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Effects of culture conditions on the mycelial growth and bioactive

Biochemical Engineering Journal33(2007)

193–201

metabolite production in submerged culture of Cordyceps militaris

Ing-Lung Shih a,Kun-Lin Tsai b,Chienyan Hsieh c,?

a Department of Environmental Engineering,Da-Yeh University,Chang-Hwa,Taiwan

b Department of Bioindustry Technology,Da-Yeh University,Chang-Hwa,Taiwan

c Department of Biotechnology,National Kaohsiung Normal University,Kao-Hsiung,Taiwan

Received24February2006;received in revised form16October2006;accepted20October2006

Abstract

The in?uence of initial pH value,various nitrogen sources,plant oils,and modes of propagation(shake-?ask and static culture)on the production of biomass,exopolysaccharide(EPS),adenosine and,in particular,cordycepin,by Cordyceps militaris CCRC32219were investigated.Optimal conditions for mycelial growth,EPS and cordycepin production were observed at relatively low pH.Amongst organic sources,yeast extract(YE) was favorable for EPS and cordycepin production,while corn steep powder(CSP)was favorable for adenosine production.A lower C/N ratio was favorable for adenosine and cordycepin production;however,too low a C/N ratio led to diminished production.All plant oils tested stimulate mycelial growth and EPS production of https://www.wendangku.net/doc/0b15384052.html,itaris,but they did not show much effect on the adenosine and cordycepin production.A two-stage fermentation process by combining shake-?ask fermentation with static culture signi?cantly enhanced cordycepin production.A Box–Behnken experimental design was employed to optimize the production of cordycepin,which showed that the optimum conditions to produce cordycepin by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219were at pH6,YE concentration of45g/l and8.0day of the shake culture followed by16days of the static culture. Under the optimized conditions,the maximum production(2214.5mg/l)of cordycepin was obtained,which is much higher than those reported up to date.

Keywords:Cordyceps militaris;Cordycepin;Adenosine;Polysaccharide;Response surface design

1.Introduction

Cordyceps militaris,an entomopathogenic fungus belonging to the class Ascomycetes,is known as one of the Chi-nese medicinal mushrooms.Some Cordyceps species have long been used for medicinal purposes in China,Japan and Korea and other oriental countries because of their various biological and pharmacological activities which were gen-erally attributed to the presence of the important bioactive ingredients such as adenosine,cordycepin and exopolysac-charides[4,14,20,24,30,35,36].Adenosine has a number of actions that merit it as a possible cardio-protective and ther-apeutic agent for chronic heart failure[16,32];cordycepin

(3 -deoxyadenosine)is a nucleoside analogue which exhibits

a broad spectrum of biological activities including antibac-terial,antifungal,antitumor,antileukemia,antiviral activities

?Corresponding author at:No.62,Shenjhong Road,Yanchao Township, Kaohsiung County824,Taiwan.Fax:+88676051353.

E-mail address:mch@https://www.wendangku.net/doc/0b15384052.html,.tw(C.Hsieh).and an immunoregulative effect[1,17,34,44];polysaccha-rides account for the anti-in?ammatory,antioxidant,antitumor, antimetastatic,immunomodulatory,hypoglycaemic,steroido-genic and hypolipidaemic effects[13,19,29,33,39,41,42].

The fruit bodies of wild https://www.wendangku.net/doc/0b15384052.html,itaris are expensive because of host speci?city and rarity in nature;they grow extremely slowly in nature,their growth is restricted to a speci?c area and their sizes are very small.Therefore,the collection of suf?cient quan-tities for extensive use as a drug remedy is prohibited.Because solid culture of mushrooms takes long time to complete a fruit-ing body,many attempts have been made to obtain useful and potent cellular or extracellular substances from a submerged mycelial culture for use in the formulation of nutraceuticals and functional foods.Submerged culture gives rise to potential advantages of higher mycelial production in a compact space and shorter time with less chance of contamination.Although submerged cultivation of mushrooms for ef?cient production of valuable metabolites has been studied extensively[6,25],sub-merged cultivation of https://www.wendangku.net/doc/0b15384052.html,itaris has scarcely been studied until recently[15,22,26–28,43].

