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研究生：	劉柏青 Liou, Po-Ching
論文名稱：	利用枯草桿菌水解台灣鯛魚鱗膠原蛋白 Hydrolysis of Collagen from Taiwan Tilapia Scales Using Bacillus sp
指導教授：	黃福永 Huang, Fu-Yung
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2008
畢業學年度：	96
語文別：	中文
論文頁數：	89
中文關鍵詞：	魚鱗、枯草桿菌、膠原蛋白、台灣鯛
外文關鍵詞：	Collagen, Scales, Bacillus sp., Taiwan Tilapia
相關次數：	點閱：73 下載：3
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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水解條件分析，使用水解的酵素種類為細菌性酵素(Bacterial protease)，品種為枯草桿菌屬(Bacillus. sp.)，本身能以魚鱗膠原蛋白作為生長之養分來源，不需額外再加入培養液。研究結果顯示，魚鱗經0.3 M NaOH除去皮膜與0.3 M HCl去灰份後，魚鱗粗膠原蛋白產率為53.09 ％，氫氧基磷灰石產率為41.31 ％。在萃取魚鱗膠原蛋白方面，溫度與醋酸濃度都會提高膠原蛋白的溶解量，膠原蛋白濃度最高可到752.3 μg/ml，經50 ℃與100 ℃處理之膠原蛋白在4 ℃二次水中凝固成膠狀的起始濃度分別是4.6 mg/ml與14.8 mg/ml。在細菌研究部分，其水解酵素有二種，分子量大小為39與42 kDa，細菌在35 ℃ pH 6～pH 8溶液中可以從魚鱗萃取出膠原蛋白，在35 ℃ pH 7～pH 8中細菌對魚鱗膠原蛋白有水解能力，而酵素水解溫度為30～40 ℃，其中反應條件以pH 8與30 ℃的結果最好，產物胺基酸組成以Glu、Gly、Ala與Pro含量較高。

This study was to investigate the method for the extraction and hydrolysis of fish scale collagen hydrolysis from Taiwan Tilapia. It was found that the yield of scales collagen was 53.09 ％ and Hydroxyapatite was 41.31 ％ after the scale was incubated with 0.3 M NaOH to decorticate the skin and with 0.3 M HCl to decalcify the Hydroxyapatite. The highest concentration of collagen extracted was 752.3 μg/ml when under 100 oC and 2 M acetic acid. The initial concentration for collagen to coagulate were 4.6 mg/ml and 14.8 mg/ml respectively for the collagen extracted at 50 ℃ and 100 ℃. When using bacteria, Bacillus sp, to extract and hydrolyze the fish scale collagen, it was found that the scale collagen can be as the nutrient without the extra culture medium. The optimum condition for bacteria to extract and the collagen from the scales was pH6 - pH 8 at 35 ℃, and it was pH 7- pH 8 at 35 ℃ for to hydrolyze scale collagen. There were two protease enzymes with the molecular weight of 39 and 42 kDa released from the bacteria. And the optimum condition for enzyme to hydrolyze the collagen was pH 7 – pH 8 at 30 ℃. In conclusion, pH 8 and 30 ℃ is the best condition in this study for enzyme protease to extract and hydrolyze the fish scale collagen, and the major amino acids obtained under this condition are Glu、Gly、Ala and Pro.

