Diversity of Host Species and Optimized Cultivation Practices for Enhanced Bioactive Compound Production in Cordyceps militaris

Abstract

Cordyceps militaris, recognized for its diverse and potent medicinal properties, plays a critical role in herbal medicine. This study investigates the genus Cordyceps, particularly focusing on C. militaris, a species widely utilized in East Asian traditional medicine for its therapeutic properties. C. militaris is rich in bioactive compounds, including cordycepin, adenosine, polysaccharides, ergosterol, and mannitol, which contribute to its broad physiological activities. The research aims to explore the diversity of host species in the cultivation of C. militaris and assess their impact on the biological activity and chemical composition of the fungus. The study highlights the variability in the efficacy of bioactive compounds due to different cultivation conditions and host species, emphasizing the need for standardized cultivation practices. Advances in genetic engineering and fermentation technology have significantly enhanced the production of these metabolites, supporting the sustainable scale-up of C. militaris cultivation. Future research should continue to explore the molecular mechanisms of its bioactive compounds, identify new clinical applications, and improve production efficiency and environmental sustainability. This comprehensive review provides insights for researchers, healthcare professionals, and cultivators on optimizing C. militaris for medicinal and commercial applications.

1. Introduction

The genus Cordyceps, known since 2000 B.C., includes fungi that parasitize insects and are highly regarded in traditional Chinese medicine (TCM) for their medicinal properties [1]. Among its species, Cordyceps militaris, classified within Ascomycetes, is recognized for its diverse and potent medicinal properties and plays a critical role in herbal medicine, particularly in East Asia [2]. It is extensively utilized both as a traditional tonic and for its therapeutic properties, which are attributed to its bioactive components like cordycepin, adenosine, polysaccharides, ergosterol, and mannitol (Figure 1) [3]. These compounds contribute to a variety of physiological activities, including antioxidant, anti-inflammatory, anti-cancer, and immunomodulating effects, making C. militaris a valuable medicinal resource [3,4,5]. The genus Cordyceps comprises over 400 species of entomopathogenic fungi, and research interest in these fungi has significantly increased, as reflected by the rise in related publications on ScienceDirect, from 34 in 2008 to 199 by 2023. This surge in research activity highlights the growing scientific and commercial interest in these fungi.

The study of C. militaris is widespread, with substantial research output from countries including Australia, China, Germany, and Japan, among others (Figure 2). This global research expansion underscores the potential of Cordyceps in various applications, particularly due to their diverse bioactive compounds [6]. However, the efficacy of these compounds can greatly vary depending on the cultivation conditions and host species, posing challenges for standardization and optimization in medical use [7,8,9]. For example, studies have shown that the antioxidant properties of C. militaris are largely due to its ability to scavenge free radicals and reduce oxidative stress, which are implicated in aging and various chronic diseases [10]. The anti-inflammatory capabilities of C. militaris have been demonstrated via its potential to suppress pro-inflammatory cytokines and modulate immune responses [11]. Additionally, its anti-cancer properties have been highlighted in studies showing the fungus’s ability to induce apoptosis in cancer cells and inhibit tumor metastasis [12]. However, these effects, while promising, are still under investigation and should be interpreted with caution.

Moreover, the immunomodulating properties of C. militaris have been linked to its polysaccharides, which are known to enhance immune responses by activating macrophages, T-cells, and natural killer (NK) cells [13]. Despite these findings, it is important to note that while C. militaris exhibits these biological activities, the results from laboratory and clinical studies should be considered as indicative of potential rather than definitive therapeutic outcomes. Therefore, ongoing research is necessary to further elucidate these mechanisms and validate the efficacy of C. militaris in clinical settings.

The life cycle of C. militaris begins when its spores infect an appropriate insect host, initiating a process where the fungus develops hyphae that consume the host’s internal organs, leading to its death and the emergence of a fruit body from the host’s remains. Known as the stroma, this fruit body houses the perithecia, which produce spores for reproduction. This intricate development of fruit bodies is significantly influenced by environmental factors like temperature, humidity, and the host’s nutritional profile, with optimal conditions ranging from 20 °C to 25 °C and relative humidity above 90% [14,15,16,17]. Nutrient-rich hosts, particularly those high in lipids and proteins, significantly enhance the growth and potency of the fruit bodies, with the host type notably impacting their size and biochemical composition. For example, lepidopteran larvae tend to yield larger, more bioactive fruit bodies compared to those grown on coleopterans [18,19].

The genetic makeup of the C. militaris strains affects their efficiency and ability to adapt to environmental stresses, influencing the timing and success of fruit body development [20]. The cultivation of C. militaris on non-insect hosts like grains and synthetic media serves as a sustainable alternative, though it results in fruit bodies with different characteristics compared to those from natural insect hosts [21]. These artificial cultivation methods facilitate controlled production and reduce dependence on natural insect populations, thereby providing a consistent supply for medicinal applications. The formation of fruit bodies is a complex biochemical process involving the breakdown of host tissues by fungal enzymes such as proteases, lipases, and chitinases [22]. The differentiation of hyphal cells into various structural and reproductive cells marks the maturation of the fruit body, culminating in the release of spores that complete the fungus’s life cycle [23]. Understanding these dynamics offers insights into the ecological and biological processes that underpin this unique fungal life cycle, guiding the optimization of C. militaris cultivation for medicinal purposes.

The cultivation of C. militaris hinges critically on the diversity of its hosts, impacting not only the yield and quality of its bioactive compounds but also the sustainability of its production methods. Host selection is pivotal, as each host contributes distinct physiological environments that influence the metabolic pathways of C. militaris, thus affecting the synthesis of crucial compounds like cordycepin and polysaccharides [24]. For instance, studies have shown that silkworm larvae, as hosts, yield higher cordycepin levels than other insects, underscoring the necessity of strategic host selection to tailor the medicinal properties of the fungus [25,26]. The range of hosts spans from natural arthropods to synthetic media designed to replicate the nutritional profile of natural hosts, providing controlled environments that enhance specific compound production [16]. This diversification in host use not only mitigates the ecological impact caused by overharvesting certain natural hosts but also presents economic opportunities by enabling large-scale, sustainable production [27].

