Optimization and Effect of Different Grain Sources on
Production of Liquid Spawn and Fruiting Body of Cordyceps militaris
Uzma Sitara1*, Parwaiz Ahmed Baloch1,
Atta Ullah Khan Pathan1 and Muhammad Ismael Bhatti2
1Food Quality & Safety
Research Institute, Southern Zone Agricultural Research Centre, Karachi-75270,
Pakistan
2Botanical Science Division,
Pakistan Museum of National History Islamabad, Pakistan
*For correspondence: uzmasitara@yahoo.com
Received 15 March 2022;
Accepted 20 July 2022; Published 25 August 2022
Abstract
Cordyceps
militaris is an expensive and edible mushroom that is being used
for therapeutic and medicinal purposes for a long time. This study was carried
out to investigate the mycelial growth of C.
militaris in a liquid medium for spawn production and fruiting body
cultivation on three substrates under different growing conditions. The
maximum, thick and the highest diameter of mycelial growth of liquid spawn was
recorded in PD (potato dextrose) containing antibiotic streptomycin with
vitamins B6 and B12 at 25 ± 1°C (relative humidity
70–75%) compared to the other three medium. Artificial cultivation of solid culture medium of soaked and unsoaked
sorghum, wheat and rice for the growth of C.
militaris was also optimized in this experiment. Among all the
substrates, soaked brown rice substrate was found the best treatment with the
highest mycelial growth, pinhead emergence, early fruiting development had
maximum weight and length in glass container procedure wrapped with polythene
sheet at 23 ± 2°C (RH, 80–85%). This is the first report in Pakistan indicated
that the modified plastic sheet glass jars could be efficiently and
economically used as a culturing container for C. militaris cultivation
and a substitute for expensive commercial growth containers. © 2022 Friends Science
Publishers
Keywords: Cordyceps militaris; Glass container; Liquid spawn; Solid
substrates; Soaked and unsoaked grains
Introduction
Cordyceps
military belongs to Ascomycota. It is cultivated on a
massive scale for commercial production all over the world (Mehra et al.
2017). Its fruiting body contains various bioactive metabolites such as
ergosterol, adenosine, exopolysaccharide and mannitol (Tatani et al. 2016; Raethong et al. 2018). Cordycepin is the most
metabolically active of these compounds and it has recently attracted a lot of
attention as anticancer, antiasthmatic, bacteriostasis, antibacterial,
anti-fungal, antiasthmatic and antihyperuricemic (Shi et al. 2020; Das
et al. 2021; Li et al. 2021; Schwenzer et al. 2021). The
percentage of cordycepin and bioactive components varies depending on the
species, the incubation and drying conditions and the chemical structure of the
fungal biomass (Rozsa et al. 2019). Cordyceps
species are well-known for their high concentrations of phytochemical
components, metabolites and biological properties (Zhong et al. 2020). It
is currently also marketed as a food and beverage supplement considering the
high option pharmacological advantages. For years, it has been widely utilized
as a traditional food stimulant and analeptic and its therapeutic
characteristics have drawn great attention (Yang et al. 2014). Various techniques for the synthesis of fungal biomass,
both on a solid substrate and in liquid surface cultures or submerged cultures,
have been developed and optimized in recent years (Rozsa et al. 2017; Kontogiannatos et al. 2021; Berovic et al. 2022). It is evident from
earlier reports that various types of additives are incorporated in the solid
media such as Sesbania leaves, rice,
brown rice, millet, rye, bean powder, cottonseed hulls, corn grains, corn cobs,
sorghum, wheat, sunflower and brewery grains (Bajwa et al. 1999; Chen et al. 2011; Shrestha et al. 2012;
Wen et al. 2014). According to researchers, various cultures exhibited
amazing impacts on the yield of mycelium and metabolic compounds of C.
militaris (Panda and Swain 2011; Yi et
al. 2014). Cereals have been commonly used in the large-scale production of
C. militaris. However, besides cereal grains, some other substrates for
the cultivation of C. militaris have also been studied. Lin et al.
