
Foote, C. S. 1976. Photosensitised oxidation and singlet oxygen : consequences in biological system. In “Free Radical in Biology,” pp.85-133. Vol. II. Eds. Pryor, W. A., Academic press., New York.
Frankel, E. N. 1980. Lipid oxidation review. Progress in Lipid Research 19(1/2):1-22.
Gonzalez-Stawinski, G. V., Parker, W., Holzknecht, Z. E., Huber, N. S., Platt, J. L. 1999. Partial sequence of human platelet heparitinase and evidence of its ability topolymerize. Biochimica et Biophysica Acta. 1429:431-438.
Hamada, M. and Kumagai, H. 1988. Chemical composition of sardine scale. Nippon Suisan Gakkaishi. 54(11):1987-1992.
Ikoma, T., Kobayashi, H., Tanaka, J., Walsh, D. and Mann, S. 2003. Physical properties of type Ⅰ collagen extracted from fish scales of Pagrus major and Oreochromis niloticas. International Journal of Biological Macromolecules. 32: 199-204.
Itoh, S., Kikuchi, M., Koyama, Y., Takakuda, K., Shinomiy, K. and Tantaka, J. 2002. Development of an artificial vertebral body using a novel biomaterals hydroxyapatite / collagen composite. Biomaterials. 23:3919-3926.
Jeon, Y. J., Byun, H. G. and Kim, S. K. 1999. Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. 35:471-478.
Kanbe, C., Klar, F., Fitzner, R., Radlanski, R. J. and Gross, U. 2002. In vitro investigation　of titanium and hydroxyapatite dental implant surface using a rat bone marrowstromal cell culture system. Biomaterials. 23:3235-3245.
Kanner, J., German, J. B. and Kinsella, J. E. 1987. Initiation of lipid peroxidation in biological systems. CRC Critical Reviews in Food Science and Nutrition. 25(4):317-363.
Kim, S. K., Kim, Y. T., Byun, H. G., Nam, K. S., Joo, D. S. and Shahidi F. 2001. Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska Pollack skin. Journal of Agricultural and Food Chemistry. 49:1984-1989.
Kimura, S., Miyauchi, Y. and Uchida, N. 1991. Scale and bone type Ⅰcollagens of carp (Cyprinus carpio). Comparative Biochemistry and Physiology. 99B: 473-476.
Konosu, S., Watanabe, K. and Shimizu, T. 1974. Distribution of nitrogenous constituents in the muscle extracts of eight species of fish. Bulletin of the Japanese Society of Scientific Fisheries. 40(9):909-915.
Konosu, S., Yamaguchi, K. and Hayashi, T. 1978. Studies on flavor components in boiled crabs-I. Amino acids and related compounds in the extracts. Bulletin of the Japanese Society of Scientific Fisheries. 41(5):505-510.
Li G. Y., Fukunaga, S., Takenouchi, K. and Nakamura, F. 2005. Comparative study of the physiological properties of collagen gelatin and collagen hydrolysate as cosmetic materials. International Journal of Cosmetic Science. 27:101-106.
Mansur, M. A., Bhadra, A., Takamura, H. and Matoba, T. 2002. Effect of processing and storage on the radical scavenging activity of horse mackerel and sardine. Fisheries Science. 68: 1390-1392.
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.
Min, D. B., Lee, S. H. and Lee, E. C. 1988. Singlet oxygen oxidation of vegetable oils. In “Flavor Chemistry of Lipid Foods,” pp. 57-97. Eds. Min, D. B. and Smouse, T. H. American Oil Chemists’ Society, Champaign, U.S.A.
Mistry, B. S. and Min, D. B. 1992. Oxidized flavor compounds in edible oil. In “Off-Flavors in Foods and Beverages,” pp. 171-209. Ed. Charalambous, G., Elsevier, Amsterdam, Netherlands.
Mizuta, S., Sakamoto, R., Nishimoto, M. and Yoshinaka, R. 2005. Identification and characterization of molecular species of collagen in ordinary muscle and skin of the Japanese flounder Paralichthys olivaceus. Food Chemistry. 90:151-156.
