大豆中genistein、daidzein、genistin及daidzin 之分離程序及以Bacillus subtilis BCRC 19679 進行生物轉換之研究

Abstract

許多研究指出大豆異黃酮具有保健生理活性，為天然的植物雌激素。已知大豆中異黃酮可分為四類共12種化合物，含量最高的為malonylglucoside形式之異黃酮，其次為glucoside形式之異黃酮，而aglycone 形式的含量最少。本研究室先前篩選得到Bacillus subtilis BCRC 19679菌株，可將genistein、 daidzein、genistin及daidzin轉化成水溶性佳之Genistein-7-O-phosphate (G7P)和Daidzein-7-O-phosphate (D7P)，推測能有效改善兩種異黃酮的生物可利用率。本研究室先前的研究係利用混和物形式進行轉化，對各別異黃酮的生物轉換尚未明瞭。本研究旨在探討Bacillus subtilis BCRC 19679對個別異黃酮生物轉化的情形，實驗分為兩部分，其一為製備genistein、daidzein、genistin及daidzin之回收程序，用以做為生物轉化之原料。另一為探討Bacillus subtilis BCRC 19679對上述異黃酮的生物轉化情形。結果顯示，中性氧化鋁可有效分離genistein及daidzein。經由本研究建立的程序，可由原料(genistein : 56 %，daidzein : 26 % )分離出genistein (純度：95 %；回收率：89 %) 及daidzein (純度：92 %；回收率：94 %)。鈣離子沉澱法可有效分離genistin及daidzin。可由原料 (genistin: 38 %，daidzin: 8 %) 得到genistin (純度：92 %；回收率：86 %) 及daidzin (純度：91 %；回收率：92 %)。分離純化程序在本研究已在本研究中被建立。 第二部分為分離出之各別異黃酮生物轉換之探討：B. subtilis BCRC 19679 對genistein有良好轉換效率，於500-2000 mg/L濃度區間，轉換率即大於90%。比轉換速率的最大值1890 μM g-1h-1發生於以500 mg/L genistein 為起始培養濃度、轉換6小時。G7P生成濃度最大值6700 μM 發生於以2000 mg/L genistein為起始培養濃度，轉換48小時，轉換率達到92 %。比生長速率最大值與G7P比生成速率有正相關性，屬於菌體連動型發酵模式。B. subtilis BCRC 19679 對daidzein有良好的轉換效率，於480-940 mg/L濃度區間，轉換率即大於 90 %。 iv 比轉換速率的最大值1400 μM g-1h-1發生於以1400 mg/L daidzein為起始培養濃度、轉換6小時。D7P生成濃度最大值4800 μM 發生於以2800 mg/L daidzein為起始培養濃度；轉換48小時，轉換率43%。比生長速率最大值與D7P比生成速率無連動性，屬於非菌體連動型發酵模式。B. subtilis BCRC 19679 對genistin有良好的轉換率，於1600-2400 mg/L genistin濃度區間，轉換率即大於90%。比轉換速率最大值340 μM g-1h-1發生於以1600 mg/L genistin 為起始濃度、轉換24小時。genistin會先轉換為genistein後，再轉換為G7P，為兩段式轉換模式。B. subtilis BCRC 19679 對daidzin之轉換率，於1540-1840 mg/L daidzin濃度區間，轉換率大於70%。比轉換速率最大值230 μM g-1h-1發生於以1840 mg/L daidzin 為起始濃度、轉換24小時。daidzin會先轉換為daidzein後，再轉換為D7P，為兩段式轉換模式，故可得知B. subtilis BCRC 19679同時具有水解glucosidic isoflavone及磷酸化aglyconic isoflavone的能力；所有以glucosidic isoflavone為基質的生物轉化組別皆有succinyl agluconic isoflavone的生成。

Soy isoflavones are the phytochemicals responsible for estrogenic activities observed in vitro and in vivo models. 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 and so on. A number of studies have revealed that the biological effects of isoflavones mainly attributed to their aglyconic forms rather than the glycosidic forms. However, aglycones have shown low bioavailability to humans due to their poor water solubility. In the previous work of our lab, a Bacillus subtilis strain designated as Bacillus subtilis BCRC 19679 showed the capability to convert daidzein and daidzin, as well as genistein and genistin into daidzein-7-O-phosphate (D7P) and genistein-7-O-phosphate (G7P). We considered that G7P and D7P could possess better bioavailability than aglyconic isoflavones. However, researches regarding individual isoflavone inclucding genistein, genistin, daidzein and daidzin that was transformed by B. subtilis BCRC 19679 are still not clear. The objective of this study is to explore the biotransformation of individual isoflavone by B. subtilis BCRC 19679. The experimental works consist of two parts. One is to develop the isolation procedure of individual isoflavone for further studies, and the other is to investigate the kinetic biotranformations of the isolated isoflavone by B. subtilis BCRC 19679. The results show neutral aluminum oxide is a promising and good-performing adsorbent for the separation of genistein and daidzein from aglyconic isoflavone mixture. After performing the separation in accordance with our procedure, genistein with 89% of recovery and 95% of purity, and daidzein 94% of recovery and 92 % of purity could be obtained from the raw material of aglyconic mixture containing 56% of genistein and 26% of daidzein. Moreover, genistin and daidzin can be separated effectively from glucosidic isoflavone mixture by means of a calcium precipitation vi process, and subsequently genistin with 86% of recovery and 92% of purity, and daidzin with 94% of recovery and 91% of purity could be obtained from the raw material of glucosidic mixture containing 38% of genistin and 8% of daidzin. Reguarding of the biotransformation of B. subtilis BCRC 19679 with each isolated isoflavone, the results showed that the bioconversion of daidzein and genistein have been going well with higher than 90% of conversion rate at the end of 48-h incubation with the concentration range of 500 to 2000 mg /L for genistein and that of 480 to 640 mg /L for daidzein Moreover, for the specific conversion rate, the maximum value of 1890 μM g-1h-1 occurred at the 6th h culture broth with an initial concentration 500 mg/L of genistein, and 1400 μM g-1h-1for daidzein, the same as occurring at the 6th h culture broth with an initial concentration 1400 mg /L of daidzein. Moreover, at the end of 48-h incubation with initial substrate levels of 2000 mg/L for genistein and that of 2800 mg/L for daidzein, the coulture broth could contain maximum levels of 6700 μM for G7P and 4800 μM for D7P, respectively, The conversion rates for genistein and daidzein were 92% and 43% respectively. For the phosphorylation process with genistein by B. subtilis BCRC 19679, we observed specific growth rate was highly correlated to specific conversion rate, whereas this phenomenon did not be obsvered when daidzein was used as the conversion substrate. For isoflavone glucosides, the bioconversion of daidzin and genistin proceeded successfully with higher than 90% of conversion rate at the concentration range of 1600 to 2400 mg/L for genistin and that higher than 70% of conversion rate of 1540-1840 mg/L for daidzin at the end of 48-h incubation.Moreover for the specific conversion rate, the maximum value of 370 μM g-1h-1 occurred at the 24th h culture broth with an initial concentration 1840 mg/L of genistin, and 230 μM g-1h-1 for daidzin the same as occurring at the 24th h culture broth with an initial concentration vii 1600 mg /L of daidzin . In this research, genistin was first hydrolyzed to aglyconic genistein, and then converted to G7P which represented a two-stage bioconversion which suggested B. subtilis BCRC 19679 enable both hydrolysis of β-glucosidic bond and phosphorylation. Besides, succinyl glucosidic isoflavones could be observed from all the culture broths that obtained from daidzin and genistin as a substrate.