(2017) investigated the ability of C.
militaris to develop on various agro-waste materials. Cottonseed shells,
corn cob particles, Italian poplar sawdust and substrates spent by Flammulina velutipes were employed to
cultivate C. militaris which exhibited greater suitability for
culturing. However, for cordycepin production, C. militaris has been commercially cultivated employing solid-state
and submerged fermentation due to Cordyceps
militaris has higher cordycepin
content compared to other species of the genus Cordyceps (Raethong et al.
2020). In another study, Amin et al. (2008) reported that the mycelium
of mushrooms cultivated in various agro-wastes could be infected, whereas that
mycelium produced in the culture medium might be free from contamination.
Mycelial development is stimulated by a growth medium, which provides quality
and year-round productivity. There has been little information available on the
effect of various parameters of temperature, humidity and incubation time on
the mycelial production of Cordyceps and
cordycepin using various solid substrates i.e. fermentation of rice, wheat and
oat. There are primarily two types of
modeling techniques for cultivating fruit bodies: those that use insects as
hosts and those that use cereal grains (mostly rice or wheat) as substrates.
The latter is more popular due to its ease of use and fruiting body products
formed with this model compete more effectively. China produces around 4000
tones of dry fruit bodies every year, utilizing at least 500,000 tones of grain
substrates (Lin et al. 2017). Artificial media have been
synthesized to extract bioactive compounds for mycelial biomass and fruiting
body production. According to Hong et al. (2010), the natural host
insects for artificial cultivations are costly, difficult to manage,
unavailable and susceptible to microbial pathogens and are threatened with
extinction in nature.
In Pakistan,
there is no any research work conducted on the production of this medicinal
mushroom therefore, this research aimed to study the effect of liquid spawn on
different soaked and unsoaked substrates of wheat, rice and sorghum and also
optimized the growth of mycelium and production of the fruiting body of C.
militaris under different growing conditions.
Materials and Methods
Collection of the fruiting body
Dry fruiting body of C.
militaris used in the present study was obtained from Pakistan Museum of
National History Islamabad, washed with 5% Sodium hypochlorite solution
three times and dried on sterilized blotter paper and incubated on PDA
supplemented with 0.5 g MgSO4 for 10 days at 25 ± 1°C in the
darkness for mycelial growth (Kang et al. 2012).
Preparation of liquid spawn from potato dextrose medium (PD)
Two hundred
grams of potato were placed into 1000 mL water, boil 25 min, cross leached with
muslin cloth afterward added glucose (30 g), peptone (5 g), yeast extract (3 g), KH2PO4
(1 g) and MgSO4.H2O (0.5 g). The material was then
autoclaved at 121°C for 15 min, cooled down subsequently from the PDA plate,
the regions of the mycelial tip of C. militaris were punched out
approximately 6 mm of PDA discs and transferred into PD. A total of four
treatments were set for PD for the growth of liquid spawn. The details of the
treatments were as:
PD 1 = PD + Streptomycin (5 mL L-1 in PD) +
vitamin B6 and B12 (0.5 mL L-1 in PD)
PD 2= PD + Streptomycin (5 mL L-1 in PD)
PD 3=PD + vitamin B6 and B12 (0.5
mL L-1 in PD)
PD 4= PD without any treatment (Control)
This mixture was cultured on a rotary shaker incubator
(Daihan Scientific, Korea) under conditions of 25 ± 1°C and darkness with
shaking at 150 rpm min-1 for 10 days and measured the diameter (cm)
of mycelium.
Determination of mycelial weight
Filtration of liquid cultures was done with pre-weighed
filter papers (What man, Germany) to separate mycelium from liquid culture of
PD, then the mycelium was weighed (g).
Preparation of soak and unsoaked grains for mycelial
growth
Grain sources consisting of brown
rice, sorghum and wheat (1000 g each) were used during the study. All grains
were rinsed three times in clean water to remove dust and other particles,
soaked in water for four hours to ensure maximum water absorption. Soaked
grains were washed again whereas unsoaked grains were washed without soaking.
