Abstract
During COVID, global wide peoples realized the importance of natural food sources and their pharmacological values and now it has been used widely. It retained the previous decades of healthy human livelihood and attention created on conventional foods and medicines. From human evolution, mushrooms played a most unique role in food and medicine. Among them, C. sinensis is a temperate loving entomopathogenic fungus at altitudes of 3000 to 14,000 MSL. It is used as energy-sourced food and medicinal uses, via. Antioxidant, antihyperlipidemic, antiviral, antiaging, antidepressant, antifatigue, hypocholesterolemic, hypotensive, vasorelaxation, aphrodisiac, chronic urinal regulators, neuroregulators, anticancer, glycemic regulators, antimicrobial, immune modulators, and functional modifiers (FMs) to better live from their production of bioactive compounds. So, this fungal source drastically reduced overexploitation, climate change, mutations, and reduction of host population rate (ghost moth). These situations are pushed into artificial cultivation by complete and seminatural systems with extended times. Thus, simplification of cultivation progress will yield a better outcome in C. sinensis productions and new generation medicines in future when accompanied with scientific approaches with knowledgeable skilled persons in mushroom technology for human livelihood.
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16.1 Introduction
Consistently, studies about “medicinal mushrooms” have always become a very interesting and important one because of their potential uses in pharmacology and its impact in global markets (Schmit and Mueller 2007). Since a 1000 years ago, fungi have been used as food and medicine (folk) by humans. In particular, 2000 mushroom species are identified as edible among 14,000 recognized mushroom species (Vikineswary et al. 2013). Due to their presence of highly nutritive values, composition of mineral states, zero fat derivatives and productions of health pack medicinal properties for improving human beings’ life from diseases (El-Ramady et al. 2022). Nowadays, fungal sources have been obtained from the two sources: natural (wild) or artificial culturing under in vitro (Chiu et al. 2016). Gathering of fungi from the wild resources causes serious difficulties due to identification, availability, nature, and environmental sustainability (Wei et al. 2021). Despite, researchers and pharma industrial companies mostly contributed in several mushroom species such as Agaricus, Antrodia, Albatrellus, Calvatia, Clitocybe, Cordyceps, Flammulina, Fomes, Fungia, Ganoderma, Inocybe, Inonotus, Lactarius, Phellinus, Pleurotus, Russula, Schizophyllum, Suillus, Trametes, Tremella, and Xerocomus (Patel and Goyal 2012). Above these mushroom species were given high attention due to their diversified production of novel anticancer, neuroregulatory, immunomodulators, anti-inflammatory, digestive, derma-care properties (pharmacological), health-improved properties (nutritional substances) derived ones (Hwang 2007). These properties are derived at very least quantities with highly input of cost and time oriented for production, standardization, and commercialization (Granato et al. 2010). Additionally, these mushroom (fungi)-yielded medicinal sources are highly valuable and biopotential with zero harm to humans than bacterial, algal, and plant species (Valverde et al. 2015). Globally, seven medicinal mushroom species play a vital role in pharmacological industries such as Hericium erinaceus (lion’s mane mushroom), Ganoderma lingzhi (reishi mushroom), Cordyceps spp. (caterpillar mushroom), Inonotus obliquus (chaga mushroom), Trametes versicolor (turkey tail mushroom), Lentinula edodes (shiitake mushroom), and Grifola frondosa (maitake mushroom) which were most contributed than other species (Litwin et al. 2020; Lovett and Leger 2017). Among them, Cordyceps spp. were attributed a crucial role in drug discoveries in pre- and post-clinical studies of human’s pharmacological activities like antioxidant (Shin et al. 2001), antihyperlipidemic (Wang et al. 2015a), antiviral (Yan et al. 2010), antiaging (Ji et al. 2009), antidepressant (Singh et al. 2014), antifatigue (Geng et al. 2017), hypocholesterolemic (Koh et al. 2003), hypotensive (Chiou et al. 2000), vasorelaxation (Wang et al. 2015b), aphrodisiac (Kashyap et al. 2016), chronic urinal regulators (Sun et al. 2019), neuroregulators (Wang et al. 2018), antioncology (Park et al. 2017), glycemic regulators (Yu et al. 2016), antimicrobial (Mamta et al. 2015), and immune modulators (Wang et al. 2011) (“FMs—functional modifiers”) to better health (Fig. 16.1). Above these functional activities were rectified by several bioactive compounds (or) structural compounds, viz. nucleosides (Shaoping et al. 2001), sterols (Zhou et al. 2009), polysaccharides (Elkhateeb et al. 2019), proteins, and related nucleic acids (Sethy et al. 2016), fatty and organic acids (Das et al. 2021), metals (Bhetwal et al. 2021), and vitamins (Holliday and Cleaver 2008). Under this circumstance, identification, characterization, mass production isolation of biometabolites, standardization, and commercialization of Cordyceps sinensis or their species was highly oriented with scientific approaches and skillfully one in pharmacological science and industries (Abdul-Rehman et al. 2022). So, given this chapter thoroughly covered about origin, economic importance, nature, distribution, habitat, morphology, and lifecycle, ethnopharmacy (eP), structural bioactive compounds (SBCs), pharmacological uses, genealogical approaches, mass production, extraction and purification progress views, toxicology, commercialization, issues, and future perspectives of Cordyceps sinensis were discussed below.
