You are on page 1of 13

Original research article

Journal of Biological and
Chemical Sciences (JBCS)

Tuli et al. (2014) vol.1, no. 1, 35-47

Optimization of fermentation conditions for cordycepin production using
Cordyceps militaris 3936
Hardeep Singh Tuli 1*, Anil K. Sharma1, Sardul Singh Sandhu2

Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala-133207,

(Haryana) India.

Department of Biological Sciences, R. D. University, Jabalpur 482001, (MP) India.

*Correspondence at
Received: November 11, 2014; Accepted: November 29, 2014


Cordycepin, an active ingredient of the insect fungus Cordyceps militaris, is a

category of compounds that exhibit significant therapeutic potential. The aim of this work was to
optimize maximum cordycepin production conditions in fermented broth of C. militaris 3936
The suitable physical and nutritional conditions for cordycepin production were investigated by
individually varying one variable at a time. The optimum culture conditions for maximum
cordycepin production (846 mg/L) were found to be at pH 5.5, temperature 25ºC, inoculum size
8 % v/v, inoculum age 72 h, incubation time 24 d and optimum culture medium included 1.5 %
dextrose, 0.8 % yeast extract, K2HPO4 0.3 %, KH2PO4 0.1 %, NaCl 0.05 %, MgSO4 0.05 % and
NaCl 0.05 %. In conclusion, present study successfully optimized cultural and nutritional
conditions for the production of cordycepin from C. militaris 3936.
Keywords: Cordyceps militaris, cordycepin, optimization, submerge fermentation

Medicinal mushrooms have been known for thousands of years to produce biometabolites which
are used or studied as possible treatment for diseases. It has been speculated that many cancerrelated deaths could be prevented or reduced by modifying our diet with mushrooms, as they
contain antioxidants [1]. Cordyceps, especially its extract contains many biologically active
compounds including Cordycepin, cordycepic acid, adenosine, exo-polysaccharides, vitamins

There are basically two fermentation techniques by which the cultivation of mycelium biomass of Cordyceps and cordycepin can be achieved including surface and submerged fermentation [1]. KH2PO4 0.1.2 gm anthrone in 100 ml of 90 % H2SO4. militaris 3936.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. no. peptone 0. 36 Scienceindoors .05 %. militaris 3936 were inoculated into 250 ml Erlenmeyer flasks with 100 ml of basal medium (glucose 1. MgSO4 0.3 %. NaCl 0. Materials and methods Procurement and maintenance of Cordyceps militaris 3936 Microbial strain of C. Till date various groups of researchers have optimized the concentrations of Carbon and Nitrogen required to produce maximal cordycepin by the surface and submerged cultivation of C. Spectrophotometric assay for quantitative estimation of cordycepin Cordycepin was extracted as per the method given in our previous study [9] and further quantified using spectrophotometric assay at 460 nm which is based on cordycepin reaction with anthrone that resulted in production of cherry red color [10].1 %.5 %. K2HPO4 0. Also the effect of some additives on cordycepin production is being evaluated by the researchers [8]. militaris [3-7]. The inoculum was prepared by punching out 5mm of PDA discs with sterilized cork borer. (2014) vol. Reagent was prepared by dissolving 0.05 %) at 25°C on a rotary shaker incubator (110 rpm) for 3 days. Keeping in view of the above facts. Out of these. Seed culture preparation The saline spore suspension of C. militaris 3936 was prepared and inoculated on PDA petriplates followed by incubation at 20°C for 7 days.5 %. The discs containing cultures of C. the work was planned to study efficient culture conditions and medium for cordycepin production using C. 1. 35-47 and enzymes. militaris 3936 was procured from IMTECH Chandigarh (India) and was regularly revived and maintained on potato dextrose agar (PDA) slants and stored at 4°C. Cordycepin or 3′-deoxyadenosine (9-(3-deoxy-β-D-ribofuranosyl) adenine) is the main active constituent which is most widely studied for its medicinal value having a broad spectrum biological activity [2].

