Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body
Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body
Yun-Fei Ko 0 1
Jian-Ching Liau 0 1
Chien-Sheng Lee 0 1
Chen-Yaw Chiu 1
Jan Martel 1
Chuan-Sheng Lin 1
Shun-Fu Tseng 1
David M. Ojcius 1
Chia-Chen Lu 1
Hsin- Chih L 1
John D. Young 0 1
0 0 Department of Respiratory Therapy, Fu Jen Catholic University , New Taipei City, Taiwan , Republic of China, 11 Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital , New Taipei City, Taiwan , Republic of China, 12 Research Center for Industry of Human Ecology, College of Human Ecology, Chang Gung University of Science and Technology , Taoyuan , Taiwan , Republic of China, 13 Graduate Institute of Health Industry and Technology, College of Human Ecology, Chang Gung University of Science and Technology , Taoyuan , Taiwan , Republic of China, 14 Laboratory of Cellular Physiology and Immunology, The Rockefeller University , New York, New York , United States of America
1 Editor: Wei-Chun Chin, University of California , Merced , UNITED STATES
The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and antitumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis' anamorph (also called Hirsutella sinensis) based on multi-
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by Primordia
Institute of New Sciences and Medicine, grant
MOST103-2320-B-182-027-MY3 from the Ministry
of Science and Technology of Taiwan, and grants
CMRPD190303, BMRPA04, and QZRPD120 from
Chang Gung Memorial Hospital. The funders had
no role in study design, data collection and
locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7,
βtubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its
optimal growth at low temperature (16ÊC) and its biochemical composition, are similar to that
of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be
used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature.
analysis, decision to publish, or preparation of the
Ophiocordyceps sinensisÐpreviously called Cordyceps sinensisÐis an Ascomycetes fungus that
grows at high altitude (3,500±5,000 m) and low temperature (~16ÊC) on the cold highlands of
the Himalayas and the Qinghai-Tibetan plateau [
]. This fungus is known for its unusual
parasitic life cycle: in late autumn, spores infect larvae of Hepialidae ghost moths in the soil,
producing a mycelium that gradually consumes the insect's internal organs; in summer, the
mycelium forms a fruiting body that protrudes from the head of the dead insect and grows
above the ground, facilitating spore dispersal and reproduction. O. sinensis is thus known in
English as the ªcaterpillar fungusº while in Chinese it is called ªwinter-worm, summer-grass,º
a name which reflects the unique life cycle of this organism.
O. sinensis has long been known in Asia as a popular folk remedy used to treat various
ailments, including cancer, fatigue, impotence, liver disease, renal dysfunction, respiratory
disease, and type 2 diabetes [
]. Recent studies have shown that O. sinensis produces a wide
range of biological effects on cultured cells and laboratory animals, including anti-aging,
antibacterial, anti-cancer, anti-diabetic, anti-fatigue, anti-inflammatory, anti-viral,
immuno-modulatory, and lipid-lowering properties [
]. In humans, O. sinensis improves renal, hepatic
and respiratory functions, delays fatigue, and reduces type 2 diabetes symptoms [
fungus has also been used as an aphrodisiac, earning it the nickname ªHimalayan Viagraº [
sinensis attracted international attention in 1993 when Chinese women athletes participating
at the National Games in Beijing broke several world records at a single distance running
event, performances which were later attributed (at least in part) to consumption of a tonic
containing the caterpillar fungus [
]. For these reasons, the fungus has emerged as a major
health supplement and tonic in recent years.
The high demand for O. sinensis fruiting bodiesÐespecially in China but also throughout
AsiaÐand the low annual production have led to overharvesting, a sharp production decline,
as well as hefty price increases on the market (e.g., top-grade fruiting bodies were sold at
$60,000/kg in 2007) [
]. O. sinensis has thus been listed as an endangered species in China
]. Unfortunately, artificial cultivation of O. sinensis fruiting bodies on a large scale has
continually failed , possibly due to the long life cycle of ghost moth insects and the absence of
environmental cues needed to induce the mushroom's fruiting process [
]. Moreover, O.
sinensis fruiting bodies harvested in nature have been shown to contain relatively high levels of
lead, arsenic, and copper [
], leading to cases of heavy metal poisoning [
]. Some O. sinensis
fruiting bodies sold on the market are also adulterated with metal in order to increase product
weight and sales profits [
]. For these reasons, much effort has been devoted to finding an
alternative that is free of contaminants and that is amenable to large-scale culture under
controlled laboratory conditions.
Several fungal species have been isolated from O. sinensis fruiting bodies collected in nature
]. This phenomenon has led to the production and sale of several incorrect strains on
the market [
]. For instance, the CS-4 strain sold in China has been shown to consist of
Paecilomyces hepiali mycelium, while other fungi such as Cordyceps militaris fruiting bodies have
been used as culture alternatives due to ease of production [
]. Confusion about the material
described as O. sinensis is also rampant in the scientific literature, with a recent study
estimating that more than three quarter of studies published on this fungus used unreliable, uncertain,
or unspecified material . Several fungal species have been proposed to represent the
anamorph mycelium of O. sinensis [
], with some authors advocating that Hirsutella sinensis
represents the sole anamorph [17±19], a claim that has been challenged by others [
no other fungus has created the level of attention and controversy seen here as far as culture,
identification or characterization is concerned.