Genetic diversity among C. militaris strains also plays a crucial role, influencing compatibility with various hosts and the efficiency of bioactive compound synthesis. Advances in molecular studies have furthered our understanding of how C. militaris adapts its metabolic pathways to optimally exploit available resources, enhancing cultivation efficacy [28]. Advances in molecular studies have furthered our understanding of how C. militaris adapts its metabolic pathways to optimally exploit available resources, enhancing cultivation efficacy [29]. Future research should continue to explore a broader spectrum of hosts and refine cultivation techniques to maximize the therapeutic potential of C. militaris, aligning with sustainable practices that ensure economic viability and environmental integrity [30]. The integration of traditional knowledge with contemporary biotechnological advancements is likely to propel the use of C. militaris in global healthcare, making its therapeutic benefits more accessible [31].

This review aims to comprehensively explore the diversity of host species utilized in the cultivation of C. militaris and to assess the influence of these hosts on the biological activity and chemical composition of the resulting fungi. Specifically, we seek to elucidate the impact of different host organisms on the yield and quality of C. militaris, which is critical for optimizing its use in medicinal and commercial applications. We intend to highlight how variations in host species can lead to differences in the levels of key bioactive compounds such as cordycepin and polysaccharides [32]. Additionally, this review will discuss sustainable cultivation practices that could mitigate the ecological impact of harvesting natural hosts and promote the conservation of biodiversity. By integrating findings from recent studies and traditional knowledge, we aim to provide insights that could guide future research and cultivation strategies. Ultimately, this review serves to inform researchers, healthcare professionals, and cultivators about the best practices and potential challenges in the cultivation of C. militaris, fostering advancements in the therapeutic use of this valuable medicinal fungus.

2. Host Diversity for Cordyceps militaris Cultivation

The cultivation of C. militaris, a fungus known for its significant medicinal properties, involves utilizing various host species, ranging from natural insect hosts to artificial substrates. The choice of host species plays a critical role in influencing fungal growth, bioactive compound production, and overall yield. Traditionally, C. militaris parasitizes various insects, particularly those belonging to the orders Lepidoptera, Coleoptera, and Hymenoptera. These natural hosts provide unique biochemical environments that facilitate fungal development and the synthesis of key metabolites, essential for the fungus’s therapeutic effects [19]. However, the reliance on natural insect hosts presents ecological and ethical concerns, prompting the exploration of alternative substrates that could sustain large-scale cultivation while maintaining or enhancing bioactive compound production (

Table 1. Impact of host diversity on C. militaris growth and bioactive compound production.

Host Type	Impact on Fungal Growth and Bioactive Compounds	Ref.
Lepidoptera (e.g., ghost moth)	Rich in lipids and proteins, conducive to substantial fungal growth and high levels of cordycepin	[33]
Coleoptera (e.g., beetles)	Chitinous exoskeleton aids in producing chitinase, affecting anti-inflammatory and anti-tumor activities	[34]
Hymenoptera (e.g., wasps, bees)	Supplies unique fatty acids and sterols, enhancing pharmacological value	[35]
Grains (e.g., rice, wheat)	More sustainable and consistent but yields lower levels of key metabolites like cordycepin compared to natural hosts	[36]
Synthetic media	Tailored with specific nutrients to control growth conditions, enhancing understanding of metabolic pathways	[37]

).

Lepidoptera hosts, such as the ghost moth, are particularly rich in lipids and proteins, which are conducive to substantial fungal growth and high cordycepin levels—a compound known for its anti-cancer and immunomodulatory properties [2]. Similarly, Coleoptera, like beetles, contribute a chitinous exoskeleton that supports the production of chitinase, an enzyme crucial for fungal structural integrity and immune-enhancing effects [38,39]. Hymenoptera, including wasps and bees, provide unique fatty acids and sterols that may enhance the pharmacological value of C. militaris extracts [40]. Despite the benefits of these natural hosts, their overharvesting raises sustainability concerns, necessitating the development of alternative substrates such as grains, synthetic media, and plant-based materials. Grains like rice and wheat offer a more sustainable and consistent output, although they typically yield lower levels of key metabolites such as cordycepin compared to insect hosts [37,41]. Synthetic media, tailored with specific nutrients, allow precise control over growth conditions, facilitating a better understanding of metabolic pathways involved in bioactive compound synthesis [42,43].

The choice of host species not only impacts the yield of bioactive compounds but also affects the quality and consistency of C. militaris products (

Table 2. Yields and bioactive compounds of Cordycepin from various host species of C. militaris.

Host Species	Yield of Cordycepin (mg/g Dry Weight)	Bioactive Compounds	Ref.
Bombyx mori Pupae (silkworm pupae)	4.37 ± 2.32	Cordycepin, Polysaccharides, Adenosine	[44]
Brown rice	2.89 ± 1.99	Primarily Polysaccharides	[44]
Brown rice paste, beerwort, and soybean meal juice	2.17 ± 0.09	-	[45]
Soybean	8.33 ± 0.44	Cordycepin	[46]
Chickpea	11.12 ± 0.76	-	[46]
Black bean	10.43 ± 0.37	-	[46]
Mung bean	6.64 ± 0.14	-	[46]
Potato Dextrose	1.16 ± 1.23	Low Levels of Cordycepin	[44]
Wheat standard substrate and pupal (Bombyx mori Pupae) injection	~1.2	-	[47]
Generation of ΔMAT1-1-2; injection of 107 ΔMAT1-1-2xΜAΤ1-2 spores/mL into the Chinese Tussah Silkworm pupae	Up to 16.77	-	[48]
Cicada Larvae	-	Cordycepin, peptides, mannitol	[49]
Pupa Substrate	-	Cordycepin, polysaccharides, vitreoscilla hemoglobin	[47]
Beetles	-	Lower levels of cordycepin, ergosterol	[50]
Soybean Powder	-	Cordycepin	[46]
Wheat Bran	-	Moderate levels of cordycepin, various vitamins	[51]
Potato Dextrose	-	Low levels of cordycepin	[44]
Synthetic Media	-	Designed to mimic natural substrates, varies based on formulation	[52]