Both soaked and unsoaked grains were filled @ 30 g into three different treated
spawn glass jars and supplemented with PD.
Preparation
of PD with egg mixture basal medium for substrate
Two blended eggs and glucose (30
g), peptone (5 g), yeast extract (3 g), KH2PO4 (1 g),
MgSO4.H2O (0.5 g), vitamin B6 and B12
(0.5 mg) were added in 1000 mL potato broth and mixed well with hand beater for ten minutes.
Container selection for optimization of fruiting body
cultivation
Three types of treatments were selected for cultivation
and optimization of the fruiting body in a glass container.
A = A glass container covered with filter paper.
B = A glass jar with a hole in the plastic lid (A hole
was punched in the plastic lid about 1.5 cm in diameter and sealed with
cotton).
C = A glass container covered with a polythene sheet.
Autoclaving of the supplemented glass jar
Soaked and unsoaked grains were filled into each glass
jar with 20 g grain and 30 mL PD basal medium and sterilized in autoclave at
121°C for 15 min. After cooling, each glass Table
1: Mycelial growth of C. militaris under different
treatments in liquid spawn
Treatment |
Diameter (cm) |
Days
Required for spawn completion |
Thickness
of mycelium (mm) |
Fresh
Biomass (g/200 mL) liquid culture |
|
PD 1 |
PD + Streptomycin+ vitamin B6
and B12 |
4.5±0.03 |
11 ± 0.00 |
5.2 ± 0.02 |
10.2 ± 0.04 |
PD 2 |
PD +Streptomycin |
4.1±0.05 |
15 ± 0.01 |
4.3 ± 0.03 |
8.3 ± 0.05 |
PD 3 |
PD+ vitamin B6 and B12 |
3.8±0.02 |
20 ± 0.01 |
4.1 ± 0.02 |
6.5 ± 0.03 |
PD 4 |
PD (Control) |
3.5±0.03 |
25 ± 0.03 |
2.5 ± 0.03 |
5.1 ± 0.02 |
Table 2: Effect of
different soaked and unsoaked substrates on the average growth and development
of C. militaris at 23 ± 2°C
Substrate |
Treatments |
Mycelial growth time (days) |
pinhead occurrence time (days) |
fruiting body mature time (days) |
length of fruiting (cm) |
|
Soaked |
Wheat |
plastic cap (T1) |
6 |
40 |
70 |
2 ± 0.03 |
filter paper (T2) |
7 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T3) |
5 |
35 |
65 |
3 ± 0.03 |
||
Sorghum |
plastic cap (T4) |
6 |
36 |
60 |
2.5 ± 0.05 |
|
filter paper (T5) |
6 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T6) |
6 |
32 |
58 |
3 ± 0.08 |
||
Brown rice |
plastic cap (T7) |
4 |
30 |
56 |
3.3 ± 0.08 |
|
filter paper (T8) |
5 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T9) |
4 |
27 |
52 |
3.8 ± 0.05 |
||
Un soaked |
Wheat |
plastic cap (T10) |
7 |
50 |
85 |
1.2 ± 0.03 |
filter paper (T11) |
9 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T12) |
6 |
46 |
82 |
1.5 ± 0.05 |
||
Sorghum |
plastic cap (T13) |
6 |
45 |
80 |
1.7 ± 0.08 |
|
filter paper (T14) |
7 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T15) |
6 |
42 |
77 |
1.8 ± 0.03 |
||
Brown rice |
plastic cap (T16) |
6 |
40 |
75 |
2 ± 0.03 |
|
filter paper (T17) |
6 |
0 |
0 |
0 ± 0.00 |
||
polythene sheet (T18) |
5 |
38 |
71 |
2.5 ± 0.05 |
jar was inoculated with 10 mL liquid spawn of C.
militaris, incubated at 25 ± 1°C under dark conditions and replicated 5
times. The diameter of the mycelium growth was measured after every 5 days for
25 days. After a period of 12 days were kept out of the dark and placed in
artificial light (400–500 lux) for about 12 h per day to initiate fruiting
under temperature between 23 ± 2°C for 60 days. After fruiting fresh fruiting
bodies were collected, weighted (mg) and measured (cm).