2 / 21
In the present study, we report the isolation of a mycelium from fresh O. sinensis fruiting
body obtained in Tibet. Identification of the mycelium species was based on multi-locus
sequence typing (MLST) of several fungal genes (ITS, nrLSU, nrSSU, RPB1, RPB2, MCM7,
βtubulin, EF-1α, and ATP6) [
], a strategy which to our knowledge has not been used
previously to validate the isolation of O. sinensis' anamorph. We show that the isolated mycelium
strain closely matches the characteristics of wild O. sinensis fruiting bodies in terms of DNA
sequences, culture conditions, and biochemical composition. The isolated mycelium thus
represents a useful alternative for the production of health supplements containing the caterpillar
Fresh O. sinensis fruiting bodies were purchased from a local vendor in the Naqu prefecture of
Tibet in August 1999, a time when the organism had not yet been listed as an endangered
]. Therefore, no specific permissions were required in this case. For the isolation of O.
sinensis (H. sinensis) strain CGB 999335, a fresh O. sinensis fruiting body (stroma section; 0.6±
0.8 g) was briefly washed with sterile water, prior to immersion in 1% sodium hypochlorite
(NaClO) for 1 min. Following subsequent wash with sterile water, the fruiting body was cut
into small pieces (2±5 mm long) with a sterile scalpel and the pieces were placed in a bottle
containing 5 ml of sterile water. The solution was homogenized with a blender prior to
dilution 10 to 100× in sterile water. A small aliquot (100 μl) of the diluted solution was inoculated
onto potato dextrose agar (PDA; 4 g/l potato extract, 20 g/l dextrose; 20 g/l agar) and cultured
aseptically at various temperatures (10±35ÊC) for several days. Cultures were observed
periodically and filamentous fungal colonies were selected and re-inoculated at least five times onto
PDA plates to remove possible contaminants. A colony was selected and cultured in potato
dextrose broth (PDB; same composition as PDA but without agar) at 18ÊC with gentle mixing.
Stock culture was maintained at ±80ÊC in 10% glycerol (v/v).
Culture of H. sinensis mycelium
Mycelium colonies were cultured in FM1 liquid culture medium (20 g/l dextrose, 12 g/l yeast
extract, 0.5 g/l K2HPO4, 0.25 g/l MgSO4·7 H2O, 0.05g/l FeSO4·7 H2O) or 1±10% (w/v)
soybean broth at 18ÊC with gentle mixing for several days. Culture was performed at various
temperatures (12±22ÊC). In some experiments, pH was adjusted (pH 4.2±8.0) with 1 M HCl or
NaOH prior to culture. Mycelium cells were harvested by centrifugation at 3,400×g for 10 min
using an Allegra 25R centrifuge (Beckman Coulter, Brea, CA). Mycelium cells were washed
twice with double distilled water, prior to drying in an oven at 105ÊC. Dried mycelium powder
was weighed to determine the amount of biomass following culture.
Mycelium cells from fresh liquid culture were visualized with an Olympus IX70 inverted
optical microscope (Tokyo, Japan) equipped with a dark-field condenser; a Nikon Eclipse 80i
upright optical microscope (Tokyo, Japan); or a Nikon SMZ1500 stereoscopic zoom
microscope. Photography was taken with Nikon D100 and E995 digital cameras.
PCR and DNA sequencing
Total genomic DNA was extracted from mycelium using glass beads as described before [
PCR amplification and sequencing of ITS1-5.8S-ITS2 rDNA, nrSSU, nrLSU, RPB1, RPB2 and
3 / 21
MCM7 amplicons were conducted as previously described [
]. Amplification of EF-1α,
βtubulin and mtATP6 was performed based on established protocols [
]. The primers used are
listed in S1 Table. PCR products were sequenced by Genomics BioSci & Tech (Taipei, Taiwan).
Sequence alignment and phylogenetic analysis
BLASTN was used to identify sequences of highest homology (NCBI, Bethesda, MD).
Sequences including Ophiocordycipitaceae, Bionectriaceae, Hypocreaceae, and Nectriaceae
sensu lato and related species were obtained for 5-gene-based MLST (S2 Table; nrSSU, nrLSU,
RPB1, RPB2, and EF-1α) and single-gene-based phylogenetic tree analysis (S3 Table). Raw
sequences were aligned and gaps were excluded using ClustalW. The Molecular Evolutionary
Genetics Analysis software (MEGA, version 6.06) was used to perform phylogenetic analysis.
For 5-gene-based MLST analysis, evolutionary history was inferred using the maximum
composite likelihood (MCL) method based on the Tamura-Nei model and phylogenetic tree of the
heuristic search was obtained using the neighbor-joining and BioNJ algorithms to obtain a
matrix of pairwise distances estimated using the MCL approach inferred from 500 bootstrap
replicates [23±25]. For single gene-based phylogenetic analysis, the evolutionary history was
inferred using the neighbor-joining method [
] combined with the MCL-based evolutionary
distance estimation [
]. The percentage of replicate trees in which the associated taxa
clustered together is shown next to the branches of the bootstrap consensus tree as before [
Novel DNA sequences of O. sinensis (H. sinensis) CGB 999335 were deposited in the NCBI
database (KU058601, KU239984±KU239991).
Energy and chemical analysis
Determination of the content of organic compounds and elements in dried HSM CGB999335
mycelium and O. sinensis fruiting bodies was performed by SGS Taiwan (New Taipei City,
Taiwan) using standard procedures.
High-performance liquid chromatography analysis
The high-performance liquid chromatography (HPLC) system (Waters, Milford, MA)
consisted of a series 600 controller, a series 717 plus autosampler, and a series 996
photodiodearray detector, connected to a cartridge column (GL Sciences, Tokyo, Japan; average particle
size of 5 μm) and a Cosmosil packed 5C18-MS-II column (Nacalai, San Diego, CA; internal
diameter of 4.6×250 mm; average particle size of 5 μm). The mobile phase consisted of buffer
A (2.5% methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.3) and buffer B (20%
methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.1). Elution started with 100%
buffer A and consisted of the following linear gradient steps: 0±10 min, 0±25% buffer B; 10±20
min, 25±40% buffer B; 20±60 min, 40±100% buffer B. A flow rate of 0.9 ml/min and an
injection volume of 20 μl was used. Temperature of the column was maintained at 25ÊC. Detection
was done at a wavelength of 260 nm. Deionised water used for preparation of the HPLC
mobile phase and sample dilution was prepared with the Milli-Q purification system
(Millipore, Bedford, MA). Nitrogenous bases, nucleosides, HPLC-grade methanol, and ammonium
dihydrogen phosphate were obtained from Sigma-Aldrich (St. Louis, MO).
