Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps...
15
Research Article Optimization of Large-Scale Culture Conditions for the Production of Cordycepin with Cordyceps militaris by Liquid Static Culture Chao Kang, 1,2 Ting-Chi Wen, 1 Ji-Chuan Kang, 1 Ze-Bing Meng, 1 Guang-Rong Li, 1 and Kevin D. Hyde 3,4 1 e Engineering and Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang, Guizhou 550025, China 2 Institute of Biology, Guizhou Academy of Sciences, Guiyang, Guizhou 550009, China 3 Institute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, ailand 4 Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11442, Saudi Arabia Correspondence should be addressed to Ji-Chuan Kang; [email protected] Received 14 February 2014; Accepted 8 April 2014; Published 23 June 2014 Academic Editor: Rajesh Jeewon Copyright © 2014 Chao Kang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cordycepin is one of the most important bioactive compounds produced by species of Cordyceps sensu lato, but it is hard to produce large amounts of this substance in industrial production. In this work, single factor design, Plackett-Burman design, and central composite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepin production. Using these culture conditions, a maximum production of cordycepin was 2008.48 mg/L for 700 mL working volume in the 1000 mL glass jars and total content of cordycepin reached 1405.94 mg/bottle. is method provides an effective way for increasing the cordycepin production at a large scale. e strategies used in this study could have a wide application in other fermentation processes. 1. Introduction Cordyceps militaris is an entomopathogenic fungus belonging to Ascomycota, Sordariomycetidae, Hypocreales, and Cordy- cipitaceae [1] and is one of the most important traditional Chinese medicinal mushrooms. Cordyceps militaris is the type species of Cordyceps, which internally parasitizes larva or pupa of lepidopteran insects and forms fruiting bodies on their insect hosts. Cordyceps militaris has long been rec- ognized as a desirable alternative for natural Ophiocordyceps sinensis [2] as it has been given Chinese Licence number Z20030034/35. is is because the gathering of Ophiocordy- ceps sinensis is causing substantial reductions in populations [3]. Cordyceps militaris produces many bioactive compounds, including polysaccharides, cordycepin, adenosine, amino acid, organic selenium, ergosterol, sterols, cordycepic acid, superoxide dismutase (SOD), and multivitamins [4, 5]. Cordycepin (3 -deoxyadenosine), a nucleoside analog, was first isolated from C. militaris [6] and is one of the species most important biologically active metabolites. It has been regarded as a medicinal agent responsible for immunological regulation [7], anticancer [8], antifungus [9], antivirus [10], antileukemia [11, 12], and antihyperlipidemia [13] activities. Cordycepin is also a Phase I/II clinical stage drug candidate for treatment of refractory acute lymphoblastic leukemia (ALL) patients who express the enzyme terminal deoxynu- cleotidyl transferase (TdT) (http://www.ClinicalTrials.Gov verified by OncoVista, Inc., 2009). In previous work, cordycepin has been synthesized by chemical [14, 15] and microbial fermentation using C. mili- taris [6] or Aspergillus nidulans [16, 17]. Solid-state fermen- tation [18, 19], submerged culture [4, 20–24], and surface liquid culture [25–27] have been used in microbial fermenta- tion of cordycepin. Cordycepin obtained through chemistry Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 510627, 15 pages http://dx.doi.org/10.1155/2014/510627
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Transcript of Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps...
Research ArticleOptimization of Large-Scale CultureConditions for the Production of Cordycepin withCordyceps militaris by Liquid Static Culture
Chao Kang12 Ting-Chi Wen1 Ji-Chuan Kang1 Ze-Bing Meng1
Guang-Rong Li1 and Kevin D Hyde34
1 The Engineering and Research Center of Southwest Bio-Pharmaceutical Resources Ministry of EducationGuizhou University Guiyang Guizhou 550025 China
2 Institute of Biology Guizhou Academy of Sciences Guiyang Guizhou 550009 China3 Institute of Excellence in Fungal Research School of Science Mae Fah Luang University Chiang Rai 57100 Thailand4Botany and Microbiology Department College of Science King Saud University Riyadh 11442 Saudi Arabia
Correspondence should be addressed to Ji-Chuan Kang bcecjckanggzueducn
Received 14 February 2014 Accepted 8 April 2014 Published 23 June 2014
Academic Editor Rajesh Jeewon
Copyright copy 2014 Chao Kang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Cordycepin is one of themost important bioactive compounds produced by species ofCordyceps sensu lato but it is hard to producelarge amounts of this substance in industrial production In this work single factor design Plackett-Burman design and centralcomposite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepinproduction Using these culture conditions a maximum production of cordycepin was 200848mgL for 700mL working volumein the 1000mL glass jars and total content of cordycepin reached 140594mgbottle This method provides an effective way forincreasing the cordycepin production at a large scale The strategies used in this study could have a wide application in otherfermentation processes
1 Introduction
Cordycepsmilitaris is an entomopathogenic fungus belongingto Ascomycota Sordariomycetidae Hypocreales and Cordy-cipitaceae [1] and is one of the most important traditionalChinese medicinal mushrooms Cordyceps militaris is thetype species of Cordyceps which internally parasitizes larvaor pupa of lepidopteran insects and forms fruiting bodieson their insect hosts Cordyceps militaris has long been rec-ognized as a desirable alternative for natural Ophiocordycepssinensis [2] as it has been given Chinese Licence numberZ2003003435 This is because the gathering of Ophiocordy-ceps sinensis is causing substantial reductions in populations[3]Cordycepsmilitaris producesmany bioactive compoundsincluding polysaccharides cordycepin adenosine aminoacid organic selenium ergosterol sterols cordycepic acidsuperoxide dismutase (SOD) and multivitamins [4 5]
Cordycepin (31015840-deoxyadenosine) a nucleoside analogwas first isolated fromCmilitaris [6] and is one of the speciesmost important biologically active metabolites It has beenregarded as a medicinal agent responsible for immunologicalregulation [7] anticancer [8] antifungus [9] antivirus [10]antileukemia [11 12] and antihyperlipidemia [13] activitiesCordycepin is also a Phase III clinical stage drug candidatefor treatment of refractory acute lymphoblastic leukemia(ALL) patients who express the enzyme terminal deoxynu-cleotidyl transferase (TdT) (httpwwwClinicalTrialsGovverified by OncoVista Inc 2009)
In previous work cordycepin has been synthesized bychemical [14 15] and microbial fermentation using C mili-taris [6] or Aspergillus nidulans [16 17] Solid-state fermen-tation [18 19] submerged culture [4 20ndash24] and surfaceliquid culture [25ndash27] have been used in microbial fermenta-tion of cordycepin Cordycepin obtained through chemistry
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 510627 15 pageshttpdxdoiorg1011552014510627
2 The Scientific World Journal
pathways is hard to purify and the cost is much higherthan through biology fermentation Thus a major need is toimprove the biology methodology [28] Fermentation timeis too long and is difficult to achieve large scale productionvia solid-state fermentation [18 19] Productivity is generallylow the costs are high and fermentation processes are easilycontaminated in submerged culture in large fermenters [4 2021 29] Productivity in surface culture techniques is higheras compared to other methods [29 30] and the cost is lower[23] New technologies such as spacemutation treatment andhigh-energy ion beam irradiation have been used to obtainbetter Cordycepin producing novel mutants of C militarisThe resulting mutants were higher cordycepin producesthan the wild strain [30 31] Bu et al [20] reported thatthe cordycepin in C militaris was substantially increased bythe elicitor of Phytophthora sp Research result showed thatglucose and yeast extract were effective media componentsfor improved cordycepin production by C militaris [3233] There have been other studies using different cultureconditions [21 24 25 32] culture medium and additives[4 22ndash24 26 27] for the production of cordycepin via liquidculture However as far as we know these reports studiedcordycepin production in 250mL or 500mL Erlenmeyerflasks and there have beenno reports to improved cordycepinproduction using static liquid culture in 1000mL glass jarsThe latter process is a good way to scale up large scalecordycepin production from the laboratory to industry
In this study the effects of working volume carbonsources nitrogen sources inorganic salts growth factornucleoside analogue and amino acid additions were studiedin order to improve the cordycepin production by static liquidculture of C militaris (strain CGMCC2459) in 1000mL glassjars The results suggested that the optimization mediumconditions were helpful for improved large scale cordycepinproduction
2 Materials and Methods
21 Microorganism and Seed Culture The isolate of C mil-itaris (strain CGMCC2459) used in the present study wascollected from Mt Qingcheng in Sichuan Province ChinaThe microorganism was maintained on potato dextrose agar(PDA) slants Slants were incubated at 25∘C for 7 days andthen stored at 4∘C The seed culture was grown in a 250mLflask containing 70mL of basal medium (sucrose 20 gL pep-tone 20 gL KH
2PO41 gL andMgSO
4sdot7H2O05 gL) at 25∘C
on a rotary shaker incubator at 150 revmin for 5 days [24]
22 Basal Medium and Static Culture of Glass Jars Thebasal medium composition for the fermentation was asfollows sucrose 20 gL peptone 20 gL KH
2PO41 gL and
MgSO4sdot7H2O 05 gL The pH was not adjusted followed
by autoclaving for 30min on the 121∘C The static cultureexperiments were performed in 1000mL glass jars (innerdiameter 110mm height 150mm) containing basal mediumafter inoculating with 10 (vv the biomass dry weight ofseed culture is 54mgmL) of the seed cultureThe culture wasincubated at 25∘C without moving for 35 days and sampleswere collected at the end of the fermentation from the
glass jars for analyzing biomass dry weight and cordycepinproduction
23 Static Culture Conditions The effects of factors affect-ing cell growth and the production of cordycepin by Cmilitaris were studied using a one-factor-at-a-time methodfor static culture The effects of carbon sources on cordy-cepinproduction were studied by substituting carbon sourcessuch as sucrose lactose soluble starch and dextrin forglucose at 25∘C for 35 days Effects of nitrogen sources(yeast extract beef extract NH
4NO3 NaNO
3 NH
4Cl
casein and carbamide) and inorganic salts (MgCl2sdot6H2O