). For example, silkworm pupae are a preferred host due to their nutrient-rich profile, which enhances the production of cordycepin and polysaccharides, compounds known for their anti-inflammatory and immune-boosting properties [44]. In contrast, other hosts like cicadas and beetles present different advantages and challenges. Cicadas are effective in supporting high-quality production, whereas beetles, due to their tougher exoskeletons, may yield lower levels of cordycepin but could produce unique peptides or secondary metabolites [45]. Furthermore, substrates such as rice and wheat bran, along with plant-based options like soybean and potato dextrose, are commonly used due to their availability and cost-effectiveness, though they generally result in lower-quality bioactive compounds compared to insect hosts. This disparity is mainly attributed to differences in nutrient profiles essential for optimizing fungal metabolism.

The choice of host species not only impacts the yield of bioactive compounds but also affects the quality and consistency of C. militaris products. For example, silkworm pupae are a preferred host due to their nutrient-rich profile, which enhances the production of cordycepin and polysaccharides, compounds known for their anti-inflammatory and immune-boosting properties [44]. In contrast, other hosts like cicadas and beetles present different advantages and challenges. Cicadas are effective in supporting high-quality production, whereas beetles, due to their tougher exoskeletons, may yield lower levels of cordycepin but could produce unique peptides or secondary metabolites [49]. Furthermore, substrates such as rice and wheat bran, along with plant-based options like soybean and potato dextrose, are commonly used due to their availability and cost-effectiveness, though they generally result in lower-quality bioactive compounds compared to insect hosts. This disparity is mainly attributed to differences in nutrient profiles essential for optimizing fungal metabolism.

The selection of analytical methods, such as high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy, is critical for evaluating how host species influence the concentration of bioactive metabolites in C. militaris. While HPLC is widely employed for its precision in quantifying cordycepin, it is essential to recognize its limitations, including the need for extensive sample preparation and the potential for compound degradation during analysis. Furthermore, HPLC may not be ideal for detecting compounds at very low concentrations or within highly complex mixtures. To address these limitations, complementary techniques like GC-MS and NMR offer broader analytical capabilities, such as identifying a wider array of metabolites and providing detailed structural information about these compounds. These methods are particularly valuable for obtaining a comprehensive metabolomic profile of C. militaris cultivated on various hosts, thereby providing deeper insights into how different hosts affect metabolite production [43].

Variations in cordycepin production among different host species can be attributed to several key factors, including the nutrient profile of the host, the physiological state of the host at the time of inoculation, and the genetic strain of C. militaris used. For instance, Lepidoptera hosts, which are rich in lipids and proteins, provide essential precursors for the synthesis of bioactive compounds. This nutrient-rich environment stimulates higher metabolic activity in C. militaris, leading to increased cordycepin production. Additionally, the interaction between fungal genetics and the host’s biochemical environment plays a significant role in optimizing bioactive compound synthesis. Certain hosts, like Lepidoptera larvae, yield higher levels of cordycepin due to their abundant lipid and protein content, which are crucial for the biosynthesis of this compound. The nutritional composition of the host directly influences the metabolic pathways in C. militaris, thereby enhancing cordycepin production. Furthermore, the host’s immune response to fungal infection can trigger the synthesis of secondary metabolites as a defense mechanism by the fungus, further boosting cordycepin production [53]. These factors underscore the importance of selecting host species that naturally enhance cordycepin production, enabling the optimization of cultivation practices to achieve higher yields, reduce costs, and improve the consistency of medicinal products derived from C. militaris. However, the choice of host species extends beyond merely maximizing production; it also has significant implications for the sustainability and scalability of C. militaris cultivation. Over-reliance on specific insect hosts could lead to ecological imbalances, highlighting the need to explore alternative hosts, such as grains or synthetic media. While these alternatives may not be as effective in producing high cordycepin levels, they offer a more sustainable and controllable environment for large-scale production [33]. The successful commercialization of C. militaris products hinges on balancing production optimization with ecological and economic considerations.

Therefore, the cultivation of C. militaris is a complex process that requires careful attention to host species, environmental factors, and cultivation techniques to maximize bioactive compound production. The diverse range of potential hosts, from natural insects to artificial substrates, presents both opportunities and challenges that influence the yield, quality, and sustainability of cultivation efforts. Advancements in cultivation technology and ongoing research are essential for enhancing the therapeutic potential and commercial viability of C. militaris products. A comprehensive understanding of the relationship between host diversity and bioactive compound production is crucial for developing sustainable and effective cultivation strategies that align with the growing demand for high-quality medicinal mushrooms. Future research should continue to explore genetic and environmental manipulation techniques to support sustainable and economically viable cultivation practices, thereby enhancing the medicinal value of C. militaris products.

3. Factors Influencing Yield and Quality

The cultivation of C. militaris, recognized for its significant medicinal properties, is influenced by genetic, environmental, and host-related factors that determine both yield and quality (Figure 3). The genetic makeup of the C. militaris strain plays a pivotal role, as variability among strains affects growth rates, bioactive compound synthesis, and adaptability to cultivation conditions [30]. Advances in genetic engineering and selective breeding have enhanced traits such as increased cordycepin production and improved stress resistance.