The pH of the medium was adjusted by using 1N HCl or 1N NaOH. 48h. inoculum size.0. fermentation was carried out in 250 ml conical flasks containing 100 ml of culture medium at 5°C intervals in the range of 15 to 35 (like 15º. inoculum age. Based on the results of the inoculum size assay. 20º. 30º. Effect of incubation period Fermentation period was an important parameter for production of cordycepin from C. In this study. The flasks were incubated for 10 days in an incubator under stationary conditions. militaris 3936. incubation period and agitation) and nutritional (Carbon and nitrogen sources) conditions were optimized by varying individually each factor as described below. 4. temperature. 8. 37 Scienceindoors . (2014) vol. 25º.1.0 to 8. 1. 10 and 12 % v/v) from 72h old fungal culture broth to the production media.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. no. The culture flasks were incubated for 10 days in stationary mode followed by quantitative estimation of cordycepin using spectrophotometric assay at 460 nm. Effect of Temperature In order to determine the effective temperature for cordycepin production. 96h and 120h) were selected to study the effect of inoculum age on cordycepin production. 35-47 Optimization of fermentation conditions for cordycepin production The various cultural (pH. 6. 35º and 40ºC) ± 2°C for 10 days under stationary conditions. a specific concentration of the inoculum of different ages (24h. The fermentation was carried out in 100 ml of production medium in 250 ml conical flasks. The production rate of cordycepin was measured at regular intervals (24 h) of time using spectrophotometric assay. fermentation experiments were carried out up to 30 days under stationary conditions. Cordyceps militaris 3936 was cultivated in a 250 mL flask containing 100 mL basal medium with different pH ranges from 4. Effect of pH To determine optimal pH for cordycepin production. Effect of inoculum size and age The effect of inoculum size was studied by adding different levels of inoculums (2. The flasks were kept in stationary mode at 20°C for 10 days in an incubator.

0.1. The broth was distributed into different flasks and various carbon sources (1. Temperature.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. 1. The medium lacking nitrogen source served as a control. galactose. militaris 3936 and incubated at 20ºC for 10 days. ammonium nitrate and ammonium sulphate) nitrogen sources were studied. Furthermore. Results and discussion Optimization of cordycepin production by C. MgSO4 0. lactose. Effect of carbon sources Effects of various carbon compounds namely. fermentation was carried out in shaking (110 rpm) as well as stationary mode. fructose.5g of per liter) inoculated with 8. K2HPO4 1g.5% of concentration was added individually to the basal medium to replace the peptone. maltose. no. militaris 3936 Cordycepin production was optimized using the basal medium (glucose 15g.5g and NaCl 0. Flasks containing the strain were further incubated under sterile conditions. For each nitrogen source. militaris 3936. were kept constant. 38 Scienceindoors . (2014) vol. The best nitrogen source was further studied for its optimum required concentration. During this experiment a set of flasks were kept initially under stationary mode for 7 d followed by shake culture for 10 d and again transferred to stationary phase for 7 d.0 % (v/v) of 72 h growth of C. glucose. The medium lacking any carbon source served as a control. and cellobiose were used for studying their effect on cordycepin production by C. casein.5 %) were added into each flask prior to inoculation of the strain. Other parameters such as pH. peptone 5g. sucrose. Incubation period and agitation) and nutritional conditions (carbon and nitrogen sources) were studied by individually varying each factor. yeast extract) and inorganic (sodium nitrate. The effects of physical (pH. 35-47 Effect of agitation In order to demonstrate the effect of agitation on cordycepin production. starch. cellulose. three-stage fermentation process by combining conventional shake-flask fermentation with static culture followed by shake flask culture was also studied. Inoculum age and size. nitrogen source etc. KH2PO4 3g. temperature. peptone. The best carbon source was further studied for its optimum dosage. carbon source. Effect of nitrogen sources Nitrogen compounds of complex organic (beef extract.

Effect of inoculums size and age Inoculation is considered an important factor as production of cordycepin has been found varying due to variety of inoculum methods employed [17. no. The maximum production of cordycepin (213 mg/L) was achieved at pH 5. with dry mycelium weight 25 mg) of 72 h old inoculum of Cordyceps militaris 3936 was added to the production medium.5 (Fig. 1. 1c). growth rate and requirement of nutrient consumption for their growth [18]. A change in inoculum size from the optimum concentration 39 Scienceindoors . The optimum values of pH for various strains of C. It was observed that cordycepin production (304 mg/L) was maximum when 8% (v/v.0. (2014) vol. pH could affect mycelial cell membrane functions such as the uptake of various nutriments and products of biosynthesis [13]. 1b). The results of this experiment showed that cordycepin production was significantly affected by the pH of production medium during fermentation. 30º and 35ºC). Cordycepin production profile was studied by varying pH of the production medium from 4. 12]. 1a). In the present study.5 to 7.1. 25º. 35-47 Effect of pH pH has been known to affect the production of metabolites from many kinds of Ascomycetes and Basidiomycetes [11.0 and the results were consistent with earlier reports. Further the variation of pH among the fungal strains could exist due to differences in metabolic reactions. but declined after reaching a maximal value (Fig. In this work. Effect of temperature The effect of temperature on cordycepin production was determined by incubating the flasks at different temperatures ranging from 15 to 35ºC (like 15º. 20º. The production of cordycepin from others strains of Cordyceps was documented in the range of 20 to 30ºC [19-21]. militaris for cordycepin production were reported previously in the pH range of 4 to 7 [14-17]. militaris 3936 could produce cordycepin when pH range was 4.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. 22]. C. the production of cordycepin was examined by varying inoculum size from 2.0 to 12 % and inoculum age from 24 to 144 h. The effect of inoculum size on cordycepin production could be correlated with total dissolved oxygen in the medium. The optimal temperature for cordycepin production (279 mg/L) was found to be 25ºC (Fig. Initially the cordycepin production enhanced with the increase in inoculum size.0 to 8.