MgSO4sdot7H2O KCl ZnSO
4 CaCl
2sdot2H2O CaSO
4sdot2H2O
FeSO4sdot7H2O and K
2HPO4sdot3H2O) were also studied using
static culture Growth factors (Vitamin B1(VB1) Vita-
min B6(VB6) Vitamin B
7(VB7) Vitamin B
11(VB11) 120572-
naphthylacetic acid (NAA) 3-Indoleacetlc acid (IAA) and24-dichlorophenoxyacetic acid (24-D)) were supplementedfor 10mgL in basal media Nucleoside analogues (1 gL) andamino acids (8 gL) established in our previous study [23] asan initial concentration were separately added to the optimalconcentration of carbon and nitrogen source inorganic saltsand growth factors and cultivated at 25∘C for 35 days Allexperiments were carried out at triplicate andmean of resultsis presented
24 Analytical Methods Samples collected at 35 days fromthe glass jars were centrifuged at 2810timesg for 20min Themycelium at the bottom of tubes was washed sufficiently witha large amount of distilled water and dried to a constant dryweight at 55∘C
For analysis of extracellular cordycepin the resultingculture filtrate was obtained by centrifugation at 2810timesgfor 20min The supernatant was filtered through a 045 120583mmembrane and the filtrate was analyzed by HPLC (1100series Agilent Technology USA) Accurate quantities ofcordycepin (Sigma USA) were dissolved in distilled waterto give various concentrations for calibration The mobilephase was 10mmolL KH
2PO4 which was dissolved in
methanoldistilled water (6 94) Elution was performed at aflow rate of 10mLmin with column temperature at 45∘C andUVwavelength of 259 nmMean values were computed fromtriplicate samples
25 Plackett-Burman Design The Plackett-Burman designan effective technique for medium-component optimization[35 36] was used to select factors that significantly influ-enced hydrogen production Sucrose (119883
1) peptone (119883
2)
K2HPO4sdot3H2O (119883
4) MgSO
4sdot7H2O (119883
5) and VB
1(1198836)
were investigated as key ingredients affecting cordycepinproduction Based on the Plackett-Burman design a 15-runwas applied to evaluate eleven factors (including two virtualvariables) Each factor was prepared in two levels minus1 for lowlevel and +1 for high level Table 1 illustrates the variablesand their corresponding levels used in the experimentaldesign The values of two levels were set according toour preliminary experimental results The Plackett-Burmandesign and the response value of cordycepin production areshown in Table 2
The Scientific World Journal 3
Table 1 Range of different factors investigated with Plackett-Burman design
Symbol Variables Experimental valueLow (minus1) High (+1)
119883
1Sucrose (gL) 20 25
119883
2Peptone (gL) 20 25
119883
3Virtual 1 minus1 1
119883
4K2HPO4sdot3H2O (gL) 1 125
119883
5VB1 (gL) 10 125
119883
6MgSO4sdot7H2O (gL) 1 125
119883
7Virtual 2 minus1 1
26 Response Surface Methodology Response surface meth-odology using a central composite design was applied tobatch cultures ofCmilitaris for identifying the effects of pro-cess variables [35 36] In this study the basic nutrient (carbonsources nitrogen sources inorganic salts and growth factors)and additives (amino acid nucleoside analogue) were studiedfor cordycepin production using static liquid culture Inthe first test a three-factor five-level central compositedesign with 20 runs was employed Tested variables (sucroseK2HPO4sdot3H2O and MgSO
4sdot7H2O) were denoted as 119883
1 1198834
and 1198836 respectively and each of them was assessed at five
different levels combining factorial points (minus1 minus1) axialpoints (minus16818 +16818) and central point (0) as shown inTable 3 Based on the above results another test a three-factor five-level central composite design with 20 runs wasemployed Tested variables (amino acid nucleoside analogueand culture time) were denoted as 119860 119861 and 119862 respectivelyand each of them was assessed at five different levels com-bining factorial points (minus1 +1) axial points (minus16818 +16818)and central point (0) as shown in Table 4
27 Statistical Analysis Dry weight and cordycepin produc-tion are expressed as means plusmn SD An analysis of variance(ANOVA) followed by Tukeyrsquos test was applied for multiplecomparisons of significant analyses at 119875 lt 005 Statisticaldata analyses were performed in SPSS version 170 softwarepacket Design-Expert Version 805b software package (Stat-Ease Inc Minneapolis USA) was used for designing exper-iments as well as for regression and graphical analysis of theexperimental data obtained
3 Results and Discussion
31 Effects of Working Volume on the Biomass and CordycepinProduction Dissolved oxygen concentration is the key factorin the medium for cell growth and metabolite biosynthesis[21] Dissolved oxygen does not only have an importantfunction in the respiratory chain but also in metabolite com-position [37 38] A previous study showed that the highestcordycepin production and productivity were obtained atlower dissolved oxygen levels [21] Masuda et al [25] alsoreported that a lower medium depth was most efficient forcordycepin production in C militaris by surface culture
100
100
200
200
300
300
0
400
400
500
500
600
600
700
700
800
800
900
Working volume (mL)
Cor
dyce
pin
prod
uctio
n (m
gL)
Tota
l con
tent
of c
ordy
cepi
n (m
g)
Cordycepin productionTotal content of cordycepinBiomass dry weight
Biom
ass d
ry w
eigh
t (g)
0
2
4
6
8
10
Figure 1 Effects of working volume on the production of cordy-cepin total production of cordycepin and biomass dry weight(total content ofcordycepin (mg) = cordycepin production (mgL)times working volume (mL))
In this study we tried to establish the most efficientworking volume of medium for improved cordycepin pro-duction Cultures ofC militariswere prepared at the workingvolumes of 100 to 900mL (corresponding to a medium depthof 126 to 1131 cm) As shown in Figure 1 cordycepin pro-duction reduced gradually with increasing working volumeof the medium from 100 to 700mL However there was nosignificant difference in cordycepin production in differentworking volumes Obviously the highest working volume of900mL did not help in cordycepin production The resultindicates that there is an upper dissolved oxygen limit inthe medium for cordycepin production [21] Lower workingvolumes result in higher cordycepin productivity with thehighest peak (46333 plusmn 5672mg of cordycepin) producedat using 700mL of media Changes in biomass values weresmall (between 300 and 700mL) because of the restricted areaand thickness of the mycelial mat In order to obtain highercordycepin production the most effective medium amountwas 700mL (corresponding to an 88 cmmedium depth) andused as the media volume for next experiment
32 Effects of Carbon and Nitrogen Sources on CordycepinProduction To find a suitable carbon source for C militariscordycepin production we added various carbon sources ata concentration of 20 gL to the sugar-free basal mediumGlucose was previously found to be an excellent precur-sor of cordycepin production [39] However as shown inFigure 2(a) sucrose and lactose proved to be better carbonsources for cordycepin production than glucose in thisstudy Cordycepin production reached 84363 plusmn 6670mgLof sucrose and 82372 plusmn 8564mgL of lactose respectivelyTherefore sucrose was selected as the main carbon source inthe remaining experiment
4 The Scientific World Journal
Table 2 Plackett-Burman design and response values
Runs Experimental value119884 (mgL) Cordycepin production
119883
1119883
2119883
3119883
4119883
5119883
6119883
7
1 1 minus1 1 minus1 minus1 minus1 1 81236 plusmn 2683
2 1 1 minus1 1 minus1 minus1 minus1 139518 plusmn 84
3 minus1 1 1 minus1 1 minus1 minus1 90025 plusmn 1029
4 1 minus1 1 1 minus1 1 minus1 80245 plusmn 4543
5 1 1 minus1 1 1 minus1 1 109766 plusmn 2557
6 1 1 1 minus1 1 1 minus1 84587 plusmn 2494
7 minus1 1 1 1 minus1 1 1 78635 plusmn 761
8 minus1 minus1 1 1 1 minus1 1 80508 plusmn 291
9 minus1 minus1 minus1 1 1 1 minus1 92048 plusmn 1621
10 1 minus1 minus1 minus1 1 1 1 69401 plusmn 7951
11 minus1 1 minus1 minus1 minus1 1 1 49728 plusmn 444
12 minus1 minus1 minus1 minus1 minus1 minus1 minus1 59283 plusmn 1613
13 0 0 0 0 0 0 0 113414 plusmn 259
14 0 0 0 0 0 0 0 110021 plusmn 008
15 0 0 0 0 0 0 0 113356 plusmn 185
Table 3 Factors and levels of central composite design for carbonsources and inorganic salts
Symbol Variables Code levelminus16818 minus1 0 1 16818
119883
1Sucrose (gL) 31821 10 20 30 368179
119883
4K2HPO4sdot3H2O (gL) 01591 05 1 15 18409
119883
6MgSO4sdot7H2O (gL) 01591 05 1 15 18409
In previous work nitrogen showed a regulating roleimportant in cordycepin production and had two effects [40]One effect was negative since in excess N promoted a fastermycelial growth and consequently diverted the source ofcarbon toward energy and biomass production The othereffect was positive because a moderate input contributedto the maintenance of citric acid productive biomass [40]To investigate the effect of nitrogen sources on cordycepinproduction in C militaris various compounds containingnitrogen (inorganic and organic nitrogen) were added indi-vidually to nitrogen free basal medium at a concentration of20 gL Among the 8 nitrogen sources tested peptone yeastextract beef extract casein and NH
4NO3were favorable
to the cordycepin production (Figure 2(b)) Organic nitro-gen was advantageous to both growth and biosynthesis ofmetabolites The result is consistent with the experimentaldata reported [18] and showed that maximum cordycepinproduction resulted when the peptone was used as a nitrogensource
33 Effects of Inorganic Salt and Growth Factor on the Cordy-cepin Production Inorganic ion was one of the most impor-tant nutrition components ofmedium for themycelial growth[41] In order to investigate the effects of inorganic salt for thecordycepin production inCmilitaris we tested nine types (at1 gL) of inorganic salts (Figure 3(a)) Media with only 20 gLglucose and 20 gL peptone were used as the control The
Table 4 Factors and levels of central composite design for aminoacid nucleoside analogue and time
Symbol Variables Code levelminus16818 minus1 0 1 16818
119860 Hypoxanthine (gL) 053 1 5 9 1053119861 L-alanine (gL) 527 8 12 16 1872119862 Culture time (days) minus009 4 10 16 2009
highest cordycepin production (112030 plusmn 10528mgL) byC militaris was observed in medium when K
2HPO4sdot3H2O
was used as an inorganic salt KH2PO4 MgSO
4sdot7H2O
KCl and MgCl2sdot6H2O were also useful inorganic salts At
last MgSO4sdot7H2O and K
2HPO4sdot3H2O were recognized as
favorable bioelements for production of cordycepinGrowth factor is essential for growth response andmetab-
olite production [42] In order to find the optimal growthfactor for cordycepin production C militaris was culturedin a basal medium with different vitamins and plant growthhormones in static liquid culture Cordycepin productionincreased in media with added 10mgL of VB
1 NAA
and VB11
(Figure 3(b)) Maximum cordycepin production(115934 plusmn 10901mgL) occurred when VB
1was used as the