194I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201

In this study,the nutritional requirements and culture con-

ditions for the submerged culture of https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219

were investigated in an attempt to obtain optimal production

of mycelia,polysaccharides,adenosine and cordycepin by this

specie.In addition,statistical experimental design(SES)strat-

egy was applied to optimize cultural conditions for cordycepin

production by https://www.wendangku.net/doc/0b15384052.html,itaris.The SES strategy has been increas-

ingly used at various phases of an optimization process in

fermentation[9,31];it is a powerful technique for testing mul-

tiple process variables because fewer experimental trials are

needed in comparison to the experiments carried out by conven-

tional“one-factor-at-a-time”technique.Besides,interactions

between variables can be identi?ed and quantitated by such a

technique[2].The application of SES strategy for the optimiza-

tion of exo-polysaccharide production by https://www.wendangku.net/doc/0b15384052.html,itaris NG3has

been described[38];however,the application of SES strategy on

optimization of cordycepin production by https://www.wendangku.net/doc/0b15384052.html,itaris is scant.

2.Materials and methods

2.1.Microorganism and seed culture

https://www.wendangku.net/doc/0b15384052.html,itaris(Linnaeus)Link CCRC32219(=ATCC26848) used in the present study was obtained from the Culture Collec-

tion and Research Center(CCRC)Taiwan.The microorganism

was maintained on potato dextrose agar(PDA)slants and sub-

cultured every month.Slants were incubated at25?C for7

days and then stored at4?C.The seed culture medium con-

sisted of the following components:glucose,40g/l;corn steep

powder(CSP),10g/l;KH2PO4,0.5g/l;K2HPO4,0.5g/l;

MgSO4·7H2O,0.5g/l;FeSO4·7H2O,0.1g/l.The mycelia of C. militaris were transferred to the seed culture medium by punch-ing out about6mm diameter agar discs from culture grown on

PDA plates;three discs were used to inoculate100ml liquid

media.The seed culture was incubated in a250-ml Erlenmeyer

?ask at25?C on a rotary shaker incubator at150rpm for5days

[26,28].

2.2.Flask culture conditions

The medium composition for the fermentation was as fol-

lows:glucose,40g/l;corn steep powder(CSP),10g/l;KH2PO4,

0.5g/l;K2HPO4,0.5g/l;MgSO4·7H2O,0.5g/l;FeSO4·7H2O, 0.1g/l.The pH was initially adjusted to6,followed by autoclav-ing.The?ask culture experiments were performed in250-ml ?asks containing100ml of medium after inoculating with5% (v/v)of the seed culture.The culture was incubated at25?C on a rotary shaker incubator at150rpm,and samples were col-lected at various intervals from the shake?asks for analyzing biomass dry weight,exo-polysaccharides(EPS),intracellular polysaccharides(IPS),adenosine,and cordycepin production.

The effects of factors affecting cell growth and the production

of components such as EPS,IPS,adenosine,and cordycepin by

https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219were studied using shake?ask culture on rotary incubator shaker(Wisdom721SR-Incubator-Shaker) as described above.Effects of nitrogen sources on https://www.wendangku.net/doc/0b15384052.html,itaris culture were studied by substituting nitrogen sources such as peptone(PE),yeast extract(YE),Ammonium chloride(AC),

and Ammonium phosphate(AP)for corn steep powder(CSP)

in a one-at-a-time fashion.Effects of plant oils on https://www.wendangku.net/doc/0b15384052.html,itaris

culture were also studied using shake?ask culture as described

above.Plant oils(soy,peanut,coconut,sun-?ower and olive oils)

were supplemented,all at volume fractions of1%in liquid media

(4%glucose,1%CSP and0.05%K2HPO4,0.05%K2HPO4,

0.05%MgSO4·7H2O,0.01%FeSO4·7H2O)and the culture was cultivated at25?C on a rotary shaker incubator at150rpm.The

effects of initial pH(4.0–7.0)on the culture of https://www.wendangku.net/doc/0b15384052.html,itaris were

also investigated.All experiments were carried out at least in

duplicate,and the results were averaged.

2.3.Analytical methods

2.3.1.Determination of mycelia dry weight,pH and

residual sugar

Samples collected at various intervals from shake?asks were

centrifuged at6000×g for10min,and the supernatant was

?ltered through a pre-weighted Whatman?lter paper No.2

(Whatman International Ltd.,Maidstone,UK).The resulting

culture?ltrate was for measuring pH value,analysis of residual

sugar,polysaccharide,adenosine and cordycepin using methods

described below.The centrifuged mycelia was washed suf?-

ciently with a large amount of distilled water,collected by

?ltration through the above Whatman?lter paper and freeze-

dried to a constant dry weight.Residual sugar concentration

was measured by a phenol-sulfuric acid method[5].