Experiments were performed in triplicate. Results are expressed as means ± standard errors
(SE). Statistical significance was evaluated using Student's t-test and a significance threshold
4 / 21
Characteristics of the mycelium isolated from O. sinensis fruiting body
Given the repeated isolation of fungal contaminants from O. sinensis fruiting bodies [
the controversy surrounding the identification of O. sinensis and its anamorph [
aimed to culture a mycelium from fresh fruiting bodies of O. sinensis (obtained in Naqu
Prefecture, Tibet). The O. sinensis specimen consisted of the characteristic caterpillar shell from
which a fruiting body of O. sinensis had protruded (an example of a dried specimen is shown
in Fig 1A). After gentle wash, the stroma of the fruiting body was cut into small pieces and
incubated onto potato dextrose agar (PDA) at low temperature (10±20ÊC), in order to mimic
the low temperature at which O. sinensis grows in the wild. After serial passages of single
colonies, we isolated a mycelium that produced diffuse, white colonies with dense aerial mycelium
and regular margins on PDA medium (Fig 1B, 28 days of culture). Under optical microscopy,
we observed that mycelium cells were hyaline, branched and smooth-walled (Fig 1C).
We cultivated the mycelium in FM1 liquid medium, which is entirely soluble and free of
debris (Fig 1D, left tube). Optical microscopy images of HSM mycelium cultured in FM1
showed abundant interlaced, branched, and hyaline mycelium cells with thin cell walls and
intercellular septa (Fig 1E). In contrast, when the mycelium was cultured in soybean broth (Fig
1D, tube on the right)Ða medium commonly used to culture Cordyceps-related mycelium [28±
30]Ðundissolved material and culture debris were observed among mycelium cells (Fig 1F).
Fig 1. Culture of H. sinensis mycelium derived from O. sinensis fruiting body. (A) O. sinensis fruiting body or stroma (top) protruding from the shell of a
caterpillar insect (bottom) was obtained in the Naqu prefecture in Tibet. HSM strain CGB 999335 was isolated from a similar fruiting body. (B) Colony of HSM
strain CGB 999335 cultured for 28 days at 18ÊC on PDA agar. (C) CGB 999335 mycelium observed under optical microscopy. (D) Sterile FM1 liquid medium
used to culture CGB 999335 mycelium in the present study (left tube; containing 1.2% of yeast extract as a source of nitrogen) and sterile liquid 1.2% (w/v)
soybean broth commonly used in other laboratories (tube on the right). Notice the pellet of undissolved powder in the tube on the right. See the Methods
section for more details. (E) Dark-field optical microscopy image of CGB 999335 mycelium cultured in FM1 medium. (F) CGB 999335 mycelium cultured in
soybean broth seen in D (tube on the right). Undissolved, brown material can be seen among mycelial cells.
5 / 21
Identification of H. sinensis using multi-locus sequence typing
To identify the mycelium species, we amplified internal transcribed spacer regions 1 and 2
(ITS1 and ITS2) and 5.8S rRNA by PCR and sequenced the obtained amplicons, a technique
used in the past to identify fungal species [
]. BLASTN search revealed that the best
sequence match was O. sinensis strain HMAS:173825 (S1 Fig; 100% identity). The
neighborjoining statistical method was used to build a phylogenetic tree and to compare the 62
ITS5.8S-rRNA sequences with the highest level of homology (Fig 2). While recent nomenclature
guidelines encourage the use of ªone fungus, one nameº , we refer to the isolated mycelium
as O. sinensis (H. sinensis mycelium) HSM strain CGB999335, or in short HSM CGB999335, in
order to provide additional information about the strain. We found that HSM CGB999335
clustered with other O. sinensis strains (Fig 2, EFCC 7287 and CO18), suggesting a common
We further confirmed the identity of HSM CGB999335 by amplifying and sequencing eight
additional housekeeping genes used for the identification of fungal species [
genes sequenced included the small and large 18S nuclear ribosomal RNA subunits (nrSSU
and nrLSU), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2),
mini-chromosome maintenance complex component 7 (MCM7), β-tubulin, translation
elongation factor 1-alpha (TEF1-α), and mitochondria ATPase synthase subunit 6 (ATP6). Results
were analyzed using BLASTN and the neighbor-joining statistical method (see Methods).
Based on the genes analyzed, HSM CGB999335 showed a high level of homology to other
O. sinensis isolates deposited in the database (S1±S17 Figs). Notably, HSM CGB999335 showed
high homology to O. sinensis isolate CO18 (S7±S17 Figs), a legitimate caterpillar fungus strain
isolated on the Qinghai-Tibetan plateau and whose genome has been partially sequenced
(GenBank: KE659607.1) [
]. As shown in S1±S4, S7 and S8 Figs, HSM CGB999335 showed
100% identity to several O. sinensis isolates for several genes, including ITS-5.8S-rRNA (strain
HMAS:173825), nrSSU (strain SJL0809), nrLSU (strain SJL0809), RPB1 (strain EFCC 7287),
MCM7 (strain CO18), and β-tubulin (strain CO18). High identity scores were also obtained
for RPB2 (S5 Fig, 99.7% identity) and TEF1-α (S6 Fig, 99.8% identity) of O. sinensis isolate
YN07-8, as well as for ATP6 of O. sinensis isolate CO18 (S9 Fig, 99% identity). Phylogenetic
trees showing the evolutionary relationships between HSM CGB999335 and related species for
nrSSU, nrLSU, RPB1, RPB2, TEF-1α, MCM7, β-tubulin and ATP6 are shown in S10±S17 Figs.
A phylogenetic tree based on a five-gene dataset (nrSSU, nrLSU, RPB1, RPB2, EF-1a) and
the 141 most homologous species showed that HSM CGB999335 has the highest homology to
O. sinensis strain CO18 (Fig 3). Based on these results, we conclude that the isolated HSM
CGB999335 mycelium is most related to O. sinensis fruiting bodies harvested in nature. These
results are in agreement with previous studies showing that the anamorph of O. sinensis
corresponds to H. sinensis [
Optimal culture conditions of O. sinensis mycelium
To analyze the growth characteristics of the HSM CGB999335 strain, we established a liquid
culture using the liquid FM1 medium. This allowed us to precisely monitor the growth
characteristics of the mycelium in addition to producing a sufficient amount of biomass for
subsequent analysis. We cultured HSM CGB999335 at different temperatures from 12 to 24ÊC in
order to determine the temperature that produces optimal growth. HSM CGB999335
produced optimal growth at 16ÊC (Fig 4), consistent with previous observations that O. sinensis
mycelium grows best at 15 and 18ÊC [
]. HSM biomass gradually decreased at temperatures
above 16ÊC and limited growth was noticed at higher temperatures, consistent with previous
work showing that O. sinensis does not grow at temperatures above 25ÊC [
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Fig 2. 5.8S-ITS rDNA phylogenetic tree of Ophiocordyceps species. The evolutionary relationship of
Ophiocordyceps 5.8S-ITS rDNA genes was determined using the neighbor-joining method [
]. Evolutionary distances
were assessed using the maximum composite likelihood (MCL) method. The bootstrap consensus tree, which represents
the evolutionary relationship of the analyzed taxa, was inferred from 500 replicates as before [
corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate
trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches.