Morimura, S., Nagata, H., Fahmi, A., Shigematsu, T., Kida, K. and Uemura, Y. 2002. Development of an effective process for utilization of collagen from livestock and fish waste. Process Biochemistry. 37: 1403-1412.
Nagai, T. and Suzuki, N. 2000. Isolation of collagen from fish waste material-skin bone and fins. Food Chemistry. 68: 277-281.
Nagai, T., Yamashita, E., Taniguchi, K., Kanamori, N. and Suzuki, N. 2001. Isolation and characterization of collagen from the outer skin waste material of cuttlefish (Sepia lycidas). Food Chemistry, 72:425-429.
Nagai, T., Izumi, M. and Ishii, M. 2004. Fish scale collagen. Preparation and partial　characterization. International Journal of Food Science and Technology. 39: 293-244.
Namiki, M. 1990. Antioxidants/antimutagens in food. CRC Critical Reviews in Food Science and Nutrition. 29:273-300.
Nawar, W. W. 1985. Lipids. In “Food Chemistry” Ed. By Fennema, O. R. pp. 139-244. Marcel Dekker Inc., New York.
Nomura, Y., Sakai, H., Ishii, Y. and Shirai, K. 1996. Preparation and some properties of type Ⅰ collagen from fish scales. Bioscience Biotechnology and Biochemistry. 60:2092-2094.
Ogawa, M., Moody, M. W., Portier, R. J., Bell, J., Schexnayder, M. A. and Losso, J. K. 2003. Biochemical properties of black drum and sheepshead seabream skin collagen. Journal of Agricultural and Food Chemistry. 51:8088-8092.
Ogawa, M., Moody, M. W., Bell, J. and Portier, R. J. 2004. Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus). Food Chemistry. 88: 495-501.
Okamoto, G., Hayase, F. and Kato, H. 1992. Scavenging of active oxygen species by glycated proteins. Bioscience Biotechnology and Biochemistry. 56:928-931.
Oyaizu, M. 1988. Antioxidative activities of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography. Nippon Shokuhin Kogyo Gakkaishi. 35(11):771-775.
Queiroz, A. C., Santos, J. D., Monteiro, F. J. and Gibson I. R. 2001. Adsorption and release studies of sodium ampicillin from hydroxyapatite and glass-reinforced　hydroxyapatite composites. Biomaterials. 22:1393-1400.
Senaratne, L. S., Park, P. J. and Kim, S. K. 2006. Isolation and characterization of collagen from brown backed toadfish (Lagocephalus gloveri) skin. Bioresource Technology, 97:191-197.
Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry. 40:945-948.
Suetsuna, K. 1999. Separation and identification of antioxidant peptides from proteolytic digest of dried bonito. Nippon Suisan Gakkaishi. 65(1):92-96.
Suetsuna, K. 2000. Antioxidant peptides from the protease digest of prawn (Penaeus japonicus ) muscle. Marine Biotechnology. 2:5-10.
Suetsuna, K., Maekawa, K., Chen, J. R., Harada, K., Hamada, M. and Shin, S. U. 2004. Separation and Identification of antioxidative peptides from peptic digest of fish scale collagen. Journal of National Fisheries University. 52(2): 57-62.
Takenaka, S., Okada, A., Iwai, K. and Ayaki, Y. 2003. Separation of collagen from fish scales by treatment of dilute hydrochloric acid aqueous solution. Nippon Shokuhin Kagaku Kogaku Kaishi. 50(2):67-71.
Takenaka, A., Kawakami, A., Adachi, M., Morita, N. and Ayaki, Y. 2004. Separation of collagen by decalcification of red sea-bream scales. Nippon Shokuhin Kagaku Kogaku Kaishi. 51(11): 572-576.
Tiselius, A., Hjerten, S. and Levin, O. 1965. Protein chromatography on calcium　phosphate columns. Archives of Biochemistry and Biophysics. 65:132-155.
Wassel, D. T. H., Hall, R. C. and Embery, G. 1995. Adsorption of bovine serum　albumin onto hydroxyapatite. Biomaterials. 16: 697-702.