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研究生:鍾朝淳
研究生(外文):Jung, Chao Chuen
論文名稱:海參體壁中膠原蛋白的萃取與特性分析
論文名稱(外文):Extraction and Property Analysis of Collagen from the Body Wall of Sea Cucumber Holothuria cinerascens
指導教授:柯文慶柯文慶引用關係
指導教授(外文):Ko, Wen-Ching
口試委員:謝昌衛黃至盛
口試委員(外文):Hsieh, Chung-WeiHwang, Jyh-Sheng
口試日期:2012-07-12
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:78
中文關鍵詞:海參膠原蛋白萃取特性分析
外文關鍵詞:Sea cucumberCollagenExtractionCharacterization
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本研究以黑赤星海參體壁為原料萃取胃蛋白酶可溶性膠原蛋白 (pepsin–solubilized collagen, PSC),並與吳郭魚魚皮和豬皮所萃取之膠原蛋白進行特性分析及比較。一般成分分析中,黑赤星海參之水分含量最高,達 85.84 %,而粗脂肪與粗蛋白質含量分別為 0.30 % 與 10.29 %,可以看出海參屬於一種高蛋白、低脂肪的無脊椎動物。以粗膠原白產率來看,豬皮最高,達 26.12 %,其次為魚皮,海參最低。
市販海參大多為乾品復水,本研究發現乾品復水後的海參無法萃出膠原蛋白,推測可能乾燥過程或復水過程受熱,造成蛋白質變性所致。
以 SDS–PAGE 分析三物種萃取之膠原蛋白皆屬於第 I 型,就分子量言,海參膠原蛋白最小 (80~90 kD);紫外光–可見光光譜分析顯示三物種皆在 230 nm出現最大吸收波峰,此乃蛋白質含有C=O, COOH和CONH2之官能基產生的;以傅立葉轉換紅外線光譜顯示三物種萃取之膠原蛋白皆有 amide A、I、II、III,此為蛋白質主要官能基產生的吸收波峰;胺基酸組成分析顯示三物種萃取之膠原蛋白,均以甘胺酸 (31 %)、脯胺酸 (9~12 %) 以及丙胺酸 (10~12 %) 之含量最高;示差掃描熱分析顯示三物種中以黑赤星海參膠原蛋白熱穩定性最低,此應與其生長溫度有關;另三物種膠原蛋白之保濕性及吸水性均較對照組 (甘油) 為佳,表示萃取之膠原蛋白其親水性基團含量較多。
綜合以上結果顯示,海參膠原蛋白之萃取雖然成本高、產率低,但就其基本特性言,海參膠原蛋白優於其他兩物種。唯需進一步研究,使於加工或其他應用上均能保持所萃膠原蛋白的特性,以最佳利用。

The body wall of sea cucumber was used as raw materials for extraction of pepsin-solubilized collagen (PSC) and to compare from that from the skin of tilapia and porcine. On proximate compositions, sea cucumber showed the most moisture content 85.84%, lower crude fat 0.3% and crude protein 10.29 %. Sea cucumbers belong to one of the high-protein and low-fat invertebrates. The yield of crude collagen from porcine skin 26.12 % was the highest, while sea cucumber showed the lowest value. The sea cucumbers placed on the market were almost treated via drying and rehydration processes. Collagen of the market products could not be extracted according the same method. Protein denaturation was speculated during treatment.
According to the profiles of SDS-PAGE analyses, the collagens from the three species were type I. Sea cucumber showed the smallest molecular weight (80~90 kDa) among the three products. Maximum peak shown at 230 nm on UV-vis spectroscopy profiles indicated that the collagens contain aromatic amino acids with C=O, COOH and CONH2 groups. FTIR spectroscopy showed that the collagen from the three species have amide A、I、II、III peaks which were generated by the main functional groups in the proteins. The dominant amino acids of the three extracted collagen were glycine (31%), proline (9~12%) and alanine (10~12%). Differential scanning calorimetry showed the collagen from sea cucumber had the lowest thermal stability. This may related to their growth circumstances. The three collagens exhibited better moisture-retention and moisture-absorption capacity than glycerol, indicating that the collagen molecules are rich in hydrophilic groups.
In conclusion, the extraction of collagen from sea cucumber was expensive cost and low yield. But its basic characteristics were better than the others. Further studies were required to retain properties of the extracted collagen during processing or other applications.