Evolutionary distances (i.e., number of base substitutions per site) were computed using the maximum composite
likelihood method [
To determine whether pH has any effect on mycelium growth, we cultured HSM in FM1
liquid culture media at various pH, ranging between 4.2 to 8.0. After five days of culture, we
observed that the culture medium with a pH of 6.2 produced the highest amount of mycelium
biomass (Fig 5). These results are in agreement with previous observations that O.
sinensisderived mycelium grows best at pH 6 [
We also analyzed the amount of biomass produced after several days in culture. Using a
temperature of 16ÊC and pH 6.2, we observed that a culture time of 8 days produced the
highest amount of mycelium biomass (Fig 6). No further increase or decline of biomass was noted
after this period (Fig 6). Taken together, these results suggest that the HSM CGB999335 strain
possesses growth characteristics similar to that of O. sinensis mycelium characterized in
previous studies and the fruiting body harvested in the wild. Culture conditions may thus be critical
for the isolation of O. sinensis mycelium.
In order to characterize the HSM CGB999335 strain isolated here, we compared its
composition with that of O. sinensis fruiting bodies. The HSM strain showed higher levels of energy,
proteins, lipids, and superoxide dismutase compared with O. sinensis fruiting bodies (Table 1).
On the other hand, HSM showed lower levels of carbohydrates and water than the fruiting
bodies, while the amount of polysaccharides was similar in both samples (Table 1). HSM
showed low levels of saturated fatty acids and sugars, while these molecules were not detected
in O. sinensis fruiting bodies (Table 1). These observations suggest that the composition of
HSM CGB999335 is comparable to that of O. sinensis fruiting bodies.
Nucsleosides have been described as major active compounds responsible for the biological
effects of O. sinensis [
]. We therefore compared the content of nitrogenous bases and
nucleosides in HSM CGB999335 and O. sinensis fruiting bodies by using high-performance liquid
chromatography (HPLC). Pure nucleosides and nitrogenous bases were processed in parallel
as positive controls. Chromatograms of O. sinensis fruiting bodies revealed the presence of
uracil, guanine, uridine, guanosine, and adenosine (Fig 7B). Notably, the chromatogram of HSM
CGB999335 showed highly similar peaks (Fig 7C vs. 7B), with minor variations in intensity.
For comparison, we also processed O. sinensis fruiting bodies in complex with the moth insect
(which is usually used to prepare TCM remedies); similar nucleosides and nitrogenous bases
were found in this case as well, although peak intensities were relatively lower than for the
fruiting bodies or HSM (Fig 7A vs 7B and 7C).
By measuring the area under the curves for each HPLC peak (Fig 7), we obtained a
quantitative analysis of each nucleoside and nitrogenous base (Table 2). Except for uridine, HSM
CGB999335 showed a higher content of nucleosides and nitrogenous bases compared with O.
sinensis fruiting bodies (Table 2). By contrast, uracil was significantly higher in HSM
CGB999335, up to 4.51 fold, in contrast with the O. sinensis fruiting bodies (Table 2). The level
of adenosine in HSM CGB999335 was higher compared with the O. sinensis fruiting bodies
(Table 2). Of note, cordycepin was not detected in any of the samples studied here. Based on
8 / 21
Fig 3. Multi-locus sequence typing-based phylogenetic analysis of Ophiocordycipitaceae,
Bionectriaceae, Hypocreaceae and Nectriaceae for a five-gene dataset. The evolutionary relationship of
a five-gene dataset (nrSSU, nrLSU, RPB1, RPB2, EF-1a) was determined using the maximum likelihood
method based on the Tamura-Nei model [
]. Initial tree(s) for the heuristic search were obtained using the
neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum
composite likelihood (MCL) approach. Shown here is the bootstrap consensus tree inferred from 500
bootstrap replicates. The percentage of replicate tree that clustered with associated taxa is indicated.
these observations, we conclude that the nucleoside and nitrogenous base composition of
HSM CGB999335 and O. sinensis fruiting bodies is highly similar.
Mushrooms grown in nature tend to assimilate various elements from the soil, including
heavy metals [
]. We compared the elemental composition of HSM CGB999335 with that
of O. sinensis fruiting bodies. We observed that HSM CGB999335 contains higher levels of
potassium, zinc, calcium and sodium compared with O. sinensis fruiting bodies (Table 3).
However, HSM harbors lower levels of magnesium, iron, and manganese (Table 3). Of note,
HSM contains less than 10 ppm of heavy metals (e.g., lead, arsenic, mercury, cadmium, and
copper; data not shown) and chromium and selenium were not detected in our samples
Fig 4. Effect of temperature on the culture of H. sinensis mycelium. CGB 999335 mycelium was cultured in liquid FM1
medium with mixing for eight days at the temperature indicated. Mycelium cells were obtained by centrifugation, followed by
drying and measurement of biomass weight.
10 / 21
Fig 5. Culture of H. sinensis mycelium at various pH. CGB 999335 mycelium was cultured in liquid FM1 medium at 16ÊC
with mixing. Prior to culture, the pH of the culture medium was adjusted to the indicated value by adding 1 M HCl or NaOH.
After five days of culture, mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass
We also analyzed the content of amino acids and other related organic compounds in HSM
CGB999335 and O. sinensis fruiting bodies (Table 4). A total of 17 amino acids were detected
in both samples, including the essential amino acids histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, and valine (Table 4). HSM contained
higher levels of most of the compounds tested (n = 39), with the exceptions of seven
compounds which were found at higher levels in O. sinensis fruiting bodies (Table 4;
2-aminoisobutyric acid, arginine, ethanolamine, ornithine, phenylalanine, phosphoethanolamine, and
serine). Taken together, these observations indicate that the compositions of HSM
CGB999335 and O. sinensis fruiting bodies are strikingly similar.
