A novel method for highly efficient biotransformation and separation of isoflavone aglycones from soybean with high-speed counter-current chromatography

Abstract

A novel bioreactor with high-speed counter-current chromatography (HSCCC) was established for the efficient biotransformation of isoflavone aglycones from soybeans. Further, the transformed isoflavone aglycones were separated by HSCCC with continuous injection mode. β-Glucosidase and snailase were chosen as the optimum enzymes for HSCCC bioreactor biotransformation. Compared with snailase, β-glucosidase showed the advantages in both enzymatic hydrolysis efficiency and the influence to retention of the stationary phase and 4 mL/min were selected as the optimum elution flow-rate. The transformed isoflavone aglycones were separated by HSCCC with a solvent system n-hexane/ethyl acetate/ethanol/water (1:1.6:1:1.6, v/v). From 200 mg crude samples, 82 mg of daidzein, 48 mg of glycitein and 24 mg of genistein were separated with purities over 95%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by EI-MS and 1H-NMR. Our results suggest that HSCCC could be developed as a bioreactor in the preparation of isoflavone aglycones from soybeans for the facility to immobilize and recycle the enzyme.