ORIGINAL ARTICLE

Enrichment of biologically active 18-β glycyrrhetinic acidin Glycyrrhiza glabra root by solid state fermentation

Makhmur Ahmad & Mohammad Jalaluddin & Bibhu Prasad Panda

Received: 24 November 2012 /Accepted: 1 August 2013 /Published online: 24 August 2013# Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract This paper describes the in situ bioconversion ofglycyrrhizin ofGlycyrrhiza glabra root to 18-β glycyrrhetinicacid by solid state fermentation. Fermentation was carriedout with two different fungal strains, Penicillium chrysogenumand Rhizopus oryzae. The solid state fermentation was carriedout under stationary state and under rotating state. Penicilliumchrysogenum is a better producer of 18-β glycyrrhetinic acidthan Rhizopus oryzae . The induced P. chrysogenum seedculture produces higher 18-β glycyrrhetinic acid with2.955 mg g−1 and maximum β-glucuronidase activity of3,583.8 U ml−1 under stationary solid state fermentation. Themycelium growth and bioconversion rate is highest at pHof 5.5 and 4.5, respectively. G. glabra root supplementedwith a solution of dextrose 9 g l−1, MnSO4·H2O 3 g l−1 and(NH4)2SO4 0.540 g l−1 produces 48.580 mg of 18-βglycyrrhetinic acid per gram of G. glabra root, i.e. 86.74 %bioconversion by P. chrysogenum in 96 h under stationarystate solid state fermentation.

Keywords Glycyrrhizin . 18-β glycyrrhetinic acid . In situbioconversion . Solid state fermentation

Introduction

Glycyrrhiza glabra root, commonly known as sweet root andlicorice , is widely used in the Indian traditional system ofmedicine. In Ayurveda , it is used to relieve “Vata” and“Kapha” disorders and is popularly known as Yashtimadhu.It is also prescribed by “Vaidyas” for treating conditionsattributed to expectorant, emollient, anti-inflammatory, anti-viral, anti-hepatotoxic and antibacterial conditions (Shanker

et al. 2007). The major active component of G. glabra root issaponin, known as glycyrrhizin (GL), a triterpene glycoside,also known as glycyrrhizic acid (Farag et al. 2012). It has asimilar structure and activity as the adrenal steroids. It inhibitscyclooxygenase activity and prostaglandin formation, as wellas indirectly inhibiting platelet aggregation, all factors in theinflammatory process, by inhibiting phospholipase A2 activ-ity. GL is potentially valuable for HIV therapy, and inhibitsHIV replication by inhibiting a cellular Casein Kinase IIenzyme (Shabani et al. 2009).

Among all the important phytoconstituents ofG. glabra , i.e.liquiritic acid, glabranins A and B, glycyrrhetol, glabrolide,formononetin, liquiritin, isoliquiritin, and other phenolic com-pounds (Suman et al. 2009), the pharmacological activity is dueto its triterpene aglycone 18-β glycyrrhetinic acid (GA) and, inlesser measures, to its glycoside GL (Hansen et al. 1999; Liet al. 2008). After oral absorption, GL hydrolyzes to GA andglucuronic acid within the gastrointestinal tract by enzymes/microorganisms through an unknown metabolic pathway.GA is absorbed into the systemic circulation and producespharmacological action. Further, it is metabolized to 3β-monoglucuronyl-18β-glycyrrhetinic acid (3-MGA) in the liv-er and excreted out from the body in the urine. The biologicalactivity of GA is 20 times more potent than GL (Lu et al.2006). Because of lower absorption, the GL molecule is notadequately transported into the liver while GA is rapidlyabsorbed and transported via carrier molecules to the liver.Therefore, hydrolysis of the glycoside molecule GL will yielda more potent and easily absorbable aglycone fraction, GA(Hattori et al. 1985; Akao et al. 1991). It has been confirmedthat GA is a 200–1,000 times more potent inhibitor of 11-β-hydroxysteroid dehydrogenase involved in corticosteroidmetabolism than GL (Ploeger et al. 2001; Obolentseva et al.1999). Therefore, scientists are now trying to produce GA fromGL by biocatalysis using microbial strains or by immobilizedβ-glucuronidase enzyme and by bioprocess under in vitromeans to obtain GA from G. glabra root.