Identification of the mycelium anamorph of O. sinensis has been controversial, mainly due to
contamination of fruiting bodies by various fungal species [
] and the difficulty in cultivating
fruiting bodies in vitro [
]. A mycelium culture that can be used as an alternative for the
declining production of O. sinensis fruiting bodies is highly needed. We report here the
isolation of a mycelium from O. sinensis fruiting bodies harvested on the Qinghai-Tibetan plateau.
Using a comprehensive PCR-based MLST analysis, we confirmed that the mycelium isolate
11 / 21
Fig 6. Culture of H. sinensis mycelium with time. CGB 999335 mycelium was cultured in liquid FM1 medium with mixing at
16ÊC for the time indicated. Mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass
corresponds to the anamorph of O. sinensis as shown by the high level of DNA sequence
homology with O. sinensis sequences deposited in the NCBI database, including a legitimate O.
sinensis strain isolated on the Qinghai-Tibetan plateau (i.e., CO18 [
]). To our knowledge,
O. sinensis Fruiting Body (per 100 g)
ND (<0.3 g)
< 12 g
1.5 × 105 U
12 / 21
Fig 7. HPLC chromatograms of H. sinensis mycelium and natural Ophiocordyceps specimens. HPLC chromatogram of (A) O. sinensis fruiting body
and insect; (B) O. sinensis fruiting body; and (C) CGB 999335 mycelium. HPLC was performed on a reverse-phase column as described in Methods. Peaks
were identified based on the use of pure standard compounds processed under the same conditions. Nucleosides and nitrogenous bases were monitored
using a UV detector. The results shown are representative of experiments performed in triplicate. Peak intensities are given in Table 2.
CGB 999335 Mycelium
O. sinensis Fruiting Body
O. sinensis + Insect
Nucleoside peaks correspond to those shown in Fig 7.
13 / 21
this work is the first independent isolation and characterization of the anamorph of O. sinensis
outside of China that is based on in-depth DNA sequencing analysis. Notably, the HSM
CGB999335 isolate shows growth characteristics and a biochemical composition similar to
that of O. sinensis fruiting bodies found in the wild, further supporting our conclusion that this
mycelium corresponds to the anamorph stage of the caterpillar fungus.
Previous studies performed on the HSM CGB999335 strain characterized here have shown
that this strain produces several beneficial effects on cultured cells and laboratory animals. For
instance, we observed earlier that an ethanol extract of the same HSM strain suppressed
interleukin-1β secretion and inflammasome activation in human macrophages [
]. The ethanol
extract also reduced bleomycin-induced lung injury, inflammation and fibrosis in mice [
indicating that the extract may be used to treat conditions associated with chronic
inflammation. Recently, we observed that a water extract of the HSM strain enhanced the cytotoxic
activity of natural killer cells against cancer cells, whereas the ethanol extract of the strain
reduced cytotoxicity [
]. These observations led us to propose that water and ethanol extracts
of medicinal mushrooms may produce opposite effects on immune cells. In another study,
Shang et al. showed that the HSM strain characterized here reduces the growth of
hepatocellular carcinoma in nude mice [
]. Furthermore, Wu et al. showed that the same HSM strain
reduces fatigue in mice as shown by increased time to exhaustion in swimming experiments
]. Similar immunomodulatory, anti-cancer and anti-fatigue effects have been reported for
O. sinensis fruiting bodies [
], further supporting the view that the HSM CGB999335 strain
isolated here may be used as a substitute for the rare and expensive fruiting bodies found in
We observed that HSM CGB999335 grows best at low temperature (16ÊC), which is similar
to previous observations on the optimal growth of O. sinensis mycelium [
]. Previous studies
claimed that O. sinensis-derived mycelium can be cultured at various temperatures ranging
from 18 to 30ÊC (see the studies described in ref. [
]). For instance, mycelium characterized
as P. hepiali [
] and Tolypocladium sp. [
] produced abundant growth at 25ÊC. On the other
hand, we observed that growth temperatures of 15±20ÊC represent a limiting range of
temperatures for the isolation of the mycelium anamorph of O. sinensis, a finding which is consistent
with previous studies [
]. These results suggest that the conditions of isolation, especially the
low temperature and the use of fresh fruiting bodies, may be crucial for the isolation of O.
Culture of O. sinensis-derived mycelium for commercial purpose has usually been
performed using liquid cell culture media containing corn extract, milk powder, silkworm pupa,
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soybean extract, wheat bran, or yeast extract [28±30,43±45]. When prepared at 1±10% (w/v) in
water, these culture media may harbor a pellet of insoluble matter, even prior to culture of the
fungus (see for instance the tube on the right in Fig 1D). Following culture of the mycelium for
several days, the pellet of undissolved culture medium initially present in solution is only
partially consumed by mycelium cells, a process that leaves corn, milk, silkworm, soybean, wheat
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or yeast residues in the final mycelium culture (Fig 1F). This strategy is likely to decrease not
only the purity of the final mycelium extract but also the biological effects it produces on
animals and humans. In addition, individuals who consume mycelium products cultured this way
may develop allergies to culture medium residues, a phenomenon reported earlier for
mycelium cultured in silkworm-containing media [
]. In contrast, we used a fully-soluble liquid
culture medium (Fig 1D, left tube), a strategy that may favor maximal product yield, purity
and efficacy, in addition to reducing the likelihood of allergic reactions.
Cordycepin has been proposed to represent a major active compound of O. sinensis. We
did not detect this compound in any of the samples submitted to HPLC analysis (Fig 7), in
spite of appropriate controls processed under the same conditions. This observation suggests
that a reevaluation of the role of cordycepin in O. sinensis fruiting bodies and mycelium is
needed. Species such as C. militaris contain cordycepin while O. sinensis fruiting bodies and
cultured mycelium contained minor traces or insignificant amounts of the compound [
suggesting the possibility that mislabeled O. sinensis samples may have been used in past
studies in which cordycepin was detected at relatively high concentrations. Some authors suggested
that nucleosides could be used to evaluate the quality of Cordyceps specimens [
Accordingly, adenosine may be used as a marker to evaluate the quality of the mycelium isolated and
final O. sinensis products available on the market.
