The reaction mixtures were extracted twice with 200 μL of water-saturated n-butanol. The n-butanol fraction was evaporated to generate the crude saponin fraction with a rotary vacuum evaporator (N-1000V, EYELA, Tokyo, Japan). Crude saponin was dissolved in 50 μL of methanol, which was subjected

to TLC and HPLC determination. The samples were then passed through a 0.45 μm PTFE syringe filter (Whatman, Brentford, Middlesex, UK) prior to injection. TLC was conducted on silica gel 60F254 plates. A solvent mixture of chloroform:methanol:water (65:35:10 v/v/v, lower phase) was used Baf-A1 solubility dmso as the developing solvent. The spots were detected by spraying with 10% sulfuric acid followed by heating under a lamp flame until the spots became clearly visible. Ginsenosides and transformed ginsenosides were identified and assayed via comparison with known ginsenoside standards. HPLC was conducted using an Agilent 1100 system (Agilent Technologies) at a detection wavelength of 203 nm. The column used was a reverse-phase column (C18, 4.6 mm × 150 mm, 5 μm) and an injection volume of sample was 20 μL. The mobile phase utilized gradient conditions with solvents A (CH3CN:H2O = 100:0) and B (CH3CN:H2O = 14:86).

The solvent A and B ratios were as follows: [20% A (0 min)]; 20% A (5 min); 30% A (10 min); 30% A (15 min); 60% A (20 min); 60% A (23 min); 0% A (25 min)] with a 1.2 mL/min flow rate. Each experiment was individually repeated three selleck compound times. All data were assigned for purposes of comparison and an analysis of variance (ANOVA) was carried out by using SPSS version 8.0 (SPSS Inc., Chicago, IL, USA). A p-value of MTMR9 <0.05 was considered significant. Aspergillus species are known as a useful source of β-glucosidase and A. niger is by far the most efficient β-glucosidase producer among the microorganisms investigated thus far. Changes in the growth and β-glucosidase activity of A. niger KCCM 11239 on potato dextrose broth medium at 30°C were evaluated under

aerobic conditions (data not shown). Very little β-glucosidase was detected in the culture broth until 12 d, but then the activity dramatically increased and reached to a maximum level (197.7 U/mL) after approximately 16 d. After that time, it appeared that the activity was slightly decreased by protease existing in culture broth. From the results, it is presumed that a production pattern of β-glucosidase is nongrowth associated type. The microbial conversion of ginsenoside Rb1 was prepared by inoculating ginsenoside Rb1 into precultured suspensions, followed by 16 d of incubation of the mixture at 30°C and 200 rpm. The microbial conversion was checked via TLC at 2-d intervals. During the 2-d growth period, much of the enzyme seemed to be produced, as evidenced by the observation of increased ginsenoside Rg3 levels. Fig. 1 shows that most of the ginsenoside Rb1 was converted to ginsenoside Rg3 and generated less polar metabolites after 4 d of incubation.