Statistical analysis
The data were statistically assessed using one-way
analysis of variance (ANOVA) on version 20.0 software (SPSS Inc., Chicago,
USA). All values were expressed as mean ± standard error (SE) as suggested by Wiengmoon et al. (2019).
Results
Effect of different treatments of
liquid spawn on mycelial growth of C. militaris
The maximum, thick and the highest diameter (4.5 ± 0.03
cm) of mycelial growth in liquid spawn was observed in PD containing antibiotic
streptomycin with vitamins (PD1) at 25 ±1°C (RH, 70–75) with the highest
biomass (10.2 ± 0.04 g) within 11 days whereas, the minimum (5.1 ± 0.02) was
noticed under PD4 treatment. The order followed as PD1 < PD2 < PD3 <
PD4 (Table 1). The fungal mycelium of C. militaris was white, dense and
more or less completely covered the surface of the culture broth (Fig. 1).
Effect of soaked and unsoaked
grain sources on mycelium growth of C. militaris
C. militaris showed varied mycelial growth on the three different
kinds of grains. In terms of mycelial growth, it took 4 to 9 days to cover the
whole soaked and unsoaked substrates of rice, wheat and sorghum. The result
showed that the earliest mycelial growth was recorded for soaked brown rice for
the treatment (T7) and T9. Initial pinhead development was noticed for T9 and
late for T1 on rice and wheat substrate respectively. In comparison with rice
media, pinheads took a longer time to mature for wheat and sorghum substrate.
However, the lengths of fruit bodies on rice media were recorded greater than
those on the other groups. The highest length of fruiting bodies was recorded
for T9 and the lowest under T10 whereas, no growth was observed for T2, T5, T8,
T11, T14 and T17 (Table 2). Among the three kinds of soaked and unsoaked substrates,
the highest yield of 16.3 g and 13.3 g from soaked rice substrate was recorded
in T9 and T7 respectively followed by the second-highest yield of 13 g (T6)
under sorghum whereas, the lowest yields (8 g) were recorded for wheat (T3). In
the case of unsoaked substrate T18, T13 and T12 yielded the highest fruiting
body for brown rice, sorghum and wheat respectively. The highest fresh weight
(16.3 g) of C. militaris was recorded
for soaked rice (T9) and the lowest (2.1 g) for unsoaked wheat grain (T10), (Fig. 2).
Fig. 1: Mycelial
growth of C. militaris affected by different PD treatments in liquid
spawn. (PD 1 = PD + Streptomycin + vitamin B6 and B12, PD 2= PD + Streptomycin,
PD 3=PD + vitamin B6 and B12 PD 4= Control)
Fig. 2: Effect of
different soaked and unsoaked substrates on the yield of C. militaris
(T1,T4,T7,T10,T13,T16(plastic
cap),T2,T5,T8,T11,T14,T17(Filter paper),T3,T6,T9,T12,T15,T18(polythene
sheet)
Effect of different glass jars on the fruit body
production of C. militaris
The result showed that amongst all treatments of glass
jar the fastest mycelial growth, early pin head development, fruiting body
maturity time, length and weight of fruiting bodies of C. militaris were recorded in glass jars sealed with polythene
sheet for soaked and unsoaked substrates of rice, sorghum and wheat followed by
glass jars capped with plastic lids. However, no growth of C. militaris was noticed in a glass jar covered with filter paper
(Fig. 3).