封面內頁
簽名頁
中文摘要iii
英文摘要v
誌謝vii
目錄ix
圖目錄xii
表目錄xiv

1. 前言1
2. 文獻回顧3
2.1 膠原蛋白之簡介3
2.2 膠原蛋白之萃取4
2.3 膠原蛋白之基本結構4
2.4 膠原蛋白之應用8
2.4.1食品領域8
2.4.2化妝品領域
2.4.3生物醫學領域9
2.5 海參的介紹11
2.5.1 海參分類與介紹11
2.5.2 海參採集與乾燥品製造12
2.5.3近年海參的研究15
2.5.4 黑赤星海參17
3. 材料與方法18
3.1 材料18
3.1.1 黑赤星海參18
3.1.2 吳郭魚18
3.1.3 豬皮18
3.2 試藥20
3.3 儀器21
3.4 實驗方法23
3.4.1 海參膠原蛋白萃取23
3.4.2 吳郭魚魚皮膠原蛋白萃取23
3.4.3 豬皮膠原蛋白萃取24
3.4.4 基本成分分析28
3.4.4.1 粗蛋白質28
3.4.4.2 粗脂肪28
3.4.4.3 水分29
3.4.4.4 灰分29
3.4.5 產率30
3.4.6 SDS-polyacrylamide gel electrophoresis30
3.4.6.1 試劑配製30
3.4.6.2 蛋白質變性32
3.4.6.3 鑄膠32
3.4.6.4 電泳32
3.4.6.5 膠體染色及脫色33
3.4.7 胺基酸分析 (Amino acid composition)35
3.4.8 傅立葉轉換紅外線光譜儀分析 (FTIR)35
3.4.9 紫外光-可見光光譜35
3.4.10 示差掃描量熱分析法 (DSC)36
3.4.11 膠原蛋白之溶解度測定36
3.4.11.1 pH 值對膠原蛋白溶解性之影響36
3.4.11.2 NaCl 對膠原蛋白溶解度之影響36
3.4.12 保濕性及吸水性測定37
4. 結果與討論38
4.1 成分分析38
4.2 膠原蛋白之產率 (%)40
4.3 膠原蛋白分子量測定 (SDS-PAGE)42
4.4 紫外光–可見光光譜44
4.5 胺基酸組成分析48
4.6 傅立葉轉換紅外線光譜分析50
4.7 膠原蛋白之溶解度測定55
4.8 示差掃描量熱儀分析 (DSC)58
4.9 吸水性及保濕性測定62
5. 結論67
參考文獻68

圖目錄

圖1. 膠原蛋白分子之三股螺旋結構7
圖2. 海參乾品製作流程14
圖3. 黑赤星海參之圖鑑19
圖4. 黑赤星海參、魚皮以及豬皮膠原蛋白分析之架構圖22
圖5. 海參體壁之膠原蛋白萃取流程圖25
圖6. 吳郭魚魚皮膠原蛋白萃取之流程圖26
圖7. 豬皮膠原蛋白萃取之流程圖27
圖8. 黑赤星海參體壁、吳郭魚魚皮以及豬皮膠原蛋白之SDS-PAGE43
圖9. 黑赤星海參膠原蛋白之紫外光–可見光光譜45
圖10.魚皮膠原蛋白之紫外光–可見光光譜46
圖11.豬皮膠原蛋白之紫外光–可見光光譜47
圖12.牛腱之第 I 型膠原蛋白之傅立葉轉換紅外線光譜圖譜51
圖13.黑赤星海參膠原蛋白之傅立葉轉換紅外線光譜 52
圖14.吳郭魚魚皮膠原蛋白之傅立葉轉換紅外線光譜 53
圖15.豬皮膠原蛋白之傅立葉轉換紅外線光譜54
圖16.黑赤星海參、魚皮及豬皮膠原蛋白於不同 pH 值下之溶解度測定56
圖17.黑赤星海參、魚皮及豬皮膠原蛋白於不同鹽濃度下之溶解度測定57
圖18.黑赤星海參膠原蛋白之示差掃描量熱儀分析59
圖19.吳郭魚魚皮膠原蛋之示差掃描量熱儀分析60
圖20.豬皮膠原蛋白之示差掃描量熱儀分析61
圖21.海參體壁、魚皮及豬皮膠原蛋白之吸水性 (RH 43 %)63
圖22.海參體壁、魚皮及豬皮膠原蛋白之吸水性 (RH 81 %)64
圖23.海參體壁、魚皮及豬皮膠原蛋白之保濕性 (RH 43 %)65
圖24.海參體壁、魚皮及豬皮膠原蛋白之保濕性 (RH 81 %)66

表目錄

表1.膠原蛋白之分類及分佈6
表2.SDS-PAGE分析所用試劑之比例34
表3.黑赤星海參體壁之一般成分分析39
表4.黑赤星海參體壁、吳郭魚魚皮以及豬皮膠原蛋白之產率41
表5.黑赤星海參體壁、吳郭魚魚皮以及豬皮膠原蛋白之胺基酸49




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