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研究生:姚軼俊
研究生(外文):Yi-jun Yao
論文名稱:固定化β-葡萄糖苷酶應用於黑豆漿中大豆異黃酮去醣基之研究
論文名稱(外文):Immobilized β-Glucosidase for Isoflavone Deglycosylation in Black Soymilk
指導教授:游若篍
指導教授(外文):Roch-Chui Yu
口試委員:周正俊劉啟德陳冠翰
口試委員(外文):Cheng-Chun ChouChi-Te LiuJimmy Chen
口試日期:2013-07-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:80
中文關鍵詞:黑豆漿異黃酮去醣基化β-葡萄糖苷酶酵素固定化尼龍纖維素凝膠
外文關鍵詞:black soymilkisoflavone deglycosylationβ-glucosidaseenzyme immobilizationNyloncellulose beadssol-gel
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大豆異黃酮是存在於大豆中的天然黃酮類化合物。研究發現,攝入足量的大豆異黃酮可以有效降低罹患骨質疏鬆症及婦女更年期綜合症的風險;在抗癌、防癌及預防心血管疾病等方面也有重要的功用。另有文獻指出,在人體內醣苷型的異黃酮不能直接被吸收,而是經由小腸中細菌產生的β- glucosidase水解去醣基後才進入血液,發揮各種生理作用。然而,人體小腸中的菌相基於個體差異活性會有很大差別,因此,若能直接攝入不帶醣基的苷元型異黃酮,則能有效提高其利用效率。本實驗之目的即是建立可將黑豆漿中大豆異黃酮醣苷轉化之固定化酵素系統。將β- glucosidase固定化於尼龍、纖維素以及凝膠等三種載體,實驗結果顯示每40 cm3載體上能固定超過40 mg之酵素,經酵素動力學測試後得到尼龍、纖維素以及凝膠系統其酵素動力學參數KM值分別為2.5042 ± 0.1901, 1.3826 ± 0.2043 和1.5889 ± 0.1152。以p-NPG作為基質測試固定化酵素活性後可知尼龍及纖維素系統具有較佳的反應穩定性。進一步使用HPLC分析固定化酵素應用於黑豆漿中異黃酮的轉化效率後得到纖維素系統為最佳反應體系,其最適作用溫度為50oC,並且可以在30分鐘之內利用40cm3含有酵素的載體將50 ml黑豆漿中的異黃酮全部轉化為去醣基化形式。在經過15次反應以及15天之後此系統仍然可維持其60%的酵素活性。

The studies of isoflavone have recently drawn tremendous attention due to its potential health-enhancing benefits, including reduction of cardiovascular disease, prevention of cancer, protection of osteoporosis, and antioxidant activities. Researches revealed that isoflavones in their aglycone forms exhibit higher biological activity and are more metabolically active that can be absorbed faster in greater amounts than their glycosides. This study is conducted to evaluate the feasibility of soymilk isoflavone conversion using an immobilized β-glucosidase enzyme system. β-glucosidase was immobilized on nylon pellets, cellulose beads, and sol-gel with the loading that over 40 mg of enzyme on 40cm3 of carriers. The KM value of these three enzymatic systems were determined to be 2.5042 ± 0.1901, 1.3826 ± 0.2043 and 1.5889 ± 0.1152, respectively. Nylon and cellulose system were proved more effective for the conversion of pNPG to p-nitrophenol. And with further determination, cellulose system exhibited a significant efficiency for isoflavone deglycosylation in black soymilk. The optimum temperature was 50 OC and complete reaction can be reached in 30 min. The amount changes of isoflavone in black soymilk after deglycosylation under different temperature were analyzed by HPLC. The reusability and storage stability were further verified. The enzymatic system can be used more than 15 cycles and 15 days which still retained 60% of the enzyme activity.

