經由生物轉化程序生產金雀異黃酮磷酸酯之研究

Abstract

存在於黃豆中的異黃酮是黃豆的二級代謝物，可與人體雌激素受體 (estrogens receptor) 結合，被稱為植物性雌激素 (phytoestrogens)。黃豆異黃酮依其化學結構不同，可分成四大類: malonyl-glucosides、acetyl-glucosides、glucosides和aglycones，其中以不帶醣基之異黃酮 (aglycones) 有較佳之生理活性。許多文獻指出屬於aglycones類的金雀異黃酮 (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，根據 Biopharmaceutics Classification System (BCS) 分類，genistein 屬於第四類化合物─低水溶性，低穿透性，造成 genistein之生物可利用率 (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)。 G7P不僅水溶性佳，且可增加genistein之生物可利用率。本研究透過 B. subtilis BCRC 80517 進行生物轉化，以 genistein 作為轉化基質，來生產 G7P，並探討放大發酵規模來評估生產 G7P的可行性。首先探討 5 公升發酵槽生產 G7P 的培養條件，進而探討利用控制溶氧、攪拌速率及消泡劑的使用等，採用饋料批次 (fed-batch) 發酵生產方式，有最佳的 G7P 轉換效率及產量，可於發酵時間 48 小時內，將 35 公克 genistein 轉化生成 G7P。放大至 50 公升，可於發酵時間 48 小時內，將 1260公克 genistein 轉化生成 G7P。

Isoflavones are a group of plant secondary metabolites that occur mostly in the subfamily Papilionoideae of the Leguminosae. Soybean (Glycine max) is the most abundant source of isoflavones. To date, 12 natural isoflavones have been found in soybeans, consisting of 3 types of isoflavone aglycones (namely daidzein, genistein and glycitein), and their corresponding conjugates with glucose, acetylglucose and malonylglucose. Genistein 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. This study aimed to scale the bioconversion process up from 5-L fermenter to 50-L fermenter for the production G7P, and developing a feasible and promising process for recovering G7P from fermentation broth. At first step, we reexamed the culture medium for production G7P in 5-L fermentation tank. And the results showed that the fed-batch fermentation process can achieve the best G7P conversion efficiency and production yield, with 35g genistein could be converted to G7P in 48 hours during fermentation process. Bioconversion process scale up from 5-L to 50-L can convert 1260 g genistein to G7P within 48 hours during fermentation process. Based on the results of this study, it was shown that G7P can increase the production yield by bioconversion process scale up.