The previous investigation showed that oxygen supply plays an important role in the cell growth and production of bio-metabolite by the higher fungus [14. The cordycepin production was measured at regular intervals along with dry mycelium weight. The incubation period was found to be directly related to the production of cordycepin from C. Whereas the rate of cordycepin production was not fallen but it increased significantly up to 24 days and stopped further with prolonged fermentation time. 27].25]. Effect of incubation period C. inoculum age of 72 h was found to be optimum for cordycepin production (Fig. militaris CCRC 32219 significantly enhanced by two-stage fermentation process [7. In the present study. Effect of agitation It is well-known that aeration is a critical factor for cell growth and metabolite production by aerobic microbial cultures. Thus. effect of different fermentation conditions such as shake flask. After that the DMW didn’t show any significant change and fallen slowly up to 30 days of cultivation time.1. 2a). Generally up to 10% (v/v) inoculum of fungal culture was found to be sufficient for cordycepin production in various strains of Cordyceps [17. 1d) which is consistent with earlier reports in literature [18]. stationary flasks and stationary+ shake+ stationary flasks was 40 Scienceindoors . The highest yield of mycelium (14. Inoculum age is known as crucial biological parameter for the production of bioactive metabolites from microorganisms.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. no. In the present study. 24.93 g/L) and cordycepin (592 mg/L) were obtained at 18 and 24 days of fermentation respectively (Fig. 1. 26]. 35-47 resulted in reduction in cordycepin production. (2014) vol. militaris 3936. 23]. Results of the present study were found to be consistent with earlier reports [17. 20]. It was found that the production of ganoderic acid from Ganoderma lucidum and cordycepin from C. militaris 3936 was inoculated into culture medium in 250 ml conical flask and incubated at 25ºC for a period of 30 days. A survey of literature revealed that 72-120 h old inoculum culture is best suited for higher production of bioactive metabolites from fungal cultures [20-21. a two-stage fermentation process by combining conventional shake-flask fermentation with static culture was proposed. A higher inoculum age is not commonly preferred at industrial scale. The dry mycelium weight (DMW) and cordycepin production was found to be increased from 0 to 18 days.

Journal of Biological and Chemical Sciences (JBCS) Tuli et al. 1: (a) Effect of pH on cordycepin production (mg/L) by C. militaris 3936 for cordycepin production (mg/L). (d) Effect of inoculation age on cordycepin production (mg/L) by C. (c) Effect of inoculum size on cordycepin production (mg/L) by C. militaris 3936. The results revealed that cordycepin production was higher in stationary+ shake+ stationary (654 mg/L) mode followed by stationary (591 mg/L) and then shaking (271 mg/L) (Fig. 41 Scienceindoors . no. 35-47 studied. (b) Effect of temperature on C.1. (2014) vol. militaris 3936. 1. Fig. 2b). militaris 3936.

The results showed that concentration of dextrose significantly affects the cordycepin production.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. increase in dextrose concentrations lead to decline in cordycepin production (Fig. Various carbon sources (glucose. militaris 3936. maltose. cellulose and cellobiose) were studied to evaluate their effects on cordycepin production from Cordyceps militaris 3936.5 to 5. 1. no. Carbon source in the medium affects the overall growth and metabolism of the microorganism. It was found that maximum cordycepin production (651 mg/L) could be achieved at 4% concentration of dextrose. 2: (a) Effect of incubation period on cordycepin production (mg/L) and dry mycelium weight by C. galactose. (b) Effect of agitation on cordycepin production (mg/L) by C. 35-47 Fig. A set of flasks devoid of any carbon source were taken as control.0 %). (2014) vol. 3b). lactose. The results are 42 Scienceindoors . sucrose. starch. 3a). The data from the study revealed that maximum cordycepin production was obtained in the presence of dextrose (647 mg/L) cellobiose>Lactose>Fructose>Galactose>Cellulose followed (Fig. militaris 3936. by starch>Sucrose>Maltose> Furthermore the optimum concentration of dextrose was investigated for cordycepin production using various concentrations of dextrose (0.1. Further. Effect of carbon source Carbon and nitrogen sources are the fundamental requirements for the growth of microorganisms. fructose.

