本研究探討台灣鯛(Taiman Tilapia)魚鱗之膠原蛋白萃取條件以及膠原蛋白物化特性的鑑定。結果顯示，將魚鱗片粉碎後的粉末，在鹽酸萃取溶液(pH 2)中，加入0.5 mg/ml胃蛋白酶可大大地提高膠原蛋白的產率。約10克的魚鱗粉末可萃取出0.08克膠原蛋白，萃取率約為0.8％。物化特性的鑑定分析則以胺基酸分析(Amino acid analysis)、圓二色光譜(Circular Dichroism)、SDS電泳、示差熱量掃描分析(Differential Scanning Calorimetry)等方法分析測定胺基酸組成、二級結構變化、變性溫度及分子量。胺基酸分析結果顯示台灣鯛魚鱗膠原蛋白中，Hydroxyproline佔魚鱗膠原蛋白11.23 %。在SDS-PAGE電泳圖顯示台灣鯛魚鱗主要由α1、α2之單體、β(雙體) 及γ(三聚體)組成，屬於第一型膠原蛋白，且50 kDa以下之多肽沒有出現，代表Telopeptide已在萃取過程去除。為了提升熱變性溫度，用dehydrothermal treatment (DHT) 共價交聯方法於125℃及150℃來穩定蛋白質的結構，利用掃描式電子顯微鏡(SEM)觀察其表面結構以及ninhydrin (NHN) assay評估不同溫度處理後對膠原蛋白結構交聯程度的影響。由SEM中觀察膠原蛋白的crosslinking隨著DHT的溫度升高，在150℃時，纖維更加交錯複雜，使得表面結構的孔洞減少，可達到高強度、高穩定之特性。在NHN assay評估上，125℃交聯處理後的魚鱗膠原蛋白交聯指數為31.34% ；150℃交聯指數為55.97%。在未來的應用上，可以增加生物組織材料的機械強度，以及抵抗人體內的免疫系統及酵素的攻擊。

The aim of the study was to investigate the extraction conditions of collagen extracted from Taiwan tilapia scale and the analysis of its physical-chemical properties. Experiment result shows that add few pepsin in HCl extraction solution (pH 2) could highly increase the production rate of collagen. The identify of physical-chemical properties includes using amino acid analysis, circular dichroism (CD), sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC) to determine the amino acid composition, secondary structure changing with denaturation, denature temperature (Td) and molecular weight. Amino acid analysis indicates that Taiwan tilapia scale collagen contains 11.23% hydroxyproline. SDS-PAGE electrophoresis result shows this collagen was mainly composed of α1, α2 monomer, β dimer and γ trimer, which is related to type I collagen, and no polypeptides bands below 50 kDa represent that Telopeptide was removed during extraction process.
The dehydrothermal treatment (DHT) stable the collagen structure, which can increase the denature temperature (Td). The scanning electron microscope (SEM) is to observe and compare the surface structure changing, and ninhydrin (NHN) assay results showed that DHT at125 ℃ the cross-linking index was 31.34%; it was 55.97% for DHT at 150 ℃. The cross-linking of collagen has the characteristics of high intensity and high stability. In application, it can promote mechanical strength of biomaterials and can resist immune system and enzyme attack in human body.

1.Tanaka, M. C., and Shimokomaki, M. (1996) Collagen types in mechanically deboned chicken meat. J. Food Biochem., 20 ,215 - 225.
2.許富銀，鄭明鎮，王盈錦。1998。膠原蛋白在醫學上的應用。生物產業9：21-26。
3.Yunoki, S., Nagai, N., and Suzuki,T. (2004) Novel biomaterial from reinforced salmon collagen gel prepared by fibril formation and cross-linking. J. Biosci. Bioeng., 98 , 40-47.
4.Nagai, N., Yunoki, S., Suzuki,T., Sakata, M., Tajima, K., and Munekata, M. (2004) Application of cross-linked salmon atelocollagen to the scaffold of human periodontal ligament cells. J. Biosci. Bioeng. , 97, 389 - 394.
5.陳世輝。2004。膠原蛋白讓你水噹噹。科學發展380,12 - 17。
6.Swatschek, D., Schatton, W., Muller, W., and Kreuter, J. (2002) Microparticles derived from marine s pong collagen (SCMPs) : preparation, characterization and suitability for dermal delivery of all-trans retinol. Eur. J. Pharm. Biopharm., 54, 125 – 133.
7.Yunoki, S., Suzuki, T., and Takai, M. (2003) Stabilization of denaturation temperature collagen from fish by physical cross-linking methods. J. Biosci. Bioeng., 96, 575- 577.
8.Nimni, M. E. (1988) Collagen volume 1 : Biochemistry. CRC press, Inc. Florida.
