ACTA BIOLOGICA SLOVENICA

LJUBLJANA 2014 Vol. 57, [t. 2: 45–52

Cordycepin production by Cordyceps militaris cultivation

on spent brewery grains
Proizvodnja kordicepina z gojenjem glive Cordyceps militaris na
pivovarskih tropinah

Andrej Gregori

Institute for Natural Sciences (Zavod za naravoslovje), Ulica bratov Učakar 108,
SI-1000 Ljubljana, Slovenia
Correspondence: andrej.gregori@zanaravo.com

Abstract: This is a first report on C. militaris mycelia and fruiting bodies culti-
vation on solid-state containing spent brewery grains (SBG). Five different strains of
C. militaris were cultivated on substrates containing rye grains and 0 to 60% SBG.
Stromata formation on SBG containing substrates was noticed with two C. militaris
strains. All strains failed to grow on substrates containing SBG amounts higher than
50%. Highest (10.42 mg/g) cordycepin concentration in cultivating substrate was
determined with strain CM2 on 50% SBG. One gram of CM11 strain fungal biomass
was able to produce 787.11 mg/g of cordycepin. SBG as a byproduct represent a
readily available, low price substrate for cordycepin solid-state production. Obtained
concentrations of cordycepin are so far the highest reported concentrations obtained
on solid-state substrates therefore we can talk about cordycepin hyperproduction.

Keywords: Cordyceps militaris, spent brewery grains, cordycepin, cultivation,

medicinal mushrooms

Izvleček: Trosnjake in podgobje glive Cordyceps militaris smo gojili na trdih

substratih, ki so vsebovali pivovarske tropine. Pet različnih sevov glive C. militaris
je preraščalo na substratih sestavljenih iz različnih razmerij rži in pivovarskih tropin.
Trosnjaki so zrasli pri dveh sevih C. militaris. Noben od sevov ni preraščal substrata z
vsebnostjo pivovarskih tropin večjo od 50%. Najvišjo koncentracijo kordicepina (10,42
mg/g) v substratu smo določili pri sevu CM2 na substratu s 50% pivovarskih tropin. En
gram glivne biomase seva CM11 je proizvedel 787,11 mg/g kordicepina. Pivovarske
tropine kot stranski produkt predstavljajo lahko dostopen in poceni substrat primeren
za proizvodnjo kordicepina. Dosežene koncentracije kordicepina so po dosedaj znanih
podatkih najvišje, zato lahko upravičeno govorimo o hiperprodukciji kordicepina.