Several O. sinensis-related products have been commercialized on the market [
]. On the
other hand, it appears unlikely that these products, which in some cases correspond to species
different from O. sinensis or H. sinensis, all produce the same effects on laboratory animals and
humans. The use of different species may be due to the fact that identification of O. sinensis is
often based solely on morphological criteria or isolation of mycelium from O. sinensis fruiting
bodies, in the absence of DNA-based analysis. For instance, recent studies have reported the
isolation of several mycelium strains from O. sinensis fruiting bodies harvested in the wild
], but identification of the fruiting bodies and mycelium species was based on
morphological observations alone and no DNA analysis was provided. Given that DNA analysis of
multiple barcode genes provides a reliable method for identifying fungal species, we believe
that the platform established in the present study may be used, in combination with
morphological observations, to identify and study O. sinensis strains as well as other fungi.
Using microbiological techniques and DNA phylogenetic analysis, we have isolated and
cultured the mycelium anamorph of O. sinensis fruiting bodies found in the wild. The growth
conditions and chemical composition of this mycelium strain are similar to the O. sinensis
fruiting bodies. Moreover, in vitro culture produces a mycelium that is free of contaminants
(of fungal or microbial origin), pesticides, or heavy metals, and that is
unadulteratedÐcharacteristics that are highly advantageous compared with some fruiting bodies available on the
market. Investigations are currently under way to verify the full extent of the functional effects
of the mycelium strain in laboratory animals. Further studies are also needed to verify the
effects of this mycelium preparation for the prevention and treatment of human diseases.
S1 Fig. Alignment of ITS-5.8S-rRNA sequences for HSM CGB999335 and O. sinensis
voucher HMAS:173825. Search was performed using BLASTN. O. sinensis voucher
HMAS:173825 18S ribosomal RNA gene, partial sequence; internal transcribed spacer
ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal
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RNA gene, partial sequence. Sequence ID: EU570952.1.
S2 Fig. Alignment of nrSSU sequences for HSM CGB999335 and O. sinensis strain
SJL0809. O. sinensis strain SJL0809 18S ribosomal RNA gene, partial sequence. Sequence ID:
S3 Fig. Alignment of nrLSU sequences for HSM CGB999335 and O. sinensis strain
SJL0809. O. sinensis strain SJL0809 28S ribosomal RNA gene, partial sequence. Sequence ID:
S4 Fig. Alignment of RPB1 sequences for HSM CGB999335 and O. sinensis strain EFCC
7287. O. sinensis strain EFCC 7287 DNA-dependent RNA polymerase II largest subunit
(RPB1) gene, partial coding DNA sequence. Sequence ID: EF468874.1.
S5 Fig. Alignment of RPB2 sequences for HSM CGB999335 and O. sinensis strain YN07-8.
O. sinensis isolate YN07-8 DNA-dependent RNA polymerase II second largest subunit (RPB2)
gene, partial. Sequence ID: JX968012.1.
S6 Fig. Alignment of TEF-1α sequences for HSM CGB999335 and O. sinensis strain
YN078. O. sinensis isolate YN07-8 TEF-1α gene, partial coding DNA sequence. Sequence ID:
S7 Fig. Alignment of MCM7 sequences for HSM CGB999335 and O. sinensis strain CO18.
O. sinensis CO18 contig_1827, whole genome shotgun sequence. Sequence ID:
S8 Fig. Alignment of β-tubulin sequences for HSM CGB999335 and O. sinensis strain
CO18. O. sinensis CO18 contig_1023, whole genome shotgun sequence. Sequence ID:
S9 Fig. Alignment of ATP6 sequences for HSM CGB999335 and O. sinensis strain CO18.
O. sinensis CO18 contig_6466, whole genome shotgun sequence. Sequence ID:
S10 Fig. Phylogenetic tree of nrSSU for Ophiocordyceps sinensis (Hirsutella sinensis)
CGB999335 and related species. Evolutionary history was inferred using the
neighbor-joining method and phylogenetic trees were built using the MEGA software. Bootstrap consensus
tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa
analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates
are collapsed. The percentages of replicate trees in which the associated taxa clustered together
in the bootstrap test (500 replicates) are shown next to the branches. Evolutionary distances
were computed using the maximum composite likelihood method and are expressed as units
of number of base substitutions per site.
17 / 21
S11 Fig. Phylogenetic tree of nrLSU sequences for HSM CGB999335 and related species.
The tree was built as in S10 Fig.
S12 Fig. Phylogenetic tree of RPB1 sequences for HSM CGB999335 and related species.
Analysis was performed as in S10 Fig.
S13 Fig. Phylogenetic tree of RPB2 sequences for HSM CGB999335 and related species.
Analysis was performed as in S10 Fig.
S14 Fig. Phylogenetic tree of TEF-1α sequences for HSM CGB999335 and related species.
Analysis was done as in S10 Fig.
S15 Fig. Phylogenetic tree of MCM7 sequences for HSM CGB999335 and related species.
Analysis was performed as in S10 Fig.
S16 Fig. Phylogenetic tree of β-tubulin sequences for HSM CGB999335 and related species.
Analysis was performed as in S10 Fig.
S17 Fig. Phylogenetic tree of ATP6 sequences for HSM CGB999335 and related species.
See S10 Fig for more information.
S1 Table. PCR primers used in this study
S2 Table. Specimen information and GenBank sequences used for the 5-gene phylogenetic
S3 Table. Specimen information and GenBank sequences used for the single-gene-based
We thank the personnel of Chang Gung Biotechnology and the members of the Center for
Molecular and Clinical Immunology at Chang Gung University for their assistance throughout
Conceptualization: YFK JDY.
Data curation: YFK JCL C.S. Lee CYC JM C.S. Lin SFT CCL HCL.
Formal analysis: YFK JCL C.S. Lee CYC JM C.S. Lin SFT DMO CCL HCL JDY.
Funding acquisition: YFK HCL JDY.
Investigation: JCL C.S. Lee CYC C.S. Lin SFT.
18 / 21
Methodology: YFK HCL JDY.
Resources: YFK HCL JDY.
Supervision: YFK DMO CCL HCL JDY.