摘要……………………………………………………………………I
Abstract………………………………………………………………Ⅱ
誌謝……………………………………………………………………V
目錄……………………………………………………………………VI
表目錄…………………………………………………………………Ⅸ
圖目錄…………………………………………………………………X
第一章 序論……………………………………………………………1
1.1、前言………………………………………………………………1
1.2、研究動機與目的…………………………………………………1
1.3、膠原蛋白…………………………………………………………2
1.3.1、膠原蛋白分子特性與結構……………………………………2
1.3.2、膠原蛋白之命名法則…………………………………………4
1.3.3、膠原蛋白之種類………………………………………………5
1.3.4、膠原蛋白應用…………………………………………………5
    A.在化妝品中常使用的方式……………………………………6
1.3.5、膠原蛋白水解產物……………………………………………9
    A.植物性酵素……………………………………………………9
    B.動物性酵素……………………………………………………10
    C.維生物性酵素…………………………………………………10
1.4、明膠………………………………………………………………10
1.4.1、明膠之分子結構及化學特性…………………………………10
1.4.2、明膠生產製程…………………………………………………12
1.4.3、明膠應用在食品工業上的各項特性與應用…………………13
    A.溶解度…………………………………………………………13
    B.凝膠性…………………………………………………………14
    C.起泡性…………………………………………………………15
    D.保護膠體/結晶性修飾…………………………………………15
    E.乳化性…………………………………………………………16
    F.儲存穩定性……………………………………………………16
    G.微膠囊化製劑…………………………………………………16
    H.食品穩定劑……………………………………………………17
    I.果汁澄清劑……………………………………………………17
1.5、魚鱗成分與利用…………………………………………………17
1.5.1、魚類之膠原蛋白相關研究……………………………………17
1.5.2、魚鱗組成成分…………………………………………………18
    A.膠原蛋白之一般性質…………………………………………18
    B.氫氧基磷灰石之ㄧ般性質……………………………………19
1.5.3、魚鱗之完全利用………………………………………………20
    A.魚鱗粉…………………………………………………………20
    B.魚鱗膠原蛋白…………………………………………………20
    C.氫氧基磷灰石在未來的利用方向……………………………21
1.6、分析膠原蛋白之方法……………………………………………21
1.6.1、蛋白質定量法…………………………………………………21
1.6.2、聚丙烯醯胺膠体電泳…………………………………………22
    A.電泳原理………………………………………………………22
    B.PAGE 種類………………………………………………………23
    C.PAGE 膠体的組成………………………………………………24
    D.染色及乾燥……………………………………………………25
1.7、台灣鯛簡介………………………………………………………25
1.7.1、台灣鯛由來……………………………………………………25
1.7.2、台灣產銷概況…………………………………………………27
1.7.3、台灣鯛外貿年史………………………………………………28
第二章 實驗藥品與方法………………………………………………30
2.1、實驗藥品…………………………………………………………30
2.1.1、藥品與材料配置………………………………………………31
2.2、儀器設備…………………………………………………………33
2.3、實驗方法…………………………………………………………34
2.3.1、台灣鯛魚麟膠原蛋白之前處理………………………………34
2.3.2、氫氧基磷灰石製備……………………………………………35
2.3.3、台灣鯛魚麟膠原蛋白之水萃條件……………………………36
2.3.4、原蛋白在4℃下的溶解性測試 ………………………………36
2.3.5、菌種保存及培養………………………………………………37
    A.菌種保存………………………………………………………37
    B.菌種培養………………………………………………………37
    C.菌體濃度定量…………………………………………………38
2.3.6、蛋白質定量……………………………………………………38
2.3.7、細菌水解臺灣鯛魚麟之pH條件探討…………………………39
2.3.8、酵素水解膠原蛋白之pH值與溫度探討………………………40
2.3.9、一維電泳分析…………………………………………………42
    A.膠片製作流程…………………………………………………42
    B.電泳分析………………………………………………………42
    C.膠片染色………………………………………………………43
    D.膠片乾燥………………………………………………………44
第三章 實驗結果與討論………………………………………………45
3.1、台灣鯛魚麟膠原蛋白之前處理…………………………………45
3.2、氫氧基磷灰石製備………………………………………………47
3.3、台灣鯛魚麟膠原蛋白之水萃取條件……………………………48
3.4、膠原蛋白在4℃下的溶解性測試………………………………50
3.5、菌種培養…………………………………………………………51
3.6、細菌水解臺灣鯛魚麟之pH條件探討……………………………51
3.7、酵素水解膠原蛋白之pH值與溫度探討…………………………53
3.7.1、細菌作用酵素之探討…………………………………………53
3.7.2、酵素水解膠原蛋白之探討……………………………………54
3.8、魚鱗膠原蛋白之胺基酸組成……………………………………55
第四章 結論……………………………………………………………57
附表……………………………………………………………………59
附圖……………………………………………………………………69
參考文獻………………………………………………………………………85
                                    

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