Short Communication

Received: 11 May 2016 Revised: 20 September 2016 Accepted article published: 17 October 2016 Published online in Wiley Online Library: 29 November 2016

(wileyonlinelibrary.com) DOI 10.1002/jsfa.8097

Evaluation of different agricultural wastes for

the production of fruiting bodies and bioactive
compounds by medicinal mushroom Cordyceps
militaris
Qunying Lin,a,b† Liangkun Long,c† Liangliang Wu,a,b Fenglun Zhang,a
Shuling Wu,a Weiming Zhanga and Xiaoming Suna*

Abstract
BACKGROUND: In commercial production of Cordyceps militaris (a famous Chinese medicine), cereal grains are usually utilized as
cultivation substrates. This study aimed to evaluate the efficiency of agricultural wastes as substitute materials in the low-cost
production of C. militaris. Cottonseed shells (CS), corn cob particles (CCP), Italian poplar sawdusts (IPS) and substrates spent by
Flammulina velutipes (SS) were employed to cultivate C. militaris, using rice medium as control.

RESULTS: CS and CCP were suitable for fruit body formation of C. militaris, with yields of 22 and 20 g per bottle respectively.
Fruit bodies grown on CCP showed the highest levels of cordycepin and adenosine, up to 9.45 and 5.86 mg g−1 respectively. The
content of D-mannitol in fruit bodies obtained on CS was 120 mg g−1 (80% of the control group), followed by that on CCP, 100
mg g−1 . Fruit bodies cultivated on CCP displayed a high crude polysaccharide level of 26.9 mg g−1 , which was the closest to that
of the control group (34.5 mg g−1 ).

CONCLUSION: CS and CCP are effective substrates for the production of fruit bodies and bioactive compounds by C. militaris.
This study provides a new approach to decreasing the cost of C. militaris cultivation and dealing with these agricultural wastes.
© 2016 Society of Chemical Industry

Keywords: cottonseed shells; corn cob particles; Cordyceps militaris; artificial cultivation; cost-effective

INTRODUCTION wheat) as substrates.9 The latter is more popular because of its

Cordyceps is a genus of mushrooms popular in Asia, especially in easy operation, and fruit body products from this model prevail
China. Many of them are used as a tonic food or herbal medicine. in the market. However, raw materials for fruit body production
Modern techniques have proved that many species of Cordy- have become a problem with the development of the cultivation
ceps possess medicinal effects such as antitumor, antioxidant and industry. Over 4000 tons of dry fruit bodies are produced per year
immunomodulatory activities.1 – 4 Cordyceps militaris, as a famous in China, consuming at least 500 000 tons of cereal substrates.
species in the genus, has been explored extensively.1 This mush- Compared with agricultural wastes, cereal materials are expensive
room produces bioactive compounds and exhibits significant anti- and can be easily contaminated. Agricultural wastes have been
tumor capacity, inhibiting NB-4 and U937 cell growth and renal utilized in mushroom cultivation for a long period and exhibit
cell carcinoma (RCC). Cordycepin is the first nucleoside antibiotic good suitability and economic efficiency.10,11 However, there is no
recovered from C. militaris.5 To date, cordycepin has been regarded report about their application in the cultivation of C. militaris. In
as the main bioactive component in C. militaris through its contri-
bution to many bioactive properties.6,7 Besides cordycepin, other
bioactive components such as adenosine, D-mannitol (cordycepic ∗ Correspondence to: X Sun, Nanjing Institute for the Comprehensive Utilization
acid) and polysaccharides also show significant activities.8 There- of Wild Plants, Nanjing 210042, China. E-mail: sunxm64@163.com
fore the contents of these bioactive components play an important
† These authors contributed equally to this work
role in the quality of fruit bodies of C. militaris.
Domestication and fruit body cultivation of C. militaris have a Nanjing Institute for the Comprehensive Utilization of Wild Plants, Nanjing
been developed for more than 30 years in China. Among all 210042, China
Cordyceps mushrooms, C. militaris is the only edible fungus with
b Jiangsu Hongfeng Fruit and Vegetable Food Co., Ltd, Suqian 223700, China
fruit bodies cultivated on a large scale for commercial production
worldwide. There are mainly two sets of models for fruit body c Department of Pharmaceutical Engineering, College of Chemical Engineering,
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cultivation, using insects as hosts or cereal grains (mainly rice or Nanjing Forestry University, Nanjing 210037, China