Discussion
The present study was conducted with the purpose to
assess the suitable, profitable and inexpensive growth conditions for the
cultivation of C. militaris. The
results suggested that mycelial development of C. militaris is
influenced by cultural conditions and available nutrients. Mycelial growth
diameter on PDA medium supplemented with MgSO4 was tested in the
current study, and it was discovered that it not only increased mycelial growth
diameter but also reduced the cultivation period. Our findings were consistent
with those of Maftoun et al. (2013), who found that Potato dextrose agar
medium (PDA) is the optimal medium for high fungal mycelial development and is
extensively used as a standard for spore inoculum formation of mushrooms. Our
results were in accordance with Shrestha et al. (2006)
who tested twenty-two different types of media and classified them as deficient
or rich in nutritional elements and found that PDA and C-DOX exhibited high
fungal intensity. Similarly, Wongsorn et al. (2021) observed that
nutritionally rich PDA medium resulted in abundant mycelium development of C. militaris. The present study
suggested that the mycelial growth of C. militaris was found best in PDA
medium at 25 ± 1°C. The findings of the present study were found to be
comparable with Adnan et al. (2017) who
studied that the mycelium progression of Cordyceps was greatest
in PDA media at 20ºC and 25ºC.
During the study, the maximum, thick and highest diameter
of mycelial biomass in liquid spawn was observed in PD containing antibiotic
streptomycin with vitamins B6 and B12 in minimum days at
25 ± 1°C (RH, 70–75). In another study, vitamins B1, B6,
B8, B9 and B12 were reported to give the
significantly highest mycelium colony diameter for the production of C. militaris (Wen et al. 2011; Ma et
al. 2015; Dang et al. 2018). Our
observations also matched those of Rozsa and Apahidean (2020), who reported
that the optimal incubation temperature for obtaining the highest mycelial
biomass was in the 24–28°C Table 3: F-ratio and significant levels derived from ANOVA for
all treatments
Factors |
SS |
d.f. |
MS |
F-value |
Significance |
Substrates |
105.000 52.500 157.500 |
12 41 53 |
8.750 1.280 |
6.833 |
***0.001 |
Treatments |
8829.000 4252.500 13081.500 |
12 41 53 |
735.750 103.720 |
7.094 |
***0.001 |
Biomass of C. militaris |
494.973 1015.120 1510.093 |
5 48 53 |
98.995 21.148 |
4.681 |
***0.001 |
F = F – ratio, P = P-value (***P ≤ 0.001, ** P ≤
0.01, P ≤ 0.05,
ns=non-significant)
Fig. 3: Fruiting body
production of Cordyceps militaris in different glass jars
(A= glass jar covered with filter paper, B= glass jar
with a hole in the plastic lid, C= covered with polythene sheet.)
temperature range. Sasaki et al.
(2005) also reported that the mycelial growth of C. sinensis was better
at 20ºC and 25ºC whereas no growth was recorded at 30ºC and 35ºC. In the
present research, liquid spawn was developed in a shaking flask, which is a
low-cost approach using basic equipment technology and is appropriate for the
mass production of fungal biomass. According to Lee
et al. (2013), mycelial
biomass powder can be utilized to make several types of supplementary capsules
and tablets.
In this study, various grains like
brown rice, wheat and sorghum were included to evaluate their effects on the
mycelium production of C. militaris. The results indicated that mycelium
extensions of wheat and sorghum were significantly slower than brown rice
treatments. Mycelium densities of brown rice on grain media were compact and
somewhat compact for both wheat and sorghum. Brown rice was found to be the
most favorable to the mycelium growth and production of the fruiting body of C.
militaris. The highest and thick mycelium was obtained from brown rice and
next to sorghum and wheat. This is probably due to the relatively higher
content of starch in brown rice and addition with eggs mixture improved
mycelial fruiting production. Furthermore, brown rice can retain water to
support the growth and supplement the mycelial growth of C. militaris. Many researchers cultivated C. militaris using different varieties of rice including black
jasmine rice, white rice, brown rice, sao hai rice, black glutinous rice, Thai
jasmine rice, and sangyod brown rice (Tianzhu et al. 2015; Sornprasert et
al. 2016). In another study, Adnan et al.
