利用微生物轉化生產水溶性大豆異黃酮條件之探討

Studies on the Processes for Producing Water Soluble Soy Isoflavones by Bioconversion

林綉霞(Sao-Ha Lam)

指導教授 ： 蘇南維

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01401

摘要

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%。

關鍵字

大豆異黃酮 ； 生物轉化 ； daidzein-7-O-phosphate ； genistein-7-O-phosphate

並列摘要

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%.

並列關鍵字

Soy isoflavone ； Biotransformations ； daidzein 7-O-phosphate ； genistein 7-O-phosphate

參考文獻

何曉雯。2012年。納豆菌對大豆異黃酮轉化之研究。國立台灣大學農業化學系研究所碩士論文。

常致綱。2006年。大豆異黃酮定量方法之改良及加工方式對大豆異黃酮種類轉換之研究。國立台灣大學農業化學系研究所碩士論文。

鄭光成。2005年。利用根黴菌之深部培養法開發機能性醱酵黑豆食品之研究。國立台灣大學微生物與生化學研究所碩士論文。

何幸容。2008年。納豆菌發酵豆奶的開發及其品質評估。國立中興大學食品暨應用生物科技學系碩士論文。

Adlercreutz H, Bannwart C, Wahala K, Makela T, Brunow G, Hase T, Arosemena PJ, Kellis JT, Jr., Vickery LE (1993) Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. The Journal of steroid biochemistry and molecular biology 44: 147-153