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Cultivation of C. militaris with agricultural wastes www.soci.org

Table 1. Effect of different substrates on growth and development of Cordyceps militaris

Mycelial growth Primordium Fruit body

Substrate time (days) occurrence time (days) mature time (days) Fruit body appearance

Cottonseed shells (CS) 4 14 40 Orange-yellow, strips, 5–6 cm

Corn cob particles (CCP) 4 14 40 Orange-yellow, strips, 5–6 cm
Italian poplar sawdusts (IPS) 4 14 35 Orange-yellow, strips and
clustered strips, 3–4 cm
Substrates spent by F. velutipes (SS) 4 14 35 Orange-yellow, strips and
clustered strips, 3–4 cm
Rice (CK) 3 12 40 Orange-yellow, strips, 5–6 cm

this research, four kinds of agricultural wastes, namely cottonseed became round and were covered with spores. After maturation,
shells (CS), corn cob particles (CCP), Italian poplar sawdusts (IPS) fresh weights of fruit bodies were recorded immediately after har-
and substrates spent by the mushroom Flammulina velutipes (SS), vesting. At least five replicates were carried out for each treatment.
were evaluated for their suitability for fruit body cultivation of C.
militaris. The biomass and main bioactive compounds produced Component analysis
by C. militaris were compared among the four agricultural wastes, Fruit bodies obtained from different substrates were dried to con-
using rice medium as control. stant weight at 60 ∘ C. Dried samples were pulverized to a par-
ticle size of less than 20-mesh. For adenosine and cordycepin
extraction, 0.5 g (accurate to 0.0001 g) of dry powder was sus-
MATERIALS AND METHODS pended in 50 mL of doubly deionized water and sonicated for 3 h in
Fungal strain and liquid spawn preparation an ultrasonic bath at 50 kHz and 400 W. The supernatant obtained
Cordyceps militaris strain 20130508 was isolated from fresh fruit by centrifugation at 1740 × g for 15 min was filtered through a 0.45
bodies produced on rice medium by our laboratory and was μm membrane filter. For D-mannitol analysis, the extracting solu-
cultured at 25 ∘ C on peptone-added potato dextrose agar (PPDA) tion was 500 mL L−1 ethanol and the extraction process was the
slants. The strain was grown for 10 days and then stored at 4 ∘ C for same as described above.
stock. Liquid spawn was grown in liquid medium (GPBY) consisting High-performance liquid chromatography (HPLC) analysis was
of 20 g L−1 glucose, 5 g L−1 peptone, 3 g L−1 beef extract and 1 g L−1 carried out using an Agilent 1200 Series HPLC system (Agilent
yeast extract. A 250 mL flask containing 100 mL of GPBY medium Technologies Inc., Santa Clara, CA, USA). The separation and detec-
was inoculated with a piece (∼0.5 cm2 ) of mycelial slant on PPDA tion conditions for adenosine and cordycepin were as follows: C18
and incubated at 25 ∘ C with shaking at 120 rpm. Mycelia grew to a column (4.6 mm × 150 mm, 5 μm particle size; Agilent), mobile
maximum after 7 days, when the spawn culture was diluted with 4 phase consisting of 10 mmol L−1 KH2 PO4 buffer (pH 6.0) and
