This article was downloaded by: [The University of Manchester Library] On: 13 April 2015, At: 09:44 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Microbial Conversion of Ginsenoside Rd From Rb1 by the Fungus Mutant Aspergillus Niger Strain TH-10a a
a
b
a
a
c
a
Li Feng , Chunchun Xu , Zhuo Li , Jing Li , Yulin Dai , Hongxiang Han , Shanshan Yu & ad
Shuying Liu a
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, China
b
The Affiliated Hospital of Changchun University of Chinese Medicine
c
Jilin Agricultural University, Changchun, China
d
Changchun center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academic of Science, Changchun, China Accepted author version posted online: 01 Apr 2015.
Click for updates To cite this article: Li Feng, Chunchun Xu, Zhuo Li, Jing Li, Yulin Dai, Hongxiang Han, Shanshan Yu & Shuying Liu (2015): Microbial Conversion of Ginsenoside Rd From Rb1 by the Fungus Mutant Aspergillus Niger Strain TH-10a, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1031391 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1031391
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Microbial conversion of ginsenoside Rd from Rb1 by the fungus mutant Aspergillus niger strain TH-10a Li Feng1, Chunchun Xu1, Zhuo Li2, Jing Li1, Yulin Dai1, Hongxiang Han3, Shanshan Yu1, Shuying Liu1,4 1
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
2
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, China The Affiliated Hospital of Changchun University of Chinese Medicine 3Jilin Agricultural University, Changchun, China 4Changchun center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academic of Science, Changchun, China Corresponding author: Shanshan Yu, Shuying Liu Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China E-mail: [email protected], [email protected]
Abstract Ginsenoside Rd, one of the ginsenosides with significant pharmaceutical activities, is getting more and more attractions on its biotransformation. In this study, a novel fungus mutant the Aspergillus niger strain TH-10a, which can efficiently convert ginsenoside Rd from Rb1, was obtained through screening survival library of LiCl and UV irradiation. The transformation product ginsenoside Rd generated by removing the outer glucose residue from the position C20 of ginsenoside Rb1 was identified through the HPLC analysis. Factors for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen sources, pH values and temperatures. It showed that maximum mycelia growth could be obtained at 28oC and pH 6.0 with cellobiose and tryptone as the carbon source and the nitrogen source, respectively. And the highest transformation rate (～86%) has been achieved at 32oC and pH 5.0 with the feeding time of substrate for 48h. Also, Aspergillus niger strain TH-10a could tolerate even 40mg/ml
1
ginseng root extract as substrate with 60% bioconversion rate after 72 hours treatment at the optimal condition. Our results highlight a novel ginsenoside Rd transformation fungus and illuminate its potentially practical application in the pharmaceutical industries.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
KEYWORDS: Ginseng; Ginsenoside; Biotransformation; Bioconversion rate; Microbial conversion of ginsenoside Rd from Rb1
INTRODUCTION Ginseng, the root of Panax ginseng C.A. Meyer, is a famous traditional herb to heal disease and stay healthy for more than 2,000 years in East Asian and has become popular over the past few years in the West. [1-3] Ginsenosides, the major components in ginseng, contribute to its significantly therapeutic effects including anti-cancer, anti- inflammation and anti-oxidant. [2,4-6] Until now, more than 180 different ginsenosides have been discovered and identified in ginseng. [7]
Ginsenoside Rd was demonstrated to have anti-cancer [8] and anti-inflammation [9] activities, attenuate oxidative damage [10], protect neurons from ischemic stroke injury [11] and neurotoxic chemicals, [12] and enhance the differentiation of neural stem cell. [13] Therefore, ginsenoside Rd has significantly pharmaceutical activities which make it good drug candidates. However, due to the low content of ginsenoside Rd in ginseng, the directly isolation of it is quite difficult. Moreover, the ginsenoside Rd manufacturing by
2
chemical total synthesis is infeasible because of its complex chemical structure. Theoretically, it is practical to obtain ginsenoside Rd from Rb1 by removing the outer glucose residue at position C20, as ginsenoside Rd has the identical aglycone
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
(protopanaxadiol) with ginsenoside Rb1 which is the main component of ginseng.
Many attempts have been made to transform ginsenoside Rb1 into Rd; however the availability of the physical and chemical methods is limited due to its low specificity and serious environmental pollution. Among these methods, biotransformation is regard as the most promising method because of its high selectivity, high productivity and high yield. So far some microorganisms or enzymes have been identified to have the ability to convert ginsenoside Rd from Rb1, [14-20] the common problem is the low yield because of requirement for pure substrates, low substrate tolerance, low hydrolytic activity and further transformation of ginsenoside Rd to other compounds. Thus, it is imperative to identify new microorganism for the production of ginsenoside Rd with high specificity, high yield and high productivity.
In this study, a fungus which can transform ginsenoside Rb1 to Rd was isolated from the soil samples of ginseng roots. Then a mutant, named Aspergillus niger strain TH-10a, was obtained by LiCl and UV irradiation aiming to further improve ginsenoside Rd-hydrolyzing activity of the strain. Also the optimal parameters for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen
3
sources, pH and temperature. Considering the large scale preparation of ginsenoside Rd, ginseng root extract which is relatively cheap was used instead of the pure ginsenoside Rb1. Also the impact of the substrate concentration on the bioconversion rate was investigated. Our research indicated that Aspergillus niger strain TH-10a has good
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
application prospect in the industrial production of ginsenoside Rd.
MATERIALS AND METHODS Materials Ginseng root extract was purchased from ZeLang Healthtech Co. Ltd., China. Acetonitrile and methanol of HPLC grade were from Fisher (Waltham, MA). Standard ginsenosides Rd and Rb1 were obtained from Jilin University (Changchun, China).
The composition of the potato dextrose agar (PDA) medium was as follows: 2% (w/v) agar, 2% (w/v) glucose and 20% (v/v) potato extract prepared by adding 200 g freshly sliced potatoes to 1000 ml of water, boiling for about 30 min, and filtering through cotton cloth.
The composition of the pNPG medium was as follows: 0.2% (w/v) pNPG, 0.4% (w/v) (NH4)2SO4, 0.05% (w/v) MgSO4·7H2O, 0.1% (w/v) KH2PO4, 0.2% (w/v) NaCl, 1.5% (w/v) agar.
4
The initial fermentation medium contained: 3% (w/v) glucose, 3% (w/v) peptone, 1% (w/v) (NH4)2SO4.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
All media were sterilized at 121oC for 20 min under 100 KPa pressure.
The initial pH value was set as 7.0.
STRAIN AND MUTAGENESIS Soil samples from 5–10 cm below ground were collected from ginseng field (Fusong, China). After 10-3-10-7 dilution with sterile water, 1 g of soil samples was spread on the PDA plates added with 2 g/l of ginseng root extract. The ginsenoside Rd-hydrolyzing ability of the pure cultures were screened by HPLC analysis. The selected pure culture was examined for its color, shape and size of the colony and observed for its morphology characteristics under light microscope.
