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標題: | 經由微生物轉化程序探討金雀異黃酮磷酸酯之生產與回收 Studies on the Production and Recovery of Genistein 7-O-phosphate via a Biotransformation Process |
作者: | Kung-Hao Kuo 郭功浩 |
指導教授: | 蘇南維(Nan-Wei Su) |
關鍵字: | 發酵槽,枯草桿菌,生物轉化,異黃酮磷酸酯衍生物,膨脹床吸附,鈉鹽製備,膜過濾, Bacillus subtilis,isoflavone,biotransformation,bioreactor,expanded bed adsorption,genistein 7-O-phosphate,sodium salts,membrane filtration, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 異黃酮(isoflavone)是植物中的二次代謝物,其化學結構與人體雌激素(estradiol)相似,因此具有雌激素的活性,被稱為植物雌激素(phytoestrogen)。異黃酮依化學結構不同,可分成四大類: malonyl-glucosides、acetyl-glucosides、glucosides和aglycones。而其中以不帶醣基之異黃酮(aglycones)有較佳之生理活性。近年來,許多文獻指出屬於aglycones類的genistein具有預防骨質疏鬆、抗心血管疾病、乳癌及前列腺癌等多項生理活性。然而,genistein不溶於水的特性,使其生物可利用率(bioavailability)不佳。本研究室先前篩選出Bacillus subtilis BCRC 80517菌株可對genistein進行磷酸酯化修飾,形成genistein 7-O-phosphate (G7P),轉換後產物G7P可大幅提升genistein的水溶性,經動物實驗證實,可增加genistein之生物可利用率。然而,先前並未利用發酵槽進行genistein生物轉換的探討及放大規模純化。因此,本論文即以此為基礎,進行後續的研究。
本研究分為三部分。第一部分,利用5 L通氣攪拌式發酵槽放大發酵製程生產G7P。結果顯示,發酵槽以最適化批次(batch)式發酵生產,最高投入轉換基質genistein濃度為18.52 mM,可於發酵時間30小時,達到100%的完全轉換,生成G7P 18.87 mM,相較搖瓶振盪培養,提高轉換基質genistein濃度達9倍之多。再者,以最佳二次性饋料(fed-batch)式發酵生產,可於發酵時間48小時內,使37.0 mM之轉換基質genistein達100%之完全轉換,生成G7P 38.21 mM,相較搖瓶振盪培養,提高轉換基質濃度達18倍之多。半連續式發酵結果顯示,最適種菌保留量為15%,前兩個批次發酵皆能維持100%之完全轉換,於第三個批次開始轉換率大幅下降。第二部分,建立自發酵液回收G7P之最適化程序。結果顯示,利用微過濾膜進行發酵液之濃縮,濃縮液以三倍體積乙酸乙酯萃,減壓濃縮至乾後,回溶於90%乙醇溶液,再以1 N NaOH溶液調鹼,使G7P-2Na析出,析出產物G7P-2Na經隔夜烘乾後,G7P-2Na之純度及回收率為89% (w/w)與67%;通過微過濾膜之濾液,以HP-20膨脹床管柱進行純化,經過一倍管柱體積之pH 1、5% NaCl水溶夜清洗再以兩倍管柱體積之0.1 N NaOH水溶液進行脫附,脫附液經凍乾,得到產物G7P-2Na之回收率及純度為90%及72%。第三部分,評估G7P-2Na之降解動力學與物理特性。結果顯示,G7P-2Na粉末之降解為一級反應,於常溫下非常安定,幾乎不會降解,G7P-2Na粉末之活化能為24.61 kcal/mole;將G7P-2Na添加於糖水中,G7P-2Na之活化能(Ea)下降至14.06 kcal/mol,推算儲存於23.5 °C下之半衰期(T0.5)及降解20%(T0.8)之值,分別為35天及11天。Genistein、G7P和G7P-2Na在25oC下水中的溶解度分別為4.7、1.2×105 mg/L與1.1×108 mg/L。顯示G7P之溶解度比genistein高出2萬5千倍之多,然而,G7P-2Na之溶解度又比G7P高出1000倍之多。G7P若以鈉鹽形式存在其熔點為289oC,相較G7P之熔點高出57 oC。本研究結果顯示,G7P確實具有潛力放大規模生產,開發為新穎性之保健產品。 Genistein, one of the polyphenolic second metabolites in soybeans, has a number of pharmacological and biological activities; however, low water solubility and poor bioavailability limit its use. Our previous study revealed a water-soluble phosphate conjugate of genistein 7-O-phosphate (G7P), generated by biotransformation of Bacillus subtilis BCRC 80517 with genistein. In pharmacokinetics studies, G7P greatly improved the maximum plasma level and bioavailability of its corresponding genistein in vivo. However, the present studies only use shake flasks to produce G7P. This study aimed to scaling the fermentation process up from shake flasks to laboratory-scale bioreactor for the production of G7P, and developing a feasible and promising process for recovering G7P from fermentation broth. In the first part of this thesis, a fermentation process for G7P production by Bacillus subtilis BCRC 80517 was successfully scaled up from 500 mL shake flask to 5 L stirred tank bioreactor. At first, through the optimal batch fermentation process, at the end of 30 h incubation time, the bioconversion rate of genistein were 100%, and the biotransformed product G7P in the harvested broth were 18.87 mM, compare to the previous flask studies, the concentration of G7P enhanced up to 9-fold. Second, we have developed a successful fed batch fermentation strategy, which can produce up to 38.21 mM of G7P at the end of 48 h incubation time, enhanced the productivity of G7P by 18 times compare with the previous flask studies. To further increase the G7P productivity, the fed batch fermentation process was repeated 4 times with cell reused. The results showed that repeated fed batch fermentation was effective in improving the fermentation efficiency and maintaining high G7P productivity for the first and second batch fermentation. In the second part of this thesis, we developed a feasible and promising process for recovering G7P from fermentation broth. At first, using micro-filtration for fermentation broth concentrating, the retentate part through EA extraction and pH adjustion, we could obtained G7P with 67% recovery and 89% (w/w) in purity. The permeate from membrane filtration through HP-20 expanded bed adsorption, washing and 0.1N NaOH solution desorption, G7P with 90% recovery and 72% (w/w) in purity was obtained. At the third part of this thesis, according to the Handbook of Pharmaceutical Salts, we chosed sodium ion as salt formers to form G7P-2Na. The results of storage test showed that G7P-2Na’s activation energy (Ea) was 24.61, exhibiting very stable characteristics. When the G7P-2Na was added to the syrup, the G7P-2Na’s activation energy (Ea) was dropped to 14.06 kcal/mol. The water solubility at 25oC of genistein, G7P and G7P-2Na were 4.7, 1.2×105 mg/L, and 1.1×108 mg/L respectively. The water solubility of G7P were 25,000-fold higher than genistein, however G7P-2Na were 1000-fold much higher than G7P. At the end of the studies, we found that the melting point of G7P-2Na were 289 oC higher than the 232 oC of G7P. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77703 |
DOI: | 10.6342/NTU201703264 |
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顯示於系所單位: | 農業化學系 |
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