2.3.2.Measurements of extracellular and intracellular polysaccharides

The resulting culture?ltrate obtained as described above was

mixed with four volumes of95%(v/v)ethanol,stirred vigorously

and left overnight at4?C.The precipitated exopolysaccharide

(EPS)was recovered by centrifugation at10,290×g for10min

(HERMLE,model Z160M)and the supernatant was discarded

[10].The precipitate of crude EPS was lyophilized and sus-

pended in1N NaOH at60?C for1h,and the reducing sugar in

the supernatant was measured by a phenol-sulfuric acid method

[5].Intracellular polysaccharides(IPS)were extracted from

dried mycelia(100mg)by suspending the mycelia in10ml dis-

tilled water and autoclaving for15min[10].The amount of IPS

was then determined by the procedure used for EPS measure-

ment as described above.

2.3.3.Measurement of adenosine and cordycepin by HPLC

Accurate amounts of adenosine(Sigma,USA)and cordy-

cepin(Sigma,USA)were dissolved in a mobile phase solution,

to give various concentrations for calibration.For the analysis of

extracellular adenosine and cordycepin,The resulting culture?l-

trate obtained as described above was mixed with15%methanol

(1:1,v/v),followed by centrifugation at6000×g for15min,the

supernatant was?ltered through a0.45?m membrane(Milli-

pore,USA)and the?ltrate was analyzed by HPLC.The HPLC

system was composed of a Hitachi L-6200solvent delivery con-

troller with a RHEODYNE M-4250injector,a Hitachi4250

UV–vis detector,a Hitachi-D-2500Chromato-integrator,and a

I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201195 reverse phase C18column(ACE,UK,250mm×4.6mm,5?m

particle size).The mobile phase was a mixture of methanol and

0.02M potassium dihydrogenphosphate(15:85).The injection

volume was20?l,the column temperature was40?C,the?ow

rate was set at1ml/min and the eluent was monitored at254nm

[22].For the analysis of extracellular adenosine and cordycepin,

dried mycelia(500mg)obtained above was suspended in5ml

distilled water and autoclaved for1h,followed by?ltration

through a0.45?m membrane(Millipore,USA).The resulting

?ltrate was extracted and analyzed using the same procedure as

that for intracellular adenosine and cordycepin[23].

2.4.Response surface methodology(RSM),the

Box–Behnken design

Box–Behnken design(BBD)[3]was conducted to optimize

cordycepin production by https://www.wendangku.net/doc/0b15384052.html,itaris.This design is a response

surface design suitable for3or more factors;it is rotatable(or

near rotatable)and requires only three levels(coded as?1,0,

and+1)of each factor.This procedure is suitable for explor-

ing quadratic response surfaces and constructing second-order

polynomial models;it creates designs with desirable statisti-

cal properties but,most importantly,with only a fraction of the

experiments required for a three-level factorial.For a four-factor,

three-level design,the experimental trials were given by a set of

points at the midpoint of each edge of a multidimensional cube

and three replication of center points,resulting in a total num-

ber of27experiments.The matrix corresponding to the BBD

is shown in Table5,together with the observed experimental

data.The BBD experimental results were?tted with a second-

order polynomial equation(Eq.(1))by a multiple regression

technique:

Y=β0+

i=1

βi x i+

i=1

βii x2i+

i=1

βij x i x j(1)

Y is the predicted response(cordycepin yield in this study,mg/l),β0,βi,βii,βij are constant coef?cients,and x i,x j are the coded independent variables or factors.

The quality of?t of the second-order model equation was expressed by the coef?cient of determination R2,and its statis-tical signi?cance was determined by an F-test.The signi?cance of the regression coef?cients was tested by a t-test.The com-puter software used was Statistica(version5.0)by Statsoft,Inc. (Tulsa,OK,USA).