5ml of conidial suspension mixture, which was pretreated with 0.15% (w/v) LiCl for 8h, was used for UV irradiation (30 W, 10 cm, 20-60 s). [21] This exposure produced a survival rate lower than 50%. As the pNP formed yellow transparent zones after react with Na2CO3, 0.1 ml of treated suspension was diluted and spread on the pNPG plate to screen mutants that can hydrolyze pNPG. Subsequently, the screened mutants were selected for their ability to convert ginsenoside Rd from Rb1 by HPLC analysis.
5
PHYLOGENETIC ANALYSIS Sequencing of the ITS rDNA gene was completed by the Beijing DingGuo ChangSheng BioTechnology Co. Ltd., China. The ITS rDNA gene sequences of the related strains
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
were from the website http://www.ncbi.nlm.nih.gov/. The phylogenetic tree was constructed using MEGA 4.1 program. [22] Confidence levels for the branches were obtained by the bootstrap analysis with 1,000 replicates.
BIOTRANSFORMATION Preparation of spore suspension was performed through washing 5d-old culture on PDA slants and violently shaking for 1 min. 3% (v/v) of fungal spore suspension were incubated in 30 ml of initial fermentation medium shaking at 28°C with 130 rpm. The final spore concentration was adjusted to 5 ×106 spores/ml. After 48 h, ginsenoside Rb1 (in steriled water and filtered through a 0.2 nm filter) was added into the flasks to a final concentration of 0.6 mg/ml. After incubation for 3 or 7 days, the culture was filtered and centrifuged. HPLC was used to analyze the supernatant extracted with n-butanol.
PROCESS OPTIMIZATION Single-factor experiments were carried out using the initial fermentation medium which contained 3% (w/v) glucose, 3% (w/v) peptone and 1% (w/v) (NH4)2SO4. Factors of carbon sources (glucose, CMC, starch, cellobiose, lactose, sucrose and maltose, at
6
concentration of 3%, w/v), nitrogen sources (peptone, yeast powder, soybean powder, beef extract, ammonium nitrate and ammonium acetate, at concentrations of 3%, w/v), pH and temperature, which may affect the mycelia growth and ginsenoside Rd production were investigated. The effect of the feeding time (12, 24, 48 and 72h) of the substrate on
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
the bioconversion rate was evaluated. Also, the ginseng root extract was used as substrate instead of the pure ginsenoside Rb1, the ginsenoside Rd production efficiency of Aspergillus niger strain TH-10a was tested with six GRE concentrations (10 mg/ml, 20 mg/ml, 30 mg/ml, 40mg/ml, 50mg/ml and 60 mg/ml).
HPLC ASSAY The reaction mixture was extracted with water-saturated n-butanol, evaporated in vacuo and the residue was dissolved in CH3OH and applied to the HPLC analysis. The separation was carried out on a C18 column (250×4.6 mm, i.d., 5μm, Agilentcorp, USA). The mobiles used were water (A) and acetonitrile (B). The gradient elution of mobile was 0-5 min 15% B, 5-13 min 19% B, 13-16 min 25% B, 16-20 min 36% B, 20-25 min 45% B, 25-28 min 65% B, 28-35 min 80% B and 35-40 min 100% B. The flow rate and detection wavelength were set as 1 ml/min and 203 nm respectively.
RESULTS Identification Of The Strain TH-10
7
The color of the aerial mycelium of TH-10 was turned from white to black during culture. As seen in Figure 1, under light microscope, branching and septate hyphae could be observed and the conidia in sporangium were black.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
The ITS rDNA gene sequence of TH-10 was aligned to those closest taxonomic species. As seen in Figure 2, the phylogenetic analysis showed that strain TH-10 was grouped with Aspergillus species and it displayed the highest degree of ITS rRNA gene sequences similarities with the Aspergillus niger strain MUM05.13 (JF838357) (99%), Aspergillus niger strain WM10.74 (HQ014696) (99%) and Aspergillus niger strain WM10.68 (HQ014690) (99%).
Through morphology characteristics and phylogenetic analysis, we suggest that the strain TH-10 should belong to Aspergillus niger and be named as Aspergillus niger strain TH-10.
MUTAGENESIS UV mutagenesis was carried out to further improve the hydrolyzing activity of TH-10. About 50 mutant strains were selected using pNPG plates. Then, the mutants were screened for their ability to transform ginsenoside Rb1 to Rd. The most efficient strain, named as Aspergillus niger strain TH-10a, could convert ginsenoside Rb1 to Rd with the bioconversion rate increased from 26.2% to 33.3%.
8
BIOCONVERSION OF GINSENOSIDE RB1 BY ASPERGILLUS NIGER STRAIN TH-10A HPLC was used to analyse conversion product by Aspergillus niger strain TH-10a. The
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
peaks were identified through comparison with ginsenosides standards in retention time. As shown in Figure 3, the peaks with retention time 24.23 and 25.83 were equivalent to ginsenosides Rb1 and Rd, respectively. After 3 days of transformation by the Aspergillus niger strain TH-10a, the peak for ginsenoside Rb1 significantly decreased and a new peak with a retention time consistent with ginsenoside Rd appeared. After 7 days, more than 90% ginsenoside Rb1 was converted to ginsenoside Rd. Moreover, ginsenoside CK, F2, Rg3 and Rh2, which are possible metabolites of ginsenoside Rd, were not detected. Hence, ginsenoside Rd was the only metabolite of ginsenoside Rb1 by Aspergillus niger strain TH-10a.
EFFECT OF CARBON AND NITROGEN SOURCES The effects of different carbon sources on the mycelia growth and ginsenoside Rd production were determined. The results are presented in Figure 4A, among the tested different carbon sources, mycelial growth and bioconversion rate reached their maximum values in the cellobiose-containing medium. In the cellobiose-containing medium, the bioconversion rate and mecelial dry weight raised to about 42% and 9 g/L, respectively,
9
compared with glucose-containing medium; whereas no ginsenoside Rd production was detected when CMC, starch, lactose, sucrose and maltose were used as carbon sources.
The effect of different nitrogen sources in the forms of organic or inorganic on mycelia
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
growth and ginsenoside Rd production by Aspergillus niger strain TH-10a was also studied. The results are presented in Figure 4B; a maximum of 10 mg/L mecelial dry weight was obtained in the yeast powder-containing medium, in which the bioconversion rate reached as high as 45%. Also, the mycelial biomass and bioconversion rate for inorganic nitrogen sources was lower than that for organic nitrogen sources.
