Study On Biotransformation Of Swertiamarin By Aspergillus Niger

Three fungi (Aspergillus niger, Aspergillus oryzae and Trichoderma koningii) and two intestinal bacteria (Lactobacillus brevis CICC 6004 and Escherichia coli CICC 10032) were applied to the biotransformation of swertiamarin. The result showed that only the three fungi are able to transform swertiamarin into three metabolites. Of the three fungi, Aspergillus niger was found to has the highest ability of metabolizing swertiamarin (approximately 36%).The purification of the metabolites was carried out by liquid-liquid extraction followed by semi-preparative HPLC separation. As a result, 11 mg of M1 (96% in purification) and 6 mg of M3 (95%in purification) were obtained. The structures of two metabolites were identified by NMR, high resolution MS, UV and IR spectra as (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c]pyridin-1-one and erythrocentaurin. Erythrocentaurin is a known active compound and the former was a novel compound. The following study of the in vitro anti-bacteria action showed that the novel compound has an activity of inhibiting the growth of Staphylococcus aureus and Escherichia coli.Theβ-glucosidase participating in the transformation of swertiamarin was isolated and purified from the broth of Aspergillus niger. The molecular weight of this enzyme was determined as approximately 88 KDa. The optimal pH and temperature were tested as 5.0-6.0 and 50-60℃, respectively. The enzyme was stable under the temperature lower than 60℃for 12 h. Mg2+ and Mn2+ activate, but Ca2+, Zn2+, Fe3+ and Cu2+ inhibit the enzyme. The enzyme has a high affinity atρ-NPG and cellobiose, but swertiamarin was proved to be a poor substrate of the enzyme. The inhibition constant of glucose on the enzyme was 0.25mM, which suggested that theβ-glucosidase could be completely inhibited by high concentration of glucose.The optimization of medium showed that the optimal concentrations of components of medium for producing M1 were MgSO4·7H2O 5.14mg/mL, MnSO4·4H2O 3.42 mg/mL, glucose 9.95mg/mL, peptone 9.24mg/mL and pH 5.80, and the biotransformation ratio for M1 was 17.64 %. The optimal concentrations of components of medium for producing M3 were MgSO4·7H2O 4.09mg/mL, MnSO4·4H2O 6.07mg/mL, glucose 7.37mg/mL, yeast extracts 7.37mg/mL and pH 5.73, and biotransformation ratio for M3 was 8.81 %.