growth factor
34 Screening of Important Variables Using Plackett-BurmanDesign The data (Table 2) indicated wide variation in cordy-cepin production in the 15 tests The data suggested that pro-cess optimization is important for improving the efficiency ofcordycepin production Analysis of the regression coefficientsand 119905 values of 7 factors (Table 5) showed that 119883
1 1198832 1198834
and 1198835had positive effects on cordycepin production 119883
6
had negative effects The variable affects with a confidencelevel above 95are considered as significant factors Based onthese results three factors (119883
1 sucrose 119883
4 K2HPO4sdot3H2O
The Scientific World Journal 5
Control Glucose Dextrin Sucrose Lactose Soluble
Carbon source
0
100
200
300
400
500
600
700
800
900lowastlowast lowastlowast
starch
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
Cordycepin production
(a)
0
100
200
300
400
500
600
700
800
1000
900
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Pept
one
Yeas
t ext
ract
Beef
extr
act
Case
in
NH
4Cl
NH
4N
O3
NaN
O3
Carb
amid
e
Nitrogen sources
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Cordycepin production
(b)
Figure 2 Effects of carbon sources and nitrogen sources on the production of cordycepin carbon sources (a) nitrogen sources (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
Con
trol
K 2H
PO4 KH
2PO
4
middot3H
2O
MgC
l 2middot6
H2O
MgS
O4middot7
H2O
ZnSO
CaSO
4middot2
H2O
CaCl
2
4
middot2H
2O
KCl
FeSO
4middot7
H2O
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowast
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production
Inorganic salts
(a)
Cordycepin production
Growth factor
Control IAA VC VB1 NAA VB1124-D
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
lowast
lowastlowast
(b)
Figure 3 Effects of inorganic salt and growth factors on the production of cordycepin inorganic salt (a) growth factors (b) lowast5 significancelevel (test group versus control group) lowastlowast
1 significance level (test group versus control group)
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
2 The Scientific World Journal
pathways is hard to purify and the cost is much higherthan through biology fermentation Thus a major need is toimprove the biology methodology [28] Fermentation timeis too long and is difficult to achieve large scale productionvia solid-state fermentation [18 19] Productivity is generallylow the costs are high and fermentation processes are easilycontaminated in submerged culture in large fermenters [4 2021 29] Productivity in surface culture techniques is higheras compared to other methods [29 30] and the cost is lower[23] New technologies such as spacemutation treatment andhigh-energy ion beam irradiation have been used to obtainbetter Cordycepin producing novel mutants of C militarisThe resulting mutants were higher cordycepin producesthan the wild strain [30 31] Bu et al [20] reported thatthe cordycepin in C militaris was substantially increased bythe elicitor of Phytophthora sp Research result showed thatglucose and yeast extract were effective media componentsfor improved cordycepin production by C militaris [3233] There have been other studies using different cultureconditions [21 24 25 32] culture medium and additives[4 22ndash24 26 27] for the production of cordycepin via liquidculture However as far as we know these reports studiedcordycepin production in 250mL or 500mL Erlenmeyerflasks and there have beenno reports to improved cordycepinproduction using static liquid culture in 1000mL glass jarsThe latter process is a good way to scale up large scalecordycepin production from the laboratory to industry
In this study the effects of working volume carbonsources nitrogen sources inorganic salts growth factornucleoside analogue and amino acid additions were studiedin order to improve the cordycepin production by static liquidculture of C militaris (strain CGMCC2459) in 1000mL glassjars The results suggested that the optimization mediumconditions were helpful for improved large scale cordycepinproduction
2 Materials and Methods
21 Microorganism and Seed Culture The isolate of C mil-itaris (strain CGMCC2459) used in the present study wascollected from Mt Qingcheng in Sichuan Province ChinaThe microorganism was maintained on potato dextrose agar(PDA) slants Slants were incubated at 25∘C for 7 days andthen stored at 4∘C The seed culture was grown in a 250mLflask containing 70mL of basal medium (sucrose 20 gL pep-tone 20 gL KH
2PO41 gL andMgSO
4sdot7H2O05 gL) at 25∘C
on a rotary shaker incubator at 150 revmin for 5 days [24]
22 Basal Medium and Static Culture of Glass Jars Thebasal medium composition for the fermentation was asfollows sucrose 20 gL peptone 20 gL KH
2PO41 gL and
MgSO4sdot7H2O 05 gL The pH was not adjusted followed
by autoclaving for 30min on the 121∘C The static cultureexperiments were performed in 1000mL glass jars (innerdiameter 110mm height 150mm) containing basal mediumafter inoculating with 10 (vv the biomass dry weight ofseed culture is 54mgmL) of the seed cultureThe culture wasincubated at 25∘C without moving for 35 days and sampleswere collected at the end of the fermentation from the
glass jars for analyzing biomass dry weight and cordycepinproduction
23 Static Culture Conditions The effects of factors affect-ing cell growth and the production of cordycepin by Cmilitaris were studied using a one-factor-at-a-time methodfor static culture The effects of carbon sources on cordy-cepinproduction were studied by substituting carbon sourcessuch as sucrose lactose soluble starch and dextrin forglucose at 25∘C for 35 days Effects of nitrogen sources(yeast extract beef extract NH
4NO3 NaNO
3 NH
4Cl
casein and carbamide) and inorganic salts (MgCl2sdot6H2O
MgSO4sdot7H2O KCl ZnSO
4 CaCl
2sdot2H2O CaSO
4sdot2H2O
FeSO4sdot7H2O and K
2HPO4sdot3H2O) were also studied using
static culture Growth factors (Vitamin B1(VB1) Vita-
min B6(VB6) Vitamin B
7(VB7) Vitamin B
11(VB11) 120572-
naphthylacetic acid (NAA) 3-Indoleacetlc acid (IAA) and24-dichlorophenoxyacetic acid (24-D)) were supplementedfor 10mgL in basal media Nucleoside analogues (1 gL) andamino acids (8 gL) established in our previous study [23] asan initial concentration were separately added to the optimalconcentration of carbon and nitrogen source inorganic saltsand growth factors and cultivated at 25∘C for 35 days Allexperiments were carried out at triplicate andmean of resultsis presented
24 Analytical Methods Samples collected at 35 days fromthe glass jars were centrifuged at 2810timesg for 20min Themycelium at the bottom of tubes was washed sufficiently witha large amount of distilled water and dried to a constant dryweight at 55∘C
For analysis of extracellular cordycepin the resultingculture filtrate was obtained by centrifugation at 2810timesgfor 20min The supernatant was filtered through a 045 120583mmembrane and the filtrate was analyzed by HPLC (1100series Agilent Technology USA) Accurate quantities ofcordycepin (Sigma USA) were dissolved in distilled waterto give various concentrations for calibration The mobilephase was 10mmolL KH
2PO4 which was dissolved in
methanoldistilled water (6 94) Elution was performed at aflow rate of 10mLmin with column temperature at 45∘C andUVwavelength of 259 nmMean values were computed fromtriplicate samples
25 Plackett-Burman Design The Plackett-Burman designan effective technique for medium-component optimization[35 36] was used to select factors that significantly influ-enced hydrogen production Sucrose (119883
1) peptone (119883
2)
K2HPO4sdot3H2O (119883
4) MgSO
4sdot7H2O (119883
5) and VB
1(1198836)
were investigated as key ingredients affecting cordycepinproduction Based on the Plackett-Burman design a 15-runwas applied to evaluate eleven factors (including two virtualvariables) Each factor was prepared in two levels minus1 for lowlevel and +1 for high level Table 1 illustrates the variablesand their corresponding levels used in the experimentaldesign The values of two levels were set according toour preliminary experimental results The Plackett-Burmandesign and the response value of cordycepin production areshown in Table 2
The Scientific World Journal 3
Table 1 Range of different factors investigated with Plackett-Burman design
Symbol Variables Experimental valueLow (minus1) High (+1)
119883
1Sucrose (gL) 20 25
119883
2Peptone (gL) 20 25
119883
3Virtual 1 minus1 1
119883
4K2HPO4sdot3H2O (gL) 1 125
119883
5VB1 (gL) 10 125
119883
6MgSO4sdot7H2O (gL) 1 125
119883
7Virtual 2 minus1 1
26 Response Surface Methodology Response surface meth-odology using a central composite design was applied tobatch cultures ofCmilitaris for identifying the effects of pro-cess variables [35 36] In this study the basic nutrient (carbonsources nitrogen sources inorganic salts and growth factors)and additives (amino acid nucleoside analogue) were studiedfor cordycepin production using static liquid culture Inthe first test a three-factor five-level central compositedesign with 20 runs was employed Tested variables (sucroseK2HPO4sdot3H2O and MgSO
4sdot7H2O) were denoted as 119883
1 1198834
and 1198836 respectively and each of them was assessed at five
different levels combining factorial points (minus1 minus1) axialpoints (minus16818 +16818) and central point (0) as shown inTable 3 Based on the above results another test a three-factor five-level central composite design with 20 runs wasemployed Tested variables (amino acid nucleoside analogueand culture time) were denoted as 119860 119861 and 119862 respectivelyand each of them was assessed at five different levels com-bining factorial points (minus1 +1) axial points (minus16818 +16818)and central point (0) as shown in Table 4
27 Statistical Analysis Dry weight and cordycepin produc-tion are expressed as means plusmn SD An analysis of variance(ANOVA) followed by Tukeyrsquos test was applied for multiplecomparisons of significant analyses at 119875 lt 005 Statisticaldata analyses were performed in SPSS version 170 softwarepacket Design-Expert Version 805b software package (Stat-Ease Inc Minneapolis USA) was used for designing exper-iments as well as for regression and graphical analysis of theexperimental data obtained
3 Results and Discussion
31 Effects of Working Volume on the Biomass and CordycepinProduction Dissolved oxygen concentration is the key factorin the medium for cell growth and metabolite biosynthesis[21] Dissolved oxygen does not only have an importantfunction in the respiratory chain but also in metabolite com-position [37 38] A previous study showed that the highestcordycepin production and productivity were obtained atlower dissolved oxygen levels [21] Masuda et al [25] alsoreported that a lower medium depth was most efficient forcordycepin production in C militaris by surface culture
100
100
200
200
300
300
0
400
400
500
500
600
600
700
700
800
800
900
Working volume (mL)
Cor
dyce
pin
prod
uctio
n (m
gL)
Tota
l con
tent
of c
ordy