EFFECT OF TEMPERATURE AND PH The impact of temperature on the mycelia growth and bioconversion rate was studied and Aspergillus niger strain TH-10a was cultured with different temperatures ranging from 22 to 42oC. As seen in Figure 4C, the optimal temperatures were determined as 28oC for mycelial growth and 32oC for bioconversion rate. Furthermore, the values remained relatively high in the temperature range of 22-28oC for mycelial biomass and 28-32oC for bioconversion rate. However above 28oC, the mycelial biomass decreased quickly, while below 28oC or above 32oC, the bioconversion rate decreased more than 30%. Hence, temperature has a great effect on the mycelial biomass and bioconversion rate of Aspergillus niger strain TH-10a.
10
The impact of pH on mycelia growth and bioconversion rate was also investigated. The results are presented in Figure 4D, the mecelial dry weight altered little within a wide pH range of 3-8 and reached a maximum of about 10 mg/l at pH 6.0. The bioconversion rate maintained high values in the narrow pH range of 5-6 and reached a maximum of 82% at
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
pH 5.0. The optimal pH for ginsenoside Rd production by Aspergillus niger strain TH-10a was similar to those by the previously reported microorganisms that can transform ginsenoside Rb1 to Rd. [14, 16, 19-20] Moreover, pH has a great influence on the preparation of ginsenoside Rd by Aspergillus niger strain TH-10a.
OPTIMISATION OF CONDITIONS FOR GINSENOSIDE RD BIOTRANSFORMATION The effect of feeding time of the substrate on the bioconversion rate was studied. Ginsenoside Rb1 was fed into the cultures which have been preincubated for different times. It can be seen in Figure 5A, the optimal feeding time was 48 h when the conversion rate was higher than 80%.
The time course on the bioconversion process of the ginsenosides by Aspergillus niger strain TH-10a was investigated via HPLC (Figure 6). At the initial substrate concentration of 0.6 mg/ml, 0.52 mg/ml of ginsenoside Rd was produced after 3 days. The yield of ginsenoside Rd reached 87%, which is the highest ever reported.
11
To better apply Aspergillus niger strain TH-10a for the production of ginsenoside Rd, GRE was used because it is relatively abundant and cheap than pure ginsenoside Rb1. The bioconversion rates were tested with six GRE concentrations. The results are presented in Figure 5B, bioconversion rate reached its maximum value (～60%) at the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
GRE concentration of 40 mg/ml. Furthermore, the bioconversion rate was still higher than 50% at the GRE concentration range of 10-40 mg/ml.
DISCUSSION Ginsenoside Rd is a potential drug candidate due to its outstanding therapeutic functions. Ginsenoside Rd was demonstrated to have anti-cancer [8] and anti-inflammation [9] activities, attenuate oxidative damage [10], protect neurons from ischemic stroke injury [11] and neurotoxic chemicals, [12] enhance the differentiation of neural stem cell. [13] Although attempts to produce ginsenoside Rd have been made using microorganisms, such as Rhizopus stolonifer, [16] Curvularia lunata, [16] Caulobacter leidyia, [23] Acremonium strictum, [14] Cladosporium fulvum, [19] but the yields were low because of requirement for pure substrates, low substrate tolerance, low hydrolytic activity and further transformation of ginsenoside Rd to other compounds. Moreover, some of the researches lacked of quantifiable measure of the biotransformation process. Thus, microorganisms with the desirable characteristics including high ginsenoside Rd-hydrolyzing activity and high specificity are required for pharmaceutical industry. In our study, forty ginsenoside Rd-hydrolyzing fungi were successfully purified from the soil of ginseng roots. The
12
strain TH-10, named Aspergillus niger strain TH-10, gave the most efficient bioconversion of ginsenoside Rb1 to Rd.
UV mutagenesis was applied to enhance the ginsenoside Rd-hydrolyzing activity of the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
wild strain. HPLC was used for the quantitative study on the conversion process by the mutant Aspergillus sp. TH-10a (Figure. 3). Under the optimal condition, it was found that 0.6 mg/ml ginsenoside Rb1 was transformed into 0.52 mg/ml ginsenoside Rd after 3 days. The yield of ginsenoside Rd reached 87%, which is the highest compared with the previous reports.
The strain Aspergillus sp. TH-10a hydrolyzed ginsenoside Rb1 by removing the outer glucose attached to the C-20 position of ginsenoside Rb1. Furthermore, the strain Aspergillus sp. TH-10a displayed high selectivity as no side reaction was detected during the bioconversion process. Hence, high selectivity of Aspergillus sp. TH-10a greatly meets the demand for the large scale preparation of ginsenoside Rd.
The optimal parameters for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen sources, pH and temperature. Among the tested carbon sources, only cellobiose could improve mycelial growth and increase ginsenoside Rd yield. These results showed that the secreted glycoside hydrolase of Aspergillus niger strain TH-10a might be an inducible enzyme which was induced by cellobiose. Some
13
signal molecules could enhance the synthesis of the secreted glycoside hydrolase by the transmembrane signal transmission function. Polysaccharides, such as CMC and starch with big molecular weights, are unable to cross the cell membranes. In contrast, disaccharides such as cellobiose, ususlly derived from polysaccharides, could cross the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
cell membranes and act as signal molecules.
So far the minor ginsenoside F2 and Rg3 have been produced as 100 g unit using biotransformation methods. [24-25] Thus it is necessary to develop effective methods for the large scale preparation of ginsenoside Rd to increase the economical feasibility and fulfill the industrial demand. Since substrates are a major proportion of product costs, ginseng root extract instead of pure ginsenoside Rb1 was used as substrate in our experiments which can be efficiently separated from ginseng and is much cheaper. Ginsenoside Rd production efficiency of Aspergillus niger strain TH-10a was tested with six GRE concentrations within 3 days. 40mg/ml GRE was selected, which was higher than 20 mg/ml used in the bioconversion of ginsenoside Rb1 to Rd by the Paecilomyces bainier 229-7 in which the saponin from Panax notoginseng leaves was used as substrate, [26]
and also higher than the ginsenoside Rb1 concentrations of 0.25 and 0.02 mg/ml by
the Cladosporium fulvum [19] and A. strictum, [14] respectively. The substrate used in the biotransformation of ginsenoside Rd by the Cladosporium fulvum and A. strictum was pure ginsenoside Rb1. In addition, Li et al reported that at concentrations of 30–60 mg/ml, the bioconversion time was delayed to 5-7 days, thus high substrate concentrations would
14
prolong the conversion time. In our experiments, at substrate concentration of 60 mg/ml, the bioconversion rate was still as high as 40% with the bioconversion time of 3 days. Hence, the biotransformation time in our experiment was shorter than these strains.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
ACKNOWLEDGMENTS This work was supported by Scientific and Technological Poject of Ji Lin Province (No:20130101127JC and No: 20130303101YY).
