本研究探討台灣鯛(Taiwan Tilapia)魚鱗之膠原蛋白萃取方式及膠原蛋白特性分析，最後是膠原蛋白與核糖交聯作用，得到熱穩定性較高的膠原蛋白。研究結果顯示，在萃取魚鱗膠原蛋白方面，反應時間與pepsin濃度都會提高膠原蛋白的溶解量，最佳萃取條件為pH 2反應時間二十四小時以上以及pepsin濃度(1 mg/ml)，粗萃取率0.864%。在鑑定膠原蛋白特性分析方面，分別利用Hydroxyproline定量法、紫外可見光譜儀、傅立葉轉換紅外線光譜分析儀、黏度計、熱示差掃描分析儀、圓二色光譜儀、掃描式電子顯微鏡，實驗結果：Hydroxyproline定量法得到膠原蛋白濃度為0.94(mg/ml)，紫外可見光譜儀測得出His+Tyr(230 nm)的訊號，傅立葉轉換紅外線光譜測得出膠原蛋白3個胺類的訊號，分別為1661 cm-1,1546 cm-1,1239cm-1，黏度計得知液態的膠原蛋白(0.3 mg/ml)的Td值為33℃，DSC得知固態的膠原蛋白的Td值為73℃，CD得到膠原蛋白三股螺旋的正223 nm跟負202 nm訊號。在核糖交聯作用方面，最佳交聯作用條件為反應時間八天、膠原蛋白(2 mg/ml)以及核糖濃度(0.2 M)，產物經FT-IR光譜測定，實驗結果顯示除了有膠原蛋白(1661 cm-1、1546 cm-1、1239 cm-1)也有核糖的C-O,C-C stretching Vibrations 和C-O-H,C-C-O bending vibrations訊號(1075 cm-1)，在DSC中，交聯後的膠原蛋白Td值為143℃，與交聯前的膠原蛋白相比，明顯地提升了70℃，顯示出交聯後的膠原蛋白熱穩定性有提升許多，交聯八天膠原蛋白的交聯指數為85.5％，最後利用膠原酶來做膠原蛋白降解的反應，再來測Hydroxyproline含量來證實交聯後的膠原蛋白抵抗性有增加，實驗結果顯示，未交聯前的膠原蛋白在降解反應3小時後完全被降解，而交聯後的膠原蛋白即使降解反應8小時也沒有被降解完全。

This study was to investigate the extraction methods and the characterization of fish scale collagen from Taiwan Tilapia. The main objective was to focus on the cross-linkage with ribose and to characterize the thermal stability of this cross-linked collagen/ribose film. It was found that the extraction rate is dependent on the concentration of pepsin and extraction time. The optimum condition was as of in pH 2 aqueous solution, extraction time over 24 hours and catalyzed by pepsin with a concentration of mg/mL. The collagen thus extracted was characterized by using Hydroxyproline quantification method, UV and FT-IR spectroscopy, viscometer, differential scanning calorimetry (DSC), Circular Dichroism Spectroscopy (CD), scanning electronic microscope (SEM). The results showed that the concentration of the extracted collagen is 0.937 mg/mL. The band at 230 nm (Histidine) of UV spectrum, the bands at 1661 cm-1, 1546 cm-1, 1239 cm-1of IR spectrum, and the positive band at 223 nm and negative band at 202 nm (triple helix structure) analyzed by CD are the collagen characteristic. The Td of solution in the concentration of (0.3mg/ml) was 33 oC. The Td of lyophilized collagen analyzed by DSC was 73 oC. The optimum cross-linking condition was that the concentration ratio of collagen to ribose is 2mg/ml to 0.2 M and the cross-linking time is 8 hours. The FT-IR spectrum of the cross-linked film showed the coexistence of collogen and ribose.The Td of the cross-linked film was 70 oC higher than that of non-cross-linked collagen film, indicating the thermal stability increased when collagen cross-linked with ribose. The cross-linking index was 85.5% for 8 days incubation in cross-linkage. And it was found that the cross-linked film was capable of resisting the degradation by collagenase up to 8 hours; while for non-cross-linked collagen, it is completely degraded within 3 hours.

1.黃玲惠,化工技術第四卷第七期,136-141,1996.

2.A. V.Persikov and J. A. Ramshaw；Amino acid propensities for the collagen triple-helix；Biochemistry., 39(48),14960-14967,2000.

3.E. A. Kersteen and R. T. Raines；Contribution of tertiary amides to the conformational stability of collagen triple helices.；Biopolymers., 59(1), 24-8, 2001.