Ključne besede: Cordyceps militaris, pivovarske tropine, kordicepin, gojenje,

zdravilne gobe
46 Acta Biologica Slovenica, 57 (2), 2014

Introduction Spent brewery grains

Spent brewery grains (SBG) are a byproduct
Cordyceps militaris of the brewing industry which remain as the outer
pericarp-seed coat layers from the original malted
C. militaris belonging to Ascomycota, is a
parasite of insects larval stage, forming fruiting barley (Hordeum vulgare) grain after barley
bodies expanding outside the insect larvae or pupae hot water extraction at 65–70°C (Mussatto et al.
(Buenz et al. 2005). C. militaris was traditionally 2006). SBG are readily available, high volume
used as a tonic and traditional folk medicine, espe- and low cost byproducts and remain a potentially
cially in East Asia (Ying et al. 1987, Holliday and more valuable resource for industrial exploitation.
Cleaver 2008, Bhandari et al. 2010). This specie Currently they are used as an animal feed. Indeed,
grows wild also in Slovenia (Ogris 2013) and in value-added products are increasingly being sought
some other European countries, but its medicinal for SBG (Robertson et al. 2010).
use in Europe has never been reported. Besides potential uses of SBG for energy via
C. militaris polysaccharides show significant intermediate pyrolysis (Mahmooda et al. 2013),
antitumor activities against cervical and liver as a potential material for coal production through
cancer cells in vitro (Yang et al. 2014), extracts of wet hydrothermal carbonization (Poerschmanna
its fruiting bodies show antioxidant, antibacterial, et al. 2014) or a potential candidate for phenolic
antifungal, antihuman tumor cell lines (Rao et al. compounds extraction (Barbosa-Pereira et al.
2010, Reis et al. 2013, Yang et al. 2014), anti- 2014), SBG have been successfully used as a
inflammatory (Won and Park 2005, Rao et al. 2010), cultivating substrate for Pleurotus ostreatus
anti-fibrotic (Nan et al. 2001), anti-angiogenetic (Gregori et al. 2008), for immobilization of kefir
(Yoo et al. 2004) and insulin secreting (Choi et and Lactobacillus casei for sourdough wheat
al. 2004) activities. This fungus is cultivated for bread making (Plessas et al. 2007), cultivation of
cordycepin (3′-deoxyadenosine), a nucleoside Lactobacillus plantarum (Gupta et al. 2013),
analogue with anti-tumour, anti-proliferative, biomass and xylitol production of Debaryomyces
anti-metastatic, insecticidal and anti-bacterial hansenii (Carvalheiro et al. 2005). Till now no
activities (Song et al. 1998). reports of SBG usage in C. militaris cultivation
In recent years C. militaris is extensively and cordycepin production was reported.
cultivated in liquid as well as solid media (Das et
al. 2010) and is the most successfully cultivated
Cordyceps species (Kobayashi 1941, Sung 1996). Materials and methods
In solid media different supplemented grain types
and seeds are used, such as millet, rye, rice, brown
Cultures cultivation and inoculum preparation
rice, bean powder, corn grains, cotton seed hulls,
sorghum, corn cobs, jowar, wheat, sunflower floral C. militaris cultures CM11, CM14 and CM15
discs (Chen and Wu 1990, Zhang and Liu 1997, were obtained from Edible Fungi Institute, Shang-
Li 2002, Holliday et al. 2004, Li et al. 2004, Zhao hai Academy of Agricultural Sciences culture
et al. 2006, Gao and Wang 2008, Wei and Huang collection, CM2 culture was kindly donated by
2009, Chen et al. 2011, Shrestha et al. 2012, Wen prof. Wu Wei from Plant Protection Institute,
et al. 2014, Yi et al. 2014). SBG so far have not Shanghai Academy of Agricultural Sciences and
being reported as a substrate component. Wu CM5 culture was obtained from culture collec-
and coworkers (2013) reported on successful C. tion of Mycomedica d.o.o., Podkoren, Slovenia.
militaris cultivation and cordycepin production All cultures were transferred to Potato Dextrose
on levan fermentation leftovers. Ni and cowork- Agar (Difco, USA) and incubated at 24°C in
ers (2009) extracted cordycepin from the spent C. complete darkness. After mycelium overgrew the
militaris substrate, concluding it as appropriate agar media, it was homogenized in a blender with
source of cordycepin with concentrations ranging 100 ml of sterile water (Wahring, USA). Liqui-
from 0.1 to 1 mg/g. fied inoculum was further used for inoculation of
cultivation substrates.
Gregori: Cordycepin production by Cordyceps militaris cultivation 47

Substrates preparation and culturing

Rye (Rebernak, Šmartno na Pohorju, Slovenia)
and spent brewery grains (Union d. d., Ljubljana,
Slovenia) were mixed in different proportions (9:1,
8:2, 7:3, 6:4, 5:5, 4:6) and filled into 720 ml glass
jars. Water was added to the mixture to achieve
65% moisture content and 100 g of substrate
weight. Substrates were prepared in triplicates.
Jars were covered with metallic lids having 14
mm hole in the middle, covered with HEPA class
14 membrane sterilized for 30 minutes at 121°C
and cooled under the flow of sterile air. During
inoculation liquid inoculum was constantly mixed
on a magnetic stirrer with 5 ml transferred into Figure 2. Sterile strain (CM11) of Cordyceps militaris
not being able to form stromata on substrate
the substrate jars. Jars were closed and incubated
containing 20% spent brewery grains and
at 24°C under cool white fluorescent light. After 80% rye
incubation the substrate and fruiting bodies were Slika 2: Sterilni sev (CM11) Cordyceps militaris ni
dried for 48 hours at 60°C and milled in a coffee zmožen tvoriti podgobja na substratu, ki
type grinder. vsebuje 20% pivovarskih tropin in 80% rži

conditions were: YMC - polyamine column (5

µm, 250 mm × 4.6 mm); solvent A - acetonitrile;
solvent B - double distilled water; linear gradient
- acetonitrile : water (v : v) - (90:10) 15 minutes
→ (86.5:13.5) 20 minutes → (75:25) 30 minutes
→ (70:30) 35 minutes; flow rate – 1 ml/minutes;
temperature - 30ºC; detective wavelength –
259 nm; injection volume – 10 µl.