Writing ± original draft: JM DMO JDY.
Writing ± review & editing: JM DMO JDY.
19 / 21
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1. Stone R ( 2008 ) Last stand for the body snatcher of the Himalayas? Science 322 : 1182 . doi: 10 .1126/ science.322.5905.1182 PMID: 19023056
2. Zhou X , Gong Z , Su Y , Lin J , Tang K ( 2009 ) Cordyceps fungi: natural products, pharmacological functions and developmental products . J Pharm Pharmacol 61 : 279 ± 291 . doi: 10 .1211/jpp/61.03.0002 PMID: 19222900
3. Zhu JS , Halpern GM , Jones K ( 1998 ) The scientific rediscovery of an ancient Chinese herbal medicine : Cordyceps sinensis: Part I. J Altern Complement Med 4 : 289 ± 303 . doi: 10 .1089/acm. 1998 . 4 . 3 -289 PMID: 9764768
4. Zhu JS , Halpern GM , Jones K ( 1998 ) The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis: Part II . J Altern Complement Med 4 : 429 ± 457 . doi: 10 .1089/acm. 1998 . 4 . 429 PMID: 9884180
5. Ng TB , Wang HX ( 2005 ) Pharmacological actions of Cordyceps, a prized folk medicine . J Pharm Pharmacol 57 : 1509 ± 1519 . doi: 10 .1211/jpp.57.12.0001 PMID: 16354395
6. Yue K , Ye M , Zhou Z , Sun W , Lin X ( 2013 ) The genus Cordyceps: a chemical and pharmacological review . J Pharm Pharmacol 65 : 474 ± 493 . doi: 10 .1111/j.2042- 7158 . 2012 . 01601 . x PMID : 23488776
7. Steinkraus DC , Whitfield JB ( 1994 ) Chinese caterpillar fungus and world record runners . Am Entomologist 40 : 235 ± 239 .
8. Zhang Y , Li E , Wang C , Li Y , Liu X ( 2012 ) Ophiocordyceps sinensis, the flagship fungus of China: terminology, life strategy and ecology . Mycology 3 : 2 ± 10 .
9. Dong C , Guo S , Wang W , Liu X ( 2015 ) Cordyceps industry in China . Mycology 6 : 121 ± 129 .
10. Hu X , Zhang YJ , Xiao GH , Zheng P , Xia YL , Zhang XY , et al. ( 2013 ) Genome survey uncovers the secrets of sex and lifestyle in caterpillar fungus . Chin Sci Bull 58 : 2846 ± 2854 .
11. Zuo HL , Chen SJ , Zhang DL , Zhao J , Yang FQ , Xia ZN ( 2013 ) Quality evaluation of natural Cordyceps sinensis from different collecting places in China by the contents of nucleosides and heavy metals . Anal Methods 5 : 5450 ± 5456 .
12. Wu TN , Yang KC , Wang CM , Lai JS , Ko KN , Chang PY , et al. ( 1996 ) Lead poisoning caused by contaminated Cordyceps, a Chinese herbal medicine: two case reports . Sci Total Environ 182 : 193 ± 195 . PMID: 8854946
13. Zhang Y , Zhang S , Wang M , Bai F , Liu X ( 2010 ) High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis . PLOS ONE 5: e15570. doi: 10.1371/journal.pone.0015570 PMID: 21179540
14. Xia F , Chen X , Guo MY , Bai XH , Liu Y , Shen GR , et al. ( 2016 ) High-throughput sequencing-based analysis of endogenetic fungal communities inhabiting the Chinese Cordyceps reveals unexpectedly high fungal diversity . Sci Rep 6 : 33437 . doi: 10 .1038/srep33437 PMID: 27625176
15. Dong CH , Yao YJ ( 2011 ) On the reliability of fungal materials used in studies on Ophiocordyceps sinensis . J Ind Microbiol Biotechnol 38 : 1027 ± 1035 . doi: 10 .1007/s10295-010 -0877-4 PMID: 20922458
16. Chioza A , Ohga S ( 2014 ) A review on fungal isolates reported as anamorphs of Ophiocordyceps sinensis . J Mycol 2014 : 913917 .
17. Chen YQ , Wang N , Qu LH , Li TH , Zhang WM ( 2001 ) Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5 .8S rDNA. Biochem Syst Ecol 29 : 597 ± 607 . PMID: 11336809
18. Liu ZY , Liang ZQ , Liu AY , Yao YJ , Hyde KD , Yu ZN ( 2002 ) Molecular evidence for teleomorph±anamorph connections in Cordyceps based on ITS-5.8S rDNA sequences . Mycol Res 106 : 1100 ± 1108 .
19. Chen YQ , Hu B , Xu F , Zhang W , Zhou H , Qu LH ( 2004 ) Genetic variation of Cordyceps sinensis, a fruitbody-producing entomopathogenic species from different geographical regions in China . FEMS Microbiol Lett 230 : 153 ± 158 . PMID: 14734179
20. Ni L , Yao YS , Gao L , Wu ZM , Tan MZ , Zhu JS ( 2014 ) Density-weighted algorithms for similarity computation and cluster tree construction in the RAPD analysis of natural Cordyceps sinensis . Am J Biomed Sci 6 : 82 ± 104 .
21. Schoch CL , Seifert KA , Huhndorf S , Robert V , Spouge JL , Levesque CA , et al. ( 2012 ) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi . Proc Natl Acad Sci U S A 109 : 6241 ± 6246 . doi: 10 .1073/pnas.1117018109 PMID: 22454494
22. Sung GH , Hywel-Jones NL , Sung JM , Luangsa-Ard JJ , Shrestha B , Spatafora JW ( 2007 ) Phylogenetic classification of Cordyceps and the clavicipitaceous fungi . Stud Mycol 57 : 5± 59 . doi: 10 .3114/sim. 2007 . 57 .01 PMID: 18490993
23. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S ( 2013 ) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0 . Mol Biol Evol 30 : 2725 ± 2729 . doi: 10 .1093/molbev/mst197 PMID: 24132122
24. Tamura K , Nei M , Kumar S ( 2004 ) Prospects for inferring very large phylogenies by using the neighborjoining method . Proc Natl Acad Sci U S A 101 : 11030 ± 11035 . doi: 10 .1073/pnas.0404206101 PMID: 15258291
25. Tamura K , Nei M ( 1993 ) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees . Mol Biol Evol 10 : 512 ± 526 . PMID: 8336541
26. Saitou N , Nei M ( 1987 ) The neighbor-joining method: a new method for reconstructing phylogenetic trees . Mol Biol Evol 4 : 406 ± 425 . PMID: 3447015
27. Felsenstein J ( 1985 ) Confidence limits on phylogenies: an approach using the bootstrap . Evolution 39 : 783 ± 791 .