REFERENCES 1. Cho, I.H. Effects of Panax ginseng in neurodegenerative diseases. J. Ginseng. Res. 2012, 36, 342-353. 2. Kang, S.; Min, H. Ginseng, the ‘Immunity Boost’: the effects of Panax ginseng on immune system. J. Ginseng. Res. 2012, 36, 354-368. 3. Qi, L.W.; Wang, C.Z.; Yuan, C.S. Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry. 2011, 72, 689-699. 4. Attele, A.S.; Wu, J.A.; Yuan, C.S. Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharmacol. 1999, 58, 1685-1693. 5. Buettner, C.; Yeh, G.Y.; Phillips, R.S.; Mittleman, M.A.; Kaptchuk, T.J. Systematic review of the effects of ginseng on cardiovascular risk factors. Ann. Pharmaco. Ther. 2006, 40, 83-95.
15
6. Kim, S.K.; Park, J.H. 2011. Trends in Ginseng Research in. J. Ginseng. Res. 2010, 35, 389-398. 7. Christensen, L.P. Ginsenosides: Chemistry, biosynthesis, analysis and potential health effects. Adv. Food. Nutr. Res. 2008, 55, 1-99.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
8. Yang, Z.G.; Sun, H.X.; Ye, Y.P. Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis. Chem. Biodivers. 2006, 3, 187-1 97. 9. Zhang, Y.X.; Wang, L.; Xiao, E.L.; Li, S.J.; Chen, J.J.; Gao, B.; Min, G.N.; Wang, Z.P.; Wu, Y.J. Ginsenoside-Rd exhibits anti-inflammatory activities through elevation of antioxidant enzyme activities and inhibition of JNK and ERK activation in vivo. Int. Immunopharmacol. 2013, 17(4), 1094-100. 10. Wang, Y.; Li, X.; Wang, X.; Lau, W.; Wang, Y.; Xing, Y.; Zhang, X.; Ma, X.; Gao, F. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3β signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS. One. 2013, 8, e70956. 11. Ye, R.D.; Zhao, G.; Liu, X.F. Ginsenoside Rd for acute ischemic stroke: translating from bench to bedside. Expert. Rev. Neurother. 2013, 13(6), 603-613. 12. Lee, J.K.; Choi, S.S.; Lee, H.K.; Han, K.J.; Han, E.J.; Suh, H.W. Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice. Planta. Med. 2003, 69, 230-234.
16
13. Shi, Q.; Hao, Q.; Bouissac, J.; Lu, Y.; Tian, S. ; Luu, B. Ginsenoside Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells. Life. Sci. 2005, 76, 983-995. 14. Chen, G.T.; Yang, M.; Song, Y.; Lu, Z.Q.; Zhang, J.Q.; Huang, H.L.; Wu, L.J.; Guo,
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
D.A. Microbial transformation of ginsenoside Rb1 by Acremonium strictum. Appl. Microbiol. Biotechnol. 2008, 77, 1345-1350. 15. Chi, H.; Ji, G.E. Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms. Biotechnol. Lett. 2005, 27, 765-771. 16. Dong, A.; Ye, M.; Guo, H.; Zheng, J.; Guo, D. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata. Biotechnol. Lett. 2003, 25, 339-344. 17. Kim, M.K.; Lee, J.W.; Lee, K.Y.; Yang, D.C. Microbial conversion of major ginsenoside Rb1 to pharmaceutically active minor ginsenoside Rd. J. Microbiol. 2005, 43, 456-462. 18. Luan, H.; Liu, X. ; Qi, X. ; Hu, Y. ; Hao, D.C.; Yang, L. Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzing β-D-glucosidase from China white jade snail. Process. Biochem. 2006, 41, 1974-1980. 19. Zhao, X.S.; Wang, J.; Li, J.; Fu, L.; Gao, J.; Du, X.L.; Zhou, Y.F.; Tai, G.H. Highly selective biotransformation of ginsenoside Rb1 to Rd by the phytopathogenic fungus Cladosporium fulvum. J. Ind. Microbiol. Biotechnol. 2009, 36, 721-726.
17
20. Ye, Li.; Zhou, C.Q.; Zhou, W.; Zhou, P.; Chen, D.F.; Liu, X.H.; Shi, X.L.; Feng, M.Q. Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7. Bioresource. Technol. 2010, 101, 7872-7876. 21. Zhang, M.C. Industrial micro breeding. Beijing Science Press, Beijing. 1984.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
22. Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 2007, 24, 1596-1599. 23. Cheng, L.Q.; Kim, M.K.; Lee, J.W.; Lee, Y.J.; Yang, D.C. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia. Biotechnol. Lett. 2006, 28, 1121-1127. 24. Cui, C.H.; Kim, J.K.; Kim, S.C.; Im, W.T. Characterizaition of a ginsenoside-transforming β-glucosidase from Paenobacillus mucilaginosus and its application for enhanced production of minor ginsenoside F2. Plos. One. 2014, 9, e85727. 25. Shin, K.C.; Seo, M.J.; Oh, H.J.; Oh, D.K. Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII. Biotechnol. Lett. 2014, 36, 1287-1293. 26. Li, Y.; Zhou, C.Q.; Zhou, W.; Zhou, P.; Chen, D.F.; Liu, X.H.; Shi, X.L.; Feng, M.Q. Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7. Bioresource. Technol. 2010, 101, 7872-7876.
18
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
Figure 1 Sporangium of the Aspergillus niger strain TH-10.
19
Figure 2 Phylogenetic trees based on the ITS rRNA gene sequences, showing the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
phylogenetic relationships of the Aspergillus niger strain TH-10.
20
Figure 3 HPLC profile of transformation ginsenoside Rb1 during different periods by
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
Aspergillus niger strain TH-10a.
21
Figure 4 (A) Effect of different carbon sources on mycelia growth and ginsenoside Rd production by Aspergillus niger strain TH-10a. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (B) Effect of different nitrogen sources on mycelia growth and ginsenoside Rd production by Aspergillus niger strain
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
TH-10a. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (C) Effect of temperature on mycelial growth and ginsenoside Rd production. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (D) Effect of pH on mycelial growth and ginsenoside Rd production. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. Data were expressed as mean ±SD from three independent experiments.
22
Figure 5 (A) Effects of the time of substrate addition on biotransformation by Aspergillus niger strain TH-10a.
(B) Effects of substrate concentration on biotransformation by
Aspergillus niger strain TH-10a. Data were expressed as mean ±SD from three
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
independent experiments.
23
Figure 6 Ginsenoside Rd production from ginsenoside Rb1 by Aspergillus niger strain
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
TH-10a. Ginsenoside Rb1 (■) and ginsenoside Rd (●) were determined.