M. Ahmad :M. Jalaluddin : B. P. Panda (*)Microbial and Pharmaceutical Biotechnology Laboratory, Centre forAdvanced Research in Pharmaceutical Sciences, Faculty ofPharmacy, Jamia Hamdard, New Delhi 110062, Indiae-mail: bibhu_panda31@rediffmail.com

Ann Microbiol (2014) 64:683–688DOI 10.1007/s13213-013-0703-0

The objective of the present research is to enrichG. glabraroot with GA by the enzyme β-glucuronidase produced dur-ing solid state fermentation (SSF) of powdered root withPenicillium chrysogenum and Rhizopus oryzae.

Materials and methods

Plant materials, microorganisms, chemicals and bioreactors

The roots ofG. glabra were collected from the local market ofNewDelhi. The crude drug was authenticated by a taxonomist,Dr H.B. Singh, chief scientist, NISCAIR, CSIR, New Delhi,India (specimen voucher no/NISCAIR/RHMD/consult/2012-2013/2085-92). Fungal strains, i.e. Penicilliumchrysogenum and Rhizopus oryzae , were isolated form JamiaHamdard University campus and identified in the Indian typeculture collection (ITCC), Plant Pathology Division, IndianAgriculture Research Institute, PUSA, New Delhi, India. Thefungal strains were grown and maintained on potato dextroseagar slants. Phenolphthalein-β-D-glucuronide, GA, and GLwere obtained from Sigma Aldrich, Banglore, India. Highperformance liquid chromatography (HPLC) grade solventwas obtained from Merck, Bombay, India. All the chemicals,reagents, and microbiological medium were obtained from Hi-Media, Bombay, India. Solid state bioreactors (GROWTEK)were procured from Tarsons, Kolkata, India.

Preparation of induced fungal seed culture

Induced fungal (induction of β-glucuronidase enzyme) seedcultures of Penicillium chrysogenum and Rhizopus oryzaewere prepared by culturing fungal mycelium in potato dex-trose broth containing 2 % water extract ofG . glabra root for48 h at 27 °C at 180 rpm in a rotary shaker.

Solid state fermentation

Stationary SSF (surface fermentation) of G. glabra root wascarried out in a solid state bioreactor (GROWTEK) (Bhanjaet al. 2007). CrudeG. glabra root dried in air and powdered ina grinding mill and was sieved through a 10/44 sieve to give acoarse powder (Liao et al. 2012). Powdered G. glabra root(10 g) was placed over the float of the bioreactor and 80 ml of0.5 % NaCl was placed at the bottom of the bioreactor (thesolid to liquid ratio was pre-optimized for better sterilizationand microbial growth). The medium was sterilized at 121 °Cfor 15 min. Fungal seed culture (induced/uninduced) wasinoculated over the solid powdered G. glabra root underaseptic conditions. The bioreactor was incubated at 27 °C ina humidity chamber (75 % RH) for SSF.

Rotating SSF ofG. glabra root was carried out in a rotatingstate fermentor. For this, powdered G. glabra root (40 g) waskept inside rotating fermentation vessels of 500 ml capacity.To these, 50 ml of production medium (optimized) wereadded. The medium was sterilized at 121 °C for 15 min.Then, 2 ml of fungal seed culture (induced/uninduced) wereinoculated under aseptic conditions and incubated at 27 °C,75 % RH under rotation of 4 rpm for SSF.

Extraction of GA and GL

The extraction of produced GA and unconverted GL fromfermented G. glabra root was carried out under biphasic con-dition by using a mixture of chloroform and water (50:50 v/v).The chloroform layer containing GA and the aqueous layercontaining GL was collected and evaporated to 1 ml. Both thephases were analyzed by HPLC for quantification of GL andGA (Siracusa et al. 2011).