Multiclinical beneficial approaches of Cordyceps spp. for human health. (Courtesy: Liu et al. (2015))
16.1.1 Origin
From 145.0 million years ago (Cretaceous period), this fungal species lived in wild hosted with entomopathogenic nature (Gibson et al. 2014). On time, they secreted a wide spectrum of secondary biometabolites during their infection and proliferation in insects (Asaia et al. 2012; Chakraborty et al. 2014a). In 1843, scientifically named as Sphaeria sinensis by Miles Berkeley, later it has renamed as Cordyceps spp. in 1878 by Andrea Saccardo (Nikoh 2000). Until 2007, it was called Cordyceps sinensis; now, it is also renamed Ophiocordyceps sinensis and otherwise commonly called “caterpillar fungus”/“yarsha gamboo”/“keera jhar”/“keera ghas” (Arora et al. 2008). It belongs to one of the 700 species on Ascomycota/Pyrenomycetes/Hypocreales/Clavicipitaceae. The word Cordyceps originated from the Greek word “Kordyle” (club) and Latin word of “ceps” (head) (Olatunji et al. 2018). Finally, the word Cordyceps denotes the fruiting body of the fungus coming out to form a mummified body of caterpillar structured one (Wu et al. 2015).
16.1.2 Economic Importance
Naturally, Cordyceps spp. are highly ecosystem-oriented growing mushroom species compared to other medicinal mushrooms. Therefore, collection of large-scale levels of this mushroom is a daunting task (Hardeep et al. 2014). However, an age group of 15–65-year-old men and women is contributed to collect this mushroom for domestic-level income in countries like Nepal, China, Bhutan, Tibet Himalayan regions, and northeastern states of India. The wild-collected mushroom (Cordyceps sinensis) is sold at Indian market at Rs. 1,00,000/kg (Sharma 2004). Depending upon the demand, availability, and supply, the price level is also increased in the global market. Worldwide, Cordyceps-based pharmaceutical industries estimated global market value is 5–11 billion US$ (Shrestha 2012). In China, this is mushroom sold under the name “soft gold” at 35,000–60,000 $/kg. In Korea, Japan, and Thailand, several species of Cordyceps are used as functional foods with a price of $ 5.8/gram (Winkler 2008). Most of the North-Eastern states of Indian tribal (>3,00,000 families) livelihood and their income source depending by the collection of C. sinensis (Panda and Chandra Swain 2011).
16.1.3 Nature, Distribution, and Habitat
Cordyceps sinensis is a rare ubiquitous living entomopathogenic fungi, around 700 species of Cordyceps; 20 species were documented as parasitize to “Elaphomyces” genus (temperate, subarctic forest ecosystem living ones) (Baral et al. 2015). Interestingly, remaining some species such as C. sinensis, C. ophioglossoides, C. militaries, C. gracilis, C. sobolifera, C. subsessilis, C. gunnii, C. cicadae, C. tuberculate, C. scarabaeicola, C. minuta, C. myrmecophila, C. canadensis, C. nutans, C. agriota, C. ishikariensis, C. konnoana C. nigrella, C. pruinose, and C. tricentric were highly predominant pathogenic to insect orders of Arachnida, Coleoptera, Hemiptera, Hymenoptera, Isoptera, and Lepidoptera (Pal and Misra 2018). Among them, C. sinensis parasitizes the larval of “swift or ghost moths” (Hepialidae); this is specially found on the deep forests and meadows of Himalayan regions in India, Tibet, Nepal, and China at mean sea level of 3000–5000 m (Grehan and Ismavel 2017) and also is distributed in Bhutan, Japan, Korea, Thailand, Vietnam (Asia), Europe, and American countries (Fig. 16.2) (Elkhateeb and Daba 2022). In India, it is presented in highest altitude subalpine regions like Kumaun and Garhwal Himalaya (Pradhan et al. 2020). The constituents of biometabolites (quality and quantity) in Cordyceps spp. were changed due to the presence of geographical and climatic factors such as level of altitudes, low temperature, high precipitation, relative humidity, lack of O2, and exposure of sunlight (Arora and Singh 2009).