Temperature is a critical environmental factor in cultivating C. militaris, influencing both fungal growth and the production of bioactive compounds. The optimal temperature range for cultivating C. militaris is between 20 °C and 25 °C [54]. Within this range, the fungus achieves optimal growth and metabolite production, including key compounds like cordycepin. Deviations from this temperature range can significantly impact fungal metabolism, affecting both the yield and quality of the final product. Lower temperatures may slow growth and metabolite production, while higher temperatures may inhibit growth or lead to the production of different metabolites [55]. Therefore, maintaining controlled environmental conditions is vital to ensure consistent, high-quality yields of C. militaris

Humidity plays a significant role in the cultivation of C. militaris, markedly influencing fungal growth and metabolite production [56]. Optimal humidity levels, generally between 60% and 80%, are crucial for promoting healthy mycelial growth and maximizing the production of bioactive compounds such as cordycepin and polysaccharides. Proper humidity levels ensure that the fungus does not dry out, potentially impeding growth and decreasing metabolite synthesis. Conversely, excessively high humidity can increase contamination risks and may lead to suboptimal growth conditions [16].

Light significantly influences the production of C. militaris by affecting metabolic responses and synthesizing key bioactive compounds, such as cordycepin and carotenoids, essential for the fungus’s medicinal properties [26]. Exposure to specific light conditions, especially variations in light intensity and wavelength, activates genetic pathways crucial for these compounds’ synthesis, impacting both the quantity and quality of the fungal biomass [57]. For instance, light-induced transcriptional changes in genes involved in metabolic pathways are critical for adapting fungal metabolism to optimal light conditions, which enhances growth and secondary metabolite synthesis. Managing light exposure is therefore vital in optimizing both the yield and concentration of bioactive compounds, thereby improving the efficacy and commercial value of the harvested product. Particularly, exposure to blue light via LED irradiation for 8 h per day has been shown to significantly increase both biomass production and the level of cordycepin in liquid-cultured C. militaris, while short-wavelength light enhances the total carotenoid content in the fruiting bodies [58]. General recommendations suggest using moderate light intensities around 400–500 lux to promote growth phases and the development of fruiting bodies under controlled conditions, although more precise details would require access to specific research results or cultivation guidelines.

CO₂ levels significantly impact the cultivation of C. militaris, affecting both growth and bioactive compound production. Research shows that maintaining elevated CO₂ concentrations during the early growth phase can increase biomass and enhance the yield of valuable metabolites such as cordycepin [59]. High CO₂ levels (up to 5000 ppm) have been shown to improve the growth rate and size of the fruiting bodies, essential for the commercial harvesting of bioactive compounds. Optimizing CO₂ levels also helps regulate fungal metabolism, directly affecting the synthesis of secondary metabolites. Managing CO₂ concentration involves careful monitoring and adjustments within controlled cultivation environments, requiring advanced ventilation and CO₂ regulation systems to maintain desired atmospheric conditions [60]. The benefits of such precise control include not only increased yield and quality but also enhanced consistency in production, crucial for meeting the stringent standards required for pharmaceutical applications of C. militaris.

Environmental factors like temperature, humidity, light, and CO₂ levels are crucial; optimal conditions typically range from 18 °C to 25 °C with 80–95% humidity, and deviations can significantly impact fungal metabolism and secondary metabolite production. The choice of host is critical as it provides essential nutrients and influences the environmental context for fungal growth. Insect hosts generally offer richer nutrients compared to plant-based or synthetic media, enhancing growth and improving the quality of the final product. Additionally, the methods used for host preparation and sterilization affect contamination rates and nutrient availability, where improper sterilization can degrade essential nutrients, negatively impacting yield and quality.

4. Selection of Host Species

4.1. Genetic Variability

Genetic variability plays a critical role in the cultivation of C. militaris, significantly influencing the fungus’s ability to adapt to various hosts and optimize the production of bioactive compounds. The genetic diversity within C. militaris strains is substantial, resulting in variations in morphological and physiological traits that directly impact the fungus’s infection efficiency, growth rate, and the synthesis of essential bioactive metabolites. This genetic diversity, which includes multiple chromosomes, is characterized by variations such as single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), both of which drive phenotypic diversity [61]. Spontaneous mutations, sexual reproduction via spore-producing fruit bodies, and geographical isolation further contribute to this diversity, leading to local adaptations and, ultimately, speciation [62]. Understanding this genetic variability is crucial for cultivation practices, as different C. militaris strains may exhibit better adaptation to specific hosts, enhancing both yield and the production of bioactive compounds. Moreover, genetic diversity can provide resilience against environmental stresses, a vital factor for ensuring consistent and sustainable cultivation [31].

The genetic basis for the production of bioactive compounds such as cordycepin and polysaccharides is well-established, with certain strains of C. militaris being naturally predisposed to higher levels of metabolite production. By selecting and cultivating these genetically superior strains, it is possible to optimize the synthesis of these valuable compounds, thereby improving the overall efficacy of C. militaris-based products [30]. To harness this genetic variability, modern methodologies such as genome sequencing, quantitative trait loci (QTL) mapping, and CRISPR/Cas9 genetic engineering have been employed. These tools allow researchers to link specific genetic traits with desirable phenotypic characteristics, such as increased cordycepin production or enhanced growth rates, facilitating the targeted improvement of C. militaris strains [63]. For example, CRISPR/Cas9 has been successfully used to knock out or modify specific genes within the C. militaris genome, resulting in strains that exhibit improved resistance to environmental stressors or enhanced bioactive compound production. Such genetic modifications can be strategically applied to tailor C. militaris strains for specific medicinal or commercial purposes.

