納豆菌對大豆異黃酮轉化之研究

Abstract

近來大豆異黃酮對人體的保健功效獲得全世界的關注。大豆中的異黃酮可分為四大類：glucosides、malonylglucosides、acetylglucosides 及aglycones。在這些異黃酮中，僅有aglycones 具有生物活性，而glucosides 須經由腸道微生物 轉化成aglycones 之型式才可被腸道吸收。因此大多數研究著力於以酵素水解或藉由微生物將glucoside conjugates 轉化成isoflavone aglycones，但本研究發現isoflavone aglycones 可進一步被微生物轉化。 本研究從市售納豆篩選出24 株納豆菌株Bacillus subtilis strains，以生物轉化方式轉化glucosides，發現glucosides 會被轉化為aglycones，有23 株菌株可進一步的轉化aglycones 產生新的成分。將發酵液以HPLC 分析，發現aglycones轉化之物質可能為新的異黃酮衍生物(derivatives)，暫且命名為NDA1、NDA2。 以液相層析質譜質譜儀(LC-MS/MS)分析，並與aglycones 的質譜結果比較，結果顯示這兩種未知成分NDA1、NDA2 確實為aglycones 的衍生物，NDA1之m/z: 335 (M+H+)，NDA2 之m/z: 351 (M+H+)。以NMR 鑑定NDA1 為Daidzein-7-O-phosphate；NDA2 為Genistein-7-O-phosphate。Daidzein-7-O-phosphate 與Genistein-7-O-phosphate 的水溶性比aglycones 佳，有利於後續之開發利用。

Isoflavones in soybean gained worldwide attention because of their benefits for human health. There are four chemical forms of natural isoflavones existing in soybean namely glucosides, malonylglucosides, acetylglucosides, and aglycones. Among these isoflavones, only aglycones rather than predominant glucosidic conjugates show their bioactivities to human. Glucosidic conjugates have to be transformed into their corresponding aglycone prior adsorption and utilization by intestinal microflora in human. Hence, most of the relevant studies were devoted on the conversion of glucosidic conjugates of isoflavone into their corresponding aglycones, no matter by hydrolysis using glucosidic enzymes or directly fermented/conversed through microorganisms. In this study, 24 strains of Bacillus subtilis natto selected from commercial products of natto were employed to conduct the conversion of soy isoflavones. According to the HPLC chromatograms of isoflavone analysis, several new components were observed and considered to be possible novel derivatives of isoflavones based on the growth of peaks representing novel derivatives along with the decline of peaks representing isoflavone aglycones. Almost all of the strains used in this study were able to converse soy isoflavone aglycones to the corresponding novel derivatives. Two predominant novel derivatives namely NDA1 and NDA2 were considered with a relationship to daidzein and genistein, respectively. The identification of NDA1 and NDA2 was conducted by LC-MS/MS and NMR spectra. The results indicated that NDA1 was possible a daidzein conjugate with an italic 335 (M+H+), and NDA2 was a genistein conjugate with an italic 351 (M+H+). The possible chemical formula of NDA1 and NDA2 were characterized respectively as daidzein-7-O-phosphate and genistein-7-O-phosphate by NMR. The two novel isoflavone conjugates with better solubility than their original forms aglycone would be advantageous to further application.