臺灣博碩士論文加值系統

植物固醇(Phytosterol)為ㄧ植物內生性之三萜類(Triterpenes)，自然界中的三萜類有4000多種，而植物固醇就占了100種以上。近年來，植物固醇酯類被發現能藉由抑制小腸中膽固醇的吸收而降低血清膽固醇的含量，因此被應用在食用油脂以及機能性的食品中。植物固醇酯類的來源在傳統上除了萃取之外還能用化學合成法製造，但在講求天然與健康的現代飲食觀念下，以生物催化法所合成的植物固醇酯類更能符合這個要求。
故本研究主要是選用脂解酵素Lipase AY (from Candida rugosa)催化飽和之穀固醇(Sitostanol)與丁酸酐(Butyric anhydride)進行酯化反應，合成植物固醇酯類(Sitostanyl butyrate)，並利用反應曲面法(Response surface methodology；RSM)及五階層四變數之中心混層實驗設計法(Central composite rotatable design；CCRD)，分別探討反應時間(2–10 h)、反應溫度(25–65 ºC)、基質莫耳比(1.0–3.0；Acyl noner/Alcohol)及酵素用量(10–50%w/w, by wt. of sitostanol)等反應參數對莫耳轉換率之影響及求得植物固醇酯類之最優化合成條件。研究結果顯示，在有機系統下，脂解酵素Lipase AY可成功地催化植物固醇酯類之合成，在反應時間6.2小時、溫度28.8 ºC、酵素用量41.6%及基質莫耳數比為2..0時可得到54.5%的最優化產率。

Phytosterols (Plant sterol) are members of the “triterpene” family of natural products, which includes more than 100 different phytosterols and more than 4000 other types of triterpenes. Recently, phytosteryl ester have been found to be effective in lowering plasma cholesterol concentration by inhibiting the absorption of cholesterol in the small intestine. Therefore, phytosteryl esters are frequently added to cooking oil and function food. Traditionally, it has been isolated from natural sources or produced by chemical synthesis. Nowadays, people focus more on natural products for health benefits. Therefore, the biosynthesis of such esters by lipase-catalyzed chemical reactions under mild conditions has become more attractive to modern industries.
The present work focuses on the ability of lipase from Candida rugosa (Lipase AY) to catalyze the esterification of sitostanol with butyric anhydride. Response surface methodology (RSM) and five-level-four-factor central composite rotatable design (CCRD) were adopted to evaluate the effects of synthesis variables, such as reaction time (2–10 h), temperature (25–65 ºC), substrate molar ratio (1–3; Acyl noner/Alcohol), enzyme amount (10–50%w/w, by wt. of sitostanol) on percentage molar conversion of sitostanyl butyrate.
The results showed that sitostanyl butyrate can been successfully synthesized by Lipase AY in solvent system. The optimum molar conversion of sitostanyl butyrate was 54.4% at 6.2 h、28.8 ºC、41.6% enzyme amount and substrate molar ratio of 2

目錄

封面內頁
簽名頁
授權書.. iii
中文摘要 iv
英文摘要 v
誌謝 vii
目錄 viii
圖目錄 xi
表目錄 xii

第一章 緒論 1
第二章 文獻回顧 5
2.1 植物固醇之簡介 5
2.2 植物固醇酯類 6
2.2.1 植物固醇酯類之簡介與現況 6
2.2.2 植物固醇酯類之生理效應 7
2.2.3 植物固醇酯類之降膽固醇機制 10
2.2.4 植物固醇酯類之安全性 11
2.2.5 植物固醇酯類之合成 12
2.3 脂解酵素 …….15
2.3.1 Lipase AY之簡介 16
2.3.2 脂解酵素之專一性 16
2.3.3 影響酵素活性之環境因子 18
2.4 反應曲面法之應用 19
2.4.1 反應曲面法之原理 19
2.4.2 中心混層設計 20
2.4.3 回應曲面模式適切性之統計檢驗 21
第三章 材料與方法 28
3.1 實驗材料 28
3.1.1 儀器設備 28
3.1.2 藥品 28
3.2 實驗設計與方法 29
3.2.1 實驗設計 29
3.2.2 酵素活性測定 29
2.2.3 酵素含水量測定 30
2.2.4 Sitostanly butyrate的合成方法 30
3.2.5 萃取與分析 31
3.2.6 統計分析 31
第四章 結果與討論 36
4.1 實驗結果 36
4.1.1 時間對莫耳轉換率之影響 37
4.1.2 溫度對莫耳轉換率之影響 37
4.1.3 酵素用量對莫耳轉換率之影響 38
4.1.4 基質莫耳數比對莫耳轉換率之影響 39
4.2 最優化合成之研究 40
4.3 綜合討論 41
4.3.1 時間對反應之影響 42
4.3.2 溫度對反應之影響 42
4.3.3 酵素用量對反應之影響 43
4.3.4 有機溶劑之選擇 44
第五章 結論 53
參考文獻 54
附錄一 DL50自動電位滴定儀操作方法 60
附錄二 Karl Fischer操作方法 62
附錄三 植物固醇酯類合成之相關文獻 64
附錄四 SAS之input數據 65














圖目錄

圖 1-1以脂解酵素催化氫化穀固醇與丁酸酐之酯化反應..........4
圖 2-1常見之植物固醇與膽固醇之結構................................25
圖2-2三甘油酯以sn-命名的代表分子.............................26
圖2-3中心混層設計法之星點及中心點補充實驗圖...............27
圖3-1氣相層析儀分析sitostanyl butyrate之標準圖譜.........35
圖 4-1反應時間對sitostanyl butyrate合成產率之影響，反應
條為：反應溫度45 C、酵素用量30% sitostanol重以
及基質莫耳數比為2.0(Acyl doner/Alcohol)...................48
圖 4-2酵素用量與反應時間對sitostanyl butyrate莫耳轉換率
影響之反應曲面圖.........................................................49
圖 4-3酵素用量與反應溫度對Sitostanyl butyrate莫耳轉換率
影響之反應曲面圖.........................................................50
圖 4-4酵素用量與反應溫度對sitostanyl butyrate莫耳轉換率
影響之反應曲面圖.........................................................51
圖 4-5 Sitostanyl butyrate百分比莫耳轉換率之等高線圖。在
等高線圖內之數字是用來表示不同的反應條件下之重量
轉換率...........................................................................52





表目錄

表 2-1植物固醇相關上市產品ㄧ覽表......................................23
表 2-2中心混層設計之補充實驗.............................................24
表 3-1五階層四變數中心混層實驗設計反應參數實驗值之範
圍..................................................................................33
表 3-2 Sitostanyl butyrate之五階層四變數中心混層實驗設計
數據..............................................................................34
表 4-1 Sitostanyl butyrate百分比莫耳轉換率對合成變數之變
異數分析.......................................................................45
表 4-2 Sitostanyl butyrate合成變數之聯合檢測分析..............46
表 4-3利用脊形分析評估合成sitostanyl butyrate莫耳轉換率
之最大值.....................................................................47

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