Global distribution of Cordyceps sinensis a diagrammatic view. Area, viz. (1) Asia; (2) Europe; (3) USA and Canada; (4) Australia
16.1.3.1 Morphology and Life Cycle
During early spring, C. sinensis mycelium originates from an insect larva (Hepialus armoricanus) and pervaded on the whole body itself and ends to club-like cap consisted of two parts: a fungal endosclerotium (within caterpillar body) and stroma (Lo et al. 2013). The upper part (stroma) is dark brown or black, when the yellow color is fresh and longer (4–10 cm) than caterpillars (Fig. 16.3). It grows from the larval head and is clavate, sublanceolate (or) fusiform, unique from the stipe (slender, glabrous, and longitudinally ridged) (Toledo et al. 2013). Naturally, C. sinensis life cycle comprises three phases, viz., infection, parasitism, and saprophytism (Guo et al. 2017). When the fungus grows on the immature larvae (host), usually it lies around 6 inches below the ground surface. As the fungus proceeds into maturity, they are heads up from underground to above soil. It consumes >90% of the infected insect larvae. The production of fungal sclerotia leads the host larvae from rigid and rapidly mummifying the hosts. Later, the sclerotial structure becomes dormant, after overwintering the fungus ruptures the host body, a sexual fruiting body arises (perithecial stroma) from the larval head that rises upward to emerge from the earth and is joined to the sclerotia (dead larva) underground to complete the cycle and finally reaches the weight of Cordyceps about 300–500 mg (Zeng et al. 2006).
Morphology of Cordyceps sinensis
16.1.4 Ethnopharmacy (eP)
From human evolution, impacts of several diseases and disorders were cured by natural sources such as plants, animals, and algal and fungal derivatives. So, intake of mushrooms as pharmacological use has followed from the genesis of before civilic (Stamets and Zwickey 2014). Cordyceps sinensis has been utilized in traditional Chinese medicine (TCM) as tonic, syrup, powder, paste, pellets, and capsules mixed with milk (or) liquor (or) hot water in morning or evening for curing diseases like bronchitis, cough, cold, diarrhea, fatigue, headache, rheumatism, and enhancer of sexual potency of male and females. Mostly, these recommendations were based on the utilization of the “trial and error” method by local folk practitioners (Choda 2017). Additionally, it is also used as a highly nutritive food source due to the presence of essential amino acids and vitamins (B1, B2, B12, and K) (Zhu et al. 1998), slow-releasing glycemic (carbohydrates), dietary fibers, and minerals (Yalin et al. 2006). Additionally, several species like C. militaris (anticancer) (Shrestha and Sung 2005), C. pruinose (stomach diseases) (Ng and Wang 2005), C. bassiana (derma care & biopesticide) (Sung et al. 2006), C. cicadae (infantile convulsion) (Choda 2017), C. guangdongensis (inflammatory disorders, avian flu, regulation of menstrual cycle & antiaging) (Wang et al. 2021). Till now, C. sinensis is sold in capsule form for curing aphrodisiac by a commercially available name “Himalayan Viagra” (or) “Himalayan Gold” (Baral 2017).
16.1.5 Structural Bioactive Compounds (SBCs)
Due to the availability, pharmacological value, and demand, C. sinensis is a highly variable nature and it requires unique set of climatic conditions for their growth, development, and maturity including collection also (Shrestha 2011). The structural bioactive compounds were mostly obtained from the well-developed mycelium through extraction by using different solvents. The obtaining biochemical compounds differed from natural and cultured ones due to the exposure of ecosystem (Au et al. 2011). Until now, these biochemical compounds have been documented as a diversifiable natured one including nucleosides and nucleobases, nucleotides, polysaccharides, sterols and fatty acids, proteins, amino acids and polypeptides, cordymin, cordycedipeptide, cordyceamides A and B, and tryptophan (Fig. 16.4) (Zhang et al. 1991). Above these compounds, quality and quantity check were determined the varietal superiority among the cultured species (Chen et al. 2011).
