臺灣博碩士論文加值系統

大豆異黃酮為植物雌激素重要來源之ㄧ。目前已有不少研究指出大豆異黃酮具有保健生理活性功效。異黃酮素主要有12種衍生物，於大豆中含量最高的為malonylglucosides形式之異黃酮，其次為β-glucosides形式之異黃酮，然而此兩類結構並非人體所能吸收之結構，若能將此類異黃酮轉換成人體較易吸收利用之aglyocnoes，則可提高大豆異黃酮的利用性。
根據本研究室先前關於大豆異黃酮之研究中發現，凍乾大豆粉於40℃熱反應，malonylglucosides含量會明顯減少，而aglycones含量則有上升的趨勢，因此推測大豆中存在著可水解malonylglucosides之相關酵素，因而著手進行對大豆異黃酮相關水解酵素之研究。
欲進行相關水解酵素之研究，需製備可供酵素作用之異黃酮素為反應基質，因此本論文亦包括精製純化malonylglucosides與glucosides等兩類異黃酮素的方法。
本研究第一部分為isoflavone malonylglucosidess及isoflavone glucosides之分離純化。結果發現，將大豆粉以60%甲醇萃取大豆異黃酮，將其濃縮去除溶劑後通過HP-20疏水性管柱，水洗去除雜質，然後以40%甲醇洗出malonylglucosides部份；接著改以70%甲醇洗出殘餘malonylglucosides及glucosdies，於95℃下加熱迴流8小時，使malonylglucosides全部轉變成glucosides，即得isoflavone glucosides。
另一方面，本研究初步試驗結果發現β-glucosidase存在於豆渣(okara)中。將分離洗淨之豆渣以3% NaCl(aq)萃取、將萃取物以UF濃縮、以50-60%丙酮沉澱、經DTT(1,4-Dithioerythritol)還原後進行CM-Sepharose CL-6B陽離子交換層析及膠體過濾而得純化之β-glucosidase。此β-glucosidase之分子量為67kD之monomer，最適溫度為45℃，最適pH為4.5；溫度穩定性佳，於70℃加熱1小時仍有85%之酵素活性，而pH穩定性以接近pH 6.0的情況最好。在醣類與金屬離子抑制方面， 1 mM Hg2+能完全抑制此酵素之活性。此純化之β-glucosidase可將glucosides全部轉換為algycones，對malonylglucosides則沒有作用。顯示此酵素為對glucosides具有專一性之酵素。

