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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. |