28. Bao T , Yue D ( 1997 ) Method for preparing fermentation product of Cordyceps, and use thereof . Chinese Patent CN1215754A.
29. Dong CH , Yao YJ ( 2005 ) Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture . J Appl Microbiol 99 : 483± 492 . doi: 10 .1111/j.1365- 2672 . 2005 . 02640 . x PMID : 16108789
30. Li G ( 2010 ) Normal temperature fermentation Hirsutella hepialid Chen et Shen, mutation breeding method and fermentation process thereof . Chinese Patent CN101372672A.
31. Hawksworth DL ( 2011 ) A new dawn for the naming of fungi: impacts of decisions made in Melbourne in July 2011 on the future publication and regulation of fungal names . IMA Fungus 2 : 155 ± 162 . doi: 10 . 5598/imafungus. 2011 . 02 .02.06 PMID: 22679600
32. Sneath PHA , Sokal RR ( 1973 ) Numerical Taxonomy: the Principles and Practice of Numerical Classification . San Francisco: W. H. Freeman and Company.
33. Schmitt I , Crespo A , Divakar PK , Fankhauser JD , Herman-Sackett E , Kalb K , et al. ( 2009 ) New primers for promising single-copy genes in fungal phylogenetics and systematics . Persoonia 23 : 35 ± 40 . doi: 10 .3767/003158509X470602 PMID: 20198159
34. Kalac P ( 2013 ) A review of chemical composition and nutritional value of wild-growing and cultivated mushrooms . J Sci Food Agric 93 : 209 ± 218 . doi: 10 .1002/jsfa.5960 PMID: 23172575
35. Wang XM , Zhang J , Wu LH , Zhao YL , Li T , Li JQ , et al. ( 2014 ) A mini-review of chemical composition and nutritional value of edible wild-grown mushroom from China . Food Chem 151 : 279 ± 285 . doi: 10 . 1016/j.foodchem. 2013 . 11 .062 PMID: 24423533
36. Huang TT , Chong KY , Ojcius DM , Wu YH , Ko YF , Wu CY , et al. ( 2013 ) Hirsutella sinensis mycelium suppresses interleukin-1β and interleukin-18 secretion by inhibiting both canonical and non-canonical inflammasomes . Sci Rep 3 : 1374 . doi: 10 .1038/srep01374 PMID: 23459183
37. Huang TT , Lai HC , Ko YF , Ojcius DM , Lan YW , Martel J , et al. ( 2015 ) Hirsutella sinensis mycelium attenuates bleomycin-induced pulmonary inflammation and fibrosis in vivo . Sci Rep 5 : 15282 . doi: 10 . 1038/srep15282 PMID: 26497260
38. Lu CC , Hsu YJ , Chang CJ , Lin CS , Martel J , Ojcius DM , et al. ( 2016 ) Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity . Innate Immun 22 : 522 ± 533 . doi: 10 .1177/1753425916661402 PMID: 27469258
39. Shang HS , Chou J , Wu MF , Shih YL , Yeh MY , Chung HY , et al. ( 2014 ) Evaluation of Hirsutella sinensis mycelium on food safety and anti-hepatoma activity in an animal model . In Vivo 28 : 811 ± 817 . PMID: 25189893
40. Wu LY , Wu MF , Lu HF , Liu CH , Lee CH , Chen YL , et al. ( 2015 ) Evaluation of Hirsutella sinensis mycelium for antifatigue effect . In Vivo 29 : 263 ± 267 . PMID: 25792655
41. Chioza A , Ohga S ( 2013 ) Mycelial growth of Paecilomyces hepiali in various agar media and yield of fruit bodies in rice based media . Adv Microbiol 3 .
42. Leung PH , Zhang QX , Wu JY ( 2006 ) Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis . J Appl Microbiol 101 : 275± 283 . doi: 10 .1111/j.1365- 2672 . 2006 . 02930 . x PMID : 16882134
43. Dai L , Lan J ( 1994 ) Producing process of Chinese caterpillar fungus hypha fermentation . Chinese Patent CN1095103A.
44. Yu Y ( 1997 ) Deep fermentation technology for Cordyceps sinensis Sacc . Chinese Patent CN1036531C.
45. Jia J , Wu C , Yang J ( 2008 ) Method for producing Chinese caterpillar fungus by fermentation . Chinese Patent CN101407767A.
46. Choi GS , Shin YS , Kim JE , Ye YM , Park HS ( 2010 ) Five cases of food allergy to vegetable worm (Cordyceps sinensis) showing cross-reactivity with silkworm pupae . Allergy 65 : 1196 ± 1204 . doi: 10 .1111/j. 1398- 9995 . 2009 . 02300 . x PMID : 20557303
47. Li SP , Yang FQ , Tsim KW ( 2006 ) Quality control of Cordyceps sinensis, a valued traditional Chinese medicine . J Pharm Biomed Anal 41 : 1571 ± 1584 . doi: 10 .1016/j.jpba. 2006 . 01 .046 PMID: 16504449
48. Yu L , Zhao J , Li SP , Fan H , Hong M , Wang YT , et al. ( 2006 ) Quality evaluation of Cordyceps through simultaneous determination of eleven nucleosides and bases by RP-HPLC . J Sep Sci 29 : 953 ± 958 . PMID: 16833227
49. Barseghyan GS , Holliday JC , Price TC , Madison LM , Wasser SP ( 2011 ) Growth and cultural-morphological characteristics of vegetative mycelia of medicinal caterpillar fungus Ophiocordyceps sinensis G. H. Sung et al. ( Ascomycetes) isolates from Tibetan plateau ( P.R.China ) . Int J Med Mushrooms 13 : 565 ± 581 . PMID: 22181845