Soybean isoflavones, a kind of phytoestrogen, have been reported to possess a variety of physiological activities and a potential health benefit. Malonylglucoside is the most abundant isoflavone derivatives in soybeans, and glucoisde is the second abundant group. However, aglycones were reported as the most easily absorbed among the various isoflavone derivatives. Therefore, malonylglucosides and glucosides were desired to be converted to aglycones before consumption.
According to our previous studies on soybean isoflavones, we found that the content of malonylglucosides were decreased and aglycones were increased obviously when ground soybean suspension incubated at 40℃. This result suggested that the enzyme existing in soybean could hydrolyze malonylglucosides. Consequently, the aim of this study was to investigate the isoflavone-related hydrolyzing enzymes.
In this study, isolation and purification of isoflavones was also carried out to prepare the substrates for enzyme reactions. Isoflavones in soybeans were extracted with 60% methanol. After removing methanol by cacuum evaporation , the aqueous was applied to a HP-20 column, After washing with water, the counn was eluted with 40% methanol solution to recover malonylglucosides. Afterwards, the residual malonylglucosides and glucosides were eluted with 70% methanol solution, followed by heating at 95℃ for 8 hour to convert malonylglucosides to glucosides completely.
β-glucosidase discovered in okara was extracted with 3% sodium choloride solution, followed by subjecting to UF concentration, precipitation with 50-60% acetone , DTT reduction, cation exchange absorption and gel filtration. The β-glucosidase was estimated to be a monomer with a mass of 67kD. The optimum temperature was 45℃, and the optimum pH was 4.5. Thermal stability was measured at 40-70℃. The enzyme maintained its activity by 85% at 70℃. It was relatively stable at pH 6.0. The enzyme activity was completely inhibited by 1 mM Hg+. The purified β-glucosidase could completely convert glucosides to their corresponding aglycones. While it could not catalyze the hydrolysis of malonylglucosides. The result indicated that the β-glucosidase from soybean okara was specific to glucosides but not to malonylglucosides.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
英文摘要 iv
第一章、研究動機 1
第二章、文獻整理 2
壹、大豆 2
一、大豆簡介 2
二、大豆之一般組成分 3
三、大豆之機能性成分 5
1. 黃豆蛋白質 5
2. 豆清蛋白 5
3. 大豆卵磷脂 5
4. 植物固醇 6
5. 大豆異黃酮 8
貳、異黃酮之生理活性 10
一、異黃酮於人體內的吸收與代謝 10
二、異黃酮的生物利用率 13
三、異黃酮素之生理功效 17
1. 抗氧化能力 17
2. 抗癌作用 17
3. 改善更年期婦女之不適症狀 18
4. 預防心血管疾病 18
参、熱處理加工對大豆異黃酮的影響 19
肆、大豆中的酵素 22
伍、異黃酮之分離純化方法 28
第三章、材料與方法 35
壹、實驗架構 35
貳、實驗材料與器材 35
参、實驗方法 37
一、大豆異黃酮之精製純化 37
1. 大豆之前處理 37
2. 大豆中異黃酮之萃取 37
3. 大豆異黃酮之分離 37
二、大豆異黃酮之含量分析 38
三、大豆異黃酮含量計算方式 40
四、大豆中β-glucosidase之研究 42
1. 大豆之前處理 42
2. 大豆中β-glucosidase之純化步驟 42
3. β-glucosidase之最適溫度及最適pH 44
4. β-glucosidase之溫度與pH 穩定性 45
5. 金屬離子、醣類及化合物對β-glucosidase之影響 45
6. β-glucosidase與大豆異黃酮素之反應 46
第四章、結果與討論 47
壹、大豆中含有異黃酮相關水解酵素之探討 47
貳、大豆異黃酮之分離 49
参、大豆中β-glucosidase之研究 57
一、β-glucosidase之純化 57
二、β-glucosidase之特性 68
1. 最適溫度及pH 68
2. 溫度及pH穩定性 68
3. 金屬離子、醣類及EDTA對於β-glucosidase活性的影響 69
4. β-glucosidase與大豆異黃酮之反應 70
第五章、結論 78
第六章、參考文獻 79
圖目錄
圖2.1植物固醇之結構 7
圖2.2大豆中異黃酮之化學結構式 9
圖2.3異黃酮代謝物與雌激素代謝物化學結構式之比較 11
圖2.4異黃酮之代謝物排泄途徑及腸道微生物之影響 12
圖2.5大豆食品中genistein化學形式之轉換 14
圖2.6人體對異黃酮之吸收代謝簡要圖示 16
圖2.7加工溫度對大豆異黃酮化合物的影響 20
圖2.8加熱對豆漿中大豆異黃酮化合物轉換的影響 21
圖2.9雞豆中之花黃素異黃酮與雞豆異黃酮之共軛反應以及推測可能之代謝反應 26
圖2.10雞豆中異黃酮共軛物酵素調控示意圖 27
圖2.11由大豆產物中萃取及分離出異黃酮之流程圖 30
圖2.12由葛根中萃取及分離出異黃酮之流程圖 31
圖3.1實驗架構圖 35
圖3.2大豆異黃酮HPLC分析之流洗梯度 39
圖4.1 40℃熱處理對大豆均質液中異黃酮轉換之影響 48
圖4.2大豆粉60%甲醇粗萃物之HPLC分析圖譜 50
圖4.3通過HP-20管柱之40%甲醇洗出液HPLC分析圖譜 51
圖4.4通過HP-20管柱70%甲醇水溶液洗出部分之HPLC分析 52
圖4.5通過HP-20管柱之70%甲醇流出液於95℃加熱8小時後HPLC分析圖譜 53
圖4.6 Malonylglucosides產物 56
圖4.7 Glucosides產物 56
圖4.8不同鹽濃度對酵素活性之影響 59
圖4.9各丙酮沉澱中蛋白質之β-glucosidase酵素活性之相對含量 60
圖4.10未還原之β-glucosidase之CM-Sepharose CL-6B陽離子交換層析圖譜 61
圖4.11未還原之β-glucosidase之膠體過濾層析圖譜 62
圖4.12還原之β-glucosidase之CM-Sepharose CL-6B陽離子交換層析圖譜 63
圖4.13還原之β-glucosidase之膠體過濾層析圖譜 64
圖4.14電泳分析 65
圖4.15溫度對β-glucosidase活性之影響 71
圖4.16 pH對β-glucosidase活性之影響 72
圖4.17 β-glucosidase對溫度之穩定性 73
圖4.18 β-glucosidase對pH之穩定性 74
圖4.19 β-glucosidase與glucosides反應前後之HPLC圖譜 76
圖4.20 β-glucosidase與malonylglucosides反應前後之HPLC圖譜 77

表目錄
表2.1黃豆之一般組成分及礦物質含量 4
表2.2各類大豆食品中異黃酮之含量 24
表3.1本研究計算大豆異黃酮含量所採用之RRF與分子量 41
表4.1大豆異黃酮粗萃液及其HP-20管柱區分中之異黃酮含量 54
表4.2大豆異黃酮分離純化之效果 55
表4.3未還原之β-glucosidase純化表 66
表4.4還原之β-glucosidase純化表 67
表4.5各種金屬離子、醣類及EDTA對β-glucosidase酵素活性之影響 75

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