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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