cepi
n (m
g)
Cordycepin productionTotal content of cordycepinBiomass dry weight
Biom
ass d
ry w
eigh
t (g)
0
2
4
6
8
10
Figure 1 Effects of working volume on the production of cordy-cepin total production of cordycepin and biomass dry weight(total content ofcordycepin (mg) = cordycepin production (mgL)times working volume (mL))
In this study we tried to establish the most efficientworking volume of medium for improved cordycepin pro-duction Cultures ofC militariswere prepared at the workingvolumes of 100 to 900mL (corresponding to a medium depthof 126 to 1131 cm) As shown in Figure 1 cordycepin pro-duction reduced gradually with increasing working volumeof the medium from 100 to 700mL However there was nosignificant difference in cordycepin production in differentworking volumes Obviously the highest working volume of900mL did not help in cordycepin production The resultindicates that there is an upper dissolved oxygen limit inthe medium for cordycepin production [21] Lower workingvolumes result in higher cordycepin productivity with thehighest peak (46333 plusmn 5672mg of cordycepin) producedat using 700mL of media Changes in biomass values weresmall (between 300 and 700mL) because of the restricted areaand thickness of the mycelial mat In order to obtain highercordycepin production the most effective medium amountwas 700mL (corresponding to an 88 cmmedium depth) andused as the media volume for next experiment
32 Effects of Carbon and Nitrogen Sources on CordycepinProduction To find a suitable carbon source for C militariscordycepin production we added various carbon sources ata concentration of 20 gL to the sugar-free basal mediumGlucose was previously found to be an excellent precur-sor of cordycepin production [39] However as shown inFigure 2(a) sucrose and lactose proved to be better carbonsources for cordycepin production than glucose in thisstudy Cordycepin production reached 84363 plusmn 6670mgLof sucrose and 82372 plusmn 8564mgL of lactose respectivelyTherefore sucrose was selected as the main carbon source inthe remaining experiment
4 The Scientific World Journal
Table 2 Plackett-Burman design and response values
Runs Experimental value119884 (mgL) Cordycepin production
119883
1119883
2119883
3119883
4119883
5119883
6119883
7
1 1 minus1 1 minus1 minus1 minus1 1 81236 plusmn 2683
2 1 1 minus1 1 minus1 minus1 minus1 139518 plusmn 84
3 minus1 1 1 minus1 1 minus1 minus1 90025 plusmn 1029
4 1 minus1 1 1 minus1 1 minus1 80245 plusmn 4543
5 1 1 minus1 1 1 minus1 1 109766 plusmn 2557
6 1 1 1 minus1 1 1 minus1 84587 plusmn 2494
7 minus1 1 1 1 minus1 1 1 78635 plusmn 761
8 minus1 minus1 1 1 1 minus1 1 80508 plusmn 291
9 minus1 minus1 minus1 1 1 1 minus1 92048 plusmn 1621
10 1 minus1 minus1 minus1 1 1 1 69401 plusmn 7951
11 minus1 1 minus1 minus1 minus1 1 1 49728 plusmn 444
12 minus1 minus1 minus1 minus1 minus1 minus1 minus1 59283 plusmn 1613
13 0 0 0 0 0 0 0 113414 plusmn 259
14 0 0 0 0 0 0 0 110021 plusmn 008
15 0 0 0 0 0 0 0 113356 plusmn 185
Table 3 Factors and levels of central composite design for carbonsources and inorganic salts
Symbol Variables Code levelminus16818 minus1 0 1 16818
119883
1Sucrose (gL) 31821 10 20 30 368179
119883
4K2HPO4sdot3H2O (gL) 01591 05 1 15 18409
119883
6MgSO4sdot7H2O (gL) 01591 05 1 15 18409
In previous work nitrogen showed a regulating roleimportant in cordycepin production and had two effects [40]One effect was negative since in excess N promoted a fastermycelial growth and consequently diverted the source ofcarbon toward energy and biomass production The othereffect was positive because a moderate input contributedto the maintenance of citric acid productive biomass [40]To investigate the effect of nitrogen sources on cordycepinproduction in C militaris various compounds containingnitrogen (inorganic and organic nitrogen) were added indi-vidually to nitrogen free basal medium at a concentration of20 gL Among the 8 nitrogen sources tested peptone yeastextract beef extract casein and NH
4NO3were favorable
to the cordycepin production (Figure 2(b)) Organic nitro-gen was advantageous to both growth and biosynthesis ofmetabolites The result is consistent with the experimentaldata reported [18] and showed that maximum cordycepinproduction resulted when the peptone was used as a nitrogensource
33 Effects of Inorganic Salt and Growth Factor on the Cordy-cepin Production Inorganic ion was one of the most impor-tant nutrition components ofmedium for themycelial growth[41] In order to investigate the effects of inorganic salt for thecordycepin production inCmilitaris we tested nine types (at1 gL) of inorganic salts (Figure 3(a)) Media with only 20 gLglucose and 20 gL peptone were used as the control The
Table 4 Factors and levels of central composite design for aminoacid nucleoside analogue and time
Symbol Variables Code levelminus16818 minus1 0 1 16818
119860 Hypoxanthine (gL) 053 1 5 9 1053119861 L-alanine (gL) 527 8 12 16 1872119862 Culture time (days) minus009 4 10 16 2009
highest cordycepin production (112030 plusmn 10528mgL) byC militaris was observed in medium when K
2HPO4sdot3H2O
was used as an inorganic salt KH2PO4 MgSO
4sdot7H2O
KCl and MgCl2sdot6H2O were also useful inorganic salts At
last MgSO4sdot7H2O and K
2HPO4sdot3H2O were recognized as
favorable bioelements for production of cordycepinGrowth factor is essential for growth response andmetab-
olite production [42] In order to find the optimal growthfactor for cordycepin production C militaris was culturedin a basal medium with different vitamins and plant growthhormones in static liquid culture Cordycepin productionincreased in media with added 10mgL of VB
1 NAA
and VB11
(Figure 3(b)) Maximum cordycepin production(115934 plusmn 10901mgL) occurred when VB
1was used as the
growth factor
34 Screening of Important Variables Using Plackett-BurmanDesign The data (Table 2) indicated wide variation in cordy-cepin production in the 15 tests The data suggested that pro-cess optimization is important for improving the efficiency ofcordycepin production Analysis of the regression coefficientsand 119905 values of 7 factors (Table 5) showed that 119883
1 1198832 1198834
and 1198835had positive effects on cordycepin production 119883
6
had negative effects The variable affects with a confidencelevel above 95are considered as significant factors Based onthese results three factors (119883
1 sucrose 119883
4 K2HPO4sdot3H2O
The Scientific World Journal 5
Control Glucose Dextrin Sucrose Lactose Soluble
Carbon source
0
100
200
300
400
500
600
700
800
900lowastlowast lowastlowast
starch
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
Cordycepin production
(a)
0
100
200
300
400
500
600
700
800
1000
900
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Pept
one
Yeas
t ext
ract
Beef
extr
act
Case
in
NH
4Cl
NH
4N
O3
NaN
O3
Carb
amid
e
Nitrogen sources
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Cordycepin production
(b)
Figure 2 Effects of carbon sources and nitrogen sources on the production of cordycepin carbon sources (a) nitrogen sources (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
Con
trol
K 2H
PO4 KH
2PO
4
middot3H
2O
MgC
l 2middot6
H2O
MgS
O4middot7
H2O
ZnSO
CaSO
4middot2
H2O
CaCl
2
4
middot2H
2O
KCl
FeSO
4middot7
H2O
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowast
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production
Inorganic salts
(a)
Cordycepin production
Growth factor
Control IAA VC VB1 NAA VB1124-D
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
lowast
lowastlowast
(b)
Figure 3 Effects of inorganic salt and growth factors on the production of cordycepin inorganic salt (a) growth factors (b) lowast5 significancelevel (test group versus control group) lowastlowast
1 significance level (test group versus control group)
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 3
Table 1 Range of different factors investigated with Plackett-Burman design
Symbol Variables Experimental valueLow (minus1) High (+1)
119883
1Sucrose (gL) 20 25
119883
2Peptone (gL) 20 25
119883
3Virtual 1 minus1 1
119883
4K2HPO4sdot3H2O (gL) 1 125
119883
5VB1 (gL) 10 125
119883
6MgSO4sdot7H2O (gL) 1 125
119883
7Virtual 2 minus1 1
26 Response Surface Methodology Response surface meth-odology using a central composite design was applied tobatch cultures ofCmilitaris for identifying the effects of pro-cess variables [35 36] In this study the basic nutrient (carbonsources nitrogen sources inorganic salts and growth factors)and additives (amino acid nucleoside analogue) were studiedfor cordycepin production using static liquid culture Inthe first test a three-factor five-level central compositedesign with 20 runs was employed Tested variables (sucroseK2HPO4sdot3H2O and MgSO
4sdot7H2O) were denoted as 119883
1 1198834
and 1198836 respectively and each of them was assessed at five
different levels combining factorial points (minus1 minus1) axialpoints (minus16818 +16818) and central point (0) as shown inTable 3 Based on the above results another test a three-factor five-level central composite design with 20 runs wasemployed Tested variables (amino acid nucleoside analogueand culture time) were denoted as 119860 119861 and 119862 respectivelyand each of them was assessed at five different levels com-bining factorial points (minus1 +1) axial points (minus16818 +16818)and central point (0) as shown in Table 4
27 Statistical Analysis Dry weight and cordycepin produc-tion are expressed as means plusmn SD An analysis of variance(ANOVA) followed by Tukeyrsquos test was applied for multiplecomparisons of significant analyses at 119875 lt 005 Statisticaldata analyses were performed in SPSS version 170 softwarepacket Design-Expert Version 805b software package (Stat-Ease Inc Minneapolis USA) was used for designing exper-iments as well as for regression and graphical analysis of theexperimental data obtained
3 Results and Discussion
31 Effects of Working Volume on the Biomass and CordycepinProduction Dissolved oxygen concentration is the key factorin the medium for cell growth and metabolite biosynthesis[21] Dissolved oxygen does not only have an importantfunction in the respiratory chain but also in metabolite com-position [37 38] A previous study showed that the highestcordycepin production and productivity were obtained atlower dissolved oxygen levels [21] Masuda et al [25] alsoreported that a lower medium depth was most efficient forcordycepin production in C militaris by surface culture
100
100
200
200
300
300
0
400
400
500
500
600
600
700
700
800
800
900
Working volume (mL)
Cor
dyce
pin
prod
uctio
n (m
gL)
Tota
l con
tent
of c
ordy
cepi
n (m
g)
Cordycepin productionTotal content of cordycepinBiomass dry weight
Biom
ass d
ry w
eigh
t (g)
0
2
4
6
8
10
Figure 1 Effects of working volume on the production of cordy-cepin total production of cordycepin and biomass dry weight(total content ofcordycepin (mg) = cordycepin production (mgL)times working volume (mL))