Structural bioactive compounds of Cordyceps sinensis
16.1.6 Pharmacological Uses
Naturally, the obtained bioactive compounds are reported as diversified with unique characteristics of pharmacological nature. So, presenting bioactive compounds are different within species due to growing nature (Mizuno 1995). The bioactive compounds (structural) viz., nucleosides and nucleobases, nucleotides; polysaccharides, sterols and fatty acids, proteins, amino acids and cyclic polypeptides (large quantities compared to other molecules) are mostly predominant better against human pathological sciences (Table 16.1) (Chen et al. 2013).
16.1.6.1 Nucleosides and Nucleobases
It is one of the most important base compounds of Cordyceps spp., nearly more than 11 different nucleosides are extracted from mycelium of C. sinensis such as adenine, cytosine, guanine, hypoxanthine, thymine and uracil (6-nucleobases), adenosine, cytidine, guanosine, inosine, and thymidine (Yang et al. 2010); among them, guanosine has highest contribution ratio (quantity) to others, and these molecules are always present in nature of reciprocal linkages and mostly contributed through (purinergic/pyrimidine receptors and glial cells) regulators of CNS (central nervous system) in human physiology (Liu et al. 2015). Nucleobases of purine and pyrimidine play as a remarkable marker for nutrition values of Cordyceps spp. (Yang et al. 2009).
16.1.6.1.1 Adenosines
Except abovementioned several nucleosides are found in specific deoxyuridine structures like adenosine, 2′-deoxyadenosine 2′ 3′-dideoxyadenosine, hydroxyethyl-adenosine, 3′-deoxyadenosine (cordycepin), pentostatin, N6-(2-hydroxy ethyl)-adenosine, tenellin, militarinones, fumosorinone, farinosones, oosporein, beauveriolides, beauvericin, cordycepin triphosphate, guanidine, and deoxyguanidine. During extraction, adenosine compounds most affected the time (extension) in natural C. sinensis (Shiao et al. 1994). Especially, adenosine act as an energy transfer and signal transductant molecules in intracells of cardiovascular tissues, anti-inflammatory properties, and antitumour (ROS-mediated mitochondrial membrane dysfunction) inducing and anticonvulsant activities through neuromodulators in human beings (Tescarollo et al. 2020). Specifically, these functions were mediated through activation of “GPCRs” (G-protein-coupled receptors), namely A1, A2A, A2B, and A3. By impulse of neurons and signal transduction to brain accompanied with nervous system and regulates psychological functions, viz. self-fear, immune modulation, anxiety, locomotion, and depression (Bockaert et al. 2010).
16.1.6.1.2 Cordycepin
Cordycepin is prominently found in the lowest amount in two species of Cordyceps such as C. sinensis and C. militaris and cannot be found in cultured ones (Tsai et al. 2010). Based on structure, cordycepin is 3′-deoxyadenosine and cordycepic acid (CA) is D-mannitol. The 3′-deoxyadenosine is derived from the absence of one O2 molecule at third position carbon of ribose sugar with molecular weight of 251.24 Da. (228–231 °C—melting point) commonly extracted by the way of using acetonitrile and H2O mixed ratio (5:95 v/v) at a flow rate of 1.0 mL/min (Hyun 2008). Under clinical diagnosis, cordycepin widely used as analgesic properties, immune booster, antitumour, antiglycemic, antiviral, antibacterial, anti-inflammatory, and neurostimulation stimulates steroidogenesis and pesticides (Phull et al. 2022).
16.1.6.1.3 Nucleotides
Prominently, three nucleotides are constituted commonly as adenosine-5′-monophosphate (AMP), guanosine-5′-monophosphate (GMP), and uridine 5′-monophosphate (UMP) with amphoteric molecules, base, and phosphoric acid (Lindequist et al. 2005). Reporting above these nucleotides were documented as enhancing the immune system of body, influence of fat metabolism, improve the gastrointestinal functions, inhibit urethral inflammation, regulates blood circulation and brain activeness (Lin and Li 2011).
