Optimization of Fermentation Conditions for Cordycepin Production Using

Journal of Biological and Chemical Sciences (JBCS) Tuli et al. (2014) vol.1, no. 1, 35-47

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Optimization of fermentation conditions for cordycepin production using

Cordyceps militaris 3936

Hardeep Singh Tuli1*, Anil K. Sharma1, Sardul Singh Sandhu2

1Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala-133207,

(Haryana) India.

2Department of Biological Sciences, R. D. University, Jabalpur 482001, (MP) India.

*Correspondence at [email protected]

Received: November 11, 2014; Accepted: November 29, 2014

Abstract: 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

Introduction

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 cancer-

related 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

Original research article


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and enzymes. Out of these, 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].

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].

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. militaris [3-7]. Also the effect of some additives on cordycepin production is being evaluated

by the researchers [8]. Keeping in view of the above facts, the work was planned to study

efficient culture conditions and medium for cordycepin production using C. militaris 3936.

Materials and methods

Procurement and maintenance of Cordyceps militaris 3936

Microbial strain of C. 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.

Seed culture preparation

The saline spore suspension of C. militaris 3936 was prepared and inoculated on PDA petri-

plates followed by incubation at 20°C for 7 days. The inoculum was prepared by punching out

5mm of PDA discs with sterilized cork borer. The discs containing cultures of C. militaris 3936

were inoculated into 250 ml Erlenmeyer flasks with 100 ml of basal medium (glucose 1.5 %,

peptone 0.5 %, KH2PO4 0.3 %, K2HPO4 0.1 %, MgSO4 0.05 %, NaCl 0.05 %) at 25°C on a

rotary shaker incubator (110 rpm) for 3 days.

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]. Reagent was prepared by dissolving

0.2 gm anthrone in 100 ml of 90 % H2SO4.


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Optimization of fermentation conditions for cordycepin production

The various cultural (pH, temperature, inoculum size, inoculum age, incubation period and

agitation) and nutritional (Carbon and nitrogen sources) conditions were optimized by varying

individually each factor as described below.

Effect of pH

To determine optimal pH for cordycepin production, Cordyceps militaris 3936 was cultivated in

a 250 mL flask containing 100 mL basal medium with different pH ranges from 4.0 to 8.0. The

pH of the medium was adjusted by using 1N HCl or 1N NaOH. The flasks were kept in

stationary mode at 20°C for 10 days in an incubator.

Effect of Temperature

In order to determine the effective temperature for cordycepin production, 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, 20º, 25º, 30º, 35º and 40ºC) ± 2°C for 10 days under stationary

conditions.

Effect of inoculum size and age

The effect of inoculum size was studied by adding different levels of inoculums (2, 4, 6, 8, 10

and 12 % v/v) from 72h old fungal culture broth to the production media. The culture flasks were

incubated for 10 days in stationary mode followed by quantitative estimation of cordycepin using

spectrophotometric assay at 460 nm.

Based on the results of the inoculum size assay, a specific concentration of the inoculum of

different ages (24h, 48h, 96h and 120h) were selected to study the effect of inoculum age on

cordycepin production. The fermentation was carried out in 100 ml of production medium in 250

ml conical flasks. The flasks were incubated for 10 days in an incubator under stationary

conditions.

Effect of incubation period

Fermentation period was an important parameter for production of cordycepin from C. militaris

3936. In this study, fermentation experiments were carried out up to 30 days under stationary

conditions. The production rate of cordycepin was measured at regular intervals (24 h) of time

using spectrophotometric assay.


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Effect of agitation

In order to demonstrate the effect of agitation on cordycepin production, fermentation was

carried out in shaking (110 rpm) as well as stationary mode. Furthermore, three-stage

fermentation process by combining conventional shake-flask fermentation with static culture

followed by shake flask culture was also studied. 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. Other parameters such as pH, temperature, carbon source,

nitrogen source etc. were kept constant.

Effect of carbon sources

Effects of various carbon compounds namely, glucose, fructose, galactose, sucrose, maltose,

lactose, starch, cellulose, and cellobiose were used for studying their effect on cordycepin

production by C. militaris 3936. The broth was distributed into different flasks and various

carbon sources (1.5 %) were added into each flask prior to inoculation of the strain. The medium

lacking any carbon source served as a control. Flasks containing the strain were further incubated

under sterile conditions. The best carbon source was further studied for its optimum dosage.