volumes of sterile distilled water and used to inoculate media for methanol at 85:15 (v/v) ratio, flow rate of 0.8 mL min−1 , column
fruit body production. temperature of 30 ∘ C and detection at wavelength of 260 nm
with UV–visible detector. The analysis of cordycepic acid from the
Substrate preparation extracts was carried out at 35 ∘ C (column temperature) and 35
CS, CCP (ground to 4-mesh), IPS and SS were separately mixed with ∘ C (refractive index detector temperature) with a mobile phase
wheat bran and rice bran at ratios of 8:1:1 (w/w/w) and moistened consisting of deionized water at a flow rate of 0.6 mL min−1 .
with nutrient liquid consisting of 20 g L−1 glucose and 5 g L−1 The injection volume in the HPLC system was 20 μL. The crude
peptone. The water content of substrates was adjusted to 650 g polysaccharide contents of fruit bodies were determined by the
kg−1 . Substrates containing 40 g of dry material were filled into phenol–sulfuric acid method.12
bottles and pressed evenly. Rice media served as control (CK),
containing 20 g of rice and 35 mL of nutrient liquid per bottle. All
bottles were sealed with polypropylene films and sterilized at 121 RESULTS
∘ C for 30 min. Growth of C. militaris on different substrates
Cordyceps militaris showed similar mycelial growth on the four
Inoculation and fruit body cultivation different agricultural waste substrates. In terms of mycelial growth,
Spawn culture stored in a sterilized spray bottle was inoculated it took 4 days to cover the whole substrates CS, CCP, IPS and
evenly into each bottle of medium at the rate of 10 mL per bot- SS. After 14 days, primordia occurred on these substrates. In
tle. All cultures were incubated for a certain time in the dark at comparison with rice media, primordia took a longer time to occur.
20 ∘ C and 60–70% relative humidity. When mycelia grew over Fruit bodies matured 5 days later on CS, CCP and rice media than
the medium surface, the incubating room was illuminated by flu- on IPS and SS. However, the lengths of fruit bodies on CS, CCP
orescent light at an intensity of 500–800 lux for 10 h per day. and rice media, recorded as 5–6 cm, were greater than those on
Meanwhile, daily ventilation for a 30 min period was carried out the other groups (Table 1). All fruit bodies cultured on substrates
every day. When the aerial and surface vegetative hyphae turned tested had normal orange-yellow strip shapes (Fig. 1).
to orange-yellow, the polypropylene seals were punctured with
3 mm holes for better air exchange. Numbers of days until the Fruit body production of C. militaris on different substrates
first appearance of primordia in media were recorded. The culti- Among the four kinds of agricultural wastes, the CS substrate gave
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vation conditions were maintained until the tops of fruit bodies the highest yield of 22 g per bottle, which was 84.6% of the control