16.1.6.2 Polysaccharides
Cordyceps spp. contains a diverse nature of polysaccharides; usually, fruiting bodies of Cordyceps consist of 3–8% of polysaccharides consisting of extracellular and intracellular at molecular weight of 16 kDa (Li et al. 2002). It is a compound nature of sugars (monosaccharides and polysaccharides) such as arabinose, fructose, glucose, galactose, mannose, mannitol, rhamnose, ribose, sorbose, and xylose. Monosaccharides mainly contributed to the organism’s growth and maturity; remaining polysaccharides (APS, CPS-1 and CPS-2) others (cordycepic acid) exhibited toward pharmacological activities for human health (Guan et al. 2010). The acid polysaccharide (APS) at ratio of (3.3:2.3:1)—(mannose:glucose:galactose) triggering protective from internal cell injuries (Zheng et al. 2022); a water-soluble polysaccharide (CPS-1) was a glucomano-galactan sugars of glucose:mannose:galactose at 2.8:2.9:1 ratio in C. sinensis. This CPS-1 especially stimulates insulin secretion and maintains the level (insulin metabolism) in the pancreatic gland and cures diabetes (Li et al. 2006). Finally, CPS-2 (combo residues of mannose, glucose, and galactose with ratio 4:11:1, mostly contributed to internal wounding of blood vessels from regulation of PDGF-BB (platelet-derived growth factor—dimeric glycoprotein) (Chen et al. 2010).
16.1.6.2.1 Cordycepic Acid
In early days, CA (cordycepic acid) possessed an isomer of quinic acid, but later it is confirmed as D-mannitol by the variation presented in forming dextrorotatory lactones and usually 7–29% in Cordyceps spp. (Chatterjee et al. 1957). Cordycepic acid plays a viable role in treating diuretic, liver fibrosis, plasma osmoregulation, and synthesis of free radicals for better blood circulation and free of breathiness (Guo and Friedman 2007).
16.1.6.3 Sterols and Fatty Acids
Normally, sterols at low quantity level in all edible mushrooms. In Cordyceps sinensis, sterols have been identified in the form of ergosterol, ergosterol-3, ergosterol peroxide 3-sitosterol, daucosterol, and campesterol (Bok et al. 1999). In fungus, ergosterol contains a different molecular weight as 1.4 mg/g (vegetative stage) and 10.6 mg/g (fruiting stage) (Li et al. 2004). Additionally, two types of fatty acids (saturated and unsaturated) were found in Cordyceps spp. Usually, different fatty acids like docosanoic acid, linoleic acid, palmitic acid, pentadecanoic acid, oleic acid, and stearic acid were documented in C. sinensis. These act as osmoregulation and lipid metabolism in blood (Jerzy Jedrejko et al. 2021).
16.1.6.4 Proteins
Generally, proteins rate varies in different stages like dead larvae (29.1%), fruiting body (30.4%), and mycelial decomposition (14.8%). Mostly, proteins like spermidine, putrescine, flazin, cadaverine, perloyrine, and l-tryptophan were presented in the form with various nature and additionally CSDNase (deoxyribonuclease) and CSP (serine protease) at intracellular and extracellular (Yang et al. 2011). DNase mainly contributed in growth of mycelium by endogenous nature. CSP is an extracellular protease enzyme of single polypeptide chain with molecular weight of 31 kDa. It maintains blood serum albumin level and therapeutic effects on cardiovascular functions (Ye et al. 2004).
16.1.6.5 Amino Acids and Cyclic Polypeptides
C. sinensis consists of numerous amino acids like glutamic acid and aspartic acid in larval stage and cyclic polypeptides such as cyclo-(Gly-Pro), cyclo-(Leu-Pro), cyclo-(Phe-Pro), cyclo-(Val-Pro), and cyclo-(Thr-Leu) which were presented in all stages (Mishra and Upadhyay 2011). Among them, cyclo-(Leu-Pro) and (Phe-Pro) were confirmed as antimicrobial and antimutagenic properties against vancomycin-resistant Enterococcus and pathogenic yeasts. Including these, cyclodipeptides inhibit the synthesis of aflatoxins (Rhee 2004). A cordymin is a putative beneficial peptide isolated from C. sinensis which is highly recommended for diabetic osteopenia and regulates blood serum glucose level (Vestergaard et al. 2009; Qian et al. 2012).