Quantification of cordycepin produced by

fungal biomass
Figure 1: Cordyceps militaris (CM2 strain) forming
stromata on substrates containing 20% spent
For determination of ergosterol concentration
brewery grains and 80% rye in control sample 0.2 g of dry C. militaris mycelia
Slika 1: Cordyceps militaris (sev CM2) tvori podgobje cultivated on PDA media was extracted in 5 ml
na substratu, ki vsebuje 20% pivovarskih of cold absolute ethanol following a modified
tropin in 80% rži protocol by Martin and coworkers (1990). For
test samples biomass determination one gram of
grinded material was extracted in absolute etha-
nol (10 ml) for 30 minutes at 4ºC, centrifuged at
Cordycepin analysis
10000 g for 10 minutes and filtered through a 0.22
10 ml of 20% ethanol was added to 200 mg µm membrane filter (Macherey Nagel).
of powdered sample, and extracted for 2 hours in Analysis was performed on a Waters HPLC
ultrasonic water bath. The supernatant was centri- system equipped with PDA 996 detector, 2690
fuged at 14000 g for 10 minutes and filtered through Separation Module and Nucleosil C18, 250 ×
0.22 µm membrane filter (Macherey Nagel). 4.6 mm, 5 µm column. Ergosterol was eluted at
The system consisted of Waters 2695 HPLC isocratic conditions of 50% methanol and 50%
system equipped with UV detector. The working acetonitrile at a flow rate of 1.5 ml/min and
48 Acta Biologica Slovenica, 57 (2), 2014

identified with help of standard retention time and containing SBG amounts of 60% and higher. When
the specific absorption triple peak characteristic transferred onto SBG containing substrates all
for ergosterol between 260 nm and 300 nm. For strains except CM11 and CM14 formed mycelia
quantification a calibration curve was employed with very strong rhyzomorphic primordia forming
using purified (Nylund et al. 1992) ergosterol characteristics. Stromata formation was noticed
standard (Sigma, Germany). Ergosterol content with strain CM2 (0, 10, 20, 30, 40 and 50% SBG)
was calculated using calibration curve for fungal and CM5 (10, 20, 30, and 50% SBG) (Figure 1).
mycelia and ergosterol. In all strains except CM11 the increase in
Two parameters were calculated for deter- CCS is noticed with the increase of portions of
mination of cultivation process effectiveness – SBG in the cultivation substrates. Only in strain
cordycepin content in substrate (CCS) and fungal CM5 CCS the decrease from 8.90 to 6.64 mg/g
biomass cordycepin production (FBCP). CCS was was observed in 50% SBG substrate. Maximum
calculated per substrate weight and shows the CCS (10.42 mg/g) was obtained with strain CM2
end concentration of cordycepin in the substrate cultivated on 50% SBG substrate (Figure 3).
(w/w). FBCP shows cordycepin production ability The highest FBCP (787.11 mg/g) was observed
of certain fungal biomass/mycelia quantity (w/w). with strain CM11 cultivated on 0% SBG drasti-
cally reduced (to 305.75 mg/g) with addition of
SBG to the substrate (Figure 4). The same FBCP
Statistical analysis
reduction trend at SBG addition was noticed
The data were evaluated by ANOVA (program with CM5 strain. FBCP stayed the highest at all
past 2.16) and significance accepted at p < 0.05. SBG concentrations compared to other strains,
the second in FBCP was CM5 strain followed by
CM2, CM14 and CM15. The lowest FBCP was
Results obtained with CM15 strain on average (Figure 2).
The comparison of different C. militaris strains
C. militaris mycelia overgrew all the tested showed that they react differently to SBG addition
substrate mixtures but failed to grow on substrates to the substrate, with CM2 being the strongest

Figure 3: Average cordycepin content in substrates (CCS) containing rye and spent brewery grains overgrown
with Cordyceps militaris. Columns within a treatment marked with different letters are significantly
different
Slika 3: Povprečna vsebnost kordicepina v substratih (CCS), ki vsebuje rž in pivovarske tropine preraščene z
glivo Cordyceps militaris. Stolpci znotraj enega obravnavanja, ki so označeni z različnimi črkami, se
značilno razlikujejo
Gregori: Cordycepin production by Cordyceps militaris cultivation 49