9.Harrington, W. F., and Von Hippel, P. H. (1961) The structure of collagen and gelatin. Advan. Protein Chem., 16 , 1 – 138.
10.Piez, K. A., Lewis, M. S., Martin, G. R., and Gross, J. (1961) Subunits of the collagen molecule. Biophys. Acta., 53, 596 - 598.
11.郭秋分。2001。以類風濕性關節炎動物模式研究水母膠原蛋白之經口免疫寬容。碩士論文。海洋大學。基隆。台灣。
12.Myllyharju, J., and Kivirikko, K. I. ( 2004) Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet., 20 , 33 - 43.
13.端木粱，李建雄，翁郁嘉，黃淑姿。1991。生物化學。藝軒出版公司。台北市。
14.Junqueira and Carneiro, Basic Histology, Text and Atlas,10th
15.劉曉芸,組織中膠原蛋白含量之分析,國立成功大學生物科技研究所,2003
16.洪雅萍。2004。膠原蛋白產品的功效。科學發展380,30 - 35。
17.林詠凱。2001。雞腳膠原蛋白與明膠之萃取及特性之研究。碩士論文。
18.黃玲惠,化工技術第四卷第七期,136-141,1996.
19.洪偉章與陳榮秀,化妝品科技概論,高立圖書,1997.
20.王棣,利用豬皮膠原蛋白酵素水解物試製機能性配料之研究,東海大學畜牧研究所,1994.
21.洪偉章、李金枝與陳榮秀,化裝品原料及功能,藝軒圖書出版社,1998.
22.賴志行,魚鱗膠原蛋白之萃取及其酵素水解物抗氧化性與角質細胞增生效果之探討，國立台灣海洋大學食品研究所,2006.
23.石凱元,酵素水解牛第一型膠原蛋白之研究,國立成功大學化學研究所,2003.
24.Choi, C., Son, G. M., Cho, Y. J., Chun, S. S., Lim, S. I., and Seok, Y. R.(1992) Purification and characteristics of bromelain from Korean pineapple. J. Korean Agric. Chem. Sot., 35, 23-29.
25. Kang, C. K., and Rice, E. E.(1970) Degradation of various meat fractions by tenderizing enzymes. J. Food Sci, 35： 563-567.
26. Rattrie, N. W., and Regenstein J. M. (1977) Action of crude papain on actin and myosin heany chains isolated from chicken breast muscle. J. Food Sci.,42, 1159-1163.
27.Brooks, B. A., Klasing, K. C., and Regenstein, J.M. (1985) Effect of antemortem injected crude papain in chicken breast muscle. J. Food Sci., 50, 1370-1374.
28. Sachedev, G.P., and Fruton, J. S. (1970) Secondary enzyme substrate interactions and specificity of pepsin. Biochemistry., 9,4465-4470.
29. 鄭靜桂,蛋白質之水解與水解液之利用,食品工業月刊, 29,10-17,1997.
30. Prince, J. F., and Schweight, B. S.(1971) The science of meat and meat products, 2nd edition ,112-120,W. H. Freeman and Company, San Francisco.
31.周繼發，豬皮膠原蛋白及化學與酵素修飾物機能性之研究,國立台灣大學畜牧學研究所,1987.
32.Miyata, T., Taira, T. and Noishiki, Y.(1992) Collagen engineering for biomaterial use. Clin. Mater.,9(3-4), 139-148.
33.Choi, S.S., and Regenstein, J.M. (2000) Physiochemical and sensory characteristics of fish gelatin; Journal of Food Science., 65, 194-99.
34.Piez, A., Gross, J.M. (1960) The amino acid composition of some fish collagens: the relation between composition and structure; Journal of Biological Chemistry., 235,995-998.
35.Michona, C, Vigourouxb, F., Boulenguerb, P. and Cuveliera, G. (2000) Gelatin/iotacarrageenan interactions in non-gelling conditions; Food Hydrocolloids., 14, 203-208.
36.Miller, E. J., and R. K. Rhode. 1982. Preparation and characterization of different types collagen. Method Enzymol. 82:33-65.
37.Sikorski, Z. E., Scott, D. N., and Buisson, D. H.(1984) The role of collagen in the quality and processing of fish. Critical reviews in food science and nutrition. 20(4),301-343.
38.Nagai, T. and Suzuki, N. (2000) Isolation of collagen from fish waste material-skin bone and fins.Food Chemistry.68, 277-281.