Looking forward, future research in C. militaris cultivation is likely to focus on expanding genetic databases to identify additional markers linked to key traits, as well as refining genetic engineering techniques to achieve even more precise modifications. Advances in CRISPR/Cas9 technology, in particular, hold significant potential for the continued improvement of C. militaris strains. For instance, recent studies have demonstrated the utility of CRISPR/Cas9 in enhancing cordycepin production by targeting the biosynthetic pathways involved in nucleotide metabolism, which is directly linked to cordycepin synthesis. Another promising area of research involves the use of CRISPR/Cas9 to increase the efficiency of fungal adaptation to non-traditional hosts, such as synthetic media or agricultural by-products, thereby improving the sustainability of large-scale cultivation efforts. By focusing on these genetic engineering strategies, researchers can optimize the production of bioactive compounds in C. militaris, making it a more viable and potent medicinal resource. These advancements underscore the critical importance of genetic understanding in maximizing the therapeutic potential of C. militaris and ensuring its commercial success.

4.2. Geographic Origin

Factors such as climate, altitude, and local ecosystem diversity impact the survival, reproductive success, metabolic pathways, and stress responses of the fungus, thereby affecting the quality and quantity of medicinal compounds produced. For example, optimal growth temperatures for C. militaris are between 20 °C and 25 °C with relative humidity around 90%; regions that naturally provide these conditions support better fungal development and higher yields of compounds like cordycepin and polysaccharides [64]. Additionally, higher altitudes expose the fungus to lower temperatures and increased UV radiation, inducing stress responses such as the synthesis of protective antioxidants, which enhance the therapeutic efficacy of the harvested compounds [38]. The local biodiversity and soil composition also dictate the types of hosts available, influencing nutrient uptake and the specific metabolic pathways activated in the fungus, leading to unique bioactive compound profiles [65].

The genetic makeup of C. militaris varies across different geographic regions, impacting its adaptability, health, and productivity. Diverse genetic traits within populations from varied regions contribute to differences in resistance to pathogens, adaptation to environmental stressors, and metabolic functions. Geographic isolation can lead to genetic drift or localized adaptations, where populations evolve distinct genetic markers that confer advantages in specific environments, optimizing resistance to local climatic stressors or host utilization [66]. Studies reveal that molecular adaptations in C. militaris strains vary by geographic location. These strains may express different gene sets, particularly those involved in stress response and secondary metabolism. Such variations optimize growth and survival under diverse environmental conditions. [67].

Advanced genomic, transcriptomic, and metabolomic technologies are critical for understanding how geographic origin influences C. militaris. Techniques such as genome sequencing of strains from various origins help identify genetic variations linked to phenotypic differences and local adaptations [68]. Metabolomic profiling provides insights into the metabolic pathways influenced by geographic factors and their impact on compound synthesis [69]. Field studies examining the natural habitats of C. militaris enhance our understanding of the ecological dynamics that influence fungal populations, including interactions with hosts and competitors [70]. This knowledge is invaluable for optimizing cultivation practices and developing conservation strategies, enabling the selection of strains adapted to specific climates or efficient in producing desired compounds, thereby enhancing yield and quality in commercial farming and aiding in the conservation of wild populations via informed sustainable harvesting and habitat preservation efforts.

4.3. Environmental Conditions

Environmental conditions are crucial for the cultivation and biological activity of C. militaris, a valued medicinal fungus. Temperature is fundamental to fungal metabolism and development, with an optimal range of 18 °C to 25 °C fostering robust growth and fruiting body production. Deviations from this range can impair metabolic activity and development, affecting polysaccharide content and cordycepin production, where higher temperatures may accelerate metabolism but degrade sensitive compounds [71]. Relative humidity (RH) should be maintained at 80–95% to prevent mycelial desiccation and support healthy fruit body development, as lower humidity leads to a dry substrate and higher moisture can encourage contaminant growth [68]. Light exposure also plays a critical role, with a required photoperiod of 12 h of light and 12 h of darkness to promote optimal morphogenesis and secondary metabolite synthesis [50]. Substrate quality, encompassing nutrient composition and physical properties like porosity and water-holding capacity, is essential for nutrient uptake and efficient gas exchange, crucial for mycelial growth [72]. The interplay between these environmental factors can compound their effects, such as the interaction between temperature and humidity influencing water evaporation and consequently mycelial growth rates and fruiting body development [73]. For commercial cultivation, understanding and optimizing these environmental conditions are paramount, as they directly impact yield, fruit body quality, and bioactive compound production. Advanced cultivation facilities often employ automated systems to monitor and adjust these parameters, ensuring consistent product quality and reducing the risk of crop failure due to environmental fluctuations. Future research should focus on refining these parameters to enhance cultivation techniques and yields, particularly in controlled environment agriculture settings, to maximize the medicinal potential of C. militaris.

Traditional isolation methods for C. militaris often involve culturing surface sterilized internal tissues from wild fruit bodies or infected insects on nutrient media such as potato dextrose agar (PDA) or malt extract agar (MEA). Although straightforward, these methods are prone to significant contamination risks, which can lead to variability in the genetic and metabolic profiles of the isolates, potentially compromising their utility in research and commercial applications [74]. An alternative approach involves spore isolation from mature fruit bodies, offering the advantage of selecting genetically diverse strains beneficial for breeding programs. However, this technique also presents contamination challenges from unwanted microorganisms [75]. To mitigate these risks, rigorous sterilization procedures, such as treating fruit bodies with ethanol or sodium hypochlorite followed by rinsing with sterile water, are essential. Performing the isolation process within a laminar flow hood further minimizes airborne contaminants. The use of selective media that inhibits the growth of common contaminants while promoting the growth of C. militaris spores, along with the incorporation of media supplements like antibiotics or antifungal agents, can enhance the purity of the isolates [76]. Modern techniques such as single-spore isolation, hyphal tip culture, and flow cytometry with cell sorting offer more precise and high-throughput options, ensuring genetic uniformity, minimizing contamination, and facilitating the selection of traits like high metabolic activity or stress resistance [33,77].