16.1.7 Genealogical Approaches
A fungus Hirsutella sinensis is an anamorph (asexual) stage of C. sinensis. Till now, molecular diagnosis is critical for this medicinal fungus. The fruiting body development is still unknown (Zhong et al. 2010). From internal transcribed spacer analysis, Cordyceps spp. yielded a fragment size of 539 bp (Zhang et al. 2010). Up to now, Cordyceps spp. characterized by genealogical approaches through functional protein genes, transcriptional factors such as Zn2Cys6—type (fruiting body initiation), mitogen-activated protein kinase gene (MAPK gene) (Zheng et al. 2011), FKS 1 gene (induction of β-1,3 glucan synthase enzyme) for immunomodulator synthesis (Ujita et al. 2006), Cu, Zn SOD 1 gene (SOD 1) for stimulating anti-inflammatory properties (C. militaris) (Park et al. 2005), Two cuticle degrading serine protease genes, csp 1 and csp 2 essential role play in development of infection in host and pathogenesis of C. sinensis (Zhang et al. 2008). These kinds of several genes like glucanase, proteinase, and cyclic peptide synthase genes are involved in characterization of Cordyceps spp. (Liu et al. 2017).
16.1.8 Mass Production of C. sinensis
16.1.8.1 Technological Approaches
For the last 50 years, the Cordyceps sinensis has played a major role in global food and pharmaceutical industries due to their importance of beneficial bioconstituents. So, the demand and distributional availability have increased rapidly. It gives benefit to wild mushroom collectors and their socioeconomic status (Singha et al. 2020). Thus, global wide C. sinensis gets a great attention for mass production by artificial conditions. In early periods, it met several issues that were attained to low production with sustainability nature. During 1982, a successful attempt of cultivation of Cordyceps is carried out in Chinese academy of medical sciences. After several explorations of mass multiplication studies, two prominent methods were identified. It is complete artificial and semi-natural cultivation; both are different in their progresses viz., complete artificial: reared larvae are inoculated with cultured strains and the infected larvae are fed indoors. Later 1–2 years, C. sinensis has been harvested. In this pattern, all processes are conducted under artificial conditions only. It was more efficient than seminatural by survival rate of larvae and shortening growth period (early maturity) of C. sinensis, but cost is so much. In a seminatural method, the infected larvae were released to the natural ecosystem, liberally allowing them to grow in free conditions. After 3–5 years, the fungus is harvested in those areas. It consumes natural resources but reduces the cost of input and takes much time. Both these methodologies are not familiar in C. sinensis mass production and not followed by a huge level of commercialization. Additionally, weather factorsinfluence the cultivation and yield from initial to harvest. C. sinensis cultivation consisted of three phases, viz. selection of host insects, preparation of culture, and inoculation of culture in to host and harvest (Qin et al. 2018).
16.1.8.2 Selection of Host Insects
In 1965, Hepialus armoricanus (ghost moth) was identified as the main host of the C. sinensis; till now, 50 species have been identified for host insects in Hepialus. The species have a wide distribution with 2500–5100 MSL with subalpine to alpine grasslands (Chu 1956). Natural vegetation and climatic factors decided the host population and occurrence, although nowadays the population diversity occurred due to deforestation and global warming (Outhwaite et al. 2022).
16.1.8.3 Artificial Rearing
Generally, sex ratio of (Hepialus) ghost moth female is always higher than male and mating is mostly at 6–9.30 pm. Female moths mated one time with their life span, but male attained 2–3 times. Normally, females lay 5–45 eggs each time and then die immediately. These eggs were collected, and they were used for further progress. Hepialus moth had stages of egg, larva, pupa, and adult. Eggs kept at a temperature of >10 °C with warm humid conditions (30–40 days for incubation), when hatching the egg shells turned from white to black in color. The larva (milk white, 2 mm length) mostly stayed in soil at a depth of 15 cm. During maturity, larvae are pale red in colour, beige body and length about 4–5 cm. Tender tuber roots or infantile buds are fed to larvae. In the month of June–July, the pupal stage is present. During rearing, abiotic factors such as temperature (15–20 °C/incubation; 10–18 °C/larval stage), humidity (36–45%/larval stage), and soil moisture maintained at 42–45%) are considered (Buenz et al. 2005).
16.1.8.4 Preparation of Culture
16.1.8.4.1 Isolation and Collection of Ascospores
The establishment of anamorph stage of C. sinensis (Hirsutella sinensis) plays a critical role in cultivation of C. sinensis. A viable strain from different climatic (temperate) zones was collected, and then, ascospores, stromata, and endosclerotia were collected which were used for culturing an anamorph stage of the fungus by using specific media, viz. 1% peptone potato dextrose agar, S31 agar, glycerol meal peptone agar, milk agar, and confirmative by Sabouraud agar (SAB). Ninety percent of fungus confirmed on their conidial stage. The ascospores (fruiting bodies) by aseptic needle into sheathing sterile bags were collected and were kept under 0–4 °C (Liu et al. 2002).