Figure 4. Average Cordyceps militaris fungal biomass for cordycepin production (FBCP) on substrates containing
spent brewery grains
Slika 4: Povprečna biomasa glive Cordyceps militaris za proizvodnjo kordicepina (FBCP) na substratih (CCS),
ki vsebuje pivovarske tropine

CCS producer and third strongest FBCP producer. C. militaris characteristics (white color without
CM11 was the strongest FBCP producer (787.11 stromata forming ability) noticed with CM11 strain
mg/g), meaning that 1 g of CM11 biomass can were reported by Sreshtha et al. (2012) and is by
produce up to 787.11 mg of intra and extracellular this author linked to strain degeneration (Figure 2).
cordycepin (Figure 4). This could mean that CM11 is a degenerated
C. militaris strain, capable of producing high FBCP
on rye substrate only. At the same time CM11 is
Discussion the only strain of which CCS is not drastically
influenced by addition of SBG to the substrate.
According to our results SBG addition into Holliday and coworkers (2004) reported 2.25
C. militaris cultivation substrate very effectively mg/g CCS in commercial C. sinensis products
increased CCS and at the same time decreased obtained through solid-state cultivation and 0.65
FBCP. CCS hyperproduction in SBG containing mg/g cordycepin in wild collected C. sinensis
substrates could be caused by higher concentra- stromata. Ni and coworkers (2009) reported
tions of low molecular compounds in SBG (simple 0.1 to 1 mg/g CCS content in spent C. militaris
sugars and other fermentation products produced cultivating substrates, Wen and coworkers (2014)
through the brewing process), compared to unfer- optimized solid-state composition for C. militaris
mented rye grains. cultivation and achieved CCS of 9.17 mg/g. All
Many different chemically defined substrate reported concentrations are lower compared to
supplements are used in commercial C. militaris results (10.42 mg/g) obtained in our research,
cultivation, with some researchers (Xie et al. 2009) showing SBG are a superior, readily available
reporting natural substrate components such as and low cost substrate for cordycepin production
brown rice, malt and soybean being better sources through C. militaris cultivation.
of nutrition for C. militaris in comparison to chemi- Why all strains failed to grow on substrates
cally defined media. This suggests high cordycepin containing SBG amounts of 60% and higher is
concentrations in SBG containing substrates could still unknown. This phenomenon could be linked
be achieved because SBG is a complex material with higher nitrogen content reported by Gao and
composed of only natural components. coworkers (2000) to suppress C. militaris growth.
50 Acta Biologica Slovenica, 57 (2), 2014

Conclusion Povzetek

SBG represent a readily available, low price Pivovarske tropine predstavljajo lahko
substrate for cordycepin solid-state production. dosegljiv, cenen substrat za proizvodnjo kordicepi-
Here reported concentrations of cordycepin are na na trdih substratih. Proizvedene koncentracije
so far the highest reported concentrations (10.42 kordicepina v substratu so najvišje (10,42 mg/g)
mg/g) obtained on solid-state substrates. koncentracije znane iz objav do sedaj.
Use of SBG for cordycepin production by Uporaba pivovarskih tropin za produkcijo
C. militaris is shown here as a very effective kordicepina z gojenjem glive C. militaris je v
technique for producing high value food additive članku predstavljena kot enostavna metoda za
or medicated animal feed - with just drying SBG proizvodnjo hrane, prehranskih dopolnil ali krme
processed through C. militaris cultivation. z visoko vsebnostjo kordicepina.
Further research are needed to determine the Za hiperprodukcijo kordicepina so potrebne
exact components and/or physical properties caus- nadaljnje raziskave za določitev ključnih karakter-
ing cordycepin hyperproduction in SBG contain- istik pivovarksih tropin ter optimizacijo gojitvenih
ing substrates and for optimization of cultivation parametrov kot so temperatura, svetloba, trajanje
parameters such as temperature, incubation time, inkubacije in prezračevanje.
light and aeration. Opisane tehnike gojenja C. militaris so že v
Here described technique of SBG usage is procesu optimizacije in komercializacije za namene
already in the process of optimization and com- proizvodnje prehranskih dopolnil in medicinirane
mercialization focusing on high cordycepin content krme z visoko vsebnostjo kordicepina.
in food and feed production.

Acknowledgement

Author would like to thank dr. Yanfang Liu

for performing the cordycepin analysis and help-
ing with the manuscript and Matej Stražišar for
providing experimental spent brewery grains.

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