3.Results and discussion

3.1.Effect of pH on cell growth and the production of metabolites

It has been reported that acidic pH was more suitable for mycelial growth and production of metabolites for many kinds of ascomycetes and basidiomycetes,including Cordyceps sp. [10,26].The effects of initial pH on the maximal production of mycelia biomass and various metabolites by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC 32219are shown in Table1,and the kinetic pro?les of the mycelia biomass,glucose consumption,pH variation and cordy-cepin production in the medium are shown in Fig.1.It is seen that the maximal mycelial concentration for pH4,5,6and7 was15.2,14.1,13.9and13.6g/l obtained on day36,30,30 and30,respectively(Table1and Fig.1(a)).It is worthy of not-ing that the pHs of the media were lower than the initial pHs while the cell growth increased in the consumption of glucose (Fig.1(b)and(c)).These suggest that the cell growth might be due to the high consumption of the carbon sources,which led to production of organic acid and consequently reduced the pH in the media[7,37].The maximal EPS production was1.1,1.1, 0.9and1.0g/l,which corresponds to EPS productivity of0.19, 0.19,0.23and0.06g/(l d)for pH4,5,6,and7,respectively.This is in contrast with the facts that the maximum EPS production (0.29g/l)was at an initial pH6.0for https://www.wendangku.net/doc/0b15384052.html,itaris C738[15],and the maximum EPS production(1.25g/l)was at an initial pH8.0 for https://www.wendangku.net/doc/0b15384052.html,itaris NG3[14].The maximal adenosine production in the culture broth was50.1,40.5,80.6and109.7mg/l,which cor-responds to productivity of10.0,8.1,13.4and21.8mg/(l d)for pH4,5,6,and7,respectively.The production of adenosine by Cordyceps sp.in submerged cultivation was scarcely studied; however,trace levels of adenosine were reported in the sub-merged cultivation of C.sinensis CCRC36421[11];they were 2.0and2.7?g/g in the mycelium and culture broth,respectively. In addition,no adenosine was detected in the medium?ltrate

Table1

The effects of initial pH on the maximal cell growth and the production of various metabolites by Cordyceps militaris a

Cell growth and metabolites Initial pH in the medium

4567

Mycelia dry weight(g/l)15.2(d36)b14.1(d30)13.9(d30)13.6(d30) EPS production(g/l)c 1.1 1.10.9 1.0

EPS productivity(g/(l d))0.190.190.230.06 Adenosine production(mg/l)c50.140.580.6109.7 Adenosine productivity(mg/(l d))10.08.113.421.8 Cordycepin production(mg/l)c315.2190.1185.2138.3 Cordycepin productivity(mg/(l d))12.67.67.4 5.5 EPS:exopolysaccharide.

a Data shown are average values of duplicate.

b Culture time indicated in the parenthesis was when the maximum cell mass was reached.

c Data shown are the values of maximum production;Adenosine an

d Cordycepin ar

e extracellular.

196I.-L.Shih et al./Biochemical Engineering Journal 33(2007)

193–201

Fig.1.Kinetic pro?les of pH effects on Cordyceps militaris cultivation (data shown are average values of triplicate).

of the https://www.wendangku.net/doc/0b15384052.html,itaris NBRC 9787culture [23].The extracellular cordycepin production by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC 32219initiated slightly after the start of the cell growth and reached the maxi-mum of 315.2,190.1,185.2and 138.3mg/l,which corresponds to productivity of 12.6,7.6,7.41,5.5mg/(l d),for pH 4,5,6,and 7,respectively (Fig.1(d)and Table 1).To the best of our knowl-edge,the pH effects on the cordycepin production in the liquid medium of https://www.wendangku.net/doc/0b15384052.html,itaris as described in this study are reported for the ?rst in the literature,although the effects of dissolved oxygen (DO),carbon sources and carbon/nitrogen on the cordy-cepin production in the submerged cultivation of https://www.wendangku.net/doc/0b15384052.html,itaris have been investigated by many investigators [21,22].It should be noted that the direct comparison of metabolite productions of various Cordyceps strains from the various studies in the lit-erature was dif?cult because the nutrient components and the culture conditions used were not exactly the same.3.2.Effect of nitrogen sources and plant oils on cell growth and the production of metabolites

The effects of nitrogen sources on the mycelial growth and the production of various metabolites by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC 32219in submerged cultivation are shown in Table 2.In comparison with organic nitrogen sources (PE,YE,CSP),inorganic nitro-gen sources (NH 4Cl,NH 4H 2PO 4)gave rise to relatively lower mycelial growth.This is consistent with what was previously suggested that most basiomycetes prefer complex organic nitro-gen sources for their growth in submerged cultures [12].Several

https://www.wendangku.net/doc/0b15384052.html,itaris species such as https://www.wendangku.net/doc/0b15384052.html,itaris C738and https://www.wendangku.net/doc/0b15384052.html,itaris NG3have been shown to have very poor mycelial growth in inor-ganic nitrogen sources [14,15].Therefore,only organic nitrogen sources were examined for their effects on the production of exopolysaccharide,adenosine and cordycepin by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC 32219in the medium.Amongst organic sources,YE yielded the best EPS production (1.5g/l);in contrast,it was pre-viously reported that the best nitrogen source for EPS production

