Extraction and determination of the converted
products
At the end of the transformation period, contents of each
flask (cells and medium) were acidified with 1 N HCl to pH
2. The reaction mixture was extracted twice with double its
volume of ethyl acetate. Then, the organic layer was
washed three times with water to get rid of any residual
HCl, dried over anhydrous sodium sulphate and
evaporated to dryness in vacuo to give solid residue (test
material) (Kim et al. 1999). The residue was dissolved in
chloroform - methanol mixture (1:1) and mounted on TLC
plates. The plate was first chromatographed for GA and 3-
oxo-GA with solvent system A, and secondly for GL with
solvent system B (Akao et al. 1991). GL, GA and 3-oxoglycyrrhetic
acid (3-oxo-GA) were detected on TLC plates
under UV light or by acid charring (10 % H2SO4, 110°C,
10 min).These compounds were quantitatively analyzed
with TLC scanner (Shimadzu CS-9000 dual wave-length
flying spot, thin layer chromato-scanner, Japan) at λs
=250 nm and λr = 400, by using calibration lines obtained
from authentic samples (Hattori et al. 1985).