5. Bioactive Compounds in C. militaris

The antioxidant capabilities of C. militaris are highlighted by its ability to scavenge free radicals, thereby mitigating oxidative stress, which contributes to aging and chronic diseases. Studies have shown that extracts of C. militaris can enhance the activity of antioxidant enzymes and reduce lipid peroxidation [78]. The anti-inflammatory properties are significant, with extracts shown to inhibit the production of pro-inflammatory cytokines like TNF-α and IL-6 in cultured macrophages, which are integral to inflammatory processes [65]. These effects are primarily attributed to cordycepin, which disrupts signaling pathways that trigger inflammation (

Table 3. Bioactive compounds in C. militaris: influential factors.

Bioactive Compound	Influential Factor	Effects	Ref.
Polysaccharides	Cultivation substrate, extraction method	Immunomodulating, antitumor, antioxidant	[8]
Beta-glucan	Host species, extraction method	Enhances immune response, lowers cholesterol	[79]
Cordycepin	Host species, cultivation conditions	Anti-cancer, anti-inflammatory, potential antiviral	[7]
Adenosine	Cultivation methods, enzyme activity	Energy metabolism, neurotransmission, cardiovascular protection	[16]
Sterols	Host species, growth stage	Anti-inflammatory, cholesterol management	[65]
Saponins	Cultivation substrate	Antifungal, antitumor, immunomodulatory	[79]
Triterpenoids	Host species, extraction process	Hepatoprotective, anti-inflammatory, antiviral	[80]
Ergosterol	Cultivation environment	Antioxidant, precursor for Vitamin D2 synthesis	[81]
Mannitol	Host nutrient availability	Diuretic, free radical scavenging	[81]
γ-Aminobutyric acid (GABA)	Fermentation conditions	Reduces anxiety, enhances mood, improves sleep	[82]
Ergothioneine	Specific enzymatic pathways	Antioxidant, cellular protector against oxidative stress	[83]

).

Moreover, C. militaris is celebrated for its anti-cancer properties; cordycepin induces apoptosis in various cancer cell lines and inhibits cell migration and invasion, crucial for cancer metastasis [84]. Ongoing clinical trials aim to harness these properties in cancer therapy by targeting multiple pathways involved in cancer progression. The immunomodulatory effects are equally pivotal, with polysaccharides in C. militaris enhancing immune response by activating macrophages, boosting lymphocyte proliferation, and promoting cytokine production, making it a valuable natural treatment for immune-compromised individuals [85]. The adaptogenic properties of C. militaris support the body’s ability to resist stressors and modulate stress response systems, potentially alleviating the impacts of physical and mental stress. This positions C. militaris not just as a therapeutic agent but also as a preventive one, helping to maintain overall health and well-being.

Despite the promising therapeutic applications, further clinical research is essential to fully understand the mechanisms of action, optimal dosages, and long-term safety of C. militaris. Future studies should explore the synergistic effects of C. militaris with other treatments and its efficacy in managing various diseases. Thus, C. militaris presents a compelling blend of traditional uses backed by modern scientific research. Its bioactive compounds provide significant health benefits, positioning it as a potential multifunctional agent in health promotion and disease prevention. As research advances, integrating C. militaris into mainstream medicine could provide an alternative or adjunct to conventional therapies, emphasizing the importance of natural products in future pharmacotherapy.

6. Industrial Production of C. militaris

6.1. Overview of Production Methods

The production of C. militaris involves several key stages, each requiring specific equipment to ensure efficiency and quality. The primary methods of cultivation are solid-state fermentation (SSF), liquid fermentation (submerged culture), and hybrid systems. In SSF, grain substrates like rice or wheat bran are used, necessitating equipment for substrate preparation such as mixers, steamers for sterilization, and incubation chambers to maintain controlled environmental conditions [86]. This method simulates the natural growth conditions of the fungus and is favored for its simplicity and low capital requirement. Conversely, liquid fermentation requires bioreactors that provide precise control over parameters like temperature, pH, and oxygen levels [87]. These systems are automated with sensors and pumps to manage the addition of nutrients and maintain optimal growth conditions. Hybrid systems combine these methods, starting with liquid culture to rapidly expand biomass before transferring to solid substrates to boost secondary metabolite production, necessitating equipment for both methods (Figure 4).

The choice of production method significantly influences the potential output and associated costs. SSF is typically less expensive to set up but may offer lower yields compared to liquid systems. Average yields for SSF can range from 0.1 to 0.5 g of dry weight per liter of substrate, depending on the strain and substrate composition. The cost of establishing an SSF setup can vary but generally remains low, focusing mainly on the cost of substrates and simple fermentation setups [87]. Liquid fermentation, while more costly in terms of initial setup due to the need for sophisticated fermenters and control systems, allows for higher biomass production. Yields from liquid fermentation can reach 10 to 20 g/L, with operational costs driven by nutrient media and energy consumption for maintaining fermentation conditions. The capital investment for a moderate-sized liquid fermentation facility can range from USD 100,000 to 500,000, with operational costs varying based on the scale and efficiency of the processes.

Hybrid systems aim to maximize the benefits of both SSF and liquid fermentation, facilitating an initial liquid culture phase followed by a transfer to solid-state conditions. This method can enhance the yield of specific bioactive compounds by 10–15% compared to individual methods. The setup cost for hybrid systems is significant, often exceeding the sum of individual setups, but they offer flexibility in production based on demand for different compounds. Operational costs for hybrid systems are higher due to the complexity and the need for additional steps in transferring cultures and maintaining two types of fermentation environments. Economic viability across these methods depends not only on the production outputs and costs but also on the market value of the compounds produced. Cordycepin, for instance, has a high market value that can offset the higher costs of production in liquid or hybrid systems. The profitability of each method thus depends on balancing the higher yield and quality of bioactive compounds against the increased investment and operational costs.