Table 2

The effects of various nitrogen sources on the cell growth and the production of various metabolites by https://www.wendangku.net/doc/0b15384052.html,itaris a Cell growth and metabolites

Nitrogen sources b PE

YE CSP Mycelia dry weight (g/l)18.222.315.5EPS production (g/l)0.4 1.50.9EPS productivity (g/(l d))0.130.500.29Adenosine production (mg/l)c

50.050.063.7Adenosine productivity (mg/(l d))12.516.712.7Cordycepin production (mg/l)c

90154.8135.0Cordycepin productivity (mg/(l d))

7.51

7.2

11.2

EPS:exopolysaccharide;PE:peptone;YE:yeast extract;CSP:corn steep pow-der.

a Data shown are average values of duplicate.

b Data for inorgani

c nitrogen sources (NH 4Cl,NH 4H 2PO 4

)are not shown

in table;mycelia dry weight for NH 4Cl and NH 4H 2PO 4are 10.2and 15.1g/l,respectively;EPS,adenosine and cordycepin for inorganic nitrogen sources were not determined.

c Data shown are the values of maximum production.

I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201197 Table3

The effects of yeast extract concentrations at various pH on the cell growth and the production of various metabolites by https://www.wendangku.net/doc/0b15384052.html,itaris a

Cell growth and metabolites Yeast extract(YE)concentrations(g/l)(at initial various pHs)b

10(6)15(6)30(6)30(5)30(4)

Mycelia dry weight(g/l)22.316.57.813.09.8 Adenosine production(mg/l)c50.070.055.3100.580.3 Cordycepin production(mg/l)c154.8650.2590.01041.5820.5

YE:yeast extract.

a Data shown are average values of duplicate.

b Values in parenthesis were initial pHs in the medium.

c Data shown are the values of maximum production;adenosine an

d cordycepin ar

e extracellular.

in the liquid culture of https://www.wendangku.net/doc/0b15384052.html,itaris C738and https://www.wendangku.net/doc/0b15384052.html,itaris NG3 was polypeptone and corn steep powder,respectively[14,15].As shown in Table2,the highest adenosine production(63.7mg/l) in the medium was achieved when CSP was the nitrogen source; however,the highest adenosine productivity(16.7mg/(l d))was obtained when YE was the nitrogen source.The highest cordy-cepin production(154.8mg/l)and productivity(17.2mg/(l d)) were obtained when YE was used.The systematic study of nitro-gen sources on the production of cordycepin in the submerged culture was rarely seen,although there was a report indicating that peptone was a good nitrogen source for cordycepin produc-tion by a strain of https://www.wendangku.net/doc/0b15384052.html,itaris in submerged cultivation[22].In addition,a PE+YE mixture was found to be effective for cordy-cepin production by https://www.wendangku.net/doc/0b15384052.html,itaris NBRC9787in a surface culture [23].

Based on the above results,YE was selected as a suitable nitrogen source for further studies.Table3summarized the effects of initial YE concentrations on the cell growth,adeno-sine and cordycepin production by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219. The mycelial growth decreased with the increase of YE con-centration when the initial pH was kept at6;the mycelia dry weigh was22.3,16.5and7.8mg/l when YE concentration was 10,15,and30g/l,corresponding to the C/N ratio(mass ratio) of1/1,1/1.5,1/3,respectively.A previous study concerning the effect of C/N ratio(mass ratio)on cell growth of a https://www.wendangku.net/doc/0b15384052.html,i-taris strain showed that a higher C/N ratio was preferred for the mycelial growth[26],an observation that accord with what was found in this work.The in?uence of C/N ratio on the produc-tion of adenosine and cordycepin by https://www.wendangku.net/doc/0b15384052.html,itaris strain has not been evaluated until now;the adenosine production was50.0, 70.0and55.3mg/l and cordycepin production was154.8,650.2, and590.0mg/l when YE concentration was10,15,and30g/l, corresponding to the C/N ratio(mass ratio)of1/1,1/1.5,1/3, respectively.This indicates that a lower C/N ratio is favorable for adenosine and cordycepin production;however,high con-centration of nitrogen source leading to a too low C/N ratio will inhibit their productions instead.Nevertheless,the data in Table3also reveal that the reduction of adenosine and cordy-cepin production by a signi?cantly low C/N ratio can be offset by careful adjustment of medium pH.When in a medium con-taining glucose(10g/l),yeast extract(30g/l)at pH5,the highest cordycepin production obtained was1041.5mg/l,which was the highest cordycepin production ever reported in the literature. The above results indicate that the cordycepin biosynthesis is controlled by the balance between the culture conditions and nutrient concentrations.