4.Bailey, A. J., and Light, N. D..；Connective Tissue In Meat And Meat Products.；London & New York: Elsevier Applied Science. 1989

5.Z. Deyl and I. Miksik；Advanced separation methods for collagen parent alpha-chains, their polymers and fragments.；J Chromatogr B Biomed Sci. Appl., 739(1), 3-31, 2000.

6.楊人龍,台灣鯛魚皮膠原蛋白、明膠和膠原蛋白水解液之特性研究與比較,國立成功大學化學研究所,2008.

7.Burjanadze TV；New analysis of the phylogenetic change of collagen thermostability.；Biopolymers 53:523-528,2000

8.Nimni, M..；Collagen volume 1 : Biochemistry.；CRC press. Florida. 1988

9.Christopher S Foster, Christine M Gosden and Youqiang Q Ke；Primer: tissue fixation and preservation for optimal molecular analysis of urologic tissues；Nature Clinical Practice Urology 3, 268-278,2006

10.Pitaru, Sandu (Tel-Aviv, IL) ;Noff, Matityahu (Rehovot, IL)；Collagen-based matrix；United States Patent 5955438 ,1999

11.Allen J. Bailey , Robert Gordon Paul, Lynda Knott；Mechanisms of maturation and ageing of collagen；Mechanisms of Ageing and Development,106,1-56,1998

12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.

13.C. Choi, G. M. Son, Y. J. Cho, S. S. Chun, S. I. Lim and Y. R. Seok；Purification and characteristics of bromelain from Korean pineapple；J. Korean Agric. Chem. Sot., 35, 23-29, 1992.

14.C. K .Kang and E. E. Rice；Degradation of various meat fractions by tenderizing enzymes；J .Food Sci, 35, 563-567, 1970.

15.N. W. Rattrie and J.M. Regenstein；Action of crude papain on actin and myosin heavy chains isolated from chicken breast muscle；J .Food Sci., 42, 1159-1163, 1977.

16.B. A. Brooks, K. C. Klasing and J.M. Regenstein；Effects of antemortem injected crude papain in chicken muscle；J. Food Sci., 50, 1370-1374, 1985.

17.李佳鴻,膠原蛋白的酵素水解產物之功能性評估,實踐大學食品營養研究所,2005.

18.G. P. Sachedev and J. S. Fruton；Secondary enzyme substrate interactions and specificity of pepsin；Biochistry., 9, 4465-4470,1970.

19.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.

20.陳威廷,纖維素水解菌之培養策略與纖維素水解酵素之鑑定,國立成功大學化學工程研究所,2004.

21.劉柏青,利用枯草桿菌水解台灣鯛魚鱗膠原蛋白,國立成功大學化學研究所,2008.

22.M. C. Tanaka and M. Shimokomaki；Collagen types in mechanically deboned chicken meat；J. Food Biochem., 20, 215-225, 1996.

23.吳坤鍵,利用水母膠原蛋白生產保健機能胜之研究,實踐大學食品營養研究所,2003.

24.W. Konigsberg, J. Goldstein and R. J. Hill；The structure of human haemoglobin with pepsin；J. Biol. Chem., 238, 2028-2033,1963.

25.T. Nagai, T. Nakamura, T. Nakagawa, H. Fujiki, K. Nakao and T. Yano；Collagen of edible jellyfish exumbrella；Journal of the Science of Food and Agriculture., 79, 855-858, 1999.

26. Z. E. Sikorski, D. N. Scott and D. H. Buisson；The role of collagen in the quality and processing of fish；Critical Reviews in Food Science and Nutrition., 20(4), 301-343, 1984.

27.黃婉君、周照仁、朱玉灼,鹼處理對吳郭魚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,2003.

28.周照仁、朱玉灼、蕭靖寰,海鱺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,2003.

29.劉雅馨,翻車魨膠原蛋白的萃取及熱穩定性改善之研究,國立東華大學生物技術研究所,2005.

30.方俊凱,台灣鯛魚皮膠原蛋白之萃取及其特性鑑定,國立海洋大學食品科學研究所,2006.

31. T. Nagai and N. Suzuki；Isolation of collagen from fish waste material-skin bone and fins. Food Chemistry., 68, 277-281, 2000.

32. M. Ogawa, M. W. Moody, R. T. Poriter, J. Bell, M. A. Schexnayder and J. K. Losso；Biochemical properties of black drum and sheepshead seabream skin collagen；Journal of Agricultureal and Food Chemistry., 51, 8088-8092, 2003.