Continuous advancements in biotechnological tools and optimization of fermentation parameters are critical for reducing costs and enhancing yields. Genetic engineering, for example, offers prospects for strain improvement that could significantly boost production efficiency [88]. Moreover, ongoing research into cheaper substrate alternatives and energy-efficient fermentation processes could further enhance the economic viability of cultivating C. militaris commercially. As market demand for natural health products continues to grow, optimizing production methods to maximize yields while minimizing costs will be crucial for maintaining competitiveness in the biotechnology sector.

6.2. Differences between Natural and Industrial Production of C. militaris

The distinction between natural and industrial production of C. militaris underscores significant contrasts in terms of yield, bioactive compound quality, and practical viability, all of which are critical to understanding the broader implications for its use in medicinal and commercial applications [89]. Naturally occurring Cordyceps, primarily sourced from the wild via insect larvae, has been highly valued for its exceptional medicinal properties, largely due to its rich profile of bioactive compounds, including cordycepin and polysaccharides. However, the natural collection of C. militaris is fraught with challenges, such as ecological sensitivity, limited availability, high harvesting costs, and significant sustainability concerns. The specific environmental conditions required for natural growth—found primarily in high-altitude regions of Asia—lead to inconsistent and often low yields, further complicating large-scale collection efforts and raising ethical and ecological concerns over the depletion of natural resources [90].

In contrast, industrial production methods, like solid-state fermentation (SSF) and liquid fermentation (LF) offer several advantages in scalability and control over environmental factors such as substrate composition, temperature, and humidity, which are essential for optimizing fungal growth and metabolite production [91]. Industrial cultivation allows for the consistent production of high Cordyceps biomass, which enables standardized extract quality, making it more suitable for pharmaceutical and nutraceutical applications. For example, industrial processes can enhance the yields of key metabolites like cordycepin and polysaccharides, which are crucial for therapeutic use. Despite these advantages, industrial cultivation does not always replicate the bioactive compound spectrum found in naturally harvested Cordyceps. Variations in the concentrations of secondary metabolites are common in industrially cultivated C. militaris, potentially affecting their therapeutic efficacy when compared to natural counterparts. This discrepancy highlights the ongoing need for advancements in biotechnology, including genetic modification and growth medium optimization, to narrow the quality gap between natural and industrial sources [92]. The challenge lies in balancing the benefits of high yield and sustainability offered by industrial production with the superior quality associated with naturally sourced Cordyceps, particularly as recognized in traditional medicine.

Moreover, the scalability and consistency of bioactive compound production in C. militaris are directly influenced by the choice of host species used in cultivation [93]. The exploration of diverse host species presents both opportunities and challenges. On one hand, utilizing various host species can significantly enhance the synthesis of bioactive compounds, which are critical for the medicinal efficacy of C. militaris [94]. This approach supports ecological sustainability by reducing the reliance on a single host species, thereby mitigating the risks associated with monoculture practices, such as increased susceptibility to disease or environmental changes. Economically, employing a variety of host species can lower the cost of raw materials and expand market opportunities, enabling producers to meet specific health-related consumer demands. On the other hand, the use of diverse host species introduces significant challenges in standardizing cultivation methods. Different hosts require different growth conditions, leading to variability in the bioactive compound profiles of the harvested products [95]. This variability complicates the standardization of cultivation protocols and can negatively impact the quality and medicinal effectiveness of C. militaris extracts. Inconsistent product quality can undermine consumer trust and reduce the marketability of C. militaris products, posing a substantial barrier to their widespread commercial adoption.

The reliance on various host species also poses challenges in scaling production while maintaining consistency. The need to optimize growth conditions for each specific host increases operational costs and requires significant investments in infrastructure and specialized training. Furthermore, ecological and ethical concerns, such as the sustainability of using certain natural hosts, complicate the scalability of production [96]. For instance, over-reliance on specific insect hosts could lead to ecological imbalances and the depletion of these species, raising ethical concerns about the long-term viability of such practices. These limitations could hinder the widespread commercial adoption of diverse host species for C. militaris cultivation. Therefore, a thorough discussion of these challenges is essential for understanding the broader implications of using multiple host species and ensuring the long-term viability and consistency of bioactive compound production in C. militaris [92].

While the use of diverse host species in the cultivation of C. militaris presents opportunities to enhance bioactive compound production, it also introduces significant challenges that must be carefully considered. One of the primary issues is the difficulty in standardizing cultivation methods across different hosts, which can lead to variability in the growth conditions and subsequently, in the bioactive compound profiles of the harvested products. These variations complicate the standardization of cultivation protocols, potentially affecting the quality and medicinal effectiveness of C. militaris extracts [92]. Inconsistent product quality can undermine consumer trust and negatively impact the marketability of the products. Moreover, the reliance on a variety of hosts poses challenges in scaling production while maintaining consistency. The need to optimize growth conditions for each specific host increases operational costs and requires significant investments in infrastructure and specialized training. Additionally, ecological and ethical concerns, such as the sustainability of using certain natural hosts, further complicate the scalability of production. These limitations could hinder the widespread commercial adoption of diverse host species for C. militaris cultivation. A thorough discussion of these challenges is essential to understanding the broader implications of using multiple host species and ensuring the long-term viability and consistency of bioactive compound production in C. militaris.

In addition to the challenges associated with host selection, further complexities arise from the interplay of genetic, environmental, and host-related factors. Genetic variability among C. militaris strains plays a crucial role in influencing growth rates and the synthesis of bioactive compounds. For example, certain strains may be naturally predisposed to higher cordycepin production, while others may excel in producing polysaccharides. Environmental conditions, such as temperature and humidity, are equally critical; deviations from optimal conditions can significantly affect fungal metabolism and the production of secondary metabolites. The selection and preparation of the host species significantly impact the availability of nutrients and the environmental conditions for fungal growth, which in turn influences the bioavailability of nutrients essential for optimal fungal development. These factors collectively determine the fungus’s adaptation to cultivation conditions, which is crucial for achieving high yields and premium quality [65,93]. The successful integration of C. militaris into mainstream medicine and functional foods requires careful consideration of these variables to ensure consistency and efficacy in the final products.

