Effects of culture conditions on the mycelial growth and bioactive

Biochemical Engineering Journal33(2007)

193–201

Effects of culture conditions on the mycelial growth and bioactive

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 influence of initial pH value,various nitrogen sources,plant oils,and modes of propagation(shake-flask 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 itaris,but they did not show much effect on the adenosine and cordycepin production.A two-stage fermentation process by combining shake-flask fermentation with static culture significantly 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 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.

©2006Elsevier B.V.All rights reserved.

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@.tw(C.Hsieh).and an immunoregulative effect[1,17,34,44];polysaccha-rides account for the anti-inflammatory,antioxidant,antitumor, antimetastatic,immunomodulatory,hypoglycaemic,steroido-genic and hypolipidaemic effects[13,19,29,33,39,41,42].

The fruit bodies of wild itaris are expensive because of host specificity and rarity in nature;they grow extremely slowly in nature,their growth is restricted to a specific area and their sizes are very small.Therefore,the collection of sufficient 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 efficient production of valuable metabolites has been studied extensively[6,25],sub-merged cultivation of itaris has scarcely been studied until recently[15,22,26–28,43].

1369-703X/$–see front matter©2006Elsevier B.V.All rights reserved. doi:10.1016/j.bej.2006.10.019