Plant oils,which were usually used as antifoam agents in fer-mentation,have been reported to be bene?cial for the mycelial growth and increased production of bioactive metabolites of sev-eral medicinal mushrooms[40].Park and coworkers reported previously that the exopolysaccharide production and mycelial growth in the submerged culture of a strain of https://www.wendangku.net/doc/0b15384052.html,itaris strain were substantially increased(almost tripled)by supplement of certain vegetable oils into the medium[27].The effects of soy, peanut,coconut,sun-?ower and olive oils were investigated in submerged fermentation of https://www.wendangku.net/doc/0b15384052.html,itaris,all at volume fractions of1%.It was seen that all plant oils tested stimulated mycelial growth and exopolysaccharide production of https://www.wendangku.net/doc/0b15384052.html,itaris to some extent.In particular,sun?ower oil at the level of1%led to an increase in mycelial growth and exopolysaccharide produc-tion in6d of cultivation,from10.5to22.5g/l and from0.8 to1.5g/l,respectively.In contrast,a previous report showed that the stimulatory effect of mycelial growth of https://www.wendangku.net/doc/0b15384052.html,itaris is greater with olive oil,while exopolysaccharide production is greater with sun?ower oil[27].However,the plant oils tested did not show much effect on the adenosine and cordycepin produc-tion(data not shown).The stimulation of cell growth by plant oils was attributed to a partial incorporation of lipids in the cell membrane,thereby facilitating the uptake of nutrients from the medium[40].However,the mechanism by which the plant oils affect the cell growth of https://www.wendangku.net/doc/0b15384052.html,itaris is yet to be determined. 3.3.Shake-?ask and static culture of https://www.wendangku.net/doc/0b15384052.html,itaris CCRC 32219

It is well-known that oxygen availability is critical to cell growth and metabolite formation by aerobic cell cultures.The previous investigation in the effect of oxygen supply on the cell growth and production of ganoderic acid(GA)by the higher fungus Ganoderma lucidum indicated that the favorable oxygen supply for cell growth and GA biosynthesis was quite different in that the cell growth was signi?cantly limited at low dissolved oxygen level(10%DO),while the content of polysaccharide and GA was higher than that at high dissolved oxygen level(25% DO)[8].Thus,a two-stage fermentation process by combining conventional shake-?ask fermentation with static culture was proposed and carried out that signi?cantly enhanced GA pro-duction[8].To investigate the effects of different fermentation

198I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201

Table4

Cell growth and metabolite production in shake and static culture of https://www.wendangku.net/doc/0b15384052.html,itaris a

Cell growth and metabolites Culture type

Shake Static Shake b+static Mycelia dry weight(g/l)15.5(36d)c14.0(30d)c–d Cordycepin production(mg/l)135.0220.31103.0 Cordycepin productivity(mg/(l d))11.29.234.4

a Data shown are average values of duplicate.

b Culture was shaken for6d followed by stati

c culture.

c Culture time indicate

d in th

e parenthesis was when the maximum cell mass was reached.

d–indicated that data were not determined.

methods on the mycelial growth and cordycepin production by https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219,the cultivation was carried out in three different ways,they are a shake-?ask culture,a static culture,and a two-stage(conventional shake-?ask fermentation followed by static culture)culture,respectively.As shown in Table4, the maximum mycelial production(15.5g/l)was obtained at day36of cultivation in the shake-?ask culture;in contrast,the maximum mycelial production(14.0g/l)was obtained at day30 of cultivation in the static culture.The maximum mycelial pro-duction in the two-stage cultivation was not determined because of the fact that the mycelia sunk to the bottom of?ask after the shaking stop,which led to the dif?culty of accurate sampling. The cordycepin production and productivity were220.3mg/l, 9.2mg/(l d)and135.0mg/l,11.2mg/(l d)for the static and the shake-?ask culture,respectively.Obviously,the maximal cordy-cepin production is higher in the static culture than that in the shake-?ask culture although the productivity is slightly lower in the static culture than that in the shake-?ask culture.However, the two-stage fermentation signi?cantly enhanced cordycepin production and productivity(1103.0mg/l and34.4mg/(l d)) which is in comparison with those(220.3mg/l,9.2mg/(l d) and135.0mg/l,11.2mg/(l d))of the static and the shake-?ask culture.This observation is consistent with what was found for GA formation by the two-stage culture of Ganoderma lucidum [8].In the observation of cell morphology,cellular speci?c oxygen uptake,and GA production of G.lucidum,it was seen that there were close relations among pellet size,cellular oxygen uptake and GA production in that large pellet size led to low speci?c oxygen uptake rate(Sp.OUR)and high GA production[8].However,the relationships among cell morphol-ogy,oxygen uptake and codycepin production still need to be determined.