Effect of nitrogen sources

Nitrogen compounds of complex organic (beef extract, casein, peptone, yeast extract) and

inorganic (sodium nitrate, ammonium nitrate and ammonium sulphate) nitrogen sources were

studied. For each nitrogen source, 0.5% of concentration was added individually to the basal

medium to replace the peptone. The medium lacking nitrogen source served as a control. The

best nitrogen source was further studied for its optimum required concentration.

Results and discussion

Optimization of cordycepin production by C. militaris 3936

Cordycepin production was optimized using the basal medium (glucose 15g, peptone 5g,

KH2PO4 3g, K2HPO4 1g, MgSO4 0.5g and NaCl 0.5g of per liter) inoculated with 8.0 % (v/v) of

72 h growth of C. militaris 3936 and incubated at 20ºC for 10 days. The effects of physical (pH,

Temperature, Inoculum age and size, Incubation period and agitation) and nutritional conditions

(carbon and nitrogen sources) were studied by individually varying each factor.


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Effect of pH

pH has been known to affect the production of metabolites from many kinds of Ascomycetes and

Basidiomycetes [11, 12]. Cordycepin production profile was studied by varying pH of the

production medium from 4.0 to 8.0. The results of this experiment showed that cordycepin

production was significantly affected by the pH of production medium during fermentation. pH

could affect mycelial cell membrane functions such as the uptake of various nutriments and

products of biosynthesis [13]. The optimum values of pH for various strains of C. militaris for

cordycepin production were reported previously in the pH range of 4 to 7 [14-17]. Further the

variation of pH among the fungal strains could exist due to differences in metabolic reactions,

growth rate and requirement of nutrient consumption for their growth [18]. In this work, C.

militaris 3936 could produce cordycepin when pH range was 4.5 to 7.0 and the results were

consistent with earlier reports. The maximum production of cordycepin (213 mg/L) was

achieved at pH 5.5 (Fig. 1a).

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, 25º, 30º and 35ºC). The optimal

temperature for cordycepin production (279 mg/L) was found to be 25ºC (Fig. 1b). The

production of cordycepin from others strains of Cordyceps was documented in the range of 20 to

30ºC [19-21].

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, 22]. In the present study, the

production of cordycepin was examined by varying inoculum size from 2.0 to 12 % and

inoculum age from 24 to 144 h. It was observed that cordycepin production (304 mg/L) was

maximum when 8% (v/v; with dry mycelium weight 25 mg) of 72 h old inoculum of Cordyceps

militaris 3936 was added to the production medium. Initially the cordycepin production

enhanced with the increase in inoculum size, but declined after reaching a maximal value (Fig.

1c). The effect of inoculum size on cordycepin production could be correlated with total

dissolved oxygen in the medium. A change in inoculum size from the optimum concentration


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resulted in reduction in cordycepin production. Generally up to 10% (v/v) inoculum of fungal

culture was found to be sufficient for cordycepin production in various strains of Cordyceps [17,

20].

Inoculum age is known as crucial biological parameter for the production of bioactive

metabolites from microorganisms. 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,

23]. A higher inoculum age is not commonly preferred at industrial scale. In the present study,

inoculum age of 72 h was found to be optimum for cordycepin production (Fig. 1d) which is

consistent with earlier reports in literature [18].

Effect of incubation period

C. militaris 3936 was inoculated into culture medium in 250 ml conical flask and incubated at

25ºC for a period of 30 days. The cordycepin production was measured at regular intervals along

with dry mycelium weight. The dry mycelium weight (DMW) and cordycepin production was

found to be increased from 0 to 18 days. After that the DMW didn’t show any significant change

and fallen slowly up to 30 days of cultivation time. Whereas the rate of cordycepin production

was not fallen but it increased significantly up to 24 days and stopped further with prolonged

fermentation time. The highest yield of mycelium (14.93 g/L) and cordycepin (592 mg/L) were

obtained at 18 and 24 days of fermentation respectively (Fig. 2a). The incubation period was

found to be directly related to the production of cordycepin from C. militaris 3936. Results of the

present study were found to be consistent with earlier reports [17, 24- 25].

Effect of agitation

It is well-known that aeration is a critical factor for cell growth and metabolite production by

aerobic microbial cultures. 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, 26].