(2017) investigated the most efficient method for boosting cordycepin
production on a wide scale and discovered that rice medium had the greatest
cordycepin production (814.60 mg g-1) followed by oat (638.85
mg g-1) and wheat (565.20 mg g-1) medium respectively. The mycelium colony diameter of C. militaris in different grains was
significantly affected by substrate type while larger surface area and spore of
substrates were responsible for enhanced mycelium growth rate (Sofi et al. 2014). According to Qin and Han
(2013), the Cordyceps can
be grown in many types of solid culture medium, the cultivation rate and volume
of the hyphae were greatest in a solid culture medium of rice as the solid
substrate in contrast with other solid media. The artificial growth of C. militaris was effectively conducted
by Pathania and Sagar (2014) under
lab-scale cultivation trials on wheat and maize grains substrates. Wu et al. (2022) examine the impact of
seven-grain substrates on the development of C. militaris fruiting bodies using solid-state fermentation and
observed that brown rice and buckwheat had greater levels of adenosine and
pentostatin.
The present experiment was
performed by culturing the C. militaris on three different substrates
with three modifications of glass jars covered with filter papers, plastic
sheets and plastic lids. The results showed that amongst all treatments the
highest mycelial growth rate and fresh yield of C. militaris were recorded in glass jars concealed with a polythene
sheet for soaked and unsoaked substrates of rice, sorghum and wheat. This happened
due to the favorable effect of temperature, light and humidity on glass jars
covered with a transparent plastic sheet as compared to others i.e. plastic
lids and filter papers. This indicated that the modified plastic sheet jars
could be beneficially used as culturing jars for Cordyceps cultivation
and could be a substitute for the expensive commercial growth jars. Our results
were also in conformity with Wiengmoon et al. (2019) who confirmed
that the growth jar is the best suitable application to yield high
quality and quantity of fruiting body of C.
militaris than the culture room because it is cheap and easily manageable.
The results further showed that the highest mycelium biomass and fruiting body
production period was found best on 23 ± 2°C (RH 80–90%) in all tested
substrates. Several authors mentioned the more or less temperature of 24°C as
optimal for incubating and growing the mycelium of the C. militaris
mushroom (Mao et al. 2005; Patel and
Ingalhalli 2013), while, Guo et al.
(1998) and Masuda et al. (2007) stated
optimal temperature of 28°C. According to Mani et al. (2015), the mycelial biomass and fruiting production
were recorded optimum at 20°C, yielding 10.5 ± 0.14 and 1.75 ± 0.99 g L-1.
The temperature range for fruiting body cultivation of C. militaris was found between 18°C to 25°C. The optimal
temperature of 20°C and 25°C was also described by (Cheng et al. 2011). Yoo et al.
(2022) developed a technique for artificially cultivating C. militaris
using germinated soybeans rather than pupae as a protein source at 25°C.
The present study found that brown
rice was found best substrate for the mycelium growth and production of C.
militaris in soaked and unsoaked treatments in glass jars wrapped with a
polythene sheet. The analysis of variance demonstrated that the mycelial growth
of C. militaris in different treatments at 0.01 level showed a
significant difference and its effect on different soaked and unsoaked
substrates and fruiting body production were found highly significantly
different for all levels (P ≤ 0.001)
(Table 3). Based on the production and quality of fruiting bodies, brown rice
substrate remained suitable for the cultivation of C. militaris similar to those observed on rice substrate, the other
next suitable substitute substrate was sorghum, which supported the moderate
production of fruit bodies, however, wheat proved the lowermost in the
cultivation of C. militaris.
Conclusion
It
is concluded from the results obtained that the mycelial and fruiting growth
rates of C. militaris were significantly highest and fastest on the rice
substrate incubated in the polythene glass jars followed by jars covered with
plastic lids and filter papers. Further, using a polythene sheet could reduce
production expenses and help protect the environment. However, further research in Pakistan is necessary to optimize
cultivation factors such as light, incubation time, aeration and temperature.
Acknowledgments
The
author is grateful to the laboratory attendant of Food quality and safety
research institute Mr. Zafar Hamid Khan and Mr. Israr Ahmed Ansari (Scientific
Assistant) for their technical support during the study period.
Author Contributions
US
and PAB planned and performed the experiments, US and MIB interpreted and
reviewed the results, US and AK statistically analyzed the data and revised the
result
Conflicts of Interest
The
authors declare no conflict of interest.
Ethics Approval
Not
applicable in this paper
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