3.4.Optimization of cordycepin production by RSM

(Box–Behnken design)

It was reported that the complexities and uncertainties associ-ated the large-scale fungi fermentation usually come from lack of knowledge of the sophisticated interactions among various factors.Our preliminary data shown above indicated that several major variables affecting the performance of the culture in terms of cordycepin production;they are the initial pH of the medium, the concentration of yeast extract,cultivation time of shake-?ask and static culture.Once the variable having the greatest in?u-ence on the response were identi?ed from the above results of one-factor-at-a-time strategy,Box–Behnken design(BBD)was used to optimize the levels of these variables.The four factors are initial pH(X1),YE concentration(X2),time of shake culture (X3),and time of static culture(X4).The levels of the variables for the BBD experiments were chosen in reconciliation with the data of our previous experiments on cordycepin production by C. militaris CCRC32219.The BBD design and the corresponding experimental data are shown in Table5.By applying multiple regression analysis on the experimental data,the experimen-tal results of the BBD design were?tted with a second-order polynomial equation(Eq.(1)).The results of the regression analysis are shown in Table6,and the second-order polyno-mial equation obtained for cordycepin production is shown in Eq.(2).

Table5

Result of four factor,Box–Behnken experimental design

Trial no.X1X2(%)X3(day)X4(day)Cordycepin(mg/l) 16 4.5610647.2

24 4.5610146.0

36 1.5610441.1

44 1.5610396.1 553816547.1 653416133.4 75384843.0 85344187.8 953610435.8 1063616741.6 1143616930.3 126364619.3 134364850.4

145 4.58101375.6

155 1.5810774.6

165 4.5410662.8

175 1.5410276.8 1853610759.9 19638101095.5 20438101024.1 2163410151.8 224341098.2

235 4.561668.4

245 1.5616109.6

255 4.564815.8

265 1.56444.4 2753610570.7

X1:initial pH of the medium;X2:YE concentration;X3:time of shake culture; X4:time of static culture.

I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201199 Table6

Regression analysis of the Box–Behnken design experiments

Parameter Coef?cient Standard error T ratio Probability Intercept?239,406*46693.28*?5.12720*.035998* X193,360*18492.60* 5.04851*.037067* X12?9,153*1844.66*?4.96206*.038296* X248,296*11150.26* 4.33134*.049388* X22?8,546*1817.62*?4.70181*.042380* X357,537*12329.32* 4.66668*.042980* X32?4,570*1019.26*?4.48401*.046308* X4782521.00 1.50057.272264

X24?9 2.35?3.97710.057795 X1×X2?18,5434419.26?4.19586.052379 X1×X3?22,585*4929.65*?4.58151*.044486* X1×X4?228192.32?1.18766.356897 X2×X3?158278.01?.56732.627687 X2×X41451.95.27515.809018 X3×X4?5 6.73?.74157.535606 Statistical experimental design:R2=0.98436;Adj:0.79669,four3-level factors,1blocks,27runs;MS residual=26499.19;DV:cordycpein.X1,initial pH of the medium;X2,YE concentration;X3,time of shake culture;X4,time of static culture.

*P<0.05.

Second-order model equation:

Y(mg/l)=93,360X1?9153X21+48,296X2?8546X22

+57,537X3?4570X23+782X4?9X24

?18,543X1X2?22,585X1X3?228X1X4

?158X2X3+14X2X4?5X3X4?239,406(2) This?t of the model was checked by the coef?cient of deter-mination R2,which was calculated to be0.984,indicating that 98.4%of the variability in the response could be explained by the model.The test statistics F values for the overall regression is signi?cant at the upper5%level,which further supported that the second-order model is very adequate in approximating the response surface of the experimental design.After performing the transformation of Eq.(2)to its canonical form,the optimum combination was found to be following:pH6;YE concentra-tion,45g/l;time of shake-?ask culture,8.0days;time of static culture,16days.The model predicted a maximum response of3162.7mg/l cordycepin yield.Veri?cation of the calculated maximum was done with experiments that were performed in the culture media representing the optimum combination found,and the cordycepin yield of2214.5mg/l(average of three repeats) was obtained.Although the measured value did not justify the predicted value of the response model,it is however signi?cantly higher than that,increased form1041.5to2214.5mg/l,obtained by the one-factor-at-a-time method described above.

