Cordycepin is the principal bioactive compound produced by Cordyceps militaris and exhibits diverse pharmacological properties. However, cordycepin production is highly sensitive to cultivation conditions, leading to substantially variable production amounts and challenges in process optimization. An interpretable machine learning framework was established in this study to predict the cordycepin produced by C. militaris cultivated on Pinus densiflora sawdust. Three key cultivation parameters—input weight, growth weight, and particle size—were quantified using submerged mycelial culture. The cordycepin content was measured via high-performance liquid chromatography. Four predictive models (random forest, support vector machine, XGBoost, and artificial neural network) were optimized through a randomized hyperparameter search and evaluated using internal validation and Tropsha’s external quantitative structure-activity relationship criteria. The validation accuracy of XGBoost was the highest (root mean square error = 42.67 μg/mL), whereas the external performance of random forest was the most reliable (R² = 0.898). Shapley additive explanations revealed that input weight most strongly influenced cordycepin production, followed by growth weight and particle size, with distinct nonlinear and interaction-driven effects among the cultivation variables. Kernel density and dependence analyses confirmed the occurrence of multimodal production regimes associated with the substrate loading and particle size characteristics. Finally, the best-performing model was deployed through a streamlit-based graphical user interface, enabling the real-time prediction of cordycepin concentration with a 95% confidence interval. The results collectively demonstrate the utility of interpretable AI-driven modeling for unveiling complex biological responses, providing a practical decision-support tool for optimizing cordycepin production in fungal biotechnologies.

The medicinal fungus Cordyceps militaris is recognized for producing cordycepin, a bioactive nucleoside with anticancer, immunomodulatory, and antioxidant properties. However, conventional culture media often entail high production costs and limited sustainability, prompting the search for alternative nutrient sources. This study evaluated onion, green onion, and garlic peel extracts—agricultural by-products rich in flavonoids, phenolics, and sulfur-containing antioxidants—as sustainable substrates for enhancing mycelial biomass and cordycepin biosynthesis in C. militaris. Liquid cultures supplemented with peel extracts (1–5%) were assessed for growth, cordycepin production (HPLC), and antioxidant activity (DPPH assay). Onion peel extract (OPE) showed the strongest growth-promoting effect, yielding 8.2 g/L of biomass at 5% and achieving a 19% increase in cordycepin concentration at 3% compared with the control. Antioxidant activity strongly correlated with cordycepin accumulation (R = 0.96, p < 0.001), indicating that secondary metabolite production contributed significantly to radicalscavenging capacity. Response surface methodology using a Box–Behnken design revealed that extract concentration, pH, and incubation period significantly influenced cordycepin production (p < 0.05), with the quadratic model showing excellent fit (R² = 0.9924). Optimal conditions were identified as 3% extract concentration, pH 6.0, and 12 days of incubation, under which cordycepin reached 0.995 mg/L, substantially higher than the control (0.693 mg/L). These findings demonstrate that agricultural by-product extracts, particularly onion peel, can serve as effective and economical substrates for enhancing cordycepin biosynthesis while supporting sustainable bioprocessing strategies in C. militaris cultivation.

This study was conducted to investigate the potential use of Cordyceps militaris spent mushroom substrate as a natural antimicrobial agent. The fruiting body and spent mushroom substrate of C. militaris were extracted into 80% ethanol, and the antimicrobial activity was compared. Both extracts (1 mg/disc) exhibited clear antimicrobial activity against Pseudomonas aeruginosa in the agar diffusion assay. The MIC values of the fruiting body and spent substrate extracts against P. aeruginosa were determined to be 0.4 mg/ml and 0.6 mg/ml, respectively, indicating a bacteriostatic action. The inhibitory effects of the C. militarisspent mushroom substrate extract on biofilm formation of P. aeruginosa were determined by biofilm biomass staining and qRT-PCR analysis. The biofilm biomass and cell growth of P. aeruginosa in the cultures treated with 0.2–2.0 mg/ml of extracts were significantly decreasedin a concentration-dependent manner. The qRT-PCR analysis showed that the lasI and lasR gene expression associated to quorum sensing (QS) in the cultures treated with 0.2–2.0 mg/ml of extracts were suppressed in a concentration-dependent manner. Based on the above results, it can be concluded that extracts from C. militaris spent mushroom substrate can be used as an antimicrobial agent derived from natural materials, as demonstrated by the bacteriostatic action and inhibition of biofilm formation of P. aeruginosa.