Analysis of GA and GL

Samples containing GL and GA were analyzed by high per-formance liquid chromatography (Shimadzu, Japan). Thechromatography was carried out by a column Lichrospher100 RP C18 (temperature 25 °C), the mobile phase consistingofmethanol:water (85:15 v/v) at a flow rate of 1 mlmin−1 withrun time 10 min. Detection of GL and GAwas carried out bythe UV detector at 254 nm (Wang et al. 2010).

β -glucuronidase enzyme assay

β -glucuronidase activity in fermented G. glabra root wasmeasured at 37 °C with 3 mM phenolphthalein-β-D-glucuro-nide, buffered with 75mMpotassium phosphate (pH 6.8) with1.0 % bovine serum albumin. The enzymatic reaction wasstopped by adding 200 mM glycine buffer solution pH 10.4.The amount of phenolphthalein released was monitored at540 nm by a microplate reader (Biotek, USA). Calibrationcurves were prepared for a standard solution of phenolphtha-lein (Combie et al. 1982).

Biomass estimation

The fungal biomass was measured by the colony-forming unit(CFU) method. To the 3 ml of sterile water, 2 g of fermentedmass was added and vortexed for 5 min. The supernatant wasgrown in ox-bile medium (ox-bile 3 %, malt extract 2 %, agar2 %; pH was adjusted to 5.5).

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Results and discussion

Effect of inducer on GA and enzyme

Bioconversion of GL to GA and β-glucuronidase activity wasstudied by induced and uninduced fungal strains in fermentedG. glabra under SSF. Induced Penicillium chrysogenum pro-duced 2.530 mg g−1 of GA with β-glucuronidase activity of3,583.8 Uml−1, while Rhizopus oryzae produced 1.549mg g−1

of GA with β-glucuronidase activity of 2,307.6 U ml−1.

The SSF was carried out by uninduced R. oryzae and P.chrysogenum producing 0.315 and 0.900 mg g−1 of GA withenzyme activity of 61 and 569 U ml−1, respectively. The aboveexperiment suggests that the higher the β-glucuronidase activ-ity the greater the biotransformation of GL to GA and thegreater the production of GA. The enzyme activity was higherin induced P. chrysogenum and R. oryzae than uninducedfungal strains which suggests that β-glucuronidase enzyme isan inducible enzyme.

Effect of pH on bioconversion rate

Under stationary state, bioconversion kinetics of GL to GA byP. chrysogenum and R. oryzae under SSF was studied atdifferent medium (optimized) pH (4.0, 4.5, 5.0, 5.5, and 6.0)under stationary state using the GROWTEK bioreactor. Thehighest concentration of GA (2.955 mg g−1) was obtained byP. chrysogenum at fermentation pH of 4.5 and in 120 h whilefurther fermentation decreased the GA level (Fig. 1a). Rhizopusoryzae under SSF condition produced 1.530 mg g−1 of GA atpH 4.5 in 96 h fermentation in the GROWTEK bioreactor and,with further increase in the fermentation time, the GA concen-tration decreased (Fig. 1b). From the above experimental result,it is clear that P. chrysogenum is the better producer of GA thanR. oryzae . Bioconversion of GL to GA is dependent on the pH

of the fermentation media, since pH is the major controllingfactor for the better enzyme activity. For both the fungal strains,the optimum pH was found to be 4.5 which suggests that β-glucuronidase activity was at a maximum at pH 4.5. FurtherGA concentration is highest at a particular fermentation time,and the concentration decreased as the fermentation time in-creased, thus suggesting that the produced GA is not stable andit is converted or degraded to other phytochemicals; therefore,harvesting time for GA must be predetermined in order toreduce the loss of GA.