24
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Microbial Conversion of Ginsenoside Rd From Rb1 by the Fungus Mutant Aspergillus Niger Strain TH-10a a
a
b
a
a
c
a
Li Feng , Chunchun Xu , Zhuo Li , Jing Li , Yulin Dai , Hongxiang Han , Shanshan Yu & ad
Shuying Liu a
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, China
b
The Affiliated Hospital of Changchun University of Chinese Medicine
c
Jilin Agricultural University, Changchun, China
d
Changchun center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academic of Science, Changchun, China Accepted author version posted online: 01 Apr 2015.
Click for updates To cite this article: Li Feng, Chunchun Xu, Zhuo Li, Jing Li, Yulin Dai, Hongxiang Han, Shanshan Yu & Shuying Liu (2015): Microbial Conversion of Ginsenoside Rd From Rb1 by the Fungus Mutant Aspergillus Niger Strain TH-10a, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1031391 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1031391
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Microbial conversion of ginsenoside Rd from Rb1 by the fungus mutant Aspergillus niger strain TH-10a Li Feng1, Chunchun Xu1, Zhuo Li2, Jing Li1, Yulin Dai1, Hongxiang Han3, Shanshan Yu1, Shuying Liu1,4 1
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
2
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, China The Affiliated Hospital of Changchun University of Chinese Medicine 3Jilin Agricultural University, Changchun, China 4Changchun center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academic of Science, Changchun, China Corresponding author: Shanshan Yu, Shuying Liu Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China E-mail: [email protected], [email protected]
Abstract Ginsenoside Rd, one of the ginsenosides with significant pharmaceutical activities, is getting more and more attractions on its biotransformation. In this study, a novel fungus mutant the Aspergillus niger strain TH-10a, which can efficiently convert ginsenoside Rd from Rb1, was obtained through screening survival library of LiCl and UV irradiation. The transformation product ginsenoside Rd generated by removing the outer glucose residue from the position C20 of ginsenoside Rb1 was identified through the HPLC analysis. Factors for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen sources, pH values and temperatures. It showed that maximum mycelia growth could be obtained at 28oC and pH 6.0 with cellobiose and tryptone as the carbon source and the nitrogen source, respectively. And the highest transformation rate (～86%) has been achieved at 32oC and pH 5.0 with the feeding time of substrate for 48h. Also, Aspergillus niger strain TH-10a could tolerate even 40mg/ml
1
ginseng root extract as substrate with 60% bioconversion rate after 72 hours treatment at the optimal condition. Our results highlight a novel ginsenoside Rd transformation fungus and illuminate its potentially practical application in the pharmaceutical industries.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
KEYWORDS: Ginseng; Ginsenoside; Biotransformation; Bioconversion rate; Microbial conversion of ginsenoside Rd from Rb1
INTRODUCTION Ginseng, the root of Panax ginseng C.A. Meyer, is a famous traditional herb to heal disease and stay healthy for more than 2,000 years in East Asian and has become popular over the past few years in the West. [1-3] Ginsenosides, the major components in ginseng, contribute to its significantly therapeutic effects including anti-cancer, anti- inflammation and anti-oxidant. [2,4-6] Until now, more than 180 different ginsenosides have been discovered and identified in ginseng. [7]
Ginsenoside Rd was demonstrated to have anti-cancer [8] and anti-inflammation [9] activities, attenuate oxidative damage [10], protect neurons from ischemic stroke injury [11] and neurotoxic chemicals, [12] and enhance the differentiation of neural stem cell. [13] Therefore, ginsenoside Rd has significantly pharmaceutical activities which make it good drug candidates. However, due to the low content of ginsenoside Rd in ginseng, the directly isolation of it is quite difficult. Moreover, the ginsenoside Rd manufacturing by
2
chemical total synthesis is infeasible because of its complex chemical structure. Theoretically, it is practical to obtain ginsenoside Rd from Rb1 by removing the outer glucose residue at position C20, as ginsenoside Rd has the identical aglycone
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
(protopanaxadiol) with ginsenoside Rb1 which is the main component of ginseng.
Many attempts have been made to transform ginsenoside Rb1 into Rd; however the availability of the physical and chemical methods is limited due to its low specificity and serious environmental pollution. Among these methods, biotransformation is regard as the most promising method because of its high selectivity, high productivity and high yield. So far some microorganisms or enzymes have been identified to have the ability to convert ginsenoside Rd from Rb1, [14-20] the common problem is the low yield because of requirement for pure substrates, low substrate tolerance, low hydrolytic activity and further transformation of ginsenoside Rd to other compounds. Thus, it is imperative to identify new microorganism for the production of ginsenoside Rd with high specificity, high yield and high productivity.
In this study, a fungus which can transform ginsenoside Rb1 to Rd was isolated from the soil samples of ginseng roots. Then a mutant, named Aspergillus niger strain TH-10a, was obtained by LiCl and UV irradiation aiming to further improve ginsenoside Rd-hydrolyzing activity of the strain. Also the optimal parameters for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen
3
sources, pH and temperature. Considering the large scale preparation of ginsenoside Rd, ginseng root extract which is relatively cheap was used instead of the pure ginsenoside Rb1. Also the impact of the substrate concentration on the bioconversion rate was investigated. Our research indicated that Aspergillus niger strain TH-10a has good
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
application prospect in the industrial production of ginsenoside Rd.
MATERIALS AND METHODS Materials Ginseng root extract was purchased from ZeLang Healthtech Co. Ltd., China. Acetonitrile and methanol of HPLC grade were from Fisher (Waltham, MA). Standard ginsenosides Rd and Rb1 were obtained from Jilin University (Changchun, China).
The composition of the potato dextrose agar (PDA) medium was as follows: 2% (w/v) agar, 2% (w/v) glucose and 20% (v/v) potato extract prepared by adding 200 g freshly sliced potatoes to 1000 ml of water, boiling for about 30 min, and filtering through cotton cloth.
The composition of the pNPG medium was as follows: 0.2% (w/v) pNPG, 0.4% (w/v) (NH4)2SO4, 0.05% (w/v) MgSO4·7H2O, 0.1% (w/v) KH2PO4, 0.2% (w/v) NaCl, 1.5% (w/v) agar.
4
The initial fermentation medium contained: 3% (w/v) glucose, 3% (w/v) peptone, 1% (w/v) (NH4)2SO4.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
All media were sterilized at 121oC for 20 min under 100 KPa pressure.
The initial pH value was set as 7.0.
STRAIN AND MUTAGENESIS Soil samples from 5–10 cm below ground were collected from ginseng field (Fusong, China). After 10-3-10-7 dilution with sterile water, 1 g of soil samples was spread on the PDA plates added with 2 g/l of ginseng root extract. The ginsenoside Rd-hydrolyzing ability of the pure cultures were screened by HPLC analysis. The selected pure culture was examined for its color, shape and size of the colony and observed for its morphology characteristics under light microscope.