In this study we tried to establish the most efficientworking volume of medium for improved cordycepin pro-duction Cultures ofC militariswere prepared at the workingvolumes of 100 to 900mL (corresponding to a medium depthof 126 to 1131 cm) As shown in Figure 1 cordycepin pro-duction reduced gradually with increasing working volumeof the medium from 100 to 700mL However there was nosignificant difference in cordycepin production in differentworking volumes Obviously the highest working volume of900mL did not help in cordycepin production The resultindicates that there is an upper dissolved oxygen limit inthe medium for cordycepin production [21] Lower workingvolumes result in higher cordycepin productivity with thehighest peak (46333 plusmn 5672mg of cordycepin) producedat using 700mL of media Changes in biomass values weresmall (between 300 and 700mL) because of the restricted areaand thickness of the mycelial mat In order to obtain highercordycepin production the most effective medium amountwas 700mL (corresponding to an 88 cmmedium depth) andused as the media volume for next experiment
32 Effects of Carbon and Nitrogen Sources on CordycepinProduction To find a suitable carbon source for C militariscordycepin production we added various carbon sources ata concentration of 20 gL to the sugar-free basal mediumGlucose was previously found to be an excellent precur-sor of cordycepin production [39] However as shown inFigure 2(a) sucrose and lactose proved to be better carbonsources for cordycepin production than glucose in thisstudy Cordycepin production reached 84363 plusmn 6670mgLof sucrose and 82372 plusmn 8564mgL of lactose respectivelyTherefore sucrose was selected as the main carbon source inthe remaining experiment
4 The Scientific World Journal
Table 2 Plackett-Burman design and response values
Runs Experimental value119884 (mgL) Cordycepin production
119883
1119883
2119883
3119883
4119883
5119883
6119883
7
1 1 minus1 1 minus1 minus1 minus1 1 81236 plusmn 2683
2 1 1 minus1 1 minus1 minus1 minus1 139518 plusmn 84
3 minus1 1 1 minus1 1 minus1 minus1 90025 plusmn 1029
4 1 minus1 1 1 minus1 1 minus1 80245 plusmn 4543
5 1 1 minus1 1 1 minus1 1 109766 plusmn 2557
6 1 1 1 minus1 1 1 minus1 84587 plusmn 2494
7 minus1 1 1 1 minus1 1 1 78635 plusmn 761
8 minus1 minus1 1 1 1 minus1 1 80508 plusmn 291
9 minus1 minus1 minus1 1 1 1 minus1 92048 plusmn 1621
10 1 minus1 minus1 minus1 1 1 1 69401 plusmn 7951
11 minus1 1 minus1 minus1 minus1 1 1 49728 plusmn 444
12 minus1 minus1 minus1 minus1 minus1 minus1 minus1 59283 plusmn 1613
13 0 0 0 0 0 0 0 113414 plusmn 259
14 0 0 0 0 0 0 0 110021 plusmn 008
15 0 0 0 0 0 0 0 113356 plusmn 185
Table 3 Factors and levels of central composite design for carbonsources and inorganic salts
Symbol Variables Code levelminus16818 minus1 0 1 16818
119883
1Sucrose (gL) 31821 10 20 30 368179
119883
4K2HPO4sdot3H2O (gL) 01591 05 1 15 18409
119883
6MgSO4sdot7H2O (gL) 01591 05 1 15 18409
In previous work nitrogen showed a regulating roleimportant in cordycepin production and had two effects [40]One effect was negative since in excess N promoted a fastermycelial growth and consequently diverted the source ofcarbon toward energy and biomass production The othereffect was positive because a moderate input contributedto the maintenance of citric acid productive biomass [40]To investigate the effect of nitrogen sources on cordycepinproduction in C militaris various compounds containingnitrogen (inorganic and organic nitrogen) were added indi-vidually to nitrogen free basal medium at a concentration of20 gL Among the 8 nitrogen sources tested peptone yeastextract beef extract casein and NH
4NO3were favorable
to the cordycepin production (Figure 2(b)) Organic nitro-gen was advantageous to both growth and biosynthesis ofmetabolites The result is consistent with the experimentaldata reported [18] and showed that maximum cordycepinproduction resulted when the peptone was used as a nitrogensource
33 Effects of Inorganic Salt and Growth Factor on the Cordy-cepin Production Inorganic ion was one of the most impor-tant nutrition components ofmedium for themycelial growth[41] In order to investigate the effects of inorganic salt for thecordycepin production inCmilitaris we tested nine types (at1 gL) of inorganic salts (Figure 3(a)) Media with only 20 gLglucose and 20 gL peptone were used as the control The
Table 4 Factors and levels of central composite design for aminoacid nucleoside analogue and time
Symbol Variables Code levelminus16818 minus1 0 1 16818
119860 Hypoxanthine (gL) 053 1 5 9 1053119861 L-alanine (gL) 527 8 12 16 1872119862 Culture time (days) minus009 4 10 16 2009
highest cordycepin production (112030 plusmn 10528mgL) byC militaris was observed in medium when K
2HPO4sdot3H2O
was used as an inorganic salt KH2PO4 MgSO
4sdot7H2O
KCl and MgCl2sdot6H2O were also useful inorganic salts At
last MgSO4sdot7H2O and K
2HPO4sdot3H2O were recognized as
favorable bioelements for production of cordycepinGrowth factor is essential for growth response andmetab-
olite production [42] In order to find the optimal growthfactor for cordycepin production C militaris was culturedin a basal medium with different vitamins and plant growthhormones in static liquid culture Cordycepin productionincreased in media with added 10mgL of VB
1 NAA
and VB11
(Figure 3(b)) Maximum cordycepin production(115934 plusmn 10901mgL) occurred when VB
1was used as the
growth factor
34 Screening of Important Variables Using Plackett-BurmanDesign The data (Table 2) indicated wide variation in cordy-cepin production in the 15 tests The data suggested that pro-cess optimization is important for improving the efficiency ofcordycepin production Analysis of the regression coefficientsand 119905 values of 7 factors (Table 5) showed that 119883
1 1198832 1198834
and 1198835had positive effects on cordycepin production 119883
6
had negative effects The variable affects with a confidencelevel above 95are considered as significant factors Based onthese results three factors (119883
1 sucrose 119883
4 K2HPO4sdot3H2O
The Scientific World Journal 5
Control Glucose Dextrin Sucrose Lactose Soluble
Carbon source
0
100
200
300
400
500
600
700
800
900lowastlowast lowastlowast
starch
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
Cordycepin production
(a)
0
100
200
300
400
500
600
700
800
1000
900
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Pept
one
Yeas
t ext
ract
Beef
extr
act
Case
in
NH
4Cl
NH
4N
O3
NaN
O3
Carb
amid
e
Nitrogen sources
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Cordycepin production
(b)
Figure 2 Effects of carbon sources and nitrogen sources on the production of cordycepin carbon sources (a) nitrogen sources (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
Con
trol
K 2H
PO4 KH
2PO
4
middot3H
2O
MgC
l 2middot6
H2O
MgS
O4middot7
H2O
ZnSO
CaSO
4middot2
H2O
CaCl
2
4
middot2H
2O
KCl
FeSO
4middot7
H2O
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowast
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production
Inorganic salts
(a)
Cordycepin production
Growth factor
Control IAA VC VB1 NAA VB1124-D
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
lowast
lowastlowast
(b)
Figure 3 Effects of inorganic salt and growth factors on the production of cordycepin inorganic salt (a) growth factors (b) lowast5 significancelevel (test group versus control group) lowastlowast
1 significance level (test group versus control group)
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
4 The Scientific World Journal
Table 2 Plackett-Burman design and response values
Runs Experimental value119884 (mgL) Cordycepin production
119883
1119883
2119883
3119883
4119883
5119883
6119883
7
1 1 minus1 1 minus1 minus1 minus1 1 81236 plusmn 2683
2 1 1 minus1 1 minus1 minus1 minus1 139518 plusmn 84
3 minus1 1 1 minus1 1 minus1 minus1 90025 plusmn 1029
4 1 minus1 1 1 minus1 1 minus1 80245 plusmn 4543
5 1 1 minus1 1 1 minus1 1 109766 plusmn 2557
6 1 1 1 minus1 1 1 minus1 84587 plusmn 2494
7 minus1 1 1 1 minus1 1 1 78635 plusmn 761
8 minus1 minus1 1 1 1 minus1 1 80508 plusmn 291
9 minus1 minus1 minus1 1 1 1 minus1 92048 plusmn 1621
10 1 minus1 minus1 minus1 1 1 1 69401 plusmn 7951
11 minus1 1 minus1 minus1 minus1 1 1 49728 plusmn 444
12 minus1 minus1 minus1 minus1 minus1 minus1 minus1 59283 plusmn 1613
13 0 0 0 0 0 0 0 113414 plusmn 259
14 0 0 0 0 0 0 0 110021 plusmn 008
15 0 0 0 0 0 0 0 113356 plusmn 185
Table 3 Factors and levels of central composite design for carbonsources and inorganic salts
Symbol Variables Code levelminus16818 minus1 0 1 16818
119883
1Sucrose (gL) 31821 10 20 30 368179
119883
4K2HPO4sdot3H2O (gL) 01591 05 1 15 18409
119883
6MgSO4sdot7H2O (gL) 01591 05 1 15 18409
In previous work nitrogen showed a regulating roleimportant in cordycepin production and had two effects [40]One effect was negative since in excess N promoted a fastermycelial growth and consequently diverted the source ofcarbon toward energy and biomass production The othereffect was positive because a moderate input contributedto the maintenance of citric acid productive biomass [40]To investigate the effect of nitrogen sources on cordycepinproduction in C militaris various compounds containingnitrogen (inorganic and organic nitrogen) were added indi-vidually to nitrogen free basal medium at a concentration of20 gL Among the 8 nitrogen sources tested peptone yeastextract beef extract casein and NH
4NO3were favorable
to the cordycepin production (Figure 2(b)) Organic nitro-gen was advantageous to both growth and biosynthesis ofmetabolites The result is consistent with the experimentaldata reported [18] and showed that maximum cordycepinproduction resulted when the peptone was used as a nitrogensource
33 Effects of Inorganic Salt and Growth Factor on the Cordy-cepin Production Inorganic ion was one of the most impor-tant nutrition components ofmedium for themycelial growth[41] In order to investigate the effects of inorganic salt for thecordycepin production inCmilitaris we tested nine types (at1 gL) of inorganic salts (Figure 3(a)) Media with only 20 gLglucose and 20 gL peptone were used as the control The
Table 4 Factors and levels of central composite design for aminoacid nucleoside analogue and time
Symbol Variables Code levelminus16818 minus1 0 1 16818
119860 Hypoxanthine (gL) 053 1 5 9 1053119861 L-alanine (gL) 527 8 12 16 1872119862 Culture time (days) minus009 4 10 16 2009
highest cordycepin production (112030 plusmn 10528mgL) byC militaris was observed in medium when K
2HPO4sdot3H2O
was used as an inorganic salt KH2PO4 MgSO
4sdot7H2O
KCl and MgCl2sdot6H2O were also useful inorganic salts At
last MgSO4sdot7H2O and K
2HPO4sdot3H2O were recognized as
favorable bioelements for production of cordycepinGrowth factor is essential for growth response andmetab-
olite production [42] In order to find the optimal growthfactor for cordycepin production C militaris was culturedin a basal medium with different vitamins and plant growthhormones in static liquid culture Cordycepin productionincreased in media with added 10mgL of VB