39.Ogawa, M., Moody, M. W., Poriter, R. T., Bell, J., Schexnayder, M. A., and Losso, J. K. (2003) Biochemical properties of black drum and sheepshead seabream skin collagen；Journal of Agricultureal and Food Chemistry., 51, 8088-8092
40.Mizuta, S., Sakamoto, R., Nishimoto, M., and Yoshinaka, R.(2005)Identification and characterization of molecular species of collagen in ordinary muscle and skin of Japanese flounder Paralichthys olivaceus；Food Chemistry., 90, 151-156.
41.Kimura, S., Miyauchi, Y., and Uchida, N. (1991)Scale and bone type Ⅰ collagens of carp(Cyprinus carpio)；Comparative Biochemistry and Physiology., 99B, 473-476.
42.Nomura, Y., Sakai, H., Ishii, Y., and Shirai, K.(1996) Preparation and some properties of typeⅠ collagen from fish scales；Bioscience Biotechnology and Biochemistry., 60, 2092-2094, 1996.
43.Ogawa, M ., Moody, M. W. , Bell, J., and Portier, R. J.(2004) Biochemical properties of bone and scale isolated from the subtropical fish black drum (pogonia cromis) and sheepshead seabream(Archosargus probatocephalus)；Food Chemistry., 88, 495-501.
44.吳清熊,水產利用化學,國立編譯館,1997.
45.Nagai, T., Izumi M.,ad M. Ishii(2004)Fish scale collagen preparation and partial characterization；International Journal of Food Science and Technology., 39, 293-344.
46.Hamada, M.,and H. Kumagai(1988)Chemical composition of sardine scale；Nippon Suisan Gakkaishi., 54(11), 1987-1992.
47.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.
48. Hill, F. (1966) The solubility of intramuscular collagen in meatanimals of various ages；J. Food Sci., 31(2), 161-165.
49.吳安邦、鄭慧文、陳淑茹、林伶紅、呂炫堃、林慶文、陳朝洋。1999。
50.Pachence, J. M. 1996. Collagen-based devices for soft tissue repair. J. Biomed. Mater. Res. 33: 35-40.
51.Lin, Y. K., and D. C. Liu. 2005b. Comparison of physico-chemical properties of type I collagen from different species. Food Chemistry. Submitted
52.Pati, F., Adhikari, B., and Dhara, S.(2010) Isolation and characterization of fish scale collagen of higher thermal stability. Bioresource Technology, 101,3737-3742
53.Yunoki, S., Nagai, N., and Suzuki,T. (2004) Novel biomaterial from reinforced salmon collagen gel prepared by fibril formation and cross-linking. J. Biosci. Bioeng., 98 , 40-47.
54. Yunoki, S., Suzuki, T., and Takai, M. (2003) Stabilization of denaturation temperature collagen from fish by physical cross-linking methods. J. Biosci. Bioeng., 96 ,575- 577.
55.藍正文。2002。膠原蛋白做為骨母細胞分離與生長基材之研究。博士論文。陽明大學。台北。台灣。
56.Edward, C.A., and O’Brien, W.D., (1980) Modified assay for determination of hydroxproline in a tissue hydrolyzate. Clin. Chim. Acta., 104 : 161-167.
57. Kesava G., R. et al.(1996) A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues. Clinical Biochemistry, 29,225-229.
58. Anderson ,S.M.L.et al.(1991) Sircil Assay:A method for quantification of collagen. Biochem. Soc. Trans., 19,48-54.
59.Reddy, G. K. , and Enwemeka, C. S. (1996) A simplified method for the analysis of hydroxyproline in biological tissue. Clin. Biochem. , 29 : 225 - 229.
60.唐紹祖, DNA Aptamer 掌性異構物辨識之熱力學與機制探討,國立中 央大學生物資訊與系統生物研究所,2008
61.Sreerama, N. and Woody, R. W. (2004) Computation and analysis of protein circular dichroism spectra. Methods in Enzymology , 383, 318-351.
62.Fasman, G. D. (1996) Circular Dichroism and the conformation analysis of biomolecules. Plenum Press.
63.賴志行,魚鱗膠原蛋白之萃取及其酵素水解物抗氧化性與角質細胞增生效果探討,國立台灣海洋大學食品研究所,2006.
64.Nalinanon S., Soottawat Benjakul, Wonnop Visessanguan, Hideki Kishimura (2007) Use of pepsin for collagen extraction from the skin of bigeye snapper. Food Chemistry104, 593-601.
65.Skierka El╴zbieta, Maria Sadowska.(2007)The influence ofdifferent acids and pepsine on the extractability of collagen from the skin of Baltic cod(Gadus morhua). Food Chemistry105,1302-1306.
66.Stenzel Kurt H., Teruo Miyata, Albert L. Rubin.(1974) Collagena as a biomateriai. Annu. Rev. Biophys. Bioeng.3:231-253.