16.1.8.4.2 Isolation of Culture and Mass Multiplication
Mostly, mass production of C. sinensis mycelia is obtained by the method of submerged cultivation. The collected ascospores are inoculated into liquid media (glucose 1.25%, sucrose 1.25%, peptone 0.02%, 0.06 g of 5% yeast powder, 0.025% KH2PO4, 0.012 g of MgSO4.7H2O, and 0.002 g of 5% vitamin B1, and are kept in neutral pH with 24 °C for 8 days. Finally, it reached a biomass of 19.5 g/L (Liu et al. 2002). Compositional alteration of media by 20% potato, 0.08% beef extract, 0.2% peptone, 0.1% KH2PO4, 0.1% MgSO4.7H2O, 1.5% sucrose, and 2.5% glucose at 23 °C with 130 rpm/min on 4 days yielded better outcome in mycelial mass weight more than 2 times. After formation of fruiting bodies (ascospores, stroma and perithecium) by keeping under 8–17 °C for 7 months (Yue et al. 2013).
16.1.8.4.3 Inoculation of Culture to Host
The matured fruiting bodies, cultures are inoculated into cultured larvae of H. armoricanus under aseptic conditions in laboratory, after infected larvae keep under well-drained moist pits with favorite feeds under field conditions with monitoring for recorded mortality and survival rate. Later, it generates the full genesis of C. sinensis with fruiting bodies of stroma collected in respective 1–2 years later (Yuan et al. 2022).
16.1.8.4.4 Mechanisms of Infection
The fungal spores infected the larva in soil, and the following year, it died and turned to fruiting bodies. When made an infection by C. sinensis, it attacks the hemocelom of host larvae and makes small fragments of hyphae. After it proliferates as individual and entirely covers the hemocoel and later turned as cystid form of (exoskeleton body wall with hard) sclerotium and sprouts stroma from buccal cavity, all these established due to synthesis of lytic enzymes and mycotoxins (Shashidhar et al. 2013).
16.1.8.4.5 Extraction and Purification
Extraction, purification, and standardization of biometabolites or structural bioactive compounds (nucleosides and polysaccharides) from the fungus are final process of commercial cultivation of C. sinensis and their pharma industries. Commonly, there are several methodologies available for above this process used by polarity, molecular weight-based like water extracts, ethanol, petroleum ether, acetone, methanol, ethyl acetate, and pressurized liquid extractions (PLE) (Xin Chen et al. 2013). Furthermore, several methodologies are involved for the determination of nucleosides (high performance liquid chromatography (HPLC), capillary electrophoresis mass spectroscopy (CEMS), liquid chromatography/electrospray ionization (LC/ESI-MS), ion-pairing reverse phase liquid chromatography (IR-RP-LC-MS) and polysaccharides (PMP-pre column derivation (monosaccharides), proteins (2D PAGEs, GCMS, periodate-oxidation and Smith degradation and nuclear magnetic resonance spectroscopy (NMRS) are followed (Nie et al. 2011; Feng et al. 2017).
16.1.9 Toxicology
Naturally, 1000 species of fungi act as an “insect pathogen” from families like Cordycipitaceae, Ophiocodycipitaceae, and Clavicipitatceae. Above these families consisted species of mushrooms have highly potential nature of pharmacological uses to humans (Chen et al. 2020). On the other hand, frequently taking these mushrooms or their metabolites in the way of food and medicines provide a slow release of toxic substances and are harmful to human physiology. There are numerous bioactive compounds transformed as toxic to later stages when we uptake regularly. These are listed in “Table 16.2” with their impacts including (Hatton et al. 2018).
16.2 Production Cost Analysis
In each and every investment is mostly for the output/benefit/net return for every all industries in global wide thought and need. Mushroom production acts as secondary-based sector for farmers during crop failure or additional returns (Singh et al. 2001). It applicable in tropical and subtropical natured mushroom cultivation, but Cordyceps spp. are highly depended on the climatic favored ones, and it influenced the production enhancement (Rawat et al. 2020). Furthermore, several reports recorded the production cost analysis, and its outcome is beneficial to artificial cultivators of Cordyceps spp. (Table 16.3), (Singh et al. 2010).