Thus, a two-stage fermentation process by combining conventional shake-flask fermentation

with static culture was proposed. It was found that the production of ganoderic acid from

Ganoderma lucidum and cordycepin from C. militaris CCRC 32219 significantly enhanced by

two-stage fermentation process [7, 27]. In the present study, effect of different fermentation

conditions such as shake flask, stationary flasks and stationary+ shake+ stationary flasks was


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studied. 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. 2b).

Fig. 1: (a) Effect of pH on cordycepin production (mg/L) by C. militaris 3936. (b) Effect of

temperature on C. militaris 3936 for cordycepin production (mg/L). (c) Effect of inoculum size

on cordycepin production (mg/L) by C. militaris 3936. (d) Effect of inoculation age on

cordycepin production (mg/L) by C. militaris 3936.


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Fig. 2: (a) Effect of incubation period on cordycepin production (mg/L) and dry mycelium

weight by C. militaris 3936. (b) Effect of agitation on cordycepin production (mg/L) by C.

militaris 3936.

Effect of carbon source

Carbon and nitrogen sources are the fundamental requirements for the growth of

microorganisms. Carbon source in the medium affects the overall growth and metabolism of the

microorganism. Various carbon sources (glucose, galactose, fructose, maltose, sucrose, lactose,

starch, cellulose and cellobiose) were studied to evaluate their effects on cordycepin production

from Cordyceps militaris 3936. A set of flasks devoid of any carbon source were taken as

control. The data from the study revealed that maximum cordycepin production was obtained in

the presence of dextrose (647 mg/L) followed by starch>Sucrose>Maltose>

cellobiose>Lactose>Fructose>Galactose>Cellulose (Fig. 3a). Furthermore the optimum

concentration of dextrose was investigated for cordycepin production using various

concentrations of dextrose (0.5 to 5.0 %). The results showed that concentration of dextrose

significantly affects the cordycepin production. It was found that maximum cordycepin

production (651 mg/L) could be achieved at 4% concentration of dextrose. Further, increase in

dextrose concentrations lead to decline in cordycepin production (Fig. 3b). The results are


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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. militaris [3, 17, 21, 23].

However Das et al., (2010) investigated the optimum production of cordycepin from mutated

strain of C. militaris at a concentration of 8.62 % dextrose [16]. Other carbon sources such as

fructose and sucrose have also been studied for cordycepin production from some other strains of

C. militaris [4, 16].

Effect of nitrogen source

The effects of nitrogen sources on the production of cordycepin by C. militaris 3936 have been

shown in fig. 3c. In comparison with inorganic nitrogen sources (sodium nitrate, ammonium

nitrate and ammonium sulphate), organic nitrogen sources (beef extract, casein, peptone, yeast

extract) gave rise to relatively higher cordycepin production. This is consistent with what was

previously suggested that most basiomycetes prefer complex organic nitrogen sources for their

growth in submerged cultures [5]. Several C. militaris strains such as C. militaris C738 and C.

militaris NG3 have been shown to have very poor mycelial growth in inorganic nitrogen sources

[6, 7]. In the present study, among the organic nitrogen sources, yeast extract was found to be

very effective for cordycepin (709 mg/L) production by C. militaris 3936.

Keeping in view of above results, yeast extract was selected as a suitable nitrogen source for

further studies. Fig. 3d, summarized the effects of various yeast extract concentrations on the

cordycepin production. The highest cordycepin production (846 mg/L) was obtained at 0.8%

concentration of yeast extract.

Conclusion

Cordycepin is considered a natural bioactive metabolite with a promising therapeutic potential.

Efforts are being made by the researchers to enhance cordycepin production from the Genus

Cordyceps. 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. The study would be extremely helpful to the scientific community especially to

the researchers working on cordycepin production from C. militaris 3936.


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Fig. 3: (a) Effect of various carbon sources on cordycepin production (mg/L) by C. militaris

3936. (b) Effect of different concentrations of dextrose on cordycepin production (mg/L) by C.

militaris 3936. (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. militaris 3936.

Conflict of interest

There are no potential conflicts of interest among the authors regarding the publication of this

manuscript.

Acknowledgments

The authors would like to acknowledge M.M. University Mullana (Ambala) for providing the

requisite facilities to perform this study


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Optimization of Fermentation Conditions for Cordycepin Production Using