1 NAA
and VB11
(Figure 3(b)) Maximum cordycepin production(115934 plusmn 10901mgL) occurred when VB
1was used as the
growth factor
34 Screening of Important Variables Using Plackett-BurmanDesign The data (Table 2) indicated wide variation in cordy-cepin production in the 15 tests The data suggested that pro-cess optimization is important for improving the efficiency ofcordycepin production Analysis of the regression coefficientsand 119905 values of 7 factors (Table 5) showed that 119883
1 1198832 1198834
and 1198835had positive effects on cordycepin production 119883
6
had negative effects The variable affects with a confidencelevel above 95are considered as significant factors Based onthese results three factors (119883
1 sucrose 119883
4 K2HPO4sdot3H2O
The Scientific World Journal 5
Control Glucose Dextrin Sucrose Lactose Soluble
Carbon source
0
100
200
300
400
500
600
700
800
900lowastlowast lowastlowast
starch
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
Cordycepin production
(a)
0
100
200
300
400
500
600
700
800
1000
900
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Pept
one
Yeas
t ext
ract
Beef
extr
act
Case
in
NH
4Cl
NH
4N
O3
NaN
O3
Carb
amid
e
Nitrogen sources
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Cordycepin production
(b)
Figure 2 Effects of carbon sources and nitrogen sources on the production of cordycepin carbon sources (a) nitrogen sources (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
Con
trol
K 2H
PO4 KH
2PO
4
middot3H
2O
MgC
l 2middot6
H2O
MgS
O4middot7
H2O
ZnSO
CaSO
4middot2
H2O
CaCl
2
4
middot2H
2O
KCl
FeSO
4middot7
H2O
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowast
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production
Inorganic salts
(a)
Cordycepin production
Growth factor
Control IAA VC VB1 NAA VB1124-D
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
lowast
lowastlowast
(b)
Figure 3 Effects of inorganic salt and growth factors on the production of cordycepin inorganic salt (a) growth factors (b) lowast5 significancelevel (test group versus control group) lowastlowast
1 significance level (test group versus control group)
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 5
Control Glucose Dextrin Sucrose Lactose Soluble
Carbon source
0
100
200
300
400
500
600
700
800
900lowastlowast lowastlowast
starch
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
Cordycepin production
(a)
0
100
200
300
400
500
600
700
800
1000
900
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Pept
one
Yeas
t ext
ract
Beef
extr
act
Case
in
NH
4Cl
NH
4N
O3
NaN
O3
Carb
amid
e
Nitrogen sources
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Cordycepin production
(b)
Figure 2 Effects of carbon sources and nitrogen sources on the production of cordycepin carbon sources (a) nitrogen sources (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
Con
trol
K 2H
PO4 KH
2PO
4
middot3H
2O
MgC
l 2middot6
H2O
MgS
O4middot7
H2O
ZnSO
CaSO
4middot2
H2O
CaCl
2
4
middot2H
2O
KCl
FeSO
4middot7
H2O
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowast
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production
Inorganic salts
(a)
Cordycepin production
Growth factor
Control IAA VC VB1 NAA VB1124-D
0
200
400
600
800
1000
1200
Cor
dyce
pin
prod
uctio
n (m
gL)
lowast
lowast
lowastlowast
(b)
Figure 3 Effects of inorganic salt and growth factors on the production of cordycepin inorganic salt (a) growth factors (b) lowast5 significancelevel (test group versus control group) lowastlowast
1 significance level (test group versus control group)
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
6 The Scientific World Journal
Table 5 Results of regression analysis of Plackett-Burman design
Symbol Regression analysisEffect Coefficient Standard error 119879 119875
84582 3286 2574 0000
lowastlowast
119883
119088 9544 3286 290 0027
lowast
119883
214923 7462 3286 227 0064
119883
3minus4085 minus2042 3286 minus062 0557
119883
424410 12205 3286 371 0010
lowast
119883
56281 3141 3286 096 0376
119883
6minus17615 minus8808 3286 minus268 0037
lowast
119883
7minus12739 minus6369 3286 minus194 0101
Ct Pt 27682 7348 377 0009
lowastlowast
lowast5 significance level lowastlowast1 significance level119883
1ndash1198837are symbols shown in Table 1
Table 6 Experimental design and responses of the central composite design for carbon sources and inorganic salts
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119883
1119883
4119883
6119883
1119883
4119883
6
1 minus1 minus1 minus1 108055 plusmn 10969 11 0 0 0 139943 plusmn 12444
2 1 minus1 minus1 135948 plusmn 1261 12 0 0 0 141543 plusmn 4213
3 minus1 1 minus1 115859 plusmn 1215 13 0 0 0 148742 plusmn 1638
4 1 1 minus1 128990 plusmn 4700 14 0 0 0 140943 plusmn 15522
5 minus1 minus1 1 98055 plusmn 3472 15 minus16818 0 0 87691 plusmn 1669
6 1 minus1 1 109748 plusmn 5242 16 16818 0 0 129000 plusmn 1471
7 minus1 1 1 98795 plusmn 289 17 0 minus16818 0 111057 plusmn 15763
8 1 1 1 111738 plusmn 11684 18 0 16818 0 134466 plusmn 6554
9 0 0 0 148538 plusmn 1219 19 0 0 minus16818 95839 plusmn 22414
10 0 0 0 133348 plusmn 9422 20 0 0 16818 95800 plusmn 7582
and 1198836 MgSO
4sdot7H2O) were considered as significant for
cordycepin production by static liquid culture methodology
35 Optimization by Response Surface Methodology for Car-bon Sources and Inorganic Salts In order to evaluate theinfluence of medium component on cordycepin productionsucrose K
2HPO4sdot3H2O and MgSO
4sdot7H2O should be exam-
ined The levels of variables for central composite designexperiments were selected according to the above results ofPlackett-Burman design Table 6 shows the detailed experi-mental design and results Regression analysis was performedto fit the response function (cordycepin production) withthe experimental data From the variables obtained (Table 6)the model is expressed by (1) which represents cordycepinproduction (119884
1) as a function of sucrose (119883
1) K2HPO4sdot3H2O
(1198834) and MgSO
4sdot7H2O (1198836) concentrations
119884
1= 141968 minus 9895119883
1+ 3145119883
4minus 5168119883
6
minus 1689119883
1119883
4minus 2048119883
1119883
6+ 236119883
4119883
6
minus 10603119883
2
1minus 5506119883
2
4minus 15031119883
2
6
(1)
Results of 119865-test analysis of variance (ANOVA) showedthat the regressionwas statistically significant at 95 and 99confidence levels (Table 7) The ldquo119865 valuerdquo of the model was921 and the value of ldquoProb gt 119865rdquo lt 001 indicated that the
model was significant In this case linear terms of 1198831and
quadratic terms of119883211198832411988326were significant ofmodel terms
for cordycepin productionThe ldquoLack of Fit F valuerdquo of 00903implied that the ldquoLack of Fitrdquo was not significant relative tothe pure error (119875 gt 005) The Pred-1198772 of 03183 was not asclose to the Adj-1198772 of 07954 as one might normally expectThe result suggested that some factors were not considered inthe model However the ldquoAdeq Precisionrdquo of 8173 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (1) is shown inFigure 4 It is evident that cordycepin production reachedits maximum at a combination of coded level (119883
1 sucrose
level 047 1198834 K2HPO4sdot3H2O level 021 119883
6 MgSO
4sdot7H2O
level minus020) when using canonical analysis of the Design-Expert Version 805b software packageThemodel predicteda maximum response of 145143mgL cordycepin productionat levels of sucrose 247 gL K
2HPO4sdot3H2O 111 gL and
MgSO4sdot7H2O 090 gL as optimized medium components
36 Effects of Nucleoside Analogue and Amino Acid onthe Production of Cordycepin Chassy and Suhadolnik [43]reported that adenine and adenosine were precursors forcordycepin synthesis Amino acids were regarded as the bestsubstance for improved cordycepin production [4 23] Basedon these results among 10 different kinds of nucleoside
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 7
Table 7 ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 6507119864 + 005 9 7230077 921 00009
lowastlowast
119883
1-1198831
1337119864 + 005 1 1337119864 + 005 1703 00021
lowastlowast
119883
4-1198834
1350430 1 1350430 172 02190
119883
6-1198836
3647748 1 3647748 465 00565
119883
1119883
4228255 1 228255 029 06016
119883
1119883
6335689 1 335689 043 05279
119883
4119883
64438 1 4438 5653119864 minus 003 09416
119883
1
21620119864 + 005 1 1620119864 + 005 2063 00011
lowastlowast
119883
4
24368518 1 4368518 556 00400
lowastlowast
119883
6
23256119864 + 005 1 3256119864 + 005 4147 lt00001lowastlowast
Residual 7851373 10 785137
Lack of Fit 6167032 5 1233406 366 00903
Pure Error 1684341 5 336868
Cor Total 7292119864 + 005 19
1198772= 08923 CV = 734 Pred-1198772 = 03183 Adj-1198772 = 07954 Adeq Precision = 8173 lowast5 significance level lowastlowast1 significance level
Table 8 Experimental design and responses of the central composite design for amino acid nucleoside analogue and time
Run Variables Code119884 (mgL) Cordycepin production Run Variables Code
119884 (mgL) Cordycepin production119860 119861 119862 119860 119861 119862
1 minus1 minus1 minus1 138301 plusmn 4153 11 0 0 0 204125 plusmn 5470
2 1 minus1 minus1 142252 plusmn 3941 12 0 0 0 202097 plusmn 7370
3 minus1 1 minus1 121688 plusmn 869 13 0 0 0 199818 plusmn 4948
4 1 1 minus1 185751 plusmn 16486 14 0 0 0 206860 plusmn 7279
5 minus1 minus1 1 121687 plusmn 25338 15 minus16818 0 0 159014 plusmn 22214
6 1 minus1 1 111118 plusmn 17050 16 16818 0 0 157390 plusmn 77616
7 minus1 1 1 153605 plusmn 7517 17 0 minus16818 0 163644 plusmn 6523
8 1 1 1 85170 plusmn 1701 18 0 16818 0 152798 plusmn 17746
9 0 0 0 207327 plusmn 6585 19 0 0 minus16818 121114 plusmn 8258
10 0 0 0 174309 plusmn 1481 20 0 0 16818 67697 plusmn 14274
analogue were supplemented for 1 gL in this study As shownin Figure 5(a) cordycepin production increased obviouslyin the medium with hypoxanthine thymine and thymi-dine additives The highest production of cordycepin wasachieved when hypoxanthine was used as the nucleosideanalogue Hypoxanthinersquos molecular structure is similar topurine bases found in cordycepin Substituent on purinebases structure is ndashOH on hypoxanthine rather than ndashNH
2
The ndashOH should be replaced in metabolic pathways Inaddition among 14 different amino acids were tested for8 gL As shown in Figure 5(b) L-alanine can improve cordy-cepin production Previous research showed that adenineadenosine and glycine were good additives for increasedcordycepin production [4 23 26 27] L-alanine may be animportant nutritional element for C militaris or componentof cordycepin production Hypoxanthine and L-alanine werethe best additives to promote cordycepin production in thisstudy
37 Optimization by Response Surface Methodology for AminoAcid Nucleoside Analogue and Fermentation Time Simi-larly central composite design was also applied to study
