臺灣博碩士論文加值系統

本研究的目的在建立台灣鯛膠原蛋白的製備及其所得之膠原蛋白物化特性的鑑定。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、變性溫度Td (Denature temperture)等方法分析測定胺基酸、二級結構、變性溫度及分子量。以了解所得之膠原蛋白的完整性以及熱安定性。
胺基酸分析結果顯示的台灣鯛魚皮膠原蛋白沒有任何的Cystine 存在，故萃取過程中端肽已去除。Proline 及 Hydroxyproline 總合在魚皮去端肽膠原蛋白為20%。與小牛皮之21.5%接近。Hydroxyproline 及 Hydroxylysine顯示的氧化羥基化程度為中等程度。在SDS-PAGE電泳圖顯示台灣鯛主要由α1、α2之單體、β(雙體) 及γ(三聚體)組成，且50 kDa以下之多肽沒有出現，代表Telopeptide已在萃取過程去除。由圓二色光譜的顯示測定台灣鯛膠原蛋白三股螺旋之比例為80.4％。Td的結果顯示在無鹽離子影響之情況下，台灣鯛膠原蛋白之耐熱性可達33.2℃。就安定性而言，純化之台灣鯛膠原蛋白低於未純化之台灣鯛膠原蛋白。

The aim of this study was to establish the isolation conditions to characterize the physical and properties of collagen from the skin waste material of hybrid tilapia (black tilapia (Oreochromis mossambicus) cross red tilapia (Oreochromis nilotica)) by solvent extraction.

The physical-chemical properties include amino acid analysis, circular dichroism (CD) , sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), denaturation temperture (Td) to characterize, and thermal denaturation temperature of hybrid tilapia skin collagen.
The results showed that hybrid tilapia collagen have no cystine, it indicated that telopeptide was removd. Proline and hydroxyproline contents in hybrid tilapia collagen is 20%. Hydroxyproline and hydroxylysine were oxidized hydroxylation a medium with compare to other fish species.
SDS-PAGE include the molecular weight of the band were at 100 kDa, 150 kDa and 250 kDa. composed of α1, α2 momomer , β dimmer, and γ trimer. No bands of molecular weight less than 50 Kda showed the telopeptide right have been removed because it has two α1 and one α2 band only. CD is use to collagen triplehelix ratio measurement is 80.4%, The Td (denature temperature) in hybrid tilapia is 33.2 ℃ with no ion influence. The range of Melting temperature(Tm) is 10-11℃.
At FTIR (Fourier transform infrared) show The positions of amide
bands A Merged around amide bands B Have stable comformation for all the blends and only slightly different for other collagen. By the stability, the purification samples lower than the unpurification ones.

誌謝..........................................................................................................Ⅰ目錄..........................................................................................................Ⅲ
圖目錄.................................................................................................... Ⅶ
表目錄..................................................................................................Ⅸ
中文摘要..............................................................................................ⅩⅠ
英文摘要............................................................................................ⅩⅡ
第一章 緒論..............................................................................................1
第二章 文獻回顧......................................................................................3
2.1 膠原蛋白之性質.................................................................................3
2.1.1 膠原蛋白的結構............................................................................3
(1) 連續結構.................................................................................4
(2) 高等結構.................................................................................7
2.1.2 膠原蛋白的生合成......................................................................12
2.1.3 膠原蛋白的分類..........................................................................15
(1) 膠原蛋白第一型...................................................................17
2.1.4 膠原蛋白一般來源及其分佈......................................................18
(1) 膠原蛋白的交鏈...................................................................19
(2) 膠原蛋白之降解...................................................................21
2.1.5 膠原蛋白的發展應用..................................................................23
2.2 魚類膠原蛋白相關之研究...............................................................28
2.3 台灣鯛介紹.......................................................................................35
第三章 台灣鯛魚皮膠原蛋白的萃取流程及特性................................36
3.1、實驗材料、儀器設備與藥品.........................................................36
3.1.1、萃取實驗................................................................................36
3.1.2、特性分析實驗.............................................................................37
3.2 實驗流程...........................................................................................39
3.3 萃取條件實驗方法...........................................................................40
3.3.1 台灣鯛膠原蛋白的萃取條件建立..............................................40
(1) 脂質及色素之去除....................................................40
(2) 膨潤實驗........................................................................42
3.4特性分析實驗方法.....................................................................44
3.4.1胺基酸定量分析(Amino acid analysis).....................................44
(1) 胺基酸定量分析儀原理.............................................................44
3.4.2 膠電泳.................................................................................45
SDS電泳..........................................................................45
3.4.3圓二色光譜 (Circular Dichroism；CD)........................................47
(1) 圓二色光譜儀測量原理.............................................................47
(2) 蛋白質或多胜肽的圓二色光譜.................................................50
3.4.3.1 圓二色光譜操作.......................................................................53
(1) CD測定部份.....................................................................53
(2) CD分析部份........................................................................54
第四章 結果與討論...............................................................................55
4.1萃取部分...........................................................................................55
(1) 脂質及色素之去除...................................................................55
(2) 膨潤試驗...................................................................................58
4.2 特性分析部分................................................................................. 61
4.2.1 胺基酸定量分析 (Amino acid analysis)......................................61
(1) 魚皮、未去端肽膠原蛋白及去端肽膠原蛋白胺基酸總量差異
................................................................................................................61
(2) 台灣鯛魚皮去端肽膠原蛋白Hydroxyproline+Proline胺基酸差異........................................................................................................65
(3) 台灣鯛魚皮去端肽膠原蛋白Hydroxyproline+ Hydroxylysine胺基酸差異............................................................................................69
4.2.2 蛋白質電泳（SDS–PAGE）....................................................72
(1) 台灣鯛魚皮之膠原蛋白端肽去除測定.................................72
(2) 台灣鯛魚皮去端肽膠原蛋白類型判定.................................72
4.2.3 圓二色光譜(Circular Dichroism) ...............................................75
(1) 台灣鯛魚皮去端肽膠原蛋白之三股螺旋比例分析..............75
(2) 台灣鯛魚皮去端肽膠原蛋白之變性溫度測定......................78
第五章 結論.........................................................................................83
第六章 參考文獻.................................................................................84
附錄.......................................................................................................90

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