3d. 35-47 consistent with the earlier reports in which maximum yield of cordycepin was obtained in the range of 4 to 6 % dextrose as carbon source from various strains of C. (2010) investigated the optimum production of cordycepin from mutated strain of C. 7]. militaris C738 and C. militaris 3936. 1. yeast extract was found to be very effective for cordycepin (709 mg/L) production by C. 21. 16]. 23]. yeast extract) gave rise to relatively higher cordycepin production. Other carbon sources such as fructose and sucrose have also been studied for cordycepin production from some other strains of C. among the organic nitrogen sources. militaris NG3 have been shown to have very poor mycelial growth in inorganic nitrogen sources [6. In comparison with inorganic nitrogen sources (sodium nitrate. militaris at a concentration of 8. However Das et al. Keeping in view of above results.8% concentration of yeast extract. Effect of nitrogen source The effects of nitrogen sources on the production of cordycepin by C. organic nitrogen sources (beef extract. ammonium nitrate and ammonium sulphate). 43 Scienceindoors .. The study would be extremely helpful to the scientific community especially to the researchers working on cordycepin production from C. Efforts are being made by the researchers to enhance cordycepin production from the Genus Cordyceps.62 % dextrose [16]. Fig. summarized the effects of various yeast extract concentrations on the cordycepin production. no. 3c. Conclusion Cordycepin is considered a natural bioactive metabolite with a promising therapeutic potential. yeast extract was selected as a suitable nitrogen source for further studies. We studied the effects of various physical and nutritional conditions on cordycepin production which lead us to produce maximum cordycepin up to 846 mg/L using Cordyceps militaris 3936. Several C. militaris 3936 have been shown in fig. militaris strains such as C.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. militaris 3936. militaris [4. 17.1. This is consistent with what was previously suggested that most basiomycetes prefer complex organic nitrogen sources for their growth in submerged cultures [5]. The highest cordycepin production (846 mg/L) was obtained at 0. In the present study. (2014) vol. casein. militaris [3. peptone.

(c) Effect of various nitrogen sources on cordycepin production (mg/L) by C. militaris 3936. (d) Effect of different concentrations of yeast extract on cordycepin production (mg/L) by C. Conflict of interest There are no potential conflicts of interest among the authors regarding the publication of this manuscript. militaris 3936. Acknowledgments The authors would like to acknowledge M. militaris 3936. (b) Effect of different concentrations of dextrose on cordycepin production (mg/L) by C.1. 35-47 Fig. University Mullana (Ambala) for providing the requisite facilities to perform this study 44 Scienceindoors . militaris 3936. (2014) vol. no.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. 1. 3: (a) Effect of various carbon sources on cordycepin production (mg/L) by C.M.

Sadhu SS. cordycepin from Cordyceps militaris 3936. Optimization of extraction conditions and antimicrobial potential of a bioactive metabolite. Korean J Mycol 25: 133–142. 6. Chen WB and Zhang S (2011). Sakurai A and Sakakibara M (2009). Enzyme Microb Technol 39(4): 641–646. Spring FS. Kredich NM and Guarino AJ (1960). Sakurai A and Sakakibara. Mycosystema 30(2): 229-234. 7. Tuli HS. Hwang HJ. WJPPS 3: 1525-1535.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. M (2006). Effects of additives on cordycepin production using a Cordyceps militaris mutant induced by ion beam irradiation. Xu CP. 9. Sharma AK (2014). 3 Biotech 4: 1-12. Nature 166: 949–954. An improved method of isolation and determination of cordycepin. Urabe E. 10. 1. J Appl Microbiol 94: 120–126. Production and characterization of exopolysaccharides from an enthomopathogenic fungus Cordyceps militaris NG3. and Hutchinson SA (1950). Masuda M. Cultural condition for the mycelial growth of Phellinus igniarius on chemically defined medium and grains. Sandhu SS. Tuli HS. Jung IC. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin. Xu CP. a metabolic product isolated from cultures of Cordyceps militaris (Linn.1. Biochim Biophys Acla 41: 361-363. Choi JW and Yum JW (2003). 4. Park KS and Lee JS (1997). Kim SH. no. Sung JM. Optimization of submerged culture process for the production of biomass and exo-polysaccharides by Cordyceps militaris C738. 5. Lee JS. Kim SW. Kim SW. Du M. Production of cordycepin by surface culture using the medicinal mushroom Cordyceps militaris. 2. Park S. Choi JW. Manson W. 8. Biotechnol Prog 19: 428–435. Hwang HJ.) Link. Cunningham KG. Sharma AK and Kashayap D (2014). Liquid culture conditions for promoting cordycepin secreted from Cordyceps militaris mycelia. Kim SY. Kwon YI. Cordycepin. 35-47 References 1. Das SK. Afr J Biotechnol 8(13): 3041–3047. 45 Scienceindoors . Zhong SM. Masuda M. (2014) vol. Kim CW and Yun JW (2003). 3.