中文摘要 ..i
Abstract ..ii
目錄 ..iii
表 ..vii
圖 ..viii
壹、 前言 ..1
貳、 文獻整理 ..2
一、 大豆異黃酮 ..2
(一) 大豆異黃酮的組成與結構 ..2
(二) 大豆異黃酮的理化性質 ..2
(三) 大豆異黃酮的生理功能 ..3
1. 大豆異黃酮的抗氧化功能 ..3
2. 大豆異黃酮的植物雌激素功能 ..5
3. 大豆異黃酮的抗菌功能 ..6
4. 其他功能 ..6
(四) 異黃酮之吸收代謝 ..7
二、 黑豆中大豆異黃酮的含量及其轉化 ..7
三、 β-葡萄醣苷酵素 ..8
(一) β-葡萄醣苷酵素的來源 ..9
(二) β-葡萄醣苷酵素的生化特性 ..9
四、 酵素固定化 ..10
(一) 酵素固定化方法 ..10
1. 物理吸附法 ..10
2. 共價結合法 ..10
3. 交聯法 ..11
4. 酵素包埋 ..11
(二) 載體材料 ..13
1. 無機載體 ..13
2. 有機高分子載體 ..14
3. 複合載體 ..14
參、 材料與方法 ..16
一、 實驗架構 ..16
二、 實驗材料 ..19
(一) 黑豆 ..19
(二) 試驗藥品 ..19
(三) 儀器設備 ..20
三、 實驗方法 ..21
(一) 尼龍載體修飾 ..21
(二) 尼龍載體與酵素共價結合 ..21
(三) 纖維素載體合成與修飾 ..21
(四) 纖維素載體與酵素共價結合 ..22
(五) 固定化酵素量檢測 ..22
(六) 載體表面共價鍵分析 (ESCA) ..22
(七) 凝膠 (sol-gel)—酵素系統製備 ..23
(八) 載體表面結構掃描電鏡分析 (SEM) ..23
(九) 固定化酵素最適反應溫度測試 ..24
(十) 固定化酵素活性測試 ..24
(十一) 酵素動力學參數測定 ..24
(十二) 黑豆漿的製備 ..24
(十三) 大豆異黃酮之定量分析 ..25
(十四) 統計分析 ..26
肆、 結果與討論 ..27
一、 β-葡萄糖苷酶固定於尼龍、纖維素及凝膠載體 ..27
(一)、 β-葡萄糖苷酶吸光值標準曲線之製作 ..27
(二)、三種載體上固定之酵素含量 ..29
(三)、β-葡萄糖苷酶固定於尼龍、纖維素載體之化學鍵結 ..29
(四)、β-葡萄糖苷酶固定前後載體表面之微觀結構 ..34
二、 固定化酵素最適作用溫度 ..38
(一)、p-NPG水解分析及p-nitrophenol吸光值標準曲線 38
(二)、固定化前後酵素在不同作用溫度下之酵素活性 ..38
三、 固定化酵素活性測試 ..41
(一)、尼龍載體 ..41
(二)、纖維素載體 ..41
(三)、凝膠載體 ..44
四、 酵素動力學測試 ..44
(一)、尼龍載體 ..44
(二)、纖維素載體 ..46
(三)、凝膠載體 ..46
五、 尼龍載體與黑豆漿中異黃酮之作用 ..48
(一)、異黃酮素分析及標準曲線之製作 ..48
(二)、經尼龍—酵素催化後黑豆漿中異黃酮含量之變化 ..51
六、 纖維素—酵素系統催化黑豆漿中異黃酮轉化之最適作用溫 ..51
七、 纖維素—酵素系統催化黑豆漿中異黃酮轉化之反應效率 ..54
(一)、經纖維素—酵素系統催化不同時間後黑豆漿中異黃酮素含量之變 ..54
(二)、纖維素—酵素系統催化黑豆漿中大豆異黃酮轉化之酵素動力學測試 ..54
八、 纖維素—酵素系統之反應穩定性 ..59
(一)、重複批次反應穩定性 ..59
(二)、儲存穩定性 ..59
伍、 結論與展望 ..62
陸、 參考文獻 ..64
個人履歷..80

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