Cordycepin was?rst extracted from https://www.wendangku.net/doc/0b15384052.html,itaris[4]and then found to be present in C.sinensis and C.kyushuensis[20]. Although cordycepin can be synthesized chemically,such a route is cumbersome and requires complicated separation that leads to low yield and the use of a large volume of harmful organic solvents.Submerged cultivation of Cordyceps is seen as a promising alternative to chemical synthesis and soil culti-vation for cordycepin production.However,very low levels of cordycepin were previously produced in mycelium and culture broth during submerged cultivation of Cordyceps sp.;7.1mg/l of cordycepin was reported in submerged cultivation of Cordy-ceps on a laboratory bioreactor scale[11].Several attempts have been made to optimize the production of cordycepin by https://www.wendangku.net/doc/0b15384052.html,-itaris.Recently,a submerged culture method of https://www.wendangku.net/doc/0b15384052.html,itaris for cordycepin production on a commercial scale using a two-stage dissolved oxygen control has been developed,which remarkably improved the production ef?ciency;moderate cordycepin pro-duction and productivity titers of188.3mg/l and14.5mg/(l d) were obtained[21].To enhance further the cordycepin pro-duction by submerged cultivation of https://www.wendangku.net/doc/0b15384052.html,itaris,the effects of carbon sources and carbon/nitrogen ratios were investigated using central composite design and response surface analysis which resulted in high cordycepin production(345.4±8.5mg/l) and productivity(19.2±0.5mg/(l d))[22].The previous highest production(640mg/l)and productivity(32mg/(l d))of cordy-cepin were reported by Masuda and coworkers in the study of a surface culture using https://www.wendangku.net/doc/0b15384052.html,itaris NBRC9787[23].The maxi-mum production(2214.5mg/l)and productivity(92.3mg/(l d)) of cordycepin obtained in the present study are signi?cantly higher than those reported to date[11,21–23].

https://www.wendangku.net/doc/0b15384052.html,parison of extracellular and intracellular adenosine and cordycepin

In the investigating the production of cordycepin by surface culture using https://www.wendangku.net/doc/0b15384052.html,itaris NBRC9787,Masuda and coworkers measured the production of cordycepin and adenosine in the?l-trate and the extract[23].Their results revealed that most(98%) of the cordycepin synthesized existed in the culture?ltrate and only a little amount(2%)existed in the mycelia;adenosine was not detectable in the culture?ltrate but it was detected with same level of cordycepin in the mycelia.These results indicated that the cordycepin synthesized by https://www.wendangku.net/doc/0b15384052.html,itaris is immediately and selectively secreted into its medium.Previously Lennon and Suhadolnik[18]proposed that the formation of cordycepin

200I.-L.Shih et al./Biochemical Engineering Journal33(2007)193–201

proceeds by the enzymatic reduction of adenosine.Hereafter,it was suggested that there was no need to follow both compounds in the mycelia,since98%of the synthesized cordycepin was found to exist in the culture medium.The cordycepin and adenosine productions in the culture?ltrate and the mycelial extract of https://www.wendangku.net/doc/0b15384052.html,itaris CCRC32219after9d of the submerged cultivation were80.5,40.4mg/l and70.2,20.3mg/l, respectively.Apparently,these results did not accord with those of Masuda et al.[23].Whether the discrepancy is due to the difference of culture methods(surface culture versus submerged culture)between the two studies or it is due to the different mechanisms involving cordycepin synthesis between the two different https://www.wendangku.net/doc/0b15384052.html,itaris strains remains to be further investigated.

4.Conclusion

The present study indicates that the cordycepin biosynthe-sis is controlled by the balance between the culture conditions and nutrient concentrations.Factors such as initial pH,YE con-centration,time of shake-?ask culture and time of static culture having the greatest in?uence on the cordycepin production were identi?ed.According to the Box–Behnken experimental design and response surface analysis,a two-stage fermentation pro-cess by combining8d of shake-?ask fermentation with16d of static culture at pH6and YE concentration of45g/l signi?cantly enhanced cordycepin production and productivity.Under opti-mized condition,the maximum production(2214.5mg/l)and productivity(92.3mg/(l d))of cordycepin were obtained;they are much higher than those reported to date.The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of https://www.wendangku.net/doc/0b15384052.html,itaris for the production of bioactive metabolites,and it is useful in regulation and optimization of https://www.wendangku.net/doc/0b15384052.html,itaris culture for ef?cient production of valuable nutraceuticals.

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