5ml of conidial suspension mixture, which was pretreated with 0.15% (w/v) LiCl for 8h, was used for UV irradiation (30 W, 10 cm, 20-60 s). [21] This exposure produced a survival rate lower than 50%. As the pNP formed yellow transparent zones after react with Na2CO3, 0.1 ml of treated suspension was diluted and spread on the pNPG plate to screen mutants that can hydrolyze pNPG. Subsequently, the screened mutants were selected for their ability to convert ginsenoside Rd from Rb1 by HPLC analysis.
5
PHYLOGENETIC ANALYSIS Sequencing of the ITS rDNA gene was completed by the Beijing DingGuo ChangSheng BioTechnology Co. Ltd., China. The ITS rDNA gene sequences of the related strains
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
were from the website http://www.ncbi.nlm.nih.gov/. The phylogenetic tree was constructed using MEGA 4.1 program. [22] Confidence levels for the branches were obtained by the bootstrap analysis with 1,000 replicates.
BIOTRANSFORMATION Preparation of spore suspension was performed through washing 5d-old culture on PDA slants and violently shaking for 1 min. 3% (v/v) of fungal spore suspension were incubated in 30 ml of initial fermentation medium shaking at 28°C with 130 rpm. The final spore concentration was adjusted to 5 ×106 spores/ml. After 48 h, ginsenoside Rb1 (in steriled water and filtered through a 0.2 nm filter) was added into the flasks to a final concentration of 0.6 mg/ml. After incubation for 3 or 7 days, the culture was filtered and centrifuged. HPLC was used to analyze the supernatant extracted with n-butanol.
PROCESS OPTIMIZATION Single-factor experiments were carried out using the initial fermentation medium which contained 3% (w/v) glucose, 3% (w/v) peptone and 1% (w/v) (NH4)2SO4. Factors of carbon sources (glucose, CMC, starch, cellobiose, lactose, sucrose and maltose, at
6
concentration of 3%, w/v), nitrogen sources (peptone, yeast powder, soybean powder, beef extract, ammonium nitrate and ammonium acetate, at concentrations of 3%, w/v), pH and temperature, which may affect the mycelia growth and ginsenoside Rd production were investigated. The effect of the feeding time (12, 24, 48 and 72h) of the substrate on
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
the bioconversion rate was evaluated. Also, the ginseng root extract was used as substrate instead of the pure ginsenoside Rb1, the ginsenoside Rd production efficiency of Aspergillus niger strain TH-10a was tested with six GRE concentrations (10 mg/ml, 20 mg/ml, 30 mg/ml, 40mg/ml, 50mg/ml and 60 mg/ml).
HPLC ASSAY The reaction mixture was extracted with water-saturated n-butanol, evaporated in vacuo and the residue was dissolved in CH3OH and applied to the HPLC analysis. The separation was carried out on a C18 column (250×4.6 mm, i.d., 5μm, Agilentcorp, USA). The mobiles used were water (A) and acetonitrile (B). The gradient elution of mobile was 0-5 min 15% B, 5-13 min 19% B, 13-16 min 25% B, 16-20 min 36% B, 20-25 min 45% B, 25-28 min 65% B, 28-35 min 80% B and 35-40 min 100% B. The flow rate and detection wavelength were set as 1 ml/min and 203 nm respectively.
RESULTS Identification Of The Strain TH-10
7
The color of the aerial mycelium of TH-10 was turned from white to black during culture. As seen in Figure 1, under light microscope, branching and septate hyphae could be observed and the conidia in sporangium were black.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
The ITS rDNA gene sequence of TH-10 was aligned to those closest taxonomic species. As seen in Figure 2, the phylogenetic analysis showed that strain TH-10 was grouped with Aspergillus species and it displayed the highest degree of ITS rRNA gene sequences similarities with the Aspergillus niger strain MUM05.13 (JF838357) (99%), Aspergillus niger strain WM10.74 (HQ014696) (99%) and Aspergillus niger strain WM10.68 (HQ014690) (99%).
Through morphology characteristics and phylogenetic analysis, we suggest that the strain TH-10 should belong to Aspergillus niger and be named as Aspergillus niger strain TH-10.
MUTAGENESIS UV mutagenesis was carried out to further improve the hydrolyzing activity of TH-10. About 50 mutant strains were selected using pNPG plates. Then, the mutants were screened for their ability to transform ginsenoside Rb1 to Rd. The most efficient strain, named as Aspergillus niger strain TH-10a, could convert ginsenoside Rb1 to Rd with the bioconversion rate increased from 26.2% to 33.3%.
8
BIOCONVERSION OF GINSENOSIDE RB1 BY ASPERGILLUS NIGER STRAIN TH-10A HPLC was used to analyse conversion product by Aspergillus niger strain TH-10a. The
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
peaks were identified through comparison with ginsenosides standards in retention time. As shown in Figure 3, the peaks with retention time 24.23 and 25.83 were equivalent to ginsenosides Rb1 and Rd, respectively. After 3 days of transformation by the Aspergillus niger strain TH-10a, the peak for ginsenoside Rb1 significantly decreased and a new peak with a retention time consistent with ginsenoside Rd appeared. After 7 days, more than 90% ginsenoside Rb1 was converted to ginsenoside Rd. Moreover, ginsenoside CK, F2, Rg3 and Rh2, which are possible metabolites of ginsenoside Rd, were not detected. Hence, ginsenoside Rd was the only metabolite of ginsenoside Rb1 by Aspergillus niger strain TH-10a.
EFFECT OF CARBON AND NITROGEN SOURCES The effects of different carbon sources on the mycelia growth and ginsenoside Rd production were determined. The results are presented in Figure 4A, among the tested different carbon sources, mycelial growth and bioconversion rate reached their maximum values in the cellobiose-containing medium. In the cellobiose-containing medium, the bioconversion rate and mecelial dry weight raised to about 42% and 9 g/L, respectively,
9
compared with glucose-containing medium; whereas no ginsenoside Rd production was detected when CMC, starch, lactose, sucrose and maltose were used as carbon sources.
The effect of different nitrogen sources in the forms of organic or inorganic on mycelia
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
growth and ginsenoside Rd production by Aspergillus niger strain TH-10a was also studied. The results are presented in Figure 4B; a maximum of 10 mg/L mecelial dry weight was obtained in the yeast powder-containing medium, in which the bioconversion rate reached as high as 45%. Also, the mycelial biomass and bioconversion rate for inorganic nitrogen sources was lower than that for organic nitrogen sources.