Gerlach SR. Mycosystema 30(2): 229-234. Optimization of submerged culture conditions for the mycelia growth and exo-biopolymer production by Cordyceps militaris. Keawsompong S. Park JP.1. Medium optimization for polysaccharide production of Cordyceps sinensis. Kang JC. Liquid culture conditions for promoting cordycepin secreted from Cordyceps militaris mycelia. Thai J Agric Sci 42(4): 219-225. Appl Biochem Biotechnol 120: 145– 157. Meng ZB. 15. Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. 14. Shih IL. Sivichai S and Hywel-Jones NL (2009). Du M. Kang C. Effect of Temperature on Cordycepin Production in Cordyceps militaris. Jiapeng T. Scientific World J 4: 1-15. Giuseppin MLF and Hunik J (1998). Hwang HJ and Yun JW (2001). Wen TC. 18. Li GR and Hyde KD (2014). Hung LT. Effects of ammonium feeding on the production of bioactive metabolites (cordycepin and exopolysaccharides) in mycelial culture of a Cordyceps sinensis fungus. Gerlach D. Biochem Eng J 33(3): 193–201. J Appl Microbiol 103: 1942–1949. 19. Influence of reactor systems on the morphology of Aspergillus awamori. Chen WB and Zhang S (2011). Kim SW. 17. (2014) vol. 12. Hanh VT. 16. 35-47 11. 1.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. Siedenberg D. Optimization of Large-scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid static culture. Schtigerl K. 46 Scienceindoors . Optimization of fermentation conditions and purification of cordycepin from Cordyceps militaris. Tsai MJ. Lett Appl Microbiol 33: 76–81. Application of neural network and cluster analysis for characterization of fungal morphology. Yiting L and Li Z (2014). Hsu TH. Prep Biochem Biotechnol 44: 90– 106. Tsai KL and Hsieh C (2007). Chang DM and Lo CT (2005). Leung PH and Wu JY (2007). Hsieh C. no. Process Biochem 33: 601–615. Zhong SM. 13.

Hatashita M. Biotechnol Prog 20(5): 1408–1413. Kang C. (2014) vol. J Food Agric Environ 11 (3&4): 534-538. Chauvatcharin S and Zhong JJ (2005). Mao XB and Zhong JJ (2004). Optimization of carbon source and carbon nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. 47 Scienceindoors . Das SK. Process Biochem 40(5): 1667–1672. Das SK. Hatashita M. Eksriwong T. no. 35-47 20. 23. Das SK. Effects of Inoculation on production of anticancer drug cordycepin in surface liquid culture using Cordyceps militaris: A minor factor may greatly affect the result. 27. Qian YX and Lei BX (2012). Fang QH and Zhong JJ (2002). Production of cordycepin by Cordyceps militaris using submerged liquid culture: Optimization of the culture medium and repeated batch fermentation. 1. Sakurai A and Sakakibara M (2009). Mycosystem 31(3): 389–397. 26. Masuda M.1. 21. Fang TT.Journal of Biological and Chemical Sciences (JBCS) Tuli et al. Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation. Wen TC. Masuda M. Hyper-production of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors. Biotechnol Prog 18: 51–54. Effects of additives and different culture conditions on cordycepin production by the medicinal fungus Cordyceps militaris. Mao XB. 24. Afr J Biotechnol 8(13): 3041–3047. Zhang JG. 25. Li QL and Wei ZJ (2013). Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum. Indian J biotechnol 9: 427-430. Masuda M. 22. Sakurai A and Sakakibara M (2010). Process Biochem 45(1): 129–132. Kang JC. Effects of additives on cordycepin production using a Cordyceps militaris mutant induced by ion beam irradiation. Sakurai A and Sakakibara M (2010).