Under rotating state, bioconversion kinetics of GL to GAby P. chrysogenum and R. oryzae under SSF was studiedat different pH (4.0, 4.5, 5.0, 5.5, and 6.0) under rotat-ing state by using a solid state fermentor. At 96 h of fermen-tation, 2.530 mg g−1 of GA was produced at pH 5.5 by P.chrysogenum with enzyme activity of 3653 U ml−1, and withfurther fermentation there was a decrease in GA concentra-tion. With R. oryzae , maximum bioconversion was observedat 120 h with 1.545 mg g−1 of GA with enzyme activity of2,307 U ml−1.

From the above experiment, it is concluded that biocon-version was higher under stationary state SSF than rotatingstate SSF, suggesting that surface culture is better for biocon-version of phytomolecules.

The advantage of using the GROWTEK bioreactor in-cludes the accumulation of heat being directly related to thethickness of the solid substrate (Rathbun and Shuler 1983). Asthe solid substrate is in direct contact with the liquid medium,the heat can be transferred to themedium, which is a good heatconductor. It is postulated that oxygen uptake in SSF isdirectly from the gas phase and to a lesser extent from theliquid associated with the solids (Krishna 2005), whereasoxygen uptake in the GROWTEKbioreactor is from the liquidpresent below the float as well as from the gas phase, so masstransfer was better in modified SSF. Again, due to the direct

Fig. 1 Amount of GA produced at different pH (pH 4 ; pH 4.5 ; pH 5 ; pH 5.5 ; pH 6 ) by (a) Penicillium chrysogenum and(b) Rhizopus oryzae under solid state fermentation

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contact of the liquid with the substrate in modified SSF, theproduct leaches out from the bed into the liquid mediumreducing the product accumulation in the bed of the substrate,and thereby protecting the organism from feedback inhibitionor catabolite repression. These might be the probable condi-tions that improved the enzyme activity in the GROWTEKbioreactor.

Change in pH of the medium and fungal biomassduring fermentation

The pH of the fermentation media gradually increased asthe fermentation time increased and it reached 7.2 at 168 hof SSF with R. oryzae . However, with P. chrysogenum , adecrease in pH was observed and it reached 6 at 168 h. Theabove experimental results suggest that there is continuingchange in pH of the fermentation media during the SSF.However, optimum fungal growth (biomass) for R. oryzaeand P. chrysogenum is at pH of 5.5 and 5.7, respectively,and optimum pH at which bioconversion occurs is 4.5 forboth the fungal strains. This suggests that there is a need fortwo-stage pH control during SSF. Initially, medium pHmust be set at 5.5 (0 h) for optimum fungal growth, andafter fungal strains attain growth, the pH of the mediummust be controlled at 4.5 (120 h) for better bioconversion ofGL to GA.

Optimization of bioconversion rate by medium engineering

Since P. chrysogenum is a better fungal strain for bioconver-sion of GL toGA, the solid state media for it was optimized byresponse surface methodology (RSM).

Experimental design for SSF was according to the Box-Behnken design tool of RSM by Design Expert softwarev.7.1.3 (Stat-Ease, USA) for the selected three nutrient param-eters of dextrose, MnSO4·H2O, and (NH4)2SO4. The variouslevels of nutrients are summarized in Table 1. Relative effectsof two variables on the response were identified from contourplots. An optimum value of the factors for maximum produc-tion of GA was determined by the point prediction tool ofDesign Expert 7.1.3 software. To identify the concentration ofkey nutrients influencing GA production, a flask method was

used. Three medium components [dextrose, (NH4)2SO4 andMnSO4·H2O)] were selected for the optimization study. Anexperimental design of 17 runs containing 5 central pointswas made according to Box-Behnken design of responsesurface methodology for the three selected medium pa-rameters. The individual and interactive effects of thesenutrient variables were studied by conducting experimentalfermentation runs at different levels (Table 1) of all three

Table 2 Box-Behnken experimental design design and amount of GAproduced (actual & predicted) by Penicillium chrysogenum

Run Dextrose(g l−1)

(NH4)2SO4

(g l−1)MnSO4·H2O(g l−1)