EFFECT OF TEMPERATURE AND PH The impact of temperature on the mycelia growth and bioconversion rate was studied and Aspergillus niger strain TH-10a was cultured with different temperatures ranging from 22 to 42oC. As seen in Figure 4C, the optimal temperatures were determined as 28oC for mycelial growth and 32oC for bioconversion rate. Furthermore, the values remained relatively high in the temperature range of 22-28oC for mycelial biomass and 28-32oC for bioconversion rate. However above 28oC, the mycelial biomass decreased quickly, while below 28oC or above 32oC, the bioconversion rate decreased more than 30%. Hence, temperature has a great effect on the mycelial biomass and bioconversion rate of Aspergillus niger strain TH-10a.
10
The impact of pH on mycelia growth and bioconversion rate was also investigated. The results are presented in Figure 4D, the mecelial dry weight altered little within a wide pH range of 3-8 and reached a maximum of about 10 mg/l at pH 6.0. The bioconversion rate maintained high values in the narrow pH range of 5-6 and reached a maximum of 82% at
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
pH 5.0. The optimal pH for ginsenoside Rd production by Aspergillus niger strain TH-10a was similar to those by the previously reported microorganisms that can transform ginsenoside Rb1 to Rd. [14, 16, 19-20] Moreover, pH has a great influence on the preparation of ginsenoside Rd by Aspergillus niger strain TH-10a.
OPTIMISATION OF CONDITIONS FOR GINSENOSIDE RD BIOTRANSFORMATION The effect of feeding time of the substrate on the bioconversion rate was studied. Ginsenoside Rb1 was fed into the cultures which have been preincubated for different times. It can be seen in Figure 5A, the optimal feeding time was 48 h when the conversion rate was higher than 80%.
The time course on the bioconversion process of the ginsenosides by Aspergillus niger strain TH-10a was investigated via HPLC (Figure 6). At the initial substrate concentration of 0.6 mg/ml, 0.52 mg/ml of ginsenoside Rd was produced after 3 days. The yield of ginsenoside Rd reached 87%, which is the highest ever reported.
11
To better apply Aspergillus niger strain TH-10a for the production of ginsenoside Rd, GRE was used because it is relatively abundant and cheap than pure ginsenoside Rb1. The bioconversion rates were tested with six GRE concentrations. The results are presented in Figure 5B, bioconversion rate reached its maximum value (～60%) at the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
GRE concentration of 40 mg/ml. Furthermore, the bioconversion rate was still higher than 50% at the GRE concentration range of 10-40 mg/ml.
DISCUSSION Ginsenoside Rd is a potential drug candidate due to its outstanding therapeutic functions. Ginsenoside Rd was demonstrated to have anti-cancer [8] and anti-inflammation [9] activities, attenuate oxidative damage [10], protect neurons from ischemic stroke injury [11] and neurotoxic chemicals, [12] enhance the differentiation of neural stem cell. [13] Although attempts to produce ginsenoside Rd have been made using microorganisms, such as Rhizopus stolonifer, [16] Curvularia lunata, [16] Caulobacter leidyia, [23] Acremonium strictum, [14] Cladosporium fulvum, [19] but the yields were low because of requirement for pure substrates, low substrate tolerance, low hydrolytic activity and further transformation of ginsenoside Rd to other compounds. Moreover, some of the researches lacked of quantifiable measure of the biotransformation process. Thus, microorganisms with the desirable characteristics including high ginsenoside Rd-hydrolyzing activity and high specificity are required for pharmaceutical industry. In our study, forty ginsenoside Rd-hydrolyzing fungi were successfully purified from the soil of ginseng roots. The
12
strain TH-10, named Aspergillus niger strain TH-10, gave the most efficient bioconversion of ginsenoside Rb1 to Rd.
UV mutagenesis was applied to enhance the ginsenoside Rd-hydrolyzing activity of the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
wild strain. HPLC was used for the quantitative study on the conversion process by the mutant Aspergillus sp. TH-10a (Figure. 3). Under the optimal condition, it was found that 0.6 mg/ml ginsenoside Rb1 was transformed into 0.52 mg/ml ginsenoside Rd after 3 days. The yield of ginsenoside Rd reached 87%, which is the highest compared with the previous reports.
The strain Aspergillus sp. TH-10a hydrolyzed ginsenoside Rb1 by removing the outer glucose attached to the C-20 position of ginsenoside Rb1. Furthermore, the strain Aspergillus sp. TH-10a displayed high selectivity as no side reaction was detected during the bioconversion process. Hence, high selectivity of Aspergillus sp. TH-10a greatly meets the demand for the large scale preparation of ginsenoside Rd.
The optimal parameters for the microbial culture and biotransformation were investigated upon the carbon sources, the nitrogen sources, pH and temperature. Among the tested carbon sources, only cellobiose could improve mycelial growth and increase ginsenoside Rd yield. These results showed that the secreted glycoside hydrolase of Aspergillus niger strain TH-10a might be an inducible enzyme which was induced by cellobiose. Some
13
signal molecules could enhance the synthesis of the secreted glycoside hydrolase by the transmembrane signal transmission function. Polysaccharides, such as CMC and starch with big molecular weights, are unable to cross the cell membranes. In contrast, disaccharides such as cellobiose, ususlly derived from polysaccharides, could cross the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
cell membranes and act as signal molecules.
So far the minor ginsenoside F2 and Rg3 have been produced as 100 g unit using biotransformation methods. [24-25] Thus it is necessary to develop effective methods for the large scale preparation of ginsenoside Rd to increase the economical feasibility and fulfill the industrial demand. Since substrates are a major proportion of product costs, ginseng root extract instead of pure ginsenoside Rb1 was used as substrate in our experiments which can be efficiently separated from ginseng and is much cheaper. Ginsenoside Rd production efficiency of Aspergillus niger strain TH-10a was tested with six GRE concentrations within 3 days. 40mg/ml GRE was selected, which was higher than 20 mg/ml used in the bioconversion of ginsenoside Rb1 to Rd by the Paecilomyces bainier 229-7 in which the saponin from Panax notoginseng leaves was used as substrate, [26]
and also higher than the ginsenoside Rb1 concentrations of 0.25 and 0.02 mg/ml by
the Cladosporium fulvum [19] and A. strictum, [14] respectively. The substrate used in the biotransformation of ginsenoside Rd by the Cladosporium fulvum and A. strictum was pure ginsenoside Rb1. In addition, Li et al reported that at concentrations of 30–60 mg/ml, the bioconversion time was delayed to 5-7 days, thus high substrate concentrations would
14
prolong the conversion time. In our experiments, at substrate concentration of 60 mg/ml, the bioconversion rate was still as high as 40% with the bioconversion time of 3 days. Hence, the biotransformation time in our experiment was shorter than these strains.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
ACKNOWLEDGMENTS This work was supported by Scientific and Technological Poject of Ji Lin Province (No:20130101127JC and No: 20130303101YY).