the significant factors (hypoxanthine L-alanine and culturetime) and their optimal levels Figure 6 shows the morpho-logical characteristics ofCmilitaris in 1000mL glass jars afterfermentation by static liquid fermentation Table 8 shows thedetailed experimental design and results Regression analysiswas performed to fit the response function (cordycepinproduction) with the experimental data From the variablesobtained (Table 9) the model was expressed by (2) whichrepresented cordycepin production (119884
2) as a function of
hypoxanthine (119860) L-alanine (119861) and culture time (119862 time)concentrations
119884
2= 199113 minus 1005119860 + 1070119861 minus 15102119862
+ 281119860119861 minus 18377119860119862 minus 2614119861119862
minus 14609119860
2minus 14603119861
2minus 37165119862
2
(2)
Results of 119865-test analysis of variance (ANOVA) showedthat the regression was statistically significant at 95 and99 confidence level (Table 9) The ldquo119865 valuerdquo of the modelwas 1191 and the value of ldquoProb gt 119865rdquo lt 001 indicatedthat the model was significant In this case linear terms
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
8 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)K2 HPO
4 middot3H2 O (gL)
K2H
PO4middot3
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
Prediction 145143
1200
1400
1400
Cordycepin production (mgL)
Sucrose (gL)
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
X1 = A sucroseX2 = B K2HPO4
Actual factorC MgSO4 = 100
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1000
1100
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
Cordycepin production (mgL)
150
130
110
090
070
050 1000
1500
2000
2500
3000
Sucrose (gL)
Sucrose (gL)
MgSO4 middot7H
2 O (gL)
MgS
O4middot7
H2O
(gL
)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1200
1200
1100
1400
Prediction 145143
(b)
Figure 4 Continued
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 9
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted148742
876911
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points 148742
876911
1100
1000
1200
1300
1400
1500
Cor
dyce
pin
prod
uctio
n (m
gL)
150
130
110
090
070
050 050
070
090
110
130
150
K2HPO4middot3H2O (gL)K 2
HPO 4middot3
H 2O (gL)
1000 1500 2000 2500 3000
050
070
090
110
130
150
1300
1300
1200
1400
Cordycepin production (mgL)
MgSO4 middot7H
2 O (gL)
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
X1 =
A sucroseX2 =
B K2HPO4 Actual factorC MgSO4
= 2000
MgS
O4middot7
H2O
(gL
)
Prediction 145143
(c)
Figure 4 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) sucrose and K
2HPO4sdot3H2O (b) sucrose and MgSO
4sdot7H2O (c) K
2HPO4sdot3H2O and
MgSO4sdot7H2O
Table 9 ANOVA for response surface quadratic polynomial model for amino acid nucleoside analogue and time
Source Sum of quares df Mean Square 119865-value 119875-value Prob gt 119865Model 2899119864 + 006 9 3221119864 + 005 1191 00003lowastlowast
119860-119860 137839 1 137839 0051 08260119861-119861 156406 1 156406 0058 08148119862-119862 3115119864 + 005 1 3115119864 + 005 1151 00068lowast
119860119861 6306 1 6306 2331119864 minus 003 09624119860119862 2702119864 + 005 1 2702119864 + 005 999 00102lowast
119861119862 546849 1 546849 020 06626119860
23076119864 + 005 1 3076119864 + 005 1137 00071lowastlowast
119861
23073119864 + 005 1 3073119864 + 005 1136 00071lowastlowast
119862
21990119864 + 006 1 1990119864 + 006 7358 lt00001lowastlowast
Residual 2705119864 + 005 10 2705363Lack of Fit 1928119864 + 005 5 3856198 248 01707Pure Error 7772641 5 1554528Cor Total 3169119864 + 006 191198772 = 09146 CV = 1070 Pred-1198772 = 04162 Adj-1198772 = 08378 Adeq Precision = 11222 lowast5 significance level lowastlowast1 significance level
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
10 The Scientific World Journal
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Nucleoside analogue
Con
trol
Aden
ine
Aden
osin
e
Urid
ine
Hyp
oxan
thin
e
Thym
ine
Cytid
ine
Thym
idin
e
Cyto
sine
Gua
nine
Gua
nosin
e
Cordycepin production
lowastlowastlowast
lowast
(a)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Cor
dyce
pin
prod
uctio
n (m
gL)
Con
trol
Asp
artic
acid
L-hy
drox
ypro
line
L-ar
gini
neL-
glut
amin
eL-
prol
ine
Lysin
eG
lyci
neL-
glut
amic
acid
L-m
ethi
onin
eL-
alan
ine
Asp
arag
ine
Cyte
ine
L-va
line
L-iso
leuc
ine
Amino acids
lowastlowast
lowast
Cordycepin production
(b)
Figure 5 Effects of nucleoside analogue and amino acid on the production of cordycepin nucleoside analogue (a) amino acid (b) lowast5significance level (test group versus control group) lowastlowast
1 significance level (test group versus control group)
5 significance level (controlgroup versus test group)
1 significance level (control group versus test group)
of 119862 interactive terms of 119860119862 and quadratic terms of 1198602119861
2 and 1198622 were significant in model terms for cordycepinproduction The ldquoLack of Fit F valuerdquo of 01707 implied thatthe ldquoLack of Fitrdquo was not significant relative to the pure error(119875 gt 005) The Pred-1198772 of 04162 was not as close to theAdj-1198772 of 08378 as one might normally expect The resultsuggested that some factors were not also considered in themodel However the ldquoAdeq Precisionrdquo of 11222 indicatedthat the model was adequate for prediction production ofcordycepin
The response surface plot obtained from (2) is shown inFigure 7 It is evident that cordycepin production reachedits maximum with a combination of coded level (119860 hypox-anthine level 011 119861 L-alanine level 006 119862 time levelminus023) by canonical analysis of the Design-Expert Version805b software package The model predicted a maximumresponse of 200848mgL cordycepin production at levelsof hypoxanthine 545 gL L-alanine 1223 gL and time 86days (in the practical test 8 days) as optimized mediumcomponents
In previous work the orthogonal design method [44ndash46] Box-Behnken design [34 47] and central compositedesign [31] were used to optimize culture conditions forcordycepin production by Cordyceps sp These experimentaldesigns have been successfully used to optimize medium forthe mycelial growth and microbial metabolite production inliquid culture processes In this study static liquid cultureconditions are optimized for the cordycepin production usingresponse surface methodology and are an effective way to
enhance the productivity of cordycepin and biomass in Cmilitaris
38 Verification Experiments and Batch Culture Based onthe results of response surface methodology the optimizedmedium was prepared as follows peptone 20 gL sucrose247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O 090 gL
VB110mgL hypoxanthine 545 gL and L-alanine 1223 gL
Five experiments were performed to confirm the aboveoptimal culture requirements The data were 201115mgL200069mgL 198922mgL 19696mgL and 206137mgLrespectively The average cordycepin production was200641 plusmn 3437mgL The experimental values wereparticularly close to the predicted values (200848mgL)The result confirmed the model suited the predictive ofhyperproduction of cordycepin by C militaris in static liquidculture Batch culture was carried for cordycepin productionunder optimized culture conditions (Figure 8)
39 In Vitro Cordycepin Production Using Liquid Culture inOther Studies The highest report for cordycepin productionwas 14300mgL by Masuda et al [29] (Table 10) In ourexperiment cordycepin production at 200848mgL waslower However a maximum total content of cordycepin(140594mg) was achieved in our study This is a secondhigher report of cordycepin production in one single fer-menter The results showed that the culture conditions willprovide an effective way for increasing cordycepin produc-tion
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 11
Table 10 Cordycepin production in the medium by liquid culture in different studies
No MethodologyWorking volume of
the mediumvv (mLmL)
Cordycepinproduction (mgL)
Total content ofcordycepin in one
bottle (mg)References
1 Submerged culture 50250 2457 125 Mao et al [32]
2 Submerged culture 50250 4205 2103 Mao and Zhong[21]
3 Surface liquid culture 100500 640 64 Masuda et al [25]4 Shaking + Static 100250 22145 22145 Shih et al [34]5 Surface liquid culture 100500 2500 250 Masuda et al [26]6 Surface liquid culture 100500 3100 310 Das et al [30]7 Surface liquid culture 100500 8570 857 Das et al [27]8 Submerged culture 100500 164421 16442 Wen et al [23]9 Dark + Shaking 100500 1015 1015 Kang et al [24]10 Surface liquid culture 150500 14300 2145 Masuda et al [29]11 Static liquid culture 7001000 200848 140594 In this study
Figure 6 Morphology of C militaris (strain CGMCC2459) in 7001000mL glass jars at the end of the fermentation process by responsesurface methodology symbols in photos indicated 20 runs
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
12 The Scientific World Journal
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1400
1400
1200
1200
1000
1000
800 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
800
1800
1800
1900
1800
1800
Cordycepin production (mgL)
L-Alanine (gL)
L-A
lani
ne (g
L)
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
X1 =
C timeX2 =
A Hypoxanthine Actual factorB L-alanine = 1000
207327
67697
Prediction 200848
(a)
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 100
300
500
700
900
Hypoxanthine (gL)
Hypoxanthine (gL)100 300 500 700 900
400
1600
1800
1800
14001600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
X1 =
B L-AlanineX2 =
A Hypoxanthine Actual factorC Time = 1200
207327
67697
Prediction 200848
(b)
Figure 7 Continued
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 13
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points above predictedDesign points below predicted207327
67697
Design-Expert SoftwareFactor Coding ActualCordycepin productionDesign points
12525
1000
1505
17575
2010
Cor
dyce
pin
prod
uctio
n (m
gL)
1600
1600
1300
1300
1000
1000
700
700
400 800
1000
1200
1400
1600
L-Alanine (gL)
L-Alanine (gL)800 1000 1200 1400 1600
400
1600
1800
1800
1600
2000
Cordycepin production (mgL)
Time (days)
Tim
e (da
ys)
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
X1 =
A HypoxanthineX2 =
A Hypoxanthine Actual factorC Time = 500
207327
67697
Prediction 200848
(c)
Figure 7 Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C militaris (strainCGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine (b) hypoxanthine and time (c) L-alanine and time
Figure 8 Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C militaris (strainCGMCC2459)
4 Conclusion
In this work single factor design Plackett-Burman designand central composite design were employed to establishthe key factors and identify optimal culture conditionswhich improved cordycepin production by C militarisCGMCC2459 Optimal media contained peptone 20 gLsucrose 247 gL K
2HPO4sdot3H2O 111 gL MgSO
4sdot7H2O
090 gL VB110mgL hypoxanthine 545 gL and L-alanine