Concentration of GA(mg g−1)

Actualvalue

Predictedvalue

1 6 2 0.5 30.217 30.217

2 9 1 0.5 48.489 41.924

3 9 2 0 27.273 24.164

4 3 3 0.5 11.714 18.278

5 6 3 1 26.516 16.843

6 9 3 0.5 46.995 48.457

7 6 2 0.5 30.217 30.217

8 9 2 1 34.031 42.241

9 6 1 1 39.105 37.458

10 6 1 0 6.425 16.098

11 3 2 0 26.061 17.850

12 6 3 0 20.802 22.448

13 3 1 0.5 40.537 39.074

14 3 2 1 12.418 15.526

15 6 2 0.5 30.217 30.217

16 6 2 0.5 30.217 30.217

17 6 2 0.5 30.217 30.217

Fig. 2 Perturbation plot showing the effect of all nutrient parameters onGA production by Penicillium chrysogenum, where A , B , and C repre-sents dextrose, (NH4)2SO4, and MnSO4·H2O, respectively

Table 1 Levels of me-dium parameters used inBox-Behnken design

Nutrient parameter(g l−1)

Levels

−1 0 +1

Dextrose 3 6 9

(NH4)2SO4 1 2 3

MnSO4·H2O 0 0.5 1

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parameters. The fermentation response was measured interms of GA production in solid fermented medium afterthe fermentation process was over. The results of the ex-perimental data and simulated values are listed in Table 2.Data collected for GA concentration in each experimentalfermentation run were analyzed by using the softwareDesign Expert 7.1.3 and fitted into a multiple nonlinearregression and resulted model proposes following equation(in coded factors).

GA production by P:chrysogenum;GA mg g−1

¼ 30:217þ 8:25725A–3:56613Bþ 3:938625C

þ 6:83225ABþ 5:10025 AC–6:7415BC

þ 4:22525 A2 þ 2:4915B2–9:4965C2

Where A, B, and C represents dextrose, (NH4)2SO4, andMnSO4·H2O, respectively, in g l−1.

The effect of all nutrient parameters on GA production wascompared with the help of perturbation plots (Fig. 2). Thelines in the graph represent the influence and sensitivity of therespective factor for GA production. This nonlinear modelresulted in three response surface graphs (Fig. 3).

The analysis of variance (ANOVA) of the regression for GAproduction by P. chrysogenum under SSF is summarized inTable 3. All the responses were analyzed for determining theoptimized value of the each nutrient parameter by the pointprediction tool of Design Expert 7.1.1 software for maximumGA production. The optimum values of dextrose,MnSO4·H2O,and (NH4)2SO4 were determined at 9, 3, and 0.540 g l−1,respectively, and predicts 48.580 mg g−1 (86.749 %) of GAproduction by P. chrysogenum inG. glabra root which is betterthan the reported results of 65 % (El-Refai et al. 2012) and78.3 % (Amin et al. 2010).

The present concept of solid state biotransformation of thephyto-molecules under in situ conditions is entirely newand needs to be applied in other biotransformation process-es involved in the direct bioconversion of plant moleculesin whole plants instead of using pure molecules or plantextracts. However, the byproducts of the fungal cell andother transformed products produced during the SSF arerequired for analysis.

Acknowledgment The authors acknowledge DST and UGC Govern-ment of India for providing financial support to our laboratory under FISTand SAP.

Table 3 Analysis of variance of the predicted model for production ofGA by Penicillium chrysogenum

Regression Residual Lack of fit

Sum of squares 1,702.651 437.176 437.1757

Df 9 7 3

Mean squares 189.1834 62.454 145.7252

F value 3.0292

P 0.0790

Coefficient correlation (r2) 0.795696

Coefficient of variation 27.3368

Adequate precision value 5.433

Fig. 3 Response surface plots showing the relative effect of two mediumparameters on GA production while keeping other nutrients at a constantlevel by Penicillium chrysogenum (a–c)

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