REFERENCES 1. Cho, I.H. Effects of Panax ginseng in neurodegenerative diseases. J. Ginseng. Res. 2012, 36, 342-353. 2. Kang, S.; Min, H. Ginseng, the ‘Immunity Boost’: the effects of Panax ginseng on immune system. J. Ginseng. Res. 2012, 36, 354-368. 3. Qi, L.W.; Wang, C.Z.; Yuan, C.S. Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry. 2011, 72, 689-699. 4. Attele, A.S.; Wu, J.A.; Yuan, C.S. Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharmacol. 1999, 58, 1685-1693. 5. Buettner, C.; Yeh, G.Y.; Phillips, R.S.; Mittleman, M.A.; Kaptchuk, T.J. Systematic review of the effects of ginseng on cardiovascular risk factors. Ann. Pharmaco. Ther. 2006, 40, 83-95.
15
6. Kim, S.K.; Park, J.H. 2011. Trends in Ginseng Research in. J. Ginseng. Res. 2010, 35, 389-398. 7. Christensen, L.P. Ginsenosides: Chemistry, biosynthesis, analysis and potential health effects. Adv. Food. Nutr. Res. 2008, 55, 1-99.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
8. Yang, Z.G.; Sun, H.X.; Ye, Y.P. Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis. Chem. Biodivers. 2006, 3, 187-1 97. 9. Zhang, Y.X.; Wang, L.; Xiao, E.L.; Li, S.J.; Chen, J.J.; Gao, B.; Min, G.N.; Wang, Z.P.; Wu, Y.J. Ginsenoside-Rd exhibits anti-inflammatory activities through elevation of antioxidant enzyme activities and inhibition of JNK and ERK activation in vivo. Int. Immunopharmacol. 2013, 17(4), 1094-100. 10. Wang, Y.; Li, X.; Wang, X.; Lau, W.; Wang, Y.; Xing, Y.; Zhang, X.; Ma, X.; Gao, F. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3β signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS. One. 2013, 8, e70956. 11. Ye, R.D.; Zhao, G.; Liu, X.F. Ginsenoside Rd for acute ischemic stroke: translating from bench to bedside. Expert. Rev. Neurother. 2013, 13(6), 603-613. 12. Lee, J.K.; Choi, S.S.; Lee, H.K.; Han, K.J.; Han, E.J.; Suh, H.W. Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice. Planta. Med. 2003, 69, 230-234.
16
13. Shi, Q.; Hao, Q.; Bouissac, J.; Lu, Y.; Tian, S. ; Luu, B. Ginsenoside Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells. Life. Sci. 2005, 76, 983-995. 14. Chen, G.T.; Yang, M.; Song, Y.; Lu, Z.Q.; Zhang, J.Q.; Huang, H.L.; Wu, L.J.; Guo,
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
D.A. Microbial transformation of ginsenoside Rb1 by Acremonium strictum. Appl. Microbiol. Biotechnol. 2008, 77, 1345-1350. 15. Chi, H.; Ji, G.E. Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms. Biotechnol. Lett. 2005, 27, 765-771. 16. Dong, A.; Ye, M.; Guo, H.; Zheng, J.; Guo, D. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata. Biotechnol. Lett. 2003, 25, 339-344. 17. Kim, M.K.; Lee, J.W.; Lee, K.Y.; Yang, D.C. Microbial conversion of major ginsenoside Rb1 to pharmaceutically active minor ginsenoside Rd. J. Microbiol. 2005, 43, 456-462. 18. Luan, H.; Liu, X. ; Qi, X. ; Hu, Y. ; Hao, D.C.; Yang, L. Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzing β-D-glucosidase from China white jade snail. Process. Biochem. 2006, 41, 1974-1980. 19. Zhao, X.S.; Wang, J.; Li, J.; Fu, L.; Gao, J.; Du, X.L.; Zhou, Y.F.; Tai, G.H. Highly selective biotransformation of ginsenoside Rb1 to Rd by the phytopathogenic fungus Cladosporium fulvum. J. Ind. Microbiol. Biotechnol. 2009, 36, 721-726.
17
20. Ye, Li.; Zhou, C.Q.; Zhou, W.; Zhou, P.; Chen, D.F.; Liu, X.H.; Shi, X.L.; Feng, M.Q. Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7. Bioresource. Technol. 2010, 101, 7872-7876. 21. Zhang, M.C. Industrial micro breeding. Beijing Science Press, Beijing. 1984.
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
22. Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 2007, 24, 1596-1599. 23. Cheng, L.Q.; Kim, M.K.; Lee, J.W.; Lee, Y.J.; Yang, D.C. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia. Biotechnol. Lett. 2006, 28, 1121-1127. 24. Cui, C.H.; Kim, J.K.; Kim, S.C.; Im, W.T. Characterizaition of a ginsenoside-transforming β-glucosidase from Paenobacillus mucilaginosus and its application for enhanced production of minor ginsenoside F2. Plos. One. 2014, 9, e85727. 25. Shin, K.C.; Seo, M.J.; Oh, H.J.; Oh, D.K. Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII. Biotechnol. Lett. 2014, 36, 1287-1293. 26. Li, Y.; Zhou, C.Q.; Zhou, W.; Zhou, P.; Chen, D.F.; Liu, X.H.; Shi, X.L.; Feng, M.Q. Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces bainier 229-7. Bioresource. Technol. 2010, 101, 7872-7876.
18
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
Figure 1 Sporangium of the Aspergillus niger strain TH-10.
19
Figure 2 Phylogenetic trees based on the ITS rRNA gene sequences, showing the
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
phylogenetic relationships of the Aspergillus niger strain TH-10.
20
Figure 3 HPLC profile of transformation ginsenoside Rb1 during different periods by
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
Aspergillus niger strain TH-10a.
21
Figure 4 (A) Effect of different carbon sources on mycelia growth and ginsenoside Rd production by Aspergillus niger strain TH-10a. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (B) Effect of different nitrogen sources on mycelia growth and ginsenoside Rd production by Aspergillus niger strain
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
TH-10a. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (C) Effect of temperature on mycelial growth and ginsenoside Rd production. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. (D) Effect of pH on mycelial growth and ginsenoside Rd production. Bioconversion rate (black column) and mycelial dry weight (gray column) were determined. Data were expressed as mean ±SD from three independent experiments.
22
Figure 5 (A) Effects of the time of substrate addition on biotransformation by Aspergillus niger strain TH-10a.
(B) Effects of substrate concentration on biotransformation by
Aspergillus niger strain TH-10a. Data were expressed as mean ±SD from three
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
independent experiments.
23
Figure 6 Ginsenoside Rd production from ginsenoside Rb1 by Aspergillus niger strain
Downloaded by [The University of Manchester Library] at 09:44 13 April 2015
TH-10a. Ginsenoside Rb1 (■) and ginsenoside Rd (●) were determined.
24