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 www.soci.org Q Lin et al.

Figure 1. Fruit bodies of Cordyceps militaris grown on cottonseed shells (CS), corn cob particles (CCP), Italian poplar sawdusts (IPS), substrates spent by
Flammulina velutipes (SS) and rice (Rice).

bodies produced on CCP contained 100 mg g−1 D-mannitol, while

fruit bodies from IPS and SS showed quite low levels of D-mannitol,
80 and 75 mg g−1 respectively (Fig. 3C).
Crude polysaccharide levels in fruit bodies cultured on CCP and
CS were 26.9 and 23.4 mg g−1 respectively. Both were lower than
the control (34.5 mg g−1 ). The levels of crude polysaccharides in
fruit bodies from IPS and SS were less than half of the control group
(Fig. 3D).

DISCUSSION
Cereals with the addition of some organic substances have been
widely used in commercial production of C. militaris.9 However,
besides cereal grains, some other materials suitable for culture of
C. militaris have also been found. Wu et al.13 proved that spent
medium from levan fermentation can be used as substrate to
Figure 2. Fruit body production of Cordyceps militaris on different sub- cultivate C. militaris. This is consistent with the results obtained
strates. Fresh weights of fruit bodies were determined immediately after in the present study, proving that C. militaris is able to grow on
harvesting. Error bars represent standard deviations from four indepen- different kinds of substrates. CCP substrate has been used on a
dent experiments. large scale for the production of F. velutipes, Pleurotus ostreatus and
Pleurotus eryngii.14,15 These applications show the advantages of
group (Fig. 2). The CCP group had the second highest yield of 20 g this raw material. The size of particles can be adjusted according
per bottle. The fruit body yields of IPS and SS groups were only to the requirement of good air permeability. Corn cobs can hold
53.8% of the control group, with both giving a yield of 14 g per water to support the growth of mushrooms. When it comes to
bottle. Based on the cost of substrates, CS and CCP were found to nutrition, corn cobs contain 100–200 g kg−1 starch and 20 g
be viable substrates for fruit body cultivation of C. militaris. kg−1 protein, which could supplement the mycelial growth of C.
militaris.14,16 CCP exhibited greater suitability to culturing of C.
Bioactive compound contents in fruit bodies of C. militaris militaris based on the above observations. This is probably due to
Four main bioactive components, cordycepin, adenosine, the relatively higher content of starch in corn cobs.16 Among the
D-mannitol and polysaccharides, in fruit bodies were analyzed four kinds of substrates tested in this study, the similarity between
and compared among the different agricultural waste substrates. corn cob particles and cereal grains led to good production of fruit
Fruit bodies produced with CCP had the highest cordycepin con- bodies of C. militaris.
tent of 9.45 mg g−1 , which was higher than the control group (8.92 The active components in fruit bodies of C. militaris were affected
mg g−1 ). A cordycepin content of 8.6 mg g−1 was assayed in fruit by various factors, especially the grain substrate. The highest
bodies produced on CS substrate. Low cordycepin contents of content of adenosine in fruit bodies of C. militaris on millet
2.70 and 2.60 mg g−1 were detected in fruit bodies produced on substrate was detected on day 40 of cultivation.17 Light and
IPS and SS respectively (Fig. 3A). other factors also affected the contents of adenosine, cordycepin,
Fruit bodies produced on CCP substrate accumulated the high- D-mannitol and exopolysaccharide.12 Pink light proved to be opti-
est level of adenosine (5.86 mg g−1 ), which was 128% of the control mal for cordycepin accumulation.18 There are many reports on
group (4.57 mg g−1 ). The adenosine content in fruit bodies reduced improving cordycepin production by various factors. Ferrous sul-
to 3.98 mg g−1 when using CS as cultivation substrate. Only 1.22 fate addition could improve cordycepin production.19 The salts
and 1.49 mg g−1 adenosine contents were assayed in fruit bodies of deep ocean water could also promote the production of
harvested from IPS and SS substrates respectively (Fig. 3B). cordycepin.20 When cordycepin biosynthesis was investigated,
The content of D-mannitol detected in fruit bodies cultivated on it was discovered that glucose-methanol-choline oxidoreductase
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CS was 120 mg g−1 , 20% lower than the control (150 mg g−1 ). Fruit and telomerase reverse transcriptase were involved in cordycepin

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Cultivation of C. militaris with agricultural wastes www.soci.org

Figure 3. Comparison of contents of (A) cordycepin, (B) adenosine, (C) D-mannitol and (D) polysaccharides in fruit bodies of Cordyceps militaris cultivated
on different substrates. Dried fruit body samples were used to analyze the contents of different bioactive compounds. Error bars represent standard
deviations from four independent experiments.

formation.21 In the present study, the cordycepin and adenosine with low contents of active components. In conclusion, using CCP
contents, up to 9.45 and 5.86 mg g−1 respectively, in fruit bod- or CS as substrate in the cultivation of C. militaris could decrease
ies obtained from CCP were higher than those reported by Lim the production cost and help protect the environment.
et al.17 Despite the proof that cordycepin biosynthesis is due to
the involvement of glucose-methanol-choline oxidoreductase and
telomerase reverse transcriptase, the regulation was not clear.21 ACKNOWLEDGEMENTS
The reasons for this discrepancy are not clear. The feasibility of
This research was supported by the Foundation of ‘12th
improvement needs further investigation. The findings obtained
Five-Year’ National Science and Technology Program (Grant
in this research support the possible use of corn cobs in culture of
No. 2013BAD16B07) and the New Project of Agriculture of Jiangsu
C. militaris, decreasing the production cost to a greater extent.
Province (Grant No. SXGC[2014]265).

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