الفهرس 
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Abstract
Licorice root contains triterpenoid saponins (4-20% of dry weight, mostly glycyrrhizin (GL) a mixture of potassium and calcium salts of glycyrrhizic acid (also known as glycyrrhizinic acid and a glycoside of glycyrrhetinic acid). Glycyrrhizic acid is a molecule composed of a hyDROPhilic part, two molecules of glucuronic acid, and a hyDROPhobic fragment, glycyrrhetinic acid. This is 50 times as sweet as sugar. By hydrolyzing one molecule of glucuronic acid, GL is transformed
into glycyrrhetinic acid mono-glucuronide (GAMG), which has several
advantages over GL, as a sweetener with high sweetness and low calorie;
sweetener is 941 times of the sucrose, and 5 times of the GL. 18-
glycyrrhetinic (GA) acid has shown anti-inflammatory properties, a better
hepatoprotective effects than GL in in vitro study, anti-ulcer, antihyperglycemic
by lowered plasma glucose with a simultaneous increase in the
insulin secretion, anti-viral, anti-allergic and anti-bacterial.
In the present study, licorice roots were used to separate the
Glycyrrhizic acid (as the fermentation inducible substrate) and the amount of
GL crude extract was 5.38 g per 100 g ground licorice roots. With a purity of
64.44 % was estimated by using HPLC.
Trichosporon jirovecii yeast was used for GA production using GL
extracted from licorice roots. Nine independent factors including initial pH,
temperature, GL concentration, GL addition time, incubation time, glucose
concentration, yeast extract, corn steep liquor (CSL) and aeration ratio were
surveyed and effective variables for GA production were determined using a
Plackett-Burman design. Results analysis showed that incubation time of 7
days; pH 6; yeast extract 0.3 %; glucose 1.0 % and CSL 0.8 % were the most
Summary
94
significant values for the highest level of GA production respectively and the
highest GA concentration content measured was 114 mg using 0.6 % GL.
The central composite design (CCD) combined with response surface
methodology (RSM) was used to optimize the conditions for GA production
from GL by the Trichosporon jirovecii yeast. Five effective variables (which
obtained from previous Plackett-Burman design) including: initial pH,
incubation time, glucose conc., yeast extract conc. and corn steep liquor conc.
were screened in 32 runs.
The highest record GA accumulation (158.0 mg) was achieved by the
trial no. 30 at the 7th day of incubation on using 0.6 % GL, with a GA yield of
71.43 % with an increase of 28.85 %, more than that obtaining by the
Plackett-Burman design.
pH, 7; incubation time, 7 days; glucose, 1 %; yeast extract, 0.3 % and
CSL, 0.8 % were the most significant values of the tested parameters using
the CCD for the GA production.
The enzyme activity of -glucuronidase was measured by -
nitrophenyl ß-D-glucuronide, and the highest activity was 0.591 mU. / ml.
One major peak from fractions 40 to 64 was obtained during the
purification of crude GA using silica gel column equilibrium with the
methanol and chloroform. The infrared (IR) spectroscopy was showed
absorption band at 3443.28 cm-1 in sample which mainly related to hydroxyl
group (–OH) functional group, other absorption band at 1642.09 cm-1 which
mainly related to C=O group which ranging from 1630 to 1900 cm-1. There
was absorption band at 1460.81 cm-1 related to C=C and other peaks at
2924.52, 2856.06 cm-1. And a molecular fragment for the GA was at m/z 470
with relative intensities 8.86 % using mass spectroscopy.
Summary
95
One fraction that was precipitated from the fermented broth by 60 %
ammonium sulphate reacted with the chromogenic substrate X-gluc (5-bromo
-4-chloro-3-indolyl -D-glucuronic acid) substrate at 500 g concentration
using acetate buffer pH 5.0. The blue color intensity was increased after the
7th day of incubation.
Conjugated bilirubin (CB) was used as a substrate for the enzyme ß-Dglucuronidase;
this to confirm the Trichosporon jirovecii capability for its
production. Maximum lowering in CB values with marked significant (p
0.05) were observed at pH 6.0 and pH 7.0 with 500 g partially purified -
glucuronidase enzyme and the CB concentrations in serum samples were
decreased from (10.39 and 10.94 mg/ 100 ml) to (8.82 and 9.11 mg/ 100 ml)
respectively.
Unconjugated bilirubin (UCB) values were increased under the acid
and neutral pH values and the highest UCB value was measured at pH 7.0
with marked significant difference than control without enzyme, and UCB
concentration was increased from 1.68 to 2.83 mg/ 100 ml.
The antimicrobial activities of the methanolic extract of licorice roots,
crude extract of GL, crude extract of GA and GA purified fraction were
examined against four Gram positive and Gram negative bacteria cultures,
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and
Bacillus subtilis using the zone of growth inhibition test and the results was
evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) reagent. GA in
both fractions and methanolic extract showed antibacterial activity with gram
negative and gram positive bacteria and GL showed antibacterial activity with
gram positive bacteria only. The lowest MIC which could inhibit microbial
growth was recorded using 20 g of the purified extract of GA against E. coli
and B. subtilis.