Moreover, the biological activity of C. militaris is profoundly influenced by the strain of the fungus, the cultivation conditions, the host substrate, and post-harvest processing techniques [97]. These elements collectively determine the types and amounts of bioactive compounds produced, which are critical for the therapeutic efficacy of C. militaris in various applications. To maximize the therapeutic potential of C. militaris in beverages and other products, it is vital to meticulously manage these factors, optimizing growth conditions and compound synthesis while selecting suitable host substrates to enhance the production of targeted bioactive compounds. Post-harvest processing methods, such as drying and extraction, further influence the stability and bioavailability of these compounds, requiring careful handling to preserve their medicinal properties. For instance, improper drying techniques can lead to the degradation of heat-sensitive compounds like cordycepin, while suboptimal extraction methods may fail to fully capture the range of bioactive compounds present in C. militaris. Therefore, effective management of these factors is essential for enhancing the utility of C. militaris in both traditional and contemporary medical applications [65,93].

This complex scenario underscores the need for a balanced approach that leverages the advantages of both natural and industrial cultivation methods. Future research should focus on optimizing cultivation techniques that combine the best aspects of both approaches, potentially via hybrid cultivation systems that offer a compromise between the high bioactive quality of natural sources and the high yield and consistency of industrial production. Additionally, the exploration of diverse host species for C. militaris cultivation should be approached with a focus on sustainability, cost-effectiveness, and the ethical implications of large-scale production. By integrating traditional knowledge with contemporary biotechnological advancements, the cultivation of C. militaris can be optimized to support its use in functional beverages and other health-promoting products, thereby enhancing its accessibility and impact on global health and wellness.

7. Research Prospects

Optimizing cultivation conditions for C. militaris is pivotal for maximizing the production of its bioactive compounds, thereby enhancing its therapeutic and commercial viability. Various environmental factors, including substrate type, temperature, humidity, light, and aeration, significantly influence the growth and metabolite output of this fungus. Here are specific strategies and research directions for improving cultivation conditions:

• Substrate Optimization: Utilizing substrates enriched with complex carbohydrates or proteins can enhance the levels of specific metabolites such as polysaccharides and bioactive peptides, crucial for the medicinal properties of C. militaris. Investigating different substrate combinations and their effects on metabolite synthesis can lead to more effective and economical cultivation practices.
• Controlled Environmental Conditions:

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  Maintaining temperatures between 20 and 22 °C is essential for optimal fungal growth and metabolite production.

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  Controlling humidity and light exposure is critical to optimize fungal metabolism and secondary metabolite synthesis. Specific light regimes can be employed to stimulate the production of targeted bioactive compounds.

• Biotechnological Advances:

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  Modifying genetic pathways in C. militaris via genetic engineering can enhance the production of targeted metabolites like cordycepin. This approach can also improve the strain’s resilience to environmental stressors, thereby increasing overall yield.

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  Both solid-state and submerged fermentation technologies can be optimized to improve biomass production and consistency of bioactive compounds. Advances in these technologies can also reduce production costs and enhance scalability.

• Bioreactor Utilization: Employing bioreactors in submerged fermentation setups allows for automated and precise monitoring of growth conditions, significantly boosting production efficiency and consistency in bioactive compound profiles. Bioreactors can provide controlled environments conducive to large-scale production, ensuring high-quality outputs.

These strategies underscore the necessity of meticulous management of cultivation parameters to ensure robust fungal growth and significantly influence the quality and quantity of bioactive compounds produced. Future research should focus on further integrating these biotechnological approaches to refine cultivation techniques, thereby improving the efficiency and sustainability of C. militaris production. This will ensure it remains a sustainable and economically viable natural health product to meet the increasing global demand.

Moreover, exploring the synergistic effects of combining C. militaris with other functional ingredients could open new avenues for product development. Research into consumer preferences and market trends can guide the creation of innovative products that deliver both health benefits and sensory appeal. By continuing to advance cultivation and processing techniques, the industry can harness the full potential of C. militaris, providing consumers with accessible and effective health-promoting products.

8. Conclusions

Research on C. militaris has significantly advanced our understanding of its potential in health supplements and medicine, underpinned by its production of bioactive compounds such as cordycepin, polysaccharides, and triterpenoids. These compounds are known for their immunomodulatory, anti-cancer, anti-inflammatory, and antioxidant properties. Advances in genetic engineering and fermentation technology have greatly enhanced the production of these metabolites. The growth and metabolite profile of C. militaris are notably influenced by substrate type and environmental conditions such as temperature and humidity.

Moreover, the sustainable scale-up of C. militaris cultivation has been a focus, with strategies including the use of agricultural by-products and renewable energy in production processes to mitigate environmental impacts. This not only supports economic viability but also aligns with global sustainability efforts. C. militaris is proving invaluable in pharmaceuticals and nutraceuticals due to its therapeutic potential. Future research is expected to further explore the molecular mechanisms of its bioactive compounds, identify new clinical applications, and enhance production efficacy and environmental sustainability.

Continued innovations in biotechnological cultivation and sustainability measures are likely to significantly expand their role in health-related applications. Efforts to understand and improve C. militaris cultivation conditions, alongside advances in bioreactor technology, could standardize production, ensuring consistency in the quality of bioactive compounds. Such developments are essential for integrating this medicinal fungus more extensively into preventive health strategies and therapeutic regimes, highlighting its importance in both traditional and contemporary medical practices.