Cordyceps militaris is widely used in China, Korea, and other Asian countries as both a traditional medicinal ingredient and an edible fungus. This study aimed to optimize the growth conditions and fruiting body production of C. militaris by investigating various culture media and physical parameters such as pH, aeration, illumination, temperature, spawn materials, and oat–sawdust-based substrate formulations. After a 7-day incubation period, oats with a pH of 6.0, under sealed and illuminated conditions at 32°C, demonstrated the most effective mycelial growth. Substrates consisting of 70% oat and 30% sawdust had the shortest incubation time of 30.5 days for fruiting body formation. The basidiospores showed a typical germination pattern where the sporidium produced a single germ tube that elongated, and branched to form monokaryotic primary mycelia. In conclusion, using oats as a substrate in the cultivation of C. militaris could reduce production costs and help protect the environment.

Cordyceps militaris (C. militaris) is a unique and valuable medicinal fungus belonging to the Cordyceps species. C. militaris is the only fungus that contains cordycepin which is a biologically active compound. In previous studies, light-emitting diodes (LEDs) are known to be effective in increasing cordycepin content, but metabolic profiling of LED-stimulated C. militaris has not been confirmed. Metabolic profiling is essential to understanding the metabolic regulation of cordycepin. This study studied the physiologically active secondary metabolites of C. militaris according to the presence or absence of stimulation of LEDs through GC-MS analysis. Most of the metabolites were detected in both samples, but there was a clear difference in the detected concentration. In particular, C. militaris had a significant difference in amino acid levels when stimulated with LEDs. Our results suggested that LEDs could stimulate amino acid synthesis in C. militaris mycelium to increase the cordycepin content.

This study aimed to verify the whitening effect of Cordyceps militaris, which is distributed in several countries worldwide, including Korea, Japan, and China, and has various medical effects. To screen the efficacy of C. militaris, the inhibitory activity of tyrosinase, which was 66% at a concentration of 1 mg/mL, was measured. Thereafter, the survival rate of melanoma cells was measured, and cell experiments were conducted at a concentration of 90% or more in which C. militaris was not toxic to cells. After measuring the inhibitory effect of TRP-1, TRP-2, tyrosinase protein, and mRNA expression, which are factors influencing melanin synthesis, C. militaris was found to decrease in all factors, with an expression level that was significantly lower compared to quercetin. This confirmed that C. militaris stimulated with LED has excellent whitening activity and can be used as a functional whitening cosmetics material.

To investigate the anti-inflammatory activity of submerged culture using Cordyceps militaris mycelium, culture-including mycelia was extracted and lyophilized into postbiotics (hot-water extract; CM-HW). HW was fractionated into crude polysaccharide (CM-CP) by ethanol precipitation, and CM-CP was further dialyzed into CM-DCP by dialysis with running water using 12~14 kDa dialysis tube. When the cytotoxicity of subfractions against cells was assessed, no subfraction had a cytotoxic impact that was substantially different from the control groups. In an inflammatory model using LPS-stimulated RAW 264.7 cells, CM-DCP significantly decreased IL-6 and MCP-1 production levels compared to the LPS-control group. CM-DCP also inhibited IL-6 and IL-8 secretion in HaCaT keratinocytes stimulated with TNF-α and IFN-γ. In the meanwhile, the neutral sugar content and mannose ratio of anti-inflammatory CM-DCP were higher than the other fractions, and CM-DCP contained β-1,3/1,6-glucan of 216.1 mg/g. High pressure size exclusion chromatography revealed that CM-DCP contained molecules with a molecular weight range of 5.6 to 144.0 kDa. In conclusion, postbiotics of C. militaris mycelium significantly promoted anti-inflammatory activity, suggesting that neutral polysaccharides including Glc and Man contribute to the anti-inflammation in RAW 264.7 or HaCaT cells.

Owing to its excellent nutritional value, eggs are among the most important components of the human diet. Gender and environmental factors, such as feed composition, may alter the nutritional profile and quality of eggs. Feed additives have recently been used to enhance the health and productivity of hens, which has resulted in the production of higher-quality eggs. The fungus Cordyceps militaris, a well-established source of traditional medicines, contains potential bioactive metabolites, which prompted us to examine the effects of C. militaris-supplemented diets on the quality of hens’ eggs. The hens of two species (Gallus gallus domesticus and Araucana) were fed with one of three different diets: a control diet and diets supplemented with 2% or 5% of C. militaris. Egg quality was determined by measuring the Haugh Unit, yolk color, and shell thickness. In addition, egg and shell densities together with the ratio of yolk to albumen were calculated. Eggshell thickness and yolk color were both enhanced by the addition of C. militaris, whereas Haugh Unit values were somewhat reduced. Egg size, eggshell weight, and yolk and albumen production were all enhanced by C. militaris supplementation. Notably, in hens fed the 2% C. militaris-supplemented diet, enhancement was more evident in the yolk than in the albumen. The overall quality of the egg yolk was enhanced when 2% C. militaris was added to the hens' diet, which led to increases in both yolk color and quantity. Eggshell thickness and weight were also higher among eggs laid by hens fed the supplemented diets. Although these effects differed depending on the chicken species, we established that, in general, C. militaris contributes to improving egg quality.