16.2.1 Commercialization
Till now, the wild mushroom collectors of Cordyceps spp. have increased day by day due to the market value and demand (Chakraborty et al. 2014b). Additionally, several multinational pharma industries took this collection and cultivation progressed due to their pharmacological nature and also available in “online mart” in different names like raw fungus, dry powder, organic capsules, and liquid drinks from the manufacturer like “Himalayan Herbs” (Paterson 2008). Globally, it costs approximately 6.7 US$ and Indian market rate at Rs.100/− per piece and 1000/− for 10 grams (Gupta and Karkala 2017). This wild collection trade is a big setup of a network from local gatherers (uncountable)—broker or agent (local market)—regional broker/agent (district)—wholesaler (state/national—market)—international (multinational pharma industries) (Fig. 16.5) (Sharma et al. 2017). Globally, Cordyceps spp. products are high priced in nature, so mostly it has been used by celebrities, players, and physicians and not by local peoples (Anita 2019).
Systematic diagram of Cordyceps sinensis commercialization from local to global
16.2.2 Issues in Cordyceps Spp.
Cordyceps sinensis is a highly temperate lovable entomopathogenic Ascomycetes fungus. So, it attained several problems (or) issues that are categorized in multiple ways through (collection, cultivation, extraction and standardization, commercialization, and intake) (Raethong et al. 2020).
16.2.2.1 During Collection
From collection to Cordyceps sinensis met several issues like, fungus attained mutation, diversity, continuous exploitation of natural sources, global warming and climate change provides a great destruction of wild species gradual to rapidly. So, gatherers met unexpected disappointments during collection by identification and poor availability (Gupta and Karkala 2017).
16.2.2.2 During Cultivation
Under cultivation process, selection of viable (host–insect) and their availability in nature to keep that ecosystem (under artificial) and maintaining the population rate (survival: mortality), parasitic to saprophytic elongation period, are daunting tasks in practically (Gupta and Karkala 2017).
16.2.2.3 During Extraction and Standardization
From the early period to now, the fungus turned as mutant and expected bioactive compounds changed in their quality and quantity. Additionally, these processes were highly cost-effective, and they consumed time also (Gupta and Karkala 2017).
16.2.2.4 During Commercialization
During commercialized conditions, C. sinensis price is not a stable one, and it depends upon changing circumstances of availability, demand, and supply. So, it is always in the “pendulum” position (Gupta and Karkala 2017).
16.2.2.5 During Intake
Anything that is excess in human life is always poisonous, and it includes physiological and psychological also. So, nowadays, frequent intake of mushrooms as functional foods and medicines gave negative outcomes in human health of social and physical life (Gupta and Karkala 2017).
16.3 Future Perspectives
Nature gives a better source for human’s life when we protect it. During disturbance, it also gives an unwanted way of impact in all stages. So, C. sinensis plays a vital role in previous to current decades also. Thus, preserving the natural ecosystem to the safest way avoided the destruction of human essential mushrooms. Additionally, continuous research on C. sinensis in the future gives a better way in human life through multidimensional scientific approaches of new species identification, and simplification of cultivation methodologies will provide better outcomes in soon (Nguyen et al. 2020).
16.4 Conclusion
During the Stone Age to now, mushrooms are used as conventional food and medicines in rural villages to global streets. In the last two decades, new diseases have emerged and created great pandemics. In these situations, pharma industries highly focused their views on available wild natural sources and their importance in human life. During that time, C. sinensis was attempting a huge familiar view on multibenefits from temperate to tropical. Currently, the species evolved several mutations due to global climate change, continuous exploitations, commercial collectors from multinational pharma-industries with unofficial thefts has created a great impact in the next step of artificial cultivation. Till now, it does not give better fulfillment in outcomes due to lack of scientific approaches, skilled persons, and unfavored changing ecosystems. Definitely, when we carry out these fields without lacking, we will gain better outcomes in C. sinensis by better nutraceutical (nutrition + pharmaceutical) ways to human livelihood with the safest thing.
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The authors thank Department of Plant Pathology, CPPS, Tamil Nadu Agricultural University, Coimbatore and Department of Plant Pathology, SRSIAT, Vedasandur, India.
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Shreedevasena, S. et al. (2022). Mass Production and Marketing of Compost Caterpillar Fungus Cordyceps sinensis. In: Amaresan, N., Dharumadurai, D., Cundell, D.R. (eds) Industrial Microbiology Based Entrepreneurship. Microorganisms for Sustainability, vol 42. Springer, Singapore. https://doi.org/10.1007/978-981-19-6664-4_16
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