33. S. Mizuta, R. Sakamoto, M. Nishimoto and R. Yoshinaka；Identification and characterization of molecular species of collagen in ordinary muscle and skin of Japanese flounder Paralichthys olivaceus；Food Chemistry., 90, 151-156, 2005.

34. I. Bae, K. Osatomi, A. Yoshida, K. Osako, A. Yamaguchi, K. Hara；Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes；Food Chemistry., 108, 49-54, 2008.

35. L. H. Wang, B. Yang, R. Wang, X. Q. Du；Extraction of pepsin-soluble collagen from grass carp (Ctenopharyngodon idella) skin using an artificial neural network；Food Chemistry., 111, 683-686, 2008.

36. L. Wang, X. X. An, F. M. Yang, Z. H. Xin, L. Y. Zhao, Q. H. Hu；Isolation and characterisation of collagens from the skin, scale and bone of deep-sea redfish (Sebastes mentella)；Food Chemistry., 108, 616-623, 2008.

37. S. Kimura；Y. Miyauchi and N. Uchida；Scale and bone type Ⅰ collagens of carp(Cyprinus carpio)；Comparative Biochemistry and Physiology., 99B, 473-476, 1991.

38.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.

39. Y. Nomura, H. Sakai, Y. Ishii and K. Shirai；Preparation and some properties of typeⅠcollagen from fish scales；Bioscience Biotechnology and Biochemistry., 60, 2092-2094, 1996.

40. T. Ikoma, H. Kobayashi, J. Tanaka, D. Walsh and S. Mann；Physical properties of typeⅠ collagen extracted from fish scales of Pagrus major and Oreochromis niloticas；International Journal of Biological Macromolecules., 32, 199-204, 2003.

41. M .Ogawa, M. W. Moody, J. Bell and R. J. Portier；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, 2004.

42. T. Nagai, M. Izumi and M. Ishii；Fish scale collagen preparation and partial characterization；International Journal of Food Science and Technology., 39, 293-344, 2004.

43. 吳清熊,水產利用化學,國立編譯館,1997.

44. M. Hamada and H. Kumagai；Chemical composition of sardine scale；Nippon Suisan Gakkaishi., 54(11), 1987-1992, 1988.

45. S. Takenaka, A. Okada, K. Iwai and Y. Ayaki；Separation of collagen from fish scales by treatment of dilute hydrochloric acid aqueous solution；Nippon Shokuhin Kagaku Kogaku Kaishi., 50(2), 67-71, 2003.

46. A. Takenaka, A. Kawakami, M. Adachi, N. Morita and Y. Ayaki；Separation of collagen by decalcification of red sea-bream scales；Nippon Shokuhin Kagaku Kogaku Kaishi., 51(11), 572-576, 2004.

47.Kesava G., R. et al.；A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues；Clinical Biochemistry, 29, 225-229, 1996.

48. Walter J.Moore,；Physical chemistry；Prentice Hall,1972

49.江晃榮，膠原蛋白的驚人療效，世茂出版社，台灣 2002

50.Sergio Torres-Giner, Jose V. Gimeno-Alca iz, Maria J. Ocio and Jose M. Lagaron；Comparative Performance of Electrospun Collagen Nanofibers Cross-linked by Means of Different Methods；ACS Appl. Mater. Interfaces, 1 (1), pp 218–223,2009

51.張俊杰；Amino Acids ＆ Biotic Resources 30(1),18-21,2008

52. Muyonga, J. H.; Cole, C. G. B.; Duodu, K. G.；Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones of young and adult Nile perch (Lates niloticus)；Food Chem.86,325−332,2004

53.Rani Roy , Adele Boskey , Lawrence J. Bonassar；Processing of type I collagen gels using nonenzymatic glycation；Biomedical Materials 93A,3,843-851,2010

54.Lu Y,Deng G,Miao F,Li Z.；Metal ion interactions with sugars. The crystal structure and FT-IR study of the NdCl3-ribose complex.；Carbohydr Res ; 338: 2913-2919,2003

55. Sung-Pei Tsai, Chien-Yang Hsieh, Chung-Yu Hsieh, Da-Ming Wang,Lynn Ling-Huei Huang, Juin-Yih Lai, Hsyue-Jen Hsieh；Preparation and Cell Compatibility Evaluation of Chitosan/Collagen Composite Scaffolds Using
Amino Acids as Crosslinking Bridges；Journal of Applied Polymer Science,105, 1774-1785,2007