臺灣博碩士論文加值系統

近年來大豆異黃酮(isoflavone)被研究發現具有改善女性更年期症狀、預防心血管疾病、預防骨質疏鬆症及癌症等功效，而其中以無配醣基之異黃酮(aglycones)效果最佳，但由於aglycones的水溶性不佳，導致生物可利用率(bioavailability)差。根據本研究室先前發現利用Bacillus subtilis FC-10能把大豆異黃酮中的aglycones轉化成水溶性佳之daidzein-7-O-phosphate (D7-P)和genistein-7-O-phosphate (G7-P)，因此推測能夠有效改善其生物可利用率。
　　本研究探討生產水溶性大豆異黃酮之生物轉換條件及產物回收之程序。分為三個部分進行，第一部分以B. subtilis FC-10分別利用daidzein及genistein進行生物轉換，所得培養液中的D7-P與G7-P再經過一連串的分離、純化步驟製備純化的D7-P及G7-P作為定量用之標準品。第二部分探討B. subtilis FC-10在培養之不同時間點加入基質與不同基質濃度之生物轉化效率。第三部分探討利用黃豆作為轉化基質之可行性。結果顯示，基質在B. subtilis FC-10的適應期間(lag phase)添加至培養液中培養，相較於在對數期(log phase)及靜止期(stationary phase)加入基質之轉化效率較佳，其轉化率可達約94%。此外，經生物轉換後之培養液利用乙酸乙酯萃取、濃縮、正己烷沉澱及HP-20大孔樹脂之分離純化步驟後，D7-P和G7-P在產物中的純度能夠從原來的2.76%提升至83.1%且回收率為97.3%。利用濕式滅菌加熱方式能有效把黃豆中之malonyl-glucosidic isoflavones轉變成β-glucosides及aglycones。比較不同處理的黃豆做為生物轉換基質的結果顯示，未經濕熱處理的黃豆基質生物轉換效率較差，為37.0%，經濕熱處理後的黃豆基質生物轉換效率較佳，為85.6%。

Soy isoflavones are the components responsible for estrogenic responses observed in vitro and in experimental animals. The beneficial effects of isoflavones include: the reduction of serum lipids, increase of bone mineral density, relief of menopausal symptoms, and chemoprevention of mammary and prostate cancer. A number of studies have revealed that the biological effects of isoflavones mainly attributed to their aglyconic forms rather than the glycosidic forms. However, many studies reported that aglycones show low bioavailability to humans because of their poor water-solubility.
In the previous work of our lab, a Bacillus subtilis strain designated as Bacillus subtilis FC-10 showed the capability on the biotransformation of daidzein and genistein into daidzein-7-O-phosphate and genistein-7-O-phosphate. The objective of this thesis is develop a procedure for recovering D7-P and G7-P from the culture broth of biotransformation, including exploring some factors on biotransformations for recovering D7-P and G7-P. Moreover, we investigate the feasibility of using soybean powder as the substrate for the transformation to produce D7-P and G7-P. The results indicated that B. subtilis FC-10 was inoculated into the initial culture medium containing the biotransformation substrate isoflavone showed the highest bioconversion rates were 95.7% for D7-P and 94.4% for G7-P. EtOAc is an efficient solvent for the extraction of D7-P and G7-P from culture broth. The purity and the recovery of the products through this operation by the extraction of 1.2 fold EtoAc for 4 times were 37.9% and 97.5%, respectively. Furthermore, the products containing D7-P and G7-P were purified by adding various ratios of n-hexane to EtOAc extract and the products were recovered from the precipitation. The purity and the recovery of D7-P and G7-P in the precipitate of 88.9% n-hexane were 47.6% and 97.2%, respectively. Then the precipitate was dissolved with little amount of deionized water and was passed through HP-20 resin column. The fraction containing the most D7-P and G7-P was collected from 15% and 30% MeOH eluates of the HP-20 resin adsorpted fraction. The purity and recovery of products containing D7-P and G7-P were 83.1% and 94.6%, respectively. Moreover, soybean powder was a feasible for D7-P and G7-P production. However, pretreatment processes of soybean substrate would seriously affected the conversion rate of D7-P and G7-P. Autoclaved treatment with 85.6% conversion rate seemed to be a better choice where as the conversion rate for soybean was only 37.0%.

中文摘要 i
目錄 I
圖目錄 IV
表目錄 VI
縮寫表 VII
第一章 前言 1
第二章 文獻整理 2
第一節、黃豆 2
第二節、植物性化學物質 3
2-1 植物性雌激素 3
2-2 類黃酮 3
第三節、大豆異黃酮 5
3-1 異黃酮之生理活性 5
3-2 大豆異黃酮之吸收與代謝 10
第四節、前驅藥物 15
第五節、生物分離與純化 15
5-1 生物分離工程之應用 15
5-2 萃取分離 16
5-3 吸附作用 17
5-4 大孔網狀聚合物吸附劑 18
5-5 離子交換 18
第六節、黃豆發酵食品 22
6-1 納豆 22
6-2 納豆激酶 23
第三章 材料與方法 25
第一節、實驗大綱 25
第二節、實驗材料 25
2-1 菌株 25
2-2 黃豆 25
2-4 試藥與溶劑 26
2-4 培養基 26
第三節、實驗儀器 29
第四節、分析方法 30
4-1 高效液相層析法之異黃酮分析條件 30
4-2 大豆異黃酮含量計算方式 30
4-3 半製備級高效液相層析儀之D7-P與G7-P之分析條件 31
第五節、實驗方法 32
5-1 利用陰離子交換樹脂分離培養液中之D7-P與G7-P 32
5-2 D7-P與G7-P標準品之製備 32
5-3 B. subtilis FC-10種菌之生長曲線 34
5-4 生產D7-P與G7-P條件之探討 34
5-5 分離與純化培養液中D7-P與G7-P 35
5-6 不同加熱條件對於黃豆中異黃酮之型態的影響 35
5-7 利用黃豆粉作為生物轉化基質之探討 36
5-8 納豆之製備(固態發酵) 36
5-9 統計分析 37
第四章 結果與討論 38
第一節、利用陰離子交換樹脂分離培養液中D7-P與G7-P 38
第二節、探討B. subtilis FC-10生物轉化之條件 41
第三節、回收及純化D7-P及G7-P 47
第四節、探討利用黃豆作為轉化基質之可行性 58
1. 分析不同台灣品種黃豆之大豆異黃酮含量 58
2. 不同加熱條件處理對大豆異黃酮之形態之改變 58
3. 以黃豆粉作為B. subtilis FC-10生物轉化基質(液態培養) 59
4. 利用B. subtilis FC-10與黃豆進行生物轉化(固態培養) 59
5. 儲藏安定性實驗 60
第五章 結論 68
第六章 參考文獻 69

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