1223 gL Hypoxanthine and L-alanine were added to theoptimal medium at 86 days Optimal incubation conditionswere 25∘C at an unaltered pH of 35 days Using theseculture conditions a maximum production of cordycepinwas 200848mgL for 700mL working volume in the1000mL glass jars and total content of cordycepin reached140594mgbottle (700mL1000mL) This method provides
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
14 The Scientific World Journal
an effective way for increasing the cordycepin production ata large scale The strategies used in this study could have awide application in other fermentation process
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Chao Kang and Ting-Chi Wen contributed equally to thiswork
Acknowledgments
This work was supported by the Agricultural Scienceand Technology Foundation of Guizhou Province (no(2011)3054) the National Natural Science Foundation ofChina (no 31200016) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (no(2012)5008)
References
[1] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007
[2] J T XuMedicinal Fungus in China Beijing Medical UniversityPeking Union Medical College Joint Press Beijing China 1997(Chinese)
[3] P E Mortimer S C Karunarathna Q Li et al ldquoPrized edibleAsian mushrooms ecology conservation and sustainabilityrdquoFungal Diversity vol 56 no 1 pp 31ndash47 2012
[4] T C Wen B X Lei J C Kang G R Li and J He ldquoEnhancedproduction of mycelial culture using additives and cordycepinby submerged in Cordyceps militarisrdquo Food and FermentationIndustries vol 35 no 8 pp 49ndash53 2009 (Chinese)
[5] H C Li P Sun and C Q Feng ldquoThe research of cordycepinas an active component in Cordycepsrdquo Journal of JinggangshanUniversity (Natural Science) vol 31 no 2 pp 93ndash96 2010(Chinese)
[6] K G Cunningham W Manson F S Spring and S AHutchinson ldquoCordycepin a metabolic product isolated fromcultures ofCordyceps militaris (Linn) linkrdquoNature vol 166 no4231 p 949 1950
[7] D D de Silva S Rapior F Fons A H Bahkali and K DHyde ldquoMedicinal mushrooms in supportive cancer therapiesan approach to anti-cancer effects and putative mechanisms ofactionrdquo Fungal Diversity vol 55 pp 1ndash35 2012
[8] N Yoshikawa S Yamada C Takeuchi et al ldquoCordycepin(31015840-deoxyadenosine) inhibits the growth of B16-BL6 mousemelanoma cells through the stimulation of adenosine A3receptor followed by glycogen synthase kinase-3120573 activationand cyclin D1 suppressionrdquo Naunyn-Schmiedebergs Archives ofPharmacology vol 377 no 4ndash6 pp 591ndash595 2008
[9] A M Sugar and R P McCaffrey ldquoAntifungal activity of3-deoxyadenosine (cordycepin)rdquo Antimicrobial Agents andChemotherapy vol 42 no 6 pp 1424ndash1427 1998
[10] K Hashimoto and B Simizu ldquoEffect of cordycepin on thereplication of western equine encephalitis virusrdquo Archives ofVirology vol 52 no 4 pp 341ndash345 1976
[11] E N Kodama R P McCaffrey K Yusa and H MitsuyaldquoAntileukemic activity and mechanism of action of cordycepinagainst terminal deoxynucleotidyl transferase-positive (TdT+)leukemic cellsrdquo Biochemical Pharmacology vol 59 no 3 pp273ndash281 2000
[12] D D de Silva S Rapior E Sudarman et al ldquoBioactivemetabolites from macrofungi ethnopharmacology biologicalactivities and chemistryrdquo Fungal Diversity vol 62 pp 1ndash402013
[13] D D de Silva S Rapior K D Hyde and A H BahkalildquoMedicinal mushrooms in prevention and control of diabetesmellitusrdquo Fungal Diversity vol 56 no 1 pp 1ndash29 2012
[14] A Todd and T L V Ulbricht ldquoDeoxynucleosides and relatedcompoundsmdashpart IX a synthesis of 31015840-deoxyadenosinerdquo Jour-nal of the Chemical Society pp 3275ndash3277 1960
[15] D W Kwon J-H Jeon C Kang and Y H Kim ldquoNewsynthesis of 31015840-deoxypurine nucleosides using samarium(III)iodide complexrdquo Tetrahedron Letters vol 44 no 43 pp 7941ndash7943 2003
[16] E A Kaczka E L Dulaney C O Gitterman H B Woodruffand K Folkers ldquoIsolation and inhibitory effects on KB cell cul-tures of 31015840-deoxyadenosine from Aspergillus nidulans (Eidam)wintrdquo Biochemical and Biophysical Research Communicationsvol 14 no 5 pp 452ndash455 1964
[17] H X Zhang W Wu W Chen X H Gao and L S TangldquoComparative analysis of cordycepin and adenosine contentsin fermentation supernatants between Aspergillus nidulans andCordyceps militarisrdquo Acta Agriculturae Shanghai vol 22 no 2pp 28ndash31 2006 (Chinese)
[18] T C Wen J C Kang B X Lei G R Li and J He ldquoEffects ofdifferent solid culture condition on fruit body and cordycepinoutput of Cordyceps militarisrdquo Guizhou Agricultural Sciencesvol 36 no 4 pp 92ndash94 2008 (Chinese)
[19] Z H Chen H Yu W B Zeng J Y Yang J Yuan and Y JChen ldquoSolid fermentation technique for cordycepin productionby Cordyceps nigrella strain cnig-56rdquo Acta Edulis Fungi vol 17no 1 pp 80ndash82 2010 (Chinese)
[20] L Bu Z Y Zhu Z Q Liang and A Y Liu ldquoUtilization offungal elicitor in increasing cordycepin of Cordyceps militarisrdquoMycosystema vol 21 no 2 pp 252ndash256 2002 (Chinese)
[21] X-B Mao and J-J Zhong ldquoHyperproduction of cordycepin bytwo-stage dissolved oxygen control in submerged cultivationof medicinal mushroom Cordyceps militaris in bioreactorsrdquoBiotechnology Progress vol 20 no 5 pp 1408ndash1413 2004
[22] X BMao and Y Q Tu ldquoEnhanced production of cordyceipn byfed batch cultivation using amino acid in Cordyceps militarisrdquoChongqing Journal of Research on Chinese Drugs and Herbs no1 pp 18ndash22 2005 (Chinese)
[23] T C Wen J C Kang B X Lei G R Li and J He ldquoEnhancedproduction of cordycepin by submerged culture using additivesin Cordyceps militarisrdquo Food Science vol 31 no 5 pp 175ndash1792010 (Chinese)
[24] C Kang T C Wen J C Kang Y X Qian and B X LeildquoEffects of additives and different culture conditions on cordy-cepin production by the medicinal fungus Cordyceps militarisrdquoMycosystem vol 31 no 3 pp 389ndash397 2012 (Chinese)
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
The Scientific World Journal 15
[25] M Masuda E Urabe A Sakurai and M Sakakibara ldquoProduc-tion of cordycepin by surface culture using themedicinalmush-room Cordyceps militarisrdquo Enzyme and Microbial Technologyvol 39 no 4 pp 641ndash646 2006
[26] MMasuda E Urabe HHonda A Sakurai andM SakakibaraldquoEnhanced production of cordycepin by surface culture usingthe medicinal mushroom Cordyceps militarisrdquo Enzyme andMicrobial Technology vol 40 no 5 pp 1199ndash1205 2007
[27] S K DasMMasuda A Sakurai andM Sakakibara ldquoEffects ofadditives on cordycepin production using a Cordyceps militarismutant induced by ion beam irradiationrdquo African Journal ofBiotechnology vol 8 no 13 pp 3041ndash3047 2009
[28] S Aman D J Anderson T J Connolly A J Crittall and G JildquoFrom adenosine to 31015840-deoxyadenosine development and scaleuprdquoOrganic Process Research and Development vol 4 no 6 pp601ndash605 2000
[29] MMasuda S K Das M Hatashita S Fujihara and A SakuraildquoEfficient production of cordycepin by the Cordyceps militarismutant G81-3 for practical userdquo Process Biochemistry vol 49pp 181ndash187 2014
[30] S K Das M Masuda M Hatashita A Sakurai and M Sakak-ibara ldquoA new approach for improving cordycepin productivityin surface liquid culture of Cordyceps militaris using high-energy ion beam irradiationrdquo Letters in Applied Microbiologyvol 47 no 6 pp 534ndash538 2008
[31] L Wen C Zhang M Xia and L Weng ldquoEffects of Cordycepsmilitaris space mutation treatment on active constituent con-tentrdquo Food Science vol 29 no 5 pp 382ndash384 2008
[32] X-B Mao T Eksriwong S Chauvatcharin and J-J ZhongldquoOptimization of carbon source and carbonnitrogen ratio forcordycepin production by submerged cultivation of medicinalmushroom Cordyceps militarisrdquo Process Biochemistry vol 40no 5 pp 1667ndash1672 2005
[33] S K Das M Masuda M Hatashita A Sakurai and MSakakibara ldquoOptimization of culture medium for cordycepinproduction using Cordyceps militaris mutant obtained by ionbeam irradiationrdquo Process Biochemistry vol 45 no 1 pp 129ndash132 2010
[34] I-L Shih K-L Tsai andCHsieh ldquoEffects of culture conditionson the mycelial growth and bioactive metabolite productionin submerged culture of Cordyceps militarisrdquo Biochemical Engi-neering Journal vol 33 no 3 pp 193ndash201 2007
[35] W Q Guo N Q Ren X J Wang et al ldquoOptimization ofsubmerged culture conditions for exo-biopolymer productionby Paecilomyces japonicardquo Bioresource Technology vol 100 pp1192ndash1196 2009
[36] J Zhou X Yu C Ding et al ldquoOptimization of phenol degrada-tion by Candida tropicalis Z-04 using Plackett-Burman designand response surface methodologyrdquo Journal of EnvironmentalSciences vol 23 no 1 pp 22ndash30 2011
[37] D F Zhao C Y Liang X Ren and S M Sun ldquoEffect ofdissolved oxygen on the biotransformation of tylosin to its acylderivativerdquo Chinese Journal of Pharmaceutical vol 37 no 3 pp162ndash164 2006 (Chinese)
[38] T Xie H Y Fang G B Zhu and G J Zhu ldquoEffect of dissolvedoxygen on glycerol production by Candida glycerinogenesrdquoChina Biotechnology vol 28 no 5 pp 65ndash70 2008 (Chinese)
[39] N M Kredich and A J Guarino ldquoStudies on the biosynthesisof cordycepinrdquo Biochimica et Biophysica Acta vol 47 no 3 pp529ndash534 1961
[40] J Pintado A Torrado M P Gonzalez and M A MuradoldquoOptimization of nutrient concentration for citric acid produc-tion by solid-state culture of Aspergillus niger on polyurethanefoamsrdquo Enzyme and Microbial Technology vol 23 no 1-2 pp149ndash156 1998
[41] J-T Bae J Sinha J-P Park C-H Song and J-W Yun ldquoOpti-mization of submerged culture conditions for exo-biopolymerproduction by Paecilomyces japonicardquo Journal of Microbiologyand Biotechnology vol 10 no 4 pp 482ndash487 2000
[42] K Lei Y Ke and N Mao ldquoThe optimization of the culturemedia for high production of cordycepin by Cordyceps militarisFjnu-01rdquo Pharmaceutical Biotechnology vol 18 no 6 pp 504ndash508 2011 (Chinese)
[43] B M Chassy and R J Suhadolnik ldquoNucleoside antibiotics IVMetabolic fate of adenosine and cordycepin by Cordyceps mili-taris during cordycepin biosynthesisrdquo Biochimica et BiophysicaActa vol 182 no 2 pp 307ndash315 1969
[44] J H Xiao D X Chen J W Liu et al ldquoOptimization ofsubmerged culture requirements for the production of mycelialgrowth and exopolysaccharide by Cordyceps jiangxiensis JXPJ0109rdquo Journal of Applied Microbiology vol 96 no 5 pp 1105ndash1116 2004
[45] J H Xiao D X Chen Y Xiao et al ldquoOptimization ofsubmerged culture conditions for mycelial polysaccharide pro-duction inCordyceps pruinosardquoProcess Biochemistry vol 39 no12 pp 2241ndash2247 2004
[46] C-H Dong and Y-J Yao ldquoNutritional requirements of mycelialgrowth of Cordyceps sinensis in submerged culturerdquo Journalof Applied Microbiology vol 99 no 3 pp 483ndash492 2005(Chinese)
[47] C Xie G Liu Z Gu G Fan L Zhang and Y Gu ldquoEffectsof culture conditions on mycelium biomass and intracellu-lar cordycepin production of Cordyceps militaris in naturalmediumrdquo Annals of Microbiology vol 59 no 2 pp 293ndash2992009
